Male meiotic spindle features that efficiently segregate paired and lagging chromosomes

Gunar Fabig; Robert Kiewisz; Norbert Lindow; James A Powers; Vanessa Cota; Luis J Quintanilla; Jan Brugués; Steffen Prohaska; Diana S Chu; Thomas Müller-Reichert

doi:10.7554/eLife.50988

. 2020 Mar 10;9:e50988. doi: 10.7554/eLife.50988

Male meiotic spindle features that efficiently segregate paired and lagging chromosomes

Gunar Fabig ^1,^✉, Robert Kiewisz ¹, Norbert Lindow ², James A Powers ³, Vanessa Cota ⁴, Luis J Quintanilla ⁴, Jan Brugués ^5,^6,⁷, Steffen Prohaska ², Diana S Chu ^4,^†, Thomas Müller-Reichert ^1,^†,^✉

Editors: Yukiko M Yamashita⁸, Anna Akhmanova⁹

PMCID: PMC7101234 PMID: 32149606

Abstract

Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a ‘tug of war’ reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.

Research organism: C. elegans

Introduction

Chromosome segregation during meiosis is regulated in each sex to produce different numbers of cells with distinct size, shape, and function. In humans, for example, up to 1500 sperm are continually generated per second via two rapid rounds of symmetric meiotic divisions. In contrast, in many organisms including humans, only oocytes that are fertilized will complete asymmetric meiotic divisions to produce one large cell and two polar bodies (El Yakoubi and Wassmann, 2017; L'Hernault, 2006; O'Donnell and O'Bryan, 2014; Severson et al., 2016; Shakes et al., 2009). While oocyte meiosis and mitosis have been studied in detail in many organisms (Bennabi et al., 2016; Müller-Reichert et al., 2010; Pintard and Bowerman, 2019), our knowledge of sperm meiotic chromosome segregation is still limited to studies in grasshoppers and crane flies using chromosome manipulation and laser microsurgery (LaFountain et al., 2011; LaFountain et al., 2012; Nicklas and Kubai, 1985; Nicklas et al., 2001; Zhang and Nicklas, 1995). Thus, despite recent alarming evidence of steep global declines in human sperm counts (Levine et al., 2017; Levine et al., 2018; Sengupta et al., 2018), little is known about the molecular mechanisms that drive male meiotic chromosome segregation required for efficiently forming healthy sperm.

The nematode Caenorhabditis elegans is an ideal model system to study sperm-specific features of chromosome segregation, as meiosis can be visualized in both sexes. C. elegans lacks a Y chromosome; thus, sex is determined by X chromosome number. Hermaphrodites have two X chromosomes (XX), while males have one (XO). This unpaired (univalent) X chromosome lags during anaphase I in males (Albertson and Thomson, 1993; Fabig et al., 2016; Madl and Herman, 1979). Previously, electron microscopy has defined the microtubule organization in female meiotic (Laband et al., 2017; Redemann et al., 2018; Srayko et al., 2006; Yu et al., 2019) and embryonic mitotic spindles (Albertson, 1984; O'Toole et al., 2003; Redemann et al., 2017; Yu et al., 2019) but a detailed study on spindle ultrastructure in spermatocytes, particularly at anaphase I showing the lagging X chromosome, is lacking.

Compared to oocytes or mitotic embryonic cells in C. elegans, spermatocytes exhibit vastly distinct features of spindle poles, chromosomes, and kinetochores (Crowder et al., 2015; Hauf and Watanabe, 2004). As in many other species, C. elegans centrosomes are present in spermatocyte meiosis (Wolf et al., 1978) and embryonic mitosis (O'Toole et al., 2003) but not in oocyte meiosis, where inter-chromosomal microtubules are reported to push chromosomes apart (Dumont et al., 2010; Laband et al., 2017; Redemann et al., 2018; Yu et al., 2019). Moreover, meiotic chromosomes in C. elegans resemble compact oblong spheres in spermatocytes and oocytes (Albertson and Thomson, 1993; Redemann et al., 2018; Shakes et al., 2009) but are long rods in mitosis (Oegema et al., 2001; Redemann et al., 2017). In addition, previous studies in C. elegans revealed the holocentric nature of meiotic and mitotic kinetochores (Albertson and Thomson, 1993; Howe et al., 2001; O'Toole et al., 2003) and the rounded structure of meiotic chromosomes (Dumont et al., 2010; Monen et al., 2005; Muscat et al., 2015; Wignall and Villeneuve, 2009). Meiotic kinetochore structure and dynamics, however, vary from mitosis, where kinetochores attach each sister to microtubules from opposite poles during the single division. Meiotic kinetochores must detach and re-attach to microtubules to allow sisters to switch from segregating to the same pole in meiosis I to opposite poles in meiosis II (Petronczki et al., 2003). In acentrosomal oocyte meiosis, this is accomplished because outer kinetochore levels dramatically decrease during anaphase I and then increase again before meiosis II (Dumont and Desai, 2012; Dumont et al., 2010). The structure and dynamics of centrosomal spermatocyte meiotic spindles, however, are largely unknown.

Different cell types also use distinct spindle structures to drive chromosome movement (McIntosh, 2017; McIntosh et al., 2012). For example, during the first centrosomal mitotic division in C. elegans, pole-to-pole separation (anaphase B) but not chromosome-to-pole shortening (anaphase A) drives chromosome movement (Nahaboo et al., 2015; Oegema et al., 2001; Scholey et al., 2016). In C. elegans acentrosomal oocyte spindles, shortening of the distance between chromosomes and poles was observed before microtubules disassemble at the acentrosomal poles (McNally et al., 2016). Pushing forces generated by microtubules assembled in the spindle midzone then drive the majority of segregation in oocyte meiosis (Laband et al., 2017; Yu et al., 2019). As yet, mechanisms that drive segregation in sperm centrosomal meiotic spindles are unknown.

To better understand sex-specific regulation of meiotic chromosome segregation and the resolution of lagging chromosomes, we quantitatively characterized the three-dimensional (3D) organization of spindles and the dynamics of chromosomes in C. elegans male spermatocytes. We applied electron tomography to produce large-scale 3D reconstructions of whole spindles in combination with a newly developed light microscopic approach for imaging chromosome and spindle dynamics in living males. Our approach defines molecular mechanisms of sperm-specific movements, focusing on the efficient segregation of both lagging and paired chromosomes.

Results

Spermatocyte meiotic spindles are distinguished by delayed segregation of the unpaired X chromosome

We developed in situ imaging within C. elegans males to visualize the dynamics of microtubules and chromosomes labeled with β-tubulin::GFP and histone::mCherry, respectively (Figure 1). Spermatocyte chromosomes arrange in a rosette pattern, with paired autosomes surrounding the unpaired X chromosome in metaphase I (Albertson and Thomson, 1993). In anaphase I, homologs segregate towards opposite poles; the unpaired X chromosome, however, remains behind and attached to microtubules connected to separating poles before resolving to one side (Figure 1A, M I, arrowheads, Figure 1—video 1; Albertson and Thomson, 1993). Thus in meiosis I, the unpaired X appears attached to both poles in contrast to the paired autosomes, each of which attaches to a single, opposite pole to enable segregation of homologs. In the second division, sister chromatids of each chromosome segregate all away from one another to opposite poles (Figure 1A,M II).

Figure 1. — (A) Time series of confocal image projections of meiosis I (M I) and meiosis II (M II) in males with microtubules (β-tubulin::GFP, green) and chromosomes (histone H2B::mCherry, red). Anaphase onset is time point zero (t = 0). White arrowheads mark the unpaired X chromosome position. Right panels show corresponding kymographs with chromosomes in white, microtubules in green. Anaphase onset is marked with a dashed orange line. Scale bars (white), 2 µm; time bar (blue), 2 min. (B) Confocal image projections of spermatocyte meiosis I in wild-type XO males, wild-type XX hermaphrodites, *tra-2*(e1094) XX males, *him-8*(e1489) XX hermaphrodites, *zim-2*(tm574) XX hermaphrodites, *zim-2*(tm574) XO males and triploid XXO males with centrosomes in green (γ-tubulin::GFP) and chromosomes in red (histone H2B::mCherry). The genotype, sex, number of autosome or X chromosome pairs, and number of unpaired chromosomes is indicated. Still images illustrate the progression of the first meiotic division over time with lagging chromosomes indicated by white arrowheads. In the corresponding kymographs (right panels), chromosomes are shown in white, spindle poles in green. Anaphase onset is marked with a dashed line (orange). Scale bars (white), 2 µm; time bar (blue), 2 min.

Lagging of chromosomes is a consequence of a lack of pairing

Next, we probed if the lagging of X may be due to a lack of having a pairing partner. Because both males and hermaphrodites undergo spermatogenesis in C. elegans, we compared spermatocytes of wild-type males (XO) to those in animals with different numbers of chromosomes (Figure 1B). First, though the unpaired X chromosome lags in wild-type XO males, paired X chromosomes in wild-type XX hermaphrodite spermatocytes did not. Although we noticed an initial delay in the segregation of a chromosome in hermaphrodite spermatocytes (n = 5/10), this slight lagging was not obvious at mid to late anaphase I. Further, we determined whether paired X chromosomes lag in males by analyzing mutants with the tra-2(e1094) mutation, which causes a somatic transformation of XX animals to males (Hodgkin and Brenner, 1977). In over 80% (n = 43/53) of tra-2(e1094) XX male spindles we did not detect lagging chromosomes during meiosis I. However, in about 20% of tra-2(e1094) spindles, we detected lagging to some extent, possibly due to improper pairing in prophase. Therefore, the majority of paired X chromosomes in male spermatocyte spindles do not lag in mid/late anaphase I, similar to paired sex chromosomes in hermaphrodite spermatocytes.

We next examined X chromosome lagging in him-8(e1489) hermaphrodite spermatocytes to eliminate the possible effect of the male soma in causing chromosomes to lag in meiosis I. A mutation of him-8 results in lack of pairing of the X; thus, pairing, synapsis, and recombination of the X chromosomes do not occur (Phillips et al., 2005). We observed lagging chromosomes in anaphase I in 70% (n = 14/20) of the analyzed him-8(e1489) spindles in hermaphrodite spermatocytes, presumably representing the two unpaired X chromosomes. This reveals that anaphase I chromosome lagging is likely caused by an inability to undergo synapsis rather than by a somatic effect of the male sex.

We further excluded that lagging is exclusive to the X chromosome by analyzing hermaphrodite and male spermatocytes with the zim-2(tm574) mutation, which prevents pairing of autosome V (Phillips and Dernburg, 2006). At least one chromosome lagged in all spindles in hermaphrodite (n = 10) and male spermatocytes (n = 5). Moreover, we created triploid males with spermatocytes containing five unpaired autosomes (Madl and Herman, 1979) and detected a massive fluorescent signal between segregating autosomes that we infer likely corresponds to the five unpaired autosomes in all spindles (n = 32). Collectively, these results show that lagging chromosomes during spermatocyte anaphase I are indeed a consequence of the lack of pairing and synapsis of any chromosome during prophase I and are not specific to sex chromosomes.

Microtubules attached to the X chromosome exert a pulling force

During anaphase I, we observed that the lagging X changes shape (Figure 2A, upper panel). To quantify this change, we calculated a shape coefficient (the ratio of length over diameter) of the X chromosome in fluorescent 3D image data over time (Figure 2A, lower panel). With this measure, stretched chromosomes have a shape coefficient greater than 1. Indeed, the X chromosome was significantly stretched early in anaphase I with a shape coefficient of 1.4 that decreased to 1.0 as it rounded up in late anaphase I (Figure 2B). This suggests that the X is under tension from pulling forces as spindle poles separate, which is released as the lagging chromosome resolves to one side.

To further assess pulling forces during anaphase, we used laser microsurgery on X chromosome-attached microtubules. We reasoned a cut on one side of the lagging X would release tension and induce segregation to the opposite side. Laser point-ablation to the bundle on one side of the X caused an immediate and continuous movement of the X towards the unablated side (n = 21/26, two examples shown; Figure 2C, Figure 2—video 1). The velocity of the X chromosome movement after the cut was variable, similar to unperturbed spindles, which also displayed variability in the speed of X chromosome movement, ranging from 0.7 to 4.9 µm/min (mean = 2.2 µm/min, n = 40) and the onset of X chromosome movement relative to anaphase onset, ranging from 1.5 to 8.5 min (mean = 4.9 min, n = 55, data not shown). We also tested cutting the microtubule bundles sequentially on each side of the X chromosome (Figure 2D, Figure 2—video 2). After initiation of movement by the first cut, the second cut on the opposite side caused a rapid shift in the direction of segregation, indicating microtubule connections are highly dynamic during anaphase I. Taken together, we conclude that kinetochore microtubules exert a pulling force on chromosomes during anaphase. This tension is most obvious on the microtubules connected to the X chromosome.

Kinetochores are not disassembled between spermatocyte meiotic divisions

A critical connection between chromosomes and microtubules required for forces that pull chromosomes during anaphase are kinetochores. However, in oocyte meiosis the localization of outer kinetochore proteins on chromosomes drops dramatically during anaphase I, allowing central spindle components to largely drive chromosome movement (Dumont and Desai, 2012; Dumont et al., 2010). We thus applied immunofluorescence microscopy to determine the localization of kinetochore components. Strikingly, the outer kinetochore proteins, KNL-1 (Desai et al., 2003), KNL-3 (Cheeseman et al., 2004) and NDC-80 (Desai et al., 2003) are retained on the poleward sides of the rounded chromosomes in a cup-shaped pattern on autosomes and X chromosome during anaphase I (Figure 2E and Appendix 1—figure 1), in contrast to their rapid depletion during oocyte meiosis. Using super-resolution microscopy, we further found kinetochores bridge interactions between chromosomes and microtubules (Appendix 1—figure 1). Outer kinetochore retention suggests that chromosome-to-pole attachments are important to drive chromosome movement during sperm anaphase.

Spermatocyte spindles maintain both end-on and lateral associations of kinetochore microtubules to chromosomes throughout meiosis

To further determine how spermatocyte spindles reorganize during anaphase, we applied large-scale electron tomography to visualize the ultrastructure of whole spindles in different stages of meiosis I with single-microtubule resolution (Redemann et al., 2017; Redemann et al., 2018; Yu et al., 2019). We segmented centrioles, microtubules, autosomes (a), and the X chromosome (x) (Figures 3 and 4, left panels). Reconstructed spindles were staged by 1) correlating the pole-to-pole and the pole-to-autosome distance with the autosome-to-autosome distance, 2) comparing our tomographic data with live-imaging data of chromosome and pole dynamics (Appendix 1—figure 2), and 3) correlating centrosome dynamics with those observed by live imaging, where centrosome volume decreased rapidly after onset of anaphase I and centrosomes flattened out before splitting into two spindle poles (Schvarzstein et al., 2013; Appendix 1—figure 3; see also Materials and methods). Accordingly, we generated: three spindles at metaphase (Figure 3A–C; Figure 3—videos 1–3), one at anaphase onset (Figure 4A; Figure 4—video 1), three complete spindles (Figure 4B–D; Figure 4—videos 2–4) and three partial spindles (Appendix 1—figure 4) at early to mid-anaphase, and one spindle at late anaphase (Figure 4E; Figure 4—video 5).

Figure 3. — (A) Early metaphase spindle (Metaphase no. 1) with unstretched chromosomes. (**B–C**) Metaphase spindles (Metaphase no. 2 and 3) with stretched chromosomes. Left panels: tomographic slices showing the centrosomes (c, with centrioles in purple), the autosomes (a), and the unpaired X chromosome (x) aligned along the spindle axis, mitochondria (m) and fibrous body-membranous organelles (fb). Mid left panels: corresponding three-dimensional models of the full spindles. Autosomes are in different shades of either blue or cyan, the X chromosome in red, centriolar microtubules in purple, microtubules within 150 nm to the chromosome surfaces in yellow, and all other microtubules in gray. Mid right panels: association of kinetochore microtubules with the kinetochores. Kinetochores are shown as semi-transparent regions around each chromosome. The part of each microtubule entering the kinetochore region around the holocentric chromosomes is shown in green. Right panels: visualization of end-on (white) *versus* lateral (orange) associations of microtubules with chromosomes. Only the parts of microtubules inside of the kinetochore region are shown. The autosome-to-autosome distance (A-A) for each reconstruction is indicated in the left column. Scale bars, 500 nm.

Figure 4. — (A) Spindle at Anaphase onset. (B) Spindle at mid anaphase (Anaphase no. 1) with a pole-to-pole distance of 2.98 µm. (C) Mid anaphase spindle (Anaphase no. 3) with a pole-to-pole distance of 3.35 µm. (D) Mid anaphase spindle (Anaphase no. 4) with a pole-to-pole distance of 3.37 µm. (E) Spindle at late anaphase (Anaphase no. 7) with a pole-to-pole distance of 5.44 µm and the X chromosome with initial segregation to one of the daughter cells. Left panels: tomographic slices showing the centrosomes (c, with centrioles in purple), the autosomes (a), and the unpaired X chromosome (x) aligned along the spindle axis, mitochondria (m) and fibrous body-membranous organelles (fb). Mid left panels: corresponding three-dimensional models illustrating the organization of the full spindle. Autosomes are in different shades of either blue or cyan, the X chromosome in red, centriolar microtubules in purple, microtubules within 150 nm to the chromosome surfaces in yellow, and all other microtubules in gray. Mid right panels: association of kinetochore microtubules with the kinetochores. Kinetochores are shown as semi-transparent regions around each chromosome. The part of each microtubule entering the kinetochore region around the holocentric chromosomes is shown in green. Right panels: visualization of end-on (white) *versus* lateral (orange) associations of microtubules with chromosomes. Only the parts of microtubules inside of the kinetochore region are shown. The autosome-to-autosome distance (A-A) for each reconstruction is indicated in the left column. Scale bars, 500 nm.

In our tomographic reconstructions, the holocentric kinetochore was visible as a ribosome-free zone around the chromosomes (Howe et al., 2001; O'Toole et al., 2003; Redemann et al., 2017) from metaphase throughout anaphase. This is in accordance with kinetochore retention during anaphase I and the association distance measured by super-resolution light microscopy (Figure 2E and Appendix 1—figure 1). Therefore, microtubules terminating or traversing in these ribosome-free zones with a width of 150 nm were considered kinetochore microtubules. The remaining microtubules were annotated as other microtubules (Figures 3 and 4 and Appendix 1—figure 4, mid left panels; mid right panels showing kinetochore microtubules only).

Next, we determined the number of kinetochore microtubules. We reconstructed ~2000 microtubules in metaphase I and ~1500 in anaphase I data sets. For metaphase I,~30–38% were kinetochore microtubules, which increased to ~45–53% for anaphase I (Table 1). Interestingly, this percentage is much higher compared to early mitosis where only 2–4% of all microtubules attach to the kinetochore (Redemann et al., 2017). We also determined the types of association kinetochore microtubules make to chromosomes (see Materials and methods). Similar to previous analysis of oocyte meiosis (Dumont et al., 2010; Laband et al., 2017; Muscat et al., 2015; Redemann et al., 2018), we recognized both a lateral and an end-on association of kinetochore microtubules to chromosomes (for details see Table 1). We found both types of association for the autosomes and the unpaired X at all reconstructed meiotic stages. This indicated to us that a complex pattern of both lateral and end-on associations is maintained throughout male meiotic progression.

Table 1. Analysis of tomographic data sets used throughout this study.

	Full data sets								Partial data sets
Spindle parameters	Metaphase no. 1	Metaphase no. 2	Metaphase no. 3	Anaphase onset	Anaphase no. 1	Anaphase no. 3	Anaphase no. 4	Anaphase no. 7	Anaphase no. 2	Anaphase no. 5	Anaphase no. 6
MTs total	1729	2406	1689	2051	1405	1540	1403	1881	(893)	(671)	(246)
MTs within 150 nm from chromosomes (KMTs)	524	912	650	794	633	821	752	944	(580)	(499)	(160)
End-on associated KMTs on X-chromosome	29	38	38	53	27	50	38	42	57	30	43
Lateral associated KMTs on X-chromosome	79	34	91	22	61	55	33	34	47	52	28
End-on associated KMTs on autosomes	154	355	199	318	106	189	181	175
Lateral associated KMTs on autosomes	262	485	321	400	437	527	500	692
Autosome-to-autosome distance [µm]	0.76	0.94	0.99	1.00	2.98	3.35	3.37	5.44	(3.14)	(3.58)	(4.47)
Autosomes1-to-X distance [µm]	0.37	0.43	0.43	0.47	1.45	1.45	1.37	1.95	(1.54)	(1.71)
Autosomes2-to-X distance [µm]	0.39	0.51	0.56	0.54	1.56	2.02	1.99	3.51	(1.72)	(1.90)
Pole-to-pole distance [µm]	3.10	3.41	3.45	3.51	4.97	5.22	4.99	7.04
Pole1-to-X distance [µm]	1.64	1.66	1.66	1.62	2.60	2.29	2.21	2.69
Pole2-to-X distance [µm]	1.48	1.76	1.80	1.90	2.39	3.04	2.77	4.38
Autosome-to-centrosome distance [µm]	1.18	1.24	1.23	1.26	1.00	0.94	0.82	0.83
Mother-to-daughter centriole distance [µm]	0.26	0.21	0.27	0.35	0.37	0.73	0.74	1.11
Original name of data set	T0391_worm13 metaphase01	T0391_worm14 metaphase	T0391_worm13 metaphase02	T0391_worm13 meta-anaphase01	T0391_worm05 anaphase02	T0391_worm08 lateanaphase	T0391_anaphase01 early	T0391_worm09 late_anaphase	T0391_worm07b	T0391_worm06	T0391_worm02
Number of sections	14	11	14	25	14	17	11	30	8	4	6
Est. tomographic volume [µm³]	102.33	94.70	107.52	115.12	113.96	131.92	101.51	268.31	36.99	18.52	30.37

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The table summarizes all microtubule numbers and distances as measured within the electron tomographic reconstructions in this study. A kinetochore microtubule (KMT) is defined as a microtubule that is at least 150 nm from the surface of a chromosome. KMTs are sub-divided into end-on and lateral associated MTs. End-on KMTs are defined as pointing towards the chromosome surface, lateral MTs are all remaining KMTs. Distances were measured between the geometric centers of autosomes (mean position of individual autosomes), centrosomes (center point of both centrioles) and centrioles (between the centers of the mother and daughter centriole). Tomographic volumes were estimated by multiplying the X-Y dimensions of each tomogram with the number of sections (with a section thickness of 300 nm).

Continuous and lengthening microtubules connect the X chromosome to centrosomes during anaphase I

One phenomenon of spermatocyte meiosis is the kinetochore microtubules that connect the lagging X to opposite spindle poles lengthen during anaphase I. This is unusual because in most centrosomal cell-types, microtubules either shorten (anaphase A) or stay the same length as poles separate (anaphase B). Further, in C. elegans mitosis, continuous microtubules do not directly connect centrosomes and chromosomes, but instead anchor into the spindle network (Redemann et al., 2017). We thus used electron tomography to determine the continuity of X-connected kinetochore microtubules during anaphase I. We found that microtubules directly connect the X to each centrosome throughout anaphase I (Figures 5A–B and 4B–E, mid right panels, Appendix 1—figure 5A and B). Further, microtubules with both end-on and lateral associations to X increased in length as X chromosome-to-pole distance increased until the X resolved to one side (Figure 5C–D). Thus, continuous microtubules connect the X chromosome to poles even as poles elongate during anaphase I, consistent with the observation that outer kinetochore protein levels are retained on the X throughout spermatocyte divisions (Figure 2E, Appendix 1—figure 1).

(A) Length distribution of end-on X chromosome-associated microtubules at different stages of meiosis I. Dots show the mean, error bars indicate the standard deviation. Dashed lines indicate a grouping of the spindles according to the meiotic stages: metaphase/anaphase onset, mid anaphase and late anaphase (see also Appendix 1—figure 2). (B) Length distribution of lateral X chromosome-associated microtubules at different stages of meiosis I. (C) Mean length of microtubules plotted for each side of the X chromosome against the respective X chromosome-to-pole distance (each tomographic data set color-coded). The values for end-on and laterally associated microtubules are given separately. The measurements were performed on all data sets as shown in Figures 3 and 4. A trend line was fitted to indicate the linear relationship between microtubule length and chromosome-to-pole distance. (D) Similar plot as in (C) but end-on (yellow) and laterally (purple) X chromosome-associated microtubules are shown. Two trend lines were fitted to the data sets to illustrate linear relationships independent of the type of association of the microtubules with the X chromosome.

Figure 5—source data 1. Measurements of microtubule lengths in spindles shown in Figures 3 and 4 used to generate data in Figure 5.

elife-50988-fig5-data1.xlsx^{(20.3KB, xlsx)}

We also observed that X-associated microtubules were curved during late anaphase I. To assess the curvature of end-on and lateral X-associated kinetochore microtubules, we measured the tortuosity of individual microtubules by calculating the ratio of the spline length over the end-to-end length (Appendix 1—figure 6A). At metaphase I and anaphase I onset, the tortuosity ratio was one, indicating kinetochore microtubules were straight. However, at anaphase, X-connected microtubules exhibited higher tortuosity, indicating higher curvature (Figures Appendix 1—figures 6B and 5C-D). In addition, laterally associated microtubules had a higher degree of tortuosity compared to end-on associated microtubules. This suggests that other cellular forces, besides those generated by pulling forces, may also be acting on microtubules connected to the lagging X during anaphase I.

Segregation of the X chromosome correlates with an asymmetry in the number of associated microtubules

To further characterize features of X chromosome lagging and resolution, we determined the ratio of microtubule length in confined volumes on each side of X for each tomographic data set (Figure 6A), which were then plotted against autosome-to-autosome distance (Figure 6B). We also determined the number of kinetochore microtubules on each side of X and calculated the ratio of these two values (Figure 6C–D). The ratio of total microtubule length and microtubule number were about one in metaphase and early anaphase, suggesting microtubules are present equally on both sides. As anaphase progresses, this ratio deviated from one, indicating less microtubules on one side that presumably enable the X to resolve to the opposing side.

Additionally, we tracked microtubules (β-tubulin::GFP) relative to chromosomes (histone H2B::mCherry) by live-cell imaging (Figure 6E, upper panel). Measuring the ratio of total GFP fluorescence in similar 3D volumes on each side of the X over time (Figure 6E, lower panel) showed a ratio of almost one at early anaphase I, indicating similar microtubule content on each side. As the X chromosome segregated to one side, we detected increased intensity on the side the X moved closer toward (Figure 6F). Thus, an asymmetry in associated microtubules correlates with X chromosome resolution, where attachments likely stochastically break as poles separate, allowing the X to resolve to the side with more associated microtubules.

Interdigitating midzone microtubules are not a prominent feature during spermatocyte anaphase progression

A hallmark of anaphase progression in C. elegans embryonic mitosis and oocyte meiosis is a structure of overlapping spindle midzone microtubules that forms between separating chromosomes known as the central spindle (Yu et al., 2019). However, the lagging X chromosome in wild-type male spermatocytes precluded detection of such a structure in either light microscopy or electron tomography. We thus examined spermatocytes in tra-2(e1094) XX males, where paired X chromosomes do not lag. By live imaging, we detected only a weak microtubule signal in early anaphase I between segregating chromosomes (Appendix 1—figure 7A). Interestingly, tra-2(e1094) males without a lagging chromosome exhibited a faster spindle elongation rate and a longer final pole-to-pole distance compared to wild-type males that have a lagging X (Appendix 1—figure 7A, B and Figure 1B). By electron tomography, we detected about 160 microtubules in the anaphase I spindle midzone of tra-2(e1094) males (Appendix 1—figure 7C; Appendix 1—figure 7—video 1). These microtubules, however, did not show an interdigitated pattern characteristic of C. elegans mitotic or oocyte meiotic spindle midzones (Yu et al., 2019). Thus, even in the absence of a lagging chromosome, male meiotic spindles do not form a typical spindle midzone of overlapping microtubules.

Appendix 1—figure 7. — (A) Time series of confocal image projections of three examples of *tra-2*(e1094) mutant spindles in meiosis I. Microtubules (β-tubulin::GFP) and chromosomes (histone H2B::mCherry) are visualized in green and red, respectively. Anaphase onset is time point zero (t = 0). Chromosome segregation is also visualized in kymographs (right panels). The start of the separation of the autosomes is indicated by a dashed line (orange). Scale bars (white), 2 µm; time bar (blue), 2 min. (B) Quantitative analysis of spindle elongation (pole-to-pole distance) over time in meiosis I in wild-type (green) and *tra-2*(e1094) (blue) spindles. Anaphase onset is time point zero (t = 0). The mean and standard deviation is given (circles and shaded areas). (C) Tomographic slice through a partial *tra-2*(e1094) mutant spindle in anaphase I (left panel). Autosomes (a), mitochondria (m) and FB-MOs (fb) are indicated. Three-dimensional model of the same *tra-2*(e1094) mutant spindle (right panel). Chromosomes are in blue, microtubules within a distance of 150 nm or closer to the chromosome surfaces in yellow and all other microtubules in white. The spindle midzone is indicated (red rectangle). Scale bars, 500 nm.

Appendix 1—figure 7—source data 1. Measurements of pole-to-pole distance over time in replicates used in Appendix 1—figure 7B.

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Further, central spindle specifiers that localize in the spindle midzone in oocyte meiosis and mitosis did not localize within the midzone during mid to late sperm meiotic anaphase I in both the presence (him-8(e1489)) or absence (tra-2(e1094)) of a lagging X chromosome (Appendix 1—figure 8A). First, Aurora B^AIR-2, a component of the chromosomal passenger complex (Davies et al., 2014; de Carvalho et al., 2008; Dumont et al., 2010; Maton et al., 2015; Schumacher et al., 1998; Severson et al., 2000), associated with separating autosomes or the lagging X during anaphase I. CLASP^CLS-2, a microtubule stabilizer (Dumont et al., 2010; Maton et al., 2015; Nahaboo et al., 2015), localized to the inside face of separating chromosomes and remained chromosome-associated during anaphase I. A centralspindlin component, MKLP1^ZEN-4, localized between separating chromosomes at very early anaphase, then to the cell membrane in a ring-like structure likely on the ingressing furrow at mid-anaphase (Powers et al., 1998; Raich et al., 1998). Similarly, using live-imaging, PRC1^SPD-1 (Mullen and Wignall, 2017; Nahaboo et al., 2015; Verbrugghe and White, 2004), a known microtubule bundling factor, initially briefly localized between segregating autosomes and around the X chromosome in very early anaphase I, but rapidly disappeared as anaphase I progressed in males and hermaphrodite spermatocytes (Appendix 1—figure 8B). Thus, spindle elongation, the segregation of autosomes, and the resolution of the X continued even without a detectable signal of PRC1^SPD-1 in between chromosomes. Overall, these results suggest that male meiotic spindles, in the presence or absence of a lagging X, do not form a ‘canonical’ midzone structure during mid-to-late anaphase I.

Spermatocyte meiotic spindles display both anaphase A and anaphase B movement

We next investigated how spermatocyte meiotic spindles drive chromosome movement over time. We measured changes in pole-to-pole (P-P), autosome-to-autosome (A-A) and pole-to-autosome (P-A) distances during both meiotic divisions using a strain with centrosomes labeled with γ-tubulin::GFP and chromosomes with H2B::mCherry. In meiosis I (Figure 7A–B; Figure 7—video 1), the pole-to-pole distance increased from 4.1 ± 0.3 µm to 8.0 ± 0.6 µm (mean ± SD; n = 31) with an elongation speed of 1.29 ± 0.36 µm/min (Figure 7E). This speed is significantly higher compared to 0.6–0.8 µm/min reported for both female meiotic divisions (McNally et al., 2016). We also found a simultaneous anaphase A-type movement in spermatocytes with pole-to-autosome distance decreased by half, from 1.6 ± 0.3 µm to 0.8 ± 0.3 µm and a speed of 0.39 ± 0.27 µm/min (Table 2). In addition, the autosome-to-autosome distance increased from 0.9 ± 0.2 µm to 6.5 ± 0.4 µm (mean ± SD; n = 31). Roughly, from this 5 µm increase in autosome-to-autosome distance, anaphase B provides about 4 µm (~80%) of separation, whereas anaphase A provides only 1 µm (~20%). Chromosome dynamics in meiosis II also exhibited anaphase A and anaphase B-type movements (Figure 7C–D and F; Figure 7—video 2; Table 2).

Figure 7. — (A) Schematic representation of metaphase and anaphase during meiosis I. Centrosomes are in green, autosomes in blue, and the univalent X chromosome in red. The pole-to-pole (P-P, green), autosome-to-autosome (A-A, red), and both pole-to-autosome distances (P-A, orange) are indicated. (B) Time series of confocal image projections of a spindle in meiosis I with centrosomes labeled with γ-tubulin::GFP (green) and chromosomes with histone H2B::mCherry (red). The separation of the centrosomes (yellow dashed line) and the autosomes (white dashed line) over time is indicated. Anaphase onset is time point zero (t = 0). Scale bar, 2 µm. (C) Schematic representation of metaphase and anaphase during meiosis II. (D) Separation of centrosomes and autosomes in meiosis II as in (F). Scale bar, 2 µm. (E) Quantitative analysis of autosome and centrosome dynamics in meiosis I show a decrease in pole-autosome distance that is characteristic of anaphase A. Anaphase onset is time point zero (t = 0). The mean and standard deviation is given (circles and shaded areas). (F) Quantitative analysis of autosome and centrosome dynamics in meiosis II. (G) Length distribution of end-on autosome-associated kinetochore microtubules at different stages of meiosis I. Dots show the mean, error bars indicate the standard deviation. Dashed lines indicate a grouping of the spindles according to the meiotic stages: metaphase/anaphase onset, mid anaphase and late anaphase (see also Appendix 1—figure 2). (H) Length distribution of laterally autosome-associated kinetochore microtubules at different stages of meiosis I.

Figure 7—source data 1. Measurements of autosome and centrosome dynamics from replicates used in Figure 7 .

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Table 2. Measurements of spindle dynamics in male meiosis.

Distance	Spindle parameter	Meiosis I		Meiosis II
Distance	Spindle parameter	Mean	SD	Mean	SD
P-P¹	Initial spindle length (metaphase)	4.1 µm	±0.3 µm	4.2 µm	±0.4 µm
	Final spindle length (end of anaphase)	8.0 µm	±0.6 µm	8.8 µm	±0.8 µm
	Initial rate (1 st minute)	1.29 μm/min	±0.36 μm/min	1.11 μm/min	±0.42 μm/min
	Duration of elongation	3–4 min		8–9 min
A-A²	Initial spindle length (metaphase)	0.9 µm	±0.2 µm	0.6 µm	±0.2 µm
	Final spindle length (end of anaphase)	6.5 µm	±0.4 µm	8.0 µm	±0.8 µm
	Initial rate (1 st minute)	2.07 µm/min	±0.37 μm/min	2.16 µm/min	±0.32 μm/min
	Duration of elongation	4–5 min		8–9 min
P-A³	Initial spindle length (metaphase)	1.6 µm	±0.3 µm	2.0 µm	±0.4 µm
	Final spindle length (end of anaphase)	0.8 µm	±0.3 µm	0.4 µm	±0.2 µm
	Initial rate (1 st minute)	−0.39 µm/min	±0.27 μm/min	−0.64 µm/min	±0.33 μm/min

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Distances: ¹P-P, pole-to-pole distance; ²A-A, autosome-to-autosome distance; ³P-A, pole-to-autosome distance. Initial spindle length is given at metaphase, final distance refers to the end of anaphase when spindle elongation plateaus. Values are given as mean values (± standard deviation, SD). The numbers of analyzed spindles are: n = 31 for meiosis I; n = 50 for meiosis II.

Taken together, spermatocyte meiotic spindles in C. elegans exhibit both anaphase A and B-type movements. This is distinct from mitosis in the early C. elegans embryo, which utilizes only anaphase B mechanisms (Oegema et al., 2001), or oocyte meiosis, which uses acentrosomal mechanisms (Dumont et al., 2010; McNally et al., 2016; Muscat et al., 2015; Redemann et al., 2018). Similar to grasshopper spermatocytes (Ris, 1949), anaphase A and B movement occurs simultaneously, with anaphase A contributing approximately one fifth to the overall chromosome displacement.

Electron tomography does not suggest a shortening of autosome-associated kinetochore microtubules during anaphase

A well-described mechanism for anaphase A (i.e. a decrease in chromosome-to-pole length) is microtubule shortening (Asbury, 2017). We thus analyzed individual kinetochore microtubule lengths in our 3D EM reconstructions. We set the metaphase I (data set Metaphase no. 1) as the earliest, since end-on kinetochore microtubules associated to autosomes were slightly longer than all other data sets (0.79 ± 0.3 µm; n = 153; Figure 7G). We speculate at this point chromosomes may not be fully under tension. In both other metaphase data sets, the length of end-on associated microtubules was 0.62 and 0.68 µm. Unexpectedly, as anaphase I progressed and the autosome-to-pole distance decreased, we observed that end-on kinetochore microtubules did not significantly shorten, remaining at 0.59–0.65 µm (Appendix 1—figure 9A and Appendix 1—figure 5E). Interestingly, the length of laterally associated microtubules did increase from 1.28 µm in metaphase to 1.44 µm in anaphase (Figure 7H, Appendix 1—figures 9B and 5F). Thus, unlike in other systems (Asbury, 2017), our tomographic analysis suggests that shortening of kinetochore microtubules does not fully account for the anaphase A observed by light microscopy.

Tension release across the spindle may contribute to autosomal anaphase A

To account for anaphase A in spermatocyte meiosis, we hypothesized that changes in the shape of chromosomes, centrosomes, and the association angles of kinetochore microtubules with autosomes induced by tension released at the metaphase to anaphase transition (Dumont and Mitchison, 2009; Gardner et al., 2005) may contribute to the decrease in chromosome-to-centrosome distance.

First, to examine the release of chromosome stretch that peaked at metaphase chromosome alignment, we measured individual autosome expansion along the spindle axis by plotting the cross-sectional areas over the chromosome distance. This generated a stretch value obtained at the Full Width at Half-Maximum (FWHM) of a Gaussian fit to the cross-sectional area along the spindle axis (Figure 8A; see Materials and methods). The autosomes of the first metaphase data set were the least stretched (0.52 ± 0.03 µm), consistent that the chromosomes in this data set were not yet under full tension. Chromosomes in metaphase data set no. two were most stretched (0.73 ± 0.12 µm). As chromosomes separated, autosomes rounded up to a value of 0.56 µm in anaphase data sets no. four and no. 7 (Figure 8B, Appendix 1—figure 5G). This is about 23% less compared to metaphase no. 2, thereby moving chromosome centers closer to the poles. Thus, the release during anaphase of chromosome stretch induced by metaphase alignment accounts for a portion of anaphase A pole-chromosome shortening.

(A) Analysis of autosome stretching in anaphase I. Schematic representation showing how the full width half maximum (FWHM) of stretching along the pole-to-pole axis for a single autosome is assessed (see also Materials and methods). (B) FWHM of chromosome stretch for all autosomes at each meiotic stage shown in Figures 3 and 4. The mean, the standard deviation and the number of measurements (n = 10) for each meiotic stage are given. Dashed lines indicate a grouping of the spindles according to the meiotic stages: metaphase/anaphase onset, mid anaphase and late anaphase (see also Appendix 1—figure 2). Additional ANOVA results are shown in Appendix 1—figure 5G. (C) Schematic depicting the determination of the distance of individual kinetochore microtubule plus ends to the closest centriole. For each kinetochore microtubule (green line), the direct distance (yellow line) from the putative plus end to the respective centriole was measured (plus ends of kinetochore microtubules are shown in circles). (D) Distance of kinetochore microtubule plus-ends to centrioles for each meiotic stage. Additional ANOVA results are shown in Appendix 1—figure 5H. (E) Analysis of the attachment angle of end-on-associated autosomal kinetochore microtubules. The schematic illustrates the defined main axis for the measurements (dashed line from the center of each autosome to the center of the centrosome). The angle (α) between each line connecting the kinetochore microtubule plus-end and the autosome center (green lines) and the main axis (dashed line) was measured for each kinetochore microtubule. (F) Plot showing the angle measurements for all data sets. Additional ANOVA results are given in Appendix 1—figure 5I.

Figure 8—source data 1. Measurements for Full Width at Half-Maximum(FWHM) of replicates in each stage shown in Figures 3 and 4 used in Figure 8.

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Second, we considered that as centrosomes split and shift from a spherical to a stretched shape, spindle poles may thus move closer to chromosomes (Appendix 1—figure 3). Because the outline of centrosomes cannot be clearly distinguished in EM data, we measured the distance of the plus-end of the kinetochore microtubules to the closest centriole (Figure 8C). This distance significantly shortened when comparing metaphase data sets (0.99–1.14 µm) to the anaphase data sets (0.78–0.95 µm; Figure 8D, Appendix 1—figure 5H), resulting in autosomes being 0.2 µm or 20% closer to centrioles.

Third, we hypothesized tension release would also alter the attachment angle of end-on kinetochore microtubules with autosomes, bringing chromosomes closer to spindle poles. We determined the angle between each kinetochore microtubule plus-end at the chromosome surface and each centrosome-chromosome axis (Figure 8E). The angle in the metaphase data sets was 37°- 41°. As autosomes rounded up during anaphase, the attachment angle in the anaphase data sets increased to 46°- 59°, bringing chromosomes closer to poles (Figure 8F, Appendix 1—figure 5I). Simple trigonometric calculations with a constant microtubule length of 0.63 µm found this increase in the attachment angle contributes about 0.17 µm or 17% shortening in chromosome-to-pole distance.

In sum, we identify three factors that contribute to pole-chromosomes shortening during anaphase: 1) a loss of chromosome stretch during anaphase, which shortens the chromosome by about 0.34 µm; 2) changes in centrosome size and shape that contributes about 0.2 µm; and 3) the opening of the attachment angle that accounts for 0.17 µm. These factors comprise ~70% of the total ~1 µm chromosome-to-pole distance shortening observed in spermatocyte meiosis, though this may be underestimated due to limitations in the tomographic reconstruction from serial semi-thick sections. Overall, our ultrastructure analysis revealed previously unknown, alternative mechanisms that contribute to anaphase-A movement.

Discussion

Prior to this work, only a handful of studies addressed spermatocyte spindle dynamics during male meiosis (Fegaras and Forer, 2018; Felt et al., 2017; Golding and Paliulis, 2011; LaFountain et al., 2011; LaFountain et al., 2012; Nicklas and Kubai, 1985; Nicklas et al., 2001; Zhang and Nicklas, 1995). Our large-scale tomographic reconstruction combined with live imaging and immunostaining in C. elegans now provides an in-depth characterization of the molecular architecture and dynamics of the male meiotic spindle.

Lagging and resolution of the X chromosome

We determined new molecular features of chromosome lagging and resolution in C. elegans. We show the absence of a pairing partner and/or the inability to pair induces any chromosome to lag. Furthermore, microtubule ‘bridges’ to lagging chromosomes consist of continuous microtubules that attach to each side and lengthen during anaphase I. Moreover, we detected both end-on and laterally-associated microtubules on the lagging X. This is distinct from embryonic mitosis, where the vast majority of microtubules make end-on attachments to rod-like chromosomes (O'Toole et al., 2003; Redemann et al., 2017). How is lengthening of X-associated microtubules achieved? Possibly, kinetochore microtubules grow at their plus ends as poles move apart. Microtubule growth at a similar rate to spindle elongation would maintain kinetochore microtubule association to the X. Alternatively, the growth rate of kinetochore microtubules could exceed the rate of spindle elongation, thus allowing minus-end directed interactions of motor proteins such as dynein (Reck-Peterson et al., 2018; Schmidt et al., 2005; Schmidt et al., 2017). Possible roles for dynein in lagging chromosome segregation, the function of end-on versus lateral associations of microtubules to the lagging X, and the influence of the meiotic kinetochore shape and connections can now be addressed in future studies in C. elegans and other systems to further understand lagging chromosome generation and resolution.

A crucial question is how lagging chromosomes resolve during anaphase I. We find the lagging X chromosome is subject to pulling forces mediated by microtubules as poles move apart (Figure 2). Our analyses support that an imbalance of pulling forces may result stochastically from continuous attachment and detachment of kinetochore microtubules. In such a ‘tug-of-war’ model, the side that maintains more connections wins (Appendix 1—figure 10A). A similar mechanism has also been suggested during chromosomal oscillations at mitotic prometaphase and metaphase (Ault et al., 1991; Skibbens et al., 1993; Soppina et al., 2009) with chromokinesins and dynein as possible candidates for switching the direction of the oscillations (Sutradhar and Paul, 2014). As for the initiation of segregation, an analogous situation is the segregation of merotelically attached mammalian kinetochores, where microtubule breakage was suggested to initiate the segregation of lagging mitotic chromosomes (Cimini et al., 2004). Additional tomographic analysis of spindles at late stages of anaphase I will be key to further support our proposed model of lagging chromosome resolution.

Importantly, many species have evolved distinct spindle structures and segregation strategies to resolve lagging of sex or unequal numbers of chromosomes (Fabig et al., 2016; Shakes et al., 2011; Winter et al., 2017). Segregation in cells with aneuploidy and chromosomal abnormalities are potential drivers of infertility (Barri et al., 2005; García-Mengual et al., 2019; Hassold and Hunt, 2001; Ioannou and Tempest, 2015) and cancer progression (Bolhaqueiro et al., 2019; Chunduri and Storchová, 2019; Ly et al., 2019). Thus, our studies can impact the understanding of partition mechanisms in other systems that segregate both paired and lagging chromosomes to efficiently and reliably generate cells with correct ploidy (Fabig et al., 2016).

Contributors of anaphase A not reliant on shortening of kinetochore microtubules

The single-microtubule resolution of electron tomography unexpectedly revealed the lengths of autosomal end-on kinetochore microtubules are largely constant during sperm anaphase. This is in contrast to the kinetochore microtubule shortening typically associated with anaphase A observed by light microscopy in many systems (Asbury, 2017). Further, we developed methods to use ultrastructural data to identify three contributors to spermatocyte anaphase A (Figure 8). First, autosomes stretched at metaphase relax from tension released by separase-mediated cleavage of cohesins at anaphase (Severson and Meyer, 2014), resulting in chromosome shape change. Second, spindle poles decrease in size and change shape as centrioles split, which also shortens distance between microtubule plus-ends and centrioles. Third, the ends of microtubules on chromosomes shift from a central to more peripheral position during anaphase, decreasing the microtubule-to-pole distance. All three factors change the relative position of centrosomes, autosomes and kinetochore microtubules to one another independent of kinetochore microtubule shortening. Microtubules can shorten during anaphase I, as observed when the X resolves to one side; thus, a shortening of a subset of microtubules that is difficult to detect by current methods may also contribute to a small portion of anaphase A movement. Nonetheless, our proposed new mechanisms can now be considered when analyzing anaphase A movement in other systems (Appendix 1—figure 10B).

Distinctions of C. elegans spindles in spermatocytes

In C. elegans, we found important differences in the molecular composition of meiotic spindles in spermatocytes compared to those in oocyte meiosis and embryonic mitosis. First, outer kinetochore proteins are retained and microtubules remain associated to chromosomes between meiotic divisions in sharp contrast to oocyte meiotic anaphase, where kinetochore levels diminish dramatically and microtubules disassemble during anaphase I progression (Dumont et al., 2010).

Second, autosome-associated kinetochore microtubules are continuous and directly attached to poles, in contrast to embryonic mitosis, where chromosome-connected microtubules end in the spindle matrix and make indirect contact with microtubules attached to poles (Redemann et al., 2017). We speculate that the direct kinetochore-to-pole connection in spermatocytes is related to the small size of spermatocytes, in contrast to the relatively large one-cell embryo (Redemann et al., 2017).

Third, we find sperm-specific differences in central spindle architecture during anaphase I that also may impact the lagging and resolution of the X chromosome. When the X lags, microtubule bridges connected to X dominate the spindle midzone. (Figures 1A and 4). With no lagging chromosome in meiosis I in tra-2(e1090) mutant males, few microtubules are found between the separated chromosomes (Figure 1B and Appendix 1—figure 7) and they lack the classical overlapping structure thought to push chromosomes apart, as observed in either acentrosomal oocyte meiosis (Dumont et al., 2010; Laband et al., 2017; Redemann et al., 2018; Yu et al., 2019) or centrosomal embryonic mitosis (Nahaboo et al., 2015; Yu et al., 2019). Furthermore, central spindle specifiers have sperm-specific localization patterns at mid-late anaphase I instead of residing strongly within the spindle midzone (Davies et al., 2014; de Carvalho et al., 2008; Dumont et al., 2010; Maton et al., 2015; Schumacher et al., 1998; Severson et al., 2000). Aurora B Kinase^AIR-2 and CLASP^CLS-2 stay associated with spermatocyte chromosomes (Appendix 1—figure 8A). Likewise, MKLP1^ZEN-4, a centralspindlin component (Powers et al., 1998; Raich et al., 1998), localizes at the ingressing furrow. PRC1^SPD-1, a microtubule bundling protein (Verbrugghe and White, 2004), while transiently present in very early anaphase becomes undetectable (Appendix 1—figure 8B). Thus, our results support that C. elegans spermatocyte meiosis forms an alternate spindle midzone structure compared with mitosis or oocyte meiosis. We speculate this may have evolved to aid lagging chromosome resolution, and it will be interesting to analyze meiotic spindle midzones in other systems with lagging sex chromosomes (Fabig et al., 2016). Alternatively, this midzone organization may stem from mechanisms that regulate cytokinesis to specify intracellular bridges that maintain the germline syncytium (Lee et al., 2018; Zhou et al., 2013).

In toto, our approach combining quantification of 3D ultrastructure of staged spindles with live imaging and immunostaining in males to identify sperm-specific features of meiosis lays the groundwork for further detailed studies on chromosome segregation and provides the necessary analytical tools for analyses on spindles in a broad range of different contexts.

Materials and methods

Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent C. elegans	N2	(Brenner, 1974)
Genetic reagent C. elegans	ANA0072	(Nahaboo et al., 2015)
Genetic reagent C. elegans	CB1489	(Herman and Kari, 1989; Phillips et al., 2005)
Genetic reagent C. elegans	CB2580	(Hodgkin, 1985)
Genetic reagent C. elegans	MAS91	(Han et al., 2015)
Genetic reagent C. elegans	MAS96	M. Srayko, Alberta		Strain maintained in the Srayko lab
Genetic reagent C. elegans	TMR17	this study		Strain maintained in the Müller-Reichert lab
Genetic reagent C. elegans	TMR18	this study		Strain maintained in the Müller-Reichert lab
Genetic reagent C. elegans	TMR26	this study		Strain maintained in the Müller-Reichert lab
Genetic reagent C. elegans	XC110	this study		Strain maintained in the Chu lab
Genetic reagent C. elegans	XC116	this study		Strain maintained in the Chu lab
Genetic reagentC. elegans	SP346	(Madl and Herman, 1979)
Genetic reagent E. coli	OP50	(Brenner, 1974)
Antibody	Rabbit polyclonal anti-NDC-80	Novus Biologicals	Novus Biologicals: 42000002; RRID:AB_10708818	1:200
Antibody	Mouse monoclonal anti-α-tubulin	Sigma-Aldrich	Sigma-Aldrich: T6199; RRID:AB_477583	1:200
Antibody	Mouse monoclonal anti-a-tubulin + FITC	Sigma-Aldrich	Sigma-Aldrich: F2168; RRID:AB_476967	1:50
Antibody	Goat polyclonal anti-rabbit + AlexaFluor 488	Invitrogen	Invitrogen: A11034; RRID:AB_2576217	1:200
Antibody	Goat polyclonal anti-mouse + AlexaFluor 488	Invitrogen	Invitrogen: A11001; RRID:AB_2534069	1:200
Antibody	Goat polyclonal anti-mouse + AlexaFluor 564	Invitrogen	Invitrogen: A11010; RRID:AB_2534077	1:200
Antibody	Donkey polyclonal anti-rabbit + Cy3	Jackson ImmunoResearch	Jackson ImmunoResearch: 711-165-152; RRID:AB_2307443	1:500
Antibody	Rabbit polyclonal anti-KNL-1	(Desai et al., 2003)		1:500
Antibody	Rabbit polyclonal anti-KNL-3	(Cheeseman et al., 2004)		1:500
Antibody	Rabbit polyclonal anti-AIR-2	(Schumacher et al., 1998)		1:200
Antibody	Rabbit polyclonal anti-CLS-2	(Espiritu et al., 2012)		1:200
Antibody	Rabbit polyclonal anti-ZEN-4	(Powers et al., 1998)		1:200
Chemical compound, drug	Polystyrene microbeads solution (0.1 µm)	Polysciences	Polysciences:00876–15
Chemical compound, drug	Hexadecene	Merck	Merck: 822064
Chemical compound, drug	BSA (fraction V)	Carl Roth	Carl Roth: 8076.2
Chemical compound, drug	Osmium tetroxide	EMS	EMS: 19100
Chemical compound, drug	Uranyl acetate	Polysciences	Polysciences: 21447–25
Chemical compound, drug	Epon/Araldite epoxy resin	EMS	EMS: 13940
Chemical compound, drug	Colloidal gold (15 nm)	BBI	BBI: EM.GC15
Other	Type-A aluminum planchette	Wohlwend	Wohlwend: 241
Other	Type-B aluminum planchette	Wohlwend	Wohlwend: 242
Software, algorithm	Code for Kymograph creation	this study		Python code provided as supplemental information
Software, algorithm	Code for Image volume resampling	this study		Python code provided as supplemental information
Software, algorithm	arivis Vision4D	Arivis AG (https://www.arivis.com/en/imaging-science/arivis-vision4d)		Versions 2.9–2.12
Software, algorithm	IMOD	(Kremer et al., 1996) (https://bio3d.colorado.edu/imod/)		Version 4.8.22
Software, algorithm	ZIBAmira	(Stalling et al., 2005) (https://amira.zib.de/)		Versions 2016.47–2017.55

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Strains and worm handling

Strains

The following strains were used in this study: N2 wild type (Brenner, 1974); ANA0072 (adeIs1 [[pMD191] mex-5p::spd-1::GFP + unc-119(+)] II; unc-119(ed3) III; ltIs37 [(pAA64) pie-1p::mCherry::his-58 + unc-119(+)] IV) (Nahaboo et al., 2015); CB1489 (him-8(e1489) IV) (Herman and Kari, 1989; Phillips et al., 2005); CB2580 (tra-2(e1094)/dpy-10(e128) II) (Hodgkin, 1985); MAS91 (unc-119(ed3) III; ItIs37[pAA64; pie-1::mCherry::HIS58]; ruIs57[pie-1::GFP::tubulin + unc-119(+)]) (Han et al., 2015); MAS96 (unc-119(ed3) III; ddIs6[tbg-1::GFP + unc-119(+)]; ltIs37[pAA64; pie-1::mCherry::HIS-58 + unc-119(+)] IV, qaIs3507[pie-1::GFP::LEM-2 + unc-119(+)]) (M. Srayko, Alberta); TMR17 (unc-119(ed3) III; ddIs6[tbg-1::GFP + unc-119(+)]; ltIs37[pAA64; pie-1::mCherry::HIS-58 + unc-119(+)] IV) (this study); TMR18 (him-8(e1489) IV; unc-119(ed3) III; ddIs6[tbg-1::GFP + unc-119(+)]; ltIs37[pAA64; pie-1::mCherry::HIS-58 + unc-119(+)] IV) (this study); TMR26 (zim-2 (tm574) IV; unc-119(ed3) III; ddIs6[tbg-1::GFP + unc-119(+)]; ltIs37[pAA64; pie-1::mCherry::HIS-58 + unc-119(+)] IV) (this study); XC110 (tra-2(e1094)/dpy-10(e128) II; unc-119(ed3) III; ItIs37[pAA64; pie-1::mCherry::HIS58] (IV); ruIs57[pie-1::GFP::tubulin + unc-119(+)]) (this study); XC116 (tra-2(e1094)/dpy-10(e128) II; ddIs6[tbg-1::GFP + unc-119(+)]; ltIs37[pAA64; pie-1::mCherry::HIS-58 + unc-119(+)] IV) (this study); SP346 (tetraploid, 4 n) (Madl and Herman, 1979).

Worm handling

Worms were grown on nematode growth medium (NGM) plates at 20°C with E. coli (OP50) as food source (Brenner, 1974). Male worms were produced by exposing L4 hermaphrodites to 30°C for 4–6 h and checking the resulting progeny for male worms after three days (Sulston and Hodgkin, 1988). Males were maintained by mating 20–30 male worms with five L4 hermaphrodites. Triploid worms were obtained by mating tetraploid hermaphrodites with males of either MAS91 or TMR17. F1 male animals were selected and imaged as described below.

Light microscopy and analysis of spindle dynamics

Light microscopy

Age-synchronized males (3 days after bleaching adult hermaphrodites fertilized by males) were placed in droplets of 1 µl polystyrene microbeads solution (diameter of 0.1 µm; Polysciences, USA) on 10% agarose pads. Samples were then covered with a coverslip and sealed with wax (Kim et al., 2013). We used a confocal spinning disk microscope (IX 83, Olympus, Japan) equipped with a 60 × 1.2 NA water immersion objective and an EMCCD camera (iXon Ultra 897, Andor, UK) for live-cell imaging. The meiotic region within single males was imaged for about one hour and a z-stack was recorded either every 20 s or 30 s. Images were then corrected for photobleaching using the Fiji software package (Schindelin et al., 2012).

Analysis of spindle dynamics

Image stacks were analyzed with the arivis Vision4D software package (arivis AG, Germany). Individual spindles were cropped and spindle poles in each frame were segmented by thresholding. The Euclidean distance of the center of mass of both spindle poles was then calculated for each time point. To produce kymographs, the original image data were resampled with a custom-made python script in arivis Vision4D (Source code 1). The spindle axes were rotated in all three dimensions to align the axis along the z-direction. As a consequence, each spindle had a comparable orientation with an isotropic voxel size of 0.1 µm and a radius of 0.9 µm around the spindle axis. All voxels were then recalculated based on the initial transformation of the axis with an extrapolation of 1 µm at each pole in the direction of the axis. As the axes of the spindles were chosen to lay in the z-dimension all images in the resampled datasets were laying orthogonally to it (x, y-plane). For the calculation of kymographs, the Gaussian weighted sum of fluorescence was calculated in each plane in 0.1 µm steps along this axis and repeated for all time points. For the analysis of chromosome movements, the two peak maxima from the kymographs of the chromosome and spindle pole fluorescence signals were then used to calculate the respective distances for each time point. These distances were then plotted against time relative to the onset of anaphase and utilized to determine the spindle characteristics, that is the pole-to-pole and autosome-to-autosome distance, the speed of segregation and the time of spindle elongation (Table 2).

Individual measurements were aligned according to the onset of anaphase and the mean distance was then calculated and plotted against time relative to anaphase onset. For characterizing the dynamic properties of spindles these mean values were then used to determine spindle length at metaphase and after anaphase. The initial speed of spindle elongation and chromosome movement was calculated by fitting a linear function to the measurements during the first minute after anaphase onset as the segregation speed slowed down continuously afterwards.

To illustrate the process of division, the spindles were resampled and rotated as described above but with a radius of 3 µm around the spindle axis and an extrapolation of 2 µm after the spindle poles (Source code 2). Then a y,z-projection over x (maximum intensity) was calculated for each time point to display the resampled volume as a plane image (Figures 1–2 and 6–7). For a comparison of microtubule density on both sides of the X chromosome facing the spindle poles, the sum of fluorescence was calculated within two cubic boxes (with a similar volume of 1 µm³) adjacent to the X chromosome in the resampled light microscopic image data. The box on the side, where the chromosome moved to at the time of segregation, was termed ‘volume 1’, the other ‘volume 2’. The ratio between both values at each time point indirectly describes the difference in the number of microtubules (Figure 6E–F).

For each data set, the visco-elastic property of the X chromosome was probed by segmenting it in a resampled 3D dataset and measuring its dimensions. Along the spindle axis, the length of the X chromosome was measured (z-dimension). Orthogonal to the z-axis, the mean values for the x- and y-dimension were calculated. A shape coefficient was then calculated (z/[(x+y)/2]) to illustrate the change of the shape of the X chromosome over time (Figure 2A–B).

The centrosomes were segmented in 3D image data from worms expressing γ-tubulin::GFP and histone H2B::mCherry with the arivis Vision4D software package by applying a cut-off threshold to the 3D image data. All fluorescence signals above the threshold were included in the segment of the centrosomes. The volume of the segments was then calculated for each frame and each centrosome individually for spindles in meiosis I and II. When centrosomes split in meiosis I and could be segmented individually both volumes were summed together for the respective frame (Appendix 1—figure 3).

Immunostaining for light microscopy

For antibody staining of C. elegans gonads, synchronized males were dissected and fixed in 1% paraformaldehyde using established protocols (Howe et al., 2001). Methanol/acetone fixation was used for immunolabeling of mitotic and meiotic embryos (Shakes et al., 2009). Primary and secondary antibodies were diluted in blocking buffer (PBS + 0.1% Tween 20 and 10 mg/ml BSA) and staining was conducted at room temperature in a humid chamber. Primary antibodies were used in overnight incubations (unless otherwise noted). Commercial sources or labs kindly providing antibodies were as listed: 1:200 rabbit anti-NDC-80 (Novus Biologicals, catalog #42000002); 1:200 mouse anti-α-tubulin (DM1A Sigma-Aldrich, catalog #T6199); 1:500 rabbit anti-KNL-1 (Desai et al., 2003); 1:500 rabbit anti-KNL-3 (Cheeseman et al., 2004); 1:200 rabbit anti-AIR-2 (Schumacher et al., 1998); 1:200 rabbit anti-CLS-2 (Espiritu et al., 2012); 1:200 rabbit anti-ZEN-4 (Powers et al., 1998); and 1:50 FITC-conjugated anti-α-tubulin (Sigma-Aldrich, #F2168). Secondary antibodies included: goat anti-rabbit AlexaFluor 488-labeled IgG (used at 1:200); goat anti-mouse AlexaFluor 488-labeled IgG (used at 1:200); goat anti-mouse AlexaFluor 564-labeled IgG (used at 1:200); and donkey anti-rabbit Cy3 (used at 1:500). DNA was visualized using DAPI at 0.1 µg/ml. Slides were prepared by using VectaShield (Vector Labs, USA) as a combined mounting and anti-fade medium. Confocal images were acquired using a Zeiss LSM710 microscope, a Zeiss LSM880 microscope, or a Leica SP8 Confocal System (Figure 2E and Appendix 1—figure 8A). Super-resolution images were collected using an OMX 3D-SIM microscope (GE Healthcare, USA) with an Olympus (Shinjuku, Japan) 100x UPlanSApo 1.4 NA objective (Olympus, Japan). Images were captured in z-steps of 0.125 µm and processed using SoftWoRx (GE Healthcare, USA) and IMARIS (Bitplane, Switzerland) 3D imaging software (Appendix 1—figure 1).

Laser microsurgery

Age-synchronized males (3 days old) were placed within a of droplet of 1 µl M9 buffer containing 1 mM levamisole and 0.1 µm polystyrene microbeads (Polysciences, USA) on a 10% agarose pad. Samples were then covered with a coverslip and sealed with wax. For imaging during laser microsurgery, we used a confocal spinning disk microscope (Ti Eclipse, Nikon, Japan) equipped with a 60 × 1.2 NA water immersion objective, a 1.5x optovar, an EMCCD camera (iXon Ultra 897, Andor, UK) and a mode-locked femtosecond Ti:sapphire laser (Chameleon Vision II, Coherent, USA) operated at a wavelength of 800 nm. After locating spindles in anaphase I within males, a single image was recorded in intervals of 1 s. Subsequently, a position for the laser cut was chosen and a single spot with a diameter of about 1.3 µm was ablated with a laser power of 150 mW and an exposure time of 30 ms. Image acquisition was continued until the X chromosome had been fully segregated (Figure 2C–D). In total, we performed 26 laser ablations. In ~80% of the experiments we observed a movement of the X towards the unablated side. For further analysis the images were corrected for photobleaching within the Fiji software package and corrected for movement using the plugin ‘image stabilizer’ (http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html; February 2008).

Specimen preparation for electron microscopy

Males were ultra-rapidly frozen using an HPF COMPACT 01 high-pressure freezer (Engineering Office M. Wohlwend, Sennwald, Switzerland). For each freezing run, five individuals were placed in a type-A aluminum planchette (100 µm deep; Wohlwend, article #241) pre-wetted with hexadecene (Merck) and then filled with M9 buffer containing 20% (w/v) BSA (Roth, Germany). The specimen holders were closed by gently placing a type-B aluminum planchette (Wohlwend, article #242) with the flat side facing the sample on top of a type-A specimen holder. The sandwiches were frozen under high pressure (~2000 bar) with a cooling rate of ~20000 °C/s (Fabig et al., 2019). Specimen holders were opened under liquid nitrogen and transferred to cryo-vials filled with anhydrous acetone containing 1% (w/v) osmium tetroxide (EMS) and 0.1% (w/v) uranyl acetate (Polysciences, USA). Freeze substitution was performed in a Leica AFS (Leica Microsystems, Austria). Samples were kept at −90°C, then warmed up to −30°C with steps of 5 °C/h, kept for 5 h at −30°C and warmed up again (steps of 5 °C/h) to 0°C. Subsequently, samples were washed three times with pure anhydrous acetone and infiltrated with Epon/Araldite (EMS, USA) epoxy resin at increasing concentrations of resin (resin:acetone: 1:3, 1:1, 3:1, then pure resin) for 2 h each step at room temperature (Müller-Reichert et al., 2003). Samples were incubated with pure resin over night and then for 4 hr. Samples were thin-layer embedded between two Teflon-coated glass slides and allowed to polymerize at 60°C for 48 h (Müller-Reichert et al., 2008). Polymerized samples were remounted on dummy blocks and semi-thin serial sections (300 nm) were cut using an EM UC6 (Leica Microsystems, Austria) ultramicrotome. Ribbons of sections were collected on Formvar-coated copper slot grids, post-stained with 2% (w/v) uranyl acetate in 70% (v/v) methanol and 0.4% (w/v) lead citrate and allowed to dry prior to inspection.

Electron tomography, microtubule segmentation and stitching of data sets

In preparation for electron tomography, both sides of the samples were coated with 15 nm-colloidal gold (BBI, UK). To select cells in meiosis, serial sections were pre-inspected at low magnification (~2900 x) using a Zeiss EM906 transmission electron microscope (Zeiss, Germany) operated at 80 kV. Serial sections containing cells/regions of interest were then transferred to a Tecnai F30 transmission electron microscope (Thermo Fischer Scientific, USA) operated at 300 kV and equipped with a US1000 CCD camera (Gatan, USA). Tilt series were acquired from −65° to +65° with 1° increments at a magnification of 4700x (pixel size 2.32 nm). Specimens were then rotated 90° to acquire a second tilt series for double-tilt electron tomography (Mastronarde, 1997). Electron tomograms were calculated using the IMOD software package (Kremer et al., 1996). As previously described (Redemann et al., 2014; Weber et al., 2012), microtubules were automatically segmented using the ZIBAmira (Zuse Institute Berlin, Germany) software package (Stalling et al., 2005).

Individual tomograms were then stitched and combined (Weber et al., 2014) to represent whole microtubule networks in 3D models (Redemann et al., 2017). Chromosomes, kinetochores and centrioles were manually segmented. Kinetochores were modeled around each chromosome by gradually increasing the chromosome volume until the area of the ribosome-free zone around each chromosome (Howe et al., 2001; O'Toole et al., 2003) was covered, giving a thickness of the male meiotic holocentric kinetochore of about 150 nm (Figures 3–4, Appendix 1—figures 4 and 7).

Analysis of tomographic data

Staging of tomographic data sets

For staging of the reconstructed spindles, we determined the autosome-to-autosome distance. We measured the distance of the individual chromosome pairs, calculated the mean of these individual distances and ordered them accordingly. As an additional criterion for staging, we took the ‚state’ of the centrosome into account, as the centrioles pre-early split in C. elegans male meiosis (see Table 1). Within each data set, the distance between the mother and the daughter centriole was determined at each spindle pole and averaged. As an example, this read-out was used to determine anaphase onset.

Classification of microtubules

First, the distance between each point of a microtubule segment and the closest point of the surface of individual chromosomes was calculated. Only microtubules within a distance of 150 nm or less were considered kinetochore microtubules as this distance was measured to be the approximate extent of the kinetochore in the electron tomograms. The kinetochore is visible in the electron tomograms as a less stained region around the chromosomes (Howe et al., 2001). Additionally, each kinetochore microtubule was assigned to the X chromosome or to one of the autosomal chromosomes according to its closest distance to the chromosome surface. As microtubules in anaphase pass between the autosomes and attach to the X chromosome after that, they were first checked for an association with the X chromosome and if there was none, further analysis was performed to check for a potential autosomal association. For each chromosome the microtubule associations were subdivided between end-on and lateral (Video 1). We defined an end-on association by extrapolating the microtubule after its end for 150 nm and checking if this extrapolated line was cutting the surface of the chromosome. If that criterion was not met, we considered the association of the microtubule with the given chromosome as lateral.

Video 1. Visualization of end-on or laterally associated kinetochore microtubules.

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This video illustrates the classification of kinetochore microtubules according to their type of association to chromosomes. Chromosomes are shown in light teal, the holocentric kinetochore area surrounding the chromosomes in dark semi-transparent teal. End-on associated kinetochore microtubules are shown in white, laterally associated microtubules in orange).

Length distribution

Furthermore, we analyzed the length distribution of microtubules. For each microtubule class in each meiotic spindle the length distribution is given (mean, standard deviation). Further, the variance among the datasets was compared using a one-way analysis of variance (ANOVA; Appendix 1—figure 5).

We also analyzed the ratio of the sum of microtubule length between two defined volumes analogous to the analysis of the light microscopic data. For that a box of 1 µm³ was placed on either side of the X chromosome facing the spindle poles. The microtubules within this box were extracted and their length was measured and summed up. The ratio of the box closer to the respective pole against the second box was calculated (Figure 6A–B). The microtubule tortuosity (microtubule spline length divided by end-end length; Appendix 1—figure 6) was measure for end-on and lateral microtubules in contact with the X chromosome.

Chromosome shape

Further, we analyzed the shape of the chromosomes in the EM data as previously described (Lindow et al., 2018). In brief, chromosomes were manually segmented and along the pole-to-pole axis of the spindle orthogonal planes were placed with 10 nm spacing. For every plane the area was calculated that intersected the individual chromosome surface. After plotting the cross-sectional area against the pole-to-pole distance a Gaussian function containing five terms was fit with MATLAB (MATLAB 2017b, The MathWorks, USA) and the full width at half maximum (FWHM) of the for each chromosome was determined and compared (Figure 8A–B). For measuring the distance between centrioles and the end-on microtubule end at the autosomes, we first selected the closest centriole at the putative microtubule minus-end. Then we extracted the position of the respective putative plus-end and calculated the Euclidean distance between the centriole and the putative plus-end (Figure 8C–D). The angle between the microtubule plus-end and the chromosome-centrosome axis was determined by calculating the vector between the respective chromosome and the centrosome and the vector between chromosome and the respective microtubule plus-end. Then the angle between both vectors was calculated (Figure 8E–F).

Acknowledgements

The authors would like to thank Dr. Michael Laue (Robert Koch Institute, Berlin, Germany) for using the COMPACT 01 (Wohlwend) high-pressure freezer, the Core Facility Cellular Imaging of the Faculty of Medicine Carl Gustav Carus (TU Dresden, Germany) and the light- and electron microscopy facilities at the MPI-CBG (Dresden, Germany) for technical assistance. The Delattre, Desai, Rose, Schumacher, and Strome labs generously provided strains or antibodies used for these studies. We are also grateful to Drs. Diane Shakes (Williamsburg VA, USA), Stefanie Redemann (Charlottesville VA, USA) and Kevin O’Connell (Bethesda MD, USA) for a critical reading of the manuscript. We would like to thank Martin Merkel, Ewa Kania, Sophia Merkel, Maura Hofmann and Isabelle Kunert for help in tomographic reconstruction and microtubule segmentation. The authors are grateful to Falko Löffler, Carola Bender and Christian Götze (arivis AG) for help with image processing in arivis Vision4D. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We acknowledge NIH grant NIH1S10OD024988-01 for the purchase on the OMX microscope. Research in the Müller-Reichert laboratory is supported by the Deutsche Forschungsgemeinschaft (MU 1423/10–1). RK received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 675737 (grant to TMü-R). Work in the Chu lab is supported by the NIH grant R03 HD093990-01A1 and the NSF Awards RUI-1817611 and DBI-1548297.

Appendix 1

(A) Plot showing centrosome volume in meiosis I over time. The 3D volume was measured in worms expressing γ-tubulin::GFP and histone H2B::mCherry. For each dataset a fixed threshold was defined to segment the outer border of the centrosome. The mean volume is plotted as a green line for unsplitted centrosomes and shown as an orange to red line for splitted centrosomes. The percentage of splitted centrosomes is indicated by this color change. For splitted centrosomes, the sum of both separated centrosomes was determined (n = 44). The standard deviation is depicted as a shaded area. (B) Centrosome volume over time (purple line) in meiosis II (n = 64).

Appendix 1—figure 3—source data 1. Measurements of centrosome volume from replicates used in Appendix 1—figure 3.

elife-50988-app1-fig3-data1.xlsx^{(11.6KB, xlsx)}

Appendix 1—figure 4. — (A) Mid anaphase spindle with a pole-to-pole distance of 3.14 µm. (B) Mid anaphase spindle with a pole-to-pole distance of 3.58 µm. (C) Mid anaphase spindle with a pole-to-pole distance of 4.47 µm. Left panels: tomographic slice showing the autosomes (a), and the univalent X chromosome (x) aligned along the spindle axis. Mitochondria (m) and fibrous body-membranous organelles (fb) are also indicated. Mid left panels: corresponding three-dimensional model illustrating the organization of the partially reconstructed spindle. Autosomes are in blue, the X chromosome in red, microtubules within a distance of 150 nm or closer to the chromosome surfaces in yellow and all other microtubules in gray. Mid right panels: association of microtubules with the kinetochores. Kinetochores are shown as semi-transparent regions around each chromosome. The part of each microtubule entering the kinetochore region around the holocentric chromosomes is in green. Right panels: visualization of end-on (white) *versus* laterally (orange) X chromosome-associated microtubules. The part of each microtubule entering the kinetochore region around the holocentric chromosomes is shown in green. The autosome-to-autosome distance (A-A) for each reconstruction is indicated in the left column. Scale bars, 500 nm.

Appendix 1—figure 5. — (A) Length of end-on X chromosome-associated kinetochore microtubule for each tomographic data set as shown in Figure 5A. The mean, the standard deviation and single measurements for each meiotic stage are given. Results of a one-way analysis of variance (ANOVA) of all data sets against each other are shown. Level of significance: * is p<=0.05; ** is p<=0.01; and *** is p<=0.001. (B) Length of lateral X chromosome-associated kinetochore microtubules corresponding to Figure 5B. (C) Tortuosity of end-on X chromosome-associated kinetochore microtubules corresponding to Appendix 1—figure 6B. (D) Tortuosity of lateral X chromosome-associated kinetochore microtubules corresponding to Appendix 1—figure 6B. (E) Length of end-on autosome-associated kinetochore microtubules corresponding to Figure 7G. (F) Length of lateral autosome-associated kinetochore microtubules corresponding to Figure 7H. (G) FWHM of the chromosome stretch for each meiotic stage corresponding to Figure 8B. (H) Distance of autosomal kinetochore microtubule plus-ends to closest centriole for each meiotic stage corresponding to Figure 8D. (I) Measurement of attachment angles of autosomal end-on associated kinetochore microtubules as given in Figure 8F.

(A) Schematic drawing illustrating the shape of X chromosome-associated kinetochore microtubules. Both end-on (yellow) and laterally associated microtubules (purple) are shown (left panels). The tortuosity of each microtubule is given by the spline length (red dotted lines; right panel) divided by the end-to-end length (black dotted lines). Dashed lines indicate a grouping of the spindles according to the meiotic stages: metaphase/anaphase onset, mid anaphase and late anaphase (see also Appendix 1—figure 2). (B) Plots showing the tortuosity of end-on (top) and laterally (bottom) associated kinetochore microtubules. The meiotic stages correspond to the data sets as shown in Figures 3 and 4. Mean, standard deviation and individual measurements are given for each data set.

Appendix 1—figure 6—source data 1. Measurements of tortuosity of end-on and lateral microtubules from replicates used in Appendix 1—figure 6.

elife-50988-app1-fig6-data1.xlsx^{(16.9KB, xlsx)}

Appendix 1—figure 8. — (A) Confocal light microscopy of the spindle midzone in *him-8*(e1489) and *tra-2*(e1094) spermatocytes. For comparison, the spindle midzone in wild-type oocytes in anaphase I is also shown. Samples were stained with antibodies against three different proteins: AuroraB^AIR-2, CLASP^CLS-2 and MKLP1^ZEN-4. Stained proteins are shown in green, chromosomes in red. Scale bar, 2 µm. (B) Time series of confocal image projections of wild-type meiosis I in males (upper row) and hermaphrodite spermatocytes (lower row). The microtubule bundling protein PRC1^SPD-1 (SPD-1::GFP, white) and the chromosomes (histone H2B::mCherry, red) are visualized. Anaphase onset is time point zero (t = 0). White arrowheads mark the position of the unpaired X chromosome in meiosis I. Chromosome segregation is also visualized in kymographs (right panels; the start of the separation of the autosomes is indicated by a dashed orange line). Scale bars (white), 2 µm; time bar (blue), 2 min.

Appendix 1—figure 9. — (A) Mean length of end-on associated microtubules plotted against the autosome-to-pole distance for all fully reconstructed spindles (as given in Figures 3 and 4). The plot shows the mean values with standard deviations. (B) Identical analysis for laterally associated microtubules.

Appendix 1—figure 9—source data 1. Measurements of pole-to-autosome and microtubule length in each stage shown in Figures 3 and 4 used in Appendix1-figure 9-source data1.

elife-50988-app1-fig9-data1.xlsx^{(10.2KB, xlsx)}

Appendix 1—figure 10. — (A) Proposed tug-of-war model for the initiation of X chromosome segregation in anaphase I in males. The X chromosome is shown in red, the holocentric kinetochore in gray, and the X chromosome-associated kinetochore microtubules in green. Left panel: At metaphase I, pulling forces at the X chromosome are in balance. Right panel: segregation of the X chromosome is initiated by an imbalance of forces, obvious by an unequal number of kinetochore microtubules associated with the opposite sides of the X chromosome. In anaphase I, the X is proposed to move to the side with more attached kinetochore microtubules. (B) Model illustrating the changes in spindle geometry. Left upper panel is metaphase; right upper panel is anaphase; lower panel shows combining anaphase A and B. Chromosomes are shown in blue, centrosomes in green, centrioles in black. Laterally associated microtubules are illustrated in orange. The end-on associated kinetochore microtubules (magenta in metaphase and green in anaphase) have the same length at both stages. The lower panel is an overlay of metaphase (magenta) with anaphase A (green, anaphase B movement was not considered) to show the relative movement of the autosomes with respect to the centrosomes. A simultaneous rounding of the autosomes, a shrinking of the volume of the centrosomes and a change in the attachment angle of the microtubules is illustrated.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Gunar Fabig, Email: gunar.fabig@tu-dresden.de.

Thomas Müller-Reichert, Email: mueller-reichert@tu-dresden.de.

Yukiko M Yamashita, University of Michigan, United States.

Anna Akhmanova, Utrecht University, Netherlands.

Funding Information

This paper was supported by the following grants:

Deutsche Forschungsgemeinschaft MU 1423/10-1 to Gunar Fabig, Thomas Müller-Reichert.
H2020 Marie Skłodowska-Curie Actions No. 675737 to Robert Kiewisz, Thomas Müller-Reichert.
National Institutes of Health R03 HD093990-01A1 to Vanessa Cota, Diana S Chu.
National Science Foundation RUI-1817611 to Vanessa Cota, Diana S Chu.
National Institutes of Health NIH1S10OD024988-01 to James A Powers.
National Science Foundation DBI-1548297 to Vanessa Cota, Diana S Chu.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing, Live imaging, electron tomography, reconstructions, data analysis, creation of Figures.

Formal analysis, Reconstruction and analysis of electron tomograms.

Software, Methodology, Software and analysis tool development for electron tomograms in ZIB Amira.

Resources, Formal analysis, Acquisition of super-resolution data with the DeltaVision OMX microscope (Appendix-Figure 1).

Formal analysis, Immunostainings (Fig. 2E).

Formal analysis, Immunostainings (Appendix-Figure 8A).

Resources, Methodology, Advice with the laser ablation experiments (Fig. 2C and D).

Software, Funding acquisition, Software and analysis tool development for electron tomograms in ZIB Amira.

Conceptualization, Supervision, Funding acquisition, Methodology, Writing - review and editing, Co-supervision.

Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Co-supervision.

Additional files

Source code 1. Kymograph.

This script needs two 3D coordinates and 3D image data as an input and creates a 2D line scan in between the two input coordinates according to the settings in the script. If a time series is used this algorithm creates a kymograph.

elife-50988-code1.py^{(16.3KB, py)}

Source code 2. Volume resampling.

This script needs two 3D coordinates and 3D image data as an input and creates a spatially reoriented and resampled 3D data set in between the two input coordinates according to the settings in the script. The center line (spindle axis) in between the two input coordinates is positioned in the z-dimension of the output data set. If a time series is used this algorithm creates a resampled 3D data set over time.

elife-50988-code2.py^{(12.6KB, py)}

Transparent reporting form

elife-50988-transrepform.pdf^{(230.8KB, pdf)}

Data availability

Data have been uploaded to the TU Dresden Open Access Repository and Archive system (OpARA) and are available as open access: https://doi.org/10.25532/OPARA-56.

The following dataset was generated:

Fabig G. 2020. Supplemental data for the publication. OpARA.

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eLife. doi: 10.7554/eLife.50988.sa1

Decision letter

Editor: Yukiko M Yamashita¹

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study provides and important and beautiful description of the C. elegans spermatocyte spindle, which exhibits interesting and unexpected features distinct from many other mitoses. Overall, the reviewers agreed that this study provides important insights into C. elegans male meiotic divisions, laying the foundation for future studies.

Decision letter after peer review:

Thank you for submitting your article "Male meiotic spindle features that efficiently segregate paired and lagging chromosomes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Essential revisions:

1) All reviewers agreed that the writing and structure of the manuscript need substantial revisions. In its current form, the manuscript lacks clarity and accuracy. Reviewers discussed that the manuscript may be improved by re-organization (suggested by reviewer #2), and more careful writing in a number of places (individual points are listed below).

2) Reviewer #3 indicated that the conclusions regarding anaphase A are not well-supported by evidence and highlighted that the segregation of the X is not being considered by the authors when suggesting that k-fiber shortening does not drive chromosome-to-pole movement in these spindles. After discussion among reviewers, the important points raised by reviewer #3 must be taken into account when presenting analysis and conclusions related to anaphase A.

During our discussion, reviewer #2 suggested the following which may help partially address reviewer #3's comments and potentially amend some of the conclusions presented.

The authors show plots of what they refer to as end-on and lateral (see comments on these terms in the detailed reviews) for the X – however, all their plots would benefit from including the X-to-pole (as well as autosome-to-pole) separation distances to help interpret what is currently only referred to as anaphase 1,2,3, 4 in the graphs. At least in the plot shown it does not look like anaphase 4 (most separated) was associated with significantly shorter MT lengths but they should address reviewer #3's comments on this point.

The authors should also separate the two spindle halves (the "winning" vs. "losing" side) in the later anaphase stages and plot lengths as a function of X-pole distance in each half-spindle; this would help in terms of assessing whether X segregation was associated with significant shortening of microtubules that they detect by tomography. Currently all these values are averaged and, as noted by reviewer #3, the modest changes and large deviations make the conclusions stated about lack of microtubule shortening questionable.

3) Reviewers recommend that the authors conduct additional live imaging analysis of anaphase B (e.g. by visualizing central spindle components and cortical force generators), as that is the dominant mechanism separating chromosomes. Any insights from the tomography analysis on anaphase B should also be discussed to provide a more balanced view of the chromosome separation mechanism. Although live imaging would be ideal, if it is not feasible, the authors can characterize the localization of central spindle proteins (SPD-1, centralspindlin, etc.) by immunofluorescence, to better demonstrate that the central spindle really doesn't exist.

Overall, while significant additional experimental analysis is not necessary, we would like authors to: (1) reorganize the writing for clarity and accuracy; (2) re-analyze existing data (or potentially add new data if available to strengthen their points) and be significantly more careful about claims related to the minor anaphase A-like movement in light of the critiques from reviewer #3; and (3) present additional characterization of the predominant chromosome separation mechanism: anaphase B.

Reviewer #1:

The manuscript by Fabig et al. describes how chromosomes are segregated on the meiosis I (and II) spindles in C. elegans spermatocytes. Using a combination of live cell imaging, electron tomography and fixed images, the authors present a beautiful description of the spermatocyte spindle. This analysis reveals some very interesting and unexpected features including novel mechanisms to account for anaphase A movement, as well as providing insight into how lagging chromosomes are segregated.

In the Introduction, the authors conflate general features of chromosome segregation with the specifics of C. elegans. For example, Introduction paragraph two, they generalize findings in C. elegans that may or may not be the same in other systems. The authors need to be more careful in their description between what is happening in C. elegans versus general features of chromosome segregation.

Subsection “Lagging of chromosomes is a consequence of a lack of pairing” paragraph three: ZIM-2 mediates the pairing/synapsis of chromosome V not IV (Phillips and Dernburg, 2006).

Subsection “Spermatocyte meiotic centrosome and spindle dynamics are distinct from that in mitosis”: What is the evidence that spindle MTs remain connected to the X chromosome?

Figure 6: Please add the color code directly to the figure (e.g., white is end-on MT interactions and gold is lateral MT interactions). Would it be possible to highlight a continuous MT from the centrosome to the X chromosome?

Appendix—figure 7: Absence of central spindle. The authors present evidence that there is no central spindle based on weak MT staining in the center (and the presence of the lagging X chromosome). This would be strengthened by staining with central spindle components such as CYK-4/ZEN-4/SPD-1 in a similar manner as was done for the inner and outer kinetochore (Figure 4).

Is there any correlation between the curved MTs and which pole the X segregates to? In the images/videos the curve is more pronounced on one side of the spindle.

Reviewer #2:

The manuscript from Fabig et al. presents live imaging analysis of male meiotic divisions in C. elegans that is complemented by electron tomographic analysis of microtubule distributions during this developmentally specialized division. This is a technically strong study with two major themes: (1) analysis of the unpaired X chromosome's segregation – lagging followed by eventual segregation to one spindle pole, and (2) description of a modest anaphase A-like movement of autosomes that is not associated with microtubule shortening but instead driven by shape changes.

The manuscript has compelling elements but is a difficult read and would greatly benefit from reorganization and more careful writing. A more challenging question is whether there is need for any type of perturbation analysis – this point can be addressed in the reviewer discussion. To enhance the manuscript's impact, I would recommend the authors organize the manuscript into two sections – the first focused on X chromosome lagging and the second briefer one on anaphase A-like autosome movement – that are bridged by the tomography (which is the most important contribution here and needs to be presented significantly earlier in the paper). Here is a recommended structure along with comments on each section:

The authors need to make it significantly easier for readers to evaluate figures and compare different datasets. All time-dependent graphs should have a common time 0, e.g. anaphase onset, and the Time axis should be labeled "Time relative to anaphase onset (s)". On 2-color images shown, the component visualized in each color should be labeled on the figure and not only in the legend. Similarly, pseudo-colored entities in the tomograms should be described on the figure with proper labels. These types of modifications will great improve accessibility of the manuscript to interested readers.

1) Merge Figure 1 and 2 into one figure showing that unpaired chromosomes lag.

The authors state that "sister chromatids of the unpaired X attach to opposite poles…"; this is not in fact supported by the data and should be removed. An interesting point that should be mentioned is that cohesion is protected between the sisters of the X in meiosis I – presumably because the lack of recombination prevented definition of an axis of cohesion removal in meiosis I.

2) Leave Figure 3 for later in the paper (see below); move Figure 4 to the supplement – this figure overlaps with prior fixed data, does not report any significant new observations and is disruptive in the flow of the manuscript.

One point here – that HIM-10 looks different from MIS12 complex and KNL-1 – is rather surprising and should be verified using CRISPR-tagged versions that have been generated for all of these components. It is possible that the staining observed with the anti-HIM-10 antibody between the homologs is non-specific.

3) Make current Figure 5 into Figure 2. This figure is focused on movement of the lagging X and will follow directly from the revised Figure 1 (see point 1 above).

As noted above, label imaged components on the figure. There is one issue with the conclusions in Figure 1A and B – the change in chromosome morphology cannot be solely attributed to loss of tension because there is also reduction in cell cycle phosphorylation between meiosis I and meiosis II. The authors should analyze their laser ablation data (especially sequences like no. 2) to assess if there is rapid relaxation after ablation. In the absence of such d ata, they need to be more cautious in the interpretation in current Figure 5A and B.

4) Make current Figure 6 (tomography) into Figure 3 – label colors on the figure and not in legend. Provide numerical summary also in the figure (can be repeated in the text).

The last column is very difficult to visualize. On a more important note, the exact criterion used to call end-on vs. lateral should be clarified here even if they were previously described. Precisely how much length of microtubule should be present in the "clear" zone for it to be designated "lateral"? This is also a potential point of confusion in that the classification does not match what these terms are used to refer to in analysis in other systems (e.g. yeast or human cells, where a lateral attachment refers to kinetochore bound on the side of a microtubule that extends far past the kinetochore). I suspect that the attachments described here may be similar to what is termed an "end-on" attachment in other systems. There is also a geometric issue here coming from the curvature of the chromosomes – the surface that is available for what is classified as an end-on attachment may limit their number.

5) Make current Figure 7 and Figure 8 into Figures 4 and 5. These continue to focus on the lagging X chromosome and its segregation but now integrate the tomography.

There are some issues with the writing and figure elements associated with this analysis. In the tortuosity analysis, is there any contribution from the cleavage furrow? Is there a furrow at the stages that are visualized or not? In current Figure 8B, what are "anaphase S1, S2, S3"? In Figure 8F, why does the ratio go up? Is it because more microtubules are formed on the "winning" side or there is a decrease in microtubules on the "losing" side? This should be clarified by looking at the measured values and not the ratio. More generally, there is not any causal relationship established by any of this analysis and the writing needs to be more circumspect. There is also an analogy to made to segregation of merotelically attached kinetochores in mitotic mammalian cells that lag and segregate to the side that ends up with more microtubules. This should be mentioned.

6) End the paper with 2 figures (Figure 6 and 7) on autosomal anaphase A (which provides ~20% of separation). Start Figure 6 with current Figure 9 to highlight that kinetochore microtubules on autosomes did not shorten and follow that with current Figure 2, based on light imaging, which shows there is modest anaphase A, as defined by reduction in distance of chromosomes to poles. Then end with what is currently Figure 10, which provides reasons for why this would be the case.

Note that the chromosome shape change and the angular change are related and not independent – thus there are 2 factors at play – the chromosome shape change and the change in the centrosome. It would be helpful if the Anaphase 1, 2, 3 and 4 were annotated to include the separation distance of the autosomes.

On a related note, 80% or more of the separation is not due to anaphase A. Is this entirely due to cortical pulling? Given their expertise, the authors should look at conserved central spindle markers (SPD-1, CYK-4, ZEN-4) and also dynein (DHC-1) – there should be endogenously tagged versions of all of these components available by now.

Reviewer #3:

This manuscript by Fabig et al. reports a detailed characterization of spermatocyte meiosis in C. elegans. The authors present data that provides some new insights into spindle organization and chromosome segregation in this system. However, I find some of their conclusions to be insufficiently supported (see points below).

1) One of the major points that this paper attempts to make is that k-fiber shortening does not drive anaphase A in spermatocytes. However, I do not think that the presented data provide strong support for this conclusion. One of the main issues is that the spermatocyte spindles are small and the poleward-facing edges of the autosomes start out very close to the poles, so the chromosomes do not move very far in anaphase A. Therefore, if there was k-fiber shortening it would be hard to detect, especially given the variability in microtubule lengths reported from the electron tomography analysis. The authors report that the "end-on" microtubules for metaphase are 0.62 +/- 0.33 µm and the "lateral" microtubules are 1.15 +/- 0.59 µm. Given this amount of variability, if some population of these microtubules shortened and helped drive movement towards the pole, it could be hard to detect. Compounding this issue, since only one metaphase spindle was analyzed, it is difficult to know whether the numbers reported for metaphase are consistent from spindle to spindle.

Additionally, the author's own data appears to suggest that k-fiber shortening could be capable of driving poleward movement in these spindles. In the case of the lagging X chromosome, the authors show nice videos of the X segregating in late anaphase (both in the normal case in Figure 1/Video 1, and in their cutting experiments in Figure 5/Video 4). In these cases (since the spindle has elongated in anaphase B), the X has to segregate over a longer distance and therefore it is easier to visualize what is happening to the microtubules/k-fibers. In these videos, it appears that once the X starts segregating to the "winning" pole, the corresponding k-fiber shortens, reeling in the chromosome. It seems unlikely to me that this k-fiber shortening mechanism to drive chromosome-to-pole movement would exist for the X chromosome, but not the autosomes. It seems more likely that the autosomes also segregate by this mechanism in anaphase A, but that it is difficult to measure because the distances are so short and the variability of the microtubule lengths is so high. Therefore, I do not find the conclusions of the authors about anaphase A mechanisms to be convincing.

2) The designation of "end-on" vs. "lateral" microtubules in Figure 6 is confusing. Many of the white microtubules designated as end-on appear to run quite far down the side of the chromosome. Therefore, they don't really appear to be "end-on". Correctly categorizing microtubules is important, because the authors make a major point about end-on microtubules not shortening during anaphase, and use this as evidence to say that k-fiber shortening does not drive anaphase A movements. But if some of the microtubules they are counting are actually running along the sides of the chromosomes and if this population of lateral microtubules do not shorten, then the authors may be missing a reduction in the length of the ones that actually are "end-on".

Related to this, in paragraph four of subsection “Tension release across the spindle may contribute to autosomal anaphase A”, the authors use their conclusion that microtubule lengths are constant at 0.63 µm between metaphase and anaphase to calculate how much altering the microtubule angle could contribute to chromosome movements (ending up with a value of 0.17 µm). However, since this measurement of microtubule length is subject to error (given the variability of microtubule length measurements and the potential issue with distinguishing end-on from lateral interactions), the calculations that led to the 0.17 µm shortening number are also in question.

3) In Figure 10, the authors report the ANOVA comparing metaphase with two of the anaphase spindles (number 3 and 4) and it appears that there is a significant difference, but they do not report the ANOVA comparison between metaphase and anaphase numbers 1 and 2 (and #2 especially does not appear to be different from metaphase). This raises the concern that variability between spindles could account for the different amounts of autosomal stretching, rather than spindle stage (metaphase vs. anaphase). This is particularly important since the authors use this data to propose that release of this stretch can account for much of the "anaphase A" pole-chromosome shortening. To me, it seems problematic to draw these conclusions when only one metaphase spindle is analyzed, and therefore it is difficult to exclude spindle-spindle differences in the autosomal FWHMs.

4) For the analysis of chromosome segregation, it would be helpful to have more information about how the distances were determined. The Experimental Procedures state "For the analysis of chromosome movements, the peak maxima of the chromosome fluorescence signals were then used to calculate the distances for each time point." This is confusing and more details on the analysis should be included so the reader can better evaluate these data – how were the centers of each autosome determined? I can imagine that this would be difficult given the resolution of the videos, and subject to error. Also, was each autosome within a given spindle measured separately, or was each segregating mass of autosomes treated as one unit (and the center of that entire mass determined)? Given potential issues in accurately determining the "center" (of either each autosome or the autosome mass), I would suggest that the authors try measuring the distance between the outer edges of the autosomes (the poleward facing sides) and the spindle pole… this would more clearly represent the distance the chromosome travels towards the spindle pole.

5) The authors state that: "Interestingly, the centrosomes remain connected to the X chromosome-connected microtubules". However, there is no figure call-out for this statement, and I can't find any figures where there is convincing evidence of this, given the resolution of the images/videos presented. I am therefore confused as to what evidence supports the subsequent statement "Thus, the separation and migration of centrosomes during anaphase I appears to be coordinated with microtubules that must maintain connections not only to segregating autosomes, but also the lagging X chromosome". Either present these data or revise these statements.

eLife. 2020 Mar 10;9:e50988. doi: 10.7554/eLife.50988.sa2

Author response

Essential revisions:

1) All reviewers agreed that the writing and structure of the manuscript need substantial revisions. In its current form, the manuscript lacks clarity and accuracy. Reviewers discussed that the manuscript may be improved by re-organization (suggested by reviewer #2), and more careful writing in a number of places (individual points are listed below).

We thank all of the reviewers for their insightful suggestions and comments. We have substantially restructured the entire manuscript mainly adhering to suggestions for reorganization by reviewer #2. In addition, we collected 2 new tomographic data sets and reanalyzed or reorganized the original tomographic data to address reviewer concerns. As suggested, we also include additional immunostaining and live imaging data to be clearer about the structure of inter-chromosomal microtubules and the extent of anaphase A (see also below). Despite the addition of new data, we streamlined the whole text as suggested by the reviewers. Importantly, we modified our conclusions to take into account all suggestions and concerns.

2) Reviewer #3 indicated that the conclusions regarding anaphase A are not well-supported by evidence and highlighted that the segregation of the X is not being considered by the authors when suggesting that k-fiber shortening does not drive chromosome-to-pole movement in these spindles. After discussion among reviewers, the important points raised by reviewer #3 must be taken into account when presenting analysis and conclusions related to anaphase A.

During our discussion, reviewer #2 suggested the following which may help partially address reviewer #3's comments and potentially amend some of the conclusions presented.

The authors show plots of what they refer to as end-on and lateral (see comments on these terms in the detailed reviews) for the X – however, all their plots would benefit from including the X-to-pole (as well as autosome-to-pole) separation distances to help interpret what is currently only referred to as anaphase 1,2,3, 4 in the graphs. At least in the plot shown it does not look like anaphase 4 (most separated) was associated with significantly shorter MT lengths but they should address reviewer #3's comments on this point.

We have made several changes to address the concerns of reviewer #3 in regard to our conclusions about anaphase A. These are described in detail below in our responses to individual reviewer comments. Our intent is not to say that there is a complete absence of kinetochore microtubule shortening but instead to show that there are alternative mechanisms that we identified that can contribute to anaphase A.

To address the suggested changes by reviewer #2, we added additional analysis to Figure 5. From what we could interpret from the suggestion, we added an additional analysis to Figure 5, which shows the microtubule length of either side of the X chromosome plotted against the respective X chromosome-to-pole distance. In Figure 5C it is possible now to distinguish the microtubule length of either side of the X chromosome in relation to the X chromosome-to-pole distance. This illustrates that for the majority of data sets the closer pole to X has also shorter microtubules and vice versa.

We also provide more detail and clarity about our staging of the reconstructed spindles. We compared our tomographic data with live-imaging data of chromosome and pole dynamics (see Appendix—figure 2). In order to keep our plots ‘readable’, we omitted the addition of the suggested X-to-pole distance or autosome-to-pole distance to each figure. All this data is also summarized in Table 1 and plotted in Figure 5D and in the additional Appendix—figure 9.

The authors should also separate the two spindle halves (the "winning" vs. "losing" side) in the later anaphase stages and plot lengths as a function of X-pole distance in each half-spindle; this would help in terms of assessing whether X segregation was associated with significant shortening of microtubules that they detect by tomography. Currently all these values are averaged and, as noted by reviewer #3, the modest changes and large deviations make the conclusions stated about lack of microtubule shortening questionable. It is important that the authors take these comments seriously when preparing their revision.

Our electron tomograms are snapshots of specific stages of meiotic divisions. Thus, assigning labels like “winning” and “losing” to each side of the single X would be speculative, which we wish to avoid. An unbiased way of analysis is to plot the mean microtubule length of microtubule from each side of the X chromosome against its X chromosome distance. As suggested, we have done this in our new Figure 5C. There is a clear linear relationship between the distance of the X chromosome to the spindle pole and the microtubule length. We also created a second new plot (Figure 5D) that color codes the identical data by the type of microtubule association to the chromosome. This way of plotting the data now illustrates nicely that both end-on and laterally associated microtubules change their length in response to the distance of the X chromosome to the respective spindle pole with similar characteristics. Again, in light of reviewer #3’s comments, we have amended the text to adjust our conclusions regarding anaphase A as described above. We have amended the text in multiple places to make this clearer which is detailed in the response below.

3) Reviewers recommend that the authors conduct additional live imaging analysis of anaphase B (e.g. by visualizing central spindle components and cortical force generators), as that is the dominant mechanism separating chromosomes. Any insights from the tomography analysis on anaphase B should also be discussed to provide a more balanced view of the chromosome separation mechanism. Although live imaging would be ideal, if it is not feasible, the authors can characterize the localization of central spindle proteins (SPD-1, centralspindlin, etc) by immunofluorescence, to better demonstrate that the central spindle really doesn't exist.

As suggested we have amended the text to better describe our characterization of sperm-specific mid-zone spindle dynamics. Our intent is to show that these dynamics are distinct from those of oocyte meiosis or mitosis, not necessarily that the central spindle doesn’t exist. Our revised manuscript now includes added immunolocalization of proteins that specify the central spindle during anaphase I to show that, instead of localizing in the spindle midzone during mid-late anaphase I as in oocyte meiosis or mitosis, they localize either on chromosomes (Aurora BAIR-2 or CLASPCLS-2) or, strikingly, at the ingressing membrane (MKLP1ZEN-4). We have also included live-imaging analysis of the localization dynamics of GFP-tagged PRC1SPD-1 during sperm meiotic anaphase I, which shows it localizes to the midzone, but its presence decreases during anaphase I progression, which is unlike oocyte meiosis and mitosis when its levels persist throughout anaphase.

Overall, while significant additional experimental analysis is not necessary, we would like authors to: (1) reorganize the writing for clarity and accuracy; […]

We have largely reorganized the paper as suggested by reviewer #2.

[…] (2) re-analyze existing data (or potentially add new data if available to strengthen their points) and be significantly more careful about claims related to the minor anaphase A-like movement in light of the critiques from reviewer #3; […]

We did significant additional analysis (see below) to thoroughly address reviewer comments. In particular, we added more data and re-analyzed previously presented data related to the minor anaphase A-like movement, which we describe in the detailed responses to individual reviewer comments below. We have also amended our claims throughout to clarify our conclusions – in particular include the possibility that shortening of kinetochore microtubules may play some role, but in combination with the new factors we describe.

[…] and (3) present additional characterization of the predominant chromosome separation mechanism: anaphase B.

We provide additional data, including immunolocalization and live-imaging of central spindle specifiers, to support that midzone spindle structures during anaphase I are distinct in sperm meiosis compared to oocyte meiosis and mitosis. We have also amended our claims throughout to clarify our conclusion that there are alternate midzone spindle structure and dynamics during sperm meiosis that support a stronger reliance on non-midzone generated forces compared to oocyte meiosis and mitosis.

Reviewer #1:

The manuscript by Fabig et al. describes how chromosomes are segregated on the meiosis I (and II) spindles in C. elegans spermatocytes. Using a combination of live cell imaging, electron tomography and fixed images, the authors present a beautiful description of the spermatocyte spindle. This analysis reveals some very interesting and unexpected features including novel mechanisms to account for anaphase A movement, as well as providing insight into how lagging chromosomes are segregated.

Thank you for this comment.

In the Introduction, the authors conflate general features of chromosome segregation with the specifics of C. elegans. For example, Introduction paragraph two, they generalize findings in C. elegans that may or may not be the same in other systems. The authors need to be more careful in their description between what is happening in C. elegans versus general features of chromosome segregation.

Thank you for pointing this out. In the revised version we have reorganized the Introduction to clarify features of chromosome segregation that pertain to C. elegans meiosis.

Subsection “Lagging of chromosomes is a consequence of a lack of pairing” paragraph three: ZIM-2 mediates the pairing/synapsis of chromosome V not IV (Phillips and Dernburg, 2006).

Thank you for pointing out this mistake in our manuscript. We have corrected this.

Subsection “Spermatocyte meiotic centrosome and spindle dynamics are distinct from that in mitosis”: What is the evidence that spindle MTs remain connected to the X chromosome?

It is traditionally hard to show, even by electron tomography, that microtubules are directly “connected’ to the holocentric C. elegans kinetochore, as the kinetochore itself is not visible as an electron-dense plaque like the kinetochore in mammalian cells. For this reason, we decided to be more cautious and change our wording to “associated’. To support the association of microtubule association to chromosomes, in the manuscript we refer here to microtubules which end or travers the ribosome-free zone around the chromosomes with at least 150 nm distance in C. elegans males (paragraph two subsection “Spermatocyte spindles maintain both end-on and lateral associations of

kinetochore microtubules to chromosomes throughout meiosis”), which is considered the holocentric kinetochore In addition to our EM data, which clearly shows that single microtubules bridge the distance between spindle poles and the X chromosome (Figure 5 and Videos 8-11), we also show in Figures 2E-F and Appendix—figure 1F that the outer kinetochore stays assembled during anaphase and is present in ~150nm distance between chromosomes (including the X) and microtubules.

Figure 6: Please add the color code directly to the figure (e.g., white is end-on MT interactions and gold is lateral MT interactions). Would it be possible to highlight a continuous MT from the centrosome to the X chromosome?

As suggested, we have added the color coding to the figures containing electron tomographic data (now Figures 3 and 4, Appendix—figure 4 and Appendix—figure 7). Actually, almost all microtubules are spanning the distance between the X and the spindle pole. For a better visualization of the connection to the poles, we modified our videos of the tomographic reconstructions (Videos 4-11) to highlight all X chromosome-associated microtubules (in cyan).

Appendix—figure 7: Absence of central spindle. The authors present evidence that there is no central spindle based on weak MT staining in the center (and the presence of the lagging X chromosome). This would be strengthened by staining with central spindle components such as CYK-4/ZEN-4/SPD-1 in a similar manner as was done for the inner and outer kinetochore (Figure 4).

As suggested, we now show additional data to strengthen the point that a “classic” central spindle with interdigitating microtubules during mid-anaphase was not observed in meiosis I in C. elegans males. First, by analyzing tra-2 XX males, which do not have a lagging X chromosome, by electron tomography, we find the microtubules appear to emanate from the poles and do not form a classic overlap, which is distinct from that observed in early mitosis or female meiosis in C. elegans (subsection “Interdigitating midzone microtubules are not a prominent feature during spermatocyte

anaphase progression”, Appendix—figure 7C). Second, we added new immunostaining data to show that the Aurora B kinase AIR-2 and the microtubule stabilizer CLASPCLS-2 remain chromosome associated in both him-8 (with a lagging X) and tra-2 (without a lagging X) animals during sperm meiotic anaphase I, in contrast to their strong midzone localization during oocyte meiosis and mitosis (Appendix—figure 8A). The centralspindlin component MKLP1ZEN-4 also shifts to localization at the ingressing cell membrane during mid-anaphase I. Further, using live-imaging we find PRC1SPD-1 exhibits transient presence in the midzone then is absent even as chromosome continue segregating during anaphase I progression (Appendix—figure 8B). A comparison of the spindle dynamics in tra-2 males with wild-type males showed that spindles without lagging chromosomes exhibit a faster pole-pole separation with a longer final spindle length (Appendix—figure 7B). This indicates that the microtubule bridge in wild-type males rather counteracts spindle elongation than promoting it. In summary, we present evidence that spermatocytes have alternate spindle structure and dynamics that do not resemble the classic spindle midzone observed in oocyte meiosis and embryonic mitosis.

Is there any correlation between the curved MTs and which pole the X segregates to? In the images/videos the curve is more pronounced on one side of the spindle.

We find that the curving of microtubules associated with the X is very interesting and also puzzling. At this stage, it is difficult to make strong statements as to which extent straight versuscurved microtubules contribute to the segregation of the X. However, we consider it important to report this phenomenon for further consideration and investigation (see Appendix—figure 6).

Reviewer #2:

The manuscript from Fabig et al. presents live imaging analysis of male meiotic divisions in C. elegans that is complemented by electron tomographic analysis of microtubule distributions during this developmentally specialized division. This is a technically strong study with two major themes: (1) analysis of the unpaired X chromosome's segregation – lagging followed by eventual segregation to one spindle pole, and (2) description of a modest anaphase A-like movement of autosomes that is not associated with microtubule shortening but instead driven by shape changes.

The manuscript has compelling elements but is a difficult read and would greatly benefit from reorganization and more careful writing. A more challenging question is whether there is need for any type of perturbation analysis – this point can be addressed in the reviewer discussion. To enhance the manuscript's impact, I would recommend the authors organize the manuscript into two sections – the first focused on X chromosome lagging and the second briefer one on anaphase A-like autosome movement – that are bridged by the tomography (which is the most important contribution here and needs to be presented significantly earlier in the paper). Here is a recommended structure along with comments on each section:

We are very grateful for the time and effort this reviewer made to make clear suggestions to improve the text. We have changed the structure largely as suggested.

The authors need to make it significantly easier for readers to evaluate figures and compare different datasets. All time-dependent graphs should have a common time 0, e.g. anaphase onset, and the Time axis should be labeled "Time relative to anaphase onset (s)".

We have made the suggested changes to the figures as suggested to address this concern. Now, time (t=0) always refers to the onset of anaphase throughout the paper, with two exceptions (Figure 2B and Figure 6F). For these two plots we used “Time relative to the onset of X segregation”. The reasons for these two exceptions are as follows: The start of X-chromosome movement varied considerably from spindle to spindle, from 1.5–8.5 minutes after anaphase onset (mean: 4.92 +/- 1.55 min). Because of this variability, we decided after careful consideration to plot the shape coefficient relative to the onset of X chromosome movement. The point of Figure 2B is to clearly indicate the change in shape of the X chromosome in response to the onset of its movement. We decided to keep the data in Figure 6F (ratio of microtubules on both sides of the X chromosome) aligned to the onset of X chromosome movement for the same reason. We tried to make this difference to the other plots of spindle dynamics clearer by relabeling the X-axis of both graphs.

On 2-color images shown, the component visualized in each color should be labeled on the figure and not only in the legend. Similarly, pseudo-colored entities in the tomograms should be described on the figure with proper labels. These types of modifications will great improve accessibility of the manuscript to interested readers.

We agree and have added legends with color coding to the individual figures (Figures 1, 2, 6 and 7) and videos (in addition to the information that is given in the figure legends).

1) Merge Figure 1 and 2 into one figure showing that unpaired chromosomes lag.

The authors state that "sister chromatids of the unpaired X attach to opposite poles…"; this is not in fact supported by the data and should be removed. An interesting point that should be mentioned is that cohesion is protected between the sisters of the X in meiosis I – presumably because the lack of recombination prevented definition of an axis of cohesion removal in meiosis I.

We have merged Figures 1 and 2 as suggested and removed the statement about the attachment of the sister chromatids. As for the second point, we feel that this information may also lack sufficient support by our data, thus we did not include it.

2) Leave Figure 3 for later in the paper (see below); move Figure 4 to the supplement – this figure overlaps with prior fixed data, does not report any significant new observations and is disruptive in the flow of the manuscript.

One point here – that HIM-10 looks different from MIS12 complex and KNL-1 – is rather surprising and should be verified using CRISPR-tagged versions that have been generated for all of these components. It is possible that the staining observed with the anti-HIM-10 antibody between the homologs is non-specific.

We largely made the suggested changes. We removed the previous Figure 4 in large parts. We also amended our text to de-emphasize distinctions in specific kinetochore protein localization. The point we would like to make is that outer kinetochore proteins (such as KNL-1, KNL-3, NDC-80) are retained on meiotic chromosomes, including X, during anaphase I, which is distinct from what occurs in oocyte meiosis (now Figure 2E). This has previously not been reported and supports that kinetochore proteins bridge chromosome-microtubule interactions during anaphase I as chromosomes are pulled apart, and important for the segregation of the X chromosome.

3) Make current Figure 5 into Figure 2. This figure is focused on movement of the lagging X and will follow directly from the revised Figure 1 (see point 1 above).

As noted above, label imaged components on the figure. There is one issue with the conclusions in Figure 1A and B – the change in chromosome morphology cannot be solely attributed to loss of tension because there is also reduction in cell cycle phosphorylation between meiosis I and meiosis II. The authors should analyze their laser ablation data (especially sequences like no. 2) to assess if there is rapid relaxation after ablation. In the absence of such data, they need to be more cautious in the interpretation in current Figure 5A and B.

The sequence of the figures and information on the color coding has been added as suggested. As for the relaxation analysis, we agree that this is an issue that needs to be taken in account. For the laser ablation we did not use 3D imaging but instead imaged a single plane with manual focusing, but with a high time resolution. Only in this way was it possible to ablate an object in the focal plane reliably. As such, we cannot analyze the shape of the X chromosome in a similar way as shown in Figure 2A and B. It was also challenging to keep the cells in focus due to the movements of the imaged worms. We also had to deal with slightly tilted random orientations of the meiotic spindles. For these technical reasons, we could not analyze the shape of the X chromosome after laser ablation, although we agree that such an analysis would be informative.

It is not clear to us how to address the comment on how reduction in cell cycle phosphorylation might change chromosome morphology between meiosis I and II. We do admit there might be multiple factors involved, though loss of tension seemed to be the most obvious contributor. Thus, due to time and space restrictions, cell cycle phosphorylation could not be analyzed or included within this study.

4) Make current Figure 6 (tomography) into Figure 3 – label colors on the figure and not in legend. Provide numerical summary also in the figure (can be repeated in the text).

The last column is very difficult to visualize. On a more important note, the exact criterion used to call end-on vs. lateral should be clarified here even if they were previously described. Precisely how much length of microtubule should be present in the "clear" zone for it to be designated "lateral"? This is also a potential point of confusion in that the classification does not match what these terms are used to refer to in analysis in other systems (e.g. yeast or human cells, where a lateral attachment refers to kinetochore bound on the side of a microtubule that extends far past the kinetochore). I suspect that the attachments described here may be similar to what is termed an "end-on" attachment in other systems. There is also a geometric issue here coming from the curvature of the chromosomes – the surface that is available for what is classified as an end-on attachment may limit their number.

We made the suggested figure change.

We are aware of the “geometric issue “that has been raised here. Indeed, end-on versuslateral association of KMTs with chromosomes is clearly defined in systems with monocentric kinetochores (for example, in budding yeast with one kinetochore microtubule per chromosome). As published for previous EM studies in C. elegans (for instance in Howe et al., 2001; O’Toole et al., 2003; Redemann et al., 2017), microtubules that are submerged in a ribosome-clear zone (representing the holocentric or diffuse kinetochore) were considered as kinetochore microtubules. We did not define a minimal distance in our analysis. Indeed, the situation is complicated by the fact that chromosomes in C. elegans meiosis are not rod-like but cup-shaded. Because of this difference in geometry, the probability of a lateral interaction of a microtubule with a chromosome is increased. In addition, and this is a very important point, the tomographic data show that the chromosome surface is not smooth but instead rather ‘rough’ or ‘curvy’. This makes the analysis even more complicated. All these considerations were taken into account when developing our approach of defining an end-on versusa lateral association. As described in detail in Materials and Methods, we modeled the chromosome surface. Then we addressed each microtubule that was at least 150 nm or closer with any part of its lattice to the surface of the chromosomes. Next, we extrapolated all microtubule ends by 150 nm and scanned for a crossing of the chromosome surface and defined the positive one as end-on associated. All other microtubules, not crossing the chromosome surface that still were 150 nm or closer next to the chromosomes were defined as laterally associated. To further illustrate this method, we added a video (see new Video 15, called out in Materials and Methods) showing both types of microtubule association to chromosomes in one 3D model at high resolution. To our mind, the developed approach is a clear und unbiased way to analyze the tomographic data. We agree that one should be more careful here in making strong conclusions. Thus, we simply want to report that both end-on and lateral associations of microtubules to chromosomes exist throughout male meiosis. We avoid strong conclusions about the function of end-on versuslateral associations as discussed in the literature for microtubules attached to a monocentric kinetochore. This issue needs to be addressed in a follow-up paper.

5) Make current Figure 7 and Figure 8 into Figures 4 and 5. These continue to focus on the lagging X chromosome and its segregation but now integrate the tomography.

There are some issues with the writing and figure elements associated with this analysis. In the tortuosity analysis, is there any contribution from the cleavage furrow? Is there a furrow at the stages that are visualized or not?

The figures have been changed as suggested.

As for the role/contribution of the ingressing furrow, we did some experiments to investigate whether the membrane plays a role here (data not shown in the paper). From the videos we recorded it was our impression that the decision in terms of direction was done before the ingressing membrane has a restrictive effect on the segregation, thus we are unable to conclude it has a clear role. In our tomograms we do not see the ingressing membrane. Even our most advanced stage (anaphase no. 7) does not show the cleavage furrow. We must collect much more data to be definitive about that issue, which we feel would be more appropriate in a follow up paper.

In current Figure 8B, what are "anaphase S1, S2, S3"? In Figure 8F, why does the ratio go up? Is it because more microtubules are formed on the "winning" side or there is a decrease in microtubules on the "losing" side? This should be clarified by looking at the measured values and not the ratio. More generally, there is not any causal relationship established by any of this analysis and the writing needs to be more circumspect.

We have clarified the description of all the data we analyzed and included these data sets to make the information available to other researchers. The data sets Anaphase S1-3 of the previous submission are additional reconstructions shown now as Appendix Material (Appendix—figure 4). We have also renamed theses data sets to anaphase no. 2, no. 5 and no. 6, to make their stage of division more clear (see Figure 6D).

We also clarified the presentation of data regarding resolution of the X chromosome. For the tomographic data, in the new Figure 6B we give the ratio of microtubule content and in Figure 6D we show the microtubule numbers for each 3D reconstruction. Indeed, the ratio indicates that one side has more microtubules, while the other one shows less microtubules. Though it would be helpful to give numbers for the “winning” and the “losing” side, we would like to point out that this is not possible for early to mid-anaphase stages by looking at the static 3D models to know which direction the X would have migrated. We were thus purposefully careful to provide just the ratio of the numbers without mentioning the direction for the tomographic data. However, to address this we provide quantification of live-imaging data to support the level of connections to the “winning” and “losing” side to further support our findings (subsection “Segregation of the X chromosome correlates with an asymmetry in the number of associated microtubules”). As shown in Figure 6E-F, we see an increase in the fluorescence intensity on one side of the X and a decrease in the intensity on the other side of the X chromosome. As a general comment, we take care in the manuscript to say the data is supportive of a model for the decision related to the direction of the X chromosome segregation. We also indicate more data sets of very late stages must be analyzed. Because each tomographic reconstruction is a tremendous amount of work, this will certainly be another “tour de force “study and we have planned already to start this kind of analysis.

There is also an analogy to made to segregation of merotelically attached kinetochores in mitotic mammalian cells that lag and segregate to the side that ends up with more microtubules. This should be mentioned.

Thank you for pointing this out. We have added a comment to address this in the Discussion.

6) End the paper with 2 figures (Figure 6 and 7) on autosomal anaphase A (which provides ~20% of separation). Start Figure 6 with current Figure 9 to highlight that kinetochore microtubules on autosomes did not shorten and follow that with current Figure 2, based on light imaging, which shows there is modest anaphase A, as defined by reduction in distance of chromosomes to poles. Then end with what is currently Figure 10, which provides reasons for why this would be the case.

Note that the chromosome shape change and the angular change are related and not independent – thus there are 2 factors at play – the chromosome shape change and the change in the centrosome. It would be helpful if the Anaphase 1, 2, 3 and 4 were annotated to include the separation distance of the autosomes.

We have changed the sequence of the figures as suggested. We have also added the requested information about the autosome-to-autosome distances in Figures 3-4 and Appendix—figure 4. This information is also given in Table 1.

On a related note, 80% or more of the separation is not due to anaphase A. Is this entirely due to cortical pulling? Given their expertise, the authors should look at conserved central spindle markers (SPD-1, CYK-4, ZEN-4) and also dynein (DHC-1) – there should be endogenously tagged versions of all of these components available by now.

Related also to comments from reviewer #3, we provide additional immunostaining data and live imaging to address this comment (subsection “Interdigitating midzone microtubules are not a prominent feature during spermatocyte anaphase progression”, Appendix—figure 7). In contrast to oocyte meiosis and mitosis, during sperm meiotic anaphase I Aurora B kinase AIR-2 and the microtubule stabilizer CLASPCLS-2 remain chromosome associated in both him-8 (with a lagging X) and tra-2 (without a lagging X) animals. Further, PRC1SPD-1 exhibits decreasing presence in the midzone during anaphase I progression while the centralspindlin component MKLP1ZEN-4 shifts to localization at the ingressing cell membrane during mid-anaphase I. These results support that spermatocytes have sperm-specific spindle structure and dynamics. This further supports a reliance on cortical forces, instead of a microtubule pushing as observed in oocyte meiosis and also in mitosis after laser microsurgery. We believe a detailed analysis of the role of cortical pulling mechanisms should be part of a further study.

Reviewer #3:

This manuscript by Fabig et al. reports a detailed characterization of spermatocyte meiosis in C. elegans. The authors present data that provides some new insights into spindle organization and chromosome segregation in this system. However, I find some of their conclusions to be insufficiently supported (see points below).

1) One of the major points that this paper attempts to make is that k-fiber shortening does not drive anaphase A in spermatocytes. However, I do not think that the presented data provide strong support for this conclusion. One of the main issues is that the spermatocyte spindles are small and the poleward-facing edges of the autosomes start out very close to the poles, so the chromosomes do not move very far in anaphase A. Therefore, if there was k-fiber shortening it would be hard to detect, especially given the variability in microtubule lengths reported from the electron tomography analysis. The authors report that the "end-on" microtubules for metaphase are 0.62 +/- 0.33 µm and the "lateral" microtubules are 1.15 +/- 0.59 µm. Given this amount of variability, if some population of these microtubules shortened and helped drive movement towards the pole, it could be hard to detect. Compounding this issue, since only one metaphase spindle was analyzed, it is difficult to know whether the numbers reported for metaphase are consistent from spindle to spindle.

We understand the reviewer’s issue with the strong conclusion that kinetochore microtubule shortening has no role in anaphase A that we detect in sperm meiosis. We were also surprised by the fact that one can see a clear anaphase A movement by light microscopy but not a clear shortening of kinetochore microtubules by electron tomography reaching single-microtubule resolution. From our analysis of the data, we believe that the spread of the data is due to the cup-shape and the ‘curvy surface’ of the autosomes (see Video 15). We also acknowledge that given this spread of the data that the reviewer points out, we cannot exclude the possibility that there could be some shortening of a subfraction of microtubules. Nonetheless, the degree to which we find that the majority of microtubules do not shorten to account for the total anaphase A distance traveled is still an interesting observation. To address the valid concerns raised by reviewer #3, we have restructured the paper and amended the text to not preclude that shortening of kinetochore microtubules may contribute in some degree to anaphase A. For example: “…our tomographic analysis suggests that shortening of kinetochore microtubules does not fully account for the anaphase A observed by light microscopy.” Also, in the Discussion we state: “Microtubules can shorten during anaphase I, as observed when the X resolves to one side; thus, a shortening of a subset of microtubules that is difficult to detect by current methods may also contribute to a small portion of anaphase A movement. Nonetheless, our proposed new mechanisms can now be considered when analyzing anaphase A movement in other systems.”

We agree with the reviewer that more metaphase data would strengthen our conclusions. To address this, we recorded and analyzed two more metaphase I tomographic data sets. These additional full data sets are now included in the manuscript. One of the new data sets (metaphase data set no. 3) is in full agreement with the already presented data. The second additional data set (metaphase no. 1), however, is slightly different. Chromosomes don’t show the typical stretching of the chromosomes known to occur at peak of metaphase immediately before anaphase onset. In addition, the pole-to-pole distance and the autosome-to-autosome distance is smaller compared to the other two data sets. For these reasons, we consider this data to represent a spindle at the prometaphase to metaphase transition or a very early metaphase.

In summary for the raised point, the conflict here remains that light microscopy shows a clear anaphase A movement (i.e. a contribution to the segregation of about 20%), whereas electron microscopy does not show a significant shortening of a large portion of the autosome-associated kinetochore microtubules, which we believe is an interesting fact. At this point, it is important to present the length measurements in a clear way and it is important to consider these values in the context of other changes in the cellular ultrastructure, of which we hope we now clearly describe and quantify to characterize other factors that can account or anaphase A displacement.

Additionally, the author's own data appears to suggest that k-fiber shortening could be capable of driving poleward movement in these spindles. In the case of the lagging X chromosome, the authors show nice videos of the X segregating in late anaphase (both in the normal case in Figure 1/Video 1, and in their cutting experiments in Figure 5/Video 4). In these cases (since the spindle has elongated in anaphase B), the X has to segregate over a longer distance and therefore it is easier to visualize what is happening to the microtubules/k-fibers. In these videos, it appears that once the X starts segregating to the "winning" pole, the corresponding k-fiber shortens, reeling in the chromosome. It seems unlikely to me that this k-fiber shortening mechanism to drive chromosome-to-pole movement would exist for the X chromosome, but not the autosomes. It seems more likely that the autosomes also segregate by this mechanism in anaphase A, but that it is difficult to measure because the distances are so short and the variability of the microtubule lengths is so high. Therefore, I do not find the conclusions of the authors about anaphase A mechanisms to be convincing.

It was not our intention to suggest that there is no kinetochore microtubule shortening possible during sperm meiosis, particularly because we did observe shortening of X-associated microtubules during later stages of anaphase I (Figure 5C). We cannot preclude that kinetochore microtubules connected to autosomes may shorten to some extent, though we provide quantitative measurements it is unlikely that this possible shortening could account for the full anaphase A displacement we find. In addition, we observe that X chromosome segregation takes place after autosomes have been partitioned to the opposite poles. During early anaphase when autosomes are separating, we actually see X-connected microtubules elongating, not shortening. We have amended the text to better reflect that we think the most likely explanation is that microtubules can grow and shorten very dynamically and depend on the balance of forces experienced by chromosomes at the time. In our revision, we simply provide evidence that the release of tension at anaphase I onset has the additional factors of tension release that contribute to anaphase A movement, not that kinetochore shortening does not occur at all. We have also amended the text to better indicate that anaphase A contributes only ~20% to the segregation of autosomes.

2) The designation of "end-on" vs. "lateral" microtubules in Figure 6 is confusing. Many of the white microtubules designated as end-on appear to run quite far down the side of the chromosome. Therefore, they don't really appear to be "end-on". Correctly categorizing microtubules is important, because the authors make a major point about end-on microtubules not shortening during anaphase, and use this as evidence to say that k-fiber shortening does not drive anaphase A movements. But if some of the microtubules they are counting are actually running along the sides of the chromosomes and if this population of lateral microtubules do not shorten, then the authors may be missing a reduction in the length of the ones that actually are "end-on".

The classification was done by the methods we mentioned earlier in response to reviewer #2 comments. As the chromosomes are round there are also end-on associated microtubules at more peripheral positions on the chromosome surface and not only at the directly pole-facing side of the chromosomes. To support this classification, we have added Video 15 to Materials and Methods. This video shows a pair of autosomes with both populations of microtubules at higher resolution. Our intention was not to show that only end-on associated microtubules contribute to the segregation. We would like to point out that both types of association persist throughout meiosis, and we have changed the text accordingly to reflect this (subsection “Spermatocyte spindles maintain both end-on and lateral associations of

kinetochore microtubules to chromosomes throughout meiosis”).

Related to this, in paragraph four of subsection “Tension release across the spindle may contribute to autosomal anaphase A”, the authors use their conclusion that microtubule lengths are constant at 0.63 µm between metaphase and anaphase to calculate how much altering the microtubule angle could contribute to chromosome movements (ending up with a value of 0.17 µm). However, since this measurement of microtubule length is subject to error (given the variability of microtubule length measurements and the potential issue with distinguishing end-on from lateral interactions), the calculations that led to the 0.17 µm shortening number are also in question.

These measurements (as all measurements) are subject to variability, which we do report. This is simply a theoretical geometric calculation to illustrate how a change in angle, while using the average microtubule lengths, could contribute to a change in pole-to-chromosome length. We do not want to draw absolute conclusions from these theoretical considerations but rather want to show that other mechanisms apart from microtubule shortening could contribute to anaphase A movements.

3) In Figure 10, the authors report the ANOVA comparing metaphase with two of the anaphase spindles (number 3 and 4) and it appears that there is a significant difference, but they do not report the ANOVA comparison between metaphase and anaphase numbers 1 and 2 (and #2 especially does not appear to be different from metaphase). This raises the concern that variability between spindles could account for the different amounts of autosomal stretching, rather than spindle stage (metaphase vs. anaphase). This is particularly important since the authors use this data to propose that release of this stretch can account for much of the "anaphase A" pole-chromosome shortening. To me, it seems problematic to draw these conclusions when only one metaphase spindle is analyzed, and therefore it is difficult to exclude spindle-spindle differences in the autosomal FWHMs.

To address this, we added two additional metaphase data sets. We have also shown all ANOVA analysis in Appendix—figure 5. In the interest of readability of Figure 8, we decided to show all significance tests in the separate Appendix—figure 5.

4) For the analysis of chromosome segregation, it would be helpful to have more information about how the distances were determined. The Experimental Procedures state "For the analysis of chromosome movements, the peak maxima of the chromosome fluorescence signals were then used to calculate the distances for each time point." This is confusing and more details on the analysis should be included so the reader can better evaluate these data – how were the centers of each autosome determined? I can imagine that this would be difficult given the resolution of the videos, and subject to error.

As suggested, we have added more information on how we determined these distances to the Materials and Methods (subsection “Analysis of spindle dynamics”). We indicate that we measured the distances in the resampled data and used a kymograph created along the spindle axis. For that, a radius of 0.9 μm was sampled around the spindle axis in 0.1 μm steps along this axis. The Gaussian weighted sum of fluorescence in each plane was calculated for each time point. The two maxima per time point of this plot were then utilized to determine the distance of the chromosomes.

Also, was each autosome within a given spindle measured separately, or was each segregating mass of autosomes treated as one unit (and the center of that entire mass determined)? Given potential issues in accurately determining the "center" (of either each autosome or the autosome mass), I would suggest that the authors try measuring the distance between the outer edges of the autosomes (the poleward facing sides) and the spindle pole… this would more clearly represent the distance the chromosome travels towards the spindle pole.

As stated above, we measured the distances between the fluorescence intensity peaks of the “segregating masses” for the autosomes. We considered this as the most robust way to determine distances because it utilizes the bulk of chromosomes in the spindle center and therefore eliminates outliers. In the suggested way of measuring the distance from the outer edge, some chromosomes might be not perfectly aligned in the segregating plate. The orientation of the spindle relative to the direction of the z-scan of the 3D imaging setup would also influence accuracy. Further, the distances in the tomographic data were determined by measuring the centers of mass of the individual chromosomes. Therefore, this makes LM and EM measurements also more comparable.

5) The authors state that: "Interestingly, the centrosomes remain connected to the X chromosome-connected microtubules". However, there is no figure call-out for this statement, and I can't find any figures where there is convincing evidence of this, given the resolution of the images/videos presented.

We agree that this paragraph was confusing. We have deleted this whole section.

I am therefore confused as to what evidence supports the subsequent statement "Thus, the separation and migration of centrosomes during anaphase I appears to be coordinated with microtubules that must maintain connections not only to segregating autosomes, but also the lagging X chromosome". Either present these data or revise these statements.

We agree that this statement was confusing. We have taken it out.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

Fabig G. 2020. Supplemental data for the publication. OpARA. [DOI]

Supplementary Materials

Figure 2—source data 1. Segregation distances and the shape of the X chromosome in replicates of anaphase I spindles analyzed in Figure 2B .

elife-50988-fig2-data1.xlsx^{(9KB, xlsx)}

Figure 5—source data 1. Measurements of microtubule lengths in spindles shown in Figures 3 and 4 used to generate data in Figure 5.

elife-50988-fig5-data1.xlsx^{(20.3KB, xlsx)}

Figure 6—source data 1. Autosome-to-autosome distances and volumetric measurements for the meiotic stages shown in Figures 3 and 4 and Appendix 1—figure 4 used to generate Figure 6B.

elife-50988-fig6-data1.xlsx^{(11.2KB, xlsx)}

Figure 7—source data 1. Measurements of autosome and centrosome dynamics from replicates used in Figure 7 .

elife-50988-fig7-data1.xlsx^{(70.1KB, xlsx)}

Figure 8—source data 1. Measurements for Full Width at Half-Maximum(FWHM) of replicates in each stage shown in Figures 3 and 4 used in Figure 8.

elife-50988-fig8-data1.xlsx^{(54KB, xlsx)}

Source code 1. Kymograph.

elife-50988-code1.py^{(16.3KB, py)}

Source code 2. Volume resampling.

elife-50988-code2.py^{(12.6KB, py)}

Transparent reporting form

elife-50988-transrepform.pdf^{(230.8KB, pdf)}

Appendix 1—figure 2—source data 1. Measurements of autosome-to-autosome and pole-to-autosome distances from replicates used in Appendix 1—figure 2.

elife-50988-app1-fig2-data1.xlsx^{(12.9KB, xlsx)}

Appendix 1—figure 3—source data 1. Measurements of centrosome volume from replicates used in Appendix 1—figure 3.

elife-50988-app1-fig3-data1.xlsx^{(11.6KB, xlsx)}

Appendix 1—figure 6—source data 1. Measurements of tortuosity of end-on and lateral microtubules from replicates used in Appendix 1—figure 6.

elife-50988-app1-fig6-data1.xlsx^{(16.9KB, xlsx)}

Appendix 1—figure 7—source data 1. Measurements of pole-to-pole distance over time in replicates used in Appendix 1—figure 7B.

elife-50988-app1-fig7-data1.xlsx^{(9.5KB, xlsx)}

Appendix 1—figure 9—source data 1. Measurements of pole-to-autosome and microtubule length in each stage shown in Figures 3 and 4 used in Appendix1-figure 9-source data1.

elife-50988-app1-fig9-data1.xlsx^{(10.2KB, xlsx)}

Data Availability Statement

Data have been uploaded to the TU Dresden Open Access Repository and Archive system (OpARA) and are available as open access: https://doi.org/10.25532/OPARA-56.

The following dataset was generated:

Fabig G. 2020. Supplemental data for the publication. OpARA.

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PERMALINK

Male meiotic spindle features that efficiently segregate paired and lagging chromosomes

Gunar Fabig

Robert Kiewisz

Norbert Lindow

James A Powers

Vanessa Cota

Luis J Quintanilla

Jan Brugués

Steffen Prohaska

Diana S Chu

Thomas Müller-Reichert

Roles

Abstract

Introduction

Results

Spermatocyte meiotic spindles are distinguished by delayed segregation of the unpaired X chromosome

Figure 1. Unpaired chromosomes lag in spermatocyte meiosis I.

Figure 1—video 1. Live-cell imaging of meiosis I and II in wild-type males.

Lagging of chromosomes is a consequence of a lack of pairing

Microtubules attached to the X chromosome exert a pulling force

Figure 2. Microtubules associated with the X chromosome exert a pulling force.

Figure 2—video 1. Laser ablation of the microtubule bridge in a spermatocyte undergoing the first meiotic division.

Figure 2—video 2. Double-cut laser ablation of the microtubule bridge in a spermatocyte undergoing first meiotic division.

Kinetochores are not disassembled between spermatocyte meiotic divisions

Spermatocyte spindles maintain both end-on and lateral associations of kinetochore microtubules to chromosomes throughout meiosis

Figure 3. Three-dimensional ultrastructure of spindles in metaphase I.

Figure 3—video 1. Full tomographic reconstruction of the metaphase I spindle in a wild-type male spermatocyte.

Figure 3—video 2. Full tomographic reconstruction of the metaphase I spindle in a wild-type male spermatocyte.

Figure 3—video 3. Full tomographic reconstruction of the metaphase I spindle in a wild-type male spermatocyte.

Figure 4. Three-dimensional ultrastructure of spindles in anaphase I.

Figure 4—video 1. Full tomographic reconstruction of the spindle in a wild-type male spermatocyte at the onset of anaphase I.

Figure 4—video 2. Full tomographic reconstruction of the anaphase I spindle in a wild-type male spermatocyte.

Figure 4—video 3. Full tomographic reconstruction of the anaphase I spindle in a wild-type male spermatocyte.

Figure 4—video 4. Full tomographic reconstruction of the anaphase I spindle in a wild-type male spermatocyte.

Figure 4—video 5. Full tomographic reconstruction of the anaphase I spindle in a wild-type male spermatocyte.

Table 1. Analysis of tomographic data sets used throughout this study.

Continuous and lengthening microtubules connect the X chromosome to centrosomes during anaphase I

Figure 5. Microtubules that connect the X chromosome to centrosomes are continuous and lengthen during anaphase I.

Segregation of the X chromosome correlates with an asymmetry in the number of associated microtubules

Figure 6. Resolution of the X chromosome to one side correlates with an asymmetry of microtubules.

Interdigitating midzone microtubules are not a prominent feature during spermatocyte anaphase progression

Appendix 1—figure 7. Organization and dynamics of tra-2(e1094) spindles in meiosis I.

Appendix 1—figure 7—video 1. Organization of tra-2 spindles in meiosis I.

Spermatocyte meiotic spindles display both anaphase A and anaphase B movement

Figure 7. Spermatocyte meiotic spindles display both anaphase A and B movement.

Figure 7—video 1. Spindle dynamics in wild-type spermatocytes in meiosis I.

Figure 7—video 2. Spindle dynamics in wild-type spermatocyte meiosis II.

Table 2. Measurements of spindle dynamics in male meiosis.

Electron tomography does not suggest a shortening of autosome-associated kinetochore microtubules during anaphase

Tension release across the spindle may contribute to autosomal anaphase A

Figure 8. Changes in spindle geometry during anaphase A.

Discussion

Lagging and resolution of the X chromosome

Contributors of anaphase A not reliant on shortening of kinetochore microtubules

Distinctions of C. elegans spindles in spermatocytes

Materials and methods

Key resources table.

Strains and worm handling

Strains

Worm handling

Light microscopy and analysis of spindle dynamics

Light microscopy

Analysis of spindle dynamics

Immunostaining for light microscopy

Laser microsurgery

Specimen preparation for electron microscopy

Electron tomography, microtubule segmentation and stitching of data sets

Analysis of tomographic data

Staging of tomographic data sets

Classification of microtubules

Video 1. Visualization of end-on or laterally associated kinetochore microtubules.

Length distribution

Chromosome shape

Acknowledgements

Appendix 1

Appendix 1—figure 1. The outer kinetochore protein NDC80 localizes between chromosomes and microtubules at spermatocyte metaphase and anaphase I.

Appendix 1—figure 2. Comparison of electron tomographic and light microscopic data.

Appendix 1—figure 3. Analysis of centrosomal volumes in meiosis I and II.

Appendix 1—figure 4. Visualization of partially reconstructed spindles in mid/late anaphase I.