Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing.

At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission.

ANCHR mediates Aurora-B-dependent abscission checkpoint control through retention of VPS4.

During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring.

The TBC/RabGAP Armus coordinates Rac1 and Rab7 functions during autophagy.

Autophagy is an evolutionarily conserved process that enables catabolic and degradative pathways. These pathways commonly depend on vesicular transport controlled by Rabs, small GTPases inactivated by TBC/RabGAPs. The Rac1 effector TBC/RabGAP Armus (TBC1D2A) is known to inhibit Rab7, a key regulator of lysosomal function. However, the precise coordination of signaling and intracellular trafficking that regulates autophagy is poorly understood. We find that overexpression of Armus induces the accumulation of enlarged autophagosomes, while Armus depletion significantly delays autophagic flux. Upon starvation-induced autophagy, Rab7 is transiently activated. This spatiotemporal regulation of Rab7 guanosine triphosphate/guanosine diphosphate cycling occurs by Armus recruitment to autophagosomes via interaction with LC3, a core autophagy regulator. Interestingly, autophagy potently inactivates Rac1. Active Rac1 competes with LC3 for interaction with Armus and thus prevents its appropriate recruitment to autophagosomes. The precise coordination between Rac1 and Rab7 activities during starvation suggests that Armus integrates autophagy with signaling and endocytic trafficking.

Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1.

Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic.

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.