The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described "heavy rafts" or non-raft membrane. Our data show that only Csk targeted into "classical" raft but not to "heavy raft" or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.

• MeSH
• buněčná membrána metabolismus   MeSH
• fosforylace MeSH
• imunoblotting MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové mikrodomény MeSH
• protoonkogenní proteiny metabolismus   MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• signální transdukce MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The plasma membrane of Saccharomyces cerevisiae contains large microdomains enriched in ergosterol, which house at least nine integral proteins, including proton symporters. The domains adopt a characteristic structure of furrow-like invaginations typically seen in freeze-fracture pictures of fungal cells. Being stable for the time comparable with the cell cycle duration, they might be considered as fixed islands (rafts) in an otherwise fluid yeast plasma membrane. Rapidly moving endocytic marker proteins avoid the microdomains; the domain-accumulated proton symporters consequently show a reduced rate of substrate-induced endocytosis and turnover.

• MeSH
• biologické modely MeSH
• buněčná membrána chemie   metabolismus   ultrastruktura   MeSH
• konfokální mikroskopie MeSH
• membránové mikrodomény chemie   metabolismus   ultrastruktura   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae chemie   metabolismus   ultrastruktura   MeSH
• transportní systémy pro bazické aminokyseliny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7 These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• Candida albicans metabolismus   MeSH
• endocytóza fyziologie   MeSH
• fosfoproteiny metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• hyfy metabolismus   MeSH
• membránové mikrodomény metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• proteinkinasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Plasma membrane of the yeast Saccharomyces cerevisiae contains stable lateral domains. We have investigated the ultrastructure of one type of domain, the membrane compartment of Can1 (MCC). In two yeast strains (nce102Delta and pil1Delta) that are defective in segregation of MCC-specific proteins, we found the plasma membrane to be devoid of the characteristic furrow-like invaginations. These are highly conserved plasma membrane structures reported in early freeze-fracture studies. Comparison of the results obtained by three different approaches - electron microscopy of freeze-etched cells, confocal microscopy of intact cells and computer simulation - shows that the number of invaginations corresponds to the number of MCC patches in the membrane of wild-type cells. In addition, neither MCC patches nor the furrow-like invaginations colocalized with the cortical ER. In mutants exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane - the furrow-like invagination.

• MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• buněčné výběžky metabolismus   ultrastruktura   MeSH
• endoplazmatické retikulum ultrasonografie   MeSH
• kompartmentace buňky MeSH
• mutace genetika   MeSH
• počítačová simulace MeSH
• povrchové vlastnosti MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   ultrastruktura   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   ultrastruktura   MeSH
• transportní systémy pro bazické aminokyseliny metabolismus   MeSH
• zalévání tkání MeSH
• Publikační typ
• práce podpořená grantem MeSH

Examples from yeast and plant cells are described that show that their plasma membrane is laterally compartmented. Distinct lateral domains encompassing both specific lipids and integral proteins coexist within the plane of the plasma membrane. The compartments are either spatially stable and include distinct sets of proteins, or they are transiently formed to accomplish diverse functions. They are not related to lipid rafts or their clusters, as defined for mammalian cells. This review summarises only well-documented compartments of plasma membranes from plants and fungi, which have been recognised using microscopic approaches. In several cases, physiological functions of the membrane compartmentation are revealed.

• MeSH
• buněčná membrána fyziologie   MeSH
• fungální proteiny fyziologie   MeSH
• houby metabolismus   fyziologie   MeSH
• membránové lipidy fyziologie   MeSH
• membránové mikrodomény fyziologie   MeSH
• membránové proteiny fyziologie   MeSH
• rostlinné proteiny fyziologie   MeSH
• rostliny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.

• MeSH
• amoniak metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• detergenty metabolismus   MeSH
• financování organizované MeSH
• kompartmentace buňky MeSH
• koncentrace vodíkových iontů MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   MeSH
• transport proteinů MeSH
• zelené fluorescenční proteiny metabolismus   MeSH

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

• MeSH
• algoritmy MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol metabolismus   MeSH
• difenylhexatrien chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční polarizace MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• buněčná membrána fyziologie   MeSH
• iontový transport fyziologie   MeSH
• kompartmentace buňky MeSH
• kvasinky MeSH
• osmolární koncentrace MeSH
• sodík fyziologie   MeSH

Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

• MeSH
• buněčná membrána metabolismus   MeSH
• kapacitace spermií * MeSH
• prasata MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• proteomika MeSH
• spermie cytologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH