Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly

Abstract

Mitotic spindle formation and chromosome segregation depend critically on kinetochore–microtubule (KT–MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

Spindly switch controls anaphase: Spindly and RZZ functions in chromosome attachment and mitotic checkpoint control

“…In human cells, however, neither Mad2 recruitment to unattached kinetochores nor its removal from bi-oriented kinetochores was strongly affected by loss of Spindly function. [32][33][34][35] The reasons for these differences are not clear at the moment but might be due to the different structures of kinetochores in these species. 91 The chromosome congression defect induced by Spindly knockdown in Drosophila was weaker than that observed in C. elegans and human cells and resembled a DHC depletion phenotype, previously shown to interfere with the onset of anaphase.…”

“…The interphase phenotype of dmSpindly knockdown cells is, however, not well understood because it does not seem to be connected to ZW10 functions in vesicle transport. 33,35 In all investigated species, however, Spindly-similar to dynein and the RZZ complex-localizes to kinetochores in early prometaphase and at spindle poles in metaphase. [33][34][35]89 Unlike SPDL-1 and hSpindly, dmSpindly re-occupied kinetochores transiently in anaphase before being completely transported to the poles, 35 suggesting that in Drosophila Spindly could be important for anaphase poleward chromosome movements.…”

“…33,35 In all investigated species, however, Spindly-similar to dynein and the RZZ complex-localizes to kinetochores in early prometaphase and at spindle poles in metaphase. [33][34][35]89 Unlike SPDL-1 and hSpindly, dmSpindly re-occupied kinetochores transiently in anaphase before being completely transported to the poles, 35 suggesting that in Drosophila Spindly could be important for anaphase poleward chromosome movements. The localization of Spindly to kinetochores depends on RZZ and a direct physical, albeit weak, interaction between the RZZ complex and Spindly could be confirmed by co-immunoprecipitation experiments.…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…The spindle assembly checkpoint proteins Mad1, Mad2, and BubR1, are removed from kinetochores upon microtubule attachment and transported along microtubules to the spindle pole (Howell et al 2000, a transition that is supposed to silence the signal emanating from kinetochores. This spindle checkpoint protein depletion system uses the dynein/dynactin motor protein and specialized dynein binding partners on kinetochores, composed of the RZZ complex (Rod, ZW10 and Zwilch) and Spindly (Barisic et al 2010;Chan et al 2009;Clute and Pines 1999;Gassmann et al 2008Gassmann et al , 2010Griffis et al 2007;Howell et al 2001;Karess 2005;Yamamoto et al 2008). However, experimental evidence suggest that these proteins cannot be the only silencing system, as cells lacking Spindly still silence the checkpoint upon chromosome alignment (Gassmann et al 2010).…”

The perpetual movements of anaphase

“…Moreover, it remains to be elucidated whether the observed reduction is a direct result of lack of dynein processive motion along K-fiber microtubules, or whether it is due to defective/unstable microtubule attachments, possibly associated with a defective SAC [103][104][105]. The recent discovery of spindly, a protein that targets dynein to kinetochores in Drosophila, C. elegans and humans, but which does not impair the recruitment of Mad2 to kinetochores [106][107][108], may prove to be an important tool for the clarification of the abovementioned issues.…”