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. 2019 Oct 2;14(10):e0223174. doi: 10.1371/journal.pone.0223174

The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array

Anna Melkov 1,#, Raju Baskar 1,#, Rotem Shachal 1, Yehonathan Alcalay 1, Uri Abdu 1,*
Editor: Maria Grazia Giansanti2
PMCID: PMC6774520  PMID: 31577833

Abstract

In the present report, we used highly elongated Drosophila bristle cells to dissect the role of dynein heavy chain (Dhc64C) in Golgi organization. We demonstrated that whereas in the bristle "somal" region Golgi units are composed of cis-, medial, and trans-Golgi compartments (“complete Golgi”), the bristle shaft contains Golgi satellites that lack the trans-Golgi compartment (hereafter referred to as “incomplete Golgi”) and which are static and localized at the base area. However, in Dhc64C mutants, the entire bristle shaft was filled with complete Golgi units containing ectopic trans-Golgi components. To further understand Golgi bristle organization, we tested the roles of microtubule (MT) polarity and the Dhc-opposing motor, kinesin heavy chain (Khc). For our surprise, we found that in Khc and Ik2Dominant-negative (DN) flies in which the polarized organization of MTs is affected, the bristle shaft was filled with complete Golgi, similarly to what is seen in Dhc64C flies. Thus, we demonstrated that MTs and the motor proteins Dhc and Khc are required for bristle Golgi organization. However, the fact that both Dhc64C and Khc flies showed similar Golgi defects calls for an additional work to elucidate the molecular mechanism describing why these factors are required for bristle Golgi organization.

Introduction

The Golgi complex possesses a highly ordered and characteristic morphology. In most mammal cells, the organelle appears as stacks of flattened cisternae that are linked by tubules to give rise to continuous structures known as ribbons [1, 2]. In most cell types, the Golgi ribbon is typically located in the perinuclear region near the centrosome. The exact localization is dependent on the microtubule (MT) and actin cytoskeleton, together with active transport or anchoring by motor proteins, such as dynein and kinesin [3]. It was, moreover, shown that MTs are closely associated with the cis-Golgi compartment [4]. In contrast, in non-neuronal Drosophila tissues, the Golgi apparatus appears as stacks dispersed in the cell cytoplasm, juxtaposed to the endoplasmic reticulum exit site (ERES), and does not form the continuous tubular organelle seen in mammalian cells [5].

Neurons, which are highly polarized cells, present a unique satellite secretory system that includes ER and Golgi outposts, preferentially localized to dendrites [68]. Although it is believed that the majority of protein synthesis, modification, and sorting takes place in the soma, it was shown that satellite Golgi at neuronal terminals is functional and may provide local membrane and protein supply [5, 6, 8, 9]. Moreover, it was demonstrated that dendritic Golgi outposts can serve as sites of de novo MT nucleation [10, 11], thus serving as microtubule-organizing centers (MTOCs). On the other hand, it was claimed that acentrosomal nucleation of MTs is only γ-tubulin-dependent and that Golgi outposts do not serve as sites for MT nucleation in neurons [12]. The appearance of distinct distal Golgi structures separated from the main Golgi apparatus and located near the nucleus mainly characterizes neuronal cells. However, discrete Golgi outposts are not only found in neurons. For instance, highly polarized mice astrocyte perivascular processes and end feet possess Golgi apparatus components [13], suggesting that the presence of Golgi outposts is not a unique feature of neurons, as was previously believed [14].

Similar to Drosophila dendrites, the bristle shaft contains acentrosomal stable and uniformly oriented MT arrays in which the minus-ends point distally (i.e., towards the growing edge of the bristle) [15]. Thus, the Drosophila bristle cell represents an excellent model for studying long-distance transport and the establishment of cellular polarity. The bristle shaft sprouts from the epithelial tissue for up to 450 μm, while the "soma", containing the polypoid nucleus, remains in the epithelial plane. During elongation, growth takes place at the tip area, which is the most dynamic part of the cell. Our lab and others have questioned the role of MT-dependent motors and asymmetrical MT organization in long-distance organelle transport [1619]. Still, the organization of the Golgi in these unique polarized cells remains to be fully elucidated.

In the present study, we describe a previously uncharacterized Golgi apparatus in bristle cells. We also addressed the role of the MT array and its MT motor proteins, dynein and kinesin, in bristle Golgi organization. We found that in the somal region containing the nucleus, the Golgi contains cis-, medial, and trans-compartments juxtaposed to the ERES, while in the bristle shaft, the Golgi is composed of ERES, cis-, and medial compartments, yet completely lacks trans-Golgi components. We thus refer this complex as incomplete Golgi. These Golgi satellites were localized to the lower shaft area and appeared static. Relying on dynein heavy chain 64C (Dhc64C) and kinesin heavy chain (Khc) mutants to follow the contributions of these molecular motor to the positioning of Golgi satellites, we found that complete Golgi units containing trans-Golgi components were ectopically localized throughout the entire bristle shaft. Next, by affecting MT polarity through mutation of the Ik2 gene, we found that complete Golgi units were localized ectopically to the entire bristle shaft; similar to what was seen in the Dhc64C and Khc mutants. Thus, our results demonstrate that organized MT polarity and the MT motor proteins Dhc64C and Khc are required for bristle Golgi organization.

Materials and methods

Drosophila stocks

Oregon-R was used as a wild-type control. The following mutant and transgenic flies were used: Dhc64C4-19/TM6B, Dhc64C8-1/TM6B [20], FRT79D Dhc64C902/TM6B [21], UAS-Ik2DN [22], UAS-LVADN [7], Khc-IR (Vienna Drosophila RNAi Center; V44337), UAS-MnII-GFP, UAS-Gal-T-RFP [7, 23], UAS-GalNacT2-YFP/TagRFP [23], and UAS-GFP-Sten [24]. Bristle expression was induced under the control of the neur-Gal4 or sca-Gal4 driver.

Dissection and preparation of pupae for live imaging

Dissection of thorax tissue containing bristles for antibody staining and removal of the pupal case for live-imaging experiments was performed as previously described [15, 18].

Scanning electron microscopy

Adult Drosophila were fixed and gradually dehydrated by immersion in increasing concentrations of ethanol (25%, 50%, 75%, and 2×100%, each for 10 min). The samples were then completely dehydrated using increasing concentrations of hexamethyldisilazane in alcohol (50%, 75%, and 2×100%, each for 2 h), air-dried overnight, placed on stubs, coated with gold, and examined with a scanning electron microscope (SEM; JEOL model JSM-5610LV).

Bristle phalloidin and antibody staining

The procedures of tissue dissection used for fixation and staining were described previously [18, 25]. Confocal images were taken using an Olympus FV1000 laser scanning confocal microscope, appearing as Z-projections in a few optical frames that together covered the bristle cell. The primary antibodies used were mouse anti-acetylated tubulin monoclonal antibodies (1:250; Sigma), and anti-dGM130 (1:500; Abcam), anti-Sec16 antibodies [26]. Secondary antibodies included Cy3-conjugated goat anti-mouse (1:100; Jackson Immunoresearch) and Alexa Fluor 488-conjugated goat anti-rabbit (1:100; Molecular Probes) antibodies. For actin staining, we used Alexa Fluor 405-conjugated phalloidin (1:250; Molecular Probes).

Organelle tracking and statistical analysis

To measure the area and density of MnII-GFP and GalNacT2-YFP-positive particles, confocal Z-stack images of maximal quality of bristle cells were obtained. A Z-stack projection of the bristle shaft was divided into two halves exactly in the middle of the shaft length, resulting in two sections of even length, with one part being close to the base (soma) and the other being distal to the base, referred to as the tip. In the case of a dynein-mutated background, the bristle appeared as two independent parts because of an extremely high signal at the tip area, relative to the base. Images were analyzed using ImageJ software, and the area of each particle was measured automatically using the "analyze particles" tool. The density of MnII and GalNacT2-positive particles was measured as the number of particles in each half of the bristle divided by the overall area of that half-bristle (i.e., the number of particles/area of the base or tip half). Statistical analysis of both parameters, i.e., particle area and density, was performed using spilt-plot ANOVA with the cell part area being considered as the within-plot treatment and the genotype being considered as the whole-plot treatment. Repeats (random factors) were nested within a genotype. Statistical analyses were performed using STATISTICA, version 10.

Results

Golgi organization in the Drosophila bristle cell

To study the organization of the Golgi in polarized cells, we used the highly elongated Drosophila bristle cell as a model system, together with a set of ERES and Golgi markers. To identify the ERES, we used either Stenosis (Sten) tagged with GFP [24] or antibody staining of Sec16 [26]. As a medial Golgi compartment marker, we followed α-mannosidase II tagged with EGFP (MnII-GFP), while RFP/GFP-tagged N-acetylgalactosaminyltransferase 2 (GalNacT2-TagRFP/GFP) and RFP-tagged Gal-T [23] served as a trans-Golgi marker. For visualization of the cis-Golgi compartment, we followed anti-dGM130 antibody staining [23].

Examination of bristle cell Golgi organization revealed that the somal region contains Golgi stacks composed of cis- (Fig 1A' and 1A''), medial- (Fig 1A–1C, 1A'', 1B'' and 1C''), trans-Golgi compartments (Fig 1B and 1B') and ERES (Fig 1C' and 1C'') scattered throughout the cytoplasm. This type of Golgi is henceforth designated as a complete Golgi unit. Thus, similar to other Drosophila tissues [5, 2729], the soma of the bristle cell contains Golgi mini-stacks and does not create higher-order structures, such as the ring-shaped organization observed in the soma of larval neurons [23]. In Drosophila larval dendritic shafts, the Golgi exists as a discrete "single compartment Golgi" or as a "Golgi mini-stack" composed of more than one compartment [23]. Examination of Golgi organization within the bristle shaft using MnII-GFP as a medial-Golgi marker revealed that the shaft contains MnII-positive particles, these particles are called hereafter Golgi satellites, (Fig 1D–1G, 1H, 1K and 1L) and are concentrated in the lower part of the bristle, with almost no such Golgi satellites being seen in the distal to soma tip area. We tested for differences in the density of Golgi satellites and the relative sizes of particles in the lower part (i.e., the half of the shaft close to the soma, designated as the base) and the upper part (i.e., the half of the bristle shaft distal to the soma, designated as the tip) of the bristle shaft (see Materials and Methods for details of the quantification process used). Such analysis of MnII-GFP -positive particles revealed that the density of Golgi satellites (i.e., the number of particles per μm2) at the base of the bristle shaft was significantly higher, as compared to the tip (For MnII-GFP, 0.18±0.05 and 0.04±0.06 particles/μm2, respectively; P<0.001, Table 1; 5 pupa, 15 bristles). In addition, the average area of each particle of both MnII-GFP was significantly different between the base and tip areas, (For MnII-GFP, 0.43±0.80 and 0.17±0.20 μm2, respectively; P<0.0015 Table 1; 5 pupa, 15 bristles)

Fig 1. Golgi organization in Drosophila bristles.

Fig 1

Confocal projections of representative WT “somal” and ~40 h APF bristle shaft areas. A-C''–Bristle “somal” region: A-A''–“Soma” of a WT bristle cell expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''Soma of a WT bristle cell expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and co-expressing GalNacT2-RFP (a trans-Golgi marker): B'–GalNacT2-RFP (a trans-Golgi marker) identifies trans-Golgi components localized throughout the bristle soma cytoplasm, B''–Merged image of green MnII-GFP and red GalNacT2-RFP showing co-localization of medial- and trans-Golgi compartments in the bristle cell soma. C-C'Soma of a WT bristle cell expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. D-L–Bristle external extension; shaft (the cell compartment emanating from soma out from the epithelial tissue plane). D-G–Bristle shaft expressing sca-Gal4::UAS-MnII-GFP co-stained with anti-dGM130 antibodies and phalloidin: D–Gray phalloidin-UV staining of actin bundles in a MnII-GFP expressing bristle, used here to highlight the cell perimeter, E–Green MnII-GFP, a medial-Golgi marker, is localized to the lower shaft area close to the somal region, F–Red anti-dGM130 antibody staining (cis-Golgi marker), G–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. H-H'–Bristle shaft co-expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and GalNacT2-RFP (a trans-Golgi marker): H–Green MnII-GFP, a medial-Golgi marker, is localized to the lower shaft area close to the somal region, H'–Red GalNacT2-RFP, indicating a trans-Golgi domain, does not enter the bristle shaft. I-L–Bristle shaft expressing sca-Gal4::UAS-MnII-GFP co-stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter, J–Red anti-Sec16 antibody staining localized to the lower shaft area close to the somal region, K–Green MnII-GFP localized to the lower shaft area close to the somal region, L–Merged image of green MnII-GFP and red anti-Sec-16-antibody staining showing co-localization of medial-Golgi and ERES components in the bristle shaft. APF-After prepupa formation. The scale bar represents 10 μm.

Table 1. Golgi outpost localization parameters in Dhc64C and khc mutant bristle shafts.

Golgi outpost (MnII-GFP) localization parameters in Drosophila bristles
Genotype Wild-type Dhc64C8-1/Dhc64C4-19 Khc- RNAi
Bristle cell area tip base tip base tip base
No. of pupae 5 5 5
No. of bristles 15 16 15
Particle area, avg (μm2) 0.17±0.20 0.43±0.80* 0.43±0.84*A 0.32±0.52A 0.20±0.52B 0.40±0.19*B
Density (particle/μm2) 0.04±0.06 0.18±0.05* 0.30±0.03A 0.43±0.16*A 0.12±0.13A 0.31±0.26*A

*—represents a significant difference of the cell part (tip/base) within each genotype; letters represent a significant difference in corresponding cell parts (tip/tip; base/base) between genotypes.

A–Significantly different from WT

B–significantly different from Dhc64C mutant. The values reflect mean±s.d. (for a detailed description of the statistical analysis performed, see Materials and Methods).

We further found that these Golgi satellites to be composed of cis- (Fig 1D–1G) and medial-Golgi compartments (Fig 1E, 1H and 1K) juxtaposed to ERES (Fig 1I–1L) and completely lacking trans-Golgi domains (Fig 1H and 1H'). To confirm the complete absence of trans-Golgi components within the bristle shaft, as observed in GalNacT2-RFP-expressing bristles, we tested for the presence of an additional trans-Golgi marker, 1,4-galactosyltransferase (Gal-T)[23, 30]. We found no Gal-T-RFP in the wild type (WT) shaft (S1 Fig). Thus, our results show that whereas the Golgi apparatus of the bristle somatic region is composed of complete Golgi units (i.e, including cis-, medial- and trans-Golgi components), the bristle shaft contained Golgi satellites completely lacking trans-Golgi components.

Dhc64C is required for Golgi organization in the bristle shaft

Given that in the Drosophila bristle, a stable MT array is organized with minus-end MT fibers pointing away from the soma [15] and with the notion that MTs and their motor proteins are required for Golgi organization [3134], we studied the role of the minus-end MT motor protein dynein in bristle Golgi organization. First, we analyzed the role of Dhc64C using the "slow dynein" hypomorphic mutant allele Dhc64C8-1 [17, 20]. We found that in these mutants, Golgi satellites were no longer concentrated at the base of the bristle shaft as in the WT (Fig 1E–1G) but were instead found throughout the entire bristle shaft (Fig 2A–2L). These ectopically localized Golgi units contained cis- (Fig 2C, 2D, 2G and 2H), medial- (Fig 2B, 2D, 2J and 2L), and trans-Golgi compartments (Fig 2F and 2H) and ERES (Fig 2K and 2L), demonstrating that in Dhc64C mutants, the bristle shaft contains complete Golgi units rather than discrete Golgi satellites. Staining of MnII-GFP-expressing Dhc64C mutants with anti-dGM130 antibodies (a cis-Golgi marker) (Fig 2C) revealed co-localization of cis- and medial-Golgi markers (Fig 2D). Likewise, co-localization of GalNacT2-YFP (a trans-Golgi marker) expressed in Dhc64C mutants with anti-dGM130 antibody staining (Fig 2F–2H) reflected co-localization of the trans- and cis-markers (Fig 2H). Also, an additional trans-Golgi marker, 1,4-galactosyltransferase (Gal-T), was found throughout Dhc64C mutant bristle shafts (S1 Fig). We thus concluded that all three compartments are co-localized. Additionally, we showed that MnII-GFP was co-localized with ERES (Fig 2L). Thus, the Golgi particles found within the Dhc64C mutant bristle are complete Golgi units. Quantitative analysis revealed that the density of MnII-GFP-positive particles was slightly higher at the base than at the tip (For MnII-GFP, 0.43±0.16 and 0.30±0.03 particles/μm2, respectively; P<0.05; Table 1; 5 pupa, 16 bristles). For GalNacT2-YFP, 0.59±0.06 and 0.24±0.02 particles/μm2, respectively; P<0.05; Table 2; 5 pupa, 16 bristles. In addition, the density of MnII-GFP-positive particles was dramatically increased in both the tip and base, as compared to the WT (P<0.002 for both tip and base; Table 1; 5 pupa, 16 bristles). Such analysis was not possible for GalNacT2-YFP due to its absence from the WT bristle shaft. Also, the average area of both MnII-GFP- and GalNacT2-YFP-positive particles was significantly larger at the tip of the bristle shaft than at the base (For MnII-GFP, 0.46±0.63 and 0.29±0.39 μm2, respectively; P<0.001; Table 1; 5 pupa, 16 bristles). For GalNacT2-YFP, 0.53±0.03 and 0.26±0.20 μm2, respectively; P<0.003; Table 2; 5 pupa, 16 bristles. Moreover, these areas of MnII-GFP were significantly (P<0.001) different from what was seen in the corresponding cell parts in WT flies (Table 1; this analysis was not possible for GalNacT2-YFP since the WT bristle did not contain any trans-compartments in the shaft to analyze, Table 2). The defects in Golgi organization in the Dhc64C mutants bristle shaft led us to check whether the somal region of the bristle was affected. The closer examination revealed that in Dhc64C mutants, the Golgi apparatus is scattered throughout the somal region (S2 Fig), as in WT.

Fig 2. Golgi organization in Dhc64C mutant bristle cell shaft.

Fig 2

Confocal projections of Dynein heavy chain-mutated background bristle shafts from ~40 h APF. A-D–Dhc64C8-1/Dhc64C902 trans-heterozygote bristle shaft expressing sca-Gal4::MnII-GFP stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: A–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: B–Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, C–Red anti-dGM130 antibody staining (cis-Golgi marker), D–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. E-H–Dhc64C8-1/Dhc64C4-19 trans-heterozygote bristle shaft expressing sca-Gal4::GalNacT2-YFP (trans-Golgi marker) co-stained with anti-dGM130 antibodies and phalloidin: E–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: F–Green GalNacT2-YFP trans-Golgi marker is ectopically localized to the entire bristle shaft area, G–Red anti-dGM130 antibody staining (cis-Golgi marker), H–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. I-L–Dhc64C8-1/Dhc64C902 trans-heterozygote bristle shaft expressing sca-Gal4::MnII-GFP, co-stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: J—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, K–Red anti-Sec16 antibody staining localized to the entire shaft area, L–Merged image of green MnII-GFP and red anti-Sec-16-antibody staining showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

Table 2. Golgi outpost localization parameters in Dhc64C and khc mutant bristle shafts.

Golgi outpost (GalNacT2-YFP) localization parameters in Drosophila bristles
Genotype Wild-type Dhc64C8-1/Dhc64C4-19 Khc- RNAi
Bristle cell area tip base tip base tip base
No. of pupae 5 5 5
No. of bristles 15 16 15
Particle area, avg (μm2) N/A N/A 0.53±0.03* 0.26±0.20 0.23±0.18 A 0.33±0.36*A
Density (particle/μm2) N/A N/A 0.24±0.02 0.59±0.06* 0.23±0.23 0.47±0.41*

*—represents a significant difference of the cell part (tip/base) within each genotype; letters represent a significant difference in corresponding cell parts (tip/tip; base/base) between genotypes.

A–Significantly different from Dhc64C mutant. The values reflect mean±s.d. (for a detailed description of the statistical analysis performed, see Materials and Methods).

N/A- Not applicable.

Lava-lamp is not required for Golgi organization in the bristle shaft

Previously it was shown that in Drosophila embryo cellularization, Golgi movement depends on cytoplasmic dynein [35]. Moreover, it was shown that the Golgin (Golgi-associated protein) Lava-lamp (Lva) mediates such dynein-based Golgi movements [35, 36]. Later, it was shown that Lva is also required for the dynamic profile of dendritic Golgi satellites [37]. Thus, we analyzed the role of Lva in bristle Golgi organization by disrupting Lva activity via over-expression of the Lva-dominant negative (LvaDN) protein [7]. We found that expression of LvaDN in bristles using neur-Gal4 but not sca-Gal4 affected bristle morphology, with the upper part of the bristle being twisted (90% of the bristle in 10 adults; Fig 3B and 3B'). On the other hand, using the same neur-Gal4, no defects in Golgi satellite organization were detected at the bristle shaft (Fig 3G–3J, S3 Fig) when compared to WT (Fig 3C–3F, S3 Fig), and no defects were found in the bristle somal region (S4 Fig).

Fig 3. Lava-lamp is not required for Golgi organization in the bristle shaft.

Fig 3

Scanning electron microscope images (A-B') of adult bristles from A–Wild type, A’–higher magnification image of the tip area showing characteristic parallel grooved surface morphology; B–neur-Gal4::UAS-LVADN fly bristle, B’—higher magnification image of the tip area of neur-Gal4::UAS-LVADN showing disrupted characteristic parallel grooved surface morphology highlighted by arrowheads. C-F- Confocal projections of neur-Gal4::UAS-MnII-GFP (Wild type) bristle shafts from ~40 h APF stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: C–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: D–Green MnII-GFP, a medial-Golgi marker, is localized to the base of bristle shaft area, E–Red anti-dGM130 antibody staining (cis-Golgi marker), F–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. G-J- Confocal projections of Lava-lampDN background bristle shafts. G-J- neur-Gal4::UAS-Lava-lampDN bristle shafts from ~40 h APF co-expressing UAS-MnII-GFP and stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: G–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: H–Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, I–Red anti-dGM130 antibody staining (cis-Golgi marker), J–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

Kinesin heavy chain is required for Golgi organization in the bristle shaft

Next, we examined the role of the plus-end-directed MT motor kinesin, and more specifically, the conventional kinesin-41, kinesin heavy chain (Khc), in bristle Golgi organization. Although dynein is believed to be the primary motor in Golgi positioning, it was shown in vertebrate cells that kinesin-light chain is present in Golgi membranes [38]. Still, the role of kinesin in Golgi trafficking is not well understood. Accordingly, we down-regulated the Khc transcript specifically in the bristle using RNAi (for simplicity, these strains are henceforth termed Khc mutants). To our surprise, we found that similar to Dhc64C mutant flies, bristles in Khc mutants also contained complete Golgi units located throughout the entire bristle shaft (Fig 4) composed of co-localized cis- (Fig 4C and 4D), medial- (Fig 4B, 4D, 4J and 4L), and trans-Golgi components (Fig 4F and 4H) and ERES (Fig 4K and 4L). However, the distribution pattern of the Golgi units in the Khc mutant bristle shafts was different from that in the Dhc64C mutants. Similar to WT and Dhc64C mutant flies (Tables 1 and 2), the density of MnII-GFP was significantly higher at the base of the bristle than at the tip (For MnII-GFP, 0.31±0.26 particle/μm2 and 0.12±0.13 particle/μm2, respectively (P<0.001); Table 1; 5 pupa, 15 bristles). Moreover, the density of MnII-positive particles at both the tip and base was significantly higher than recorded for the WT (Table 1, P<0.0001) but similar to Dhc64C. However, in contrast to Dhc64C mutants, the average area of the MnII-GFP and GalNacT2-positive particles was significantly larger at the base of the bristle shaft than at the tip (For MnII-GFP, 0.40±0.19 μm2 and 0.20±0.52 μm2, respectively. For GalNacT2-YFP, 0.33±0.36 μm2 and 0.23±0.18 μm2, respectively, Table 2, 5 pupa, 15 bristles). Also, in the Khc mutant bristle somal region, no obvious defects in Golgi organization were detected (S5 Fig).

Fig 4. Golgi organization in kinesin heavy chain-mutated bristles.

Fig 4

Confocal projections of kinesin heavy chain-mutated background bristle shafts from ~40 h APF. A-DKhc-RNAi bristle shaft expressing sca-Gal4::UAS-MnII-GFP co-stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: A–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter, B–Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, C–Red anti-dGM130 antibody staining (cis-Golgi marker), D–Merged image of green MnII-GFP and red anti-dGM130 showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. E-H–Khc-RNAi bristle shaft expressing sca-Gal4::UAS- GalNacT2-YFP (trans-Golgi marker) co-stained with anti-dGM130 antibodies and phalloidin: E–Gray phalloidin-UV staining of actin bundles in a GalNacT2-YFP-expressing bristle, used here to highlight the cell perimeter, F–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, G–Red anti-dGM130 antibody staining (cis-Golgi marker), H–Merged image of green GalNacT2-YFP and red anti-dGM130 showing co-localization of trans- and cis-Golgi compartments in a mutated bristle shaft. I-L–Khc-RNAi bristle shaft expressing sca-Gal4::UAS-MnII-GFP co-stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter, J—green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, K–Red anti-Sec16 antibody staining localized to the entire shaft area, L–merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial- and ERES components in a mutated bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

Mutation of Ik2 affects Golgi organization in the bristle shaft

We previously demonstrated that in the bristle, stable MTs are organized in an asymmetrical manner, with the minus-end pointing outwards [15]. Moreover, we found that the Spn-F/Ik2 complex is required for bristle MT polarity [39]. Thus, to test whether MT polarity is essential for bristle Golgi organization, we studied Golgi organization in flies expressing Ik2dominant-negative (Ik2DN) specifically in the bristle. Ik2DN is a defective kinase in which lysine at position 41 was changed to alanine (Ik2K41A). We and other have shown that expression of Ik2DN strongly affects bristle morphology and MT organization [15, 40, 41]. First, we found that expression with neur-Gal4 but not sca-Gal4 generated typical Ik2 bristle defects. Thus, we used the neur-Gal4 for further examinations. We found that similar to Dhc64C and Khc mutant flies, in Ik2DN mutants, the bristle contained complete Golgi units localized throughout the entire bristle shaft (Fig 5A–5L (and co-localized cis- (Fig 5C, 5D, 5G and 5H), medial- (Fig 5B, 5D, 5J and 5L) and trans-Golgi components (Fig 5F and 5H) and ERES (Fig 5K and 5L). Since we used different Gal4s for Golgi expression in Dhc64C and Khc-RNAi (sca-Gal4), in comparison to Ik2DN expression (neur-Gal4), we were not able to statistically compare Golgi parameter (area and density) between them. Instead, we compared the neur-Gal4::UAS-MnII-GFP and GalNacT2-positive particle densities in Ik2DN mutant bristle shafts versus their Gal4-UAS genetic background (neur-Gal4::UAS-MnII-GFP and GalNacT2-YFP).

Fig 5. Confocal projections of Ik2DN background bristle shafts from ~40 h APF.

Fig 5

A-D–neur-Gal4::UAS-Ik2DN bristle shaft co-expressing UAS-MnII-GFP and stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: A–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: B–Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, C–Red anti-dGM130 antibody staining (cis-Golgi marker), D–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments in the bristle shaft. E-H–neur-Gal4::UAS-Ik2DN bristle shaft co-expressing UAS-GalNacT2-YFP (trans-Golgi marker) stained with anti-dGM130 antibodies and phalloidin: E–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: F–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, G–Red anti-dGM130 antibody staining (cis-Golgi marker), H–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. I-L–neur-Gal4::UAS-Ik2DN bristle shaft co-expressing UAS-MnII-GFP stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: J—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, K–Red anti-Sec16 antibody staining is localized to the entire shaft area, L–Merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

First, we noticed that there was no difference in MnII-GFP-positive particle average area between the tip (0.32±0.23 μm2, Table 3) and base (0.32±0.28 μm2, Table 3) in the Ik2 mutant bristle shaft. The same pattern was also detected in GalNacT2-positive particle average area (tip: 0.24±0.04 μm2; base: 0.24±0.09 μm2, Table 3) from the Ik2 mutant. Interestingly, MnII-GFP-positive particles density was higher at the tip (0.26±0.10 particle/μm2, P<0.001; 5 pupa, 18 bristles, Table 3) but was similar at the base to what was noted in the WT (Table 3). In case of GalNacT2-YFP, this analysis was impossible because of its absence (Table 3). Moreover, in Ik2DN mutants no defects in Golgi organization at the somal region of the bristle could be detected (S6 Fig).

Table 3. Golgi outpost localization parameters in Ik2DN bristles.

Golgi outpost localization parameters in Drosophila bristles
Genotype Wild-type Ik2DN Wild-type Ik2DN
Golgi compartments Medial- compartment (mnII) Medial-
compartment
(mnII)
Trans-
compartment
(GalNacT2)
Trans-
compartment
(GalNacT2)
Bristle cell area Tip Base Tip Base Tip Base Tip Base
No. of pupae 5 5 5 5
No. of bristles 15 18 18 18
Particle area avg (μm2) 0.17±0.20 0.43±0.80* 0.32±0.23A 0.32±0.28 N/A N/A 0.24±0.04 0.24±0.09
Density (particle/μm2) 0.04±0.06 0.18±0.05* 0.26±0.10A 0.20±0.32A N/A N/A 0.30±0.03 0.23±0.07

*—represents a significant difference of the cell part (tip/base) within each genotype; letters represent a significant difference in corresponding cell parts (tip/tip; base/base) between genotypes.

A–Significantly different from WT. The values reflect mean±s.d. (for a detailed description of the statistical analysis performed, see Materials and Methods).

N/A- Not applicable.

Differential effects on Golgi organization among Dhc, Khc and Ik2 mutants

To understand the molecular mechanism by which Dhc64C, Khc and Ik2DN affect bristle Golgi organization; we followed the timing of Golgi organization in these mutants during bristle development. Since we used a different Gal4 for Ik2DN (neur-Gal4) than used with the Dhc64C and Khc mutants (sca-Gal4), we could only compare between Dhc64C and Khc mutants, with Ik2DN being compared to the control. We found that at early stages of WT bristle development, i.e. until 30–35 h APF (After prepupa formation), no Golgi satellites were detected in the bristle shaft in the WT (Fig 6A–6B). However, in both the Dhc64C (Fig 6D and 6E) and Khc (Fig 6G and 6H) mutants, even in the early stages of elongation, e.g., ~32 h APF, MnII-GFP positive particles could be detected throughout the bristle shaft. As mentioned above, later in the development of the Dhc64C and Khc mutants, the Golgi were spread all over the bristle shaft (Fig 6F and 6I). In Ik2DN, the timing of MnII-GFP entry into the bristle shaft was at 38 h APF in bristle development (Fig 7F).

Fig 6. Comparing developmental stages as a function of Golgi outpost localization.

Fig 6

Confocal projections of live WT developing bristle shafts expressing sca-Gal4::MnII-GFP (a medial-Golgi marker) at different elongation stages (A-C). A–~32 h APF, the developing bristle shaft shows no MnII-positive particles at this early stage of bristle development. Arrow points to the accumulation of MnII-GFP protein at the bristle tip, B–~35h APF, the developing bristle shaft shows no Golgi outposts localized to the bristle shaft. C–~39 h APF, the developing bristle shaft shows Golgi outposts beginning to accumulate at the base of the bristle shaft. D-F–Dynein heavy chain (Dhc64C8-1/Dhc64C902 trans-heterozygotes)-mutated developing bristle shafts expressing sca-Gal4::MnII-GFP: D–~32 h APF, the developing Dhc64C8-1/Dhc64C902 background bristle shaft shows MnII-positive particles filling the entire shaft area at this early stage of bristle development, E–~35 h APF, Golgi outposts are found throughout entire bristle shaft. The same is true for the following developmental stage of bristle shaft elongation: F– 39 h APF. G-I–developing bristle shafts expressing both sca-Gal4::MnII-GFP and UAS-Khc-RNAi; images show the corresponding developmental staging of Khc-depleted bristles, depicting the early appearance of Golgi outposts, G– 32 h APF, H– 35 h APF, I—At an advanced developmental stage (~39h APF), Golgi outposts localize evenly throughout the entire bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

Fig 7. Comparing developmental stages as a function of Golgi outpost localization in Ik2DN bristles.

Fig 7

Confocal projections of live WT developing bristle shafts expressing MnII-GFP by neur-Gal4 (a medial-Golgi marker) at different elongation stages (A-C). A–~32 h APF, the developing bristle shaft shows no MnII-positive particles at this early stage of bristle development. Arrow points to the accumulation of MnII-GFP protein at the bristle tip, B–~34h APF, the developing bristle shaft shows no Golgi outposts localized to the bristle shaft. Arrow points to the accumulation of MnII-GFP protein at the bristle tip, C–~38 h APF, the developing bristle shaft shows Golgi outposts beginning to accumulate at the base of the bristle shaft. D-Fneur-Gal4::UAS-Ik2DN-developing bristles co-expressing UAS-MnII-GFP, D—~32 h APF, the developing bristle shaft shows no Golgi outposts localized to the bristle shaft, E -~34 h APF, the developing bristle shaft shows no Golgi outpost accumulation, F—Developing bristle shaft at -~38 h showing Golgi outposts beginning to accumulate entire bristle shaft. APF—After prepupa formation. Scale bar, 10 μm.

Discussion

Golgi organization in Drosophila bristles

In different Drosophila tissues, the Golgi shows different organizational patterns, reflecting differences in cell type. In most Drosophila cells, the Golgi is organized as discontinuous stacks composed of ERES and cis-, medial-, and trans-Golgi compartments [5]. During spermatogenesis, the Golgi-based acroblast has a ring shape [42, 43]. In Drosophila larval neurons, the Golgi is differently organized. Here, the soma contains a high-ordered ring-shaped complete Golgi structure. In addition, similar to mammals, the dendrites contain either a discrete "single compartment Golgi" or "Golgi mini-stacks" composed of more than one Golgi compartment [23]. In our study, we showed that bristle cells contain complete Golgi stacks at the soma and incomplete Golgi composed of ERES, and cis-, and medial-Golgi compartments at the shaft. We show for the first time specifically in Drosophila that Golgi satellites can be found in cell types other than neurons. In the bristle, Golgi satellites are preferentially localized to the shaft base area. The role of these Golgi satellites remains, however, to be determined. In dendrites, Golgi outposts localize preferentially to branch points, where they are believed to serve as MTOCs (Microtubule organizing centers) [11, 44]. Thus, exact positioning of Golgi outposts is essential for establishing dendritic morphology. Additionally, in mammal neurons, Golgi outposts are more prominent during periods of intensive dendrite growth, while in mature neurons, their frequency decreases with time [14]. This points to not only tight spatial control of Golgi positioning but also to the developmental importance of the process. Here, we focused our analysis on the molecular machinery that regulates organelle positioning, although the function of Golgi satellites in the bristle cell remains to be elucidated.

The role of MTs in bristle Golgi organization

MTs are the main tracks for long-distance transport in the cell. Thus, MT array integrity and dynamics are essential for proper cellular elongation of both bristles and neurons. In highly polarized neurons, the dendrite and axon differ in terms of their MT array polarity. MTs in the axons of mammal and Drosophila neurons are oriented with their plus-ends pointed distally, while Drosophila dendrites possess MTs mainly oriented with their minus-ends pointed outwards. This difference in MT orientation is believed to provide directional cues for differential organelle transport [4547]. To confirm this hypothesis, we decided to disrupt MT array polarity through expression of mutant Ik2 protein. We found that affecting MT polarity resulted in abnormal Golgi unit localization, together with ectopic trans-Golgi domain appearance. We thus showed that polarized MTs are essential for correct Golgi organization. Specifically, the presence of polarized MTs prevents the appearance of trans-Golgi components within the bristle shaft.

Possible roles of dynein and kinesin in bristle Golgi organization

Dynein is believed to be the major Golgi architecture effector [5, 48, 49]. In Drosophila dendrites, dynein was found to be responsible for dendrite-specific Golgi outpost localization and MT orientation [7, 37, 50]. In neurons, kinesin-1 plays both indirect and direct role in dendritic selective sorting of Golgi outposts [51]. Indirectly, kinesin depletion alters axon MT orientation, which leads to ectopic Golgi outposts’ localization to axons driven by a different motor on misleading MT tracks. Directly, control of kinesin-1 auto-inhibition prevents entry of Golgi outposts into the axon [51].

We found that in Dhc64C mutant flies, bristle shaft Golgi satellites contained an ectopic trans-Golgi compartment that co-localized with cis- and medial-Golgi components. This raises the question of the role of Dhc64C in bristle Golgi organization. One explanation is that Dhc64C is required for Golgi satellite anchoring/sorting. In this model, dynein may prevent the entry of Golgi units from the soma to the shaft, while in the shaft, dynein specifically anchors the organelle to the lower zone close to soma, preventing the localization of Golgi units to the distant growth area of the cell tip.

A second explanation for the defects in Golgi unit localization observed in Dhc64C mutants could be disrupted interactions with the oppositely-directed motor, kinesin. However, taking into consideration MT array organization (i.e., with the minus-end pointed towards the tip) in a fly expressing a dynein mutant, active kinesin would be expected to return Golgi back to the soma. Yet, exactly the opposite is true. Moreover, we demonstrate that, similarly to Dhc64C flies, the bristle shaft contained an ectopic trans-Golgi compartment co-localized with cis- and medial Golgi components. In summary, our results shed new light of Golgi organization in polarized cells, such as the Drosophila bristle. Still, further study is needed to understand the molecular mechanism by which the combination of a polarized MT network with MT motor protein function affects Golgi bristle organization.

Supporting information

S1 Fig. The trans-Golgi marker Gal-T-RFP shows a localization pattern similar to that of GalNacT2-RFP/YFP.

Confocal projections of live developing bristle shafts expressing Gal-T-RFP (a trans-Golgi marker): A-B- Sca-Gal4-UAS-EB1-GFP::UAS-Gal-T-RFP (WT) developing bristle shaft showing no Gal-T-positive particles at an early stage of bristle development; AGreen EB1-GFP used to highlight the cell perimeter, B- RedGal-T-RFP. C-D—Confocal projections of dynein heavy chain-mutated background bristle shafts, Dhc64C8-1/Dhc64C4-19 trans-heterozygote bristle shaft expressing Gal-T-RFP stained with Oregon-green phalloidin; C–Red- The Gal-T-RFP (trans-Golgi) marker is localized to the entire bristle shaft area, D–Merged—green—phalloidin staining of actin bundles, used here to highlight the cell perimeter, red—Gal-T-RFP. Scale bar, 10 μm.

(EPS)

S2 Fig. Golgi organization in Dhc64C mutant bristle cell soma.

A-C'' -Confocal projections of representative of a WT bristle “somal” region from Dhc64C mutant background: A-A''–“Soma” of a dynein mutant bristle cell expressing sca-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green UAS-MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a Dhc64C mutant bristle cell expressing sca-Gal4::GalNacT2-RFP (a Trans-Golgi marker), B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–Soma of a dynein mutant bristle cell expressing sca-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

(EPS)

S3 Fig. Golgi organization in LvaDN mutant bristle shaft.

A-D- A Wild type bristle shaft expressing neur-Gal4-UAS-GalNacT2-YFP (trans-Golgi marker) stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: A–Gray phalloidin-UV staining of actin bundles in a GalNacT2-YFP expressing bristle, used here to highlight the cell perimeter: B–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, C–Red anti-dGM130 antibody staining (cis-Golgi marker), D–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. E-H- neur-UAS-Lava-lampDN bristle shaft co-expressing UAS-GalNacT2-YFP (trans-Golgi marker) and stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: E–Gray phalloidin-UV staining of actin bundles in a GalNacT2-YFP expressing bristle, used here to highlight the cell perimeter: F–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, G–Red anti-dGM130 antibody staining (cis-Golgi marker), H–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. I-L–A wild type neur-Gal4::UAS-MnII-GFP bristle shaft expressing stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: J—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, K–Red anti-Sec16 antibody staining is localized to the entire shaft area, L–Merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. M-P–neur-UAS-Lava-lampDN bristle shaft co-expressing UAS-MnII-GFP stained with anti-Sec16 (ERES) antibodies and phalloidin: M–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: N—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, O–Red anti-Sec16 antibody staining is localized to the entire shaft area, P–Merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. APF—After prepupa formation. The scale bar represents 10 μm.

(EPS)

S4 Fig. Golgi organization in LvaDN mutant bristle cell soma.

Confocal projections of representative of bristle “somal” region from WT (A-A'', C-C'', E-E'') and LvaDN mutant background: A-A''–“Soma” of a WT mutant bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–“Soma” of a LvaDN mutant bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). B'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, B''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. C-C''–Soma of a WT bristle cell expressing neur-Gal4::GalNacT2-YFP (a Trans-Golgi marker), C’- stained with anti-dGM130 antibodies (a cis-Golgi marker), C''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. D-D''–Soma of a LvaDN mutant bristle cell expressing GalNacT2-YFP (a Trans-Golgi marker), D’- stained with anti-dGM130 antibodies (a cis-Golgi marker), D''–Merged image of UAS-GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. E-E''–Soma of a WT bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): E'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, E''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. F-F''–Soma of a LvaDN mutant bristle cell expressing MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): F'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, F''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

(EPS)

S5 Fig. Golgi organization in kinesin heavy chain mutant bristle cell soma.

A-C'' -Confocal projections of representative of bristle “somal” region from kinesin heavy chain mutant background: A-A''–“Soma” of a bristle cell co-expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS-Khc-RNAi stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a bristle cell expressing sca-Gal4::UAS-GalNacT2-YFP (a Trans-Golgi marker) and UAS-Khc-RNAi, B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–Soma of a bristle cell expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS-Khc-RNAi stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

(EPS)

S6 Fig. Golgi organization in Ik2DN bristle cell soma.

A-C'' -Confocal projections of representative of bristle “somal” region from Ik2DN mutant background: A-A''–“Soma” of a bristle cell expressing neur-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS- Ik2DN stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a bristle cell expressing neur-Gal4::GalNacT2-YFP (a Trans-Golgi marker) and UAS-Ik2DN, B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–“Soma” of a bristle cell expressing neur-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS- Ik2DN stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

(EPS)

Acknowledgments

We thank VDRC Austria and the Bloomington Stock Center for generously providing fly strains. We thank Catherine Rabouille for her enlightening comments and for providing the antibodies against Sec-16.

Data Availability

All relevant data are within the manuscript; DOI: https://doi.org/10.5061/dryad.6gt43qg.

Funding Statement

This work was supported by the Israel Science Foundation (ISF) (grant 278/16) to U.A.

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Decision Letter 0

Maria Grazia Giansanti

8 Jul 2019

PONE-D-19-15627

The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array

PLOS ONE

Dear Dr. Uri Abdu

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Reviewer #2: Partly

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Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: In their manuscript Abdu and colleagues investigate the organization of Golgi stacks in Drosophila bristles. Similar to dendrites, the bristles of the fly contain Golgi outposts, which are likely used to supply processed secretory/membrane proteins at sites far from the cell's soma. This is an interesting model system, and the authors have done a nice work to characterize the microtubule dependent formation of these Golgi outposts. I believe this will be a good contribution to the literature, but suggest that the authors consider some revision as detailed below.

1. It would be good to document the number of repeats for each measurement where statistics was used (displayed in table 1).

2. The dual color staining of the Golgi in different combinations does not give this reviewer a lot of confidence that the Golgi mini-stacks are properly organized. Three or even four color images should be possible where secondary antibodies are used (e.g. for dGM130 antibody staining), showing that the Golgi (and ERES) are in fact properly polarized in a cis-trans direction.

3. The use of the IK2 dominant negative reagent is very interesting, but I had to dig deep and hard to find information on what this reagent (or for that matter the IK2 protein in this context) does. Some more information would be nice, this could be in the introduction, the results or even in the discussion sections, depending on the authors' preference, but I feel it would make the paper's message easier to understand.

4. The authors' use of the term "mini-stacks" is not correct in my opinion. Mini-stack is generally used to mean Golgi stacks that are not assembled in a ribbon, and are as such commonly used by mammalian cell biologists (although not exclusive to the mammalian system of course). Here the authors use it seemingly to mean stacks lacking a cisterna (in this case the trans cisterna). I would caution against that, and would rather use a different term.

Reviewer #2: In this manuscript, Melkov and colleagues present a series of experiments aimed to describe the organization of Golgi in highly elongated bristles upon disruption of microtubule motor protein function in Drosophila melanogaster.

The authors used fluorescently labeled transgenic lines to visualize the medial and trans-Golgi compartments, and Sec16 and GM130 antibodies to show the distribution of ERES and cis-Golgi in WT and different mutant backgrounds. They measured the distribution and composition of Golgi in the bristle shaft tip and the base region and found complete Golgi in the entire bristle shaft when they interfere with microtubule polarity.

Overall, the experimental results are interesting, but the experiments have several fundamental problems and the interpretation of the results is not satisfactory. Several questions need to be answered in order to be published in PlOS One.

Major comments:

1. To give a more definite answer regarding the involvement of microtubule polarity in the elongated bristle shaft cell the whole cell should be tested. They compare only the bristle shaft tip and base regions, but not the middle region or the soma region. How is the Golgi distribution in the soma in the different mutants? Is it possible, that the abnormal Golgi distribution is only the indirect consequences of the general problem in cell homeostasis and globally disturbed Golgi organization?

2. They used the UAS/Gal4 system to drive transgenes in the bristle. While ManII-GFP signal is strong in Fig 1A and C, it is weaker when it is co-expressed by the GalNacT2-RFP, probably due to the fact that two transgenes use the Gal4 source in those flies. It is not clear which driver did they use, the neur-Gal4 or the sca-Gal4 driver in the WT. Why do they used the Sca-Gal4 on Khc RNAi and Dhc64 mutant, but Neur-Gal4 on the Lava-lamp dominant negative background?

On the same line, they used different driver lines in the WT and the mutants when they visualized the developmental distribution of Golgi outpost in different pupal stages (Fig6). This is especially important when the authors compare statistically the Golgi localization (particle area, density) in WT and mutants (Table1).

3. Based on the fact that Golgi distribution changes during pupal development, it will be important to mention what is the age of the pupae of the WT and mutants on Fig1-5.

4. Ik2 dominant negative mutant bristles are shorter than WT, therefore the statistical analysis could be misleading when there is a much shorter bristle.

5. On page 20 they state that ~32h APF complete Golgi units could be detected, but only MnII-GFP is presented, no cis- or trans-Golgi marker is shown.

6. The 3 and 4 pupae per genotype is not enough to do proper statistical analyses. It is not clear whether they tested 4, 5 or 6 bristles altogether or per one pupae in each genotype. Especially in the case of Khc-RNAi, I suggest doubling the tested bristles.

7. Why do they used Dhc64C8-1/Dhc64C902 mutant combination to visualize the cis-Golgi and ERES distribution (Fig2A-C and G-I), but Dhc64C8-1/Dhc64C4-19 combination to test the trans-Golgi GalNacT2-YFP (Fig2D-F)?

8. It is shown the actin distribution in WT and Khc-RNAi, but not in Dhc mutant, LvaDN or Ik2DN mutants. It will be necessary to include those images too.

9. Fig3 C shows only the MnII-GFP localization, but not the cis- or trans-Golgi marker. It will be useful to include those as well.

10. Table 1 is including data from the MnII-GFP measurement, but it will be useful to include the cis- or trans-Golgi data also. Moreover, the precise description (measurement based on MnII-GFP) of Table 1 is missing.

11. There are several generalizations in the text. Organization of the Golgi is specialized in Drosophila, therefore the authors should be careful when they generalize about Golgi organization. (“kinesin is present in the Golgi membranes” (Ref. 38). There is not much information about kinesin function in Golgi trafficking, therefore, the Golgi distribution in the soma of the mutant should be included.

12. On page 22 they referred, that in the soma of the larval neurons contains the ring-shaped complete Golgi, however, it was shown that during spermatogenesis, the Golgi based acroblast organization is similar. (ref: Farkas RM. et. al. Molecular Biology of the Cell (2003) Vol.14, 190–200, Fari K et. al. Biology Open (2016) 5, 1102-1110)

In addition, the manuscript would benefit from careful editing by a native English speaker; there are missing words, sentences where the subject and verb do not agree, places where there are extra or missing commas, sentences where the word order is confusing or spelling mistakes (mammal neurons instead of mammalian neurons, etc.).

Minor comments:

1. Description of Fig4 has several confusing sentences.

2. Scale bar on Fig3 B, B’ is not correct.

3. Dhc4-19 allele is missing from the stock list of Materials and methods.

4. On Page 20. they use Khc64C, which should be Dhc64C.

5. In the “Kinesin heavy chain section”, Dhc64 is mentioned in the 7th line, but that one is the mutant, not the gene, therefore it is should be labeled correctly.

**********

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Reviewer #1: Yes: Daniel Ungar

Reviewer #2: No

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PLoS One. 2019 Oct 2;14(10):e0223174. doi: 10.1371/journal.pone.0223174.r002

Author response to Decision Letter 0


4 Sep 2019

Reviewer #1: In their manuscript Abdu and colleagues investigate the organization of Golgi stacks in Drosophila bristles. Similar to dendrites, the bristles of the fly contain Golgi outposts, which are likely used to supply processed secretory/membrane proteins at sites far from the cell's soma. This is an interesting model system, and the authors have done a nice work to characterize the microtubule dependent formation of these Golgi outposts. I believe this will be a good contribution to the literature, but suggest that the authors consider some revision as detailed below.

1. It would be good to document the number of repeats for each measurement where statistics was used (displayed in table 1).

We agree with this comment and have added the number of repeats for each experiment throughout the revised text.

2. The dual color staining of the Golgi in different combinations does not give this reviewer a lot of confidence that the Golgi mini-stacks are properly organized. Three or even four color images should be possible where secondary antibodies are used (e.g. for dGM130 antibody staining), showing that the Golgi (and ERES) are in fact properly polarized in a cis-trans direction.

In this paper, we used the different Golgi marker only for marking the different Golgi compartments and not for revealing the spatial organization of the Golgi compartments in relation to each other. Indeed, for such analysis more experiments and tools are needed, which we emphasized was not the aim in our paper.

3. The use of the IK2 dominant negative reagent is very interesting, but I had to dig deep and hard to find information on what this reagent (or for that matter the IK2 protein in this context) does. Some more information would be nice, this could be in the introduction, the results or even in the discussion sections, depending on the authors' preference, but I feel it would make the paper's message easier to understand.

We agree with this comment and have added the following description to the Results: ”Ik2DN is a defective kinase where lysine at position 41 was changed to alanine (Ik2K41A)”.

4. The authors' use of the term "mini-stacks" is not correct in my opinion. Mini-stack is generally used to mean Golgi stacks that are not assembled in a ribbon, and are as such commonly used by mammalian cell biologists (although not exclusive to the mammalian system of course). Here the authors use it seemingly to mean stacks lacking a cisterna (in this case the trans cisterna). I would caution against that, and would rather use a different term.

We agree with this comment, and indeed the bristle shaft contains Golgi stacks that lack trans cisterna and thus could not be termed mini-stacks. Thus, we decided to call this unique structure incomplete Golgi.

Reviewer #2: In this manuscript, Melkov and colleagues present a series of experiments aimed to describe the organization of Golgi in highly elongated bristles upon disruption of microtubule motor protein function in Drosophila melanogaster.

The authors used fluorescently labeled transgenic lines to visualize the medial and trans-Golgi compartments, and Sec16 and GM130 antibodies to show the distribution of ERES and cis-Golgi in WT and different mutant backgrounds. They measured the distribution and composition of Golgi in the bristle shaft tip and the base region and found complete Golgi in the entire bristle shaft when they interfere with microtubule polarity.

Overall, the experimental results are interesting, but the experiments have several fundamental problems and the interpretation of the results is not satisfactory. Several questions need to be answered in order to be published in PlOS One.

Major comments:

1. To give a more definite answer regarding the involvement of microtubule polarity in the elongated bristle shaft cell the whole cell should be tested. They compare only the bristle shaft tip and base regions, but not the middle region or the soma region. How is the Golgi distribution in the soma in the different mutants? Is it possible, that the abnormal Golgi distribution is only the indirect consequences of the general problem in cell homeostasis and globally disturbed Golgi organization?

We agree with the comment referring to revealing the localization of Golgi in the somatic region of WT, as compared to the mutants. We analyzed the localization pattern of the Golgi in the somal region of the wild type and all other mutant backgrounds. New figures were generated and since no obvious changes in the localization pattern of the Golgi were noted, we added these as Supplemental figures.

As to the localization pattern at the bristle shaft, as explained in the Materials and methods, we divided the entire bristle shaft into two parts, thus there is no need for a middle part.

(Page 7-“A Z-stack projection of the bristle shaft was divided into two halves exactly in the middle of the shaft length, resulting in two sections of even length, with one part being close to the base (soma) and the other being distal to the base, referred to as the tip”).

2. They used the UAS/Gal4 system to drive transgenes in the bristle. While ManII-GFP signal is strong in Fig 1A and C, it is weaker when it is co-expressed by the GalNacT2-RFP, probably due to the fact that two transgenes use the Gal4 source in those flies. It is not clear which driver did they use, the neur-Gal4 or the sca-Gal4 driver in the WT.

We agree with this comment and have added the Gal4 used in this figure.

Why do they used the Sca-Gal4 on Khc RNAi and Dhc64 mutant, but Neur-Gal4 on the Lava-lamp dominant negative background? On the same line, they used different driver lines in the WT and the mutants when they visualized the developmental distribution of Golgi outpost in different pupal stages (Fig6). This is especially important when the authors compare statistically the Golgi localization (particle area, density) in WT and mutants (Table1).

In this study, we alternated between the different Gal-4 lines due to technical problems. Both over-expression of Lava-lampDN and Ik2DN with sca-Gal4 was not as effective as with Neur-Gal4. We agree with the reviewer’s comment that it is difficult to compare statistically, so we omitted the statistical analysis between Ik2DN and Dhc and Khc, and just describe the effect of mutations of Ik2 on Golgi organization. Thus. the following text was added: “First, we found that expression with neur-Gal4 and not Sca-Gal4 generated typical Ik2 bristle defects. Thus, we used this Gal4 for further examinations” . Later in the text, we added: “Since we used different Gal4s for Golgi expression in Dhc64C and Khc-RNAi (Sca-Gal4), in comparison to Ik2DN expression (neur-Gal4), we were not able to statistically compare Golgi parameter (area and density) between them.”

3. Based on the fact that Golgi distribution changes during pupal development, it will be important to mention what is the age of the pupae of the WT and mutants on Fig1-5.

We agree with this comment and have added the age of the pupa in all figures.

4. Ik2 dominant negative mutant bristles are shorter than WT, therefore the statistical analysis could be misleading when there is a much shorter bristle.

Based on comment 2 of reviewer #2, we decided that we could not preform statistical analysis between Ik2 and the rest of the mutants. As such, this comment is not relevant.

5. On page 20 they state that ~32h APF complete Golgi units could be detected, but only MnII-GFP is presented, no cis- or trans-Golgi marker is shown.

We agree with this comment and have changed the text throughout figure 6 caption accordingly.

6. The 3 and 4 pupae per genotype is not enough to do proper statistical analyses. It is not clear whether they tested 4, 5 or 6 bristles altogether or per one pupae in each genotype. Especially in the case of Khc-RNAi, I suggest doubling the tested bristles.

We agree with this comment and have doubled our tested bristle analysis. Now, we have at least 5 to 6 pupa and at least 3 bristles per pupa.

7. Why do they used Dhc64C8-1/Dhc64C902 mutant combination to visualize the cis-Golgi and ERES distribution (Fig2A-C and G-I), but Dhc64C8-1/Dhc64C4-19 combination to test the trans-Golgi GalNacT2-YFP (Fig2D-F)?

There is no specific reason why we used these two different alleles since we (Melkov et al., 2016) and others (Gepner et al., 1996 and Li et al., 2008) previously showed that both alleles, Dhc64C902 and Dhc64C4-19, are strong amorphic alleles which give the exact bristle phenotypes in combinations with Dhc64C8-1

8. It is shown the actin distribution in WT and Khc-RNAi, but not in Dhc mutant, LvaDN or Ik2DN mutants. It will be necessary to include those images too.

We agree with this comment and have performed this analysis again and include actin staining.

9. Fig3 C shows only the MnII-GFP localization, but not the cis- or trans-Golgi marker. It will be useful to include those as well.

We agree with this comment and have performed this analysis. The new figure 3 now includes other Golgi domains markers.

10. Table 1 is including data from the MnII-GFP measurement, but it will be useful to include the cis- or trans-Golgi data also. Moreover, the precise description (measurement based on MnII-GFP) of Table 1 is missing.

We agree with this comment and have added a new data set for GalNacT2-YFP as well; all of these new results are summarized in Table 2.

11. There are several generalizations in the text. Organization of the Golgi is specialized in Drosophila, therefore the authors should be careful when they generalize about Golgi organization. (“kinesin is present in the Golgi membranes” (Ref. 38). There is not much information about kinesin function in Golgi trafficking, therefore, the Golgi distribution in the soma of the mutant should be included.

We agree with this comment and have changed the text accordingly. Also, as mentioned in comment #1, Golgi distribution in the soma of the mutant was also included.

12. On page 22 they referred, that in the soma of the larval neurons contains the ring-shaped complete Golgi, however, it was shown that during spermatogenesis, the Golgi based acroblast organization is similar. (ref: Farkas RM. et. al. Molecular Biology of the Cell (2003) Vol.14, 190–200, Fari K et. al. Biology Open (2016) 5, 1102-1110)

We agree with this comment and have added Golgi-based acroblast organization in sperrmatogeneis to the text. The following text and references were added to the discussion: ” “During spermatogenesis the Golgi-based acroblast has a ring shape.

In addition, the manuscript would benefit from careful editing by a native English speaker; there are missing words, sentences where the subject and verb do not agree, places where there are extra or missing commas, sentences where the word order is confusing or spelling mistakes (mammal neurons instead of mammalian neurons, etc.).

The paper was edited by a native English editor.

Minor comments:

1. Description of Fig4 has several confusing sentences.

We have changed the text

2. Scale bar on Fig3 B, B’ is not correct.

The scale bar was fixed.

3. Dhc4-19 allele is missing from the stock list of Materials and methods.

We have added this allele to the stock list of Materials and methods.

4. On Page 20. they use Khc64C, which should be Dhc64C.

We changed the text accordingly.

5. In the “Kinesin heavy chain section”, Dhc64 is mentioned in the 7th line, but that one is the mutant, not the gene, therefore it is should be labeled correctly.

We changed the text accordingly.

Attachment

Submitted filename: Response to reviewers.docx

Decision Letter 1

Maria Grazia Giansanti

17 Sep 2019

The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array

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Acceptance letter

Maria Grazia Giansanti

23 Sep 2019

PONE-D-19-15627R1

The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array

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Associated Data

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

    Supplementary Materials

    S1 Fig. The trans-Golgi marker Gal-T-RFP shows a localization pattern similar to that of GalNacT2-RFP/YFP.

    Confocal projections of live developing bristle shafts expressing Gal-T-RFP (a trans-Golgi marker): A-B- Sca-Gal4-UAS-EB1-GFP::UAS-Gal-T-RFP (WT) developing bristle shaft showing no Gal-T-positive particles at an early stage of bristle development; AGreen EB1-GFP used to highlight the cell perimeter, B- RedGal-T-RFP. C-D—Confocal projections of dynein heavy chain-mutated background bristle shafts, Dhc64C8-1/Dhc64C4-19 trans-heterozygote bristle shaft expressing Gal-T-RFP stained with Oregon-green phalloidin; C–Red- The Gal-T-RFP (trans-Golgi) marker is localized to the entire bristle shaft area, D–Merged—green—phalloidin staining of actin bundles, used here to highlight the cell perimeter, red—Gal-T-RFP. Scale bar, 10 μm.

    (EPS)

    S2 Fig. Golgi organization in Dhc64C mutant bristle cell soma.

    A-C'' -Confocal projections of representative of a WT bristle “somal” region from Dhc64C mutant background: A-A''–“Soma” of a dynein mutant bristle cell expressing sca-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green UAS-MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a Dhc64C mutant bristle cell expressing sca-Gal4::GalNacT2-RFP (a Trans-Golgi marker), B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–Soma of a dynein mutant bristle cell expressing sca-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

    (EPS)

    S3 Fig. Golgi organization in LvaDN mutant bristle shaft.

    A-D- A Wild type bristle shaft expressing neur-Gal4-UAS-GalNacT2-YFP (trans-Golgi marker) stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: A–Gray phalloidin-UV staining of actin bundles in a GalNacT2-YFP expressing bristle, used here to highlight the cell perimeter: B–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, C–Red anti-dGM130 antibody staining (cis-Golgi marker), D–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. E-H- neur-UAS-Lava-lampDN bristle shaft co-expressing UAS-GalNacT2-YFP (trans-Golgi marker) and stained with anti-dGM130 (cis-Golgi marker) antibodies and phalloidin: E–Gray phalloidin-UV staining of actin bundles in a GalNacT2-YFP expressing bristle, used here to highlight the cell perimeter: F–Green GalNacT2-YFP, a trans-Golgi marker, is ectopically localized to the entire bristle shaft area, G–Red anti-dGM130 antibody staining (cis-Golgi marker), H–Merged image of green GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of trans- and cis-Golgi compartments in the mutant bristle shaft. I-L–A wild type neur-Gal4::UAS-MnII-GFP bristle shaft expressing stained with anti-Sec16 (ERES) antibodies and phalloidin: I–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: J—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, K–Red anti-Sec16 antibody staining is localized to the entire shaft area, L–Merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. M-P–neur-UAS-Lava-lampDN bristle shaft co-expressing UAS-MnII-GFP stained with anti-Sec16 (ERES) antibodies and phalloidin: M–Gray phalloidin-UV staining of actin bundles in a MnII-GFP-expressing bristle, used here to highlight the cell perimeter: N—Green MnII-GFP, a medial-Golgi marker, is localized to the entire bristle shaft area, O–Red anti-Sec16 antibody staining is localized to the entire shaft area, P–Merged image of green MnII-GFP and red anti-Sec-16-antibody showing co-localization of medial-Golgi and ERES components in the mutant bristle shaft. APF—After prepupa formation. The scale bar represents 10 μm.

    (EPS)

    S4 Fig. Golgi organization in LvaDN mutant bristle cell soma.

    Confocal projections of representative of bristle “somal” region from WT (A-A'', C-C'', E-E'') and LvaDN mutant background: A-A''–“Soma” of a WT mutant bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–“Soma” of a LvaDN mutant bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-dGM130 antibodies (a cis-Golgi marker). B'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, B''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. C-C''–Soma of a WT bristle cell expressing neur-Gal4::GalNacT2-YFP (a Trans-Golgi marker), C’- stained with anti-dGM130 antibodies (a cis-Golgi marker), C''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. D-D''–Soma of a LvaDN mutant bristle cell expressing GalNacT2-YFP (a Trans-Golgi marker), D’- stained with anti-dGM130 antibodies (a cis-Golgi marker), D''–Merged image of UAS-GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. E-E''–Soma of a WT bristle cell expressing neur-Gal4::MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): E'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, E''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. F-F''–Soma of a LvaDN mutant bristle cell expressing MnII-GFP (a medial-Golgi marker) stained with anti-Sec16-antibodies (to identify the ERES): F'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, F''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

    (EPS)

    S5 Fig. Golgi organization in kinesin heavy chain mutant bristle cell soma.

    A-C'' -Confocal projections of representative of bristle “somal” region from kinesin heavy chain mutant background: A-A''–“Soma” of a bristle cell co-expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS-Khc-RNAi stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a bristle cell expressing sca-Gal4::UAS-GalNacT2-YFP (a Trans-Golgi marker) and UAS-Khc-RNAi, B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–Soma of a bristle cell expressing sca-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS-Khc-RNAi stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

    (EPS)

    S6 Fig. Golgi organization in Ik2DN bristle cell soma.

    A-C'' -Confocal projections of representative of bristle “somal” region from Ik2DN mutant background: A-A''–“Soma” of a bristle cell expressing neur-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS- Ik2DN stained with anti-dGM130 antibodies (a cis-Golgi marker). A'–Anti-dGM130 antibody staining showing a cis-Golgi compartment localized throughout the bristle soma cytoplasm, A''–Merged image of green MnII-GFP and red anti-dGM130 antibody staining showing co-localization of medial- and cis-Golgi compartments. B-B''–Soma of a bristle cell expressing neur-Gal4::GalNacT2-YFP (a Trans-Golgi marker) and UAS-Ik2DN, B’- stained with anti-dGM130 antibodies (a cis-Golgi marker), B''–Merged image of GalNacT2-YFP and red anti-dGM130 antibody staining showing co-localization of medial- and Trans-Golgi compartments. C-C''–“Soma” of a bristle cell expressing neur-Gal4::UAS-MnII-GFP (a medial-Golgi marker) and UAS- Ik2DN stained with anti-Sec16-antibodies (to identify the ERES): C'–Anti-Sec16 antibody staining showing ERES localized throughout the bristle soma cytoplasm, C''–Merged image of green MnII-GFP and red anti-Sec16 antibody staining showing co-localization of medial-Golgi and ERES components in the bristle cell soma. The scale bar represents 10 μm.

    (EPS)

    Attachment

    Submitted filename: Response to reviewers.docx

    Data Availability Statement

    All relevant data are within the manuscript; DOI: https://doi.org/10.5061/dryad.6gt43qg.


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