The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.

The midbody (MB) is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. MBs persist in cells long after division as midbody derivatives (MBds), but their fate is unclear. Here we show that MBds are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells, induced pluripotent stem cells (iPSCs) and potential cancer ‘stem cells’ (CSCs) in vivo and in vitro. MBd loss accompanies stem cell differentiation, and involves autophagic degradation mediated by binding of the autophagic receptor, NBR1, to the MB protein Cep55. Differentiating cells and normal dividing cells do not accumulate MBds and possess high autophagic activity. Stem cells and cancer cells accumulate MBds by evading autophagosome encapsulation and exhibit low autophagic activity. MBd enrichment enhances reprogramming to iPSCs and increases in vitro tumorigenicity of cancer cells. These results suggest unexpected roles for MBds in stem cells and CSCs.

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

SUMMARY Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is caused by mutations in the centrosome gene pericentrin (PCNT) which lead to severe pre- and post-natal growth retardation[1]. As in MOPDII patients, disruption of pericentrin (Pcnt) in mice caused a number of abnormalities including microcephaly, aberrant hemodynamics analyzed by in utero echocardiography and cardiovascular anomalies; the latter being associated with mortality, as in the human condition[1]. To identify the mechanisms underlying these defects, we tested for changes in cell and molecular function. All Pcnt−/− mouse tissues and cells examined showed spindle misorientation. This mouse phenotype was associated with misdirected ventricular septal growth in the heart, decreased proliferative symmetric divisions in brain neural progenitors and increased misoriented divisions in fibroblasts; the same phenotype was seen in fibroblasts from three MOPDII individuals. Misoriented spindles were associated with disrupted astral microtubules and near complete loss of a unique set of centrosome proteins from spindle poles (ninein, Cep215, centriolin). All these proteins appear to be crucial for microtubule anchoring and all interacted with Pcnt, suggesting that Pcnt serves as a molecular scaffold for this functionally-linked set of spindle pole proteins. Importantly, Pcnt disruption had no detectable effect on localization of proteins involved in the cortical polarity pathway (NuMA, p150glued, aPKC). Not only do these data reveal a spindle-pole-localized complex for spindle orientation, but they identify key spindle symmetry proteins involved in the pathogenesis of MOPDII.