Lipid rafts Dotaz Zobrazit nápovědu
The recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.
- MeSH
- aktivace trombocytů MeSH
- biopolymery MeSH
- buněčná membrána chemie MeSH
- cytoplazmatická granula chemie MeSH
- cytoskelet chemie MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- membránové mikrodomény chemie MeSH
- P-selektin MeSH
- PrPC proteiny krev MeSH
- trombocyty chemie ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.
- MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buňky NK cytologie metabolismus MeSH
- centrifugace - gradient hustoty MeSH
- chemotaxe leukocytů fyziologie MeSH
- detergenty farmakologie MeSH
- kultivované buňky MeSH
- lektinové receptory NK-buněk - podrodina C metabolismus MeSH
- lektinové receptory NK-buněk - podrodina K fyziologie MeSH
- lidé MeSH
- malá interferující RNA farmakologie MeSH
- membránové mikrodomény účinky léků fyziologie MeSH
- mikrofilamenta fyziologie MeSH
- mikrofilamentové proteiny antagonisté a inhibitory genetika fyziologie MeSH
- multiproteinové komplexy MeSH
- proteom MeSH
- receptory imunologické metabolismus MeSH
- RNA interference MeSH
- signální transdukce imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of lipid or membrane rafts. The idea that detergent-resistant membranes are equivalent to lipid rafts, which was generally abandoned after a decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane microdomains, meanwhile, has been verified by unequivocal independent evidence. This review summarizes the current state of research in plants and fungi with respect to common aspects of both kingdoms. In these organisms, principally immobile microdomains large enough for microscopic detection have been visualized. These microdomains are found in the context of cell-cell interactions (plant symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance of membrane microdomains.
- MeSH
- biologický transport MeSH
- buněčná membrána chemie metabolismus MeSH
- detergenty MeSH
- houby chemie metabolismus MeSH
- membránové mikrodomény chemie metabolismus MeSH
- rostlinné buňky chemie metabolismus MeSH
- rostliny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.
- MeSH
- autofagie * MeSH
- buněčné linie MeSH
- membránové mikrodomény chemie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- proteomika * MeSH
- sekvence aminokyselin MeSH
- tularemie metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.
An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
- MeSH
- anizotropie MeSH
- buněčná membrána chemie MeSH
- cholesterol chemie MeSH
- detergenty chemie MeSH
- gelová chromatografie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie MeSH
- lipidy chemie MeSH
- maleáty chemie MeSH
- mastné kyseliny chemie MeSH
- membránové mikrodomény chemie MeSH
- membránové proteiny chemie MeSH
- membrány umělé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- polymery chemie MeSH
- radiační rozptyl MeSH
- rozpustnost MeSH
- styren chemie MeSH
- světlo MeSH
- T-lymfocyty cytologie MeSH
- ultracentrifugace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.
- MeSH
- adenylátcyklasový toxin chemie metabolismus MeSH
- antigeny CD11b metabolismus MeSH
- antigeny CD18 metabolismus MeSH
- Bordetella enzymologie MeSH
- buněčná membrána enzymologie mikrobiologie MeSH
- cholesterol metabolismus MeSH
- cytosol enzymologie MeSH
- extracelulární prostor metabolismus MeSH
- lidé MeSH
- makrofágový antigen 1 metabolismus MeSH
- makrofágy metabolismus mikrobiologie MeSH
- membránové mikrodomény enzymologie mikrobiologie MeSH
- myši MeSH
- talin metabolismus MeSH
- terciární struktura proteinů MeSH
- U937 buňky MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.
- MeSH
- aktivace enzymů MeSH
- aktivace lymfocytů MeSH
- antigeny CD45 metabolismus MeSH
- buněčná membrána metabolismus MeSH
- CD4-pozitivní T-lymfocyty imunologie MeSH
- centrifugace - gradient hustoty MeSH
- detergenty farmakologie MeSH
- membránové mikrodomény metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- signální transdukce MeSH
- transport proteinů MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
