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Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the "pseudo 3D" morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s -1 ) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis., Methods: Glomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification., Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed., Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment., Competing Interests: All authors have nothing to disclose., (Copyright © 2020 by the American Society of Nephrology.)

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes., Competing Interests: MN, AJ, RP, WK, ST, BC, JF No competing interests declared, (© 2020, Narasimhan et al.)

Deformation of the plasma membrane into clathrin-coated vesicles is a critical step in clathrin-mediated endocytosis and requires the orchestrated assembly of clathrin and endocytic adaptors into a membrane-associated protein coat. The individual role of these membrane-bending and curvature-stabilizing factors is subject to current debate. As such, it is unclear whether the clathrin coat itself is stiff enough to impose curvature and if so, whether this could be effectively transferred to the membrane by the linking adaptor proteins. We have recently demonstrated that clathrin alone is sufficient to form membrane buds in vitro. Here, we use atomic force microscopy to assess the contributions of clathrin and its membrane adaptor protein 2 (AP2) to clathrin coat stiffness, which determines the mechanics of vesicle formation. We found that clathrin coats are less than 10-fold stiffer than the membrane they enclose, suggesting a delicate balance between the forces harnessed from clathrin coat formation and those required for membrane bending. We observed that clathrin adaptor protein AP2 increased the stiffness of coats formed from native clathrin, but did not affect less-flexible coats formed from clathrin lacking the light chain subunits. We thus propose that clathrin light chains are important for clathrin coat flexibility and that AP2 facilitates efficient cargo sequestration during coated vesicle formation by modulating clathrin coat stiffness., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The fibroblast growth factor FGF21 was labeled with molecularly defined gold nanoparticles (AuNPs), applied to human adipocytes, and imaged by cryo-electron tomography (cryo-ET). Most AuNPs were in pairs about 80 Å apart, on the outer cell surface. Pairs of AuNPs were also abundant inside the cells in clathrin-coated vesicles and endosomes. AuNPs were present but no longer paired in multivesicular bodies. FGF21 could thus be tracked along the endocytotic pathway. The methods developed here to visualize signaling coupled to endocytosis can be applied to a wide variety of cargo and may be extended to studies of other intracellular transactions., Competing Interests: MA, SC, GJ, RK No competing interests declared, JW, JZ, YL Employee of Amgen at the time the study was conducted. There are no other competing financial interests to declare., (© 2019, Azubel et al.)

Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions., (Copyright © 2015, American Association for the Advancement of Science.)

The internalization and subsequent endosomal trafficking of proteins and membrane along the endocytic pathway is a fundamental cellular process. Over the last two decades, this pathway has emerged to be subject to extensive regulation by phosphoinositides (PIs), phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositol. Clathrin-mediated endocytosis (CME) is the endocytic mechanism characterized in most detail. It now represents a prime example of a process spatiotemporally organized by the interplay of PI metabolizing enzymes. The most abundant PI, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂], serves as a denominator of plasma membrane identity and together with cargo proteins is instrumental for the initiation of clathrin-coated pit (CCP) formation. During later stages of the process, the generation of phosphatidylinositol-3,4-bisphosphate [PI(3,4)P₂] and the dephosphorylation of PI(4,5)P₂regulate CCP maturation and vesicle uncoating. Here we provide an overview of the mechanisms by which PIs are made and consumed to regulate CME and other endocytic pathways and how conversion of PIs en route to endosomes may be accomplished. Mutations in PI converting enzymes are linked to multiple diseases ranging from mental retardation and neurodegeneration, to inherited muscle and kidney disorders suggesting that the tight control of PI levels along the endocytic pathway plays a critical role in cell physiology. This article is part of a Special Issue entitled Phosphoinositides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Clathrin-mediated endocytosis (CME) is a key pathway for transporting cargo into cells via membrane vesicles; it plays an integral role in nutrient import, signal transduction, neurotransmission, and cellular entry of pathogens and drug-carrying nanoparticles. Because CME entails substantial local remodeling of the plasma membrane, the presence of membrane tension offers resistance to bending and hence, vesicle formation. Experiments show that in such high-tension conditions, actin dynamics is required to carry out CME successfully. In this study, we build on these pioneering experimental studies to provide fundamental mechanistic insights into the roles of two key endocytic proteins-namely, actin and BAR proteins-in driving vesicle formation in high membrane tension environment. Our study reveals an actin force-induced "snap-through instability" that triggers a rapid shape transition from a shallow invagination to a highly invaginated tubular structure. We show that the association of BAR proteins stabilizes vesicles and induces a milder instability. In addition, we present a rather counterintuitive role of BAR depolymerization in regulating the shape evolution of vesicles. We show that the dissociation of BAR proteins, supported by actin-BAR synergy, leads to considerable elongation and squeezing of vesicles. Going beyond the membrane geometry, we put forth a stress-based perspective for the onset of vesicle scission and predict the shapes and composition of detached vesicles. We present the snap-through transition and the high in-plane stress as possible explanations for the intriguing direct transformation of broad and shallow invaginations into detached vesicles in BAR mutant yeast cells.

Clathrin-mediated endocytosis involves the coordinated assembly of clathrin cages around membrane indentations, necessitating fluid-like reorganization followed by solid-like stabilization. This apparent duality in clathrin's in vivo behavior provides some indication that the physical interactions between clathrin triskelia and the membrane effect a local response that triggers fluid-solid transformations within the clathrin lattice. We develop a computational model to study the response of clathrin protein lattices to spherical deformations of the underlying flexible membrane. These deformations are similar to the shapes assumed during intracellular trafficking of nanoparticles. Through Monte Carlo simulations of clathrin-on-membrane systems, we observe that these membrane indentations give rise to a greater than normal defect density within the overlaid clathrin lattice. In many cases, the bulk surrounding lattice remains in a crystalline phase, and the extra defects are localized to the regions of large curvature. This can be explained by the fact that the in-plane elastic stress in the clathrin lattice are reduced by coupling defects to highly curved regions. The presence of defects brought about by indentation can result in the fluidization of a lattice that would otherwise be crystalline, resulting in an indentation-driven, defect-mediated phase transition. Altering subunit elasticity or membrane properties is shown to drive a similar transition, and we present phase diagrams that map out the combined effects of these parameters on clathrin lattice properties.

HeLa cell lines can be experimentally manipulated using drugs or gene-silencing techniques such as RNA interference. Fractions enriched for clathrin-coated vesicles (CCVs) can be isolated from these cell lines and used to study the effects of these manipulations on the composition of CCVs. This protocol, originally developed in the laboratory of Margaret Robinson (Cambridge, United Kingdom), describes the preparation of a HeLa cell fraction that is enriched for a mixed population of CCVs and is suitable for analysis by mass spectroscopy, western blotting, or electron microscopy., (© 2014 Cold Spring Harbor Laboratory Press.)

The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by tryptic in-gel digest and peptide extraction followed by analysis in an Orbitrap mass spectrometer., (© 2014 Cold Spring Harbor Laboratory Press.)

The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by in-solution proteolytic digest and subsequent peptide fractionation, followed by analysis in a Q Exactive mass spectrometer., (© 2014 Cold Spring Harbor Laboratory Press.)

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

We developed "fractionation profiling," a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps., (© 2014 Borner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Clathrin-coated vesicles are major carriers of vesicular traffic in eukaryotic cells. This endocytic pathway relies on cycles of clathrin coat assembly and Hsc70-mediated disassembly. Here we identify histidine residues as major determinants of lattice assembly and stability. They are located at the invariant interface between the proximal and distal segments of clathrin heavy chains, in triskelions centered on two adjacent vertices of the coated-vesicle lattice. Mutation of these histidine residues to glutamine alters the pH dependence of coat stability. We then describe single-particle fluorescence imaging experiments in which we follow the effect of these histidine mutations on susceptibility to Hsc70-dependent uncoating. Coats destabilized by these mutations require fewer Hsc70 molecules to initiate disassembly, as predicted by a model in which Hsc70 traps conformational distortions during the auxilin- and Hsc70:ATP-mediated uncoating reaction., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Methods for the subcellular fractionation and enrichment of specific intracellular compartments are essential tools in the analysis of compartment composition and function. In vitro characterization of isolated cell organelles and other endomembrane intermediates, including exploration of the compartment protein ensemble, offers strong clues of in vivo function identity. Here, we describe methodology for the isolation of clathrin-coated vesicles from Arabidopsis thaliana suspension-cultured cells on the basis of differential and density centrifugation.

Clathrin-mediated endocytosis is a major route for the retrieval of plasma-membrane cargoes, and defects of this process can cause catastrophic human dysfunctions. However, the processes governing how a clathrin-coated profile (ccp) is initiated are still murky. Despite an ever-growing cast of molecules proposed as triggers of ccp nucleation and increasingly sophisticated bioimaging techniques examining clathrin-mediated endocytosis, it is yet unknown if ccp formation is governed by a universal mechanism. A recent paper by Cocucci et al. has tracked single-molecule events to identify that stable accumulation of ccps requires the near-simultaneous arrival of two AP2 adaptors bridged by one clathrin triskelion. This commentary examines the role of AP2 in cargo-mediated endocytosis in the light of recent advances in biophotonics, chemical inhibitors and genetics, examines the claims of other molecules to be the initiators of ccp formation and proposes future directions in research into this topic. Editor's suggested further reading in BioEssays: The evolution of dynamin to regulate clathrin-mediated endocytosis Abstract Clathrin-mediated endocytosis: What works for small, also works for big Abstract., (Copyright © 2013 WILEY Periodicals, Inc.)

Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate., (Published by Elsevier Inc.)

Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the "knocksideways" system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

The function of the Golgi has long been recognized to critically depend on vesicular transport from, to, and within its cisternae, involving constant membrane fission and fusion. These processes are mediated by Arf GTPases and coat proteins, and Rabs, tethers and SNARE proteins, respectively. In this article, we describe structural studies of Golgi coats and tethers and their interactions with SNAREs and GTPases as well as insights regarding membrane traffic processes that these have provided.

Flat clathrin lattices or 'plaques' are commonly believed to be the precursors to clathrin-coated buds and vesicles. The sequence of steps carrying the flat hexagonal lattice into a highly curved polyhedral cage with exactly 12 pentagons remains elusive, however, and the large numbers of disrupted interclathrin connections in previously proposed conversion pathways make these scenarios rather unlikely. The recent notion that clathrin can make controlled small conformational transitions opens new avenues. Simulations with a self-assembling clathrin model suggest that localized conformational changes in a plaque can create sufficiently strong stresses for a dome-like fragment to break apart. The released fragment, which is strongly curved but still hexagonal, may subsequently grow into a cage by recruiting free triskelia from the cytoplasm, thus building all 12 pentagonal faces without recourse to complex topological changes. The critical assembly concentration in a slightly acidic in vitro solution is used to estimate the binding energy of a cage at 25-40 k(B) T/clathrin., (© 2011 John Wiley & Sons A/S.)

Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles.

For more than 50 years cell biologists have embraced the concept that biochemical and enzymatic analysis of isolated subcellular fractions provides insight into the function and machineries of cellular compartments including organelles. The utility of this approach has been significantly enhanced with the advent of mass spectrometry leading to the broad application of organelle proteomics. Clathrin-coated vesicles (CCVs) form at the plasma membrane where they select protein and lipid cargo for endocytic entry into cells. CCVs also form at the trans-Golgi network, where they function in protein transport from the secretory pathway to the endosomal/lysosomal system. Herein we will describe how organelle proteomics of CCVs has greatly expanded our knowledge of the machineries, mechanisms and sites of clathrin-mediated membrane trafficking., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50-100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.

The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.

Our current view on biological membranes has gradually evolved from the influential fluid mosaic model of the early 1970s to a distinctively more complex picture. Biological membranes are now assumed to encompass multiple membrane domains and a plethora of protein-lipid and protein-protein interactions that compartmentalize and temporarily order what has originally been envisioned to be mostly random. In this minireview, we will first highlight some structural principles that govern membrane domain formation and permit a classification of membrane domains. We will then focus on the still controversial issue of lipid-based membrane domains, or lipid rafts, and discuss recent advances in detecting these enigmatic structures in living cells. Finally we will evaluate biochemical approaches to characterize lipid rafts and discuss their contribution to the emerging topic of lipid raft diversity.

Coated vesicles concentrate and package cargo molecules to mediate their efficient transport between intracellular compartments. Cytosolic coat proteins such as clathrin and adaptor complexes and coat protein complex I (COPI) and COPII self-assemble to deform the membrane and interact directly with cargo molecules to capture them in nascent buds. The guanosine triphosphatases (GTPases) Arf, Sar1, and dynamin are core components of the coated vesicle machinery. These GTPases, which associate with and dissociate from donor membranes in a guanosine triphosphate-dependent manner, can also actively remodel membranes. Recent evidence suggests that, although structurally diverse, Arf family GTPases and dynamin may play mechanistically similar roles as fidelity monitors that govern cargo packaging and coated vesicle maturation and as components of the fission machinery to mediate vesicle release.

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: alpha-COP, epsilon-COP, beta'-COP, beta-COP, gamma-COP, delta-COP, and zeta-COP. Here we report the solution structure of a truncated form (residues 1-149; zeta-COP149) of human zeta-COP (total 177 residues). It is the first three-dimensional structure of a "core" subunit of the COP I F-subcomplex. The structure of zeta-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel beta-sheet, a two-stranded antiparallel beta-sheet, and five alpha-helices. The global folding of zeta-COP149 is very similar to the crystal structures of AP1-sigma1 and AP2-sigma2, directly demonstrating the structural similarity between the "core" subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of zeta-COP149 and gamma-COP have packing interfaces and relative subunit orientations similar to those of AP2-sigma2 and AP2-alpha heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.

The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.

Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane-associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre-synaptic plasma membrane during exo- and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed.

The crystallography of two-dimensional particle packings on flexible surfaces of spherical topology is investigated. Examples are viral capsids and crystalline vesicles. Computer simulations of dynamically triangulated surfaces are employed to study the shape and structure of lattice defects as a function of the Föppl-von Kármán number gamma. We find that grain-boundary scars become much more fuzzy with increasing temperature, that the size of grain-boundary scars saturates with increasing vesicle radius, and that the buckling transition shifts to higher values of gamma due to the presence of scars.

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.

Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a "mock CCV" fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.

It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type-specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.

Endocytic clathrin-coated vesicles arise through the deformation of a small region of plasma membrane encapsulated by a cytosol-oriented clathrin lattice. The coat assembles from soluble protomers in a rapid and highly cooperative process, and invagination is tightly linked to the selective enrichment of cargo molecules within the nascent bud. Recent structural and functional studies demonstrate that coat assembly, membrane deformation, local actin dynamics and the final scission event are intricately coupled, and begin to reveal how key multifunctional, modular proteins are responsible for this linkage. An emerging mechanistic theme is how sequential engagement of common interaction surfaces or network hubs can evict prior binding partners from the assembly zone to ensure vectorial progression of the coat assembly process.

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force. We model CCVs as multilayered elastic spherical shells and, from AFM measurements, estimate their bending rigidity to be 285 +/- 30 k(B)T, i.e., approximately 20 times that of either the outer clathrin cage or inner vesicle membrane. Further analysis reveals a flexible coupling between the clathrin coat and the membrane, a structural property whose modulation may affect vesicle biogenesis and cellular function.

Cells rely on the correct sorting of endocytic ligands and receptors for proper function. Early endosomes have been considered as the initial sorting station where cargos for degradation separate from those for recycling. Using live-cell imaging to monitor individual endosomes and ligand particles in real time, we have discovered a sorting mechanism that takes place prior to early endosome entry. We show that early endosomes are in fact comprised of two distinct populations: a dynamic population that is highly mobile on microtubules and matures rapidly toward late endosomes and a static population that matures much more slowly. Several cargos destined for degradation are preferentially targeted to the dynamic endosomes, whereas the recycling ligand transferrin is nonselectively delivered to all early endosomes and effectively enriched in the larger, static population. This pre-early endosome sorting process begins at clathrin-coated vesicles, depends on microtubule-dependent motility, and appears to involve endocytic adaptors.

The process of clathrin-coated vesicle (CCV) formation/disassembly involves numerous proteins that act cooperatively. Phosphorylation is an important regulatory mechanism governing protein interactions in CCVs, and many of the core and accessory proteins of the CCV machinery are reversibly phosphorylated in vivo. CK2 is highly enriched in CCVs and is capable of phosphorylating a number of peripheral membrane proteins involved in the process of clathrin-mediated endocytosis. At least some of these phosphorylation events have been shown to be inhibitory for CCV assembly, and CK2 has been shown to be inactive when associated with intact CCVs. Here we show that CCV membranes inhibit CK2 activity even after incubation in trypsin, indicating that a component of the lipid bilayer may be the inhibitory factor. Consistent with this, we showed that liposomes containing phosphatidylinositol phosphates inhibit the activity of CK2 and that CK2 binds to those liposomes. Notably, liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), a component of CCVs, bind CK2 and inhibit its activity. Furthermore, we showed that the binding of CK2 to PtdIns(4,5)P(2)-containing liposomes is via the active site of CK2, thus providing a molecular explanation for the inhibition of CK2 activity when it is bound to PtdIns(4,5)P(2)-containing liposomes. Thus CK2 is inactive in CCVs because of the fact that it is bound to the CCV membrane via an interaction between PtdIns(4,5)P(2) in the CCV membrane and the active site in CK2.

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.