InteractiveFly: GeneBrief

Zeste-white 10: Biological Overview | References

A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. Overexpression of CAL1 is associated with increased CENP-A levels at centromeres and uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration suggesting crosstalk of CENP-A loading with the regulatory machinery of mitosis. Mitosis length is influenced by the spindle assembly checkpoint (SAC), and this study found that CAL1 interacts with the SAC protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the SAC to ensure mitotic fidelity (Pauleau, 2019).

The formation of two genetically identical daughter cells with a correct and stable genome is of utmost importance during mitosis. Condensed chromosomes are attached and segregated to the opposing poles of the dividing cell at anaphase by the mitotic spindle. At the interface between the chromosomes and the spindle microtubules lies the kinetochore. This multi-protein complex is formed by the components of the KMN network (formed by the Knl1 complex, the Mis12 complex, and the Ndc80 complex) (Joglekar, 2017). The Ndc80 complex is mainly responsible for connecting microtubules with kinetochores while the Knl1 complex primarily coordinates the Spindle Assembly Checkpoint (SAC) (Musacchio, 2017). The SAC delays entry into anaphase until all chromosomes are properly attached and aligned at the metaphase plate. The metaphase to anaphase transition is controlled by the activation of Cdc20 of the APC/C, a multisubunit ubiquitin ligase that triggers the degradation of cell cycle regulators by the proteasome. The SAC proteins Mad2, BubR1, and Bub3 sequester Cdc20 by forming the Mitotic Checkpoint Complex (MCC) thereby preventing the activation of the APC/C. Besides, other proteins have been implicated in SAC activity including Bub1, Mad1, the Mps1 and Aurora B kinases, and the RZZ complex (formed by the three proteins Rough Deal (ROD), Zw10 and Zwilch (Musacchio, 2015). Finally, the Mis12 complex serves as a hub at the kinetochore interacting with all kinetochore complexes as well as with the centromere (Pauleau, 2019).

The kinetochore assembles on the centromere during mitosis, a highly specialized chromatin region that is defined by an enrichment of nucleosomes containing the histone H3 variant CENP-A, also called CID in Drosophila. In contrast to canonical histones, CENP-A deposition at centromeres is independent of DNA replication and is temporally restricted to a specific cell cycle stage, which varies between organisms: late telophase/early G1 in mammalian cultured cells, G2 in S. pombe and plants, and mitosis to G1 in Drosophila. The timing of CENP-A is particularly intriguing in Drosophila cultured cells as centromeric CENP-A is replenished during prometaphase-metaphase thus coinciding with kinetochore assembly. CENP-A loading requires the action of its dedicated chaperones: HJURP in humans, Scm3 in fungi and CAL1 in Drosophila (Chen, 2014, Erhardt, 2008, Schittenhelm, 2010). Deregulation of CENP-A and its loading machinery can result in the misincorporation of CENP-A into regions along the chromosome arms. Misincorporated CENP-A is usually rapidly degraded. If, however, CENP-A-containing nucleosomes remain at non-centromeric sites ectopic formation of functional kinetochores can occur that may lead to chromosome segregation defects and aneuploidy (Pauleau, 2019).

In Drosophila melanogaster, two other proteins are constitutively present at centromeres and essential for centromere function: the conserved protein CENP-C and the CENP-A chaperone CAL1. CENP-C has been shown to act as a linker between CENP-A nucleosomes and the Mis12 complex, therefore, providing a platform for kinetochore assembly (Przewloka, 2011). CENP-C is also implicated in CENP-A replenishment at centromeres during mitosis by recruiting CAL1 (Chen, 2009; Erhardt, 2008). CAL1 interacts with CENP-A in both pre-nucleosomal and nucleosomal complexes and is necessary for CENP-A protein stabilization via Roadkill-Cullin3-mediated mono-ubiquitination. Moreover, CAL1 has been previously shown to be the limiting factor for CENP-A centromeric incorporation in fly embryos. However, differences in centromere assembly have been reported between Drosophila cultured cells and embryos. Firstly, CENP-A loading has been shown to take place during mitotic exit in early embryos and prometaphase to early G1 in cultured cells. Second, CENP-C incorporates concomitantly to CENP-A in embryos while this time window seems to be larger in cultured cells. This study, therefore, set out to determine more precisely the function of CAL1 in CENP-A loading regulation in Drosophila cultured cells (Pauleau, 2019).

During the course of these studies, overexpression of CAL1 was found not only to increase endogenous and exogenous CENP-A abundance at centromeres, but also was found to uncouple CENP-A loading from mitosis. Strikingly, it was discovered a co-dependence of mitotic duration and accurate CENP-A loading that may be coordinated by an interaction of the CENP-A loading machinery with the SAC protein and RZZ subunit Zw10. These data suggest an intricate coordination of the spindle assembly checkpoint, CENP-A loading, and mitotic duration in order to safeguard accurate mitotic progression (Pauleau, 2019).

In Drosophila cells, CENP-A loading takes place primarily during prometaphase-metaphase. Additional turnover of CENP-A in G1 has been reported leading to the hypothesis that CENP-A could be further incorporated at this stage, which was not observe in FRAP experiments when centromeric CENP-A was bleached at the end of cytokinesis. However, the FRAP experiments and most importantly live staining of newly synthesized SNAP-CENP-A confirmed that the majority of CENP-A loading takes place during mitosis in Drosophila cultured cells (Pauleau, 2019).

In flies, CENP-A incorporation is controlled by its chaperone CAL1. It has been shown previously that co-overexpression of exogenous CENP-A and CAL1 leads to an increase of centromeric CENP-A in embryos. This study now shows that overexpression of CAL1 alone leads to increased endogenous CENP-A protein levels in Drosophila cultured cells. Ectopic incorporation of CENP-A, however, was never observed suggesting that CAL1 loads CENP-A exclusively to centromeres and that ectopic CENP-A incorporation in flies depends on alternative loading mechanisms similar to what has been suggested in human cells. Importantly, increased centromeric CENP-A levels following CAL1 overexpression correlated with faster mitosis. A similar acceleration of mitotic timing was observed when CENP-A was only mildly overexpressed, revealing a possible link between CENP-A loading and mitotic timing. Indeed, shortening mitosis duration by depleting Mad2 or BubR1 was associated with decreased CENP-A loading. However, just elongating the mitotic time window during which CENP-A can get loaded (Spindly or Cdc27 depletion, or by drug treatment) did not increase the amount of CENP-A incorporated at centromeres, showing that the length of mitosis alone is insufficient to control CENP-A amounts at centromeres. Rather, these experiments showed that only a defined amount of CENP-A can be incorporated at each mitosis probably correlating with CAL1's availability. Indeed, live analysis of CAL1-overexpressing cells allowed visualization of newly synthesized CENP-A incorporation to centromeres in all stages of the cell cycle. This strongly suggests that CAL1 controls CENP-A incorporation into centromeric chromatin both quantitatively and temporally. How exactly CENP-A levels at centromeres are sensed is unclear but this study identified the RZZ-component Zw10 as a new CAL1 interacting partner, which directly connects CENP-A loading to the SAC (Pauleau, 2019).

It has been proposed that SAC activation is a 2-steps process: at the end of G2-beginning of mitosis, before the kinetochores are assembled, cytosolic Mad1-Mad2 dimers initiate MCC formation inhibiting APC/CCdc20 and determining the timing of mitosis. After nuclear envelope disassembly, kinetochore-dependent MCC are generated and regulated by kinetochore-microtubules attachment. Therefore, the following model is suggested: efficient CENP-A loading by CAL1 during mitosis recruits Zw10 up to a threshold, which is sensed by the SAC. Low CENP-A levels at centromeres could lead to more cytosolic Mad2 thereby keeping the timer active longer. Higher CENP-A levels at centromeres during early mitosis would accelerate the recruitment of RZZ and consequently Mad2 to the kinetochores or capture microtubules more efficiently, therefore, releasing the timer and shortening mitosis duration in cells where kinetochores attach properly to the spindle microtubules. Interestingly, Nocodazole treatment did not affect CENP-A loading confirming previous observations that kinetochore attachment to the microtubule spindle does not play a role in CENP-A loading. These results are pointing further to an additional function of the SAC independent of the control of microtubule attachment. Interestingly, recent evidence shows that RZZ together with Spindly plays a central role in kinetochore expansion during early mitosis to form a fibrous corona that then compacts upon microtubule capture. Whether and -if so- how the kinetochore expansion by RZZ and spindly is involved in CENP-A loading needs to be investigated in the future (Pauleau, 2019).

Many essential components of the SAC require outer kinetochore components for their localization to centromeric regions. However, several outer components are missing from the Drosophila kinetochore and even though Mad1/2 recruitment to the kinetochore depends on the RZZ complex, the factors necessary for the localization of the RZZ to kinetochores are unknown. This study has shown that RZZ localization to the kinetochores does not require KNL1Spc105R but depends on the centromeric proteins CAL1 and CENP-A. Therefore, it is proposed that the Drosophila outer kinetochore and components of the SAC assemble through two independent pathways: the CENP-C-KMN-Bub1-Bub3/BubR1 branch or the CAL1-RZZ-Mad1/2 branch. How those two pathways communicate for the formation of MCC complexes remains to be determined. One link may be the KMN complex since Mad2 is diminished in the absence of KMN proteins. Interestingly, Spc105R mutation does not affect SAC function in fly embryos suggesting that flies rely more on the RZZ-Mad1/2 branch to engage the SAC (Pauleau, 2019).

CENP-A expression and its stability together with its dependence on the low abundant and highly specific loading factor CAL1 and the connection to mitotic events are likely interconnected cellular surveillance mechanisms to avoid misincorporation of CENP-A and, therefore, securing genome stability. How CAL1 itself is regulated to obtain such specificity is currently unknown. In conclusion, this study has shown that there is direct crosstalk between the SAC and the maintenance of centromeric chromatin, ensuring mitotic fidelity not only by controlling microtubule attachment but also by regulating the accurate composition of centromeres (Pauleau, 2019).

Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function

The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10, and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the SAC by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. To explore the importance of the Rod_C domain in RZZ function in Drosophila, a series of point mutations was generated in a stretch of 200 residues within this domain, and their phenotypes are reported in this study. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores (Menant, 2020).

Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, this study has identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. It is proposed that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. These findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation (Barbosa, 2020).

To ensure the fidelity of chromosome segregation, sister kinetochores (KTs) mediate the attachment of chromosomes to microtubules (MTs) of opposite spindle poles (amphitelic attachments). However, the initial contact of KTs with MTs is stochastic and consequently erroneous attachments-syntelic (chromosome bound to MTs from the same spindle pole) or merotelic (same KT bound to MTs from opposite poles)-can be formed during early mitosis. Thus, accurate mitosis requires a tight regulation of KT-MT turnover so mistakes are prevented or corrected and amphitelic end-on interactions are stabilized. This relies heavily on the activity of two conserved mitotic kinases, Aurora B and Polo/Plk1. Aurora B promotes the destabilization of KT-MT interactions mainly through phosphorylation of proteins of the KMN network (KNL1/Spc105, Mis12 and Ndc80), which decreases their affinity for MTs. Interestingly, it has been shown that the RZZ complex (Rod, ZW10 and Zwilch) is able to interact with Ndc80 N-terminal tail and prevent the adjacent calponin homology (CH) domain from binding to tubulin (Cheerambathur, 2013). This Aurora B-independent destabilizing mechanism is proposed to prevent Ndc80-mediated binding when KTs are laterally attached, hence reducing the potential for merotely during early mitosis. The RZZ additionally recruits Spindly and the minus end-directed motor dynein to KTs, thus providing the means to relieve its inhibitory effect over KT-MT attachments, as well as to ensure the timely removal of spindle assembly checkpoint (SAC) proteins from KTs. However, it remains unclear how RZZ removal by Spindly-dynein is coordinated with end-on attachment formation (Barbosa, 2020).

Polo/Plk1 activity is implicated in both stabilization and destabilization of KT-MT attachments. While the contribution to the former function has been attributed to PP2A-B56 phosphatase recruitment through Plk1-dependent BubR1 phosphorylation, the mechanism underlying Polo/Plk1 destabilizing activity remains unclear. Interestingly, Polo/Plk1 KT localization and activity decrease from early mitosis to metaphase, concurrent with an increase in KT-MT stability. Moreover, high Plk1 activity at KTs was shown to correlate with decreased stability of KT-MT attachments during prometaphase, but the underlying molecular mechanisms have only been marginally addressed (Barbosa, 2020).

This study describes the mitotic effect of expressing a constitutively active Polo-kinase mutant (PoloT182D) in Drosophila neuroblasts and cultured S2 cells. The expression of PoloT182D causes persistent KT-MT instability and congression defects, extends mitotic timing associated with SAC activation and increases chromosome mis-segregation. A small-scale candidate-based RNAi screen was designed to identify partners/pathways that are affected by constitutive Polo activity in the Drosophila eye epithelium. The screen revealed that downregulation of the RZZ subunit Rod rescues the defects resulting from PoloT182D expression. PoloT182D causes permanent accumulation of the RZZ complex at KTs, which is associated with a delay in achieving stable biorientation. Accordingly, Rod depletion rescues the time required for establishing end-on KT-MT attachments and for chromosome congression. This study further demonstrates that Polo phosphorylates the dynein-adaptor Spindly to decrease its affinity for the RZZ. This in turn prevents dynein-dependent stripping of RZZ from KTs, hence causing a delay in the formation of stable end-on attachments. The findings provide a mechanism for the destabilizing action of Polo/Plk1 over KT-MT attachments. A model is proposed in which Polo/Plk1 activity fine-tunes the RZZ-Spindly-dynein module throughout mitosis to ensure the fidelity of KT-MT attachments and chromosome segregation (Barbosa, 2020).

KT-MT attachments at metaphase must be sufficiently stable to satisfy the spindle assembly checkpoint and sustain chromatid segregation during anaphase. On the other hand, during prometaphase, MTs must be able to rapidly detach from KTs to allow efficient correction of erroneous attachments. How KTs regulate the balance of MTs stabilizing and destabilizing forces during successive mitotic stages has remained unclear. This study shows that Polo kinase plays a critical role in this process through control of the RZZ-Spindly-dynein module at KTs. Polo-mediated phosphorylation of Spindly on Ser499 results in a transient increase in RZZ accumulation at KTs, which inhibits stable end-on attachments and likely minimizes merotely in early mitosis. However, permanent Spindly Ser499 phosphorylation is deleterious for mitotic fidelity since it prevents stable KT biorientation and timely chromosome congression (Barbosa, 2020).

Polo/Plk1 has been implicated in the stabilization of KT-MT attachments. Intriguingly, however, attachments are most stable during metaphase, when Polo/Plk1 activity is reduced. Maintaining Polo active in Drosophila larval neuroblasts markedly decreases the stability of KT-MT interactions, which is in line with previous observations in RPE-cultured cells. It is important to mention that insc-GaL4- driven expression of PoloWT and PoloT182D consistently yielded higher levels of the latter protein. This, however, unlikely explains the different phenotypic consequences observed in these neuroblasts, as analogous experiments with S2 cells expressing equivalent levels of PoloWT and PoloT182D mimic the decrease in the efficiency of chromosome congression and KT-MT stability when constitutively active Polo is expressed. Moreover, previous work has shown that Drosophila S2 cells depleted of Polo accumulated hyperstable attachments and that this phenotype was not exclusively attributed to reduced Aurora B activity. A requirement for Polo in fine-tuning the RZZ-Spindly-dynein axis offers a mechanistic explanation for these observations. During early mitosis, high levels of active Polo at KTs ensure that as soon as Spindly is recruited to RZZ, it is efficiently phosphorylated on Ser499. This promptly reduces Spindly affinity towards Zwilch, sensitizing RZZ-uncoupled Spindly for dynein-mediated transport away from KTs. As a result, the RZZ complex is retained at KTs to levels that normally inhibit the formation of stable end-on attachments by the Ndc80 complex (Cheerambathur, 2013) and maintain the SAC signalling active. Rod interacts with the basic tail of Ndc80 and, in this way, precludes binding of MTs to the calponin homology domain of Ndc80 (Cheerambathur, 2013). Thus, the conversion of lateral attachments preferentially formed at early stages of mitosis into stable amphitelic interactions that are essential for faithful chromosome segregation requires the relief of this Rod-mediated inhibitory mechanism. Evidence is provided that a decrease in Polo activity and, consequently, in Spindly phosphorylation, is critical for this transition by allowing the RZZ to fully engage with the Spindly-dynein complex and to be stripped from KTs. This raises the question of how and when Polo activity and Ser499 phosphorylation are antagonized to allow timely formation of stable end-on attachments. PP2A-B56 phosphatase may have a role in this process, since impairing its association with BubR1 was recently shown to dramatically increase the frequency of laterally attached KTs in human cells. However, because BubR1-PP2A-B56 is already present at high levels on early mitotic KTs, it was reason that additional mechanisms must operate to prevent premature end-on conversion. It is plausible that the switch is determined by the levels of cyclin A, which have been shown to function as a timer in prometaphase to destabilize attachments and facilitate error correction. Since Cdk1/CycA is able to phosphorylate human Spindly in vitro, it is hypothesized that this phosphorylation primes Spindly for Polo binding and increases Ser499 phosphorylation to levels that surpass the opposing phosphatase activity. As mitosis progresses, degradation of Cyclin A tips the balance towards Ser499 dephosphorylation, hence favouring stabilization of end-on attachments. This concurs with an increase in tension across KTs that allows the recruitment of PP1, whose role in Polo T-loop dephosphorylation has been described (Barbosa, 2020).

Although the Polo-phosphorylation site in Drosophila Spindly is not conserved in vertebrates, additional residues conforming to Polo/Plk1 consensus signature are present within the same domain, hinting that an analogous regulatory mechanism may take place in these organisms. Interestingly, Ser499 lies within motif that is conserved among different dynein-adaptors. Two other conserved domains have also been recently described for a number of adaptors and shown to act as regulatory modules involved in the interaction with dynein. Thus, it is envision that the motif identified in this study might provide an additional level of regulation in controlling dynein-adaptor complex formation (Barbosa, 2020).

The results suggest that Polo-mediated phosphorylation of Spindly on Ser499 uncouples dynein-mediated transport of the RZZ complex from Spindly. Moreover, it is proposed that phosphorylation of Ser499 causes Spindly C-terminal domain to elicit a negative regulatory action over the N-terminus Zwilch binding domain. In line with these results, it has been recently shown that intramolecular interactions occur within Spindly, causing it to fold on itself at different regions (Sacristan, 2018). Spindly C-terminal region could be involved in facilitating these interactions since it is thought to be of disordered nature (Sacristan, 2018). This structural organization resembles that of BicD/BicD2, a dynein-adaptor which is predicted to share with Spindly a similar mechanism of interaction with dynein. It is therefore noteworthy that Polo has been shown to activate BicD-dynein transport during oogenesis. Furthermore, several point mutations in BicD/BicD2 were shown to hyperactivate dynein for cargo transport. It will be interesting to establish whether Spindly Ser499 phosphorylation could also impact on dynein complex motility/processivity (Barbosa, 2020).

Long-lasting Polo activation or permanent Spindly Ser499 phosphorylation stalls KTs in labile interactions with MTs. The data confirm a destabilizing role for Polo in KT-MT attachments which has also been shown to operate through the control the kinase exerts over the recruitment and activation of Aurora B and the MT depolymerizing motor Kif2b . Hence, high levels of active Polo in early mitosis ensure efficient correction of merotelic and syntelic attachments, errors that typically occur upon nuclear envelope breakdown as a result of stochastic interactions between KTs and spindle MTs. Paradoxically, Plk1 activity has also been implicated in stabilization of KT-MT attachments through phosphorylation of BubR1. A model is envisioned where these apparently antagonistic Polo-directed inputs are not mutually exclusive but rather cooperate to establish proper attachments. Phosphorylation of BubR1 by Polo/Plk1 in prometaphase promotes the accumulation of PP2A-B56, which opposes Aurora B destabilizing phosphorylations on Ndc80. This is important to allow binding of MTs to the Ndc80 complex during the end-on conversion process, tipping the balance against the KT-MT destabilizing environment, particularly when Cyclin A levels drop. The observation that disrupting Plk1 activity rescues the attachment defects otherwise generated by depletion of PP2A-B56 strongly argues in favour of this integrated model for Polo-regulated stabilizing and destabilizing forces (Barbosa, 2020).

In summary, these findings demonstrate that the RZZ-Spindly-dynein module is tightly regulated by Polo kinase to ensure accurate chromosome segregation. Spindly phosphorylation by Polo on early mitotic KTs ensures RZZ-mediated inhibition of end-on interactions, hence preventing premature stabilization of erroneous attachments. As mitosis progresses, decreased Polo-kinase activity and concurrent Spindly dephosphorylation render the RZZ prone for removal from KTs by Spindly-dynein. This alleviates RZZ antagonism of MT binding by the Ndc80 complex, thus allowing timely conversion of labile lateral interactions into stable amphitelic attachments ensuring proper sister chromatid segregation (Barbosa, 2020).

RZZ and Mad1 dynamics in Drosophila mitosis

The presence or absence of Mad1 at kinetochores is a major determinant of spindle assembly checkpoint (SAC) activity, the surveillance mechanism that delays anaphase onset if one or more kinetochores remain unattached to spindle fibers. Among the factors regulating the levels of Mad1 at kinetochores is the Rod, Zw10, and Zwilch (RZZ) complex, which is required for Mad1 recruitment through a mechanism that remains unknown. The relative dynamics and interactions of Mad1 and RZZ at kinetochores have not been extensively investigated, although Mad1 has been reported to be stably recruited to unattached kinetochores. This study directly compare Mad1-green fluorescent protein (GFP) turnover dynamics on unattached Drosophila kinetochores with that of RZZ, tagged either with GFP-Rod or GFP-Zw10. Nearly 40 % of kinetochore-bound Mad1 has a significant dynamic component, turning over with a half-life of 12 s. RZZ in contrast is essentially stable on unattached kinetochores. In addition, this study reports that a fraction of RZZ and Mad1 can co-immunoprecipitate, indicating that the genetically determined recruitment hierarchy (in which Mad1 depends on RZZ) may reflect a physical association of the two complexes (Defachelles, 2015).

The Drosophila RZZ complex - roles in membrane trafficking and cytokinesis

The Zw10 protein, in the context of the conserved Rod-Zwilch-Zw10 (RZZ) complex, is a kinetochore component required for proper activity of the spindle assembly checkpoint in both Drosophila and mammals. In mammalian and yeast cells, the Zw10 homologues, together with the conserved RINT1/Tip20p and NAG/Sec39p proteins, form a second complex involved in vesicle transport between Golgi and ER. However, it is currently unknown whether Zw10 and the NAG family member Rod are also involved in Drosophila membrane trafficking. This study shows that Zw10 is enriched at both the Golgi stacks and the ER of Drosophila spermatocytes. Rod is concentrated at the Golgi but not at the ER, whereas Zwilch does not accumulate in any membrane compartment. Mutations in zw10 and RNAi against the Drosophila homologue of RINT1 (rint1) cause strong defects in Golgi morphology and reduce the number of Golgi stacks. Mutations in rod also affect Golgi morphology, whereas zwilch mutants do not exhibit gross Golgi defects. Loss of either Zw10 or Rint1 results in frequent failures of spermatocyte cytokinesis, whereas Rod or Zwilch are not required for this process. Spermatocytes lacking zw10 or rint1 function assemble regular central spindles and acto-myosin rings, but furrow ingression halts prematurely due to defective plasma membrane addition. Collectively, these results suggest that Zw10 and Rint1 cooperate in the ER-Golgi trafficking and in plasma membrane formation during spermatocyte cytokinesis. These findings further suggest that Rod plays a Golgi-related function that is not required for spermatocyte cytokinesis (Wainman, 2012).

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).

In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).

To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav⁺ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).

These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype. cav homozygotes and cav¹/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).

The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).

It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).

SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).

Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).

To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).

Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).

It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).

Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).

Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).

It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).

Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).

Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores. Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, a new protein (Spindly) has been identified that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. A human homologue of Spindly serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. These findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores (Griffis, 2007).

The spindle assembly checkpoint (SAC) is critical for preventing the onset of anaphase until all chromosomes are aligned on the metaphase plate. A single misaligned kinetochore is sufficient to generate a wait anaphase signal, thereby ensuring that all sister chromatids segregate to opposite ends of the spindle and are equally distributed to the daughter cells. Failure of the SAC can lead to premature anaphase onset and aneuploidy. Such defects can have consequences for a whole organism; mice that lack a full complement of SAC genes have more frequent DNA segregation errors and are more susceptible to tumor development (Griffis, 2007).

The presence of the SAC was initially inferred from observations that cells delay in metaphase when meiotic sex chromosomes fail to pair and align or after the spindle is perturbed by either microtubule poisons or microsurgery. Molecules responsible for the SAC were later identified in yeast genetic screens and named Mad1, -2, and -3 (Mad for mitotic arrest deficient) and Bub1, -2, and -3 (Bub for budding unperturbed by benzimidazole). Subsequent work showed that these proteins together with the MPS1 kinase form distinct complexes that target to the kinetochore. Two additional metazoan checkpoint proteins, Zw10 and Rough Deal (Rod), were later isolated as cell cycle mutants in Drosophila melanogaster. These two proteins, together with a third protein called Zwilch, form a complex (Rod-Zw10-Zwilch complex [RZZ]) that regulates the levels of Mad1 and Mad2 on the kinetochore (Griffis, 2007).

Ultimately, the SAC pathway must lead to inhibition of the anaphase-promoting complex (APC), a multisubunit ubiquitin E3 ligase that targets multiple mitotic regulators (e.g., mitotic cyclins as well as the securin protein that inhibits the cleavage of cohesin molecules) for proteosome degradation to allow mitotic exit. Several studies have shown that localization of the checkpoint proteins to misaligned kinetochores is essential for establishing the SAC and keeping the APC inhibited, most likely by generating a diffusible signal that inhibits the APC. The nature of the diffusible signal is still subject to debate. However, a current model suggests that the kinetochore-bound Mad1-Mad2 complex acts as a template that coverts the free, inactive Mad2 to an active form that can diffuse away from the kinetochore and bind to and sequester Cdc20, a regulatory component of the APC (Griffis, 2007).

The capture of microtubules by the kinetochore and the downstream activity of two different microtubule motors are required for silencing the SAC in metazoans. One of these motors is the kinesin centromere protein (CENP) E, which may act as a tension sensor that, when stretched, inactivates the BubR1-dependent inhibition of Cdc20. The second motor is dynein, which transports Mad1, Mad2, and RZZ from the kinetochore to the spindle pole. Dynein-based removal of Mad1 and Mad2 from the kinetochore may disrupt the template mechanism that generates the active Mad2 that inhibits the APC (for review see Musacchio, 2007). After inhibition or depletion of dynein or its cofactors, metazoan cells arrest in metaphase with correctly aligned chromosomes and high levels of kinetochore-bound Mad1, Mad2, and RZZ (Griffis, 2007).

Resolving the mechanism of dynein recruitment to kinetochores is important for understanding how kinetochore-microtubule binding ultimately leads to inactivation of the SAC. Currently, it is thought that dynein is brought to the kinetochore by binding directly to dynactin (a multisubunit complex required for multiple dynein functions), which, in turn, binds to the Zw10 subunit of the RZZ complex. Lis1, another dynein cofactor, also has been proposed to play a role in targeting dynein to kinetochores. Dynactin, Lis1, and Zw10 are not kinetochore-specific factors, as they are involved in targeting dynein to multiple other locations in the cell. It has not been clearly established whether dynactin and Lis1 are sufficient for targeting dynein to kinetochores or whether other proteins might be involved (Griffis, 2007).

To find new proteins that might participate in the SAC, an automated 7,200 gene mitotic index RNAi screen was undertaken in S2 cells. This screen uncovered a novel gene, which was also identified in an independent screen of genes involved in S2 cell spreading and morphology. This protein (termed Spindly) localizes to microtubule plus ends in interphase and to kinetochores during mitosis. Cells depleted of Spindly arrest in metaphase with high levels of Mad2 and Rod on aligned kinetochores, a defect caused by a failure to recruit dynein to the kinetochore. However, Spindly is not required for other dynein functions during interphase and mitosis. A human homologue of Spindly, which is similarly involved in recruiting dynein to kinetochores, was identifed. Thus, these results have uncovered a novel conserved dynein regulator that is involved specifically in dynein's function in silencing the SAC (Griffis, 2007).

An RNAi screen has identified Spindly as an essential factor for docking dynein to the kinetochore. Spindly is recruited to the kinetochore in an RZZ-dependent manner, and there, together with dynactin, Spindly recruits dynein to the outermost region of the kinetochore. The dynein motor complex then transports Spindly along with Mad2 and the RZZ complex to the spindle poles to inactivate the SAC. A Spindly homologue plays a similar role in human cells, revealing a conserved dynein kinetochore targeting mechanism in invertebrates and vertebrates. These data provide new insight into the mechanism and importance of recruiting dynein to the kinetochore to inactivate the SAC. Spindly also plays a role in maintaining S2 cell morphology during interphase and localizes to the growing ends of microtubules (Griffis, 2007).

The depletion of Spindly creates several mitotic defects that appear to reflect a loss of dynein activity exclusively at the kinetochore. Metaphase arrest is the most evident defect observed after the RNAi-mediated depletion of Spindly in Drosophila or human cells. This metaphase arrest phenotype is most likely explained by the absence of kinetochore-bound dynein in Spindly-depleted cells, and, indeed, the data support a model proposes that kinetochore-bound dynein is required for transporting Mad2 from the kinetochore to inactivate the SAC. Nevertheless, the possibility that the mitotic delay seen after dynein or Spindly depletion is caused by another kinetochore aberration that keeps the checkpoint activated. However, Spindly-depleted cells ultimately overcome metaphase arrest, as seen in live cell imaging experiments and by the modest increases in the mitotic indices of Spindly-depleted S2 and HeLa cells (three- to seven-fold and two-fold, respectively). The mechanism of slippage from this metaphase arrest is not clear, but it might involve proteins (e.g., p31 comet) that silence the SAC by disrupting the interaction between Mad2 and Cdc20 (Griffis, 2007).

In addition to mitotic arrest, chromosomes in Spindly- and dynein-depleted S2 cells require a longer time to align on the metaphase plate. This result may be attributable either to the displacement of CLIP-190 (a microtubule tip-binding protein) from kinetochores after Spindly or dynein depletion or the loss of dynein-mediated lateral attachments to microtubules in early prometaphase. In HeLa cells, a defect in chromosome alignment was noticed after Hs Spindly depletion, which also has been observed after the depletion of dynein (perhaps mediated through a loss of kinetochore-bound CLIP-170) (Griffis, 2007).

Thus, the spectrum of mitotic defects observed in Spindly-depleted cells is consistent with a loss of dynein function specifically at the kinetochore. Spindly depletion does not produce any other defects seen after dynein depletion, such as centrosome detachment and spindle defocusing. Dynactin is another protein that is required for recruiting dynein to kinetochores, but it is important for other mitotic and interphase dynein functions. Depletion of the RZZ complex inhibits the kinetochore recruitment of dynein, but this also prevents Mad1 and Mad2 recruitment and reduces kinetochore tension to a greater degree than Spindly or dynein depletion alone. Thus, Spindly depletion appears to be the most specific means identified to date for interfering with dynein function only at the kinetochore (Griffis, 2007).

These findings provide new insight into how dynein localizes to kinetochores. Previous studies have led to a model in which dynactin binds to the RZZ complex and then, either alone or in collaboration with Lis1, recruits dynein to the kinetochore. Because it was found that both dynactin and Spindly are required for dynein localization to kinetochores, an updated model is proposed in which Spindly and dynactin target to the kinetochore independently and work together to recruit dynein (Griffis, 2007).

Thus, dynein recruitment to the kinetochore may involve multiple weak interactions. Consistent with the possibility of weak interactions, endogenous dynein, dynactin, and Rod did not coprecipitate with GFP in pull-down experiments, and Spindly did not coenrich with these proteins in sucrose gradient fractions. Lis1 is not included in the dynein localization model, since it was found that Lis1 RNAi does not block dynein recruitment to the kinetochore (using a colchicine treatment localization assay), although Lis1 depletion does cause a mitotic delay and substantial increase in GFP-Spindly on aligned kinetochores. Thus, a role is favored for Lis1 in dynein activity but not in recruiting dynein to the kinetochore (Griffis, 2007).

Spindly's role in the spreading morphology of S2 cells makes it unusual among proteins involved in silencing the SAC (including dynein and dynactin), which did not produce phenotypes in the interphase morphology screen. The Spindly RNAi interphase phenotype of defective actin morphology and the formation of extensive microtubule projections is still not understood. However, a clue may be Spindly's dynamic localization to the growing microtubule plus end. Other plus end-binding proteins (+TIPs) interact with signaling molecules that regulate cell shape, one example being the binding and recruitment of RhoGEF2 to the microtubule plus end by EB1. Spindly may similarly interact with and carry an actin regulatory molecule to the cortex, but this hypothesis will require identifying proteins that interact with Spindly during interphase (Griffis, 2007).

The mechanism of Spindly recruitment to the microtubule plus end also warrants further investigation. This interaction must be regulated by the cell cycle because GFP-Spindly no longer tracks along microtubule tips in prometaphase. Seven consensus CDK1 phosphorylation sites are present in the positively charged C-terminal repeats of Spindly, and phosphorylation of these sites could reverse the charge of these repeats and regulate the transition from microtubule tip binding to kinetochore binding at the onset of mitosis (Griffis, 2007).

Motor proteins must be guided to the correct subcellular site to execute their biological function. To carry out the multitude of transport activities required in eukaryotic cells, metazoans have evolved numerous kinesin motors (25 genes in Drosophila) with distinct domains that dictate their localization and regulation. In contrast, a single cytoplasmic DHC performs numerous roles in interphase and mitosis, suggesting that additional regulatory factors guide dynein to specific cargoes (e.g., organelles, mRNAs, and vesicles). The main dynein-associated proteins (the dynactin complex, Lis1, and NudEL) are involved in dynein function at many sites and, thus, do not appear to be cargo specific. Zw10 was initially thought to specifically regulate the recruitment of dynein-dynactin to the kinetochore, but it now also appears to play an essential role in targeting dynein to membrane-bound organelles. Bicaudal D is another multifunctional adaptor molecule that has a role in the dynein-based transport of multiple cargoes such as RNA, vesicles, and nuclei. Perhaps the most site-specific dynein recruitment factor is the Saccharomyces cerevisiae Num1 protein that binds to the DIC Pac11p to target the motor to the cortex of daughter cells, where it pulls the nucleus into the bud neck. However, dynein only serves this one function in yeast compared with its plethora of activities in metazoans, and Num1p homologues have yet to be identified in higher eukaryotes (Griffis, 2007 and references therein).

Spindly appears to be a highly selective dynein-recruiting factor, and, unlike other dynein cofactors, it does not appear to be involved in the motor's nonkinetochore functions in mitosis (e.g., pole focusing) or in interphase (e.g., endosome transport). However, the mechanism by which Spindly recruits dynein to the kinetochore remains to be elucidated. Observations that Spindly moves from kinetochores to the spindle poles as discrete punctae strongly suggests that it may incorporate into a large and somewhat stable particle that contains the RZZ complex, Mad1-Mad2, dynein, and likely additional proteins. Therefore, Spindly not only serves to recruit dynein to the kinetochore but also is part of a cargo that dynein transports. Future studies will be needed to better understand the protein composition of these transport particles and the contacts that Spindly makes within them (Griffis, 2007).

Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis

Multiple approaches were used to investigate (1) the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and (2) conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. It is concluded that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG (Sun, 2007).

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/Dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. This study generated and/or characterized mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, an improved time-lapse video microscopy technique was developed that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, GFP-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: initially promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both C. elegans and mammals. It is concluded that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release (Siller, 2005).

This study shows that both Lis1 and Gl are enriched on centrosomes/spindle poles in wild type neuroblasts, and Lis1/Gl are required for centrosome separation in prophase neuroblasts. A role for centrosome separation has been reported for dynein in Drosophila embryos, dynein in mammalian cells, and dynein/dynactin/Lis1 in C. elegans blastomeres. However, the exact mechanism by which they promote centrosome separation is unclear. One proposed model suggests that dynein may promote centrosome separation by generating pulling forces on astral MTs attached to the cortex or cytoplasmic structures. Alternatively, dynein associated with the nuclear envelope may exert pulling forces on astral MTs to promote centrosome separation. No GFP-Lis1 was detected on the nuclear envelope or at the neuroblast cortex, although it is possible that high cytoplasmic levels mask low levels of Lis1/dynactin at these sites. Thus, it remains unclear how Lis1/Gl promotes centrosome separation in neuroblasts. Centrosome separation is not completely blocked in Lis1 or Gl mutant neuroblasts, either due to residual amounts of maternal protein or due to the presence of a Lis1/dynactin/dynein-independent pathway. Interestingly, cortical non-muscle myosin II has been shown to contribute to centrosome separation in some cell types, raising the possibility that Lis1/dynactin/dynein and myosin II play partially redundant roles in neuroblast centrosome separation (Siller, 2005).

These observations further support a role for centrosomal/spindle pole-associated Lis1/Gl in spindle assembly, spindle pole focusing, and centrosome attachment in prometaphase and metaphase neuroblasts. Detachment of centrosomes from the spindle has been observed in dynein mutants in Drosophila, and in mammalian cells with reduced dynein or dynactin function. These findings show that Lis1 and dynactin act as cofactors for dynein-dependent focusing of spindle poles and attachment of spindle MTs minusends to centrosomes. In vertebrate cells dynein/dynactin is thought to contribute to focusing of spindle poles and attaching MT-minus ends to centrosomes by transporting pericentriolar proteins and MT-binding proteins, such as NuMA, to centrosomes. Although no clear NuMA orthologue is encoded in the Drosophila genome, a dynein/dynactin/Lis1 complex may contribute to spindle pole focusing by concentrating other MT cross-linking proteins with NuMA-like function at spindle MT minus ends (Siller, 2005).

Gl and Lis1 mutant neuroblasts occasionally form multipolar spindles and have more than two centrosome-like Centrosomin/gamma-tubulin structures. Due to the lack of Drosophila centriolar markers it was not possible to determine whether these extra centrosomelike structures contained centrioles. Multipolar spindles have also been observed in mammalian cells overexpressing Lis1 protein or in which Lis1 function was reduced. Time-lapse analysis of Lis1 mutant neuroblasts reveal occasional co-segregation of both centrosomes into the neuroblast as a consequence of incomplete centrosome separation and centrosome detachment from the spindle. Such a mis-segregation event may be followed by duplication of both centrosomes during the next cell cycle leading to supernumerary centrosomes. Alternatively, extra centrosomes in Lis1 and Gl mutant neuroblasts may be due to uncoupling of centrosome duplication from the cell cycle or centrosome fragmentation (Siller, 2005).

Time-lapse imaging experiments show that loss of Lis1/Gl in neuroblasts results in extension of both prometaphase and metaphase. Prometaphase in Lis1 mutant neuroblasts is characterized by delayed congression of chromosomes to the equatorial plate: this is likely to be largely due to inefficient kinetochore capturing as an indirect result of spindle assembly defects. Importantly, in Lis1 mutant neuroblasts congression of all chromosomes into a tight metaphase plate eventually occurs, suggesting that Lis1/Gl are not absolutely critical for MT/kinetochore attachment per se (Siller, 2005).

In addition, severe delays were observed in metaphase-to-anaphase transition. A few of these neuroblasts showed individual chromosomes that were transiently lost from and recongressed to the metaphase plate. Thus, consistent with findings in mammalian cells, Lis1 appears to play some role in maintaining stable chromosome alignment in metaphase neuroblasts. However, in contrast to mammalian studies, it was found that loss of Lis1 function causes delays in metaphase-to-anaphase transition even when all chromosomes stay aligned in a tight metaphase plate. Thus, mitotic checkpoint activity remains high even after apparent bipolar kinetochore attachment. Two defects appear to contribute to prolonged checkpoint activity in Lis1 mutant metaphase neuroblasts: reduced inter-kinetochore tension and failure to transport checkpoint proteins (e.g., Rod) off kinetochores. Reduced inter-kinetochore tension may be due to lack of Lis1/dynactin on kinetochores or on spindle pole/MTs (which may affect forces acting on kinetochore pairs as a consequence of altered spindle morphology or MT dynamics). Defects in Rod checkpoint protein transport off kinetochores can be explained as a direct consequence of depletion of kinetochore-associated Lis1/dynactin/dynein motor complex, which in wild type cells is loaded with Rod at kinetochores. However, previous studies have indicated that Rod and Zw10 are removed from kinetochores in response to inter-kinetochore tension not MT-attachment. Therefore, in addition to its direct role as a 'carrier', Lis1/dynactin/dynein may also play an indirect role in modulating Rod transport by generating the inter-kinetochore tension required to trigger initiation of Rod streaming (Siller, 2005).

In summary, the data is consistent with and extends a model recently proposed for dynein function in checkpoint protein transport in Drosophila and mammalian cells. According to this model a Lis1/dynactin/dyneinRod/Zw10 complex, pre-assembled on unattached kinetochores, is critical for timely anaphase onset by promoting poleward streaming of checkpoint proteins away from kinetochores after correct kinetochore-MT attachment has occurred. The data demonstrate that in Drosophila, the Lis1 protein is an obligate component in this process. Although the Lis1-binding proteins NudE/Nudel have been implicated in facilitating dynein-dependent checkpoint protein transport, it remains to be directly tested whether Lis1 has a similar function in mammalian cells (Siller, 2005).

What is the link between Rod/Zw10 and Mad2 in mitotic checkpoint function? Two recent studies demonstrate that the Rod/Zw10 complex is required for efficient recruitment of Mad2 to unattached kinetochores in mammalian cells and Drosophila neuroblasts, and that Mad2 and Rod colocalize during poleward transport along kMTs in Drosophila neuroblasts. Although a physical link between the Rod/Zw10 complex and Mad2 has not been discovered, an attractive model is that Rod/Zw10 links Mad2 to the Lis1/dynactin/dynein complex during poleward checkpoint protein transport (Siller, 2005).

Epistasis of Lis1/dynactin localization at kinetochores Lis1/dynactin localization is regulated differently in worm and mammalian cells. In mammalian cells, dynactin is required for Lis1 kinetochore association, but Lis1 is not required for dynactin localization. Whereas in C. elegans, Lis1 localizes to kinetochores independently of dynactin. Surprisingly, a third mechanism is found in Drosophila neuroblasts, where Lis1 and dynactin (Gl) are co-dependent for their localization to kinetochores. In neuroblasts, Lis1 may have a 'structural' role in recruiting dynein/dynactin to the kinetochore, in addition to stimulating dynein/dynactin activity. Thus, despite the conservation of the physical interaction between Lis1/dynein/dynactin, subcellular localization of these proteins can be regulated differently in various organisms (Siller, 2005).

Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. This study simultaneously followed GFP-Mad2 and RFP-Rod and found they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores (Buffin, 2005).

To gain insight into the role of Rod/Zw10 relative to other checkpoint proteins, a study of fluorescently tagged (GFP and mRFP1 Rod (CG1569), Mad2 (CG17498), and BubR1 (CG7838) in a single cell type, the Drosophila larval neuroblast. All three fusion proteins are controlled by their natural promotors, and all three retain their biological activity (Buffin, 2005).

Consistent with earlier reports, Rod and BubR1 are cytoplasmic in interphase, whereas Mad2 is associated with the nucleoplasm and nuclear envelope. In fly neuroblasts, as in Hela cells but unlike in the marsupial cell line PtK, BubR1 is the first to accumulate on kinetochores during prophase. Mad2 and Rod begin to label kinetochores only during nuclear-envelope breakdown (NEB), easily recognized by the invasion of Rod into the nucleoplasm. The first kinetochore-associated Mad2 signals above the nucleoplasmic background are seen simultaneously with the first Rod signal (Buffin, 2005).

In prometaphase, the kinetochores brightly label with all three proteins. Because cytoplasmic Mad2 signal is consistently higher than either BubR1 or Rod, Mad2 kinetochore labeling appears relatively less prominent. As the kinetochores capture MTs, Mad2 and Rod both are transported poleward. This process, called 'shedding', requires dynein/dynactin and may be important for shutting off the checkpoint once MTs are properly attached (Buffin, 2005).

These live images reveal a robustness that was not evident for Mad2 transport in earlier studies in PtK cells and Drosophila cells , although it can be seen sometimes even by immunostaining. It is difficult to quantify these signals, but the films clearly show that new cytosolic Mad2 is continuously recruited to kinetochores even after MT capture and replaces that lost to shedding; the total Mad2 signal on kinetochore microtubules (KMTs) over the duration of prometaphase and metaphase is far greater than the original kinetochore-associated signal. This is particularly evident where metaphase is prolonged. Thus Mad2, like Rod, establishes a flux of recruitment to and shedding from attached kinetochores (Buffin, 2005).

GFP-Rod and RFP-Mad2 show a near-perfect coincidence of signal in prometaphase and early metaphase, not only on kinetochores but also along the KMTs. The overall patterns of the two proteins are superimposable. Where discrete particles of GFP-Mad2 could be followed, they always contained RFP-Rod. These results suggest that Mad2 and Rod/Zw10 remain associated as they leave the kinetochore along the KMTs (Buffin, 2005).

By late metaphase, Mad2 signal has essentially disappeared from kinetochores and is only faintly visible on the spindle above the cytoplasmic Mad2 background, whereas Rod shedding continues robustly up to anaphase onset. In larval neuroblasts, the timing of NEB to anaphase onset is typically 7-12 min, of which metaphase lasts 2-8 min. There does not appear to be much delay between Mad2 disappearance from the spindle and anaphase onset. On average, Mad2 is gone less than 1 min prior to anaphase, and sometimes just seconds before. This contrasts with the situation in PtK cells, where anaphase occurs on average 10 min after the disappearance of the last detectable Mad2 signal. The significance of this difference is for now unclear. It may reflect simply an adaptation to the very rapid mitosis in flies (7-12 min NEB-anaphase, compared to 25 min after alignment of the last chromosome for Ptk cells). Alternatively, it may reflect a more fundamental difference in the way the spindle checkpoint is turned off (Buffin, 2005).

The behavior of Mad2 and Rod was distinguishable from that of BubR1 in several ways. BubR1 remained tightly associated with kinetochores and was not detectable along the spindle after MT capture. Although in PtK cells BubR1 may be transported from kinetochores to poles after energy depletion, in normal fly neuroblasts shedding does not appear to be a major route by which BubR1 levels are reduced on attached kinetochores. Moreover, close inspection of in vivo double-labeled cells revealed that, as the metaphase plate develops, BubR1 becomes enriched in a kinetochore domain slightly internal to that of Rod and Mad2 (Buffin, 2005).

Rod/Zw10, dynein/dynactin, Mad2 and BubR1, and all the transient kinetochore proteins are normally classified as outer-domain kinetochore components, and indeed they all form enlarged crescents around the MT-free kinetochores. The outer domain can be further subdivided into a more interior 'outer plate' which appears to be the MT attachment site as well as the location of BubR1, and an outer fibrous corona that is believed to contain Rod/Zw10, dynein/dynactin, and CenpE. The relative locations of the various checkpoint proteins have not been compared in attached kinetochores of living cells. The observation that Mad2 colocalizes with Rod but not with BubR1 is the first demonstration that Mad2 is part of the corona (Buffin, 2005).

The different locations of Mad2 and BubR1 are consistent with certain distinct features of their behavior. For example, Mad2 accumulation is highly sensitive to MT attachment and is depleted from kinetochores by shedding along KMTs. BubR1 by contrast is not depleted significantly by shedding and responds more to changes in tension. If this correlation holds, perhaps other proteins with robust shedding (for example, CenpF will prove to colocalize in the corona with Mad2, Rod/Zw10, and dynein (Buffin, 2005).

In summary, Mad2 and Rod/Zw10 behavior on kinetochores and spindles are qualitatively closely linked. They are simultaneously recruited and are shed together during prometaphase and early metaphase. BubR1, by contrast, is independently recruited to a different kinetochore domain and does not undergo detectable shedding (Buffin, 2005).

To further probe the relationship of Mad2 and Rod/Zw10, the behavior of GFP-Mad2 was examined in rod and zw10 null-mutant cells. Given the importance of dynein-dynactin for shedding and the role of Rod/Zw10 in dynein recruitment, it was anticipated that rod or zw10 mutants would show abnormal retention of Mad2 on kinetochores. In fact, however, in these cells kinetochore-associated GFP-Mad2 was significantly reduced, although Mad2 was still prominent on interphase rod nuclei. The reduction of kinetochore-associated Mad2 was evident in every rod or zw10 mutant cell examined, although the extent of reduction was somewhat variable. In three of 15 rod cells (20%) filmed from NEB to anaphase onset, no kinetochore-associated Mad2 was detectable above the cytoplasmic background at any stage. In the rest, a weak signal was briefly detectable on some kinetochores during prometaphase. Quantitation of these signals revealed that the kinetochore intensity in rod cells was only about 20% above the cytoplasmic level, at their maximum, whereas in wild-type cells kinetochore Mad2 signals averaged 4.4-fold higher than cytoplasmic signals. Depolymerizing microtubules with colchicine, which normally elevates kinetochore levels of checkpoint proteins, including Mad2, did not increase Mad2 kinetochore signals in rod cells. These observations indicated that Mad2 requires the Rod/Zw10 complex to achieve its normal levels on kinetochores. An earlier report did not find that inactivating Rod by antibody injection of Hela cells had any effect on Mad2 recruitment, although the antibody did block Rod recruitment at the kinetochore and did lead to premature mitotic exit. The discrepancy with the current results may be due to the different methodologies employed (Buffin, 2005).

Several other checkpoint proteins were examined in rod and zw10 mutants. BubR1 and Bub3 were still present. Mps1 and Bub1 were also unaffected by rod mutants. Thus, the requirement for Rod/Zw10 seems to be specific to Mad2. By contrast, treatments that remove Mad2 from kinetochores in vertebrate cells have no effect on Rod/Zw10 (Buffin, 2005).

It was possible that the failure of rod and zw10 mutant cells to recruit Mad2 was caused by the premature degradation of cyclin B in these checkpoint-defective cells; perhaps Mad2 cannot bind kinetochores when cyclinB/cdc2 kinase activity is low. To test this possibility, Mad2 behavior was examined in cells doubly mutant for rod and imaginal discs arrested (ida), the gene encoding APC5, a component of the APC/C. ida cells arrest in M phase with consistently elevated cyclin B. The ida phenotype is epistatic to rod: i.e., ida rod double mutants do not exit mitosis, and they retain elevated cyclin B (Buffin, 2005).

In ida cells, chromosomes are frequently found unattached to spindles, and Mad2 accumulation on kinetochores is therefore prominent even without colchicine. Significantly, in ida rod or ida zw10 double mutants, Mad2 signal on kinetochores was greatly reduced, just as in rod or zw10 mutants alone. This result argues that the Rod/Zw10 complex is physically required, directly or indirectly, for normal Mad2 accumulation on kinetochores (Buffin, 2005).

This study has shown that many aspects of Mad2 behavior are intimately associated with the Rod/Zw10 complex. Rod/Zw10 accompanies Mad2 as it accumulates on unattached kinetochores and as it leaves kinetochores after MT attachment, and in the absence of Rod/Zw10, little or no Mad2 accumulates on kinetochores. Given that Rod/Zw10 is also required for dynein/dynactin recruitment, which removes Mad2 from attached kinetochores, one can say that the entire kinetochore cycle of Mad2 depends, directly or indirectly, on Rod/Zw10. The checkpoint defect of rod and zw10 mutants is now presumably explained by this failure to recruit Mad2. These results suggest that Rod/Zw10 is physically interacting with a complex containing Mad2 (or Mad1, see below) throughout mitosis. However, two-hybrid screening, immunoaffinity columns, and coimmunoprecipitation experiments have not revealed any interaction between Rod/Zw10 and Mad1 or Mad2. Thus, unlike dynein/dynactin, Mad1/Mad2 may be binding only indirectly to Rod/Zw10, perhaps via an unknown protein. Alternatively, there may be direct interactions between Rod/Zw10 and Mad1/Mad2, but only under native conditions on intact kinetochores (Buffin, 2005).

Kinetochore recruitment of Mad2 initially occurs as part of a complex with Mad1, to which it is tightly bound even in interphase. The Mad1/Mad2 complex is relatively stable at unattached kinetochores, but a second Mad2 population, which depends on the first, turns over rapidly and presumably becomes an activated form, the 'wait anaphase' signal. Once MTs have attached, however, the Mad1/Mad2 complex is rapidly depleted, at least partially by dynein-mediated shedding along KMTs, and this is believed to be part of the mechanism that extinguishes the checkpoint signal. It is therefore likely that the Rod/Zw10 complex is exerting its effect on the Mad1/Mad2 complex and not on Mad2 alone. Recent work in Hela cells supports this contention by showing that depletion of Zw10 by RNAi reduces both Mad1 and Mad2 recruitment to unattached kinetochores (Buffin, 2005).

It is unclear what kinetochore components constitute the Mad1/Mad2 'binding site'. The hierarchy of kinetochore assembly has been studied in several model systems, not always with consistent results. However, it appears that the Ndc80 complex, Bub1, and Mps1 kinase activity are required for the subsequent assembly of Mad1/Mad2 on kinetochores. Conversely, interfering with Mps1 or the Ndc80 complex in Hela cells has no effect on Rod or dynein recruitment, and rod and zw10 mutants have no effect on BubR1, Bub3, CenpMeta (the fly homolog of CenpE), Bub1, or Mps1 (this study), nor in all likelihood on the Ndc80 complex (in rod mutants, chromosomes are efficiently captured by MTs and congress). Thus Rod/Zw10, with Ndc80 complex, Bub1, and Mps1, all contribute to Mad2 kinetochore recruitment. The role of Rod/Zw10 may be to enhance the affinity of Mad1/Mad2 for its binding site (because some Mad2 binds even in rod mutants), increasing its stability on kinetochores prior to MT capture, perhaps by interacting with Ndc80 complex (Buffin, 2005).

The current results also demonstrate that, just like Rod/Zw10, Mad1/Mad2 is continuously recruited to and then released from kinetochores, even following MT capture, and only disappears from spindles just prior to anaphase onset. This differs significantly from the behavior reported in vertebrate cells, in which MT capture appears to shut off new Mad2 recruitment. The difference need not conflict with the basic model in which kinetochore Mad2 generates the anaphase inhibitor. In both cases, there is a rapid decline, perhaps below a critical threshold, in the net steady-state abundance of Mad2 on attached kinetochores. Alternatively, MT capture may render the remaining kinetochore-associated Mad2 inactive. Perhaps the difference is in the rate of Mad2 recruitment in the two cell types. Even in PtK cells there is some evidence that Mad2 is capable of recruitment to attached kinetochores: If dynein activity (and therefore shedding) is blocked after chromosome alignment, Mad2 eventually reaccumulates at attached kinetochores, suggesting that prior to dynein inhibition, Mad2 was being recruited and immediately shed from these kinetochores. This continuous recruitment of Mad1/Mad2 to attached kinetochores may ensure that it will always be available to begin generating anaphase inhibitor should one or more MTs inadvertently detach. At the same time, the continued presence of Rod/Zw10 ensures the dynein levels required both to remove unneeded Mad1/Mad2 and, later, to power anaphase movement (Buffin, 2005).

Rough Deal and Zw10 are required for the metaphase checkpoint in Drosophila

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. This study shows that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast (Basto, 2000).

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or have failed to complete steps during mitosis, including those involving kinetochore function. Bub1 is an evolutionarily conserved mitotic checkpoint control protein that is present in organisms as diverse as yeast and humans. Bub1 inhibits ubiquitin ligase activity of anaphase promoting complex (APC) preventing mitosis until all chromosomes are correctly attached to the mitotic spindle. Drosophila Bub1 (see below: The gene described in this section of the Interactive Fly is more properly termed BudR1) localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants (Basu, 1999).

The relationship between Bub1 and other kinetochore components was investigated. Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes (detected by anti 3F3/2 antibody) at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies (Basu, 1999).

Both human Bub1 and Drosophila Bud1 associate strongly with the kinetochores of chromosomes unattached to the spindle prior to anaphase onset of normal mitosis, and with all the kinetochores in cells treated with microtubule depolymerizing drugs. Reduced amounts of both proteins are also found at the kinetochores of chromosomes either at the metaphase plate or being pulled toward the poles at anaphase. Near null mutations in the gene encoding this Drosophila protein cause phenotypes indicating an abrogation of the spindle checkpoint. These same mutations abolish the ability of another checkpoint component, Drosophila Bub3, to localize to the kinetochores (Basu, 1998). This latter finding fits well with a wealth of data substantiating an intimate relationship between Bub1 and Bub3. Taken together, these observations in Drosophila provide strong evidence for the conservation of Bub1 function throughout evolution (Basu, 1999).

In S. cerevisiae, bub and mad genes are nonessential in the absence of microtubule depolymerizing agents, though the growth of mutant cells is slowed (Roberts, 1994). In S. pombe, bub1 null mutants are viable, though some abnormalities in chromosome segregation are observable during mitosis (Bernard, 1998). In marked contrast, loss of bub1 function in Drosophila leads to lethality at the larval/pupal transition. Lethality at this stage has been observed for many mutations affecting essential cell cycle components, presumably because maternally supplied stores of protein obtained from a nonmutant mother are exhausted by this point in development. Examination of brain neuroblasts dissected from dying third instar bub1 homozygous mutant larvae has thus facilitated an analysis of how loss of checkpoint function affects cell division in a multicellular organism. In the description below, the possibility cannot be excluded that some aspects of the phenotype reported are indirect consequences of problems encountered in earlier cell divisions. However, it should be noted that all embryonic divisions appear to be normal, and essentially all bub1 mutant animals hatch into larvae that survive until the third instar. Since there is very little cell division in the larval brain before the third instar, the number of cell divisions that could take place between the exhaustion of maternal stores of Bub1 protein and the time of analysis is limited. Moreover, these phenotypes are quite specific to bub1 mutants, and have not been observed in analysis of many other mitotic mutants in Drosophila (Basu, 1999).

Treatment of bub1 mutant neuroblasts with colchicine causes precocious sister chromatid separation, instead of the prometaphase arrest with attached sister chromatids typical of wild-type neuroblasts. This phenotype is a predictable property of mutations affecting the operation of the spindle checkpoint, since it indicates that bub1 mutant neuroblasts attempt to enter anaphase despite the absence of a functional spindle (Basu, 1999).

More interesting are the effects of bub1 mutations on normal cell division in neuroblasts that have not been treated with microtubule depolymerizing drugs. Observations suggest that bub1 mutant neuroblasts enter anaphase prematurely even in these untreated cells. In particular, the ratio of metaphase figures to anaphase figures is decreased 5-10-fold in bub1 brains relative to wild-type brains. This result is consistent with studies showing that microinjection of Mad2 antibodies into mammalian cells causes premature sister chromatid separation and entry into anaphase. Interestingly, loss of Bub1 in Drosophila generates a sharp decrease in mitotic index. This finding could be explained by an accelerated transit through mitosis as has been suggested for mammalian cell cultures expressing dominant negative forms of mouse Bub1 (Taylor, 1997). However, it is also possible that the lowered mitotic index reflects the assumption of an apoptotic fate by many neuroblasts in the brain (Basu, 1999).

A high proportion of anaphases in untreated bub1 mutant brains show a variety of aberrations, including extensive chromatin bridging, lagging chromosomes (most likely leading to aneuploidy), and chromosome fragmentation. These aberrations are interpreted as further evidence for the precocious entry into anaphase. In this view, the proper synchronization of different aspects of sister chromatid separation at the metaphase/anaphase transition does not occurred. It is well known that the forces holding sister chromatids together along their arms are separable from the forces joining sister chromatids at their centromeres. For example, acentric chromosome fragments in irradiated grasshopper neuroblasts remain associated until the onset of anaphase. In addition, sister chromatid cohesion along the arms can also be disrupted independently of centromeric cohesion through treatment with hypotonic solutions. It is hypothesized that absence of bub1 function leads to loss of cohesive forces at the centromere before the separation of sister chromatids along their arms is completed. Thus, the chromatin bridging and fragmentation seen in bub1 mutant anaphases most likely reflect a failure to resolve concatenated sister chromatid DNAs along the arms at a time at which the centromeres have already separated and are being pulled toward the poles. In support of this interpretation, mutations in the Drosophila gene barren, which encodes a chromosome-associated protein that interacts with topoisomerase II, cause substantial chromatin bridging during anaphase of late embryonic divisions (Basu, 1999).

A striking feature of Drosophila bub1 mutants is the occurrence of significantly elevated frequencies of apoptotic nuclei in larval brains. This result was unexpected, since expression of a dominant negative form of mouse Bub1 has been reported to reduce the frequency of apoptotic nuclei in nocodazole-treated cells (Taylor, 1997), implying that loss of checkpoint function prevents the apoptotic response. The reasons for the apparent dichotomy between the results in Drosophila and those from mouse tissue culture cells are not clear. These effects could be organism or cell type-specific, or the differences could reflect unusual consequences of the dominant negative forms of Bub1 utilized in the mouse study (Basu, 1999).

A strong possibility for the high level of apoptotic cells seen in bub1 mutant brains emerges from findings that the chromosomes in many mutant anaphase figures are extensively fragmented. It has been well documented that chromosome breakage in Drosophila is normally a cell lethal event preventing entry into the next round of mitosis. The larval brains were examined of a number of new, relatively uncharacterized mitotic mutants that cause massive chromosome fragmentation, and these uniformly have high levels of apoptotic cells. Moreover, the induction of chromosome breakage with the FLP/FRT system is also associated with apoptosis. Apoptosis (or in fact any aspect of the bub1 mutant phenotype) cannot be an indirect consequence of aneuploidy, because brains from zw10 and rod mutants, which have many aneuploid cells, do not show the massive apoptotic response (or any of the cell cycle defects) generated by bub1 mutants. Regardless of the mechanism underlying the induction of apoptosis in bub1 mutant brains, it is clear that loss of spindle checkpoint function does not prevent a cell's entry into the apoptotic pathway (Basu, 1999).

In yeast, Bub1 acts as a kinase that can phosphorylate both itself and Bub3 (Roberts, 1994). Because Bub1's cell cycle distribution parallels that of 3F3/2 phosphoepitopes that appear to be intimately involved in the metaphase-anaphase transition, Bub1 has been suggested as a possible source of the kinase activity that generates these phosphoepitopes. This is not the case in Drosophila. F3/2 epitopes are strongly phosphorylated in a bub1 mutant, showing that Bub1 cannot be a significant source of 3F3/2 kinase activity in vivo. In addition, Bub3 fails to associate with the kinetochore in bub1 mutants, ruling out Bub3 as a major 3F3/2 phosphoepitope (Basu, 1999).

If Bub1 does not phosphorylate 3F3/2 phosphoepitopes, what kinase(s) can supply such an activity? A recent report indicates that ERK and MKK (extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase) localize to the kinetochore and can phosphorylate 3F3/2 phosphoepitopes. It is not clear whether this activity is direct or indirect; in any event, results indicate that Bub1 does not participate in the same 3F3/2 phosphorylation pathway (Basu, 1999 and references therein).

It was surprising to find that 3F3/2 epitopes at the kinetochores remain phosphorylated in anaphase figures from bub1 mutants. In contrast, 3F3/2 phosphoepitopes at the kinetochores are normally lost completely at the start of anaphase. The implications of this result are twofold. First, dephosphorylation of kinetochore-associated 3F3/2 epitopes is not required for the metaphase/anaphase transition, at least in a bub1 mutant background. One possibility is that 3F3/2 dephosphorylation is not, as commonly suggested, a part of the signaling pathway for anaphase onset, but is instead a downstream response to the signal. Alternatively, Bub1 may function downstream of 3F3/2 dephosphorylation in the pathway governing the metaphase–anaphase transition. A second implication of the observation is that Bub1 kinase activity is required, presumably indirectly, for the dephosphorylation of 3F3/2 epitopes at the metaphase-anaphase transition. A possible explanation for the continued phosphorylation of kinetochore-based 3F3/2 epitopes is that the accelerated transit through mitosis in bub1 mutants may not allow enough time for action of the relevant phosphatase(s) (Basu, 1999).

The localization of Bub1 to the kinetochore is not abolished by mutations in several genes encoding other kinetochore components, nor do mutations in bub1 affect the association of ZW10 or Polo proteins with the kinetochore. These findings suggest that the kinetochore may be assembled in at least two independent pathways. In one pathway, interaction between Bub1 and Bub3 is required for the kinetochore targeting of either protein (Roberts, 1994; Basu, 1998; Taylor, 1998). In a second subassembly, ZW10 and Rod proteins form a complex needed for the recruitment of the microtubule motor dynein to the kinetochore. The fact that polo mutations do not disrupt the kinetochore localization of Bub1, Bub3, or ZW10 suggests either a third independent pathway or that the kinetochore binding of Polo protein is subsequent to the recruitment of one of the two subassemblies (Basu, 1999 and references therein).

In colchicine-treated larval neuroblasts from zw10 mutants where the sister chromatids have separated prematurely, high levels of Bub1 protein remain at the kinetochores. This phenomenon is not restricted to a zw10 mutant background, since prolonged treatment of wild-type larval neuroblasts with hypotonic solution after colchicine incubation also generates precocious sister chromatid separation with continued strong Bub1 staining at the kinetochores. These observations indicate that it is possible to initiate anaphase despite the presence of the Bub1 'wait-anaphase' signal at the kinetochores. It is thus conceivable that the relative loss of Bub1 from kinetochores at metaphase and anaphase is not normally a prerequisite for anaphase onset (Basu, 1999).

Although the existence of a tension-dependent 'wait-anaphase' checkpoint in meiotic grasshopper spermatocytes has been well established, several observations suggest that such a checkpoint may not play a major role in Drosophila spermatogenesis. The presence of univalents (chromosomes without a pairing partner) does not obviously affect meiotic progression. Mutations in mei-S322 and ord, which lead to sister chromatid separation before the start of the second meiotic division, do not affect entry into anaphase II. Finally, colchicine-treated spermatocytes that cannot segregate their chromosome still exit meiosis and differentiate into spermatids (Basu, 1999).

Nevertheless, Drosophila Bub1 and Bub3 both associate strongly with the kinetochores of primary spermatocytes before metaphase of both meiotic divisions, and kinetochore staining cas be observed with antibodies against Xenopus Mad2 in prometaphase primary spermatocytes. Bub1 responds differentially to the presence and absence of tension across chromosomes during meiosis exactly as would be predicted were it acting as part of a functional spindle checkpoint. In addition, bub1 mutations have a dramatic effect on Drosophila spermatogenesis. Though it is difficult to distinguish aberrations introduced during mitotic germ line cell proliferation from those occurring during meiosis, the appearance of disrupted meiotic spindles and of multiple nuclei of uneven volume within 'onion-stage' spermatids are suggestive of defects specifically affecting meiosis (Basu, 1999).

On the basis of these observations, it is believed that a spindle checkpoint does exist in Drosophila meiotic spermatocytes, but that it operates with significantly reduced efficiency or according to different signals. The reasons underlying this apparent inefficiency remain unclear, but very likely involve part of the checkpoint pathway downstream of Bub1. One prediction of this viewpoint is that conditions that should enable the checkpoint would delay, but not completely block, cell cycle progression past the metaphase of either meiotic division. It will thus be of importance in the near future to verify this prediction through real-time observations of male meiosis in cultured spermatocytes (Basu, 1999).

ZW10 helps recruit dynactin and dynein to the kinetochore

Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, these results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset (Starr, 1998).

Search PubMed for articles about Drosophila Zw10

Barbosa, J., Martins, T., Bange, T., Tao, L., Conde, C. and Sunkel, C. (2019). Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments. Embo j: e100789. PubMed ID: 31849090

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date revised: 20 November 2020

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