scholarly journals Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

Cell Reports ◽  
2020 ◽  
Vol 30 (4) ◽  
pp. 1129-1140.e5 ◽  
Author(s):  
Ning Wu ◽  
Vitalij Cernysiov ◽  
Dominique Davidson ◽  
Hua Song ◽  
Jianlong Tang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pore Formation ◽  
Critical Role ◽  
Phosphatidylserine Exposure

scholarly journals Role of Na+/H+ exchanger regulatory factor 1 in forward trafficking of the type IIa Na+-Pi cotransporter

2015 ◽  
Vol 309 (2) ◽  
pp. F109-F119 ◽  
Author(s):  
Corey J. Ketchem ◽  
Syed J. Khundmiri ◽  
Adam E. Gaweda ◽  
Rebecca Murray ◽  
Barbara J. Clark ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Mean Square Displacement ◽  
Active Movement ◽  
Binding Motif ◽  
Late Time ◽  
Cell Fractionation ◽  
Regulatory Factor ◽  
Ok Cells

Na+/H+ exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Pi cotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pi medium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pi medium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pi medium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.

The Abcc4 Knockout Reveals An Important Role for Abcc4 in Platelet Aggregation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1141-1141
Author(s):  
Satish Babu Cheepala ◽  
Kazumasa Takenaka ◽  
Tamara I. Pestina ◽  
Carl W. Jackson ◽  
Schuetz John
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Critical Role ◽  
Antiplatelet Drugs ◽  
Null Mouse ◽  
Dense Granules

Abstract Abstract 1141 Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides. We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels. Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10. This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis

2020 ◽  
Vol 16 (12) ◽  
pp. e1009152
Author(s):  
Chakir Bello ◽  
Yasmine Smail ◽  
Vincent Sainte-Rose ◽  
Isabelle Podglajen ◽  
Alice Gilbert ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Connexin 43 ◽  
Critical Role ◽  
Neurovascular Unit ◽  
Brain Slices ◽  
Host Cells ◽  
Intermediate Filament Protein ◽  
Young Infants ◽  
The Brain

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43–dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca2+ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires co-opting of connexin plasma membrane channels.

scholarly journals The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0238452 ◽  
Author(s):  
Alexander Zhgun ◽  
Mariya Dumina ◽  
Ayrat Valiakhmetov ◽  
Mikhail Eldarov
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Critical Role ◽  
Cephalosporin C ◽  
Acremonium Chrysogenum

scholarly journals Critical role of sulfhydryl group(s) in ATP-dependent Ca2+ sequestration by the plasma membrane fraction from rat liver

FEBS Letters ◽  
1983 ◽  
Vol 163 (1) ◽  
pp. 136-139 ◽  
Author(s):  
Giorgio Bellomo ◽  
Francesca Mirabelli ◽  
Plinio Richelmi ◽  
Sten Orrenius
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Critical Role ◽  
Sulfhydryl Group ◽  
Plasma Membrane Fraction

Critical role of the plasma membrane for expression of mammalian mitochondrial side chain cleavage activity in yeast

2003 ◽  
Vol 270 (7) ◽  
pp. 1502-1514 ◽  
Author(s):  
Catherine Duport ◽  
Barbara Schoepp ◽  
Elise Chatelain ◽  
Roberto Spagnoli ◽  
Bruno Dumas ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Side Chain ◽  
Side Chain Cleavage ◽  
Cleavage Activity ◽  
Chain Cleavage

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin

2018 ◽  
Vol 475 (19) ◽  
pp. 3039-3055 ◽  
Author(s):  
Reema Kathuria ◽  
Anish Kumar Mondal ◽  
Rohan Sharma ◽  
Samarjit Bhattacharyya ◽  
Kausik Chattopadhyay
Keyword(s):  
Vibrio Cholerae ◽  
Formation Mechanism ◽  
Pore Formation ◽  
Critical Role ◽  
Lipid Vesicles ◽  
Membrane Lipid ◽  
Human Erythrocytes ◽  
Binding Ability ◽  
Cholesterol Binding

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

scholarly journals The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa.

2022 ◽  
Author(s):  
Alfredo Figueroa-Melendez ◽  
Leonora Martinez-Nunez ◽  
Adriana Maria Rico-Ramirez ◽  
Juan Manuel Martinez-Andrade ◽  
Mary Munson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Complex Function ◽  
Mass Spectrometry Analysis ◽  
Knockout Mutant ◽  
Transmission Electron Microscopy Analysis ◽  
C Terminus

The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the apices of the cells, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

scholarly journals Regulation of Plasma Membrane Sterol Homeostasis by Nonvesicular Lipid Transport

Contact ◽  
2021 ◽  
Vol 4 ◽  
pp. 251525642110424
Author(s):  
Dylan Hong Zheng Koh ◽  
Yasunori Saheki
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Structural Integrity ◽  
Critical Role ◽  
Lipid Transfer ◽  
Fusion Reactions ◽  
Lipid Transfer Proteins ◽  
Membrane Budding ◽  
Mode Of Transport

Sterol contributes to the structural integrity of cellular membranes and plays an important role in the regulation of cell signaling in eukaryotes. It is either produced in the endoplasmic reticulum or taken up from the extracellular environment. In most eukaryotic cells, however, the majority of sterol is enriched in the plasma membrane. Thus, the transport of sterol between the plasma membrane and other organelles, including the endoplasmic reticulum, is crucial for maintaining sterol homeostasis. While vesicular transport that relies on membrane budding and fusion reactions plays an important role in bulk sterol transport, this mode of transport is slow and non-selective. Growing evidence suggests a critical role of nonvesicular transport mediated by evolutionarily conserved families of lipid transfer proteins in more rapid and selective delivery of sterol. Some lipid transfer proteins act primarily at the sites of contacts formed between the endoplasmic reticulum and other organelles or the plasma membrane without membrane fusion. In this review, we describe the similarities and differences of sterol biosynthesis and uptake in mammals and yeast and discuss the role of their lipid transfer proteins in maintaining plasma membrane sterol homeostasis.

Critical role of G-gamma2 C-terminal region in its interaction with plasma membrane

2010 ◽  
Vol 163 ◽  
pp. S45
Author(s):  
Maria A. Noguera-Salvà ◽  
Xavier Busquets ◽  
Pablo V. Escribá
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Terminal Region
Sign in / Sign up

Export Citation Format

Share Document