Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Caveolin-1 (Cav-1) is a trans-membrane protein that is a major component of the caveolae structure on the plasma membrane. Cav-1 is involved in the regulation of various cellular processes, including cell growth, differentiation, endocytosis, and in particular it has been implied in cellular senescence. Here we review current knowledge about Cav-1 in cellular signaling and discuss the role of Cav-1 in aging-related diseases.
Caveolae ; Caveolin 1 ; Cell Aging ; Cell Membrane ; Endocytosis

PURPOSE: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. METHODS: We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. RESULTS: The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. CONCLUSION: MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.
Apoptosis ; Caveolin 1 ; Cell Adhesion ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; Cell Proliferation ; Cell Survival ; Cholesterol ; Detergents ; Fibronectins ; Filipin ; Flow Cytometry ; In Vitro Techniques ; Membrane Microdomains ; Membranes ; Methods ; Neoplasm Metastasis ; Nystatin ; Protein-Tyrosine Kinases ; Triple Negative Breast Neoplasms* ; Vitronectin

Galectin-4, a tandem repeat member of the β-galactoside-binding proteins, possesses two carbohydrate-recognition domains (CRD) in a single peptide chain. This lectin is mostly expressed in epithelial cells of the intestinal tract and secreted to the extracellular. The two domains have 40% similarity in amino acid sequence, but distinctly binding to various ligands. Just because the two domains bind to different ligands simultaneously, galectin-4 can be a crosslinker and crucial regulator in a large number of biological processes. Recent evidence shows that galectin-4 plays an important role in lipid raft stabilization, protein apical trafficking, cell adhesion, wound healing, intestinal inflammation, tumor progression, etc. This article reviews the physiological and pathological features of galectin-4 and its important role in such processes.
Animals ; Axons ; metabolism ; Endocytosis ; Galectin 4 ; blood ; genetics ; metabolism ; Humans ; Inflammatory Bowel Diseases ; metabolism ; pathology ; Membrane Microdomains ; metabolism ; Neoplasms ; metabolism ; pathology ; Neurons ; metabolism ; Wound Healing

This study investigated a nano drug delivery system built by one sort of modified trimethyl chitosan (TMC). The TMC was modified by cRGDyk, ligand of integrin receptor avβ3. Single factor screening was used to optimize the prescription in which the particle sizes of TMC nanoparticle (TMC NPs) and cRGDyk modified TMC nanoparticle (C-TMC NPs) were (240.3 ± 4.2) nm and (259.5 ± 3.3) nm. Electric potential of those two nanoparticles were (33.5 ± 0.8) mV and (25.7 ± 1.6) mV. Encapsulation efficiencies were (76.0 ± 2.2) % and (74.4 ± 2.0) %. Drug loading efficacies were (50.1 ± 2.1) % and (26.1 ± 1.0) %. Then the cellular uptake, uptake mechanism and transport efficacy of TMC NPs and C-TMC NPs were investigated using Caco-2 cell line. The uptake rate and accumulating drug transit dose of C-TMC NPs were 1.98 and 2.84 times higher than TMC NPs, separately. Mechanism investigations revealed that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis were involved in the intercellular uptake of both TMC NPs and C-TMC NPs. What is more, free cRGDyk could remarkably inhibit the uptake of C-TMC NPs.
Biological Transport ; Caco-2 Cells ; Caveolae ; Chitosan ; chemistry ; Clathrin ; Endocytosis ; Humans ; Integrin alphaVbeta3 ; chemistry ; Nanoparticles ; Particle Size ; Pinocytosis

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.
Actins ; Caveolae ; Caveolin 1 ; Clathrin* ; Endothelial Cells* ; GTP Phosphohydrolases ; Human Umbilical Vein Endothelial Cells ; Humans ; Microtubules ; Phosphatidylinositol 3-Kinase ; Phosphotransferases ; Polymerization ; Polymers ; Porphyromonas gingivalis* ; RNA, Small Interfering ; src-Family Kinases

The release of microvesicles(MV) is one of the critical mechanisms underlying the angiogenesis-promoting activity of mesenchymal stem cells(MSC). This study was aimed to explore the appropriate condition under which MSC releases MV. Bone marrow samples from 5 healthy adults were collected, and MSC were isolated, culture-expanded and identified. MSC at passage 5 were suspended in medium without or medium with 10% fetal(FCS) calf serum and seeded into culture dishes. The culture was separately maintained in hypoxia (1% oxygen) or normoxia (around 20% oxygen), and 20 dishes of cells (2×10(6)/dish) were used for each group. The supernatants were collected for MV harvesting. The cell number was counted with trypan blue exclusion test and the protein contents in the MV were determined. MV were identified by observation under an electron microscope. The surface markers on MV were analyzed by flow cytometry. MTT test was performed to observe the pro-proliferative activity of MV that were added into the culture of human umbilical cord vein endothelial cells at a concentration of 10 µg/ml. The results showed that the majority of MV released by MSC were with diameters of less than 100 nm, and MV took the featured membrane-like structure with a hypodense center. They expressed CD29, CD44, CD73 and CD105, while they were negative for CD31 and CD45. The increase multiples of the adherent trypan blue-resistant cells cultured in normoxia with serum, in normoxia without serum, in hypoxia with serum and hypoxia in the absence of serum were 4.05 ± 0.73, 1.77 ± 0.48, 5.80 ± 0.65 and 3.69 ± 0.85 respectively, and the estimated protein contents per 10(8) cells were 463.48 ± 138.74 µg, 1604.07 ± 445.28 µg, 2389.64 ± 476.75 µg and 3141.18 ± 353.01 µg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial cells more efficiently than those from cells in normoxia. It is concluded that hypoxia can enhance the release of microvesicles from MSC, and cultivation of MSC in hypoxia and medium without serum may provide an appropriate condition for MV harvesting.
Bone Marrow Cells ; cytology ; metabolism ; Caveolae ; metabolism ; Cell-Derived Microparticles ; metabolism ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism

OBJECTIVETo reveal the transmembrane signal pathway participating in regulating neuron functions of treating Alzheimer's disease (AD) by acupuncture.

METHODSSAMP8 mice was used for AD animal model. The effect of acupuncture method for qi benefiting, blood regulating, health supporting, and root strengthening on the amount and varieties of transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice was detected using HPLC MS/MS proteomics method.

RESULTSCompared with the control group, acupuncture increased 39 transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice, of them, 14 belonged to ionophorous protein, 8 to G protein, 8 to transmembrane signal receptor, and 9 to kinase protein. Totally 3 main cell signal pathways were involved, including G-protein-coupled receptors signal, enzyme linked receptor signal, and ion-channel mediated signal. Compared with the sham-acupuncture group, acupuncture resulted in significant increase of kinase signal protein amount. From the aspect of functions, they were dominant in regulating synapse functions relevant to cytoskeleton and secreting neurotransmitters.

CONCLUSIONThe cell biological mechanism for treating AD by acupuncture might be achieved by improving synapse functions and promoting the secretion of neurotransmitters through transmembrane signal transduction, thus improving cognitive function of AD patients.

Acupuncture Therapy ; Alzheimer Disease ; metabolism ; Animals ; Disease Models, Animal ; Female ; Male ; Membrane Microdomains ; metabolism ; Mice ; Proteomics ; Signal Transduction ; Tandem Mass Spectrometry

Caveolae are specialized lipid rafts that form flask-shaped invaginations of the plasma membrane. Many researches show that caveolae are involved in cell signaling and transport. Caveolin-1 is the major coat protein essential for the formation of caveolae. Recently, several reports indicated that the other caveolae-associated proteins, Cavins, are required for caveola formation and organization. It's worth noting that Cavin-1 could cooperate with Caveolin-1 to accommodate the structural integrity and function of caveolae. Here, we reviewed that the relationship between Cavins and Caveolins and explore the role of them in regulating caveolae.
Animals ; Caveolae ; physiology ; Caveolin 1 ; metabolism ; physiology ; Caveolins ; metabolism ; physiology ; Humans ; Membrane Proteins ; metabolism ; physiology ; RNA-Binding Proteins ; metabolism ; physiology

Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma (ATL), infects over 20 million people worldwide. About 1 million of HTLV-1-infected patients develop ATL, a highly aggressive non-Hodgkin's lymphoma without an effective therapy. The pX region of the HTLV-1 viral genome encodes an oncogenic protein, Tax, which plays a central role in transforming CD4+ T lymphocytes by deregulating oncogenic signaling pathways and promoting cell cycle progression. Expression of Tax following viral entry is critical for promoting survival and proliferation of human T cells and is required for initiation of oncogenesis. Tax exhibits diverse functions in host cells, and this oncoprotein primarily targets IκB kinase complex in the cytoplasm, resulting in persistent activation of NF-κB and upregulation of its responsive gene expressions that are crucial for T cell survival and cell cycle progression. We here review recent advances for the pathological roles of Tax in modulating IκB kinase activity. We also discuss our recent observation that Tax connects the IκB kinase complex to autophagy pathways. Understanding Tax-mediated pathogenesis will provide insights into development of new therapeutics in controlling HTLV-1-associated diseases.
Autophagy ; CD4-Positive T-Lymphocytes ; metabolism ; virology ; Cell Cycle ; Cell Transformation, Neoplastic ; genetics ; Gene Expression Regulation, Neoplastic ; Gene Products, tax ; genetics ; metabolism ; Human T-lymphotropic virus 1 ; physiology ; Humans ; I-kappa B Kinase ; genetics ; metabolism ; Leukemia-Lymphoma, Adult T-Cell ; genetics ; metabolism ; virology ; Membrane Microdomains ; metabolism ; virology ; NF-kappa B ; genetics ; metabolism ; Protein Binding ; Signal Transduction ; genetics