A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

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Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor: Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

Journal of the American Chemical Society ◽

10.1021/ja067674f ◽

2007 ◽

Vol 129 (2) ◽

pp. 268-269 ◽

Cited By ~ 30

Author(s):

Siwarutt Boonyarattanakalin ◽

Jianfang Hu ◽

Sheryl A. Dykstra-Rummel ◽

Avery August ◽

Blake R. Peterson

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Cell Surface Receptor ◽

Synthetic Cell ◽

Tissue Targeting ◽

Surface Receptor

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Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

Science Advances ◽

10.1126/sciadv.1500821 ◽

2015 ◽

Vol 1 (10) ◽

pp. e1500821 ◽

Cited By ~ 36

Author(s):

Hong-Bo Pang ◽

Gary B. Braun ◽

Erkki Ruoslahti

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Vascular Permeability ◽

Mammalian Cells ◽

Cell Penetrating Peptides ◽

Proteolytic Processing ◽

Cell Surface Receptor ◽

Neuropilin 1 ◽

Cell Penetrating ◽

Surface Receptor

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

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Specific Capture of Mammalian Cells by Cell Surface Receptor Binding to Ligand Immobilized on Gold Thin Films

Journal of Proteome Research ◽

10.1021/pr050467e ◽

2006 ◽

Vol 5 (7) ◽

pp. 1580-1585 ◽

Cited By ~ 32

Author(s):

Dora Peelen ◽

Voula Kodoyianni ◽

Jieun Lee ◽

Ting Zheng ◽

Michael R. Shortreed ◽

...

Keyword(s):

Thin Films ◽

Cell Surface ◽

Receptor Binding ◽

Mammalian Cells ◽

Cell Surface Receptor ◽

Surface Receptor ◽

Gold Thin Films

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CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

10.1101/2020.12.04.411645 ◽

2020 ◽

Author(s):

Anh Hoang Le ◽

Tamas Yelland ◽

Nikki Paul ◽

Loic Fort ◽

Savvas Nikolaou ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Ewing Sarcoma ◽

Negative Regulator ◽

Cell Surface Receptor ◽

Actin Dynamics ◽

Surface Receptor ◽

Sarcoma Cells ◽

Wave Complex

The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinocytosis maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation to regulate actin polymerisation. CYRI-A precedes RAB5A recruitment to engulfed macropinocytic cups and departs as RAB5A is recruited, consistent with a role for CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The suppression of integrin a5b1 uptake caused by the co-depletion of CYRI-A and B in Ewing sarcoma cells, leads to an enhancement of surface integrin levels and enhanced invasion and anchorage-independent growth in 3D. Thus CYRI-A is a dynamic regulator of integrin uptake via macropinocytosis, functioning together with CYRI-B to regulate integrin homeostasis on the cell surface.

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ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024605118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2024605118

Author(s):

Eric J. Schmidt ◽

Salome Funes ◽

Jeanne E. McKeon ◽

Brittany R. Morgan ◽

Sivakumar Boopathy ◽

...

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Alpha Helix ◽

Actin Dynamics ◽

Internal Dynamic ◽

Actin Assembly ◽

Loss Of Function ◽

Dynamics Simulations ◽

Induced Changes ◽

Lateral Sclerosis

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

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Faculty Opinions recommendation of Endocytic delivery of vancomycin mediated by a synthetic cell surface receptor: rescue of bacterially infected Mammalian cells and tissue targeting in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1103770.559890 ◽

2008 ◽

Author(s):

Daniel Flynn

Keyword(s):

Cell Surface ◽

Mammalian Cells ◽

Cell Surface Receptor ◽

Synthetic Cell ◽

Tissue Targeting ◽

Surface Receptor

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Rho Family Gtpase Cdc42 Is Essential for the Actin-Based Motility of Shigella in Mammalian Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.11.1905 ◽

1999 ◽

Vol 191 (11) ◽

pp. 1905-1920 ◽

Cited By ~ 46

Author(s):

Toshihiko Suzuki ◽

Hitomi Mimuro ◽

Hiroaki Miki ◽

Tadaomi Takenawa ◽

Takuya Sasaki ◽

...

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Host Cells ◽

Actin Dynamics ◽

Cloud Formation ◽

Actin Assembly ◽

Egg Extracts ◽

Rho Family ◽

Green Fluorescent

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP–depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP–actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein–tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG–N-WASP–Arp2/3 complex formed on intracellular Shigella.

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Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells.

The Journal of Cell Biology ◽

10.1083/jcb.87.1.132 ◽

1980 ◽

Vol 87 (1) ◽

pp. 132-141 ◽

Cited By ~ 207

Author(s):

J L Salisbury ◽

J S Condeelis ◽

P Satir

Keyword(s):

Cell Surface ◽

Coated Vesicles ◽

Cell Surface Receptor ◽

Coated Vesicle ◽

Vesicle Formation ◽

Rabbit Muscle ◽

Coated Pits ◽

Receptor Complexes ◽

Alternate Pathway ◽

Receptor Clusters

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42-kdalton component that comigrates with rabbit muscle actin and a 18.5-kdalton minor component that comigrates with calmodulin as well as 110-, 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.

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