Spindly: Biological Overview | References
Gene name - Spindly
Synonyms - CG15415
Cytological map position - 24A1-24A1
Function - signal transduction
Keywords - spindle assembly checkpoint,
Symbol - Spindly
FlyBase ID: FBgn0031549
Genetic map position - 2L:3,527,553..3,530,286 [-]
Classification - conserved protein; predicted coiled-coil sequences
Cellular location - cytoplasmic
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 (Howell, 2001; Wojcik, 2001). 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 (for review see Karess, 2005), 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 (Acquaviva, 2006). 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 (for review see Musacchio, 2007) 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 (Chan, 1999; Mao, 2005). 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 (De Antoni, 2005; 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; Schroer, 2004), which, in turn, binds to the Zw10 subunit of the RZZ complex (Starr, 1998). Lis1, another dynein cofactor, also has been proposed to play a role in targeting dynein to kinetochores (Dzhindzhev, 2005). 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 the model of Howell (2001), who 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 (Dzhindzhev, 2005) 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).
Search PubMed for articles about Drosophila Spindly
Acquaviva, C. and Pines, J. (2006). The anaphase-promoting complex/cyclosome: APC/C. J. Cell Sci. 119: 2401-2404. PubMed ID: PubMed ID; Online text
Chan, G. K., et al. (1999). Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J. Cell Biol. 146: 941-954. PubMed ID: 10477750
De Antoni, A., et al. (2005). The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint. Curr. Biol. 15: 214-225. PubMed ID: 15694304
Dzhindzhev, N. S., Rogers, S. L., Vale, R. D. and Ohkura. H. (2005). Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends. J. Cell Sci. 118: 3781-3790. PubMed ID: PubMed ID; Online text
Griffis, E. R., Stuurman, N. and Vale, R. D. (2007). Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. J. Cell Biol. 177(6): 1005-15. PubMed ID: 17576797
Howell, B. J., et al. (2001). Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. J. Cell Biol. 155: 1159-1172. PubMed ID: PubMed ID; Online text
Karess, R. (2005). Rod-Zw10-Zwilch: a key player in the spindle checkpoint. Trends Cell Biol. 15: 386-392. PubMed ID: 15922598
Mao, Y., Desai, A. and Cleveland, D. W. (2005). Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling. J. Cell Biol. 170: 873-880. PubMed ID: 16144904
Musacchio, A. and Salmon, E. D. (2007). The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8: 379-393. PubMed ID: 17426725
Schroer, T. A. (2004). Dynactin. Annu. Rev. Cell Dev. Biol. 20: 759-779. PubMed ID: 15473859
Starr, D. A., Williams, B. C., Hays, T. S. and Goldberg, M. L. (1998). ZW10 helps recruit dynactin and dynein to the kinetochore. J. Cell Biol. 142: 763-774. PubMed ID: 9700164
Wojcik, E., et al. (2001). Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein. Nat. Cell Biol. 3: 1001-1007. PubMed ID: 11715021
date revised: 30 March 2008
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