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. 2020 Mar 19;9:e53807. doi: 10.7554/eLife.53807

Figure 3. The deformed kinetochore-fiber’s shape indicates specialized, short-lived crosslinking to the spindle near chromosomes.

See also Figure 3—figure supplements 1–3 and Figure 3—videos 1 and 2. (A) Schematic of the assay to probe the physical basis of k-fiber anchorage in the spindle: manipulation of the outer k-fiber for 60 s and quantification of local curvature along its length. The absence of k-fiber negative curvature (1) would suggest pivoting at poles and chromosomes. K-fiber negative curvature at poles (2) or chromosomes (3) or at both (4) would suggest it is laterally anchored there and prevented from pivoting. (B) Top: Timelapse images of a representative PtK2 metaphase spindle (GFP-tubulin, grey) during a 60 s manipulation, with microneedle position (white circle) and traced manipulated k-fiber (white) displayed on the images. Scale bar = 5 μm. Time in min:sec. Bottom: Curvature mapped along highlighted k-fiber for each time point in the top panel (blue, negative curvature; red, positive curvature). This manipulation can expose contact points (asterisk) between the k-fiber and non-kMTs. (C) Local curvature of deformed k-fibers for normalized positions along the k-fiber (n = 23 cells). Most k-fibers exhibit negative curvature near the chromosome (orange), and a few show no negative curvature (grey) near the chromosome. Few k-fibers also show negative curvature near poles. Scatter plot of microneedle positions shown above (inset). (D) Percentage of k-fiber curvature profiles with negative curvature less than −0.1 1/μm, proximal to chromosomes (n = 17/23 cells) and the pole (n = 3/23 cells). (E) Schematic of two possible outcomes of manipulating the outer k-fiber at different locations along its length: either the negative curvature position (orange star) remains fixed relative to the microneedle (black circle) position (uniform anchorage along the k-fiber, Model 1) or remains fixed relative to the chromosome (specialized, non-uniform anchorage near chromosome, Model 2). (F–G) Position of the curvature maxima (microneedle, white circle) and curvature minima (negative curvature, orange star) (F) measured from the microneedle position (n = 23 cells), and (G) measured from the chromosome (n = 23 cells). Dashed lines connect the maxima (microneedle) and minima (negative curvature) for a given manipulation. The negative curvature position is tightly distributed near chromosomes, regardless of the microneedle’s position, supporting a specialized crosslinking model (Model 2, E). Plot also shows microneedle positions of manipulations that do not result in negative curvature (grey circles). (H) Top: Timelapse images of a PtK2 metaphase spindle (GFP-tubulin, grey) manipulate-and-hold experiment to probe the timescale of k-fiber reinforcement in the spindle center, performing a 60 s manipulation and then holding the microneedle (white circle) in place to measure when the negative curvature in the manipulated k-fiber (white trace) disappears (1:39, 21 s after 1:18 hold started). Scale bar = 5 μm. Time in min:sec. Bottom: Curvature mapped along highlighted k-fiber for each point in the top panel (blue, negative curvature; red, positive curvature). Negative curvature (black arrow) disappears over 21 s of holding time. (I) Curvature minima near chromosome as a function of time the microneedle has been held in place (n = 5 cells). Negative curvature disappears after holding for 20 s. Plot shows mean ± SEM (orange).

Figure 3—source data 1. This spreadsheet contains the local curvature along k-fibers manipulated over 60 s in PtK2 cells (Figure 3C), the positions of the microneedle and negative curvature with respect to the plus-end and the microneedle as well as their respective curvature values (Figure 3F–G), and the negative curvature near chromosomes during the hold time of the ‘manipulate-and-hold’ assays (Figure 3I).

Figure 3.

Figure 3—figure supplement 1. Deformed kinetochore-fibers exhibit negative curvature in 12 s manipulations.

Figure 3—figure supplement 1.

(A) Top: Timelapse images of a representative PtK2 spindle (GFP-tubulin, grey) during a 12 s manipulation.
Microneedle position (white circle) and traced manipulated k-fiber (white) displayed on the images. Scale bar = 5 μm. Time in min:sec. Bottom: Curvature mapped along highlighted k-fiber for each point in the top panel (blue, negative curvature; red, positive curvature). This manipulation can expose contact points between the k-fiber and non-kMTs (asterisk). (B) Local curvature of deformed k-fibers for normalized positions along the k-fiber (n = 7 cells). Some k-fibers exhibit negative curvature near the chromosome (orange), and others do not (grey).
Figure 3—figure supplement 1—source data 1. This spreadsheet contains the local curvature along k-fibers manipulated over 12 s in PtK2 cells (Figure 3—figure supplement 1B).
Figure 3—figure supplement 2. Tight coupling between sister kinetochore-fibers in 60 s manipulations.

Figure 3—figure supplement 2.

(A) Schematic of the two measurements made in (B–C): Inter-kinetochore distance measured between sister k-fiber plus-ends of the manipulated k-fiber, and angle measured between the sister k-fiber plus-end (opposite the manipulated k-fiber) and the pole-pole axis, measured over 60 s. (B) Change in inter-kinetochore distance in PtK2 unmanipulated (control, n = 13 kinetochore pairs from 6 cells) and manipulated (n = 8 kinetochore pairs from 8 cells) spindles, measured over 60 s. There was no significant difference in inter-kinetochore distance after manipulation (p=0.06, Mann-Whitney U test). Plot shows mean ± SEM. (C) Change in angle of sister k-fiber plus-end with respect to the pole-pole axis, in PtK2 unmanipulated (control, n = 12 k-fibers from 6 cells) and manipulated (n = 11 k-fibers from 11 cells) spindles, measured over 60 s. The sister k-fiber in manipulated spindles moves in towards the pole-pole axis (p=5×10−4, Mann-Whitney U test). Plot shows mean ± SEM.
Figure 3—figure supplement 2—source data 1. This spreadsheet contains the change in inter-kinetochore distance (Figure 3—figure supplement 2B) and angle of sister k-fiber plus-end from the pole-pole axis (Figure 3—figure supplement 2C) in unmanipulated and manipulated spindles over 60 s.
Figure 3—figure supplement 3. Non-kinetochore microtubule contacts distributed close to observed negative curvature.

Figure 3—figure supplement 3.

Frequency distribution of the distance from a contact point between the k-fiber and non-kMTs (non-kMT contact) to the curvature minima (negative curvature) position, exposed during 60 s manipulations in PtK2 spindles. The average distance between a non-kMT contact and the negative curvature near chromosomes is 1 ± 0.1 μm (mean ± SEM, n = 14 cells).
Figure 3—figure supplement 3—source data 1. This spreadsheet contains the position of negative curvature from the k-fiber plus-end, position of non-kinetochore microtubule contact from the k-fiber plus-end, and the distance between them.
Figure 3—video 1. Microneedle manipulation of a kinetochore-fiber reveals pivoting around poles and local reinforcement near chromosomes.
Download video file (45.4KB, mp4)
Microneedle manipulation of a metaphase spindle in a PtK2 cell. The microneedle (Alexa-647, white circle) pulls (time 00:00) on the spindle’s outer k-fiber (GFP-tubulin, grey) over 60 s and deforms the spindle. The k-fiber bends around the needle, pivots around the pole but does not pivot around chromosomes, instead remaining straight in the spindle center. This indicates the presence of a region of k-fiber reinforcement in the spindle center. Scale bar = 5 μm. Time in min:sec. Video was collected using a spinning disk confocal microscope, at a rate of 1 frame every 4 s before and during manipulation. Video has been set to play back at constant rate of 5 frames per second. Movie corresponds to still images from Figure 3B.
Figure 3—video 2. Manipulate-and-hold assay reveals that local reinforcement near chromosome has a 20 s lifetime.
Download video file (39.7KB, mp4)
Manipulate-and-hold experiment in a metaphase spindle in a PtK2 cell. The microneedle (Alexa-647, white circle) pulls (time 00:00) on the spindle’s outer k-fiber (GFP-tubulin, grey) over 60 s and is then held in place for 30 s. During the course of the microneedle hold, the negative curvature on the deformed k-fiber disappears in 20 s, indicating that this local, specialized reinforcement is short-lived. Scale bar = 5 μm.Time in min:sec. Video was collected using a spinning disk confocal microscope, with a variable frame rate of 1 frame every 10 s before manipulation and 1 frame per every 7 s during manipulation. Video has been set to play back at 7 frames per second despite the variable acquisition rate. Movie corresponds to still images from Figure 3H.