Capturing the mechanics of clathrin-mediated endocytosis

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.


Introduction
Endocytosis is an intriguing mechanical process by which cells internalise molecules and transport them to specific locations. Clathrin-mediated endocytosis is one of the major endocytic pathways, so named for its requirement for the protein clathrin. Through clathrinmediated endocytosis antibodies, hormones and nutrients such as cholesterol and iron, amongst other molecules, are selectively absorbed via interactions with specific receptors and intracellular adaptor proteins.
Beyond simple absorption of required substances, however, internalisation of receptors leads to control of signalling processes that has wide ranging influence on cellular function and enables a coordinated response to the changing needs of the cell.
For clathrin-mediated endocytosis to take place the plasma membrane becomes coated with clathrin and adaptor proteins and changes shape to form a clathrincoated pit (CCP). Ligand-bound receptors known as cargo are selectively included in the newly forming vesicle and once formed, the coated vesicle detaches from the membrane, travels within the cell and delivers its contents (summarised in Figure 1). The defining events in clathrin-coated vesicle formation; selection of receptors, membrane bending, progression from coated pit to fully formed vesicle and detachment from the membrane, are driven by a coordinated network involving at least 40 proteins, some of which assemble to form the clathrin coat. Central to this web of interactions are the adaptor protein AP2 and clathrin. AP2 binds to cytoplasmic receptor motifs and phosphatidylinositol 4,5-bisphosphate (PIP2) in the lipid membrane, becoming activated to an open state [1e6]. Synergistic interactions between AP2 and clathrin enable further activation of AP2 and result in clathrin assembling around the coated vesicle, with recruitment of multiple further accessory proteins, to form a scaffold with a striking polyhedral structure. Once completed, dynamin, along with other adaptor proteins, facilitates fission of the vesicle from the membrane and the clathrin coat is then rapidly removed.
This necessity to trigger assembly of coat components to form the clathrin coat in response to changing cellular needs requires us to understand the mechanical process of coat assembly and membrane bending in structural detail. Cryo-EM studies have contributed significantly to our understanding of these processes from as early as 1986 [7e11], but more recently, high resolution single particle and tomography studies have delivered key findings illuminating a series of events in the life cycle of a clathrin-coated vesicle.

Clathrin cages and AP2
The striking lattice seen around clathrin-coated vesicles results from the assembly of three-legged clathrin trimers into polyhedral structures, which are thought to stabilise the vesicle as it grows while allowing clathrin to engage with multiple binding partners that comprise the clathrin coat. These lattice structures are short-lived however, since after vesicle formation the clathrin coat is rapidly disassembled and the coat components released. Thus, the mechanical properties of the clathrin lattice must permit the seemingly paradoxical formation of a stable structure that can then be quickly dismantled. So how can such organised structures assemble so readily? Single particle cryo-EM studies have recently elucidated key structural elements important for cage lattice formation that provide some clues to answering this. In work using purified clathrin, Morris et al. [12] showed that reconstituted clathrin cages with differing geometries employed similar contacts between triskelion legs and that differences in geometry could be accommodated via flexing of the legs (Figure 2a and b). This ability to make cages of different shapes using common contact points without requiring specific leg conformations would facilitate formation of a wide range of structures without losing time waiting for the 'right' leg shape to achieve a match. This complements previous biochemical and biophysical studies that showed leg to leg contacts were weak and suggesting avidity effects would stabilise the lattice once multiple contacts had been made [13,14]. In addition the molecular model of Morris et al. [12] revealed coiled-coil interactions within the helical tripod that comprises the clathrin trimerization domain (Figure 2c) suggesting an explanation for the stability of this domain. Further detail came from the structure of a natively assembled clathrin hub (Paraan et al. [15]) which showed that the QLML residues of the QLMLT motif (critical for Hsc70 binding and clathrin uncoating [16]) in each helix of the trimerization domain interact with two crossing distal legs. This suggests that Hsc70 may be able to directly destabilise cages by disrupting this cross-linking interaction (Figure 2d), in addition to the entropic effect contributed through binding of Hsc70 to this site that adds mass to the flexible polypeptide at the clathrin C terminus [17]. Stages of clathrin-mediated endocytosis. Schematic diagram illustrating the assembly and disassembly of a clathrin-coated pit. Briefly, AP2 (and other clathrin-associated sorting proteins) binds cargo and phosphoinositide moieties in the membrane, which triggers clathrin recruitment and subsequent lattice assembly. Other adaptor proteins are recruited to the invaginating membrane to ensure stable lattice growth and close of the clathrin-coated pit. Dynamin (along with other adaptor proteins) drives membrane scission resulting in the clathrin-coated vesicle being fully internalised into the cytoplasm. Auxilin and Hsc70 are key factors mediating clathrin uncoating. The vesicle is then free and primed to fuse with an early endosome. Figure adapted from 'Clathrin-mediated endocytosis', by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates. In cells the process of clathrin assembly is coordinated with the action of adaptor proteins that may bind to selected cargo, to the lipid membrane, to one another and clathrin. Thus, the question of what drives clathrincoated vesicle formation forward is complex. Amidst this complexity the role of AP2 is critical. Its activation has been elegantly uncovered through a series of crystallographic and mechanistic studies spanning more than two decades [1e6]. Until recently a structural description of AP2 when bound to lipid membranes was still lacking. Kovtun et al.
[18] used cryo-electron tomography to determine structures of a recombinant form of AP2 (lacking the alpha hinge and appendage domain) bound to PIP2-containing phospholipid membranes and revealed the AP2 structure progressively opening in the presence of two types of receptor internalisation motif; first the YXXPHI motif and then the LLXXX motif. These results provided direct visualisation of stages of the activation mechanism of AP2 predicted by previous crystallographic and biochemical studies, offering confirmation that AP2 moves from a closed state when in solution to one that opens up to bind more effectively to membrane and cargo when those components are encountered.
Two further studies have investigated the transitions triggered in AP2 during CCV initiation and the influence of muniscins on this process. Muniscins are amongst a number of pioneer proteins that, along with AP2, arrive at the membrane prior to clathrin coat formation [27] report a 3.9 Å cryo-EM structure of the purified core of AP2 bound to the AP2-activator domain of SGIP (termed SGIP APA ) and plasma membrane substitute, heparin. The structure revealed that SGIP APA binding induces a previously uncharacterised AP2 conformation (distinct from the canonical closed and open AP2 structures [1,4]), which the authors termed the 'primed' state of AP2. Biochemical pull-down assays and disulphide crosslinking experiments using supported-lipid bilayers suggested that this primed AP2 state was compatible with AP2 in a membrane bound state susceptible to further rearrangement upon binding of YxxF-cargo.
[28] on the other hand have used eTIRF-SIM and biolayer interferometry to reveal that membrane and FCHO2 compete for AP2 binding, supporting the conclusions drawn from experiments in cells with cryo-electron tomography and single particle analysis of AP2-FCHO2 complexes. In these structural studies, density relating to portions of FCHO2 known to associate with AP2 could not be observed. The results from Zaccai et al. suggest FCHO2 and AP2 do not remain bound to each other whilst AP2 is associated with the membrane. The different conclusions arising from these two studies further emphasise the sensitivity of AP2 conformation to its environment and its intriguing ability to adopt a range of conformations and binding modes. Further work is needed to resolve this mechanism since such flexibility may well be crucial in allowing AP2 to make responses precisely tailored to the presence or otherwise of cargos, membrane, adaptors and clathrin during endocytosis.
In addition to the sequential binding events discussed above, AP2 also undergoes multiple phosphorylation events , also linked two adjacent terminal domains. Thus, it seems that multiple binding modes are a feature of the interaction of b2-appendage plus hinge with clathrin. These structural data are summarized in Figure 3.
These new structural studies also highlighted key residues at the interfaces between the b2-appendage and clathrin terminal domain. One of these, Tyr 815 on the 'sandwich' site of the b2-appendage was shown in cells to be of functional importance. Using the 'hot-wiring' method of Wood et al. [37], Smith et al. [36] showed that two binding sites on AP2, one involving Tyr815 and a second containing the clathrin box motif within the b2-appendage hinge region, were necessary to trigger hot-wired endocytosis. Taken together with the structural information from all three groups, and the significant body of work contributed by previous studies (see [34,43,44] for reviews) this supported a model whereby cross-linking of clathrin legs by the b2-appendage and hinge binding sites was a crucial element of productive coated vesicle formation.

Clathrin lattice structure in cells
The mechanical challenges that clathrin must overcome in performing its function are not limited to the assembly process itself. For decades the existence of extensive flat clathrin lattices in cells has stimulated debate [38e43] and observations that clathrin-coated vesicles can emerge from flat lattices have raised questions about how their curvature is achieved and how clathrin assembly might adapt to the changing curvature of the membrane (reviewed in [40,42,44]). Recent work by Sochacki et al. [45] has moved the debate forward by combining cryo-electron tomography with platinum replica electron microscopy (PREM) and fluorescence microscopy to examine the morphology of clathrin lattices in eight different mammalian cell lines. All cell lines contained a mixture of flat, domed and spherical patches of clathrin, with the domed intermediates having a lower curvature but the same surface area as the spherical clathrin. These findings agree with previous live cell fluorescence and electron microscopy studies that showed that flat clathrin can curve at the start of, during or after clathrin recruitment [41,46,47]. Quantitative analysis of the PREM data confirmed the presence of pentagons in flat, curved, and spherical clathrin lattice geometries, with an abundance in flat clathrin. Surprisingly, cryo-electron tomography of flat lattices revealed that these pentagon-containing assemblies are not a rigid network as has been modelled in the past [9,48] but contain many disordered and loose connections (Figure 4), challenging previous assumptions that flat clathrin lattices were complete and stable hexagonal arrays, which would have presented a higher energy barrier for rearrangement. These new structural details led the authors to propose a new model of CCV formation whereby clathrin curvature is triggered by the release of an inherent lattice flattening force (possibly imposed by binding to the substrate [45], the membrane [45] or actin [49]), which subsequently permits the loosely connected triskelia to relax into an energetically favourable geometry [45]. Interestingly a report by Tagiltsev et al. (2021) [50] independently proposes that flat clathrin lattices in cells store elastic energy and that curved clathrin-coated vesicles spontaneously emerge when removal of constraints releases that energy. Tagiltsev et al. [50] used high speed AFM methods to measure the dimensions of clathrin-coated structures in unroofed cells. Nanodissection techniques enabled the authors to remove clathrin triskelia from clathrin-coated structures and monitor the shape of structures that formed subsequently. The experimental measurements are placed in the context of calculations of the likely energy landscape defining formation of clathrin-coated structures on the cell membrane and tested against previous models proposing either constant area or constant curvature mechanisms of vesicle formation. Taken together these two reports independently propose compatible mechanisms for the formation of curved clathrin structures in cells based on complementary experimental observations.

Epsin and Hip1R lipid binding domains and their role in endocytosis
We have so far considered two central components of clathrin-mediated endocytosis, clathrin and AP2, but many further adaptor proteins contribute to the production of the clathrin-coated vesicle. Cryo-EM has recently contributed to our understanding of how two of these adaptor proteins, Sla2 and Ent1 from yeast (homologous to HIP1R and epsin in mammals), assemble on lipid membranes. Using single particle cryo-EM Lizarrondo et al. (2021) [51] resolved the structure of assembled ANTH and ENTH domains from Sla2 and Ent1 bound to PIP2. The 3.9 Å structure showed that ANTH-ENTH domain heterotetramers housed three distinct PIP2 binding sites in which PIP2 molecules were shared across ANTH and ENTH shows the side view from (a). Common and contrasting features of each binding mode are shown and commented in the panelled text. Associated cryo-electron microscopy maps and models are shown below their respective panels. Images were produced in Chimera [58] using accession codes: Smithclass 15 EMD12984 and 70m8.pdb; Smithclass 18 EMD12980, 1bpo.pdb [59] and 1e42.pdb [60]; Kovtun -EMD10754 and 6yai.pdb; and Paraan -EMD21616, 1bpo.pdb [59] and 1e42.pdb [60]. protein interfaces. Small-angle X-ray scattering and biolayer interferometry studies also demonstrated that assembly and disassembly of these complexes took place on the 100 ms time scale. Taken together this work highlighted the critical role played by PIP2 not only to enable the complex to bind to the membrane but also in promoting the rapid formation of the ANTH-ENTH complex. This supported previous suggestions for proposed positive cooperativity in the AENTH domain-membrane binding mechanism [52,53] and suggested a crucial role for PIP2 binding in control of membrane deformation by Sla2 and Ent1. Sla2 and Ent1 had previously been shown to link the endocytic machinery in yeast to the actin cytoskeleton [54]. The authors propose that lipid locking, combined with insertion of ENTH a0 amphipathic helices into the membrane [55e57], would make the Sla2/Ent1 AENTH assembly a more effective membrane anchor capable of distributing pulling forces contributed by actin polymerisation evenly over the membrane, thus facilitating membrane invagination during endocytosis.

Conclusions
The increasing sophistication of cryo-electron microscopy structure determination both through single particle analysis and tomography applications is having a profound impact on our understanding of biology. This has been exemplified in the field of clathrinmediated endocytosis where biochemically purified high-resolution structures can now be interpreted in the context of a more physiologically relevant membrane environment or within cells themselves. Determining the architecture of clathrin assemblies in vitro, clathrin-coated vesicles purified from source material and clathrin, AP2, cargo and lipid in model systems have provided a clearer picture of how AP2 interfaces with clathrin in a coated vesicle and of its mode of activation. The link shown between PIP2 binding and assembly of the ANTH and ENTH domains of Sla2 and Ent1 has further demonstrated the importance of being able to visualise membrane-bound structures to inform our understanding of function whilst emphasising the value of complementary biophysical techniques to provide dynamic information. Within cells, detailed structures of clathrin in flat lattices from cryo-electron tomography and results obtained using high speed AFM, together provide new insights into how clathrin morphology can adapt during CCV formation. In all these examples, advanced cryo-electron microscopy applications, in partnership with results from complementary biophysical and structural techniques, have captured mechanical processes driving clathrinmediated endocytosis in unprecedented detail.