Citron kinase controls a molecular network required for midbody formation in cytokinesis

Abstract

Cytokinesis partitions cytoplasmic and genomic materials at the end of cell division. Failure in this process causes polyploidy, which in turn can generate chromosomal instability, a hallmark of many cancers. Successful cytokinesis requires cooperative interaction between contractile ring and central spindle components, but how this cooperation is established is poorly understood. Here we show that Sticky (Sti), the Drosophila ortholog of the contractile ring component Citron kinase (CIT-K), interacts directly with two kinesins, Nebbish [the fly counterpart of human kinesin family member 14 (KIF14)] and Pavarotti [the Drosophila ortholog of human mitotic kinesin-like protein 1 (MKLP1)], and that in turn these kinesins interact with each other and with another central spindle protein, Fascetto [the fly ortholog of protein regulator of cytokinesis 1 (PRC1)]. Sti recruits Nebbish to the cleavage furrow, and both proteins are required for midbody formation and proper localization of Pavarotti and Fascetto. These functions require Sti kinase activity, indicating that Sti plays both structural and regulatory roles in midbody formation. Finally, we show that CIT-K's role in midbody formation is conserved in human cells. Our findings indicate that CIT-K is likely to act at the top of the midbody-formation hierarchy by connecting and regulating a molecular network of contractile ring components and microtubule-associated proteins.

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

“…Indeed, we showed that CIT-K loss alters midbody microtubule stability in sensitive cells, whereas the stability of microtubules affects the sensitivity of cytokinesis to CIT-K loss (Figure 2). In addition to the previous studies implicating CIT-K in actindependent midbody stabilization, 23,25,53 these results add strong support the notion that CIT-K plays a crucial role in the crosstalk between actin and microtubules responsible for the maturation of midbody that precedes abscission. [23][24][25]54 The activity of CIT-K on microtubule stability is most likely independent from the reported interaction between CIT-K and KIF14.…”

“…A second conclusion of our study is that midbody microtubules are a primary target of CIT-K in abscission control. Although the involvement of CIT-K in events related to microtubule dynamics has previously been suggested, 25,52 we provide for the first time evidence of a direct functional role. Indeed, we showed that CIT-K loss alters midbody microtubule stability in sensitive cells, whereas the stability of microtubules affects the sensitivity of cytokinesis to CIT-K loss (Figure 2).…”

“…Previous results indicated that the primary function of CIT-K, conserved from insects to mammals, is to promote the stability and the maturation of midbody before abscission. 15,[23][24][25] To assess whether this function may involve the stabilization of midbody microtubules, we measured the ratio of acetylated versus tyrosinated α-tubulin at the midbody of control cells and of cells lacking CIT-K. 28 Indeed, dynamic microtubules contain a relatively high amount of α-tubulin tyrosinated at its carboxyterminus, whereas stable microtubules show a high content of α-tubulin acetylated at the K40 residue. 29 We studied two cellular models characterized by different sensitivity to CIT-K loss and by different physiological relevance: cultures of proliferating neocortical neuronal progenitors obtained from control or from Cit-K knockout mice at embryonic day (E)12.5 and HeLa cells depleted of CIT-K by RNAi.…”

“…In contrast, the ratio is not significantly different from the control in nondividing progenitors and HeLa cells, and outside of the midbody in telophase HeLa cells (Supplementary Figures S1A and B). We then evaluated whether the altered ratio between tyrosinated and acetylated tubulin produced by CIT-K loss could depend on KIF14, a microtubule-binding protein that has been shown to work immediately downstream of CIT-K in Drosophila 25 and has been proposed to do so also in mammalian cells 15,25 by depleting this protein in cultured wild-type neuronal progenitors and in HeLa cells (Supplementary Figure S2A). However, cells depleted for KIF14 did not show significant differences when compared with the corresponding controls (Figures 2a and b).…”

“…23,24 However, recent results have indicated that CIT-K is also capable of binding microtubules and of promoting midbody maturation by affecting the localization of the kinesins MKLP1 and KIF14 and of the microtubule-bundling protein PRC1. 25 These results raised the possibility that the function of CIT-K may also be related to microtubule organization and that context-dependent differences in microtubule stability may condition the requirement for CIT-K during cytokinesis. In this report we show that this is indeed the case.…”