A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits.

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface.

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.

A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis.

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca2+ plays critical roles in exocytosis­endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca2+ in endocytosis have been reported; however, Ca2+ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca2+ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis­endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca2+-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca2+-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca2+ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

A clathrin-related protein FaRRP1/SCD2 integrates ABA trafficking and signaling to regulate strawberry fruit ripening.

Abscisic acid (ABA) is a critical regulator for nonclimacteric fruit ripening such as in the model plant of strawberry (Fragaria × ananassa). Although FaRRP1 is proposed to participate in clathrin-mediated endocytosis of ABA, its action molecular mechanisms in ABA signaling are not fully understood. Here, using our isolated FaRRP1 (ripening-regulation protein) and candidate ABA receptor FaPYL2 and FaABAR from strawberry fruit, a series of silico and molecular interaction analyses demonstrate that they all bind to ABA, and FaRRP1 binds both FaPYL2 and FaABAR; by contrast, the binding affinity of FaRRP1 to FaPYL2 is relatively higher. Interestingly, the binding of FaRRP1 to FaPYL2 and FaABAR affects the perception affinity to ABA. Furthermore, exogenous ABA application and FaRRP1 transgenic analyses confirm that FaRRP1 participates in clathrin-mediated endocytosis and vesicle transport. Importantly, FaRRP1, FaPYL2, and FaABAR all trigger the initiation of strawberry fruit ripening at physiological and molecular levels. In conclusion, FaRRP1 not only binds to ABA but also affects the binding affinity of FaPYL2 and FaABAR to ABA, thus promoting strawberry fruit ripening. Our findings provide novel insights into the role of FaRRP1 in ABA trafficking and signaling, at least in strawberry, a model plant for nonclimacteric fruit ripening.

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 roles of dynein and myosin VI motor proteins in endocytosis.

Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.

The TGEV Membrane Protein Interacts with HSC70 To Direct Virus Internalization through Clathrin-Mediated Endocytosis.

Coronavirus membrane protein is a major component of the viral envelope and plays a central role in the viral life cycle. Studies of the coronavirus membrane protein (M) have mainly focused on its role in viral assembly and budding, but whether M protein is involved in the initial stage of viral replication remains unclear. In this study, eight proteins in transmissible gastroenteritis virus (TGEV)-infected cells coimmunoprecipitated with monoclonal antibodies (MAb) against M protein in PK-15 cells, heat shock cognate protein 70 (HSC70), and clathrin were identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF MS). Further studies demonstrated that HSC70 and TGEV M colocalized on the cell surface in early stages of TGEV infection; specifically, HSC70 bound M protein through its substrate-binding domain (SBD) and preincubation of TGEV with anti-M serum to block the interaction of M and HSC70 reduced the internalization of TGEV, thus demonstrating that the M-HSC70 interaction mediates the internalization of TGEV. Remarkably, the process of internalization was dependent on clathrin-mediated endocytosis (CME) in PK-15 cells. Furthermore, inhibition of the ATPase activity of HSC70 reduced the efficiency of CME. Collectively, our results indicated that HSC70 is a newly identified host factor involved in TGEV infection. Taken together, our findings clearly illustrate a novel role for TGEV M protein in the viral life cycle and present a unique strategy used by HSC70 to promote TGEV infection in which the interaction with M protein directs viral internalization. These studies provide new insights into the life cycle of coronaviruses. IMPORTANCE TGEV is the causative agent of porcine diarrhea, a viral disease that economically affects the pig industry in many countries. However, the molecular mechanisms underlying viral replication remain incompletely understood. Here, we provide evidence of a previously undescribed role of M protein in viral replication during early stages. We also identified HSC70 as a new host factor affecting TGEV infection. We demonstrate that the interaction between M and HSC70 directs TGEV internalization in a manner dependent on CME, thus revealing a novel mechanism for TGEV replication. We believe that this study may change our understanding of the first steps of infection of cells with coronavirus. This study should facilitate the development of anti-TGEV therapeutic agents by targeting the host factors and may provide a new strategy for the control of porcine diarrhea.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells.

IMPORTANCE: Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Guanylate-binding protein 1 inhibits Hantaan virus infection by restricting virus entry.

Hantaan virus (HTNV) infection can cause hemorrhagic fever with renal syndrome (HFRS) in humans, and currently, there are no long-standing protective vaccines or specific antivirals available. Guanylate-binding protein 1 (GBP1) is an interferon-stimulated gene that defends against various pathogen infections. However, the function of GBP1 in HTNV infection remains unknown. Here, we describe how GBP1 prevents HTNV infection by obstructing virus entry. We found that HTNV infection induced GBP1 expression and that overexpression of GBP1 inhibited HTNV infection, while knockout of GBP1 had the opposite effect. Interestingly, GBP1 did not affect interferon (IFN) signaling during HTNV infection. Instead, GBP1 prevented HTNV from entering cells through clathrin-mediated endocytosis (CME). We also discovered that GBP1 specifically interacted with actin but not dynamin 2 (DNM2) and made it difficult for DNM2 to be recruited by actin, which may account for the suppression of CME during HTNV infection. These findings establish an antiviral role for GBP1 in inhibiting HTNV infection and help us better understand how GBP1 regulates HTNV entry and could potentially aid in developing treatments for this virus.

Insight into the role of clathrin-mediated endocytosis inhibitors in SARS-CoV-2 infection.

Emergence of SARS-CoV-2 variants warrants sustainable efforts to upgrade both the diagnostic and therapeutic protocols. Understanding the details of cellular and molecular basis of the virus-host cell interaction is essential for developing variant-independent therapeutic options. The internalization of SARS-CoV-2, into lung epithelial cells, is mediated by endocytosis, especially clathrin-mediated endocytosis (CME). Although vaccination is the gold standard strategy against viral infection, selective inhibition of endocytic proteins, complexes, and associated adaptor proteins may present a variant-independent therapeutic strategy. Although clathrin and/or dynamins are the most important proteins involved in CME, other endocytic mechanisms are clathrin and/or dynamin independent and rely on other proteins. Moreover, endocytosis implicates some subcellular structures, like plasma membrane, actin and lysosomes. Also, physiological conditions, such as pH and ion concentrations, represent an additional factor that mediates these events. Accordingly, endocytosis related proteins are potential targets for small molecules that inhibit endocytosis-mediated viral entry. This review summarizes the potential of using small molecules, targeting key proteins, participating in clathrin-dependent and -independent endocytosis, as variant-independent antiviral drugs against SARS-CoV-2 infection. The review takes two approaches. The first outlines the potential role of endocytic inhibitors in preventing endocytosis-mediated viral entry and its mechanism of action, whereas in the second computational analysis was implemented to investigate the selectivity of common inhibitors against endocytic proteins in SARS-CoV-2 endocytosis. The analysis revealed that remdesivir, methyl-ß-cyclodextrin, rottlerin, and Bis-T can effectively inhibit clathrin, HMG-CoA reductase, actin, and dynamin I GTPase and are more potent in inhibiting SARS-CoV-2 than chloroquine. CME inhibitors for SARS-CoV-2 infection remain understudied.

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

Conditional destabilization of the TPLATE complex impairs endocytic internalization.

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Nanoparticles Internalization through HIP-55-Dependent Clathrin Endocytosis Pathway.

Nanoparticles are promising tools for biomedicine. Many nanoparticles are internalized to function. Clathrin-mediated endocytosis is one of the most important mechanisms for nanoparticle internalization. However, the regulatory mechanism of clathrin-mediated nanoparticle endocytosis is still unclear. Here, we report that the adapter protein HIP-55 regulates clathrin-mediated nanoparticle endocytosis. CdSe/ZnS quantum dots (QDs), a typical nanoparticle, enter cells through the HIP-55-dependent clathrin endocytosis pathway. Both pharmacological inhibitor and genetic intervention demonstrate that QDs enter cells through clathrin-mediated endocytosis. HIP-55 can interact with clathrin and promote clathrin-mediated QDs endocytosis. Furthermore, HIP-55 ΔADF which is defective in F-actin binding fails to promote QDs endocytosis, indicating HIP-55 promotes clathrin-mediated QDs endocytosis depending on interaction with F-actin. In vivo, HIP-55 knockout also inhibits endocytosis of QDs. These findings reveal that HIP-55 acts as an intrinsic regulator for clathrin-mediated nanoparticle endocytosis, providing new insight into the nanoparticle internalization and a new strategy for nanodrug enrichment in target cells.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis.

Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.

α-Synuclein induces deficiency in clathrin-mediated endocytosis through inhibiting synaptojanin1 expression.

Parkinson's disease (PD) is an age-related chronic neurological disorder, mainly characterized by the pathological feature of α-synuclein (α-syn) aggregation, with the exact disease pathogenesis unclear. During the onset and progression of PD, synaptic dysfunction, including dysregulation of axonal transport, impaired exocytosis, and endocytosis are identified as crucial events of PD pathogenesis. It has been reported that over-expression of α-syn impairs clathrin-mediated endocytosis (CME) in the synapses. However, the underlying mechanisms still needs to be explored. In this study, we investigated the molecular events underlying the synaptic dysfunction caused by over-expression of wild-type human α-syn and its mutant form, involving series of proteins participating in CME. We found that excessive human α-syn causes impaired fission and uncoating of clathrin-coated vesicles during synaptic vesicle recycling, leading to reduced clustering of synaptic vesicles near the active zone and increased size of plasma membrane and number of endocytic intermediates. Furthermore, over-expressed human α-syn induced changes of CME-associated proteins, among which synaptojanin1 (SYNJ1) showed significant reduction in various brain regions. Over-expression of SYNJ1 in primary hippocampal neurons from α-syn transgenic mice recovered the synaptic vesicle density, clustering and endocytosis. Using fluorescence-conjugated transferrin, we demonstrated that SYNJ1 re-boosted the CME activity by restoring the phosphatidylinositol-4,5-bisphosphate homeostasis. Our data suggested that over-expression of α-syn disrupts synaptic function through interfering with vesicle recycling, which could be alleviated by re-availing of SYNJ1. Our study unrevealed a molecular mechanism of the synaptic dysfunction in PD pathogenesis and provided a potential therapeutic target for treating PD.