The localization and function of the moonlighting protein Clathrin during oocyte maturation.

Clathrin is one of the leading players in the endocytic process during oocyte maturation. Immunofluorescence and transmission electron analysis on fully-grown germinal vesicle (GV) mouse oocytes shows Clathrin localization on the cortical region with three peculiar patterns: complete, incomplete, and half-moon. The first configuration is characterized by Clathrin lattices along the cortex; the second is represented by Clathrin lattices interrupted by invaginations forming coated vesicles as an indication of active endocytosis. The half-moon profile, the less frequent but the most interesting one, refers to Clathrin lattices distributed to one-half of the cell. The in vivo analysis of organelles' positioning and cytoplasmic rearrangements, performed to understand the possible relation between endocytosis and oocyte maturation, suggests that the half-moon pattern indicates those fully-grown oocytes that may have likely undergone Germinal Vesicle Breakdown, MI, and MII. Our results show that, before oocytes undergo maturation, Clathrin localizes on the side of the cell, opposite to future spindle migration, thus marking spindle orientation in mouse oocytes.

CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115.

The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, shuttles the glucose transporter GLUT4 from the ERGIC (endoplasmic-reticulum-to-Golgi intermediate compartment) directly to an intracellular GLUT4 storage compartment (GSC), from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. We show that the C-terminal trimerization domain of CHC22 interacts with SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both N- and C-termini of the heavy chain, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi-bypass route for GLUT4 trafficking. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.

Comprehensive exploration of FCHO1 mutations: Clinical manifestations and implications across disorders.

FCH domain only 1 (FCHO1) is a key player in clathrin-mediated endocytosis, vital for various cellular processes, including immune regulation and cancer progression. However, the clinical implications of FCHO1 mutations, particularly in combined immunodeficiency, remain unclear. This systematic review aims to provide an objective analysis of the molecular genetics, clinical manifestations, and potential therapeutic targets associated with FCHO1 mutations. A systematic search following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was conducted across electronic databases up to March 25, 2024, to identify studies investigating the relationship between FCHO1 and different clinical manifestations. Eligibility criteria were applied to screen studies, and data extraction included study characteristics, reported symptoms, genetic variants, and primary outcomes. In silico analyses were performed to assess protein-protein interactions and gene expression patterns. Five studies were included, offering insights into the molecular genetics, T-cell deficiency mechanisms, clinical manifestations, and potential therapeutic targets associated with FCHO1 mutations. Molecular analyses identified specific mutations disrupting FCHO1 function, leading to impaired T-cell proliferation, cytokine production, and susceptibility to infections. Clinically, patients exhibited recurrent infections, lymphopenia, and malignancies, with allogeneic hematopoietic stem cell transplantation emerging as a therapeutic option. In silico analyses revealed potential interactions and co-expression between FCHO1 and genes involved in cancer progression and immune signaling pathways. This systematic review objectively elucidates the multifaceted role of FCHO1 in immune regulation and disease pathogenesis. Understanding the molecular mechanisms underlying FCHO1 mutations and their impact on disease manifestations is crucial for guiding clinical management and developing targeted therapeutic strategies.

Receptor-mediated endocytosis in kidney cells during physiological and pathological conditions.

Mammalian cell membranes are very dynamic where they respond to several environmental stimuli by rearranging the membrane composition by basic biological processes, including endocytosis. In this context, receptor-mediated endocytosis, either clathrin-dependent or caveolae-dependent, is involved in different physiological and pathological conditions. In the last years, an important amount of evidence has been reported that kidney function involves the modulation of different types of endocytosis, including renal protein handling. In addition, the dysfunction of the endocytic machinery is involved with the development of proteinuria as well as glomerular and tubular injuries observed in kidney diseases associated with hypertension, diabetes, and others. In this present review, we will discuss the mechanisms underlying the receptor-mediated endocytosis in different glomerular cells and proximal tubule epithelial cells as well as their modulation by different factors during physiological and pathological conditions. These findings could help to expand the current understanding regarding renal protein handling as well as identify possible new therapeutic targets to halt the progression of kidney disease.

Vesiculation pathways in clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, a patch of flat plasma membrane is internalized to form a vesicle. In mammalian cells, how the clathrin coat deforms the membrane into a vesicle remains unclear and two main hypotheses have been debated. The "constant area" hypothesis assumes that clathrin molecules initially form a flat lattice on the membrane and deform the membrane by changing its intrinsic curvature while keeping the coating area constant. The alternative "constant curvature" hypothesis assumes that the intrinsic curvature of the clathrin lattice remains constant during the formation of a vesicle while the surface area it covers increases. Previous experimental studies were unable to unambiguously determine which hypothesis is correct. In this paper, we show that these two hypotheses are only two extreme cases of a continuum of vesiculation pathways if we account for the free energies associated with clathrin assembly and curvature generation. By tracing the negative gradient of the free energy, we define vesiculation pathways in the phase space of the coating area and the intrinsic curvature of clathrin coat. Our results show that, overall, the differences in measurable membrane morphologies between the different models are not as big as expected, and the main differences are most salient at the early stage of endocytosis. Furthermore, the best fitting pathway to experimental data is not compatible with the constant-curvature model and resembles to a constant-area-like pathway where the coating area initially expands with minor changes in the intrinsic curvature, later followed by a dramatic increase in the intrinsic curvature and minor change in the coating area. Our results also suggest that experimental measurement of the tip radius and the projected area of the clathrin coat will be the key to distinguish between models.

Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants.

Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.

An overview of the safety and efficacy of LX-9211 in treating neuropathic pain conditions.

INTRODUCTION: LX-9211 is a drug designed to treat neuropathic pain conditions. It functions by inhibiting the adaptor-associated kinase 1 (AAK1) enzyme which promotes clathrin-dependent endocytosis. Preclinical studies have shown that LX-9211 does produce a reduction in nociceptive related behaviors and produces no major adverse effects in rats. Thus, LX-9211 has advanced to clinical trials to assess its safety and efficacy in humans. So far, phase 1 and phase 2 clinical trials involving patients with postherpetic neuralgia and diabetic peripheral neuropathic pain have been conducted with phase 3 trials planned in the future. AREAS COVERED: This paper highlights preclinical studies involving LX-9211 in rodents. Additionally, phase 1 clinical trials examining the safety of LX-9211 in healthy subjects as well as phase 2 studies looking at the safety and efficacy of LX-9211 compared to placebo in patients with diabetic peripheral neuropathic pain and postherpetic neuralgia are also discussed. EXPERT OPINION: In phase 1 and phase 2 clinical trials conducted so far, LX-9211 has been shown to produce few adverse effects as well as cause a significantly greater reduction in pain compared to placebo. However, more clinical studies are needed to further assess its effects in humans to ensure its safety.

Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

A clathrin mediated endocytosis scaffolding protein, Intersectin 1, changes in an isoform, brain region, and sex specific manner in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aß) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aß. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human post-mortem AD and control (CTL) frontal cortex (FC; AD n = 22, CTL n = 23) and hippocampus (HP; AD n = 34, CTL n = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aß pathology at different timepoints during aging and disease progression by immunoblot (n = 5-8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro. Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aß accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression.

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.

Dissecting the roles of dynamin and clathrin in platelet pinocytosis.

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Confocal Surface-Enhanced Raman Imaging of the Intestinal Barrier Crossing Behavior of Model Nanoplastics in Daphnia Magna.

Nanoplastics (nP) pose hazards to aquatic animals once they are ingested. Significant knowledge gaps exist regarding the nP translocation across the animal intestine, which is the first barrier between the ingested nP and the animal body. We examined the intestinal barrier crossing behavior of nP in an aquatic animal model (Daphnia magna) and determined the translocation mechanism with the help of model "core-shell" polystyrene nanoplastics (nPS) and confocal surface-enhanced Raman spectroscopy (SERS). The Raman reporter (4-mercaptobenzoic acid)-tagged gold "core" of the model nPS enables sensitive and reliable particle imaging by confocal SERS. This method detected SERS signals of model nPS concentration as low as 4.1 × 109 particles/L (equivalent to 0.27 µg/L PS "shell" concentration). The translocation was observed with the help of multilayer stacked Raman maps of SERS signals of the model nPS. With a higher concentration or longer exposure time of the model nPS, uptake and translocation of the plastic particles increased. In addition, we demonstrated that clathrin-dependent endocytosis and macropinocytosis were two major mechanisms underlying the translocation. This study contributes to a mechanistic understanding of nP translocation by using the pioneering model nPS and an analytical toolkit, which undergird further investigations into nP behavior and health effects in aquatic species.

Comprehensive Analyses of the Effects of the Small-Molecule Inhibitor of the UHM Domain in the Splicing Factor U2AF1 in Leukemia Cells.

Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1 mut , we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1wt protein.

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with ligands. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces a capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR individually with drugs prevents the recruitment of both EGFR and FGFR. Our data reveals novel crosstalk between multiple unrelated receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

Inhibition clathrin mediated endocytosis: Pitstop 1 and Pitstop 2 chimeras.

Twenty-five chimera compounds of Pitstop® 1 and 2 were synthesised and screened for their ability to block the clathrin terminal domain-amphiphysin protein-protein interaction (NTD-PPI using an ELISA) and clathrin mediated endocytosis (CME) in cells.  Library 1 was based on Pitstop 2, but no notable clathrin PPI or in-cell activity was observed.  With the Pitstop 1, 16 analogues were produced with 1,8-naphthalic imide core as a foundation.  Analogues with methylene spaced linkers and simple amides showed a modest to good range of PPI inhibition (7.6 to 42.5 mM, naphthyl 39 and 4-nitrophenyl 40 respectively) activity.  These data reveal the importance of the naphthalene sulfonate moiety, with no des-SO3 analogue displaying PPI inhibition.  This was consistent with the observed analogue docked poses within the clathrin terminal domain Site 1 binding pocket.  Further modifications targeted the naphthalene imide moiety, with the installation of 5-Br (45a), 5-OH (45c) and 5-propyl ether (45d) moieties.  Among them, the OH 45c and propyl ether 45d retained PPI inhibition, with propyl ether 45d being the most active with a PPI inhibition IC50 = 7.3 mM.  This is 2x more potent than Pitstop® 2 and 3x more potent than Pitstop 1.

CED-6/GULP and components of the clathrin-mediated endocytosis machinery act redundantly to correctly display CED-1 on the cell membrane in Caenorhabditis elegans.

CED-1 (cell death abnormal) is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in Caenorhabditis elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter include the adapter protein CED-6/GULP and the ATP-binding cassette family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the Adaptor protein 2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

SH3Ps recruit auxilin-like vesicle uncoating factors for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is an essential process of cargo uptake operating in all eukaryotes. In animals and yeast, BAR-SH3 domain proteins, endophilins and amphiphysins, function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains the BAR-SH3 domain proteins SH3P1-SH3P3, but their role is poorly understood. Here, we identify SH3Ps as functional homologs of endophilin/amphiphysin. SH3P1-SH3P3 bind to discrete foci at the plasma membrane (PM), and SH3P2 recruits late to a subset of clathrin-coated pits. The SH3P2 PM recruitment pattern is nearly identical to its interactor, a putative uncoating factor, AUXILIN-LIKE1. Notably, SH3P1-SH3P3 are required for most of AUXILIN-LIKE1 recruitment to the PM. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors rather than the uncoating phosphatases, synaptojanins. SH3P1-SH3P3 act redundantly in overall CME with the plant-specific endocytic adaptor TPLATE complex but not due to an SH3 domain in its TASH3 subunit.