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

Abstract

Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin β-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules

“…It must be noted that the number of neurons produced in CITK À/À may be strongly underestimated, because of their apoptotic death ( Fig 1E). Indeed, we observed a strong absolute reduction of BrdU-positive cells in the neuronal layers of CitK À/À mice ( Fig 1C), consistent with the previous finding that in this model apoptosis occurs especially in post-mitotic neuroblasts and neurons [53]. Altogether, these results indicate that CitK À/À developing cortex is characterized by increased cell cycle exit and increased generation of basal progenitors.…”

“…It also remains to be established how CITK may modulate We have shown that CITK affects both MT nucleation and stability. CITK stabilizes midbody microtubules by indirectly stimulating TUBB3 phosphorylation [53]. Indeed, CITK promotes midbody maturation by recruiting at the midbody the kinesins KIF14 [48,78] and MLKP1 [58], which in turn recruit the MT-crosslinking protein PRC1.…”

Investigating cytokinesis failure as a strategy in cancer therapy

“…It was recently reported that tubulin β-III (TUBB3) over-expression increased sensitivity to CIT-K depletion in HeLa cells [39]. However, our analysis indicated no correlation (R 2 = 0.019) between the level of TUBB3 expression and how cells responded to CIT-K knockdown (Figure 9C, 9E, Table 1).…”

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53

“…To address whether the molecular events induced by ZIKV infection display significant similarities with genetic microcephaly, we set out to compare the expression profiles of ZIKV-infected hNPCs 12 with the profiles of developing neural tissues obtained from three different mouse models of severe genetic microcephaly, that is, CitK−/− mice, 24 Magoh+/− mice 25 and conditional Elp3-knockout mice. 26 CitK is a ubiquitous protein implicated in midbody maturation, 27, 28, 29 whose inactivation leads to cytokinesis failure and massive apoptosis in the mammalian developing brain. 24, 30 We obtained RNA-seq data from control and CitK−/− developing cerebellum (Bianchi et al , Gene Expression Omnibus data set no.…”