Unconventional Functions for Clathrin, ESCRTs, and Other ...cshperspectives.cshlp.org/content/6/9/a017004.full.pdfthree-legged triskelion-shaped molecule made up of three clathrin

Unconventional Functions for Clathrin,ESCRTs, and Other Endocytic Regulatorsin the Cytoskeleton, Cell Cycle, Nucleus,and Beyond: Links to Human Disease

Frances M. Brodsky1, R. Thomas Sosa2, Joel A. Ybe3, and Theresa J. O’Halloran2

1Department of Bioengineering and Therapeutic Sciences, Departments of Pharmaceutical Chemistryand Microbiology and Immunology, The G.W. Hooper Foundation, University of California,San Francisco, San Francisco, California 94143-0552

2Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas,Austin, Texas 78712-1095

3Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405

Correspondence: [email protected]

The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required fortransport) proteins are well defined in endocytosis. These proteins can also participate inintracellular pathways that are independent of endocytosis and even independent of themembrane trafficking function of these proteins. These nonendocytic functions involve un-conventional biochemical interactions for some endocytic regulators, but can also exploitknown interactions for nonendocytic functions. The molecular basis for the involvementof endocytic regulators in unconventional functions that influence the cytoskeleton, cellcycle, signaling, and gene regulation are described here. Through these additional functions,endocytic regulators participate in pathways that affect infection, glucose metabolism,development, and cellular transformation, expanding their significance in human healthand disease.

The discovery and characterization of clathrin(Pearse 1975) initiated molecular definition

of the many endocytosis regulators described inthis collection, which mediate the clathrin-de-pendent and -independent pathways for mem-brane internalization (see Kirchhausen et al.2014; Mayor et al. 2014; Merrifield and Kakso-nen 2014). In accompanying reviews, we haveseen how these endocytic pathways influence

nutrition and metabolism (see Antonescu etal. 2014), signal transduction (see Bokel andBrand 2014; Di Fiore and von Zastrow 2014),neuronal function (see Morgan et al. 2013;Cosker and Segal 2014), infection and immuni-ty (see ten Broeke et al. 2013; Cossart and Hel-enius 2014), tissue polarity and development(see Eaton and Martin-Belmonte 2014; Gonza-lez-Gaitan and Julicher 2014), and migration

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

Additional Perspectives on Endocytosis available at www.cshperspectives.org

Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a017004

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and metastasis (see Mellman and Yarden 2013).Recently, it has been established that some en-docytic regulators have molecular propertiesthat expand their functions beyond endocyto-sis. These include molecular interactions thataffect the microtubule and actin cytoskeletons,nuclear translocation that influences gene regu-lation, and the formation of membrane-associ-ated scaffolds that serve as signaling and sortingplatforms. Through these diverse nonendocyticfunctions, endocytosis regulators play addition-al roles in cell division, pathogen infection, celladhesion, and oncogenesis. In this article, wereview the nonconventional behavior of endo-cytic regulators, first discussing the molecularproperties that enable their moonlighting func-tions and then discussing the cellular processesand disease states that are influenced by thesefunctions.

NOVEL INTERACTIONS ANDBIOCHEMISTRY OF ENDOCYTICREGULATORS

Clathrin

The polyhedral lattice of the conventionalclathrin coat is formed by self-assembly of thethree-legged triskelion-shaped molecule madeup of three clathrin heavy-chain (CHC17) sub-units, encoded by the CTLC gene on humanchromosome 17 (for a review of clathrin bio-chemistry, see Brodsky 2012; Kirchhausen et al.2014). The CHC17 triskelion has bound cla-thrin light-chain (CLC) subunits that span thecentral portion from the vertex to the bendin the knee. CLC confers stability to the trime-rization domain of the triskelion and rigidityto the proximal leg portion, and its binding tothe triskelion knee via a labile salt bridge canstraighten the knee to an angle that is incompat-ible with assembly, enabling negative regulationof clathrin self-assembly (Wilbur et al. 2010b).The CLC subunit also links the actin-organizinghuntingtin-interacting protein 1 (Hip1) and itsrelated mammalian protein Hip1R or the yeasthomolog Sla2p to the clathrin lattice (Brodsky2012).

Clathrin with bound CLC is the active formfor endocytosis and vesicular transport for all

eukaryotes. When vertebrates emerged, a sec-ond isoform of clathrin was enabled by geneduplication, generating the CTCL1 gene on hu-man chromosome 22 (and homologous genesin other species) encoding the CHC22 heavychain. This gene is present in most vertebratesbut has degenerated into a pseudogene in mice.The encoded CHC22 protein is 85% identicalin sequence to CHC17, with differences equal-ly distributed along the length of the protein.CHC22 trimerizes but does not bind CLC.In spite of being missing from mice, CHC22clathrin plays a key nonendocytic role in hu-mans, mediating retrograde membrane trafficfrom late endosomes to the trans-Golgi network(TGN) (Esk et al. 2010). This pathway generatesthe intracellular storage compartment for theinsulin-responsive GLUT4 glucose transporterin fat and muscle, where CHC22 is expressedat its highest levels (Vassilopoulos et al. 2009),and is a minor pathway in other tissues whereCHC22 is present at low levels. Studies to datesuggest that CHC22 clathrin is more stable inits membrane association than CHC17 and thatits biochemical behavior is different becauseof its lack of CLC association and more limitedinteraction with adaptor molecules (Liu et al.2001).

The amino-terminal domain of CHC17clathrin binds the AP1, AP2, AP3, and GGAadaptor molecules, but it can also participatein microtubule binding (Royle 2012). At cellcycle onset, Aurora kinase A activation phos-phorylates TACC3, a microtubule-stabilizingprotein. Phosphorylated TACC3 interacts withthe terminal domain of CHC17, and these pro-teins join chTOG in a complex that stabilizesmicrotubule-based structures including cen-trosomes and the kinetochore fibers of the mi-totic spindle (Booth et al. 2011; Foraker et al.2012). Thus, CHC17 decorates the mitoticspindle in mammalian cells, and its presencecontributes to centrosome integrity. Althoughthere is some debate about the molecular detailsof interaction within the CHC17–TACC3–chTOG complex, it is clear that this complexplays a cellular role in microtubule functionthat is completely distinct from the membranetraffic function of clathrin and that this role

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begins in S phase upon Aurora A kinase activa-tion (Cheeseman et al. 2011; Foraker et al.2012). Kinetochore microtubule stabilizationby the CHC17–TACC3–chTOG complex re-quires CHC17 trimerization, and the complexappears to act as a cross-bridge within microtu-bule bundles, exploiting the multimeric prop-erties of CHC17 (Booth et al. 2011). The evo-lutionary extent of CHC17 association withTACC3 and chTOG is not clear, but spindle-associated clathrin is evident in Drosophila, aswell as all vertebrates that have been examined.

CHC17 clathrin is also capable of nucleartranslocation, where it can influence tran-scriptional activation by p53. For CHC17 totranslocate, the conventional clathrin trimericmolecule must be made monomeric. Dissocia-tion of CLC and consequent destabilization ofthe carboxy-terminal trimerization domain ofCHC17 increases levels of CHC17 monomer,although this is still a minor form of CHC17in the cell. Atomic-level details of what struc-tural elements contribute to the stability ofthe CHC17 trimer were revealed by the X-raystructure of the isolated trimerization domain(CTXD) without bound CLC (Fig. 1) (PDB code3QIL) (Ybe et al. 2013). In the structure, theposition of Helix 7j is shifted by �10 A towardHelix 7h compared with lower-resolution mod-els with bound CLC (Fotin et al. 2004; Wilburet al. 2010b). In the structures with bound CLC(assembled clathrin [PDB code 1X14] [Fotinet al. 2004] and clathrin hub [PDB code 3LVG][Wilbur et al. 2010b]), the space between Helices7j and 7h is occupied by CLC density, suggestingthat the orientation of Helix 7j is dependent onCLC (Wilbur et al. 2010b; Ybe et al. 2013). TheCTXD structure raises new questions of whatcan happen to the global topology of trimericclathrin if CLC is not present or if the carboxylterminus of CLC that contributes to the stabilityof the trimerization domain (Ybe et al. 2007b) ismoved away. It is important to point out that theimpact of CLC on clathrin trimer stability variesin different organisms. CLC knockouts in Sac-charomyces cerevisiae but not in Dictyostelium(Wang et al. 2003) result in detrimerization(Chu et al. 1996; Huang et al. 1997). The detailsof how nontrimeric clathrin enters the nucleus

are not yet known, but the binding of monomer-ic CHC17 to p53 suggests that entry may befacilitated by the nuclear translocation signalin p53. It will also be important to find outwhether there is any regulatory role for CLC.The evidence so far suggests that the bindingof p53 is only possible when CLC is not boundto CHC17 (Enari et al. 2006). The detrimeriza-tion switch hypothesis (Fig. 1) offers a novelmechanism to convert the topology of clathrinfor new function in the nucleus.

Endocytic Accessory Proteins

Many endocytic accessory proteins, includingeps15 and epsin, are known to translocate tothe nucleus, although their function there re-mains poorly understood (Benmerah 2004; Pi-lecka et al. 2007). Among those with implicatedfunction is Hip1, which in addition to its rolein linking CLC to the actin cytoskeleton, can alsopiggyback on androgen receptor (AR) to get in-side the nucleus to regulate transcription (Millset al. 2005). Hip1 is a member of the HIP fami-ly of proteins that includes Hip1R (R for relat-ed), the Dictyostelium Hip homolog, and yeastSla2p. Originally identified as a binding part-ner of huntingtin in the brain (Kalchman et al.1997; Wanker et al. 1997), Hip1 is a multido-main dumbbell-shaped protein that exists as aparallel homodimer (Engqvist-Goldstein et al.2001). Hip1 binds to phospholipids throughan ANTH (AP 180 amino-terminal homology)domain in its amino terminus (Mishra et al.2001; Legendre-Guillemin et al. 2004). Hip1contains FXDXF and DPF motifs (X denotesany amino acid) to bind AP2 and has a clathrinbox (332LMDMD) that interacts with the ami-no-terminal domain of clathrin in vitro (Brettet al. 2002). The central coiled-coil of the Hip1molecule serves as a dimerization domain and isinherently flexible (Ybe et al. 2007b; Niu andYbe 2008; Wilbur et al. 2010a; Fontaine et al.2012). The structure of Hip1 (PDB 2NO2) re-veals that the CLC binding surface originallymapped to the 484DLLRKN region (Legendre-Guillemin et al. 2005) is composed of a shortsolvent-exposed hydrophobic patch that is in-terrupted by a basic patch centered on K494

Unconventional Functions of Endocytosis Regulators

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Leg 3

AB

C D

Top latch

Leg 2Leg 1

CME

Trimer/CLC MitochondrionMonomer +

dimer (with CLC)

???

Monomer/CLC

CLC freedbeforeentry

1

2

3

4

5

Nucleus

ER

pCs transition

Helix 7j position shiftHelix 7j

TX1

pCs

Bottom latch

Helix tripod distortions

Figure 1. Topology switch that disrupts clathrin trimerization. The clathrin topology switch is proposed to have atop and a bottom latch that hold the triskelion together. The formation of monomeric and dimeric clathrinrequires these latches to be opened. Monomeric clathrin can translocate to the nucleus for nonendocytic clathrinfunctions. (A) The top view of the clathrin trimerization domain structure (PDB 3QIL) shows that each heavy-chain leg contains is own topology switch comprising Helix 7j (teal), TX1 (green), and pCs (red, predicted toundergo coil-to-strand transition). The interface between neighboring legs (dashed lines) involves pCs in oneleg and TX1 in the next leg over. (B) The top latch elements of the topology switch (same color coding as in A).Helix 7j in 3QIL without the light chain is moved by �10 A compared with the same helix (gray) in a structurewith the light chain (PDB 1X14). This suggests that the light chain could be involved in determining theorientation of Helix 7j. The red strand pCs is coiled in 1X14 (also in 3LVG). This is significant because thisstretch—predicted to undergo a strand-to-helix transition—would impact the interface between the legs in A ifits conformation changes. (C) The bottom latch of the topology switch in the helix tripod of the trimerizationdomain must be broken to completely free a leg. The distortion in 3QIL is partly due to disruptions in the helixtripod (see arrows) when the helix tripod of 3QIL is compared with that of 1X14. (D) Cartoon of a eukaryoticcell shows a pool of trimeric clathrin separate from that participating in clathrin-mediated endocytosis (CME).This population detrimerizes (step 1) to yield a mixture of monomers and dimers with bound light chain (CLC,red). Dimers may be useful as is or further converted to monomeric clathrin (step 2). Monomers enter thenucleus in step 3. It is also possible that CLC is first released in the cytosol (step 4) before entry in step 5 (yellow).In this hypothesis, step 4 and 5 produce a mixed nuclear pool of monomeric clathrin with and without the lightchain. Although CLC-free monomeric clathrin can interact with p53 in the nucleus, further studies are needed tofully understand the function of nuclear clathrin.

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(Ybe et al. 2007a). There is a second CLC bind-ing determinant (K474) upstream from theDLLRKN region (Ybe et al. 2009). The car-boxy-terminal region of Hip1 (the crystal struc-ture of the Hip1R region is known; PDB 1R0D)(Brett et al. 2006) has a THATCH domain thattethers clathrin-coated vesicles to the cellularcytoskeleton. Immunoprecipitation studies in-dicate the Hip1 coiled-coil domain can bind an-drogen receptor (AR), and confocal microscopyshows a significant redistribution of Hip1 to thenucleus, where it regulates AR transcription(Mills et al. 2005).

Hip1 can also influence cellular apopto-sis when bound to an accessory protein that ispredicted to contain a death effector domain(DED). HIPPI (Hip1 protein interactor) formsa proapoptotic complex with Hip1 that somehave suggested contributes to the apoptoticdeath of specific neurons in Huntington’s dis-ease (Hackam et al. 2000; Gervais et al. 2002;Niu and Ybe 2008). The crystal structure of theHIPPI-binding domain of Hip1 (PDB 2QA7)suggests that the formation of the HIP1/HIPPIcomplex is mediated by the flexibility of Hip1(Niu and Ybe 2008). There are conflicting datain the literature about the ability of HIP1/HIPPI to activate both intrinsic (caspase-8 in-dependent) and extrinsic (caspase-8 depen-dent) cell death pathways (Hackam et al. 2000;Gervais et al. 2002). The fact that alternativesplicing of the HIP1 gene yields two splice var-iants (Chopra et al. 2000) could reconcile whyboth pro- and antiapoptotic effects involvingHip1 have been reported (Rao et al. 2001).

The Numb protein binds the AP2 endocyticadaptor, but it also translocates to the nucleus,where it has been implicated in growth controland breast cancer metastasis. Best known for itsrole in regulating the Notch signaling pathway,the Numb protein is portioned asymmetricallyinto the daughter cells during Drosophila neuro-genesis. In cells inheriting Numb, Numb directlybinds to the Notch receptor anda-adaptin of theAP2 complex, serving as a scaffold to promoteNotch internalization and degradation (Guoet al. 1996; Santolini et al. 2000). Thus, prefer-ential inheritance of Numb regulates Notch dis-tribution and signaling, thereby suppressing the

proliferative potential of the Notch signalingpathway (Berdnik et al. 2002; Wirtz-Peitz et al.2008). Numb also undergoes nucleocytoplas-mic shuttling by binding to the Mdm2 protein,allowing Numb to have a nuclear function inde-pendent of its endocytic regulation of Notchsignaling (Scita and Di Fiore 2010).

The endosomal APPL protein (adaptor pro-tein-containing PH domain, PTB domain, andleucine zipper motif ) also translocates into thenucleus. APPL1 and APPL2 function in the en-docytic pathway as effectors of the early endo-somal protein Rab5 (Miaczynska et al. 2004).After GTP-Rab5 hydrolyzes its GTP, APPL dis-sociates from the Rab5 endosomes and translo-cates into the nucleus. Although APPL1 does notcontain a nuclear localization signal (NLS), itsinteractions with nuclear proteins throughits PH domain may target it to the nucleus.APPL2 contains a PH domain and a putativeNLS that could facilitate its nuclear localization(Miaczynska et al. 2004).

In addition to endocytic roles, the clathrinassembly lymphoid myeloid (CALM) proteinalso has nuclear roles. In endocytosis CALMbinds to several components of the clathrincoat, including clathrin, AP2, and PIP2 (Ahleand Ungewickell 1986; Tebar et al. 1999; Fordet al. 2001; Huang et al. 2004). Here CALMfunctions to regulate endocytic and endosomaltrafficking. However, CALM also plays a role intranscriptional regulation. CALM does not con-tain a nuclear localization signal, yet has beenshown to undergo nuclear translocation (Vec-chi et al. 2001). This could be mediated by in-teractions between CALM and CATS (CALM-interacting protein expressed in thymus andspleen) (Archangelo et al. 2006). Despite lack-ing a canonical nuclear localization signals,CATS preferentially localizes to the nucleus.Thus, CATS may promote nuclear import ofCALM, providing a means for CALM to func-tion as a transcriptional regulator.

ESCRTs

Proteins in the ESCRT (endosomal sortingcomplex required for transport) complexes con-ventionally function in degradative pathways by





sorting ubiquitinated cargo and inducing theformation of multivesicular bodies (MVBs)(see Henne et al. 2013). In brief, this pathwayinvolves the interaction of ESCRT-0 (alsoknown as Hrs) with clathrin and sequestrationof ubiquitinated cargo at endosomes, whichtriggers sequential recruitment of complexesof Vps proteins known as ESCRT-II and -III,leading to membrane deformation and MVBbudding. This is followed by recruitment ofthe Vps4–Vta1 complex, which mediatesESCRT dissociation from membranes (Henneet al. 2011). ESCRTs also act in nonendocyticfunctions including the budding of envelopedviruses from cells, as well as promoting the finalstage of membrane scission during cytokinesis(McDonald and Martin-Serrano 2009; Hurley2010). What these seemingly disparate cellularpathways have in common is that they all sharetopologically similar fusion events. In MVB for-mation, small vesicles bud into the endosomelumen, and in viral budding, the enveloped vi-rus buds from the cytosol into an endosome-like compartment that is connected to the cellsurface. In cytokinesis, when a daughter cell sep-arates from its sister, formation of the abscissionfurrow requires a similar bending of membranes(Fig. 2). Despite topological similarities, theprecise mechanisms differ somewhat for eachprocess as the particular ESCRT complex(es)involved vary for these different pathways(Peel et al. 2011; Morita 2012).

PHYSIOLOGICAL AND DISEASE-RELATEDCONSEQUENCES

Having described the biochemistry of endocyticregulators that facilitates their nonendocyticfunctions, we now address the physiologicalpathways and disease states that are influencedby these functions. We also summarize the novelfunctions mediated by individual proteins inTable 1.

Organizing Actin for Adhesion, BacterialInfection, and Immunity

The CLC–Hip protein–actin interaction is im-portant for clathrin-mediated endocytosis from

membranes under tension such as in polarizedcells in culture and presumably most tissues(Boulant et al. 2011). CLC–Sla2p interactionis also required for endocytosis from mem-branes under turgor pressure such as the yeastplasma membrane and the contractile vacuolemembrane in Dictyostelium cells (Archangeloet al. 2006; Stavrou and O’Halloran 2006; Agha-mohammadzadeh and Ayscough 2009). The in-teraction of CLC with actin via Hip proteins andthe additional binding of Hip1R to cortactinin metazoans confer an additional role on theclathrin lattice as an actin organizer during ad-hesion and infection by large viruses or bacteria(see Cossart and Helenius 2014). These path-ways represent situations in which the clathrinlattice itself is not performing an endocyticfunction but acting as a platform to recruit theHip proteins through their interaction with

A′

AB

MVBVirus

ESCRT-I, -III

ESCRT-0–III

Midbody

ESCRT-III

C

B′

C′

Figure 2. ESCRT proteins function in similar mem-brane fission events. (Left) The endosomal sortingcomplexes required for transport (ESCRT) complex-es have nonendocytic roles in viral budding (A), mul-tivesicular body (MVB) formation (B), and cytoki-nesis (C). (Right) The outward bending of the plasmamembrane enveloped around a virus (A0) and vesiclesbudding into the lumen of multivesicular bodies (B0)require membrane fusion leading to fission. Similar-ly, ESCRTs induce closely apposed membranes (C0)to fuse and liberate the midbody during cytokinesis.

F.M. Brodsky et al.




CLC in order to influence local actin cytoskele-ton arrangements.

During the initial stages of adherens junc-tion (AJ) formation, clathrin is recruited to sitesof cell–cell interaction through its interactionwith cadherin in the mammalian junction (Bo-nazzi et al. 2012). If this recruitment is inhib-ited, actin cables do not properly form at theAJ. Normal actin morphology at the AJ alsodepends on the presence of the CLC and Hipproteins and is concomitant with tyrosinephosphorylation of CHC17. Listeria monocyto-genes exploits this pathway for intracellularinfection by binding to cadherin through itsInternalin A protein. Binding induces CHC17phosphorylation, which is required for actin re-cruitment via CLC–Hip protein interactions(Bonazzi et al. 2011). Actin then surroundsthe clathrin associated with cell-bound Listeriaand mediates internalization of the organism,which is too large to enter cells through clath-rin-coated vesicles, the latter being limited to

200 nm in diameter. CHC17 clathrin is com-parably used during formation of actin pedes-tals during pathogenic interaction of entero-pathic Escherichia coli (EPEC) with host cells.CHC17 is phosphorylated at the bacterium–host interface and recruits a clathrin-associatedlayer of actin, which forms a pedestal structurein the host cell enabling transfer of pathogenicmolecules from the attached bacterium into thehost cytoplasm (Bonazzi et al. 2011).

Tyrosine phosphorylation of CHC17 wasfirst observed in response to epidermal growthfactor (EGF) binding to its receptor and wasshown to be a function of SRC family kinasesignaling downstream from receptor tyrosinekinase activation (Wilde et al. 1999). SRC familykinase activation by the T-cell receptor andB-cell receptor (surface immunoglobulin) onlymphocytes also induces CHC17 phosphory-lation and is required for endocytic clearanceof these receptors (Stoddart et al. 2002; Crotzeret al. 2004). The target phosphorylation sites are

Table 1. Nonendocytic functions of endocytic regulators

Endocytic regulator Nonendocytic function Pathway significance

Clathrin-CHC17isoform (CLC bound)

Microtubule binding in a complex with TACC3 andchTOG on the mitotic spindle and in centrosomes

Cancer/cell cycle

Nuclear translocation and p53 transcription CancerPlatform for actin organization during viral and

bacterial infection and adherens junctionformation (dependent on CLC and Hip proteins)

Infection/development

Cytokinesis (also a role for dynamin) Cancer/cell cycle

Clathrin-CHC22isoform (no CLC)

Retrograde sorting at the endosome for cargotargeted to the TGN and to the insulin-responsiveGLUT4 compartment

Metabolism/type II diabetes

Hip1 Androgen receptor binding and nucleartranslocation for transcriptional regulation

Cancer

Apoptosis effector via HIPPI binding Cancer/development

Numb Nuclear translocation and influence on growthcontrol and p53 transcription

Cancer/development

Regulation of Notch signaling Signaling

APPL proteins Nucleosome remodeling and transcriptional control Signaling

CALM Transcriptional regulation Cancer

ESCRT complexes Abscission in cytokinesis Cell cycleVirus budding Infection





tyrosine 1477 and 1487 (Bonazzi et al. 2011).Based on their location within the proximalleg of the triskelion and the fact that dephos-phorylation is also required for receptor inter-nalization, we hypothesize that the tyrosinephosphorylation modification might “freeze”clathrin lattices at sites of signaling by prevent-ing addition of further triskelia. CHC17 phos-phorylation would thus allow use of clathrinto organize actin locally, as well as synchronizeinternalization events upon dephosphoryla-tion. In the case of the pathogenic bacteria–host-cell interactions described above, SRCfamily kinases are required and stabilized clath-rin-coated pits are observed at the bacterial–host interface, supporting the idea that bacteriaexploit this clathrin modification to organizeactin for their own purposes. Observation ofclathrin and actin at the T-cell immunologicalsynapse where the TCR signals to phosphorylateclathrin suggests a role for clathrin in local actinorganization as well as receptor clearance (Cala-bia-Linares et al. 2011).

Sorting Patches Influence Recycling,Degradation, and Metabolism

Purified CHC17 clathrin with or without theCLC will self-assemble into closed polyhedralprotein baskets, resembling the coats on clath-rin-coated vesicles isolated from brain and oth-er tissues. Electron microscopy (EM) and totalinternal reflection fluorescence (TIRF) micros-copy have revealed that CHC17 clathrin boundto the AP2 adaptor molecule also forms extend-ed flat patches at cell–substrate interfaces (Ag-geler and Werb 1982; Saffarian et al. 2009). Ad-ditionally, clathrin on sorting endosomes andlate endosomes forms nonvesicular patches. Onearly endosomes, these patches are adjacent toregions of retromer assembly at the edge of tu-bules (Popoff et al. 2007). It has been suggestedthat these clathrin patches collect cargo mole-cules that are then somehow transferred to theretromer-induced tubule. There is, however, nobiochemical evidence for interaction betweenCHC17 and the particular sorting nexins impli-cated in this pathway (McGough and Cullen2013). CHC17 is also recruited to endosomes

by ESCRT-0 (Hrs). Hrs-associated CHC17 ap-pears in EM as a double-layered coat (Raiborget al. 2002), but the details of the molecularcontributions to the morphology of this coathave yet to be established. Clathrin does notparticipate in further steps of MVB formation,which are ESCRT-mediated.

Interaction of CHC22 with sorting nexin 5(SNX5), a retromer component, was identifiedby yeast two-hybrid studies and confirmed bymammalian protein expression (Towler et al.2004). This limited biochemical analysis sug-gests that CHC22 might interact with retromerat late endosomes in an analogous function tothat observed for CHC17 at sorting endosomes.The assembly behavior of CHC22 is still undercharacterization, and it is not yet known wheth-er CHC22 exerts its function as a sorting patchor as a vesicle coat or both. Nonetheless, itsfunctional role in contributing to formation ofthe GLUT4 storage compartment (GSC) inskeletal muscle and fat cells is clearly established(Vassilopoulos et al. 2009). The GSC, describedelsewhere in this collection (see Antonescu et al.2014), is a complex network of tubules and ves-icles containing the GLUT4 glucose transporter,unique to muscle, fat, and cerebellar neurons(Bryant et al. 2002; Bakirtzi et al. 2009). TheGLUT4-containing vesicles and possibly sometubules are released to fuse with the plasmamembrane upon insulin signaling, thereby al-lowing glucose clearance from the blood intothe responsive tissues. Depletion of CHC22 pre-vents GSC formation, whereas CHC22 accumu-lates on the expanded GSC observed in muscleof insulin-resistant patients with type 2 diabetes(Vassilopoulos et al. 2009). Whether this accu-mulation contributes to pathogenesis or justreflects the accumulation of sorted cargo is un-der investigation.

Signaling and Apoptotic Functionin Development

Clathrin has recently been implicated in the or-ganization of signalosomes involved in Wntsignaling at the cell surface (Kim et al. 2013).The Wnt signaling pathway is a critical player indevelopment, tissue homeostasis, and metabo-

F.M. Brodsky et al.




lism. Wnt binding to its receptors leads to re-ceptor phosphorylation, a process that dependson formation of a signaling patch known asa signalosome. Signalosome formation is de-pendent on production of PtdIns(4,5)P2 earlyin the Wnt signaling cascade. The specificityof the AP2 adaptor for PtdIns(4,5)P2 causesclathrin accumulation at Wnt signaling sites,and this process was shown to play a role instabilizing signalosome components at the plas-ma membrane, without leading to Wnt recep-tor internalization. Thus, clathrin functions asa scaffold for signalosomes rather than a vehiclefor endocytosis.

The phenotype of Hip1-knockout mice sug-gests a role for Hip1 in late stages of spermato-genesis in the testes (Rao et al. 2001). Hip1 isalso involved in forming red blood cells, becausemutations of Hip1 lead to hematopoietic ab-normalities (Oravecz-Wilson et al. 2004). Thesedevelopmental defects may be a result of Hip1’sinfluence on apoptosis.

Scaffolds for Viral Pathogenesis

HIV exploits several aspects of the endocyticpathway. The Nef protein interacts with bothAP1 and AP2 clathrin adaptors, influencingthe endocytosis of both class I histocompati-bility molecules and CD4 (Roeth et al. 2004;Chaudhuri et al. 2007). The former contributesto immune evasion, and the latter contributesto increased infectivity of HIV particles. Clath-rin also plays a nonendocytic role in HIVparticle maturation. In this case, clathrin is in-corporated into the budding particle as a viralmatrix protein and appears to be contributingits function as a scaffold to organize viral coreproteins (Zhang et al. 2011).

Enveloped viruses engulf themselves withpatches of the cellular membrane as they as-semble and subsequently bud into either endo-some-like compartments or from the plasmamembrane, the location varying for differentviruses and different cell types (Welsch et al.2007; Benaroch et al. 2010). For these buddingpathways, most enveloped viruses, includingHIV, co-opt ESCRT proteins normally used bycells to generate multivesicular bodies (MVBs)

from endosomes. Although MVB formation re-quires the sequential action of ESCRT-0 andthen ESCRT-I–III, enveloped viruses use theirown proteins to substitute for the ESCRT-0, bydirectly linking the ESCRT-I protein TSG101 tothe viral GAG protein, and also bypass ESCRT-II by using the adaptor protein Alix to linkESCRT-III to GAG (Pornillos et al. 2003; Lan-gelier et al. 2006; Medina et al. 2011). AlthoughESCRT-II may not be used in viral budding(Langelier et al. 2006), a role for ESCRT-II pro-teins was shown in other aspects of HIV infec-tion. siRNA-mediated depletion of the ESCRT-II protein EAP30 blocked the normal traffickingof HIV-1 genomic RNA and resulted in an ac-cumulation of genomic HIV-1 RNA in the nu-cleus and juxtanuclear domains (Ghoujal et al.2012). In this case, it would appear that ESCRT-II is playing a nonendocytic role in the HIV lifecycle.

Cell Division

Temporal control of clathrin inactivation insynchronized cells showed that the interactionof the terminal domain of CHC17 with theTACC3–chTOG complex at the onset of S phaseinfluences both centrosome and spindle integ-rity during the cell cycle. If CHC17 is inacti-vated at S phase by drug-induced cross-linkingof CLC, centrosome maturation is affected dur-ing the onset of mitosis, resulting in fragmentedcentrosomes occur and multipolar spindles(Foraker et al. 2012). chTOG localization to thecentrosome was decreased upon clathrin in-activation, and because chTOG is required forcentrosome maturation (Cassimeris and Mora-bito 2004), clathrin inactivation could possiblyaffect centrosome integrity by failing to com-plex with chTOG. Spindle defects also occurupon clathrin depletion and are attributed todirect binding of clathrin to kinetochore mi-crotubules via the TACC3–chTOG complex(Booth et al. 2011). Loss of clathrin from thespindle consequently induces chromosomedysjunction (Royle et al. 2005).

Clathrin and associated proteins also playa role in cytokinesis, the last step of cell divi-sion. This role for clathrin is observed in a wide





variety of organisms from protists to plant cellsto animal cells despite widely different mecha-nisms of cell division in these cells. In plants,cell division is accomplished by building a cellwall between the two daughter cells. Here thepre-prophase band and the phragmoplast, thelocations where the cell plate will fuse together,are sites of abundant clathrin activity (Jurgens2005; Tahara et al. 2007; Karahara et al. 2009). Inanimal cells, an actomyosin contractile ring thatconstricts at the equator facilitates cell division(Pollard 2010). Live-cell imaging has shownthat after anaphase, GFP-labeled clathrin coatedstructures move toward the equator and disap-pear, suggesting a role for clathrin in remodel-ing the plasma membrane at the equator (War-ner et al. 2006). It is not known precisely whatessential role these clathrin-coated structuresplay, but live-cell imaging in Dictyostelium cellssuggests that this event is late in cytokinesis,after constriction of the actomyosin ring (Ger-ald et al. 2001). A role late in cytokinesis forclathrin and associated proteins was confirmedrecently in animal cells by showing that siRNAdepletion of several endocytic proteins blockedcompletion of abscission but not constrictionof the cleavage furrow in dividing cells (Smithand Chircop 2012). Additionally, dynamin playsa role in abscission (Joshi et al. 2010). Becauseof their ability to capture membrane and spe-cific proteins and lipids, clathrin-coated struc-tures could coordinate the massive remodelingat the equator of either plant or animal cellsand thereby establish the essential membraneor protein composition needed to complete cy-tokinesis. Several lines of evidence indicate thatrab11 recycling endosomes play crucial roles insupplying membrane at the cleavage furrow latein cytokinesis (Wilson et al. 2005). Genetic andother evidence has implicated many othermembrane trafficking proteins as key playersin cytokinesis (McKay and Burgess 2011).

During late stages of cytokinesis, proteinsin the ESCRT-III complex are recruited to themidbody complex. Here they are thought to as-sist in the severing of membranes in the inter-cellular bridge that connects the daughter cells,analogous to their role in assisting fusion inthe formation of MVBs or in budding of envel-

oped viruses, although the intercellular bridgethat occurs during cytokinesis requires fusionacross a larger distance than occurs in MVB or avirus budding (Fig. 2). Recent high-resolution,live-cell imaging of the bridge in dividing cellscoupled with electron tomography shows thatfilaments consistent with the size of filamentsmade by ESCRT-III proteins could drive thisprocess and also provide a scaffold to recruitproteins required for the removal of the mid-body (Guizetti et al. 2011). Recent evidence sug-gests that ESCRT-III complexes could also assistin regulating the final stages of cytokinesis.Members of the ESCRT-III complex promotethe Aurora B abscission checkpoint (Carltonet al. 2012), a cell regulation role that could oc-cur independently of the fusion function typi-cally associated with ESCRT complexes.

Transcriptional Function andOncogenesis

Loss of p53 is associated with oncogenesis be-cause p53 activation controls genes for growtharrest or apoptosis in response to DNA damage.Clathrin heavy chain has been shown to be ap53 activator (Enari et al. 2006). The exogenousexpression of CHC17 increases the transacti-vation of p53-responsive promoters ( p53AIP1,P21waf1, p53R2, and Noxa), whereas RNAi si-lencing reduces p53 transcriptional activity(Enari et al. 2006). There are data showingthat CHC17 recruits histone acetyltransferasep300 to p53 to promote p53-mediated tran-scription (Enari et al. 2006; Ohmori et al.2008). Nuclear fractionation and immuno-elec-tron microscopy studies detect a small fraction(�5%) of CHC17 in the nucleus (Enari et al.2006). Luciferase-based reporter assays indicatethat trimeric clathrin is not required to partic-ipate in p53-mediated transcription and thatthere is increased nuclear localization whenthe trimerization domain is artificially deleted(Ohmori et al. 2008). However, this begs thequestion: How does clathrin for the nucleusdetrimerize in the cytosol? The detrimerizationswitch hypothesis offers a way to understandhow heavy-chain legs of trimeric clathrin canbe detrimerized without removing the trimeri-

F.M. Brodsky et al.




zation domain (Fig. 1). CHC17 clathrin hub(amino acids 1074–1675) is reduced to a mix-ture of nontrimeric molecules when cysteine-1573—located in the middle of mobile Helix7j—is mutated to alanine (Ybe et al. 2013).These nontrimeric CHC17 hub molecules lo-calize to the nuclei of a variety of human cancercell lines (Ybe et al. 2013). It is not yet knownwhat, if any, role CLC plays in dislocating clath-rin heavy-chain legs for nuclear translocation.There is some evidence suggesting that CLC isinvolved because in vitro studies show that thecarboxyl terminus of CLC contributes to thestability of clathrin triskelion (Ybe et al. 2003,2007b, 2013). The proposed detrimerizationswitch (Fig. 1) has potential implications foroncogenesis if further studies continue to sup-port a connection between monomeric clathrinand p53 function.

Hip1R was the first human homolog ofSla2p to be identified as a cofactor in clathrin-mediated endocytosis (CME) (Engqvist-Gold-stein et al. 1999, 2001). Like its relative, Hip1 isalso a player in CME (Mishra et al. 2001; Raoet al. 2001; Waelter et al. 2001), but is func-tionally distinct from Hip1R because Hip1 isinvolved in human cancer. Analysis of tissuesamples revealed that Hip1 is a consistent mark-er for solid tumors, with elevated levels in celllines derived from breast, colon, kidney, lung,melanoma, ovarian, and prostate tumors (Raoet al. 2001; Hyun and Ross 2004). It was ob-served that the overexpression of Hip1 is corre-lated to the progression of prostate cancer (Raoet al. 2001). As the cancer progresses, there arechanges in the transcriptional response andexpression of androgen receptor (AR) (Chenet al. 2004). AR is a member of the nuclearhormone receptor superfamily of transcriptionfactors and has polyglutamine and polyglycinerepeats in the amino-terminal domain, whichinteract with transcriptional coregulators. Thepolyglutamine tract in AR may also facilitate itsbinding to Hip1, which can interact with pro-teins with polyglutamine repeats. Hip1 bindsto AR through its central coiled-coil domain,and stimulation by androgen causes Hip1 tobe recruited to DNA response elements (Millset al. 2005). AR-mediated transcription is di-

minished when Hip1 expression is loweredand activated when Hip1 is overexpressed (Millset al. 2005). Hip1 has a functional, but weaknuclear localization signal (NLS) in its carboxylterminus (Mills et al. 2005). The activities ofHip1 in the nucleus link this endocytic proteinto the transcriptional modulation of hormone-responsive genes. Furthermore, the ability ofHip1 to be in the nucleus raises the possibilitythat nuclear clathrin could associate with Hip1to mediate nonendocytic cellular events that wedo not yet know about. Hip1 may also influenceoncogenesis via signaling. In chronic myelomo-nocytic leukemia, a gene translocation fusesHip1 to platelet-derived growth factor b recep-tor (PDGFbR) (Ross et al. 1998).

The endocytic role of the clathrin adap-tor Numb and the importance of Numb inregulation of the Notch signaling pathway indeveloping embryos have been extensively char-acterized. However, recent studies show an ad-ditional role for Numb as a tumor suppres-sor independent of its role in endocytosis andNotch regulation. Numb binds to both p53 andthe E3 ubiquitin ligase Mdm 2, which is knownto shuttle Numb into the nucleus and is a potentregulator of the tumor suppressor p53 (Juven-Gershon et al. 1998; Colaluca et al. 2008). Ad-ditionally, Numb forms dimeric and trimericcomplexes with Mdm2 and p53, thereby inhib-iting degradation of p53 (Colaluca et al. 2008).Therefore, loss of Numb can result in increasedcell proliferation by two separate means: (1)an increase in Notch signaling, and (2) attenu-ation of p53 signaling. The biological impor-tance of Numb signaling is underscored by theobservation that about half of human breastcarcinomas display a loss of Numb-mediatedcontrol of Notch signaling (Pece et al. 2004).

Although the APPLs normally functionas Rab5 effectors in endosomal trafficking, ex-tracellular stimuli such as oxidative stress orgrowth factor internalization promotes GTPhydrolysis by rab5, resulting in release of theAPPL proteins from endosomal membranesinto the cytosol and translocation of the APPLsinto the nucleus. Once in the nucleus, bothAPPLs interact with the nucleosome remodel-ing and HDAC (histone deacetylase) multi-





protein complex, NuRD/MeCP1, suggesting arole for the APPL proteins in transcriptionalregulation. More recent studies provide directevidence for transcriptional regulation by theAPPL proteins. Nuclear-localized APPL pro-teins stimulateb-catenin/TCF-dependent tran-scription (Rashid et al. 2009). Here, shuttlingof APPLs into the nucleus results in dissociationof HDAC1, HDAC2, and b-catenin from thetranscriptional repressor Reptin. Taken togeth-er, these studies reveal an important link be-tween endocytic trafficking and signal-inducedtranscriptional activation.

Fusion of endocytic genes with other genescan lead to new roles. Chromosomal translo-cation and fusion of the CALM gene with theAF10 gene is associated with acute myeloid leu-kemia, suggesting a possible link between endo-cytic defects and oncogenesis (Dreyling et al.1998; Kumon et al. 1999; Narita et al. 1999).However, recent studies suggest that the con-tribution of CALM to CALM–AF10-mediatedoncogenic transformation is independent ofdisruption of endocytosis (Stoddart et al.2012). Rather, evidence suggests that the fu-sion with CALM leads to the oligomeriza-tion of CALM–AF10, potentially influencingtranscriptional regulation leading to myeloidleukemias. Additional studies have found thatCALM–AF10 contributes to up-regulation ofseveral DNA repair and maintenance genes(Commd3, Bmi1, Dnajc1, and Spag6) (Mulawet al. 2012). The functional consequences ofthis up-regulation have yet to be determined.

CONCLUDING REMARKS AND FUTUREDIRECTIONS

Here we describe multiple nonendocytic func-tions for endocytosis regulators that impactcytoskeleton organization, cell division, andgene regulation, as well as nonendocytic sortingpathways. From an evolutionary perspective,some of these nonconventional functions haveemerged as recent specializations from geneduplication, and others likely represent residualancient functions of endocytic proteins. Someresult from splice variants, and some are gener-ated as a result of novel posttranslational mod-

ification. It is likely that the current and futureforays in whole-genome screening and analysisof “interactomes” will reveal further unexpectedroles for endocytosis regulators in nonendo-cytic pathways. The known “extra functions”already influence pathways of infection, onco-genesis, and glucose metabolism, showing thevalue of keeping an open mind about the func-tions of endocytic regulators and their relevanceto human health and disease.

ACKNOWLEDGMENTS

Preparation of this manuscript was supportedby the following NIH grants relevant to the au-thors’ research: GM038093 and DK095663 toF.M.B., GM089896 to T.J.O., and GM064387to J.A.Y.

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F.M. Brodsky et al.




2014; doi: 10.1101/cshperspect.a017004Cold Spring Harb Perspect Biol Frances M. Brodsky, R. Thomas Sosa, Joel A. Ybe and Theresa J. O'Halloran to Human Disease

LinksRegulators in the Cytoskeleton, Cell Cycle, Nucleus, and Beyond: Unconventional Functions for Clathrin, ESCRTs, and Other Endocytic

Subject Collection Endocytosis

Endocytosis: Past, Present, and Future

ZerialSandra L. Schmid, Alexander Sorkin and Marino Clathrin-Mediated Endocytosis

Imaging and Modeling the Dynamics of

Marcel Mettlen and Gaudenz Danuser

Endosomal SystemRab Proteins and the Compartmentalization of the

Angela Wandinger-Ness and Marino ZerialClathrin-Mediated EndocytosisEndocytic Accessory Factors and Regulation of

Christien J. Merrifield and Marko Kaksonen

Regulator of Cell Polarity and Tissue DynamicsCargo Sorting in the Endocytic Pathway: A Key

Suzanne Eaton and Fernando Martin-BelmonteSystemThe Complex Ultrastructure of the Endolysosomal

Judith Klumperman and Graça Raposo

Links to Human DiseaseCytoskeleton, Cell Cycle, Nucleus, and Beyond:and Other Endocytic Regulators in the Unconventional Functions for Clathrin, ESCRTs,

et al.Frances M. Brodsky, R. Thomas Sosa, Joel A. Ybe,

Lysosome-Related OrganellesThe Biogenesis of Lysosomes and

Dieckmann, et al.J. Paul Luzio, Yvonne Hackmann, Nele M.G.

Endocytosis of Viruses and BacteriaPascale Cossart and Ari Helenius

Endocytosis, Signaling, and BeyondPier Paolo Di Fiore and Mark von Zastrow

Responds to External CuesLysosomal Adaptation: How the Lysosome

Carmine Settembre and Andrea Ballabio

Clathrin-Independent Pathways of Endocytosis

DonaldsonSatyajit Mayor, Robert G. Parton and Julie G.

MetabolismReciprocal Regulation of Endocytosis and

Amira KlipCostin N. Antonescu, Timothy E. McGraw and

SignalingThe Role of Endocytosis during Morphogenetic

Marcos Gonzalez-Gaitan and Frank Jülicher

Cooperation?Endocytosis and Autophagy: Exploitation or

Sharon A. Tooze, Adi Abada and Zvulun ElazarDiseaseRole of Endosomes and Lysosomes in Human

Frederick R. Maxfield

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Unconventional Functions for Clathrin, ESCRTs, and Other ...cshperspectives.cshlp.org/content/6/9/a017004.full.pdfthree-legged triskelion-shaped molecule made up of three clathrin