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.

ESCRT proteins and the regulation of endocytic delivery to lysosomes

2009 ◽  
Vol 37 (1) ◽  
pp. 178-180 ◽  
Author(s):  
J. Paul Luzio ◽  
Siân C. Piper ◽  
Katherine Bowers ◽  
Michael D.J. Parkinson ◽  
Paul J. Lehner ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mhc Class I ◽  
Mammalian Cells ◽  
Transport Proteins ◽  
Class I ◽  
Multivesicular Bodies ◽  
Down Regulation ◽  
Morphological Effect ◽  
Endosomal Sorting ◽  
Endocytic Compartments

In mammalian cells, there is evidence of cargo specificity in the requirement for particular ESCRT (endosomal sorting complex required for transport) proteins to sort cargo into the luminal vesicles of MVBs (multivesicular bodies). We have focussed on studying the ESCRT requirements for delivery of MHC class I to lysosomes following polyubiquitination by the Kaposi's sarcoma-associated herpesvirus protein K3. Down-regulation of polyubiquitinated cell-surface MHC class I in HeLa cells stably expressing K3 is achieved via clathrin-mediated endocytosis, followed by sorting into the luminal vesicles of MVBs and eventual delivery to lysosomes. Depletion of ESCRT-I and some ESCRT-III components interferes with this sorting and allows recycling of MHC class I to the cell surface. Depletion of ESCRT-II components has no effect on K3-mediated down-regulation of MHC class I and no gross morphological effect on endocytic compartments. Thus virally polyubiquitinated MHC class I does not require all of the ESCRT proteins in order to be sorted into the luminal vesicles of MVBs. However, there may be a further requirement for ESCRT-III proteins to ensure the efficient fusion of MVBs with lysosomes.

scholarly journals Molecular mechanism of Fast Endophilin-Mediated Endocytosis

2020 ◽  
Vol 477 (12) ◽  
pp. 2327-2345 ◽  
Author(s):  
Alessandra Casamento ◽  
Emmanuel Boucrot
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mechanism ◽  
Receptor Activation ◽  
Membrane Curvature ◽  
Surface Proteins ◽  
Resting Cells ◽  
Cellular Functions ◽  
Surface Receptors ◽  
Key Steps

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

scholarly journals Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe

1997 ◽  
Vol 328 (3) ◽  
pp. 889-895 ◽  
Author(s):  
M. Donna FELSCHOW ◽  
Zenghui MI ◽  
J. STANEK ◽  
J. FREI ◽  
W. Carl PORTER
Keyword(s):  
Cell Surface ◽  
Tumour Cell ◽  
Solid Tumour ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Protein S ◽  
Cell Types ◽  
Biochemical Properties ◽  
Surface Proteins ◽  
Polyamine Transport

Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 μM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the identified pbps may be associated with that function.

Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

2010 ◽  
Vol 15 (6) ◽  
pp. 703-709 ◽  
Author(s):  
Gregory W. Fisher ◽  
Sally A. Adler ◽  
Margaret H. Fuhrman ◽  
Alan S. Waggoner ◽  
Marcel P. Bruchez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Adrenergic Receptor ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Living Cells ◽  
Surface Proteins ◽  
Receptor Internalization ◽  
Cell Interior ◽  
Cell Surface Proteins ◽  
Signaling Systems

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

scholarly journals Find and fuse: Unsolved mysteries in sperm–egg recognition

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000953
Author(s):  
Enrica Bianchi ◽  
Gavin J. Wright
Keyword(s):  
Cell Surface ◽  
Sperm Cell ◽  
Surface Proteins ◽  
Secreted Proteins ◽  
Egg Recognition ◽  
Cell Surface Proteins ◽  
Molecular Events ◽  
Eukaryotic Species ◽  
Fundamental Process

Sexual reproduction is such a successful way of creating progeny with subtle genetic variations that the vast majority of eukaryotic species use it. In mammals, it involves the formation of highly specialised cells: the sperm in males and the egg in females, each carrying the genetic inheritance of an individual. The interaction of sperm and egg culminates with the fusion of their cell membranes, triggering the molecular events that result in the formation of a new genetically distinct organism. Although we have a good cellular description of fertilisation in mammals, many of the molecules involved remain unknown, and especially the identity and role of cell surface proteins that are responsible for sperm–egg recognition, binding, and fusion. Here, we will highlight and discuss these gaps in our knowledge and how the role of some recently discovered sperm cell surface and secreted proteins contribute to our understanding of this fundamental process.

scholarly journals CELL SURFACE PROTEINS OF CRYPTOCOCCUS NEOFORMANS MEDIATE ADHERENCE AND INTERNALISATION INTO MAMMALIAN CELLS

2019 ◽  
Vol 22 (2) ◽  
pp. 24-30
Author(s):  
Khi Khi Choo ◽  
Voon Kin Chin ◽  
Pei Pei Chong ◽  
Siong Hock Ho ◽  
Voon Chen Yong
Keyword(s):  
Cell Surface ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Surface Proteins ◽  
Cell Surface Proteins

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 Bringing order to early B cell chaos

2006 ◽  
Vol 203 (6) ◽  
pp. 1389-1389 ◽  
Author(s):  
Heather L. Van Epps
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Molecular Events ◽  
B Cell Precursors

In the early 1990s, Richard (Randy) Hardy and colleagues divided B cell precursors into subpopulations—the Hardy fractions—based on the cells' expression of various cell surface proteins. This classification helped lay the groundwork for our present-day understanding of the molecular events that control early B cell development.

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