Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance

Abstract

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division.T he ESCRT pathway functions across eukaryotes and many archaeal species, where it helps mediate (i) vesicle formation at multivesicular bodies (MVB) (1), (ii) enveloped virus budding (2), and (iii) the abscission stage of cytokinesis (3-7). These seemingly disparate biological processes all involve the resolution of thin, cytoplasm-filled membrane tubules, implying that ESCRT machinery can be recruited to different biological membranes to mediate topologically similar membrane fission events. Most ESCRT pathway proteins function as subunits of five multiprotein complexes, termed the ESCRT-0, -I, -II, -III, and VPS4 complexes. Other ESCRT factors, such as ALIX, function as discrete proteins. Classic studies in yeast have established that the ESCRT components are recruited sequentially to endosomal membranes where they assemble into higher order complexes that mediate protein sorting, membrane remodeling, and fission (8). Initially, early-acting factors such as ESCRT-I, ESCRT-II, and ALIX interact with upstream recruiting factors, concentrate protein cargoes, and help deform membranes (9). These early-acting factors recruit subunits of the ESCRT-III complex, which form filaments within the necks of membrane tubules and mediate membrane fission (10-16). ESCRT-III assemblies, in turn, recruit VPS4 ATPases, which use the energy of ATP hydrolysis to disassemble the ESCRT complexes (10-14, 17).ESCRT-III and VPS4 homologs mediate abscission in hyperthermophilic crenarchaeal species that diverged from eukaryotes several billion years ago, suggesting that cell division may have been the primordial function of the E...

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

“…Now evidence has been presented that an ESCRT-III complex containing CHMP7 as a subunit is involved in resealing of the nuclear envelope during mitosis (Olmos et al 2015;Vietri et al 2015). Earlier work also pointed to a role of CHMP7 in mitosis (Morita et al 2010). In another report, it was suggested that a novel ESCRT-III-like complex could be involved in the surveillance of nuclear pore complex (NPC) assembly at the nuclear membrane in yeast (Webster et al 2014), but it is not clear whether this complex contains a CHMP7 ortholog.…”

Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission

“…In addition, ESCRTs are known to mediate fission of small vesicles (<50 nm diameter) or viruses (<100 nm) (7), making it unclear how they could mediate largescale scission of membranes >1 μm in diameter (3,5), as exist within the intercellular bridge during cytokinetic abscission. Finally, cytokinetic failure in the absence of ESCRTs could be an indirect effect of ESCRTs' having multiple functions in cell division, including roles in maintaining the integrity of centrosomes (18) and the midbody (13).…”

The Freud-1/CC2D1A Family: Multifunctional Regulators Implicated in Mental Retardation

“…Cells lacking the ESCRT-III and VPS4 proteins displayed abnormalities in abscission, and unexpectedly, at earlier mitotic stages, with centrosome number, morphology and function being altered (Morita, et al, 2010). Given that human CC2D1A interacts with CHMP4B and CHMP4C, and that CC2D1A-targeted siRNA results in multipolar spindles, it is noteworthy that this same phenotype is evident in cells treated with siRNA to ESCRT-III/VPS4 proteins, specifically, CHMP1A, CHMP1B, CHMP2B, CHMP4B, CHMP4C, CHMP7, VPS4A, and VPS4B (Morita, et al, 2010). These cells additionally had greater numbers of centrosomes and spindles.…”

“…ESCRT-III and VPS4 proteins are localized to the centrosomes, regulating both maintenance or proliferation and cell division at the midbodies during abscission (Carlton, et al, 2008;Lindas, et al, 2008;Morita, et al, 2007;Samson, et al, 2008). Cells lacking the ESCRT-III and VPS4 proteins displayed abnormalities in abscission, and unexpectedly, at earlier mitotic stages, with centrosome number, morphology and function being altered (Morita, et al, 2010). Given that human CC2D1A interacts with CHMP4B and CHMP4C, and that CC2D1A-targeted siRNA results in multipolar spindles, it is noteworthy that this same phenotype is evident in cells treated with siRNA to ESCRT-III/VPS4 proteins, specifically, CHMP1A, CHMP1B, CHMP2B, CHMP4B, CHMP4C, CHMP7, VPS4A, and VPS4B (Morita, et al, 2010).…”