Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast. We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.     

Structural and functional coupling of Hsp90- and Sgt1-centred multi-protein complexes

Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod-like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90–Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N-terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1-dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate-specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins.     

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.     

Sgt1p contributes to cyclic AMP pathway activity and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p in budding yeast

Sgt1p is a highly conserved eucaryotic protein that is required for both SCF (Skp1p/Cdc53p-Cullin-F-box)-mediated ubiquitination and kinetochore function in yeast. We show here that Sgt1p is also involved in the cyclic AMP (cAMP) pathway in Saccharomyces cerevisiae. SGT1 is an allele-specific suppressor of cdc35-1, a thermosensitive mutation in the leucine-rich repeat domain of the adenylyl cyclase Cyr1p/Cdc35p. We demonstrate that Sgt1p and Cyr1p/Cdc35p physically interact and that the activity of the cAMP pathway is affected in an sgt1 conditional mutant. Sequence analysis suggests that Sgt1p has features of a cochaperone. Thus, Sgt1p is a novel activator of adenylyl cyclase in S. cerevisiae and may function in the assembly or the conformational activation of specific multiprotein complexes.     

Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts

Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. We performed a genetic screen to identify regulators of neuroblast self-renewal, and identified a mutation in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. We found that sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of "neuroblast tumors." Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1, or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. We propose that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors.     

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.     

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.     

Plk1 phosphorylates sgt1 at the kinetochores to promote timely kinetochore-microtubule attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.     

SGT2 and MDY2 interact with molecular chaperone YDJ1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Sgt2 was thought to be the homologue of vertebrate SGT (small glutamine tetratricopeptide repeat-containing protein). SGT has been known to interact with both Hsp70 and Hsp90. However, it was not clear whether Sgt2 might have a similar capacity. Here, we showed that Ssa1/Ssa2 (yeast heat shock cognate [Hsc]70), Hsc82 (yeast Hsp90), and Hsp104 coprecipitated with Sgt2 from yeast lysates. Another molecular chaperone, Ydj1, known to interact with Ssal and Hsc82, also coprecipitated with Sgt2. Synthetic lethality between SGT2 and YDJ1 was observed after the cells were under stress, although Sgt2 might not interact physically with Ydj1. We also found that Mdy2 interacted with the N-terminal region of Sgt2 and that Mdy2 appeared to interact physically with Ydj1. Mdy2 therefore may mediate the association of Ydj1 and Sgt2. In addition, the mating efficiency of mdy2delta, sgt2delta, and mdy2deltasgt2delta strains was reduced to a similar extent. Compared with mdy2delta and ydj1delta cells, ydj1deltamdy2delta cells, however, showed a further suppression in mating efficiency. Moreover, MDY2 interacted genetically with YDJ1. These results suggest that protein complexes containing Sgt2 and Mdy2 bring molecular chaperones together to carry out certain chaperoning functions.     

Sgt1 is required for human kinetochore assembly

Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.     

The centrosome and bipolar spindle assembly: Does one have anything to do with the other?

In vertebrate somatic cells, the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles, such as plant cells and many oocytes, bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization, both before and after nuclear envelope breakdown (NEB), is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.Key words: centrosome, centriole, mitosis, spindle, cell cycle, meiosis, plant cell, microsurgery     

Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability

We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant POLO: We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.     

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates

To determine the molecular composition of the centrosome of a higher eukaryote, we carried out a systematic nano-electrospray tandem or MALDI mass spectrometry analysis of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. We have found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

An essential step of kinetochore formation controlled by the SNARE protein Snap29

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis‐segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.     

Spindle assembly requires complete disassembly of spindle remnants from the previous cell cycle

Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.     

Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins

Abstract TPR proteins modulate the activity of molecular chaperones. Here, we describe the S. cerevisiae TPR protein Sgt2 as interaction partner of Ssa1 and Hsp104 and as a component of the GET pathway by interacting with Get5. The GET pathway mediates the sorting of tail-anchored (TA) proteins, harboring a C-terminal trans-membrane segment, to the ER membrane. S. cerevisiae sgt2Δ cells show partial defects in TA protein sorting. Sgt2 activity in vivo relies on its N- and C-terminal domains, whereas the central TPR domain and thus chaperone interactions are dispensable. We show that TA protein sorting defects are more severe in sgt2Δ get5Δ mutants compared to single knockouts. Furthermore, overproduction of Sgt2 becomes toxic to get3Δ but not to get5Δ cells. Together, these findings indicate an additional, Get5-independent role of Sgt2 in TA protein sorting, pointing to parallel pathways of substrate delivery to Get3.     

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of γ-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in γTub23C^PI mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though γ-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a γ-tubulin-independent manner in Drosophila spermatocytes. However, γ-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.