共查询到9条相似文献,搜索用时 0 毫秒
1.
Ying Wang Tadayuki Yago Nan Zhang Salim Abdisalaam George Alexandrakis William Rodgers Rodger P. McEver 《The Journal of biological chemistry》2014,289(51):35159-35171
Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. 相似文献
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García-Sáez AJ Buschhorn SB Keller H Anderluh G Simons K Schwille P 《The Journal of biological chemistry》2011,286(43):37768-37777
Pore-forming toxins have evolved to induce membrane injury by formation of pores in the target cell that alter ion homeostasis and lead to cell death. Many pore-forming toxins use cholesterol, sphingolipids, or other raft components as receptors. However, the role of plasma membrane organization for toxin action is not well understood. In this study, we have investigated cellular dynamics during the attack of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, by combining time lapse three-dimensional live cell imaging, fluorescence recovery after photobleaching, FRET, and fluorescence cross-correlation spectroscopy. Our results show that membrane binding by equinatoxin II is accompanied by extensive plasma membrane reorganization into microscopic domains that resemble coalesced lipid rafts. Pore formation by the toxin induces Ca(2+) entry into the cytosol, which is accompanied by hydrolysis of phosphatidylinositol 4,5-bisphosphate, plasma membrane blebbing, actin cytoskeleton reorganization, and inhibition of endocytosis. We propose that plasma membrane reorganization into stabilized raft domains is part of the killing strategy of equinatoxin II. 相似文献
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Aisha Shamas-Din Scott Bindner Weijia Zhu Yehudit Zaltsman Clinton Campbell Atan Gross Brian Leber David W. Andrews Cécile Fradin 《The Journal of biological chemistry》2013,288(30):22111-22127
Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death. 相似文献
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Eitaro Aihara Courtney L. Hentz Abraham M. Korman Nicholas P. J. Perry Vikram Prasad Gary E. Shull Marshall H. Montrose 《The Journal of biological chemistry》2013,288(47):33585-33597
We report that a localized intracellular and extracellular Ca2+ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca2+-sensitive protein (yellow cameleon 3.0) report that intracellular Ca2+ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca2+ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca2+ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca2+ mobilization. Indomethacin and verapamil also inhibit the luminal Ca2+ increase. Intracellular Ca2+ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca2+ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca2+ and unevenly inhibits late-phase intracellular Ca2+ mobilization. Both modes of Ca2+ chelation slow gastric repair. In plasma membrane Ca-ATPase 1+/− mice, but not plasma membrane Ca-ATPase 4−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca2+ increase. We conclude that endogenous Ca2+, mobilized by signaling pathways and transmembrane Ca2+ transport, causes increased Ca2+ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo. 相似文献
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Jasmin Schmid Heiko Dussmann Gerhardt J. Boukes Lorna Flanagan Andreas U. Lindner Carla L. O'Connor Markus Rehm Jochen H. M. Prehn Heinrich J. Huber 《The Journal of biological chemistry》2012,287(49):41546-41559
Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac−/−, similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAPAdv) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP. 相似文献
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Yoshiaki Suzuki Hisao Yamamura Susumu Ohya Yuji Imaizumi 《The Journal of biological chemistry》2013,288(51):36750-36761
L-type voltage-dependent Ca2+ channels (LVDCC) and large conductance Ca2+-activated K+ channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca2+ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca2+ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K+ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca2+ dynamics including the negative feedback, to control the arterial excitability and contractility. 相似文献
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Cardarelli F Serresi M Albanese A Bizzarri R Beltram F 《The Journal of biological chemistry》2011,286(14):12292-12299
A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin α (Impα) and importin β (Impβ) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impβ carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/β-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences. 相似文献
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Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance. 相似文献