Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane ...composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.
The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in ...propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.
Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo ...uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.
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•Highly sensitive detection of protein association with CCPs•CCPs undergo a multistep maturation process gated by a checkpoint•Early dynamin recruitment is required for CCP maturation•Compensatory mechanisms obscure EAP roles in CCP stabilization and maturation
Aguet et al. perform a high-sensitivity analysis of clathrin-coated pits (CCPs). They establish methods that circumvent many difficulties associated with low signal-to-noise ratios, enable quantitative tracking of CCP/accessory dynamics, and reveal that dynamin and the α-adaptin appendage domain of AP2 each gate different stages in the progression of clathrin-mediated endocytosis.
Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often ...results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from
clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.
The STIM1-ORAI1 pathway of store-operated Ca(2+) entry is an essential component of cellular Ca(2+) signalling. STIM1 senses depletion of intracellular Ca(2+) stores in response to physiological ...stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca(2+) channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca(2+) entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum-plasma membrane junctions before and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.
Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal ...tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.
Proliferating cells, compared with quiescent cells, are more dependent on glucose for their growth. Although glucose transport in keratinocytes is mediated largely by the Glut1 facilitative ...transporter, we found that keratinocyte-specific ablation of Glut1 did not compromise mouse skin development and homeostasis. Ex vivo metabolic profiling revealed altered sphingolipid, hexose, amino acid, and nucleotide metabolism in Glut1-deficient keratinocytes, thus suggesting metabolic adaptation. However, cultured Glut1-deficient keratinocytes displayed metabolic and oxidative stress and impaired proliferation. Similarly, Glut1 deficiency impaired in vivo keratinocyte proliferation and migration within wounded or UV-damaged mouse skin. Notably, both genetic and pharmacological Glut1 inactivation decreased hyperplasia in mouse models of psoriasis-like disease. Topical application of a Glut1 inhibitor also decreased inflammation in these models. Glut1 inhibition decreased the expression of pathology-associated genes in human psoriatic skin organoids. Thus, Glut1 is selectively required for injury- and inflammation-associated keratinocyte proliferation, and its inhibition offers a novel treatment strategy for psoriasis.
Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their ...heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.
Severe COVID-19 is characterized by an increase in the number and changes in the function of innate immune cells including neutrophils. However, it is not known how the metabolome of immune cells ...changes in patients with COVID-19. To address these questions, we analyzed the metabolome of neutrophils from patients with severe or mild COVID-19 and healthy controls. We identified widespread dysregulation of neutrophil metabolism with disease progression including in amino acid, redox, and central carbon metabolism. Metabolic changes in neutrophils from patients with severe COVID-19 were consistent with reduced activity of the glycolytic enzyme GAPDH. Inhibition of GAPDH blocked glycolysis and promoted pentose phosphate pathway activity but blunted the neutrophil respiratory burst. Inhibition of GAPDH was sufficient to cause neutrophil extracellular trap (NET) formation which required neutrophil elastase activity. GAPDH inhibition increased neutrophil pH, and blocking this increase prevented cell death and NET formation. These findings indicate that neutrophils in severe COVID-19 have an aberrant metabolism which can contribute to their dysfunction. Our work also shows that NET formation, a pathogenic feature of many inflammatory diseases, is actively suppressed in neutrophils by a cell-intrinsic mechanism controlled by GAPDH.
Signaling receptors are internalized and regulated by clathrin-mediated endocytosis (CME). Two clathrin light chain isoforms, CLCa and CLCb, are integral components of the endocytic machinery whose ...differential functions remain unknown. We report that CLCb is specifically upregulated in non-small-cell lung cancer (NSCLC) cells and is associated with poor patient prognosis. Engineered single CLCb-expressing NSCLC cells, as well as “switched” cells that predominantly express CLCb, exhibit increased rates of CME and altered clathrin-coated pit dynamics. This “adaptive CME” resulted from upregulation of dynamin-1 (Dyn1) and its activation through a positive feedback loop involving enhanced epidermal growth factor (EGF)-dependent Akt/GSK3β phosphorylation. CLCb/Dyn1-dependent adaptive CME selectively altered EGF receptor trafficking, enhanced cell migration in vitro, and increased the metastatic efficiency of NSCLC cells in vivo. We define molecular mechanisms for adaptive CME in cancer cells and a role for the reciprocal crosstalk between signaling and CME in cancer progression.
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•CLCb and Dyn1 contribute to adaptive clathrin-mediated endocytosis in cancer cells•CLCb modulates dynamin-1 via an APPL1/Akt/GSK3β signaling circuit•Upregulation of CLCb alters EGFR trafficking dependent on dynamin-1•Upregulation of CLCb increases tumor cell migration and metastasis
Signaling downstream of receptor tyrosine kinases is regulated by their trafficking. Chen et al. show that isoform-specific upregulation of clathrin light chain b and dynamin-1 in cancer cells, coupled to reciprocal crosstalk with Akt/GSK3β signaling, leads to adaptive clathrin-mediated endocytosis that alters EGFR trafficking and signaling and increases metastasis.
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