An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells

2012 ◽  
Vol 177 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Unai Silván ◽  
Céline Boiteux ◽  
Rosmarie Sütterlin ◽  
Ulrich Schroeder ◽  
Hans Georg Mannherz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Endocytic Pathway

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.

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

2005 ◽  
Vol 8 (1) ◽  
pp. 46-54 ◽  
Author(s):  
Oleg O. Glebov ◽  
Nicholas A. Bright ◽  
Benjamin J. Nichols
Keyword(s):  
Mammalian Cells ◽  
Endocytic Pathway

scholarly journals Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae.

1987 ◽  
Vol 104 (1) ◽  
pp. 67-75 ◽  
Author(s):  
M Makarow ◽  
L T Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ammonium Chloride ◽  
Mammalian Cells ◽  
Extracellular Fluid ◽  
Endocytic Pathway ◽  
Sodium Vanadate ◽  
Energy Depletion ◽  
Internal Ph ◽  
Intermediate Compartment ◽  
Cytoplasmic Compartment

Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.

scholarly journals Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

2009 ◽  
Vol 186 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Pinkesh Bhagatji ◽  
Rania Leventis ◽  
Jonathan Comeau ◽  
Mohammad Refaei ◽  
John R. Silvius
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Folate Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fundamental Property ◽  
Membrane Lipid ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Lipid Markers

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

scholarly journals Clathrin- and Arp2/3-Independent Endocytosis in the Fungal Pathogen Candida albicans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Elias Epp ◽  
Elena Nazarova ◽  
Hannah Regan ◽  
Lois M. Douglas ◽  
James B. Konopka ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Proteins ◽  
Fungal Pathogen ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Fungal Species ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Content Type ◽  
Actin Cables

ABSTRACT Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species. IMPORTANCE There is a well-established process of endocytosis that is generally used by eukaryotic cells termed clathrin-mediated endocytosis (CME). Although the details are somewhat different between lower and higher eukaryotes, CME appears to be the dominant endocytic process in all eukaryotes. While fungi such as Saccharomyces cerevisiae have proven excellent models for dissecting the molecular details of endocytosis, loss of CME is so detrimental that it has been difficult to study alternate pathways functioning in its absence. Although the fungal pathogen Candida albicans has a CME pathway that functions similarly to that of S. cerevisiae, inactivation of this pathway does not compromise growth of yeast-form C. albicans. In these cells, lipids and fluid-phase molecules are still endocytosed in an actin-dependent manner, but membrane proteins are not. Thus, C. albicans provides a powerful model for the analysis of CME-independent endocytosis in lower eukaryotes.

scholarly journals New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 899 ◽  
Author(s):  
James H. Grissom ◽  
Verónica A. Segarra ◽  
Richard J. Chi
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Endocytic Pathway ◽  
Model Organisms ◽  
Endosomal Trafficking ◽  
Endomembrane System ◽  
Recycling Endosome ◽  
New Findings

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

scholarly journals At the crossroads of homoeostasis and disease: roles of the PACS proteins in membrane traffic and apoptosis

2009 ◽  
Vol 421 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Robert T. Youker ◽  
Ujwal Shinde ◽  
Robert Day ◽  
Gary Thomas
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Virus Assembly ◽  
Membrane Traffic ◽  
Endocytic Pathway ◽  
Viral Pathogens ◽  
Viral Spread ◽  
Disease States ◽  
Organ Systems

The endomembrane system in mammalian cells has evolved over the past two billion years from a simple endocytic pathway in a single-celled primordial ancestor to complex networks supporting multicellular structures that form metazoan tissue and organ systems. The increased organellar complexity of metazoan cells requires additional trafficking machinery absent in yeast or other unicellular organisms to maintain organ homoeostasis and to process the signals that control proliferation, differentiation or the execution of cell death programmes. The PACS (phosphofurin acidic cluster sorting) proteins are one such family of multifunctional membrane traffic regulators that mediate organ homoeostasis and have important roles in diverse pathologies and disease states. This review summarizes our current knowledge of the PACS proteins, including their structure and regulation in cargo binding, their genetics, their roles in secretory and endocytic pathway traffic, interorganellar communication and how cell-death signals reprogramme the PACS proteins to regulate apoptosis. We also summarize our current understanding of how PACS genes are dysregulated in cancer and how viral pathogens ranging from HIV-1 to herpesviruses have evolved to usurp the PACS sorting machinery to promote virus assembly, viral spread and immunoevasion.

scholarly journals Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

2013 ◽  
Vol 82 (3) ◽  
pp. 1222-1233 ◽  
Author(s):  
Julie Allombert ◽  
Jean-Claude Lazzaroni ◽  
Nathalie Baïlo ◽  
Christophe Gilbert ◽  
Xavier Charpentier ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Endocytic Pathway ◽  
Intracellular Pathogen ◽  
Diguanylate Cyclase ◽  
Phagocytic Cells ◽  
Metabolizing Enzymes ◽  
Content Type ◽  
Large Repertoire ◽  
Fine Tune

ABSTRACTLegionella pneumophilais an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of theL. pneumophilaLens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication ofL. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of theLegionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest thatL. pneumophilauses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.

scholarly journals AtVPS41-mediated endocytic pathway is essential for pollen tube–stigma interaction in Arabidopsis

2016 ◽  
Vol 113 (22) ◽  
pp. 6307-6312 ◽  
Author(s):  
Lihong Hao ◽  
Jingjing Liu ◽  
Sheng Zhong ◽  
Hongya Gu ◽  
Li-Jia Qu
Keyword(s):  
Pollen Tube ◽  
Late Stage ◽  
Mammalian Cells ◽  
Sequence Similarity ◽  
Single Copy ◽  
Pollen Tubes ◽  
Endocytic Pathway ◽  
Signaling Cascades ◽  
Vacuolar Protein ◽  
Endomembrane Trafficking

In flowering plants, extensive male–female interactions are required for successful fertilization in which various signaling cascades are involved. Prevacuolar compartments (PVC) and vacuoles are two types of subcellular compartments that terminate signal transduction by sequestrating signaling molecules in yeast and mammalian cells; however, the manner in which they might be involved in male–female interactions in plants is unknown. In this study, we identified Arabidopsisthaliana vacuolar protein sorting 41 (AtVPS41), encoded by a single-copy gene with sequence similarity to yeast Vps41p, as a new factor controlling pollen tube–stigma interaction. Loss of AtVPS41 function disrupted penetration of pollen tubes into the transmitting tissue and thus led to failed male transmission. In the pollen tubes, AtVPS41 protein is associated with PVCs and the tonoplast. We demonstrate that AtVPS41 is required for the late stage of the endocytic pathway (i.e., endomembrane trafficking from PVCs to vacuoles) because internalization of cell-surface molecules was normal in the vps41-deficient pollen tubes, whereas PVC-to-vacuole trafficking was impaired. We further show that the CHCR domain is required for subcellular localization and biological functioning of AtVPS41. These results indicate that the AtVPS41-mediated late stage of the endocytic pathway is essential for pollen tube–stigma interaction in Arabidopsis.

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