scholarly journals Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

2017 ◽  
Author(s):  
Delia Bucher ◽  
Felix Frey ◽  
Kem A. Sochacki ◽  
Susann Kummer ◽  
Jan-Philip Bergeest ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Adaptor Protein ◽  
Membrane Tension ◽  
Coated Pits ◽  
Lattice Curvature ◽  
Cellular Processes ◽  
Molecular Events ◽  
Electron And Light Microscopy ◽  
Mechanical Factors ◽  
Clathrin Adaptor

AbstractAlthough essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

scholarly journals Regulation of Clathrin-Mediated Endocytosis

2018 ◽  
Vol 87 (1) ◽  
pp. 871-896 ◽  
Author(s):  
Marcel Mettlen ◽  
Ping-Hung Chen ◽  
Saipraveen Srinivasan ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Environmental Changes ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Membrane Composition ◽  
Coated Pits

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.

Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors

1999 ◽  
Vol 112 (3) ◽  
pp. 317-327 ◽  
Author(s):  
T. Sorkina ◽  
A. Bild ◽  
F. Tebar ◽  
A. Sorkin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Image Deconvolution ◽  
Protein Markers ◽  
Coated Pits ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Clathrin Adaptor

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

scholarly journals Adaptor Protein Complex 1 Mediates the Transport of Lysosomal Proteins from a Golgi-like Organelle to Peripheral Vacuoles in the Primitive EukaryoteGiardia lamblia

2004 ◽  
Vol 15 (7) ◽  
pp. 3053-3060 ◽  
Author(s):  
María C. Touz ◽  
Liudmila Kulakova ◽  
Theodore E. Nash
Keyword(s):  
Cysteine Protease ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Adaptor Protein ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Specific Amino Acid ◽  
Clathrin Adaptor ◽  
Sorting System ◽  
Lysosomal Proteins

Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Giμa). Hemagglutinin-tagged Giμa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Giμa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.

scholarly journals Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

1999 ◽  
Vol 10 (3) ◽  
pp. 677-691 ◽  
Author(s):  
Carol Pitcher ◽  
Stefan Höning ◽  
Anja Fingerhut ◽  
Katherine Bowers ◽  
Mark Marsh
Keyword(s):  
Phorbol Esters ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Differentiation Antigen ◽  
Coated Pits ◽  
Early Endosomes ◽  
Clathrin Adaptor ◽  
Cluster Of Differentiation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56 lck , undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56 lck , we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

scholarly journals Evolving models for assembling and shaping clathrin-coated pits

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Zhiming Chen ◽  
Sandra L. Schmid
Keyword(s):  
Protein Interactions ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Adaptor Protein ◽  
Structural Studies ◽  
Coated Pits ◽  
Molecular Events ◽  
Em Tomography ◽  
Evolving Models ◽  
Flexible Model

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

scholarly journals Local actin polymerization during endocytic carrier formation

2018 ◽  
Vol 46 (3) ◽  
pp. 565-576 ◽  
Author(s):  
Claudia Hinze ◽  
Emmanuel Boucrot
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Surface Proteins ◽  
Membrane Tension ◽  
Pit Formation ◽  
Molecular Events ◽  
Key Steps ◽  
Endocytic Compartments ◽  
Cargo Molecule

Extracellular macromolecules, pathogens and cell surface proteins rely on endocytosis to enter cells. Key steps of endocytic carrier formation are cargo molecule selection, plasma membrane folding and detachment from the cell surface. While dedicated proteins mediate each step, the actin cytoskeleton contributes to all. However, its role can be indirect to the actual molecular events driving endocytosis. Here, we review our understanding of the molecular steps mediating local actin polymerization during the formation of endocytic carriers. Clathrin-mediated endocytosis is the least reliant on local actin polymerization, as it is only engaged to counter forces induced by membrane tension or cytoplasmic pressure. Two opposite situations are coated pit formation in yeast and at the basolateral surface of polarized mammalian cells which are, respectively, dependent and independent on actin polymerization. Conversely, clathrin-independent endocytosis forming both nanometer [CLIC (clathrin-independent carriers)/GEEC (glycosylphosphatidylinositol (GPI)-anchored protein enriched endocytic compartments), caveolae, FEME (fast endophilin-mediated endocytosis) and IL-2β (interleukin-2β) uptake] and micrometer carriers (macropinocytosis) are dependent on actin polymerization to power local membrane deformation and carrier budding. A variety of endocytic adaptors can recruit and activate the Cdc42/N-WASP or Rac1/WAVE complexes, which, in turn, engage the Arp2/3 complex, thereby mediating local actin polymerization at the membrane. However, the molecular steps for RhoA and formin-mediated actin bundling during endocytic pit formation remain unclear.

scholarly journals Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

2002 ◽  
Vol 158 (3) ◽  
pp. 563-575 ◽  
Author(s):  
Sathyamangla V. Naga Prasad ◽  
Stéphane A. Laporte ◽  
Dean Chamberlain ◽  
Marc G. Caron ◽  
Larry Barak ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Coated Pits ◽  
Receptor Endocytosis ◽  
Sequential Binding ◽  
Β2 Adrenergic Receptor ◽  
Clathrin Adaptor ◽  
Phosphoinositide 3 Kinase ◽  
Protein Recruitment

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

scholarly journals Clathrin Adaptor AP-2 Is Essential for Early Embryonal Development

2005 ◽  
Vol 25 (21) ◽  
pp. 9318-9323 ◽  
Author(s):  
Takashi Mitsunari ◽  
Fubito Nakatsu ◽  
Noriko Shioda ◽  
Paul E. Love ◽  
Alexander Grinberg ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Adaptor Protein ◽  
Membrane Receptors ◽  
Vesicle Formation ◽  
Targeted Disruption ◽  
Gene Encoding ◽  
Homozygous Mutant ◽  
Transport Vesicle ◽  
Clathrin Adaptor ◽  
Plasma Membrane Receptors

ABSTRACT The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the μ2 subunit of AP-2 in the mouse. We found that μ2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no μ2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that μ2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.

scholarly journals Clathrin-independent pathways do not contribute significantly to endocytic flux

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Vassilis Bitsikas ◽  
Ivan R Corrêa ◽  
Benjamin J Nichols
Keyword(s):  
Mammalian Cells ◽  
High Temporal Resolution ◽  
Coated Pits ◽  
Alternative Pathways ◽  
Specific Proteins ◽  
Clathrin Adaptor ◽  
And Function ◽  
Endocytic Vesicles ◽  
Data Call ◽  
Endocytic Pathways

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.

scholarly journals Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth

2015 ◽  
Vol 112 (41) ◽  
pp. 12717-12722 ◽  
Author(s):  
Nicolas Loyer ◽  
Irina Kolotuev ◽  
Mathieu Pinot ◽  
Roland Le Borgne
Keyword(s):  
Adaptor Protein ◽  
Tissue Growth ◽  
Intercellular Adhesion Molecule ◽  
Membrane Tension ◽  
Genetic Screens ◽  
Functional Compensation ◽  
Ring Canals ◽  
Block Face ◽  
Clathrin Adaptor ◽  
E Cadherin

Intercellular bridges called “ring canals” (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs’ anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin–containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs’ anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin–dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.

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