α-Synuclein colocalizes with AP180 and affects the size of clathrin lattices.

α-Synuclein and family members ß- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αßγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.

O-GlcNAc transferase modulates the cellular endocytosis machinery by controlling the formation of clathrin-coated pits.

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked ß-GlcNAc (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

The molecular associations in clathrin-coated pit regulate ß-arrestin-mediated MAPK signaling downstream of µ-opioid receptor.

It has been thought that µ-opioid receptors (MOPs) activate the G protein-mediated analgesic pathway and ß-arrestin 2-mediated side effect pathway; however, ligands that only minimally recruit ß-arrestin 2 to MOPs may also cause opioid side effects. Moreover, such side effects have been induced in mutant mice lacking ß-arrestin 2 or expressing phosphorylation-deficient MOPs that do not recruit ß-arrestin 2. These findings raise the critical question of whether ß-arrestin 2 recruitment to MOP triggers side effects. Here, we show that ß-arrestin 1 and 2 are essential in the efficient activation of the Gi/o-mediated MAPK signaling at MOP. Moreover, the magnitude of ß-arrestin-mediated signals is not correlated with the magnitude of phosphorylation of the carboxyl-terminal of MOP, which is used to evaluate the ß-arrestin bias of a ligand. Instead, the molecular association with ß2-adaptin and clathrin heavy chain in the formation of clathrin-coated pits is essential for ß-arrestin to activate MAPK signaling. Our findings provide insights into G protein-coupled receptor-mediated signaling and further highlight a concept that the accumulation of molecules required for endocytosis is critical for activating intracellular signaling.

Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles.

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.

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.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2.

Clathrin-coated vesicles mediate membrane cargo transportation from the plasma membrane, the trans-Golgi network, the endosome, and the lysosome. Heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) are bridges that link cargo-loaded membranes to clathrin coats. Assembly of AP2 was previously considered to be spontaneous; however, a recent study found AP2 assembly is a highly orchestrated process controlled by alpha and gamma adaptin binding protein (AAGAB). Evidence shows that AAGAB controls AP1 assembly in a similar way. Insights into the orchestrated assembly process and three-dimensional structures of assembly intermediates are only emerging. Here, we describe a protocol for reconstitution and purification of the complexes containing AAGAB and AP1 or AP2 subunits, known as AP1 and AP2 hemicomplexes. Our purification routinely yields milligrams of pure complexes suitable for structural analysis by X-ray crystallography and electron microscopy.

Advances in structural, spatial, and temporal mechanics of plant endocytosis.

Endocytic trafficking underlies processes essential for plant growth and development, including the perception of and response to abiotic and extracellular stimuli, post-Golgi and exocytic trafficking, and cytokinesis. Protein adaptors and regulatory factors of clathrin-mediated endocytosis that contribute to the formation of endocytic clathrin-coated vesicles are evolutionarily conserved. Yet, work of the last ten years has identified differences between the endocytic mechanisms of plants and Opisthokonts involving the endocytic adaptor TPLATE complex, the requirement of actin during CME, and the function of clathrin-independent endocytosis in the uptake of plant-specific plasma membrane proteins. Here, we review clathrin-mediated and -independent pathways in plants and describe recent advances enabled by new proteomic and imaging methods, and conditional perturbation of endocytosis. In addition, we summarize the formation and trafficking of clathrin-coated vesicles based on temporal and structural data garnered from high-resolution quantitative imaging studies. Finally, new information about the cross-talk between endocytosis and other endomembrane trafficking pathways and organelles will also be discussed.

Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin.

During clathrin-mediated endocytosis (CME), flat plasma membrane is remodeled to produce nanometer-scale vesicles. The mechanisms underlying this remodeling are not completely understood. The ability of clathrin to bind membranes of distinct geometries casts uncertainty on its specific role in curvature generation/stabilization. Here, we used nanopatterning to produce substrates for live-cell imaging, with U-shaped features that bend the ventral plasma membrane of a cell into shapes resembling energetically unfavorable CME intermediates. This induced membrane curvature recruits CME proteins, promoting endocytosis. Upon AP2, FCHo1/2, or clathrin knockdown, CME on flat substrates is severely diminished. However, induced membrane curvature recruits CME proteins in the absence of FCHo1/2 or clathrin and rescues CME dynamics/cargo uptake after clathrin (but not AP2 or FCHo1/2) knockdown. Induced membrane curvature enhances CME protein recruitment upon branched actin assembly inhibition under elevated membrane tension. These data establish that membrane curvature assists in CME nucleation and that the essential function of clathrin during CME is to facilitate curvature evolution, rather than scaffold protein recruitment.

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis.

Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.

Clathrin coated pits as signaling platforms for Akt signaling.

Signaling by the activated epidermal growth factor receptor (EGFR) results in diverse cell fates. In this issue, Cabral-Dias et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.201808181) demonstrate how plasma membrane clathrin coated pits can act as a signaling platform for one branch of EGFR downstream signaling.

Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components.

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.

Dual clathrin and integrin signaling systems regulate growth factor receptor activation.

The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor ß5-integrin. We show that ligand-activated EGFR, Grb2, Src, and ß5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to EGF into large flat plaques and provide a signaling platform that link EGFR and ß5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/ß5-integrin axis prevents both clathrin plaque growth and dampens receptor signaling. Our study reveals a reciprocal regulation between clathrin lattices and two different receptor systems to coordinate and enhance signaling. These findings have broad implications for the regulation of growth factor signaling, adhesion, and endocytosis.

Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry.

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s).

A conserved clathrin-coated vesicle component, OsSCYL2, regulates plant innate immunity in rice.

Clathrin-mediated vesicle trafficking (CMVT) is a fundamental process in all eukaryotic species, and indispensable to organism's growth and development. Recently, it has been suggested that CMVT also plays important roles in the regulation of plant immunity. However, the molecular link between CMVT and plant immunity is largely unknown. SCY1-LIKE2 (SCYL2) is evolutionally conserved among the eukaryote species. Loss-of-function of SCYL2 in Arabidopsis led to severe growth defects. Here, we show that mutation of OsSCYL2 in rice gave rise to a novel phenotype-hypersensitive response-like (HR) cell death in a light-dependent manner. Although mutants of OsSCYL2 showed additional defects in the photosynthetic system, they exhibited enhanced resistance to bacterial pathogens. Subcellular localisation showed that OsSCYL2 localized at Golgi, trans-Golgi network and prevacuolar compartment. OsSCYL2 interacted with OsSPL28, subunit of a clathrin-associated adaptor protein that is known to regulate HR-like cell death in rice. We further showed that OsSCYL2-OsSPL28 interaction is mediated by OsCHC1. Collectively, we characterized a novel component of the CMVT pathway in the regulation of plant immunity. Our work also revealed unidentified new functions of the very conserved SCYL2. It thus may provide new breeding targets to achieve both high yield and enhanced resistance in crops.

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses.

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.

Dynamin-2 mediates clathrin-dependent endocytosis for amyloid-ß internalization in brain microvascular endothelial cells.

Dynamin is recognized as a crucial regulator for membrane fission and has three isoforms in mammals. But the expression patterns of dynamin isoforms and their roles in non-neuronal cells are incompletely understood. In this study, the expression profiles of dynamin isoforms and their roles in endocytosis was investigated in brain endothelial cells. We found that Dyn2 was expressed at highest levels, whereas the expression of Dyn1 and Dyn3 were far less than Dyn2. Live-cell imaging was used to investigate the effects of siRNA-mediated knockdown of individual dynamin isoforms on transferrin uptake, and we found that Dyn2, but not Dyn1 or Dyn3, is required for the endocytosis in brain endothelial cells. Results of dextran uptake assay showed that dynamin isoforms are not involved in the clathrin-independent fluid-phase internalization of brain endothelial cells, suggesting the specificity of the role of Dyn2 in clathrin-dependent endocytosis. Immunofluorescence and electron microscopy analysis showed that Dyn2 co-localizes with clathrin and acts at the late stage of vesicle fission in the process of endocytosis. Further results showed that Dyn2 is necessary for the basolateral-to-apical internalization of amyloid-ß into brain endothelial cells. We concluded that Dyn2, but not Dyn1 or Dyn3, mediates the clathrin-dependent endocytosis for amyloid-ß internalization particularly from basolateral to apical side into brain endothelial cells.