In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%+/-5.9% versus 57.9%+/-3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%+/-3.8% versus 24.0%+/-2.3%, P<0.0001; third round, 2.3%+/-1.6% versus 8.6%+/-1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200+/-23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1+/-10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.
Cells, Cultured ; Endocytosis ; Hippocampus ; cytology ; Neurons ; physiology ; Synaptic Vesicles ; physiology

Capsid Proteins ; metabolism ; Endocytosis ; physiology ; Virus Internalization ; Viruses ; metabolism

OBJECTIVETo investigate the protein expression of caveolin-1 in type II alveolar epithelial cells (A549) exposed to carbon black nanoparticles (CB NPs) and the role of caveolin in the endocytosis of CB NPs.

METHODSA549 cells were exposed to 0, 25, 50, 100, 200, and 400 µg/ml CB NPs for 24 h; then, trypan blue assay was applied to determine the cell viability. A549 cells were also exposed to 0, 25, 50, and 100 µg/ml CB NPs for 24 h, then, transmission electron microscopy (TEM) and flow cytometry were applied to observe the morphological change of cells and cellular side scatter (SSC), and Western blot was used to analyze the effect of CB NPs on the protein expression of caveolin-1. A549 cells were co-exposed to1 µg/ml filipin and 100 µg/ml CB NPs for 24 h, then, the cellular SSC was observed.

RESULTSCompared with controls, the A549 cells exposed to 200 and 400 µg/ml CB NPs had the cell viability decreased by 38.2% and 46.6%, respectively (P < 0.05), while those exposed to 25, 50, and 100 µg/ml CB NPs showed no significant decrease in cell vitality (P > 0.05). The protein expression of caveolin-1 was significantly higher in the cells exposed to 50 and 100 µg/ml CB NPs than in controls (P < 0.05). The TEM showed that plasmalemmal vesicles containing black particles were found in the cytoplasm of the cells exposed to 50 and 100 µg/ml CB NPs. The flow cytometry showed that the cellular SSC ratio increased from 1.007 to 1.331 as the dose of CB NPs rose within 0 ∼ 100 µg/ml and fell to 1.25 after the cells were co-exposed to1 µg/ml filipin and 100 µg/ml CB NPs.

CONCLUSIONCarbon black nanoparticles can be transferred into A549 cells by endocytosis, but caveolin-mediated endocytic pathway plays a minor role in this process.

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

Neurotransmission begins with neurotransmitter being released from synaptic vesicles. To achieve this function, synaptic vesicles endure the dynamic "release-recycle" process to maintain the function and structure of presynaptic terminal. Synaptic transmission starts with a single action potential that depolarizes axonal bouton, followed by an increase in the cytosolic calcium concentration that triggers the synaptic vesicle membrane fusion with presynaptic membrane to release neurotransmitter; then the vesicle membrane can be endocytosed for reusing afterwards. This process requires delicate regulation, intermediate steps and dynamic balances. Accumulating evidence showed that the release ability and mobility of synapses varies under different stimulations. Synaptic vesicle heterogeneity has been studied at molecular and cellular levels, hopefully leading to the identification of the relationships between structure and function and understanding how vesicle regulation affects synaptic transmission and plasticity. People are beginning to realize that different types of synapses show diverse presynaptic activities. The steady advances of technology studying synaptic vesicle recycling promote people's understanding of this field. In this review, we discuss the following three aspects of the research progresses on synaptic vesicle recycling: 1) presynaptic vesicle pools and recycling; 2) research progresses on the differences of glutamatergic and GABAergic presynaptic vesicle recycling mechanism and 3) comparison of the technologies used in studying presyanptic vesicle recycling and the latest progress in the technology development in this field.
Action Potentials ; Axons ; physiology ; Calcium ; physiology ; Endocytosis ; Humans ; Presynaptic Terminals ; physiology ; Synapses ; physiology ; Synaptic Transmission ; Synaptic Vesicles ; physiology

Cutaneous dendritic cells (DCs), Langerhans cells (LCs) and dermal dendritic cells (DDCs), are present in an immature state. The maturation of DCs is crucial for initiating an immune response. Since HLA-DM has an important role for antigen presentation, an increase in HLA-DM expression according to the maturation of blood monocyte-derived dendritic cells (MoDCs), which have similar characteristics with DDCs, is expected. Therefore, the aim of this study was to determine whether or not HLA-DM expression in MoDCs is related to maturation at each culture day (from day 0 to day 13) by flow cytometry. This was compared with the functional changes related to the maturation of MoDCs. MoDCs were generated by culturing human peripheral blood monocytes in the presence of GM-CSF and IL-4 for 7 days, which were followed by subsequent treatment with a cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6 and PGE2) for the maturation of MoDCs. The intracellular HLA-DM was expressed in the immature MoDC. A sudden 3 to 8 fold increase in the intracellular HLA-DM expression was observed after treatment with a cytokine cocktail. HLA-DM was weakly expressed on the surface of the immature MoDC, but it seemed to be decreased with maturation. This study indicated that the intracellular HLA-DM expression increased, but not on the MoDC surface during maturation. This was despite the fact that HLA-DM expression was noted not only on the surface but also in the intracellular in the MoDC.
Dendritic Cells/*immunology/physiology ; Endocytosis ; Flow Cytometry ; HLA-D Antigens/*analysis ; Human ; Monocytes/*physiology

Intersectin is an evolutionarily conserved multifunctional adaptor protein with multifunctional domains. These domains interact with components of the endocytic and exocytic pathways, such as the clathrin mediating synaptic vesicle recycling, the protein related to endocytosis via caveolae, the with-no-lysine kinases related to the regulation of renal outer medullar potassium, and the Cdc42 mediating exocytic pathway. Recently, the understanding of intersectin function in the pathogenesis of endocrine tumor and many neurodegenerative diseases such as Down syndrome, Alzheimer disease has been deepened. This article reviewed the structure and roles in endocytosis/exocytosis and diseases of intersectin.
Adaptor Proteins, Vesicular Transport ; physiology ; Endocytosis ; Exocytosis ; Humans ; Synaptic Vesicles ; physiology

The identification and use of molecular biomarkers have greatly improved the diagnosis and treatment of malignant tumors. However, a much deeper understanding of oncogenic proteins is needed for the benefit to cancer patients. The lipid raft marker proteins, flotillin-1 and flotillin-2, were first found in goldfish retinal ganglion cells during axon regeneration. They have since been found in a variety of cells, mainly on the inner surface of cell membranes, and not only act as a skeleton to provide a platform for protein-protein interactions, but also are involved in signal transduction, nerve regeneration, endocytosis, and lymphocyte activation. Previous studies have shown that flotillins are closely associated with tumor development, invasion, and metastasis. In this article, we review the functions of flotillins in relevant cell processes, their underlying mechanisms of action in a variety of tumors, and their potential applications to tumor molecular diagnosis and targeted therapy.
Animals ; Cell Differentiation ; Endocytosis ; Humans ; Membrane Proteins/physiology* ; Neoplasms/etiology* ; Nerve Regeneration

OBJECTIVE: To explore the interaction between arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles and endothelial cells, and to investigate mechanisms for endocytosis kinetics and endocytosis.
 METHODS: Human umbilical vein endothelial cells (HUVECs) were selected as the research model.Cellular uptake of HAP/Arg nanoparticles were observed by laser scanning confocal microscopy.Average fluorescence intensity of cells after ingestion with different concentrations of HAP/Arg nanoparticles were determined by flow cytometer and atomic force microscopy.
 RESULTS: The HAP/Arg nanoparticles with doped terbium existed in cytoplasm, and most of them distributed around the nucleus area after cellular uptake by HUVECs. Cellular uptake process of HAP/Arg nanoparticles in HUVECs was in a time and concentration dependent manner. 4 h and 50 mg/L was the best condition for uptake. HAP/Arg nanoparticles were easier to be up-taken into the cells than HAP nanoparticles without arginine functionalized.
 CONCLUSION HAP/Arg nanoparticles are internalized by HUVECs cells through an active transport and energy-dependent endocytosis process, and it is up-taken by cells mainly through caveolin-mediated endocytosis, but the clathrin-dependent endocytic pathway is also involved..
Arginine ; pharmacology ; Biological Transport, Active ; physiology ; Caveolins ; physiology ; Cells, Cultured ; Clathrin ; physiology ; Durapatite ; pharmacokinetics ; Endocytosis ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Nanoparticles ; metabolism

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.
Endocytosis/physiology* ; Histatins/pharmacology* ; Humans ; Membrane Proteins ; Mitochondria/metabolism* ; RNA, Small Interfering/pharmacology* ; Receptors, G-Protein-Coupled/metabolism* ; Receptors, sigma