Membrane Remodeling in Clathrin-Mediated Endocytosis Volker Haucke1,2,* and Michael M

REVIEW Membrane remodeling in clathrin-mediated endocytosis Volker Haucke1,2,* and Michael M. Kozlov3,*

ABSTRACT membrane fission and vesicle uncoating to eventually allow fusion Clathrin-mediated endocytosis is an essential cellular mechanism by of the nascent endocytic vesicle with endosomes (Box 1). which all eukaryotic cells regulate their plasma membrane composition In spite of important advances in the characterization of these to control processes ranging from cell signaling to adhesion, migration factors, key questions regarding the endocytic process remain. and morphogenesis. The formation of endocytic vesicles and For example, different models have been proposed regarding the tubules involves extensive protein-mediated remodeling of the initiation of clathrin-coated pit (CCP) formation and the onset of plasma membrane that is organized in space and time by protein– membrane curvature acquisition. Moreover, knowledge regarding protein and protein–phospholipid interactions. Recent studies the nanoscale distribution of endocytic proteins within the bilayer combining high-resolution imaging with genetic manipulations of plane during vesicle formation and the mechanisms that guide the endocytic machinery and with theoretical approaches have led their orchestrated assembly and disassembly is only now beginning to novel multifaceted phenomenological data of the temporal and to emerge. Finally, our understanding of the nature and dynamics spatial organization of the endocytic reaction. This gave rise to of membrane phospholipids (Box 2) and their associations with various – often conflicting – models as to how endocytic proteins endocytic proteins during endocytic membrane remodeling and their association with lipids regulate the endocytic protein remains limited. choreography to reshape the plasma membrane. In this Review, we Here, we review the existing data on four mutually complementing discuss these findings in light of the hypothesis that endocytic aspects of the process of endocytic vesicle generation in CME: membrane remodeling may be determined by an interplay between (1) the timecourse of shape transformations of the endocytic site, protein–protein interactions, the ability of proteins to generate and (2) the ability of endocytic proteins to generate membrane curvature, sense membrane curvature, and the ability of lipids to stabilize and (3) the spatial organization of endocytic proteins in the plane of the reinforce the generated membrane shape through adopting their endocytic site, and (4) the timecourse of endocytic protein arrival lateral distribution to the local membrane curvature. at and departure from the endocytic site. We will formulate a model of CME that accounts for all four sets of data, and, KEY WORDS: BAR domain proteins, Clathrin, Endocytosis, hopefully, will bring us closer to understanding the molecular Membrane curvature, Membrane remodeling, Vesicle mechanism of the endocytic process.

Introduction Timecourse of shape transformations of endocytic plasma Endocytosis is a cellular process that involves the formation of small membrane sites membrane vesicles or tubules to deliver cargos, such as ligand- Formation of clathrin-coated endocytic vesicles (CCVs) involves bound receptors, ion channels, transporters or other types of plasma major transformations of the local shape and topology of the membrane molecules, into the eukaryotic cell interior (Brodsky, plasma membrane. In general, formation of a CCV involves two 2012; Kaksonen and Roux, 2018; McMahon and Boucrot, 2011). processes: (1) the assembly of a clathrin coat on the membrane Clathrin-mediated endocytosis (CME) arguably is a primary surface, and, (2) the shaping of the resulting clathrin-coated endocytic mechanism and of central importance for nutrient structure (CCS) into a CCV. The latter reaction starts with the uptake, cell signaling, adhesion, migration and morphogenesis, invagination of a CCS (Fig. 1A) towards the cytoplasm and the as well as for neurotransmission, among many other processes. formation of a shallow CCP that adopts the shape of a spherical The formation of endocytic vesicles during CME is accompanied membrane segment characterized by homogeneous and isotropic by extensive remodeling of the plasma membrane that is organized curvature (Fig. 1B). Shallow pits undergo progressive invagination in space and time by endocytic proteins and their interactions into deep dome-like shapes, which are smoothly connected to with each other and with specific membrane lipids (Daumke et al., the plasma membrane by a funnel-like rim (Fig. 1C). Further 2014; Kaksonen and Roux, 2018; McMahon and Boucrot, 2011). invagination leads to formation of a spherical bud, while the rim During recent years we have acquired extensive knowledge about transforms into a distinct hourglass-like membrane neck (Fig. 1D). the CME machinery, which, apart from clathrin, comprises more Eventually, the neck undergoes fission resulting in formation of a than 50 additional cytosolic proteins that have roles in clathrin CCV (Fig. 1E). recruitment and assembly, cargo selection, membrane bending, A topic of heated discussion in the literature is the temporal relationship between these two processes, that is, between the timing of the clathrin coat assembly that can be visualized as CCS 1Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle- area growth and the dynamics of CCS shaping (curvature Straße 10, 13125 Berlin, Germany. 2Freie Universität Berlin, Department of Biology, acquisition) into a CCV. EM images of CCSs taken in the 1980s Chemistry, Pharmacy, Takustrasse 3, 14195 Berlin, Germany. 3Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, showed that CCPs coexist with, and are linked to, the edges of larger Tel Aviv 69978, Israel. flat clathrin lattices or ‘plaques’ (Heuser, 1980, 1989; Heuser and Kirchhausen, 1985; Larkin et al., 1986). Clathrin plaques were often *Authors for correspondence ([email protected]; [email protected]) seen to be surrounded by smaller clathrin domes that had emerged

V.H., 0000-0003-3119-6993; M.M.K., 0000-0003-4831-8571 out of the otherwise flat lattices (Maupin and Pollard, 1983), and Journal of Cell Science

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Box 1. Protein components of CME Box 2. Lipid-mediated regulation of CME The CME machinery comprises more than 50 additional cytosolic Endocytic protein recruitment is governed by specific membrane proteins, in addition to clathrin, that have roles in clathrin recruitment phospholipids, in particular by phosphatidylinositol 4,5-bisphosphate

and assembly, cargo selection, membrane bending, membrane fission [PI(4,5)P2] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and vesicle uncoating to eventually allow fusion of the nascent (Posor et al., 2014). While CCP nucleation strictly depends on

endocytic vesicle with endosomes (extensively reviewed in Kaksonen PI(4,5)P2 (Di Paolo and De Camilli, 2006; Kaksonen and Roux, 2018), and Roux, 2018; Kelly and Owen, 2011; McMahon and Boucrot, 2011; membrane remodeling during CCP maturation into a CCV is accompanied Merrifield and Kaksonen, 2014). In typical CCPs of 150 nm diameter by the partial conversion of the identity of the internalized membrane

these proteins are present in copy numbers that range from ∼180 for from PI(4,5)P2, a characteristic lipid of the plasma membrane that is AP-2 or CALM to ∼30–40 molecules for PI3KC2α and SNX9/18 (Borner progressively hydrolyzed during the late stages of the endocytic reaction

et al., 2012; Schoneberg et al., 2017) and 30 molecules for dynamin by the 5-phosphatases OCRL and synaptojanin, to PI(3,4)P2,whichis (Grassart et al., 2014). generated by local phosphorylation of PI(4)P underneath the clathrin- Cargo selection and clathrin recruitment are mediated by adaptors, such as coated membrane area by phosphatidylinositol 3-kinase C2a (PI3KC2α) the heterotetrameric AP-2 complex (comprising α, β, µ and σ subunits) and (Kaksonen and Roux, 2018; Posor et al., 2014). Arrival of PI3KC2α

a plethora of cargo-selective adaptors for sorting specific cargos [e.g. follows that of clathrin with some delay to trigger the PI(3,4)P2-dependent epidermal growth factor receptor substrate 15 (Eps15) and Eps15- recruitment of PX-BAR domain proteins SNX9 and SNX18. Lack of either interacting 1 and 2 (epsin 1 and 2) for ubiquitylated cargos, arrestins for PI3KC2α, or SNX9 or SNX18 stalls CME at the level of deeply invaginated G protein-coupled receptors, Dab2 and ARH for LDLR family receptors, CCPs that fail to form a narrow membrane neck, which provides a suitable CALM or AP180 for VAMPs/ synaptobrevins, or stonin 2 for substrate for endocytic vesicle scission by dynamin (Lo et al., 2017; synaptotagmins (reviewed in Kaempf and Maritzen, 2017; Kelly and Posor et al., 2013; Schoneberg et al., 2017). A similar CCP maturation Owen, 2011; Maritzen et al., 2010; Reider and Wendland, 2011; Traub, phenotype is observed in cells depleted of the N-WASP-associated

2009)]. In many cell types, the maturation of CCPs is accompanied and F-BAR domain protein FCHSD2, another effector of PI(3,4)P2 (Almeida- possibly facilitated by the assembly of a network of branched actin Souza et al., 2018). As CCPs are scissioned from the plasma membrane,

filaments mediated by neuronal Wiskott–Aldrich syndrome (N-WASP) and PI(3,4)P2 is, likely, hydrolyzed to PI(3)P, a signature lipid of the endosomal the actin-related protein (ARP) 2/3 complex (Doyon et al., 2011; Kaksonen system (He et al., 2017; Kaksonen and Roux, 2018; Posor et al., 2014). and Roux, 2018; Yoshida et al., 2018). Recent data have suggested that N- WASP is activated at the base of CCPs by the F-BAR protein FCHSD2 (Almeida-Souza et al., 2018). Filamentous (F)-actin might aid CCP maturation (Almeida-Souza et al., 2018) and/or fission of the invagination Application of correlative light and electron microscopy (CLEM) neck of late-stage CCPs that is mediated by BAR-domain-containing to BSC-1 cells, which lack long-lived clathrin-coated plaques, in proteins, such as endophilins, amphiphysins and SNX9 and SNX18, and combination with mathematical analysis led to the conclusion that the BAR domain protein-associated GTPase dynamin (Doyon et al., 2011; CCSs first grow flat and start developing curvature later on, once Kaksonen and Roux, 2018). they have accumulated about 70% of their final clathrin content (Bucher et al., 2018). Beginning from this stage, while completing the area growth, a CCS acquires the consecutive shapes of pits, scission events leading to the formation of coated vesicles tended domes and buds (Bucher et al., 2018). Another study of CCP to cluster at the edges of plaques (reviewed in Lampe et al., 2016). dynamics used polarized total internal reflection fluorescence The relative dynamics of pit and plaque formation, however, was (pol-TIRF) microscopy combined with electron, atomic force and not resolved at that time. Recent studies using state-of-the-art super-resolution optical microscopy, and measurements of the imaging techniques have led to conflicting suggestions regarding time evolution of CCSs in SKMEL-2 cells that express endogenously the origin of curved pits and flat plaques and the timecourse of tagged clathrin light chain and dynamin 2 (Scott et al., 2018). That a possible transformation of plaques into pits. According to one work revealed a variable delay between the beginning of CCS scenario, referred to as the constant curvature model, the essence assembly and the onset of curvature acquisition. For approximately of CCP maturation is the increase of its area through self-assembly half of the CCSs, an acquisition of curvature was detected already of identical clathrin elements characterized by a curved shape in at the very beginning of CCS assembly. In the other half of the the membrane plane. The curvature of a CCP remains constant events, CCSs started to grow prior to the detection of membrane throughout the whole maturation process and equal to the curvature of bending. Within these latter group of events, there was a small a single clathrin element (Cocucci et al., 2012; Kirchhausen, 2009; subset in which CCSs started to bend after having reached their Saffarian et al., 2009). In contrast, the constant area model postulates final areas, and a larger group for which the curvature acquisition that CCS transformation into a curved CCP and, further, a overlapped in time with the completion of area growth (Scott et al., spherical CCV proceeds upon conservation of the in-plane area of 2018). Delayed membrane bending after the onset but prior to the the clathrin array, while its curvature gradually increases through the completion of clathrin assembly was also described in a recent structural rearrangements of the clathrin cage (Avinoam et al., 2015; study that combined high-speed atomic force microscopy with see Box 3 for details). The essential difference between the fluorescence confocal microscopy (Yoshida et al., 2018). underlying principles of the constant area and constant curvature The lack of a firm temporal coupling between the processes of CCS models consists in the coupling between the degree of CCS area growth and shaping suggests that curvature acquisition by a indentation into the cytoplasm and the growth of its area. The growing CCS is a process that requires clathrin coat assembly, but is constant curvature model suggests that the two processes proceed not a direct consequence of it. Hence, another process must mediate in parallel, that is that they are strongly coupled to each other, the link between CCS area growth and curvature acquisition. An while the constant area model proposes that the degree of CCS example of such a process could be the accumulation of curvature- indentation that results from the curvature acquisition is fully mediating proteins, such as epsins and CALM (or its close neuronal decoupled from CCS area growth. A number of recent studies have paralog AP180, also known as SNAP91) (see below; Chen et al., provided further indications in favor of the latter model by showing 1998; Ford et al., 2002; Koo et al., 2015; Maritzen et al., 2012; Messa that CCS indentation into the cytoplasm is largely decoupled from et al., 2014; Miller et al., 2015; Scott et al., 2018; Zhang et al., 1998)

CCS area growth (Bucher et al., 2018; Scott et al., 2018). by the emerging clathrin coat. Heterogeneity between cells or cell Journal of Cell Science

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A B C D E

Fig. 1. Timecourse of shape transformations of endocytic plasma membrane sites in CME. Shown is a gallery of electron micrographs of Cos7 cells illustrating the various stages of endocytic vesicle formation in CME. Nearly flat (A) and shallowly invaginated (B) clathrin-coated pits (CCPs) characterized by homogeneous and isotropic curvature. (C) Deeply invaginated CCP with a dome-like shape that is smoothly connected to the plasma membrane by a funnel-like rim. (D) Spherical late-stage CCP bud (also referred to as Ω-shaped CCP) containing an hourglass-like membrane neck. (E) Clathrin-coated vesicle (CCV). Scale bar: 200 nm. types may be one reason for the observed variations in the delay by two equal principal curvatures, which are conventionally defined to between the onset of curvature acquisition and the timing of clathrin be negative, whereas the two principal curvatures of the neck have coat self-assembly. opposite signs, one being positive and the second being negative (see, for example, Spivak, 1970). Membrane curvature generation by endocytic proteins Among the ∼50 different proteins involved in CCV formation The architecture of an invaginating CCS has two parts with (Kaksonen and Roux, 2018; McMahon and Boucrot, 2011; fundamentally distinct shapes: the dome-like part, which has the Merrifield and Kaksonen, 2014), there is a relatively limited subset shape of a spherical segment, whose solid angle increases in the of proteins that are deemed responsible for shaping the CCS course of pit transformation into a dome-like and, then, a spherical membrane. We will refer to these proteins as the structural proteins bud (Fig. 1). In addition, there is the membrane neck, which of endocytosis. In the following section, we will first discuss the connects the CCP to the plasma membrane and has a funnel- or basic mechanisms by which proteins generate and/or stabilize hourglass-like shape depending on the stage of CCP maturation membrane curvature, and then describe the endocytic structural (Fig. 1). The spherical dome has an overall concave shape that is proteins involved in sculpting the isotropically curved dome-like isotropically curved, that is has equal curvatures in all directions part of an invaginating CCS and its anisotropically bent neck. along the membrane surface (Fig. 1). In contrast, the neck part displays a saddle-like configuration that is anisotropically curved: Membrane curvature generation the neck membrane is concave along the direction of the CCP axis, The ability of proteins to generate membrane curvature has been and convex in the perpendicular direction (Fig. 1). In geometrical extensively explored both experimentally and theoretically over the terms, the isotropically curved dome-like part of a CCP is characterized past decade (for recent reviews, see Jarsch et al., 2016; McMahon and Boucrot, 2015; Simunovic et al., 2016). Less emphasis has been put on the isotropic versus anisotropic character of the local membrane Box 3. The constant curvature versus constant area shape created by each of the specific curvature-generating proteins. models of CME Here, we will stress the latter issue since it is of importance for our The constant curvature model of CME (Cocucci et al., 2012; discussion of the shaping of the dome- and neck-like parts of CCSs. Kirchhausen, 2009; Saffarian et al., 2009) sees a CCP as composed There are two major mechanisms by which proteins can generate local of effective units, with each unit consisting of a clathrin triskelion with an membrane bending. One is the hydrophobic insertion (or wedging) underlying membrane element and having a shape of a spherical mechanism, based on shallow embedding in one of the membrane segment that is characterized by a certain curvature. These units self- leaflets of a hydrophobic or amphipathic protein domain (Zimmerberg assemble within the surrounding membrane side-by-side, similar to and Kozlov, 2006), such as N-terminal amphipathic helices (Ford mosaic pieces, into a CCS. Within this scenario, in the course of the CCS area growth, that is, as additional clathrin triskelia are added, the et al., 2002; Kozlov et al., 2014) or short hydrophobic loops structure progressively invaginates into the cytoplasm with its shape (McMahon et al., 2010). Such a mode of protein insertion results in evolving from a shallow to a dome-like CCP and further to a spherical splaying the region of lipid polar heads of the lipid monolayer with clathrin-coated bud. Importantly, the curvature of all these structures respect to the remainder of the monolayer interior, which generates a remains constant and equal to that of a single clathrin element strong tendency of the membrane to locally bulge in the direction of (Kirchhausen, 2009; Saffarian et al., 2009). the insertion. Importantly, embedding of an amphipathic helix that is a Recently, the constant curvature model was challenged by the application of a correlative fluorescence microscopy and electron few nanometers large generates an essentially isotropic curvature tomography method, which provides three-dimensional information around the insertion (Campelo et al., 2008; Kozlov et al., 2014). This about the shapes of clathrin-coated pits (Avinoam et al., 2015). is a consequence of the behavior of lipid monolayers as two- According to this study, the coated membrane area in CCPs and, dimensional fluids. Owing to this membrane property, the possible hence, the total number of clathrin molecules, do not appreciably change curvature anisotropy that has developed in the closest proximity of the throughout the entire CCP maturation and budding process, beginning inserted domain disappears at a nanometer-scale distance from the from slightly invaginated pits through completely spherical CCVs. Conservation of the area in the course of invagination means that the insertion, such that the membrane deformation around the insertion is, curvature of the structures must change all the way from zero at the onset largely, isotropic (Campelo et al., 2008). Hence, proteins generating of CCP shape acquisition up to its maximal value at the final stage of membrane curvature through the hydrophobic insertion mechanism CCV formation. This scenario is referred to as the constant area model. are particularly suited to shape the dome-like part of CCSs

characterized by an isotropically curved shape. Journal of Cell Science

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The other established mechanism of local curvature generation demonstrated and quantitatively characterized. Endocytic factors referred to as the scaffolding mechanism, underlies the action of proven to bend membranes through the hydrophobic insertion rigid hydrophilic protein domains with an intrinsically bent shape mechanism are the AP180 (ANTH) or epsin (ENTH) N-terminal (McMahon and Gallop, 2005; Zimmerberg and Kozlov, 2006). homology domain-containing proteins CALM and epsins 1 and 2 Binding of such protein domains to the polar face of the membrane (Ford et al., 2002; Messa et al., 2014; Miller et al., 2015). Depletion imposes the curvature of the protein onto the membrane patch of CALM, an abundant clathrin-associated sorting adaptor for underneath. For the scaffolding mechanism to be effective, the VAMP/synaptobrevin SNARE sorting (Koo et al., 2015; Maritzen intrinsic rigidity of the protein domain and its affinity to the et al., 2012; Miller et al., 2011), causes a twofold decrease in the membrane surface must be substantially larger than the rigidity of curvature of CCPs and CCVs, indicating that CALM can generate the membrane with respect to bending (Zimmerberg and Kozlov, membrane curvature by itself (Miller et al., 2015). In vitro studies 2006). The most common protein domains producing bent showed that curvature generation by CALM depends on an membranes by the scaffolding mechanism are the ∼10 nm-long N-terminal amphipathic helix within its ANTH domain, which dimers of bin-amphiphysin-rvs (BAR) domains (for a review, see on its own is extremely efficient at extensively tubulating Daumke et al., 2014; Frost et al., 2009; McMahon and Boucrot, 200 nm-diameter liposomes containing phosphatidylinositol 2015) that most often have a crescent-like shape due to the way the 4,5-bisphosphate [PI(4,5)P2] (Miller et al., 2015). The resulting two monomers are bound to each other. Because of its strongly tubules have a pearl-on-a-chain-like appearance with a pearl elongated shape, a BAR domain dimer is predicted to bend the diameter of ∼40 to 50 nm, similar to that of the smallest membrane underneath in the direction of the protein axis but not in endocytic CCVs found in neuronal synapses (Miller et al., 2015). other directions in the membrane plane (Schweitzer and Kozlov, A similar function may be executed by the CALM-related protein 2015). It has been computationally predicted that this curvature AP180 in presynaptic neurons (Koo et al., 2015; Zhang et al., 1998). anisotropy must propagate along the membrane to distances of up to Consistent with this, it has been shown that loss of CALM and/or tens of nanometers around the protein (Schweitzer and Kozlov, AP180 in flies, worms and mice results in abnormally sized and 2015). Hence, such scaffolding proteins can be expected to act in shaped synaptic vesicles formed by clathrin-mediated budding at shaping the CCP neck. synapses (Koo et al., 2015; Zhang et al., 1998). Epsins, similar to In principle, protein scaffolds that generate nearly isotropic CALM, are early-acting clathrin- and AP-2-associated endocytic membrane curvature may also exist. As discussed below, a proteins that contain a PI(4,5)P2-binding membrane-targeting ENTH hypothetical example of such a scaffold is the clathrin triskelion domain (Chen et al., 1998; Ford et al., 2002). Upon interaction (den Otter and Briels, 2011; Kirchhausen, 2009; Merrifield and with PI(4,5)P2, the ENTH domain forms an additional amphipathic Kaksonen, 2014; Saffarian et al., 2009). α-helix that generates local membrane curvature. Importantly, while, The ability of a protein to produce membrane curvature guarantees at first, this curvature generation by the ENTH domain leads that its affinity to membranes depends on the membrane curvature, a to conversion of the nearly flat liposomal membranes into property referred to as curvature sensitivity (Antonny, 2011; Campelo anisotropically bent thin tubules (Ford et al., 2002), at longer and Kozlov, 2014). The mechanism by which a protein senses time scales, these tubules transform into separate spherical vesicles membrane curvature is intimately related to the mechanism of with isotropic curvature (Boucrot et al., 2012), as expected for the curvature generation. Protein domains that curve membranes hydrophobic insertions. Hence, the initial tubules likely represent through the hydrophobic insertion mechanism enable relaxation of the kinetically trapped intermediates rather than the equilibrium the intra-membrane stresses generated as a result of membrane membrane shapes (Boucrot et al., 2012). Collectively, these data bending by external forces (Campelo and Kozlov, 2014). support the idea that CALM and epsins use the hydrophobic A specific microscopic mechanism of such relaxation was proposed insertion mechanisms to shape the isotropically curved dome-like to underlie the protein-mediated rescue of the hydrophobic structural part of invaginating CCPs. defects that develop in the membrane as a result of bending (Vanni Among the structural proteins that generate curvature merely et al., 2013). Stress relaxation makes protein binding more favorable. by scaffolding are endocytic F-BAR domain proteins, such as As a consequence, stronger membrane bending by external forces F-BAR domain only protein 1 or 2 (FCHo1 or FCHo2), and creates larger intra-membrane stresses, resulting in an enhanced possibly F-box protein 7 (FBP17, also known as FNBP1) and membrane-binding affinity of a membrane-inserting domain, which syndapin (Henne et al., 2010, 2007), as well as the structurally represents the essence of its curvature-sensing ability (Campelo and unrelated GTPase dynamin (Ford et al., 2011; Faelber et al., 2011; Kozlov, 2014). Different from the hydrophobic insertions, the protein Ferguson and De Camilli, 2012; Sochacki et al., 2017). The dimeric domains, which bend membranes by scaffolding, directly sense the F-BAR module displays the shape of a shallow arc whose concave curvature of the membrane surface to which they bind rather than face binds the membrane surface through attractive interaction intra-membrane stress. The largest affinity of a scaffolding protein between positively charged residues and the negatively charged must be to a membrane whose curvature matches best the intrinsic polar headgroups of acidic phospholipids (Henne et al., 2007; see curvature of the protein itself, which is the optimal curvature for also Box 2). F-BAR domains can polymerize on the membrane the protein to associate with. Binding of a protein scaffold to surface into a helical coat, in which the dimers are held together by membranes whose curvatures are larger or smaller than that of the lateral and tip-to-tip interactions (Frost et al., 2008). These coats protein is expected to be weaker than binding to a membrane with convert membranes into tubules with variable diameters between optimal curvature. 70 nm (Frost et al., 2008) and up to 130 nm (Henne et al., 2007). Since the F-BAR dimers can be described as shallow crescents Endocytic proteins involved in membrane shaping characterized by a small or almost vanishing curvature, the function The endocytic machinery exerts both the hydrophobic insertion of F-BAR proteins in CME in general and that of FCHo1/2, in and the scaffolding mechanisms of membrane shaping. For some of particular, remains a matter of debate (Hollopeter et al., 2014; the proteins that are potentially involved in sculpting CCSs, the Ma et al., 2016). Initial experimental data are consistent with the ability to generate and sense membrane curvature has been directly idea that FCHo1/2 contributes to slight membrane bending at Journal of Cell Science

4 REVIEW Journal of Cell Science (2018) 131, jcs216812. doi:10.1242/jcs.216812 the rim of shallow early-stage CCPs during the initiating steps of generate the isotropic membrane curvature that, in turn, induces CCP formation (Henne et al., 2010). Interestingly, the subunits of changes in clathrin topology to match the membrane shape underneath dynamin also show intrinsically curved arc-like shapes; they bind (for recent discussion, see Bucher et al., 2018; Scott et al., 2018). the membrane through electrostatic interactions (Faelber et al., 2011; Ford et al., 2011) and polymerize on the membrane surface Spatial organization of endocytic proteins within the plane into helical structures, which constricts the membrane into 50 nm of the endocytic site tubules (see, for example, Hinshaw, 1999). Importantly, upon GTP As discussed above, the structural proteins responsible for endocytic hydrolysis, dynamin drives fission of membrane necks and tubules membrane remodeling can be subdivided into those producing the through mechanisms that remain uncertain (Antonny et al., 2016). isotropic membrane shape that characterizes the dome-like part of Taken together, F-BAR domain proteins and dynamin use a an invaginating CCS and proteins that generate anisotropic shapes scaffolding mechanism that generates anisotropic membrane of the membrane neck connecting the CCS to the surrounding curvature and are, therefore, suitable for shaping the rims or flat membrane. Based on this essential difference between the necks of CCPs. subsets of endocytic structural proteins, it seems logical to assume Finally, dimers of N-BAR endocytic proteins, such as that these proteins are differently distributed over the CCP surface. amphiphysin and endophilin, possess both amphipathic N-terminal For instance, one would expect that dome-sculpting proteins helices and elongated crescent-like scaffolding domains. The radii of occupy the central part of a nascent CCP, whereas neck-shaping the latter are ∼10 nm (Gallop et al., 2006; Peter et al., 2004) such that proteins segregate along the CCP periphery. In addition to their their curvatures are significantly larger than those of F-BAR membrane-shaping properties, other factors may contribute to an domains (Frost et al., 2009; McMahon and Boucrot, 2015). These uneven distribution of endocytic proteins along the CCP surface. proteins have the potential to curve membranes by both the For example, endocytic proteins that sort and recruit cargo are hydrophobic insertion and scaffolding mechanisms (Gallop et al., expected to localize to the central part of the CCS structure, while 2006; Peter et al., 2004). When added to liposomes, N-BAR proteins proteins such as dynamin that are involved in fission of the generate membrane tubules that are a few tens of nanometers (i.e. 20 membrane neck at the final stage of CCV formation may be to 40 nm) thin (Peter et al., 2004; Gallop et al., 2006), and a fraction concentrated along the CCP periphery. of these is, over time, converted into small spherical vesicles Recent progress in super-resolution imaging has indeed made it (Boucrot et al., 2012). Vesiculation appears to depend largely on possible to directly study the endocytic protein distribution along the the hydrophobic insertions as it is more pronounced in the case of CCP membrane (Sochacki et al., 2017). Use of large-scale correlative endophilin, whose BAR domain dimer harbors an additional super-resolution light and transmission electron microscopy has amphipathic helix as compared to amphiphysin (Boucrot et al., enabled the mapping of the distribution of 19 key proteins involved in 2012). Because of these dual properties, endocytic N-BAR domain CME with nanometer-scale precision and provided a comprehensive proteins could, in principle, participate in shaping both the dome-like molecular architecture of endocytic structures (Sochacki et al., 2017). parts and the necks of CCSs. In the case of the N-BAR domain The most remarkable feature of these data is a clear separation protein endophilin, its dimers have been shown to form helical of endocytic proteins into three main groups according to their polymeric coats through interactions between the membrane- localization with respect to the edge of the clathrin lattice, which inserted N-terminal helices of adjacent dimers (Mim et al., 2012). covers the central dome-like part of CCPs (Fig. 2A,B). The inside These coats can convert liposomes into tubules of 25 nm and 32 nm of the clathrin-coated zone is occupied by a group of seven proteins – diameters. Based on the diameter of these endophilin-coated tubules, epsin (1/2), NECAP (1/2), HIP1R, CALM, and the transmembrane endophilin dimers would be predicted to localize to the neck of cargos transferrin receptor and VAMP/synaptobrevin 2 – which are CCPs in cells. Clearly, the interplay between the N-terminal distributed all over the clathrin lattice, similar to clathrin light chains. helices and the crescent-like scaffolds of N-BAR proteins in In the course of CCP maturation these proteins remain in the center of the process of endocytic membrane remodeling needs further the structure and change their concentrations in accordance with that exploration. of clathrin (Sochacki et al., 2017). The important structural factors, An important open question is whether the isotropic curvature such as EPS15, FCHo2, amphiphysin, endophilin, syndapin, SNX9 of the dome-like parts of CCSs is also generated or stabilized by and dynamin, localize almost exclusively at the edge of the clathrin protein scaffolds, in addition to hydrophobic insertion of epsin1/2 lattice, that is at the neck region of CCPs, similar to what has been and CALM. One possibility is that clathrin triskelia perform this observed for SNX9 by 3D-dSTORM microscopy (Schöneberg et al., function (Dannhauser and Ungewickell, 2012; Hinrichsen et al., 2017). Interestingly, the concentrations of early-acting factors, such 2006), as the 110° pucker angle at the triskelion apex matches as FCHo2 and its binding partner EPS15, decreased as CCPs matured the characteristic values of the isotropic membrane curvature of into a spherical bud with a corresponding narrowing of the membrane endocytic vesicle domes (den Otter and Briels, 2011; Kelly et al., neck. In contrast, the amounts of dynamin and the associated N-BAR 2014; Kirchhausen, 2009; Lampe et al., 2016; Saffarian et al., 2009). protein amphiphysin at the neck increased with CCP maturation However, a problem is that, in addition to the curved configurations, towards a spherical bud (Sochacki et al., 2017). Finally, several CCSs also can adopt a nearly flat geometry (den Otter and Briels, proteins, such as AP-2, DAB2, stonin2, arrestin, and intersectin, have 2011), in which the pucker angle is 90°. Moreover, for geometrical been found both in the central and the peripheral zones of CCS reasons, the transition between flat and curved configurations of (Sochacki et al., 2017). clathrin triskelia requires a substantial rearrangement of the Of note, the existence of proteins that are localized to the periphery clathrin cage that alters the ratio between the number of hexagons of the major protein coat appears to be a general feature that, in and pentagons, which might represent a serious kinetic challenge addition to clathrin coats, also characterizes the caveolin–cavin coat, (den Otter and Briels, 2011; Lampe et al., 2016). Hence, the question which is bordered by EHD2 complexes (Ludwig et al., 2013; remains as to whether clathrin subunits are able to generate membrane Yeow et al., 2017) and the Gag-containing shells of budding HIV curvature by themselves or whether the polymerized clathrin cage viruses, which are bounded by ESCRT-I complexes (Sundquist merely serves as a platform for the recruitment of other proteins, which and Krausslich, 2012). In the two latter systems, the peripheral Journal of Cell Science

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A Flat fluorescence profiles Fig. 2. Spatial organization and recruitment signatures of endocytic proteins. (A,B) Spatial organization of endocytic proteins at sites of CME. CALM* *AP2 (β) Dyn2* 1.0 (A) One-dimensional fluorescence intensity profiles (1DFLIPs) for GFP fusions of select endocytic proteins compared with that of the clathrin light chain

=79 (black curve) for flat CCSs. The vertical brown line indicates the position of the n =397 =444

n n edge of the clathrin cage. The width of the alternating pink and light brown bars 0 corresponds to 50 nm distance. The asterisks denote the terminus of the protein that contains the GFP. The number of structures included in the profile generation are given as n. Standard error is shown as the shaded area. Epsin1**DAB2 *FCHO2 (B) Shown here is a combined representation of the location of different groups 1.0 of proteins with respect to the clathrin edge. The position of the latter is indicated by a white dashed line as observed by EM. The x-axis represents the distance. =99 n =338 =245 The image is obtained by combination of 1DFLIPs of all the proteins with controls n n for flat CCSs. The 1DFLIPs are projected with their fluorescence axes depicted 0 with pixel intensity. Different colors mark groups of proteinsthat are characterized by a similar location with respect to the clathrin edge, with their names given *Epsin2 *Stonin2 *SNX9 according to their colored groupings. Scale bar: 24 nm. Reprinted from Sochacki 1.0 Average normalized intensity Average et al. (2017) with permission from Nature Cell Biology, Springer Nature. (C) Timecourse of endocytic protein recruitment to CCVs. Shown here are the =81 =94 n n =159 average recruitment signatures of select endocytic proteins normalized to their n randomized measures. The vertical black line shows time point 0, which 0 corresponds to fission of the neck connecting the endocytic vesicle to the plasma membrabe. Horizontal black lines show median randomized measures, taken as B fluorescence origin, with gray areas representing the 95% upper and lower confidence intervals. Scale bars: 20 s (horizontal) and 10×95% confidence Clathrin, clathrin (Ig) interval (vertical). Reproduced from Taylor et al. (2011), where it was published VAMP2, TFR, NECAP, CALM, under a CC-BY license (https://creativecommons.org/licenses/by/4.0/). epsin1, epsin2, epsin1 (Ig)

Hip1R, intersectin, β-arrestin, AP-2 (σ), AP-2 (β), stonin, Dab2 be completed within 60 to 120 s in most types of eukaryotic cells Dyn2, Fcho2 (FLAG), FCHo2, FCHo2 (Ig) (Cocucci et al., 2012; Kadlecova et al., 2017; Nández et al., 2014; Eps15 (FLAG), Eps15, Eps15 (Ig) Taylor et al., 2011). The specific timecourse of the assembly and shape transformations of CCSs driven by endocytic proteins and Snx9, syndapin, amphiphysin lipids (Box 2) and the characteristic spatially modulated distribution of these proteins in the membrane plane imply a certain time order in which endocytic proteins are recruited to the endocytic site. Indeed, C FCHO2 Endophilin several recent studies have explored the dynamics of endocytic Eps15 protein recruitment in mammalian cells (Bucher et al., 2018; Henne mu2 (AP2) Amph1 et al., 2010; Miller et al., 2015; Scott et al., 2018; Sochacki et al., BIN1 2017; Taylor et al., 2011). These works have demonstrated that, similar to what was shown in the seminal findings by Drubin and FCHO1 coworkers for yeast cells (Kaksonen et al., 2005), mammalian endocytic proteins can be grouped into distinct modules according to their recruitment kinetics (Taylor et al., 2011). Extensive Epsin Dynamin1 measurements of the recruitment kinetics of 34 mammalian CALM Dynamin2 endocytic proteins by using dual-color total internal reflection NECAP fluorescence microscopy (TIRF) with respect to the timing of Clathrin Myosin 1E CCV scission (Taylor et al., 2011) have defined four major (LCa) Synaptojanin endocytic protein groups that may comprise functional modules in cells (Fig. 2C). The earliest group of proteins that accumulate at the endocytic site comprises the peripherally located proteins FCHo1/2 and ESP15 and their binding partners intersectin 1/2 and AP-2, with the latter found both at the periphery and in the center protein complexes appear to spatially limit coat polymerization of the coat (Sochacki et al., 2017). The concentrations of these (Sundquist and Krausslich, 2012; Yeow et al., 2017), in addition to proteins reaches their maximal level ∼20 s before scission and other possible functions. A challenge for future research will be to decay towards the scission reaction (Taylor et al., 2011). understand whether the segregation of endocytic proteins into central According to a more detailed scenario (Henne et al., 2010), and peripheral nanodomains is a general feature of all protein coats in within this group of proteins, FCHo1/2 appear first and recruit cells and to determine whether the peripherally localized proteins Eps15 and the intersectins, which then cooperatively engage and execute functions that are common for different vesicle coats. activate the adaptor complex AP-2 (Hollopeter et al., 2014; Ma et al., 2016) through a large-scale conformational transition that Timecourse of endocytic protein recruitment to sites of aligns its lipid- and cargo-binding sites with the plasma membrane endocytosis in mammalian cells (Jackson et al., 2010). The entire process of endocytic vesicle formation from its initiation The second group of proteins that is recruited with some to vesicle separation from the plasma membrane and uncoating can delay comprises the endocytic factors found in the central part of Journal of Cell Science

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CCSs, such as clathrin, CALM (or AP180 in neurons), epsin and According to the time sequence of endocytic protein recruitment NECAP1/2. These proteins attain their maximal presence close to to sites of CME (Henne et al., 2007; Taylor et al., 2011), the the time point of the scission event and disappear right after it endocytic process is proposed to be initiated by the self-assembly (Taylor et al., 2011). of FCHo1/2 proteins (Henne et al., 2007) on the surface of the Proteins of the third group arrive and reach their maximal plasma membrane through their lateral and end-to-end interactions concentration at the endocytic site relatively late, just a few seconds (Frost et al., 2008) (but see Cocucci et al., 2012 for a different view). prior to scission, and disappear at or right after membrane scission. In in vitro experiments using liposomes in the presence of high These comprise the N-BAR domain proteins endophilin and concentrations of FCHo2 that are sufficient to cover the entire amphiphysin, as well as dynamin (Taylor et al., 2011). These available membrane surface (Frost et al., 2008; Henne et al., 2007), factors predominantly localize to the coat periphery (Sochacki FCHo2 forms helical coats molding membranes into tubules of et al., 2017) and, at the time point of their maximal recruitment, the ∼130 nm diameter (Henne et al., 2010). It is reasonable to assume concentrations of the early-recruited peripheral proteins, such as that at lower intracellular FCHo concentrations, arc-like FCHo coats FCHo2 and EPS15, are already low (Sochacki et al., 2017; Taylor are initially formed on the membrane surface (Fig. 3A), which can et al., 2011). As a consequence, the proteins of the first and third cover only a small portion of the plasma membrane and will be groups, practically, do not co-exist at the CCS edge. referred to below as FCHo arcs. The shapes of the FCHo arcs and The fourth group of proteins, which nucleate and promote the of the membrane underneath is determined by the interplay polymerization of actin filaments, such as N-WASP, ARP2/3, between the bending rigidity of the membrane and the overall and actin itself reach their maximal concentration close to fission rigidity of the FCHo arc, with the latter depending on the arc (Taylor et al., 2011). According to a recent proposal, actin thickness (the amount of proteins forming the arc). For a small polymerization is initiated primarily at the base of endocytic CCPs FCHo polymer that forms an arc, the arc rigidity must be smaller by the N-WASP-associated F-BAR domain protein FCHSD2 to than or comparable to that of the membrane. In this case, the FCHo facilitate CCP maturation (Almeida-Souza et al., 2018). In addition, arc is not expected to be able to effectively bend the membrane F-actin may aid membrane fission (Ferguson et al., 2009) and propel into a cylindrical shape, because of the internal membrane the newly formed endocytic vesicles away from the endocytic site resistance to curving. As a result, the FCHo arc, likely, lies (Nández et al., 2014). As the exact role and importance of actin may nearly flat on the membrane surface or may adopt a shape of a vary depending on cell type and conditions (e.g. membrane tension), segment of an almost flat truncated cone with an internal edge we refer the reader to excellent recent reviews on the topic (Hinze and diameter of 130 nm (Fig. 3A). The corresponding shape of the Boucrot, 2018; Kaksonen and Roux, 2018). membrane in the vicinity of the arc must be flat or slightly curved Finally, the fifth group of proteins that is recruited to CCSs (Fig. 3A). The internal edge of the FCHo arc recruits and activates comprises factors that are linked to vesicle uncoating, such as AP-2 proteins, which become concentrated near the internal the J-domain protein auxilin, a co-factor for Hsc70-mediated border of the FCHo arc. Activated AP-2 (Hollopeter et al., 2014) ATP-driven clathrin disassembly, and inositol phosphate in turn binds clathrin and activates its polymerization. FCHo arcs phosphatases, including oculocerebrorenal syndrome of Lowe may further polymerize into complete rings that direct clathrin (OCRL) and synaptojanin (Di Paolo and De Camilli, 2006; polymerization into an almost flat cage that covers the membrane He et al., 2017; Kaksonen and Roux, 2018; Nández et al., 2014; patch inside the ring (Fig. 3B), in accordance with Sochacki et al. Posor et al., 2015). (2017). The expected diameter of such clathrin arrays should, therefore, roughly correspond to the preferred internal diameter of A speculative model for endocytic vesicle formation the FCHo ring (∼130 nm), similar to what has been observed in How precisely endocytic membrane sites are progressively recent experimental data (Bucher et al., 2018). The clathrin array reshaped has remained largely enigmatic. Based on the recent formed within the FCHo ring then recruits epsin and CALM to the data regarding the timing and spatiotemporal nanoscale distribution central part of the CCS (Sochacki et al., 2017), which insert their of endocytic proteins described above, we propose the following amphipathic helices into the clathrin-coated membrane to generate speculative model for endocytic vesicle formation that integrates local isotropic curvature (Fig. 3C). The generation of membrane information from cellular and in vitro experiments, as well as curvature via positive feedback facilitates the recruitment of theoretical modeling. additional curvature-sensitive epsin and CALM molecules, leading to

AB CDE

Fig. 3. A speculative model for endocytic vesicle formation. (A) Endocytic vesicle formation is initiated by helical arc-like FCHo coats (yellow). The FCHo arc complex activates and recruits AP-2 (green) to direct clathrin assembly (black) at the FCHo arc. (B) As FCHo arcs grow into rings clathrin assemblies fill the inside of the FCHo ring. (C) Addition of curvature-inducing epsin and CALM molecules (blue) leads to the progressive invagination of the membrane within the FCHo ring into a dome-like shape. The FCHo ring remains at the periphery of this structure and, thereby, covers the forming membrane neck. (D) As the neck narrows, binding of highly curved PX-BAR and N-BAR protein scaffolds (red; SNX9, SNX18, amphiphysin, endophilin) is favored. (E) These PX-BAR and N-BAR proteins then recruit dynamin (pink) to the neck, with FCHo dissociating prior to fission. Journal of Cell Science

7 REVIEW Journal of Cell Science (2018) 131, jcs216812. doi:10.1242/jcs.216812 the progressive invagination of the membrane within the FCHo ring either of these endocytic structural proteins (Boucrot et al., 2012; into a dome-like shape, and further towards a closed spherical shape. Henne et al., 2010; Koo et al., 2015; Miller et al., 2015) would be This transformation requires additional polymerization of clathrin expected to result in shallow, possibly less stable CCSs in cells. within the ring, which should be made possible by the fast exchange Our final prediction is that progressive CCS invagination and the of clathrin triskelia between the clathrin cage and the surrounding resulting constriction of the CCP neck that is derived from the rim cytoplasm (Avinoam et al., 2015; Wu et al., 2001). area of flat or shallow CCPs will cause FCHo proteins to dissociate In the course of epsin- and CALM-driven shaping of the central from endocytic structures and to be replaced at the CCP necks by clathrin-coated part of the endocytic site, the FCHo ring remains at BAR-domain proteins, such as SNX9, SNX18, amphiphysin and the periphery of the structure and, thereby, covers the forming endophilin, which preferentially bind to highly curved membrane. membrane neck that connects the nascent bud to the surrounding Indeed, FCHo has been shown to dissociate from CCPs during late membrane. The neck becomes gradually narrower during the stages prior to membrane fission (Henne et al., 2010; Taylor et al., evolution of the central part towards a closed spherical shape 2011). However, whether FCHo rings are retained under conditions (Fig. 3D). Thinning of the neck makes it progressively unfavorable of impaired curvature acquisition (e.g. owing to depletion of epsins for FCHo proteins to bind to the membrane as it prefers the radius of and/or CALM or AP180) or neck constriction (e.g. loss of SNX9 or membrane curvature to be ∼130 nm. We predict that the progressive SNX18) has not been experimentally tested so far. incompatibility between neck diameter and curvature preference of Moreover, we suggest that similar principles to those outlined here the FCHo polymer drives its dissociation from the membrane neck. for membrane remodeling during CME may also govern other related At the same time, as the neck becomes narrower, the binding of membrane-trafficking processes, such as the formation of vesicles protein scaffolds that are more curved, such the PX-BAR and and tubules during the different pathways of clathrin-independent N-BAR domain proteins, SNX9/18, amphiphysin and endophilin, is endocytosis, caveolar membrane invagination, constitutive secretion, favored (Fig. 3E). Eventually, these factors, possibly in association and the formation and budding of vesicles and tubules at endosomes with dynamin, replace FCHo on the neck, thereby facilitating and the Golgi complex (Antonny, 2011; Bonifacino and Glick, 2004; further constriction of the neck region to eventually give rise to Faini et al., 2013; Kozlov et al., 2014). The recent development and dynamin-mediated scission and the release of a free CCV. application of genome engineering together with quantitative super- resolution and correlative light and electron microscopy approaches Predictions from the hypothetical model and outlook should allow us to test the above predictions and address these Several predictions arise from this hypothetical model for endocytic exciting questions in the near future. vesicle formation. FCHo1/2 and associated factors, such as Eps1, Eps15R and intersectin 1, direct clathrin polymerization to the Acknowledgements inside of the predicted FCHo arc, so that clathrin polymerization is We wish to thank Dr Dmytro Puchkov (Electron microscopy facility of the FMP, Berlin) for providing the electron micrographs shown in Fig. 1. initiated along the internal edge of the FCHo arc and propagates towards the center. Therefore, FCHo polymers may serve to spatially Competing interests limit the polymerization of clathrin to an area with a calibrated The authors declare no competing or financial interests. diameter of ∼130 nm. With the recent advent of novel super- resolution and correlative microscopy imaging methods (Lehmann Funding ’ et al., 2015; Liu et al., 2018; Schöneberg et al., 2017; Sochacki Work in the authors laboratories was supported by grants from the German Funding Agency Deutsche Forschungsgemeinschaft (DFG) (SFB958/A07 to V.H.), et al., 2017), testing this prediction indeed seems experimentally the Israel Science Foundation (grant 1066/15 to M.M.K.), and European Union feasible. The effective purpose of limiting clathrin polymerization consortium InCeM (to M.M.K.). to the inside of the predicted FCHo ring could be to allow the formation of a membrane neck that is free of a clathrin coat, which References facilitates the binding and boosts the activity of fission proteins, Almeida-Souza, L., Frank, R. A. W., Garcia-Nafria, J., Colussi, A., Gunawardana, N., Johnson, C. M., Yu, M., Howard, G., Andrews, B., such as endophilin and dynamin. Vallis, Y. et al. (2018). A flat BAR protein promotes actin polymerization at the However, the FCHo–Eps15–intersectin complex also stabilizes base of clathrin-coated pits. Cell 174, 325-337.e14. clathrin coats by recruiting and activating AP-2. Our model therefore Antonny, B. (2011). Mechanisms of membrane curvature sensing. Annu. Rev. Biochem. 80, 101-123. predicts that, depending on the cell type and the expression levels Antonny, B., Burd, C., De Camilli, P., Chen, E., Daumke, O., Faelber, K., of other factors that can recruit clathrin or AP-2, loss of FCHo may Ford, M., Frolov, V. A., Frost, A., Hinshaw, J. E. et al. (2016). Membrane fission result in an excessive growth of clathrin arrays, which leads by dynamin: what we know and what we need to know. EMBO J. 35, 2270-2284. to enhanced clathrin plaque formation, as reported previously Avinoam, O., Schorb, M., Beese, C. J., Briggs, J. A. and Kaksonen, M. (2015). ENDOCYTOSIS. Endocytic sites mature by continuous bending and remodeling (Ma et al., 2016; Mulkearns and Cooper, 2012), and/or to reduction of the clathrin coat. Science 348, 1369-1372. of the stability of productive CCPs, as a consequence of a partial Bonifacino, J. S. and Glick, B. S. (2004). The mechanisms of vesicle budding and inactivation of AP-2. The latter effect can be expected, in particular, fusion. Cell 116, 153-166. in cells where clathrin plaque formation, for reasons that remain to Borner, G. H. H., Antrobus, R., Hirst, J., Bhumbra, G. S., Kozik, P., Jackson, L. P., Sahlender, D. A. and Robinson, M. S. (2012). Multivariate be fully understood, does not occur (e.g. BSC-1 cells) (Cocucci proteomic profiling identifies novel accessory proteins of coated vesicles. et al., 2012; Henne et al., 2010). The budding of endocytic vesicles J. Cell Biol. 197, 141-160. from the inside of large clathrin-coated arrays is energetically costly Boucrot, E., Pick, A., Çamdere, G., Liska, N., Evergren, E., McMahon, H. T. and owing to the presence of a clathrin coat at the neck region that would Kozlov, M. M. (2012). Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124-136. need to be removed prior to vesicle scission. Hence, loss of FCHo in Brodsky, F. M. (2012). Diversity of clathrin function: new tricks for an old protein. either of the above scenarios would be expected to hamper the Annu. Rev. Cell Dev. Biol. 28, 309-336. efficiency of endocytic vesicle formation in CME. Bucher, D., Frey, F., Sochacki, K. A., Kummer, S., Bergeest, J.-P., ̈ The above model further predicts a specific and crucial endocytic Godinez, W. J., Krausslich, H.-G., Rohr, K., Taraska, J. W., Schwarz, U. S. et al. (2018). Clathrin-adaptor ratio and membrane tension function for epsins and CALM or AP180 in generating curvature regulate the flat-to-curved transition of the clathrin coat during endocytosis. inside FCHo rings to enable CCS invagination. Therefore, loss of Nat. Commun. 9, 1109. Journal of Cell Science

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