scholarly journals Cytoskeleton-dependent Membrane Domain Segregation during Neutrophil Polarization

2001 ◽  
Vol 12 (11) ◽  
pp. 3550-3562 ◽  
Author(s):  
Stéphanie Seveau ◽  
Robert J. Eddy ◽  
Frederick R. Maxfield ◽  
Lynda M. Pierini
Keyword(s):  
Plasma Membrane ◽  
Myosin Light Chain ◽  
Transmembrane Proteins ◽  
Cell Polarization ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Actin Remodeling ◽  
Cytoskeletal Rearrangements ◽  
Triton X ◽  
Detergent Resistant Membrane

On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC16, are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.

scholarly journals Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

2014 ◽  
Vol 206 (5) ◽  
pp. 609-618 ◽  
Author(s):  
Josse van Galen ◽  
Felix Campelo ◽  
Emma Martínez-Alonso ◽  
Margherita Scarpa ◽  
José Ángel Martínez-Menárguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Transmembrane Proteins ◽  
Short Chain ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Specific Membrane

Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins? In this paper, we show that disruption of SM homeostasis at the trans-Golgi network (TGN) by treatment of HeLa cells with d-ceramide-C6, which was converted together with phosphatidylcholine to short-chain SM and diacylglycerol by SM synthase, led to the segregation of Golgi-resident proteins from each other. We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other. Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

scholarly journals An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

1998 ◽  
Vol 9 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Hans de Vries ◽  
Cobi Schrage ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Insoluble Fraction ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Polarized Cells ◽  
Vesicular Stomatitis ◽  
Triton X

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

scholarly journals Cdc42 regulates neutrophil migration via crosstalk between WASp, CD11b, and microtubules

Blood ◽  
2012 ◽  
Vol 120 (17) ◽  
pp. 3563-3574 ◽  
Author(s):  
Sachin Kumar ◽  
Juying Xu ◽  
Charles Perkins ◽  
Fukun Guo ◽  
Scott Snapper ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Neutrophil Migration ◽  
Cell Polarization ◽  
Membrane Domains ◽  
Long Distance ◽  
Cellular Defense ◽  
Persistent Cell ◽  
Proper Balance ◽  
Detergent Resistant Membrane ◽  
Standing Question

Abstract Chemotaxis promotes neutrophil participation in cellular defense by enabling neutrophil migration to infected tissue and is controlled by persistent cell polarization. One long-standing question of neutrophil polarity has been how the pseudopod and the uropod are coordinated. In our previous report, we suggested that Rho GTPase Cdc42 controls neutrophil polarity through CD11b signaling at the uropod, albeit through an unknown mechanism. Here, we show that Cdc42 controls polarity, unexpectedly, via its effector WASp. Cdc42 controls WASp activation and its distant localization to the uropod. At the uropod, WASp regulates the reorganization of CD11b integrin into detergent resistant membrane domains; in turn, CD11b recruits the microtubule end binding protein EB1 to capture and stabilize microtubules at the uropod. This organization is necessary to maintain neutrophil polarity during migration and is critical for neutrophil emigration into inflamed lungs. These results suggest unrecognized mechanism of neutrophil polarity in which WASp mediates long-distance control of the uropod by Cdc42 to maintain a proper balance between the pseudopod and the uropod. Our study reveals a new function for WASp in the control of neutrophil polarity via crosstalk between CD11b and microtubules.

scholarly journals Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

2020 ◽  
Author(s):  
Tetsuo Mioka ◽  
Guo Tian ◽  
Wang Shiyao ◽  
Takuma Tsuji ◽  
Takuma Kishimoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Living Cells ◽  
Yeast Cells ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Artificial Membranes ◽  
Free Region ◽  
Large Phase ◽  
Plasma Membrane Domains

AbstractMembrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.Summary statementYeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.

scholarly journals The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

2014 ◽  
Vol 35 (1) ◽  
pp. 288-302 ◽  
Author(s):  
Wia Baron ◽  
Hande Ozgen ◽  
Bert Klunder ◽  
Jenny C. de Jonge ◽  
Anita Nomden ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Body ◽  
Proteolipid Protein ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Conformational Alteration ◽  
Syntaxin 3 ◽  
Triton X

Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLP's reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

scholarly journals Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress

2020 ◽  
Vol 21 (20) ◽  
pp. 7694
Author(s):  
Lucía Yepes-Molina ◽  
Micaela Carvajal ◽  
Maria Carmen Martínez-Ballesta
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Lower Amount ◽  
Membrane Domains ◽  
Cellular Functions ◽  
Osmotic Water ◽  
Fatty Acid Saturation ◽  
Protein Partitioning ◽  
Detergent Resistant Membranes ◽  
Detergent Resistant Membrane

Detergent-resistant membranes (DRMs) microdomains, or “raft lipids”, are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

scholarly journals Endocytosis of GPI-linked membrane folate receptor-alpha.

1996 ◽  
Vol 132 (1) ◽  
pp. 35-47 ◽  
Author(s):  
S Rijnboutt ◽  
G Jansen ◽  
G Posthuma ◽  
J B Hynes ◽  
J H Schornagel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Folate Receptor ◽  
Large Fraction ◽  
Integral Membrane Protein ◽  
Endocytic Pathway ◽  
Reduced Folate Carrier ◽  
Membrane Domains ◽  
Chemotherapeutic Drugs ◽  
Folate Receptors ◽  
Triton X

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

CD44 exhibits a cell type dependent interaction with triton X-100 insoluble, lipid rich, plasma membrane domains

1995 ◽  
Vol 108 (9) ◽  
pp. 3127-3135 ◽  
Author(s):  
S.J. Neame ◽  
C.R. Uff ◽  
H. Sheikh ◽  
S.C. Wheatley ◽  
C.M. Isacke
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Membrane Domains ◽  
Cell Type ◽  
Transmembrane Glycoprotein ◽  
Morphological Studies ◽  
Plasma Membrane Domains ◽  
Triton X

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4 degrees C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.

scholarly journals Human Immunodeficiency Virus Type 1 Assembly and Lipid Rafts: Pr55gag Associates with Membrane Domains That Are Largely Resistant to Brij98 but Sensitive to Triton X-100

2003 ◽  
Vol 77 (8) ◽  
pp. 4805-4817 ◽  
Author(s):  
Kirsi Holm ◽  
Katarzyna Weclewicz ◽  
Roger Hewson ◽  
Maarit Suomalainen
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Intracellular Membranes ◽  
Immunodeficiency Virus ◽  
Triton X

ABSTRACT The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55 gag . We have analyzed whether Pr55 gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55 gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55 gag particles (VLPs) yield buoyant Pr55 gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55gag complexes cannot be taken as a proof for raft association of Pr55 gag , since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55 gag . However, Pr55 gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55 gag . Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55 gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55 gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55 gag VLPs.

Cytoskeletal interactions regulate inducible L-selectin clustering

2005 ◽  
Vol 289 (2) ◽  
pp. C323-C332 ◽  
Author(s):  
Polly E. Mattila ◽  
Chad E. Green ◽  
Ulrich Schaff ◽  
Scott I. Simon ◽  
Bruce Walcheck
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Transmembrane Protein ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Lateral Mobility ◽  
Progressive Change ◽  
Glycosyl Phosphatidylinositol ◽  
Effector Functions ◽  
G Protein Coupled

L-selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of L-selectin and serves to increase its adhesiveness, signaling, and colocalization with β2-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin’s lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin’s effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton.

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