Chmp4c is a novel kinetochore component that is required for optimal kinetochore-microtubule attachment and spindle checkpoint signaling


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Eleni Petsalaki, Maria Dandoulaki, George Zachos

Abstract

Background

Formation of stable kinetochore-microtubule attachment is essential for accurate chromosome segregation and is mediated by the Hec1-Nuf2 kinetochore complex in human cells. In the presence of unattached kinetochores, the mitotic spindle checkpoint delays anaphase onset until all kinetochores bi-orient. Errors in spindle checkpoint signalling can lead to aneuploidy and chromosomal instability that are associated with carcinogenesis; however, how microtubule attachment is coupled with spindle checkpoint signalling is incompletely understood. Chmp4c is a component of the endosomal sorting complex required for transport (ESCRT) machinery that is required for biogenesis of multivesicular endosomes, viral budding and cytokinetic abscission; however, a role for Chmp4c independent of membrane-directed activities has not been previously reported.

Method

To investigate Chmp4c functions in the mitotic spindle, we used site directed mutagenesis, RNA silencing, replacement of endogenous proteins with transfected wild-type or mutant transgenes, in combination with confocal microscopy, live cell imaging and biochemistry techniques.

Results

Here, we show that human Chmp4c localises to kinetochores in prometaphase and is required for stable kinetochore-microtubule interactions, accurate chromosome alignment and segregation in human cell lines. Chmp4c is required for optimal localization of the RZZ (Rod, Zwilch and ZW10) spindle checkpoint protein complex to kinetochores and for anaphase delay when all kinetochores are unattached by nocodazole-treatment. Mechanistically, Chmp4c binds to ZW10 in cell extracts and in vitro to promote RZZ kinetochore-targeting. We also show that Chmp4c promotes Hec1 and Nuf2 localization to prometaphase kinetochores and associates with Hec1 and Nuf2 in cell extracts. Furthermore, Chmp4c contributes to kinetochore-microtubule stability independently of regulating Hec1-Nuf2, and directly binds and bundles microtubules in vitro.

Conclusion

These results show previously unknown molecular interactions between the ESCRT machinery and the mitotic spindle and identify Chmp4c as a novel kinetochore component that links kinetochore-microtubule attachment with ZW10-dependent spindle checkpoint signaling.

Impact statement

These results describe novel mechanisms that could protect against aneuploidy and carcinogenesis by promoting accurate chromosome segregation during mitotic cell division.