As the basic physiological function of synapses, vesicle cycling involves in many aspects of process. Among them, vesicle recycling is the basis of synaptic vesicle cycling. Studies show that clathrin mediated endocytosis is a major pathway of vesicle recycling, in which Dynamin plays an important role. Dynamin is a GTPases with molecular weight of 100 kD, which acts as "scissors" in the endocytosis, separating the clathrin coated pits from membrane. It has been found that Dynamin is associated with epilepsy, Alzheimer's disease, centronuclear myopathy, and several other neurological diseases. In this paper, we discussed the structure, function and regulation of Dynamin, and reviewed recent advance in the studies on Dynamin related diseases.
Clathrin ; physiology ; Coated Pits, Cell-Membrane ; physiology ; Dynamins ; physiology ; Endocytosis ; Humans ; Synapses ; physiology ; Synaptic Transmission ; Synaptic Vesicles ; physiology

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/metabolism

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Adaptor Proteins ; Animal ; Brain Chemistry ; Calpain/*metabolism ; Carrier Proteins ; Caspases/*metabolism ; Cattle ; Clathrin/*metabolism ; Coated Pits, Cell-Membrane/*metabolism ; Hydrolysis ; Membrane Proteins ; Molecular Weight ; Nerve Tissue Proteins/chemistry/*isolation & purification/metabolism ; Neurons/*chemistry ; Protein Binding ; Protein Conformation ; Recombinant Proteins/chemistry/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

Caveolin-2, a protein about 20 kD, is a major component of the inner surface of caveolae, small invaginations of the plasma membrane. Similar with caveolin-1 and caveolin-3, it serves as a protein marker of caveolae. Caveolin-1 and -2 are located next to each other at 7q31.1 on human chromosome, the proteins encoded are co-localized and form a stable hetero-oligomeric complex, distributing similarly in tissue and cultured cells. Caveolin-3 is located on different chromosomes but confirmed to interact with caveolin-2. Caveolin-2 is similar to caveolin-1 in many respects but differs from the latter in functional domains, especially in G-protein binding domain and caveolin scaffolding domain. The mRNAs of both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during differentiation of 3T3-L1 cells to adipocytes. Caveolin-2-deficient mice demonstrate clear pulmonary defects, with little or no change in caveolin-1 expression and caveolae formation, suggesting that caveolin-2 plays a selective role in lung functions. Caveolin-2 is also involved in lipid metabolism and human cancers.
Biomarkers ; metabolism ; Caveolae ; metabolism ; Caveolin 2 ; genetics ; metabolism ; Chromosomes, Human, Pair 7 ; Humans

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

BACKGROUND AND OBJECTIVES: The caveolin is known as a mediator of cell death or survival of injured cell and inhibitor of various signaling pathways. We examined expression of caveolin-1 involved by protein kinase A(PKA) signaling pathway in the differentiated mouse vestibular cell line(UB/UE-1) after gentamicin toxicity. MATERIALS AND METHOD: We observed caveolae in the vestibular hair cell of healthy guinea pig through electron microscope. UB/UE-1 cells were cultured at 95% CO2, 5% O2, 33DegreeC for 2days and at 95% CO2, 5% O2, 39DegreeC for 24 hours for differentiation. Cells were treated with 1 mM of gentamicin, 0.02 mM H89 (PKA inhibitor), and then incubated for 24 hours. Caveolin-1 expression was examined by western blot and PKA activity by PepTag? assay. RESULTS: Caveolae were observed in the vestibular hair cell of healthy guinea pig by electron microscope. Caveolin-1 was expressed spontaneously in differentiated UB/UE-1 cells and increased after gentamicin treatment. PKA is overactivated by gentamicin treatment. The gentamicin induced caveolin-1 expression and PKA overactivation was inhibited by H89. CONCLUSION: Our results indicate that gentamicin induced caveolin-1 expression is mediated by PKA signaling pathway. We conclude that the caveolae/caveolin through a PKA signaling pathway is the important mechanism of gentamicin induced ototoxicity.
Animals ; Blotting, Western ; Caveolae ; Caveolin 1* ; Cell Death ; Cell Line* ; Gentamicins* ; Guinea Pigs ; Hair Cells, Vestibular ; Mice ; Protein Kinases

BACKGROUND: Caveolin-1 is a principal component of caveolae membranes in vivo. Although expression of caveolae structure and expression of caveolin family, caveolin-1, -2 and -3, was known in chondrocytes, the functional role of caveolae and caveolins in chondrocytes remains unknown. In this study, we investigated the role of caveolin-1 in articular chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. Caveolin-1 cDNA was transfected to articular chondrocytes using LipofectaminePLUS. The cyclooxygenase-2 (COX-2) expression levels were determined by immunoblot analysis, immunostaining, immunohistochemistry, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. RESULTS: Ectopic expression of caveolin-1 induced COX-2 expression and activity, as indicated by immunoblot analysis and PGE2 assay. And also, overexpression of caveolin-1 stimulated activation of p38 kinase and ERK-1/ -2. Inhibition of p38 kinase and ERK-1/-2 with SB203580 and PD98059, respectively, led to a dose-dependent decrease COX-2 expression and PGE2 production in caveolin-1-transfected cells. CONCLUSION: Taken together, our data suggest that ectopic expression of caveolin-1 contributes to the expression and activity of COX-2 in articular chondrocytes through MAP kinase pathway.
Cartilage ; Caveolae ; Caveolin 1* ; Caveolins ; Chondrocytes* ; Cyclooxygenase 2 ; Digestion ; Dinoprostone ; DNA, Complementary ; Humans ; Immunohistochemistry ; Membranes ; Phosphotransferases* ; Rabbits

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

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