scholarly journals ERM proteins: The missing actin linkers in clathrin-mediated endocytosis

2018 ◽  
Author(s):  
Audun Sverre Kvalvaag ◽  
Kay Oliver Schink ◽  
Andreas Brech ◽  
Kirsten Sandvig ◽  
Sascha Pust
Keyword(s):  
Actin Polymerization ◽  
Adaptor Proteins ◽  
Coated Vesicle ◽  
Apical Surface ◽  
Coated Pits ◽  
Erm Proteins ◽  
Detection And Tracking ◽  
Subsequent Propagation ◽  
Transferrin Uptake ◽  
Mechanical Stretching

AbstractCanonical clathrin-coated pits (CCPs) are nucleated by the coordinated arrival of clathrin triskelia and AP2 adaptor proteins at phosphatidylinositol 4,5-bisphosphate (PIP2) enriched plasma membrane domains (1, 2). Subsequent propagation of the clathrin lattice helps to deform the membrane into a sharply curved pit (3). A large proportion of the initiated pits fall apart as abortive endocytic events within about 20 s, possibly due to insufficient cargo capture (4). Successful clathrin-mediated endocytosis (CME) is concluded when a clathrin-coated vesicle is released into the cell by dynamin-mediated fission of the CCP membrane neck (5, 6). A vast array of accessory proteins important for successful CME has been identified. Among these is actin, which has been shown to be required for the maturation and internalization of a subset of CCPs in human cells (7, 8). Actin dependency during CME correlates with elevated membrane tension, and CCP maturation requires actin polymerization during mitosis, in microvilli at the apical surface of polarized cells and in cells upon global mechanical stretching or hypotonic treatment (9–11). The Arp2/3 complex is thought to trigger an acute actin burst coinciding with CCP internalization, but how actin is recruited to CCPs in the first place is not known (12, 13). Here we show that the ERM (ezrin, radixin, moesin) protein family of membrane-actin linkers associates with CCPs, and that functional perturbation of ERM proteins impedes CCP maturation and reduces the rate of transferrin uptake. By total internal reflection fluorescence (TIRF) microscopy and unbiased object detection and tracking, we show that ezrin localizes to nascent CCPs and that these pits subsequently recruit actin. Based on these data, we propose a model in which activated ERM proteins recruit the initial actin filament during CME.

scholarly journals Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Matteo Biancospino ◽  
Gwen R. Buel ◽  
Carlos A. Niño ◽  
Elena Maspero ◽  
Rossella Scotto di Perrotolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coated Vesicle ◽  
Apical Surface ◽  
Myosin Vi ◽  
Coated Pits ◽  
Interacting Protein ◽  
Vesicle Budding ◽  
Polarized Epithelia ◽  
Actin Motor ◽  
Huntingtin Interacting Protein

Abstract Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

scholarly journals Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell–Substrate Adhesion

2010 ◽  
Vol 21 (17) ◽  
pp. 3070-3079 ◽  
Author(s):  
Erika M. Batchelder ◽  
Defne Yarar
Keyword(s):  
Actin Polymerization ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Cytoskeletal Dynamics ◽  
Major Route ◽  
Cellular Import ◽  
Cell Substrate Adhesion

Clathrin-dependent endocytosis is a major route for the cellular import of macromolecules and occurs at the interface between the cell and its surroundings. However, little is known about the influences of cell–substrate attachment in clathrin-coated vesicle formation. Using biochemical and imaging-based methods, we find that cell–substrate adhesion reduces the rate of endocytosis. Clathrin-coated pits (CCPs) in proximity to substrate contacts exhibit slower dynamics in comparison to CCPs found more distant from adhesions. Direct manipulation of the extracellular matrix (ECM) to modulate adhesion demonstrates that tight adhesion dramatically reduces clathrin-dependent endocytosis and extends the lifetimes of clathrin structures. This reduction is in part mediated by integrin-matrix engagement. In addition, we demonstrate that actin cytoskeletal dynamics are differentially required for efficient endocytosis, with a stronger requirement for actin polymerization in areas of adhesion. Together, these results reveal that cell–substrate adhesion regulates clathrin-dependent endocytosis and suggests that actin assembly facilitates vesicle formation at sites of adhesion.

scholarly journals Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

2011 ◽  
Vol 208 (5) ◽  
pp. 1055-1068 ◽  
Author(s):  
Bebhinn Treanor ◽  
David Depoil ◽  
Andreas Bruckbauer ◽  
Facundo D. Batista
Keyword(s):  
Actin Cytoskeleton ◽  
B Cell ◽  
Protein Function ◽  
Actin Polymerization ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Temporary Increase ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Large Family ◽  
Adaptor Proteins ◽  
Serine Phosphorylation ◽  
Binding Motif ◽  
Double Knockout ◽  
Coated Pits ◽  
Knockout Mutants ◽  
Golgi Network

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

scholarly journals Coated Vesicle-Mediated Transport and Deposition of Vitellogenic Ferritin in the Rapid Growth Phase of Snail Oocytes

1982 ◽  
Vol 53 (1) ◽  
pp. 173-191 ◽  
Author(s):  
W. BOTTKE ◽  
I. SINHA ◽  
I. KEIL
Keyword(s):  
Growth Phase ◽  
Rapid Growth ◽  
Lymnaea Stagnalis ◽  
Follicle Cells ◽  
Coated Vesicle ◽  
Secretory Cells ◽  
Basal Region ◽  
Coated Pits ◽  
Iron Storage ◽  
Basement Lamina

The main yolk component in oocytes of the pulmonate freshwater snails Planorbarius corneus L. and Lymnaea stagnalis L. consists of the iron storage protein ferritin and iron-free apoferritin. Both compounds are deposited in the yolk in the form of large paracrystalloids, tubular structures and randomly dispersed particles. In addition, the plasm contains lysosome-like inclusions with depositions of haemosiderin. Haemosiderin is interpreted as the product of proteolytic degradation of ferritin. During the rapid growth phase of the oocytes vitellogenic ferritin is transported across the basement lamina and taken up by adsorptive endocytosis via coated pits and vesicles. Formation of yolk bodies occurs by fusion of ferritin-containing vacuoles and empty vesicles that are probably derived from the Golgi apparatus. Uptake of ferritin is restricted to the basal region of the oocyte. No involvement of the follicle cells in synthesis and deposition of ferritin could be detected. Secretory cells of the midgut gland are the most likely site of synthesis of vitellogenic ferritin. Under conditions of iron overload large masses of ferritin are encountered in the basement lamina of the oocytes. However, no significant increase in the uptake of ferritin could be observed. With the use of a tannic acid-glutaraldehyde fixation procedure a hitherto unobserved filamentous or rod-like material was detected inside the lamina and in coated pits. This material is probably also taken up by the oocytes and integrated into yolk platelets. Though ferritin is a rather unusual vitellogenic protein, the mode of its uptake and deposition in the oocyte plasm is highly reminiscent of that of typical hormone-induced vitellogenins in other animal groups.

scholarly journals Multi-modal adaptor-clathrin contacts drive coated vesicle assembly

2021 ◽  
Author(s):  
Sarah M Smith ◽  
Gabrielle Larocque ◽  
Katherine M Wood ◽  
Kyle L Morris ◽  
Alan M Roseman ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Binding Sites ◽  
Potential Role ◽  
Fundamental Property ◽  
Structural Knowledge ◽  
Coated Vesicle ◽  
Functional Importance ◽  
Coated Pits ◽  
Interaction Sites ◽  
Cross Links

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the b2 subunit of AP2, and several binding sites on b2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of b2 hinge and appendage (b2HA) domains. We find that the b2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, b2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of b2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the ''sandwich site'' on the appendage, with the appendage ''platform site'' having less importance. From these studies and the work of others, we propose that b2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells

1996 ◽  
Vol 109 (12) ◽  
pp. 2927-2935 ◽  
Author(s):  
W. Shurety ◽  
N.A. Bright ◽  
J.P. Luzio
Keyword(s):  
Cell Surface ◽  
Apical Cell ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Cytochalasin D ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Acid Media ◽  
Kinase C

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited > 95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was not due to an effect on ricin movement along the microvillar surface, and may be explained by increases in formation and pinching off of clathrin coated pits at the apical cell surface.

scholarly journals Molecular Mechanisms of Receptor-Mediated Endocytosis in the Renal Proximal Tubular Epithelium

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Akihiko Saito ◽  
Hiroyoshi Sato ◽  
Noriaki Iino ◽  
Tetsuro Takeda
Keyword(s):  
Molecular Mechanisms ◽  
Adaptor Proteins ◽  
Risk Marker ◽  
Tubular Epithelium ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Other Substances ◽  
Proximal Tubular ◽  
Renal Proximal Tubular ◽  
Proximal Tubular Epithelium

Receptor-mediated endocytosis is a pivotal function of renal proximal tubule epithelial cells (PTECs) to reabsorb and metabolize substantial amounts of proteins and other substances in glomerular filtrates. The function accounts for the conservation of nutrients, including carrier-bound vitamins and trace elements, filtered by glomeruli. Impairment of the process results in a loss of such substances and development of proteinuria, an important clinical sign of kidney disease and a risk marker for cardiovascular disease. Megalin is a multiligand endocytic receptor expressed at clathrin-coated pits of PTEC, playing a central role in the process. Megalin cooperates with various membrane molecules and interacts with many intracellular adaptor proteins for endocytic trafficking. Megalin is also involved in signaling pathways in the cells. Megalin-mediated endocytic overload leads to damage of PTEC. Further studies are needed to elucidate the mechanism of megalin-mediated endocytosis and develop strategies for preventing the damage of PTEC.

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