The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

Abstract

Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7 prevents chromosome segregation and cytokinesis defects in oocytes

“…In line with a previous study, we noticed the presence of numerous ectopic microtubule asters near the cell cortex of KLP-7-depleted or -deleted oocytes (Fig. 4A,B) (Han et al, 2015). Ectopic cortical asters persisted throughout meiosis but disappeared at anaphase II onset (Fig.…”

“…We suspect that KLP-7 is required to generate this overlap by preventing excessive and/or ectopic microtubule elongation from chromosomes, where it is concentrated during meiotic anaphase (Fig. 1E) (Han et al, 2015). SPD-1 and the Centralspindlin complex (including CYK-4) have been shown to preferentially interact with overlapping microtubule plus-ends, which might explain their delocalization in the denser and disorganized central spindle that is assembled following klp-7 (RNAi) (Bieling et al, 2010;Davies et al, 2015).…”

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7MCAK prevents chromosome segregation and cytokinesis defects in oocytes

“…In the C. elegans oocyte, KLP-7 has been proposed to limit metaphase spindle pole numbers by correcting improper kinetochore-microtubule attachments, but its precise function throughout the two meiotic divisions remains elusive (Connolly et al, 2015;Han et al, 2015). Here, we show the crucial meiotic function of KLP-7 in preventing ectopic microtubule assembly that otherwise leads to spindle disorganization and chromosome segregation defects.…”

Sumoylation regulates central spindle protein dynamics during chromosome segregation in oocytes

“…It has become increasingly clear that the midbivalent ring domain does not behave as a 'static' entity. Its composition changes dramatically during metaphaseanaphase I and each protein displays a characteristic and dynamic localisation pattern (Wignall and Villeneuve, 2009;Dumont et al, 2010;Collette et al, 2011;Connolly et al, 2015;Han et al, 2015.;Muscat et al, 2015;Gigant et al, 2017). While SUMO depletion on its own does not drastically affect chromosomes segregation, it regulates the dynamic localisation of midbivalent/central-spindle proteins.…”

“…Many central-spindle proteins begin to concentrate between homologous chromosomes during prometaphase in a ring-shaped structure (hereafter ring domain), which marks the site of cohesion loss (Dumont et al, 2010;Muscat et al, 2015;Pelisch et al, 2017;Wignall and Villeneuve, 2009). The metaphase I ring domain consists of key cell division regulators including the chromosomal passenger complex (CPC) components AIR-2 AuroraB , ICP-1 IN CEN P , BIR-1 Survivin (Schumacher et al, 1998;Speliotes et al, 2000;Kaitna et al, 2002;Rogers et al, 2002;Romano et al, 2003), the checkpoint kinase BUB-1 Bub1 (Monen et al, 2005;Dumont et al, 2010), Condensin I components (Collette et al, 2011), the kinesin KLP-7 M CAK (Connolly et al, 2015;Han et al, 2015.;Gigant et al, 2017), and the chromokinesin KLP-19 Kif 4A (Wignall and Villeneuve, 2009). We recently showed that a number of these components are held together by a combination of covalent SUMO modification and non-covalent SUMO interactions (Pelisch et al, 2017).…”