scholarly journals High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

10.3791/58351 ◽  
2019 ◽  
Author(s):  
Jonathan G.T. Lam ◽  
Chi Song ◽  
Stephanie Seveau
Keyword(s):  
Plasma Membrane ◽  
High Throughput ◽  
Mammalian Cells ◽  
Membrane Resealing

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing

1990 ◽  
Vol 11 (2) ◽  
pp. 154-166 ◽  
Author(s):  
N. J. Severs ◽  
A. M. Slade ◽  
T. Powell ◽  
V. W. Twist ◽  
C. R. Green
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Cardiac Myocyte ◽  
Membrane Resealing

scholarly journals Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes

1996 ◽  
Vol 317 (3) ◽  
pp. 933-938 ◽  
Author(s):  
Meylin SUJU ◽  
Marbelly DAVILA ◽  
German POLEO ◽  
Roberto DOCAMPO ◽  
Gustavo BENAIM
Keyword(s):  
Plasma Membrane ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Human Erythrocytes ◽  
Maximal Velocity ◽  
Ethanol Intoxication ◽  
Solubilized Enzyme ◽  
Acidic Phospholipids ◽  
Additive Combination ◽  
Stimulation Of

Phosphatidylethanol is formed by ‘transphosphatidylation’ of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca2+-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca2+-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidylalcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca2+-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca2+ concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca2+-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

2001 ◽  
Vol 114 (10) ◽  
pp. 1893-1900 ◽  
Author(s):  
S. Lusa ◽  
T.S. Blom ◽  
E.L. Eskelinen ◽  
E. Kuismanen ◽  
J.E. Mansson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Ldl Cholesterol ◽  
Normal Cells ◽  
Recycling Endosomes ◽  
Regulate Protein ◽  
Niemann Pick ◽  
Accumulation Characteristic

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

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