scholarly journals Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

10.3791/57837 ◽  
2018 ◽  
Author(s):  
Stanislav Vosolsobě ◽  
Kateřina Schwarzerová ◽  
Jan Petrášek
Keyword(s):  
Plasma Membrane ◽  
Membrane Partitioning ◽  
Associated Proteins

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (10) ◽  
Author(s):  
Margarida Caldeira ◽  
Joana Ferreira ◽  
Ana Carvalho ◽  
Carlos Duarte
Keyword(s):  
Plasma Membrane ◽  
Cultured Neurons ◽  
Associated Proteins

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

Determination of permeability of yeast plasma membrane for amphiphiles

2004 ◽  
Vol 399 (1-6) ◽  
pp. 371-373
Author(s):  
D. I. Bondarenko ◽  
D. A. Aliverdieva ◽  
D. V. Mamaev ◽  
K. F. Shol’ts
Keyword(s):  
Plasma Membrane ◽  
Yeast Plasma Membrane

scholarly journals Na delivery and ENaC mediate flow regulation of collecting duct endothelin-1 production

2012 ◽  
Vol 302 (10) ◽  
pp. F1325-F1330 ◽  
Author(s):  
Meghana M. Pandit ◽  
Kevin A. Strait ◽  
Toshio Matsuda ◽  
Donald E. Kohan
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Extracellular Fluid ◽  
Na Channel ◽  
Endothelin 1 ◽  
Flow Response ◽  
Mrna Content ◽  
Static Conditions ◽  
Biologic System

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm2, followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca2+ enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca2+ exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

scholarly journals Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Mario Codemo ◽  
Sandra Muschiol ◽  
Federico Iovino ◽  
Priyanka Nannapaneni ◽  
Laura Plant ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Streptococcus Pneumoniae ◽  
Extracellular Vesicles ◽  
Factor H ◽  
Host Cells ◽  
Immunomodulatory Effects ◽  
Content Type ◽  
Associated Proteins ◽  
Tract Infections

ABSTRACTGram-positive bacteria, including the major respiratory pathogenStreptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.IMPORTANCEStreptococcus pneumoniaeis a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

scholarly journals Cell patterning by secretion-induced plasma membrane flows

2020 ◽  
Author(s):  
Veneta Gerganova ◽  
Iker Lamas ◽  
David M. Rutkowski ◽  
Aleksandar Vještica ◽  
Daniela Gallo Castro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Negative Feedback ◽  
Reaction Diffusion ◽  
Cell Patterning ◽  
Yeast Cells ◽  
Membrane Insertion ◽  
Gtpase Activating Proteins ◽  
Associated Proteins ◽  
Bulk Membrane ◽  
Polarized Exocytosis

AbstractCells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

scholarly journals The eisosome core is composed of BAR domain proteins

2011 ◽  
Vol 22 (13) ◽  
pp. 2360-2372 ◽  
Author(s):  
Agustina Olivera-Couto ◽  
Martin Graña ◽  
Laura Harispe ◽  
Pablo S. Aguilar
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Bar Domain ◽  
Macromolecular Assemblies ◽  
Surface Patches ◽  
Cytoplasmic Proteins ◽  
Associated Proteins ◽  
Core Components ◽  
Membrane Bending

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein–mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane.

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