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EMBO Reports Dec 2023The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR...
The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR domain-induced PM furrows. Here we report a novel function for the Sur7 family of tetraspanner proteins in the regulation of local PM topography. Combining TIRF imaging, STED nanoscopy, freeze-fracture EM and membrane simulations we find that Sur7 tetraspanners form multimeric strands at the edges of PM furrows, where they modulate forces exerted by BAR domain proteins at the furrow base. Loss of Sur7 tetraspanners or Sur7 displacement due to altered PIP2 homeostasis leads to increased PM invagination and a distinct form of membrane tubulation. Physiological defects associated with PM tubulation are rescued by synthetic anchoring of Sur7 to furrows. Our findings suggest a key role for tetraspanner proteins in sculpting local membrane domains. The maintenance of stable PM furrows depends on a balance between negative curvature at the base which is generated by BAR domains and positive curvature at the furrows' edges which is stabilized by strands of Sur7 tetraspanners.
Topics: Cell Membrane; Proteins
PubMed: 37902009
DOI: 10.15252/embr.202357232 -
Cells Oct 2023Glycosphingolipids (GSLs) are products of lipid glycosylation that have been implicated in the development of cardiovascular diseases. In diabetes, the adipocyte...
Glycosphingolipids (GSLs) are products of lipid glycosylation that have been implicated in the development of cardiovascular diseases. In diabetes, the adipocyte microenvironment is characterized by hyperglycemia and inflammation, resulting in high levels of GSLs. Therefore, we sought to assess the GSL content in extracellular vesicles derived from the adipose tissues (adiposomes) of obese-diabetic (OB-T2D) subjects and their impact on endothelial cell function. To this end, endothelial cells were exposed to adiposomes isolated from OB-T2D versus healthy subjects. Cells were assessed for caveolar integrity and related signaling, such as Src-kinase and caveolin-1 (cav-1) phosphorylation, and functional pathways, such as endothelial nitric oxide synthase (eNOS) activity. Compared with adiposomes from healthy subjects, OB-T2D adiposomes had higher levels of GSLs, especially LacCer and GM3; they promoted cav-1 phosphorylation coupled to an obvious loss of endothelial surface caveolae and induced eNOS-uncoupling, peroxynitrite generation, and cav-1 nitrosylation. These effects were abolished by Src kinase inhibition and were not observed in GSL-depleted adiposomes. At the functional levels, OB-T2D adiposomes reduced nitric oxide production, shear response, and albumin intake in endothelial cells and impaired flow-induced dilation in healthy arterioles. In conclusion, OB-T2D adiposomes carried a detrimental GSL cargo that disturbed endothelial caveolae and the associated signaling.
Topics: Humans; Caveolae; Endothelial Cells; Lipid Droplets; Signal Transduction; src-Family Kinases; Vascular Diseases; Diabetes Mellitus, Type 2
PubMed: 37887297
DOI: 10.3390/cells12202453 -
Proceedings of the National Academy of... Oct 2023Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within...
Munc13-1 is essential for vesicle docking and fusion at the active zone of synapses. Here, we report that Munc13-1 self-assembles into molecular clusters within diacylglycerol-rich microdomains present in phospholipid bilayers. Although the copy number of Munc13-1 molecules in these clusters has a broad distribution, a systematic Poisson analysis shows that this is most likely the result of two molecular species: monomers and mainly hexameric oligomers. Each oligomer is able to capture one vesicle independently. Hexamers have also been observed in crystals of Munc13-1 that form between opposed phospholipid bilayers [K. Grushin, R. V. Kalyana Sundaram, C. V. Sindelar, J. E. Rothman, , e2121259119 (2022)]. Mutations targeting the contacts stabilizing the crystallographic hexagons also disrupt the isolated hexamers, suggesting they are identical. Additionally, these mutations also convert vesicle binding from a cooperative to progressive mode. Our study provides an independent approach showing that Munc13-1 can form mainly hexamers on lipid bilayers each capable of vesicle capture.
Topics: SNARE Proteins; Diglycerides; Synapses; Molecular Chaperones; Phospholipids
PubMed: 37883433
DOI: 10.1073/pnas.2306086120 -
Cellular & Molecular Biology Letters Oct 2023Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1...
BACKGROUND
Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1.
METHODS
We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed.
RESULTS
EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca concentration.
CONCLUSIONS
Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.
Topics: Humans; Cell Membrane; Epidermal Growth Factor; HeLa Cells; Protein Binding; Membrane Microdomains; Adaptor Proteins, Signal Transducing; Membrane Proteins
PubMed: 37880612
DOI: 10.1186/s11658-023-00497-y -
The Journal of Biological Chemistry Dec 2023Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell...
Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.
Topics: Cell Aggregation; Extracellular Matrix; Membrane Microdomains; Monomeric GTP-Binding Proteins; Signal Transduction; MDA-MB-231 Cells; Humans; Animals; Mice; Cell Line, Tumor; Mice, Inbred BALB C; Hyaluronan Receptors; rac1 GTP-Binding Protein; Anoikis; Enzyme Activation; Neoplasm Metastasis
PubMed: 37866630
DOI: 10.1016/j.jbc.2023.105377 -
Frontiers in Immunology 2023Cellular Ca signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca increase, called Ca microdomains, constitute building... (Review)
Review
Cellular Ca signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca increase, called Ca microdomains, constitute building blocks that are differentially arranged to create cellular Ca signatures controlling physiological responses. Here, we focus on Ca microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca dynamics. Simulations are able to predict the characteristics of Ca increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca microdomains in the first seconds of T cell stimulation is emerging.
Topics: Calcium Channels; T-Lymphocytes; Cell Membrane; Endoplasmic Reticulum; Computer Simulation
PubMed: 37860008
DOI: 10.3389/fimmu.2023.1235737 -
Neurologia 2023Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft... (Review)
Review
INTRODUCTION
Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders.
DEVELOPMENT
We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease.
CONCLUSIONS
Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.
Topics: Humans; Caveolae; Membrane Microdomains; Cholesterol; Cell Membrane; Alzheimer Disease
PubMed: 37858892
DOI: 10.1016/j.nrleng.2023.10.003 -
RSC Advances Oct 2023To understand the growth mechanism of pyrolytic graphite on ideal graphite substrate by chemical vapor deposition (CVD), the process simulated by molecular dynamics is...
To understand the growth mechanism of pyrolytic graphite on ideal graphite substrate by chemical vapor deposition (CVD), the process simulated by molecular dynamics is performed. The simulation revealed that only an amorphous graphite-like membrane (a-GLM) can be obtained using the Tersoff potential function. It is revealed that the formation process for the first layer of the a-GLM initiates from the firstly deposited carbon monomers, dimers, and trimers as growth points, on which Y-shaped long chain is formed, and then the network with pores are developed, and finally the graphene-like layer with six-membered dominated rings appears after repair treatment. The following deposition simulation for more deposited carbon atoms indicates that a second layer like-graphene is more prone to superpose onto the microdomains of the first layer like-graphene until the formation of the island-like bumps, and the concave zone around the bumps will be filled by deposited atoms and modified by defect repair. The growth mechanism for pyrolytic graphite prepared by CVD can be deduced by the simulation inspiration. The optimal deposition temperature of 2400 K is acquired by evaluating the integrity of the first deposited graphene film by statistics of five-to-seven-membered carbon ring number. This study can provide theoretical references for the design of the pyrolytic graphite production process.
PubMed: 37849699
DOI: 10.1039/d3ra06430a -
Aging Cell Nov 2023Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T-type Ca 3.2-RyR axis for extracellular Ca...
Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T-type Ca 3.2-RyR axis for extracellular Ca influx to trigger Ca sparks in vascular smooth muscle cells (VSMCs). We hypothesize that the administration of senolytics, which can selectively clear senescent cells, could preserve the caveolar Ca 3.2-RyR axis in aging VSMCs. In this study, 10-month-old mice were administered the senolytics cocktail consisting of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Using VSMCs from mouse mesenteric arteries, we found that Ca sparks were diminished after caveolae disruption by methyl-β-cyclodextrin (10 mM) in cells from D + Q treated but not vehicle-treated 14-month-old mice. D + Q treatment promoted the expression of Ca 3.2 in 14-month-old mesenteric arteries. Structural analysis using electron tomography and immunofluorescence staining revealed the remodeling of caveolae and co-localization of Ca 3.2-Cav-1 in D + Q treatment aged mesenteric arteries. In keeping with theoretical observations, Ca 3.2 channel inhibition by Ni (50 μM) suppressed Ca in VSMCs from the D + Q group, with no effect observed in vehicle-treated arteries. Our study provides evidence that age-related caveolar Ca 3.2-RyR axis malfunction can be alleviated by pharmaceutical intervention targeting cellular senescence. Our findings support the potential of senolytics for ameliorating age-associated cardiovascular disease.
Topics: Animals; Mice; Cardiovascular Diseases; Caveolae; Mesenteric Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Senotherapeutics
PubMed: 37837625
DOI: 10.1111/acel.14002 -
The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.Cancers Sep 2023Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and...
Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.
PubMed: 37835445
DOI: 10.3390/cancers15194751