Molecular Mechanisms of the Membrane Sculpting ESCRT...

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Molecular Mechanisms of the Membrane Sculpting ESCRT Pathway William Mike Henne 1,2 , Harald Stenmark 3,4 , and Scott D. Emr 1,2 1 Weill Institute for Cell and Molecular Biology, Cornell University, Weill Hall, Ithaca, New York 14853 2 Department of Molecular Biologyand Genetics, Cornell University, Weill Hall, Ithaca, New York 14853 3 Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Montebello, N-0379 Oslo, Norway 4 Department of Biochemistry, Institute for Cancer Research,Oslo University Hospital, Montebello, N-0379 Oslo, Norway Correspondence: [email protected] The endosomal sorting complexes required for transport (ESCRT) drive multivesicular body (MVB) biogenesis and cytokinetic abscission. Originally identified through genetics and cell biology, more recent work has begun to elucidate the molecular mechanisms of ESCRT- mediated membrane remodeling, with special focus on the ESCRT-III complex. In particular, several light and electron microscopic studies provide high-resolution imaging of ESCRT-III rings and spirals that purportedly drive MVB morphogenesis and abscission. These studies highlight unifying principles to ESCRT-III function, in particular: (1) the ordered assembly of the ESCRT-III monomers into a heteropolymer, (2) ESCRT-III as a dynamic complex, and (3) the role of the AAA ATPase Vps4 as a contributing factor in membrane scission. Mechanistic comparisons of ESCRT-III function in MVB morphogenesis and cytokinesis suggest common mechanisms in membrane remodeling. THE ESCRT PATHWAY IN MVB BIOGENESIS AND CYTOKINESIS T he endosomal sorting complex required for transport (ESCRT) comprises five dis- tinct protein complexes denoted numerically as ESCRTs -0, -I, -II, -III, and the AAA ATPase Vps4 complex (Hurley, 2010; Babst 2011; Henne et al. 2011). They were initially identified through yeast genetics as vacuolar protein sort- ing (vps) genes whose loss blocked the delivery of vacuole hydrolases, leading to aberrant endo- somal morphology. After their initial identifi- cation, further cell biological studies revealed that these gene products constitute separate protein complexes that engage transmembrane cargoes at endosomes and convert these organ- elles into multivesicular bodies (MVBs) with the cargoes sorted within their internal vesicles. Since their discovery in 2001 (Katzmann et al. 2001), much has been learned concerning how the ESCRT pathway drives MVB morpho- genesis. One particular theme involves the divi- sion of labor shared between distinct ESCRT complexes. ESCRTs -0, -I, and -II form stable hetero-oligomers that function together to rec- Editors: Sandra L. Schmid, Alexander Sorkin, and Marino Zerial Additional Perspectives on Endocytosis available at www.cshperspectives.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016766 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a016766 1 on April 13, 2020 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/ Downloaded from

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Molecular Mechanisms of the MembraneSculpting ESCRT Pathway

William Mike Henne1,2, Harald Stenmark3,4, and Scott D. Emr1,2

1Weill Institute for Cell and Molecular Biology, Cornell University, Weill Hall, Ithaca, New York 148532Department of Molecular Biology and Genetics, Cornell University, Weill Hall, Ithaca, New York 148533Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Montebello,N-0379 Oslo, Norway

4Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello,N-0379 Oslo, Norway

Correspondence: [email protected]

The endosomal sorting complexes required for transport (ESCRT) drive multivesicular body(MVB) biogenesis and cytokinetic abscission. Originally identified through genetics and cellbiology, more recent work has begun to elucidate the molecular mechanisms of ESCRT-mediated membrane remodeling, with special focus on the ESCRT-III complex. In particular,several light and electron microscopic studies provide high-resolution imaging of ESCRT-IIIrings and spirals that purportedly drive MVB morphogenesis and abscission. These studieshighlight unifying principles to ESCRT-III function, in particular: (1) the ordered assembly ofthe ESCRT-III monomers into a heteropolymer, (2) ESCRT-III as a dynamic complex, and (3)the role of the AAA ATPase Vps4 as a contributing factor in membrane scission. Mechanisticcomparisons of ESCRT-III function in MVB morphogenesis and cytokinesis suggest commonmechanisms in membrane remodeling.

THE ESCRT PATHWAY IN MVB BIOGENESISAND CYTOKINESIS

The endosomal sorting complex requiredfor transport (ESCRT) comprises five dis-

tinct protein complexes denoted numericallyas ESCRTs -0, -I, -II, -III, and the AAA ATPaseVps4 complex (Hurley, 2010; Babst 2011;Henne et al. 2011). They were initially identifiedthrough yeast genetics as vacuolar protein sort-ing (vps) genes whose loss blocked the deliveryof vacuole hydrolases, leading to aberrant endo-somal morphology. After their initial identifi-

cation, further cell biological studies revealedthat these gene products constitute separateprotein complexes that engage transmembranecargoes at endosomes and convert these organ-elles into multivesicular bodies (MVBs) withthe cargoes sorted within their internal vesicles.

Since their discovery in 2001 (Katzmannet al. 2001), much has been learned concerninghow the ESCRT pathway drives MVB morpho-genesis. One particular theme involves the divi-sion of labor shared between distinct ESCRTcomplexes. ESCRTs -0, -I, and -II form stablehetero-oligomers that function together to rec-

Editors: Sandra L. Schmid, Alexander Sorkin, and Marino Zerial

Additional Perspectives on Endocytosis available at www.cshperspectives.org

Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016766

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ognize ubiquitin and the endosome-enrichedphospholipid, phosphatidylinositol 3-phos-phate (PtdIns(3)P) at the endosomal surface(Fig. 1A) (Raiborg et al. 2001; Katzmann et al.2003; Teo et al. 2006). In addition, ESCRT-IIinitiates the assembly of the ESCRT-III complex(Teis et al. 2008). Although also a hetero-olig-omer, ESCRT-III is unique for several reasons:(1) it lacks the ability to recognize ubiquitindirectly, although recent reports indicate ES-CRT-III-interacting protein Bro1/Alix (ALG2-interacting protein X) can directly bind ubi-quitin (Dowlatshahi et al. 2012); (2) it displaysno lipid specificity for PtdIns(3)P, implyingwhy it can directly function in membrane re-modeling at both endosomes as well as the plas-ma membrane; and (3) ESCRT-III does notform a stable complex, but rather assemblestransiently during membrane sculpting andfission events. ESCRT-III is additionally uniquebecause this membrane-remodeling machineinduces invaginations that protrude away fromthe cell cytoplasm. This is topologically invertedfrom more studied vesicle budding pathwayssuch as clathrin and COP-mediated vesicleformation, and remains a mysterious process

whose molecular mechanisms are beginning tobe addressed.

A remarkable connection between MVBbiogenesis and cytokinesis, the process by whichthe plasma membrane ingresses to divide intoseparate daughter cells, emerged when it wasfound that ESCRT proteins are recruited tothe cell midbody immediately before final ab-scission (Garrus et al. 2001; Carlton and Mar-tin-Serrano 2007; Morita et al. 2007). Addition-ally, ESCRT proteins are directly involved inHIV budding from the plasma membrane,which is topologically consistent with both celldivision and MVB morphogenesis (Garrus et al.2001). For the purposes of this review, we arefocusing on the properties of ESCRT-mediatedmembrane remodeling in MVB morphogene-sis and cytokinesis. ESCRT-I subunit Tsg101(Vps23) and Alix (Bro1) are recruited to themidbody by centrosomal protein of 55 kDa(CEP55), a known cytokinesis effector protein(Fig. 1A). Later studies established that severalESCRT-III proteins also localize to the mid-body. Furthermore, depletion of ESCRT-IIIproteins Snf7/CHMP4B or Vps2/CHMP2, ormutation of the ESCRT-III binding regions on

HIV buddingESCRT-III

Closed

Open

CEP55Tsg101

Alix

Midbody

CHMP2CHMP4

Secondingression

Lysosome(vacuole)

Firstingression

Daughtercell

α4α3 α5

α2 α1

A B

Figure 1. ESCRT-mediated membrane remodeling in cellular physiology and pathophysiology. (A) Schematic ofESCRT-III helices driving MVB cargo sorting, HIV budding, and cell division. (B) Helical model of a Snf7subunit from Saccharomyces cervisiae (top). ESCRT-III subunits undergo conformational changes from a closed(middle) to open state (bottom).

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Alix, perturbs late stages of cell division (Table1) (Carlton and Martin-Serrano 2007).

Showing that ESCRT proteins play a directrole in cytokinesis was groundbreaking, andhighlighted the fact that both MVB sortingand cytokinesis require topologically similarmembrane remodeling. However, despite iden-tifying ESCRT-III subunits as multipurpose

membrane remodeling effectors, the molecularmechanisms by which ESCRT-III drives thismembrane sculpting remained poorly under-stood. Recent high-resolution light and electronmicroscopy studies are now providing a work-ing model for how ESCRT-III achieves mem-brane sculpting. These studies are mechanisti-cally impactful because they provide direct

Table 1. ESCRT proteins in MVB sorting and cytokinesis

The complexes

Function

Necessary for

MVB sorting?

Necessary for

cytokinesis?

Disease

associationsYeast Human

ESCRT-0 Binds ubiquitinated cargo andPtdIns(3)P

CancerVps27 Hrs Yes NoHse1 STAM1/2 No No

ESCRT-I Binds ESCRT-0,-II, andubiquitinated cargo;localizes to midbody duringcell division

Cancer, AIDSVps23 Tsg101 Yes YesVps28 Vps28 Yes YesVps37 Vps37A-D Yes YesMvb12 hMvb12A, B Slight defect n.d.— UBAP1 Yes No

ESCRT-II Binds ESCRT-I and nucleatesESCRT-III; bindsubiquitinated cargo andPtdIns(3)P

Vps36 EAP45 Yes NoVps22 EAP30 Yes NoVps25 EAP20 Yes No

ESCRT-III Sculpts MVB vesicles anddrives cytokinesis

FTD3, AIDSHDParkinson’s

Vps20 CHMP6 Yes No?Snf7 CHMP4A-D Yes YesVps24 CHMP3 Yes YesVps2 CHMP2A, B Yes YesVps60 CHMP5 Yes YesBro1 Alix Yes YesIst1 IST1 No YesDid2 CHMP1 Yes Yes

AAA ATPases Drives ESCRT-III disassemblysevers MT duringabscission

AIDSVps4 Vps4A and B Yes Yes— Spastin Yes Yes HSP

Other MIT domainproteins

Vta1 LIP5 Slight defect Yes— MITD1 No Yes

Additional notableproteins

Recruits ESCRTs to midbodyphosphorylates CHMP4C;cell division checkpoint— CEP55 No Yes

— Aurora B No Yes

FTD3, frontotemporal dementia linked to chromosome 3; AIDS, acquired immune deficiency syndrome; HD, Huntington’s

disease; HSP, herditary spastic paraplegia; n.d., no data.

Mechanisms of ESCRT-Mediated Membrane Sculpting

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visualization of ESCRT-III assemblies both invitro and in vivo. The purpose of this reviewis to highlight the recent advances in the mech-anistic understanding of ESCRT-III and dis-cuss their implications for the mechanism ofESCRT-mediated membrane remodeling in dif-ferent cellular pathways. The review will firstdiscuss ESCRT-III architecture and functionin the context of MVB sorting, and then turnto recent advances in understanding its roles inmammalian cell division. Comparison betweenthese two distantly related pathways suggestsancient principles for membrane remodelingthat have been adapted over time to functionin diverse spatial and temporal scales.

ESCRT-III FUNCTION IN MVBMORPHOGENESIS

Molecular Architecture and ConformationalDynamics of ESCRT-III Subunits

Yeast expresses four “core” ESCRT-III subunits(Vps20, Snf7, Vps24, and Vps2). Humans sharethese “core” subunits and additionally have fourSnf7 paralogues (CHMP4A-D) and two Vps2paralogues (CHMP2A and B) (CHMP: chargedmultivesicular body protein). Early studies ofESCRT-III subunits established that they weresmall, soluble, and electrostatically charged pro-teins that all share a common architectural fold(Shim et al. 2007). For simplicity, they are oftenbisected into a basic amino-terminal half andacidic carboxy-terminal half. The amino-termi-nal half is highly structured and is composed offour a-helices that fold into an elongated bun-dle approximately 7 nm in length (Muziol et al.2006; Bajorek et al. 2009). This amino-terminal“core” domain is enriched with basic aminoacids that bind acidic lipids within lipid bilayers.The carboxy-terminal half, in contrast, is largelyunstructured.

Elegant biochemical work indicated that theacidic carboxy-terminal half of the proteinformed intramolecular contacts with the basicamino-terminal half, rendering ESCRT-III sub-units in an autoinhibited “closed” conforma-tional state (Fig. 1B) (Zamborlini et al. 2006).X-ray crystallographic structures of VPS24/

CHMP3, and IST1 revealed the structural basisfor this autoinhibition (Muziol et al. 2006; Ba-jorek et al. 2009). A conserved carboxy-terminalregion folds into ana-helix (denoted here asa5)forming numerous intramolecular contactsalong the amino-terminal core domain. Morerecent work showed that this autoinhibitorymechanism was present in Snf7, indicating thatit is a general principle among all “core” ESCRT-III subunits (Henne et al. 2012). Because ofthis, it is commonly accepted that ESCRT-IIIsubunits exist as “closed” monomers in thecell cytoplasm until their activation and recruit-ment to membrane surfaces during ESCRT-IIIassembly.

Displacement of the carboxy-terminal auto-inhibitory region drives both membrane bind-ing and, for at least Snf7 and Vps24, homo-oli-gomerization (Shim et al. 2007; Ghazi-Tabatabaiet al. 2008; Henne et al. 2012). Numerous ob-servations also suggest that the carboxy-ter-minal region is critical for stabilizing homo-polymers. The mechanism for this is unclear,but may involve novel intermolecular contactsformed when a5 and the flexible linker regionof one subunit reach across to contact a neigh-boring subunit. When VPS24 crystallized as anantiparallel dimer, each a5 formed intermolec-ular contacts with its opposing hVps24 mono-mer. This suggested that these regulatory helicesmay form stabilizing contacts with neighbor-ing subunits following their release from auto-inhibition (Muziol et al. 2006; Bajorek et al.2009). Similarly, GST-fusion constructs encod-ing VPS24 a5 were sufficient to interact withVPS24 core domains in in vitro pull down ex-periments, suggesting they could function as in-termolecular contacts (Zamborlini et al. 2006).In another example, deletion or mutation ofthis helix in yeast Snf7 attenuates polymer for-mation, although similar manipulation of hu-man Snf7 did not completely halt oligomeriza-tion in vivo using mammalian cells (Hansonet al. 2008; Henne et al. 2012).

Thus, it appears that the conformationalopening of ESCRT-III subunits tightly coor-dinates three different activities: (1) subunitrelease from autoinhibition, (2) promotion ofmembrane binding, and (3) polymer stabiliza-

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tion. One perplexing question is how this con-formational opening exposes the membraneinteracting surfaces of ESCRT-III subunits. Pre-vious studies suggest that the primary mem-brane interacting surfaces of these proteins arecomposed of positively charged amino acidsalong the a-helical hairpin (Muziol et al.2006). How the carboxy-terminal autoinhibi-tory region occludes these ostensibly large elec-trostatic contacts requires additional structuralstudies. One possibility is that the flexible linkerthat precedes a5 may physically mask the elec-trostatic interface that contacts membrane. Inyeast Snf7, this linker region is highly acidic,and is predicted to form electrostatic contactswith basic residues along the Snf7 core domain(Henne et al. 2012). Another open questionis how ESCRT-II and other ESCRT-III sub-units trigger the release of ESCRT-III subunitautoinhibition. ESCRT-III subunits recruit oneanother sequentially to endosomes, indicatingthat the physical contact between subunits trig-gers a conformational opening (Teis et al. 2008).How this is structurally coordinated requiresadditional study.

THE ASSEMBLY OF ESCRT-III POLYMERS:DOME VERSUS HELICAL MODELS

Following its recruitment by ESCRT-I, ESCRT-II directly binds Vps20 and initiates the sequen-tial activation of ESCRT-III subunits at the en-dosome surface (Teo et al. 2004). Notably, thisactivation follows a precise sequence of eventsallowing ESCRT-III to polymerize by a prede-termined subunit order, a process termed “or-dered assembly.” Initially, Vps20 directly bindsSnf7, which in turn homo-oligomerizes. Snf7 inturn recruits Vps24 and Vps2, which containhigh-affinity MIM motifs necessary for the re-cruitment and activation of the AAA ATPaseVps4 (Teis et al. 2008).

Several studies have begun to visualize thisstepwise ESCRT-III assembly with high-res-olution techniques. By overexpressing SNF7/CHMP4 truncations that lacked the autoin-hibitory carboxy-terminal region, Hanson etal. were the first to visualize ESCRT-III poly-mers in vivo. Using quick freeze deep-etch EM

(DEEM) microscopy, they discovered thatSNF7/CHMP4 assembled into circular arrayson the inner leaflet of the plasma membraneof COS-7 cells (Fig. 2A) (Hanson et al. 2008).These arrays were composed of loosely coiledfilaments �4 nm in diameter that extendedacross the membrane surface. Strikingly, coex-pressing SNF7/CHMP4 with an ATP-hydrolysisdefective mutant Vps4 caused the arrays tocompact into tightly packed circular lattices.These conditions also generate membranebuds that protruded outward from the plasmamembrane in a direction topologically consis-tent with MVB morphogenesis. Because Vps4recruitment is thought to follow the assemblyof Snf7 polymers, this Vps4-dependent mem-brane deformation appears consistent with theordered assembly model for ESCRT-III.

Although visually striking, the imaging ofSnf7 circular arrays did not provide an intuitiverole for Vps24 and Vps2 in ESCRT-III-mediatedmembrane remodeling. In vitro studies usingpurified VPS24/CHMP3 and VPS2/CHMP2soon provided additional details. Using a similarstrategy, studies by Lata et al. (2008) used nega-tive stain electron microscopy to show that trun-cated VPS24 and VPS2 coassemble into helicalcylinders in vitro. The outsides of these cylindersadhered to liposomes, suggesting a membrane-binding interface that was topologically consis-tent with MVB budding (Lata et al. 2008). No-tably, these cylinders terminated with domelikecaps. These observations have given rise to the“dome model” of ESCRT-III catalyzed mem-brane scission (Fig. 3) (Fabrikant et al. 2009).In this model, a Vps24/Vps2 dome lines the in-terior of a membrane bud. The protein domenaturally tapers the lipid bilayers into close prox-imity at the dome base, promoting spontaneousfission. This tapering alone may be sufficient forthe fission, or it may be catalyzed by the recruit-ment of Vps4 and subsequent disassembly of theVps24/Vps2 dome (Lata et al. 2008).

These studies provided important insightsinto the molecular architecture of individualESCRT-III polymers. However, it was unclearhow Snf7, Vps24, and Vps2 worked togetherto drive membrane remodeling. More recentwork now suggests that these proteins coas-

Mechanisms of ESCRT-Mediated Membrane Sculpting

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semble into helical polymers that drive vesiclebudding by a springlike mechanism (Henneet al. 2012). These helices are structurally dis-tinct from the flat spirals and circular arrays thatSnf7 forms as a homopolymer. Thus, Vps24 andVps2 may modulate Snf7 polymer architectureby either incorporating into a growing Snf7polymer or binding to a preassembled polymerand modulating its architecture, inducing atransition from a two-dimensional spiral intoa three-dimensional helix (Figs. 2B, 2C, and3). This transition may generate the mechani-cal force necessary for vesicle budding (Henne

et al. 2012). This model also explains why ves-icles could bud into giant unilamellar vesicles(GUVs) after addition of Snf7, but was moreefficient when Snf7, Vps24, and Vps2 were se-quentially added to GUVs (Wollert et al. 2009;Henne et al. 2012).

Vps4 INVOLVEMENT IN ESCRT-MEDIATEDMEMBRANE SCULPTING

The dome and helical models provide struc-tural explanations for ESCRT-III-induced mem-brane invagination, but do not necessarily ex-

A

0 nm 15 nm

α-Tubulin

1 µm

CHMP4B

30 nm 45 nm

200 nm

Monolayer

Grid

Edge

Microtubules17-nm FilamentsPlasma membrane

B C

D

Figure 2. High-resolution imaging of ESCRT-III assemblies. (A) Electron micrograph of circular array proteinscaffolds on membrane tubules in COS-7 cells expressing Snf7-1/CHMP4A and VPS4B(E235Q)-GFP. (Imagefrom Hanson et al. 2008; adapted, with permission, from The Rockefeller University Press# 2008.) (B) Electronmicrograph of reconstituted yeast Snf7 spirals assembled on lipid monolayers. (C) A helical polymer of recon-stituted yeast Snf7, Vps24, and Vps2, imaged by electron microscopy. (Panels B and C are from Henne et al. 2012;adapted, with permission, from Cell Press # 2012.) (D) Electron tomography of high-pressure frozen cells,which show 17-nm filaments spiraling along the constriction zone. (This image is from Guizetti et al. 2011;adapted, with permission, from the author.) Scale bars, 100 nm, unless stated otherwise.

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plain membrane fission. In the dome model, theprotein shell causes the membrane to taperdown to a highly curved termination. This hasbeen proposed to promote spontaneous fission(Fabrikant et al. 2009), although electrostatical-ly charged protein scaffolds have been proposedto stabilize high curvature and inhibit mem-brane fission (Fig. 3) (Boucrot et al. 2012). Analternative model would be that Vps4-mediateddisassembly of the ESCRT-III dome would re-lease membrane tension, promoting bucklingand subsequent fission.

Vps4 directly modulates the architectureof ESCRT-III helices in vivo (Hanson et al.2008). Expression of an ATP hydrolysis-defec-tive Vps4E235Q caused hSnf7 spirals to condenseinto tightly bundled circular arrays with mem-brane tubules sprouting from their centers.This suggested that Vps4 contributed directlyto ESCRT-III-mediated membrane remodeling.In contrast, in vitro experiments on GUVs in-dicated that Vps4 was not required for ESCRT-III-mediated vesicle biogenesis, but rather func-tioned to disassemble ESCRT-III followingvesicle fission (Wollert et al. 2009). However,at least two recent observations suggest a directrole for Vps4 in vesicle formation. First, live-celltotal internal reflection fluorescence microsco-

py (TIRFM) of HIV budding from the plasmamembrane revealed that fluorescently taggedVps4A was recruited to HIV buds before theirrelease, indicating a prefission role for Vps4(Baumgartel et al. 2011). Second, more recentreal-time imaging of GUVs suggests that ESCRT-III can drive membrane invaginations, but ves-icle release was often inefficient (Henne et al.2012). Several studies have noted that GUVsspontaneously deform and generate internalvesicles independent of ESCRT-III-mediatedremodeling (Mayers and Audhya 2011). Theaddition of ESCRT proteins may stabilize oreven promote these spontaneous deforma-tions, completing vesicle scission in a reconsti-tuted, but not physiologically relevant, manner.Thus, further experiments are needed to defin-itively show whether Vps4 is directly involvedin completing ESCRT-mediated membrane re-modeling and fission.

ESCRT COMPLEXES IN CYTOKINESIS

The observations that ESCRT components arerequired for completion of cytokinesis in plants(Spitzer et al. 2006) and in cultured mamma-lian cells (Carlton and Martin-Serrano 2007)have opened new avenues for understanding

i. ESCRT-IIIassembly

ii. Cargosequestration

iii. Budmaturation

iv. Neckconstriction

v. Membranescission

Vps4

Helicalmodel

Domemodel

Snf7filament

ESCRT-II

Vps24 andVps 2

Figure 3. Helical and dome models for ESCRT-III-mediated membrane remodeling and vesicle scission.

Mechanisms of ESCRT-Mediated Membrane Sculpting

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the membrane-sculpting properties of ESCRTs,because cytokinesis is topologically similar tobut spatially distinct from MVB budding. Themammalian cytokinesis process overlaps withtelophase in the cell division cycle and beginswith the formation of an actomyosin-drivencleavage furrow. As the cleavage furrow ingress-es, an intercellular bridge is formed between thetwo daughter cells, and eventually the daughtercells become completely separated when thisbridge resolves at one or two secondary ingres-sions—constriction zones—that form on eitherside of the electron-dense midbody during theprocess known as cytokinetic abscission. Theintercellular bridge typically has a diameter ofmore than 1 mm just before abscission and ispacked with interdigitating spindle microtu-bules, so one of the enigmas in the field ofcell division has been how secondary ingression,microtubule severing, and membrane scissionare coordinated and executed. Proposed mod-els for membrane scission have ranged fromconstriction through intracellular vesicle fusionand assembly of cortical membrane severingmachineries. The involvement of ESCRTs incytokinetic abscission offers mechanistic in-sight that has led to new views of how micro-tubule severing and membrane scission are ex-ecuted.

A key factor in mammalian cytokinesis isthe centrosomal protein CEP55, which is re-cruited to the midbody during mitotic exit asthe activity of an inhibitory kinase, Plk1, ceases(Bastos and Barr 2010). CEP55 in turn recruitsESCRT-I to the midbody via recognition ofmotif containing the amino acid sequence gly-cine-proline-proline (GPP). Interestingly, theESCRT-associated protein Alix is also recruitedto the midbody by a GPP-mediated interactionwith CEP55 (Carlton and Martin-Serrano 2007;Morita et al. 2007). Recruitment of ESCRT-Iand Alix to the midbody functions as a platformfor recruitment and activation of ESCRT-IIIwithout the need for ESCRT-II. The specificfunction of ESCRT-I in cytokinesis remainsunresolved to date. Depletion of TSG101 pro-foundly inhibits cytokinesis, but it is not clearwhether other subunits of ESCRT-I are also in-volved in cytokinesis.

The details of ESCRT-III activation duringcytokinesis are not known, but it has beenshown that Alix interacts directly with the ES-CRT-III subunit SNF7/CHMP4, and that thisinteraction is required for cytokinesis (Moritaet al. 2007). An interaction between the Bro1domain of Alix and the autoinhibitory carbox-yl-terminus of SNF7/CHMP4 might releaseautoinhibition and thereby activate ESCRT-IIIoligomerization. On the other hand, VPS20/CHMP6 also seems to play a role in cytokinesis,although it is less essential than the other 11human ESCRT-III subunits (Table 1) (Moritaet al. 2011). This raises the possibility thatESCRT-III activation during cytokinesis couldbe mechanistically related to ESCRT-III activa-tion during MVB biogenesis.

Electron microscopy of cytokinetic bridgeshas shown the presence of 17-nm helical fila-ments that spiral from the midbody towardthe constriction zones (Fig. 2D) (Guizetti et al.2011). Because these filaments are CHMP4B-dependent and because helices of CHMP4Bcan be detected by structured illumination mi-croscopy, it is tempting to speculate that the17-nm filaments consist of ESCRT-III subunits,although this remains to be formally shown.Intriguingly, filaments composed of yeast Snf7measure �4 nm in diameter, but bundle to-gether in pairs, forming protofilaments �9–12 nm in diameter (Henne et al. 2012). In fur-ther support, spiraling CHMP4B filaments�5–6 nm diameter have also been observedwhen overexpressed in COS-7 cells (Hansonet al. 2008).

Three alternative models have been pro-posed for ESCRT-mediated formation of theconstriction zone during cytokinesis (Fig. 4).According to the “spiral-ingression” model,ESCRT-III-containing spirals wind from themidbody to define the abscission zone at theend of the narrowing spiral. Here, Spastin isrecruited by CHMP1B to sever microtubules,and membrane severing is mediated directlyby the ESCRT filaments (Yang et al. 2008; Gui-zetti et al. 2011). A variant of this model is the“sliding spiral” model in which ESCRT-III fila-ments start to form at the midbody, and a spiralfilament slides along the intracellular bridge to

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form an abscission zone about 1 mm from themidbody (Elia et al. 2011). A third alternative isthe “vesicle-mediated ingression” model, inwhich FIP3-containing vesicles derived from re-cycling endosomes define the abscission zoneby causing narrowing of the intercellular bridgethrough fusion with the plasma membrane(Schiel et al. 2012). According to this lattermodel, ESCRT-III filaments are recruited afterthe abscission zone is formed. Common to thethree models is that ESCRTs mediate the finalabscission between the two daughter cells. As isthe case with scission of MVB vesicles, severalalternative explanations have been put for-ward, including membrane constriction causedby inward-directed curvature of ESCRT-III fil-aments, constriction caused by remodeling ofESCRT-III filaments, or ESCRT-III-mediatedpromotion of spontaneous lipid-driven mem-brane bulging (Fededa and Gerlich 2012). Thesemodels for cytokinesis are in agreement withcurrent models for ESCRT-mediated MVBmorphogenesis. In particular, the “spiral ingres-sion” model is consistent with the visualizationof yeast ESCRT-III spirals and helices in vitro.

As with MVB biogenesis, the ATPase activityof VPS4 is essential during cytokinesis (Carltonand Martin-Serrano 2007; Morita et al. 2007).

VPS4 is recruited at a late stage during the ab-scission process through interaction with theESCRT-III like IST1 protein (Agromayor et al.2009; Bajorek et al. 2009), which is negativelyregulated by another MIT-domain-containingprotein, MITD1 (Hadders et al. 2012; Lee etal. 2012). It is interesting to note that IST1,while being essential for mammalian cytokine-sis, is dispensable for viral budding (Agromayoret al. 2009), illustrating a point of divergencebetween these two ESCRT-III-dependent mem-brane scission processes. The recruitment ofVPS4 occurs after that of CHMP4B and co-incides with membrane cutting (Elia et al.2011). This is consistent with the possibilitythat VPS4 could provide the mechanochemicalforce for membrane severing as such, but alsowith the scenario that it could serve in ESCRT-III disassembly and recycling.

A relatively frequent event during mamma-lian cell division is the entrapment of laggingchromosomes in the intercellular bridge. Toprevent chromosomal damage during cytokine-sis, the cells are equipped with an abscissioncheckpoint termed NoCut, which senses trap-ping of lagging chromosomes and ensures thatthe time of abscission is delayed sufficiently toallow removal of residual chromatin from the

First ingression

Actomyosinring

Spiral ingression model Sliding spiral model Vesicle-mediatedingression model

Second ingression

GuideCEP55

Tsg101 (ESCRT-I)

ESCRT-III

FIP3-positiveendosome

Abscission

Figure 4. Three models for ESCRT-III-mediated abscission.

Mechanisms of ESCRT-Mediated Membrane Sculpting

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cytokinetic bridge. The chromosomal passen-ger complex (CPC), which consists of AuroraB kinase, INCENP, Survivin, and Borealin, isthe central player in the NoCut checkpoint.It has been unclear how CPC activation canbe translated into cytokinesis arrest, but recentstudies of the ESCRT-III isoform CHMP4Chave revealed an intriguing link between CPCand the abscission machinery (Carlton et al.2012). Whereas depletion of CHMP4B and oth-er ESCRT-III components inhibits or delayscytokinetic abscission, depletion of CHMP4Creduces abscission time by about 30 min inHeLa cells. CHMP4C associates with the Bor-ealin component of CPC and contains a specificinsertion, which includes a site for Aurora B-mediated phosphorylation. Aurora B-mediatedphosphorylation of this site, which is conservedin mammals but not in lower eukaryotes, pro-motes recruitment of CHMP4C to the midbodyand halts abscission. The exact mechanism ofabscission inhibition by CHMP4C remains tobe identified, but it is tempting to speculate thatphosphorylated CHMP4C engages in nonpro-ductive ESCRT-III filaments.

CONCLUDING REMARKS: REMAININGQUESTIONS AND FUTURE STUDIES

Recent studies have highlighted the remarkableability of ESCRT-III to remodel membranesin diverse cellular pathways. Using high-resolu-tion methodologies, the mechanisms that gov-ern this membrane remodeling are beginningto be understood at extreme spatial and tempo-ral detail. However, several outstanding ques-tions remain to be addressed, and will requirenew and clever techniques.

The most pressing question is how the finalmembrane fission event is coordinated. Wheth-er ESCRT-III itself is sufficient to drive this fis-sion, or whether it requires additional factorslike the energy released through ATP hydroly-sis from Vps4 is a closely linked question thatrequires further investigation. We also need abetter understanding of ESCRT-III dynamicsduring membrane remodeling. The high spatialresolution provided by transmission electronmicroscopy fails to provide real-time imaging

of ESCRT dynamics. Similarly, live-cell imagingof ESCRT-mediated cytokinesis and viral bud-ding provides excellent temporal resolution, butis limited by the spatial resolution of visiblelight. New techniques are required to better un-derstand ESCRT-III dynamics, which will hope-fully reveal how these dynamics generate mem-brane-bending forces. Such techniques may alsoprovide insights into how ESCRT-III subunitscoassemble into helices, including whetherVps24 and Vps2 integrate with Snf7 into a het-eropolymer, or bind to the surface of a Snf7homopolymer.

Further high-resolution experiments may al-so reveal other mechanistic distinctions uniqueto the ESCRT pathway. ESCRT-III drives mem-brane remodeling that pushes away from the cy-toplasm, yet it is unclear how it avoids consum-ing itself in this process. Likewise, the vesiclesthat bud into the endosomal lumen by ESCRTscontain no outer vesicle coat, yet show consistentdiameters, suggesting ESCRT complexes regu-late vesicle size. How this is achieved remains amystery, but could be attributed to the uniformsize of ESCRT-III rings, which may set the size ofvesicles by surrounding the membrane surfacethat is transformed into the vesicle (Henneet al. 2012). Other models suggest that raftlikemembrane domains that are induced by ESCRT-binding are sufficient to stabilize vesicle buddingand potentially regulate vesicle diameter (Ro-zycki et al. 2012). Another possibility is thatthe transmembrane cargoes themselves contrib-ute to vesicle morphogenesis through crowding.Here, the physical sterics of cargo packing maypromote the membrane bending initially gener-ated by ESCRTs. In line with this, experimentsusing high concentrations of GFP moleculestethered to GUVs show that clustered proteinsare sufficient to drive membrane bending, al-though the significance of this needs to beshown in living cells (Stachowiak et al. 2012).Indeed, how ubiquitinated cargoes are initiallycaptured, sequestered, and loaded into vesiclesby ESCRTs requires additional study. Recentwork shows that EGF receptors are transferredfrom ESCRT-0 to ESCRT-III by the coordinatedaction of HD-PTP/PTPN23 and UBPY (Aliet al. 2013). This implies a “hand-off” in which

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cargoes are simultaneously deubiquitinated andloaded into forming vesicles, and should be in-vestigated further.

Finally, further studies are needed to revealthe molecular architecture of ESCRT-III com-plexes at atomic resolution. Numerous electronmicrographs exist for ESCRT-III assemblies, butthese cannot reveal details like protein–proteininterfaces. Currently, crystal structures exist forportions of VPS24/CHMP3, SNF7/CHMP4,and IST1 (Muziol et al. 2006; Bajorek et al.2009; Martinelli et al. 2012). However, thereare no structures of either full-length ESCRT-III subunits, or ESCRT-III hetero-oligomers.Solving such structures will provide insightsinto the molecular interfaces along subunit–subunit contacts. Such structures may even beable to be fitted into existing protein densitiesderived from electron micrographs, revealingthe molecular architecture of ESCRT-III inhigher resolution. No doubt future studies willcontinue to reveal new and exciting propertiesof this novel membrane sculpting machinery.

ACKNOWLEDGMENTS

The authors thank members of the Emr andStenmark labs for helpful discussions. W.M.H.is sponsored by a Sam and Nancy Fleming Re-search Fellowship. S.D.E. is supported by a Cor-nell University Research Grant. H.S. is support-ed by the Research Council of Norway and theEuropean Research Council.

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2013; doi: 10.1101/cshperspect.a016766Cold Spring Harb Perspect Biol  William Mike Henne, Harald Stenmark and Scott D. Emr Molecular Mechanisms of the Membrane Sculpting ESCRT Pathway

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