scholarly journals Transient anchorage of cross-linked glycosyl-phosphatidylinositol–anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides

2006 ◽  
Vol 175 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Yun Chen ◽  
William R. Thelin ◽  
Bing Yang ◽  
Sharon L. Milgram ◽  
Ken Jacobson
Keyword(s):  
Transmembrane Protein ◽  
Cross Linking ◽  
Cholesterol Depletion ◽  
Transmembrane Conductance Regulator ◽  
Membrane Biology ◽  
Glycosyl Phosphatidylinositol ◽  
Caveolin 1 ◽  
Outer Leaflet ◽  
Membrane Components ◽  
Open Question

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol–anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle–induced clusters of both Thy-1 and CD73, a 5′ exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

scholarly journals Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

1998 ◽  
Vol 141 (4) ◽  
pp. 929-942 ◽  
Author(s):  
Thomas Harder ◽  
Peter Scheiffele ◽  
Paul Verkade ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Tyrosine Kinase ◽  
Cholesterol Depletion ◽  
Placental Alkaline Phosphatase ◽  
Glycosyl Phosphatidylinositol ◽  
Lipid Domain ◽  
Polarized Cells ◽  
T Lymphoma ◽  
Raft Domains ◽  
Membrane Components

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

scholarly journals Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

2019 ◽  
Vol 116 (12) ◽  
pp. 5550-5557 ◽  
Author(s):  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Joshua Yoon ◽  
Tim Stearns ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Cellular Level ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Motional Dynamics ◽  
Directional Transport ◽  
Bulk Behavior

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Cell-surface attachment of pedestal-forming enteropathogenicE. coliinduces a clustering of raft components and a recruitment of annexin 2

2002 ◽  
Vol 115 (1) ◽  
pp. 91-98
Author(s):  
Nicole Zobiack ◽  
Ursula Rescher ◽  
Sven Laarmann ◽  
Silke Michgehl ◽  
M. Alexander Schmidt ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Surface ◽  
Actin Binding ◽  
Surface Attachment ◽  
Glycosyl Phosphatidylinositol ◽  
Annexin 2 ◽  
Endosomal Membranes ◽  
Membrane Components ◽  
Functional Receptor ◽  
Pedestal Formation

Annexin 2 is a Ca2+-regulated membrane- and F-actin-binding protein implicated in the stabilization or regulation of membrane/cytoskeleton contacts, or both, at the plasma membrane and at early endosomal membranes. To analyze the dynamic nature of such action we investigated whether annexin 2 could be found at sites of localized actin rearrangements occurring at the plasma membrane of HeLa cells infected with noninvading enteropathogenic Escherichia coli (EPEC). We show that adherent EPEC microcolonies, which are known to induce the formation of actin-rich pedestals beneath them, specifically recruit annexin 2 to the sites of their attachment. Mutant EPEC (EPECtir), which lack a functional receptor for intimate attachment (Tir, translocated intimin receptor) and which fail to produce full pedestal formation, are still capable of recruiting annexin 2 to the bacterial contact sites. Accumulation of annexin 2 at sites of EPEC or EPECtir attachment is accompanied by a recruitment of the annexin 2 protein ligand S100A10. EPEC and EPECtir attachment also induces a concentration of cholesterol and glycosyl phosphatidylinositol-anchored proteins at sites of bacterial contact. This indicates that membrane components present in rafts or raft-like microdomains are clustered upon EPEC adherence and that annexin 2 is recruited to the cytoplasmic membrane surface of such clusters, possibly stabilizing raft patches and their linkage to the actin cytoskeleton beneath adhering EPEC.

scholarly journals What Role Does CFTR Play in Development, Differentiation, Regeneration and Cancer?

2020 ◽  
Vol 21 (9) ◽  
pp. 3133 ◽  
Author(s):  
Margarida D. Amaral ◽  
Margarida C. Quaresma ◽  
Ines Pankonien
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Transmembrane Conductance Regulator ◽  
Foetal Development ◽  
Term Survival ◽  
Mesenchymal Transition ◽  
Cellular Processes ◽  
Underlying Mechanisms ◽  
Novel Therapeutic Strategies ◽  
Open Question

One of the key features associated with the substantial increase in life expectancy for individuals with CF is an elevated predisposition to cancer, firmly established by recent studies involving large cohorts. With the recent advances in cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies and the increased long-term survival rate of individuals with cystic fibrosis (CF), this is a novel challenge emerging at the forefront of this disease. However, the mechanisms linking dysfunctional CFTR to carcinogenesis have yet to be unravelled. Clues to this challenging open question emerge from key findings in an increasing number of studies showing that CFTR plays a role in fundamental cellular processes such as foetal development, epithelial differentiation/polarization, and regeneration, as well as in epithelial–mesenchymal transition (EMT). Here, we provide state-of-the-art descriptions on the moonlight roles of CFTR in these processes, highlighting how they can contribute to novel therapeutic strategies. However, such roles are still largely unknown, so we need rapid progress in the elucidation of the underlying mechanisms to find the answers and thus tailor the most appropriate therapeutic approaches.

scholarly journals Targeting of PKCζ and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide

2008 ◽  
Vol 410 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Eric Hajduch ◽  
Sophie Turban ◽  
Xavier Le Liepvre ◽  
Soazig Le Lay ◽  
Christopher Lipina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Structural Integrity ◽  
Insulin Signalling ◽  
Protein Kinase B ◽  
Cholesterol Depletion ◽  
Crucial Step ◽  
Caveolin 1 ◽  
Kinase C ◽  
Phosphoinositide Phosphatase ◽  
Phosphatase And Tensin Homologue

Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCζ (protein kinase C-ζ), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCζ–PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3′-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCζ and PKB and suppressed kinase–caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1−/− mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.

scholarly journals Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33247-33251 ◽  
Author(s):  
Ying Wang ◽  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Cystic Fibrosis ◽  
Channel Blocker ◽  
Cross Linking ◽  
Transmembrane Conductance Regulator ◽  
Mature Protein ◽  
Transmembrane Conductance ◽  
P Glycoprotein ◽  
Δf508 Cftr ◽  
Cross Linker ◽  
Cystic Fibrosis Transmembrane

The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe508 (ΔF508). The ΔF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of ΔF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

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