Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K''s coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins.     

Adherens junctions determine the apical position of the midbody during follicular epithelial cell division

Cytokinesis is asymmetric along the apical–basal axis of epithelial cells, positioning the midbody near the apical domain. However, little is known about the mechanism and purpose of this asymmetry. We use live imaging of Drosophila follicle cell division to show that asymmetric cytokinesis does not result from intrinsic polarization of the main contractile ring components. We show that adherens junctions (AJs) maintain close contact with the apical side of the contractile ring during cytokinesis. Asymmetric distribution of AJ components within follicle cells and in the otherwise unpolarized S2 cells is sufficient to recruit the midbody, revealing that asymmetric cytokinesis is determined by apical AJs in the epithelia. We further show that ectopic midbody localization induces epithelial invaginations, shifting the position of the apical interface between daughter cells relative to the AB axis of the tissue. Thus, apical midbody localization is essential to maintain epithelial tissue architecture during proliferation.     

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference-mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis.     

Mutations of DMYPT cause over constriction of contractile rings and ring canals during Drosophila germline cyst formation

Ring canals, also known as stable intercellular bridges, are derived from the contractile rings of incomplete cytokinesis (IC) in most organisms. Formation of ring canals is necessary to generate functional eggs and sperm in multiple organisms including insects, birds, mammals and various plants. How the constriction of a contractile ring is arrested and how an arrested contractile ring is transformed into a ring canal is unknown. We describe here the function of the Drosophila melanogaster myosin binding subunit of myosin phosphatase (DMYPT) in both processes. We have found that DMYPT is highly enriched in the cytoplasm of cells undergoing IC during oogenesis. DMYPT mutations in germ cells, but not in somatic follicle cells, resulted in over-constriction of contractile rings and ring canals. This leads to formation of small ring canals and mis-regulation of centriole migration during female germline cyst formation. Our results suggest that there may be two parallel mechanisms to prevent the contractile rings from being completely closed, physical resistance and inhibition of myosin II activity via DMYPT.     

A Septin Double Ring Controls the Spatiotemporal Organization of the ESCRT Machinery in Cytokinetic Abscission

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The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8^ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8^ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8^ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development.