Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance Eiji Moritaa, Leremy A. Colfa, Mary Anne Karrena, Virginie Sandrina, Christopher K. Rodeschb, and Wesley I. Sundquista,1 aDepartment of Biochemistry and bFluorescence Microscopy Core Facility, University of Utah School of Medicine, Salt Lake City, UT 84112-5650 Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved June 7, 2010 (received for review April 30, 2010) The ESCRT pathway helps mediate the final abscission step of cyto- for abscission. This translocation event is one of many emerging kinesis in mammals and archaea. In mammals, two early acting pro- links between centrosomes and cytokinesis (reviewed in ref. 19). teins of the ESCRT pathway, ALIX and TSG101, are recruited to the Indeed, it has been proposed that centrosomes may regulate midbody through direct interactions with the phosphoprotein mammalian pathway(s) that help control the transition from mi- CEP55. CEP55 resides at the centrosome through most of the cell tosis to cytokinesis, as is the case for the “mitotic exit network,” cycle but then migrates to the midbody at the start of cytokinesis, which is organized by yeast spindle pole bodies (20). CEP55 binds suggesting that the ESCRT pathway may also have centrosomal and recruits the early-acting ALIX and ESCRT-I proteins to the links. Here, we have systematically analyzed the requirements for midbody, and these interactions are required for efficient abscis- late-acting mammalian ESCRT-III and VPS4 proteins at different sion (3, 4, 21). The roles of late-acting ESCRT factors in cytoki- stages of mitosis and cell division. We found that depletion of nesis are less well characterized, but several recent reports indicate VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) that VPS4, the ESCRT-III protein CHMP1B, and the ESCRT-III- proteins inhibited abscission. Remarkably, depletion of individual like protein IST1 can also function at midbodies (3, 4, 21–24). ESCRT-III and VPS4 proteins also altered centrosome and spindle In this study, we tested the requirements for each human pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 ESCRT-III and VPS4 protein during different stages of cell di- proteins) or monopolar spindles (CHMP2A or CHMP5) and causing vision. Cells lacking each of the late-acting ESCRT-III/VPS4 defects in chromosome segregation and nuclear morphology. VPS4 proteins exhibited abscission defects. Remarkably, cells lacking proteins concentrated at spindle poles during mitosis and then at ESCRT-III/VPS4 proteins also exhibited profound defects ear- midbodies during cytokinesis, implying that these proteins function lier in mitosis, including aberrations in centrosome number, directly at both sites. We conclude that ESCRT-III/VPS4 proteins function morphology, and function, implying that ESCRT-III/VPS4 pro- at centrosomes to help regulate their maintenance or proliferation teins perform important centrosomal functions and may also and then at midbodies during abscission, thereby helping ensure the function during other stages of mitosis. ordered progression through the different stages of cell division. Results ESCRT | CHMP | mitosis | cytokinesis | abscission ESCRT-III and VPS4 Proteins Are Required for Normal Cell Division. To test for roles in cell division, each of the 11 different human he ESCRT pathway functions across eukaryotes and many ar- ESCRT-III/CHMP proteins and both VPS4 proteins were in- chaeal species, where it helps mediate (i) vesicle formation at dividually depleted from HeLa cells using siRNA treatments. At T least four siRNAs against each ESCRT-III/VPS4 protein were multivesicular bodies (MVB) (1), (ii) enveloped virus budding (2), fi – tested for ef cacy using antibodies against the endogenous pro- and (iii) the abscission stage of cytokinesis (3 7). These seemingly teins, except in the case of CHMP4C, where suitable antibodies disparate biological processes all involve the resolution of thin, cy- fi were not available and protein depletion was therefore tested using toplasm- lled membrane tubules, implying that ESCRT machinery exogenously expressed CHMP4C. At least two effective siRNAs can be recruited to different biological membranes to mediate to- against each target protein were identified, and their specificities pologically similar membrane fission events. Most ESCRT pathway were confirmed by testing for cross-reactivity with related ESCRT- proteins function as subunits of five multiprotein complexes, termed III/VPS4 family members (Fig. S1 and Table S1). This set of the ESCRT-0, -I, -II, -III, and VPS4 complexes. Other ESCRT fac- ESCRT-III/VPS4 siRNAs was then used to test the roles of each tors, such as ALIX, function as discrete proteins. Classic studies in ESCRT-III and VPS4 protein in cell division. Measurements of yeast have established that the ESCRT components are recruited cellular DNA content and nuclear morphology were confirmed sequentially to endosomal membranes where they assemble into with at least two different siRNAs against each ESCRT-III/VPS4 higher order complexes that mediate protein sorting, membrane re- protein, but for clarity the results are reported for a single siRNA modeling, and fission (8). Initially, early-acting factors such as (except where noted). ESCRT-I, ESCRT-II, and ALIX interact with upstream recruiting As an initial screen for cell cycle defects, HeLa cells were CELL BIOLOGY depleted of individual ESCRT-III/VPS4 proteins and assayed for factors, concentrate protein cargoes, and help deform membranes fl (9). These early-acting factors recruit subunits of the ESCRT-III changes in DNA copy number using ow cytometry measurements (Fig. 1). Cells received two siRNA treatments at 24-h intervals, complex, which form filaments within the necks of membrane tu- fi – and the resulting phenotypes were measured 24 h after the second bules and mediate membrane ssion (10 16). ESCRT-III assem- treatment. Irrelevant siRNAs were used as negative controls, and blies, in turn, recruit VPS4 ATPases, which use the energy of ATP siRNAs that targeted ALIX or CEP55 were used as positive – hydrolysis to disassemble the ESCRT complexes (10 14, 17). controls for abscission defects (3, 4). As expected, depletion of ESCRT-III and VPS4 homologs mediate abscission in hyper- thermophilic crenarchaeal species that diverged from eukaryotes several billion years ago, suggesting that cell division may have Author contributions: E.M., L.A.C., C.K.R., and W.I.S. designed research; E.M., L.A.C., M.A.K., been the primordial function of the ESCRT pathway (6, 7). The and V.S. performed research; E.M., L.A.C., and W.I.S. analyzed data; and E.M., L.A.C., and ESCRT pathway also helps mediate abscission in mammals (3), W.I.S. wrote the paper. where its activities must be integrated with a number of other The authors declare no conflict of interest. important processes, including centrosome function, mitosis, and This article is a PNAS Direct Submission. contractile ring formation and constriction. As cells exit mitosis, 1To whom correspondence should be addressed. E-mail: [email protected]. the phosphoprotein CEP55 leaves centrosomes and concentrates This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. at the midbody (18), where it helps to organize factors required 1073/pnas.1005938107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1005938107 PNAS | July 20, 2010 | vol. 107 | no. 29 | 12889–12894 Downloaded by guest on September 25, 2021 ALIX or CEP55 dramatically increased the frequencies of cells A Control -ALIX -CEP55 -CHMP1A with four copy (4C) DNA content (>6-fold increases) and 8C DNA (>16-fold increases) compared with the negative controls. 1 These profiles arose because the cells went through multiple 2 rounds of mitosis without completing cytokinesis, and therefore -CHMP2B -CHMP4C -VPS4A -VPS4B ended up with multiple nuclei and multiple copies of their DNA 3 (Figs. 1 and 2). Depletion of individual ESCRT-III/VPS4 proteins similarly increased the frequency of cells with 4C DNA content (3- to 8-fold increases), implying nonredundant roles for all of the 20 m human ESCRT-III/VPS4 proteins in cell division. CHMP3 de- Multinucleated (1) Midbody (2) pletion additionally increased the fraction of cells with 8C DNA B 100 content (approximately 9-fold increase), and this phenotype 80 therefore resembled the ALIX and CEP55 controls. In contrast, 60 fi depletion of other ESCRT-III/VPS4 proteins caused a signi cant 40 increase in the fraction of cells with 4C but not 8C DNA, sug- gesting that these ESCRT-III/VPS4 proteins might perform dif- 20 ferent or additional functions in cell division. % Abscission Defects 0 -ALIX -CEP55 -VPS4A -VPS4B ESCRT-III and VPS4 Proteins Are Required for Mitosis. To identify -CHMP3 -CHMP5 -CHMP6 -CHMP7 Control 1 Control 2 -CHMP1A -CHMP2A -CHMP4A other potential ESCRT-III/VPS4 protein functions, cells were de- -CHMP1B -CHMP2B -CHMP4B -CHMP4C fi pleted of individual ESCRT-III/VPS4 proteins, xed, stained with Fragmented Nuclei (3) Unaligned Chromosomes fluorescent markers for DNA (green) and microtubules (red), and C 100 examined by confocal microscopy (Fig. 2A). As expected, depletion 80 of CEP55 or ALIX dramatically increased the frequencies of cells 60 > fl with visible midbodies ( 5-fold increases, re ecting delayed abscis- 40 sion) and multiple nuclei (>25-fold increases, reflecting failed B 20 abscission) (Fig. 2 ). Depletion of CHMP3 similarly increased % Mitotic Defects the frequencies of cells with visible midbodies and multiple nuclei 0 (10- and 16-fold increases, respectively), again indicating that this -ALIX -CEP55 -VPS4A -VPS4B ESCRT-III protein, like ALIX and CEP55, functions primarily -CHMP3 -CHMP5 -CHMP6 -CHMP7 Control 1 Control 2 -CHMP1A -CHMP2A -CHMP4A during abscission. Cells lacking CHMP2A also had similar pheno- -CHMP1B -CHMP2B -CHMP4B -CHMP4C types, but these cells exhibited other mitotic defects (described be- Fig. 2. Mitosis and cytokinesis defects in cells lacking ESCRT-III and VPS4 pro- low). Depletion of the remaining ESCRT-III/VPS4 proteins also teins.