Mechanoregulation of clathrin-mediated endocytosis.
Ontology highlight
ABSTRACT:
SUBMITTER: Ferguson JP
PROVIDER: S-EPMC5702056 | biostudies-literature | 2017 Nov
REPOSITORIES: biostudies-literature
Publications
Mechanoregulation of clathrin-mediated endocytosis.
Ferguson Joshua P JP Huber Scott D SD Willy Nathan M NM Aygün Esra E Goker Sevde S Atabey Tugba T Kural Comert C
Journal of cell science 20170918 21
We characterized the tension response of clathrin-mediated endocytosis by using various cell manipulation methodologies. Elevated tension in a cell hinders clathrin-mediated endocytosis through inhibition of <i>de novo</i> coat initiation, elongation of clathrin coat lifetimes and reduction of high-magnitude growth rates. Actin machinery supplies an inward pulling force necessary for internalization of clathrin coats under high tension. These findings suggest that the physical cues cells receive ...[more]
Similar Datasets
Project description:The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.
| S-EPMC3809577 | biostudies-literature
Project description: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.
| S-EPMC11343097 | biostudies-literature
Project description:A cell's ability to establish polarization is one of the key steps in directional migration. Upon the addition of a chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMLP), neutrophils rapidly develop a front end marked by a wide and dense actin network which is a feature of cell polarization. Despite a general understanding of bi-directional crosstalk between endocytosis and polarization, it remains unclear how clathrin-mediated endocytosis (CME) induced by chemoattractant binding to formyl peptide receptor (FPR) affects neutrophil polarization. In this work, we characterized the spatial organization of FPR and clathrin-coated pits (CCPs), the functional unit of CME, with and without fMLP and found that fMLP induced different distributions of FPR and CCPs. We further found that cells had impaired polarization induced by fMLP when CME is inhibited by small molecule inhibitors. Under these conditions, pERK, pAkt308, and pAkt473 were all severely blocked or had altered dynamics. The spatial organization between actin and two major clathrin-mediated endocytic proteins, clathrin and ?-arrestin, were distinct and supported clathrin and ?-arrestin's functional roles in mediating neutrophil polarization. Together these results suggest that CME plays a pivotal role in a complex process such as cell polarization.
| S-EPMC6157066 | biostudies-literature
Project description:Clathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.
| S-EPMC8841809 | biostudies-literature
Project description:Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000-10000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.
| S-EPMC5792084 | biostudies-literature
Project description:How does mitosis influence the critical process of endocytosis? Some experiments lead to the conclusion that endocytosis arrests completely during mitosis, whereas others indicate that endocytosis persists. We have resolved this apparent discrepancy by showing how conditions of the experiment influence its outcome. The dynamics of clathrin-coated pit formation and the uptake of transferrin are maintained in naturally dividing cells but are nearly absent in mitotic cells arrested chemically by treatment with nocodazole, S-Trityl-L-cysteine, or RO-3306. Moreover, sequentially incubating cells at 4°C and then shifting them to 37°C or to serum starvation artificially increases the amount of transferrin receptor at the surface of naturally dividing cells, leading to the incorrect conclusion that endocytosis has ceased during mitosis. Thus, our data show that endocytosis is unaffected during all stages of natural cell division.
| S-EPMC3849811 | biostudies-literature
Project description:A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXX?, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being "on" in interphase to "off" in mitosis as cells traverse the G(2)/M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.
| S-EPMC3340072 | biostudies-literature
Project description:Clathrin-mediated endocytosis is a process by which specific molecules are internalized from the cell periphery for delivery to early endosomes. The key stages in this step-wise process, from the starting point of cargo recognition, to the later stage of assembly of the clathrin coat, are dependent on weak interactions between a large network of proteins. This review discusses the structural and functional data that have improved our knowledge and understanding of the main weak molecular interactions implicated in clathrin-mediated endocytosis, with a particular focus on the two key proteins: AP2 and clathrin.
| S-EPMC5694535 | biostudies-literature
Project description:Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane-permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.
| S-EPMC7480105 | biostudies-literature
Project description:Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca(2+)-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural-functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.
| S-EPMC4718405 | biostudies-literature