Hormone secretion is mediated by Ca(2+)-regulated exocytosis. The key step of this process consists of the merger of the vesicle and the plasma membranes, leading to the formation of a fusion pore. This is an aqueous channel through which molecules stored in the vesicle lumen exit into the extracellular space on stimulation. Here we studied the effect of sub-lethal dose of aluminium onprolactin secretion in isolated rat pituitary lactotrophs with an enzyme immunoassay and by monitoring electrophysiologically the interaction of a single vesicle with the plasma membrane in real time, by monitoring membrane capacitance. After 24-h exposure to sub-lethal AlCl(3) (30 muM), the secretionof prolactin was reduced by 14+/-8% and 46+/-11% under spontaneous and K(+)-stimulated conditions, respectively. The frequency of unitary exocytotic events, recorded by the high-resolution patch-clamp monitoring of membrane capacitance, a parameter linearly related to the membrane area, underspontaneous and stimulated conditions, was decreased in aluminium-treatedcells. Moreover, while the fusion pore dwell-time was increased in the presence of aluminium, the fusion pore conductance, a measure of fusion pore diameter, was reduced, both under spontaneous and stimulated conditions. These results suggest that sub-lethal aluminium concentrations reduce prolactin secretion downstream of the stimulus secretion coupling by decreasing the frequency of unitary exocytotic events and by stabilizing the fusion pore diameter to a value smaller than prolactin molecule, thus preventing its discharge into the extracellular space.
COBISS.SI-ID: 29371609
Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in real time, by transfecting them with different GDP- and GTP-locked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobilityin the presence of wild-type and Rab mutants. Dominant-negative and dominant-positive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L, Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement, and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N, and Rab5 Q79L. Our data indicate that changes in the GDP-GTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton. (c) 2012 Wiley Periodicals, Inc.
COBISS.SI-ID: 29424345
In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-gama (IFN-gama) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. Methods The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN- Ž activationwas determined immunocytochemically by using confocal microscopy. Timelapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-gama treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using Particle TR software. Results Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-Ž induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-Ž-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-gama-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-gama-induced increase in the mobility of MHC class II compartments is IF-dependent. Conclusions Since reactivity of astrocytes isa hallmark of many CNS pathologies, it is likely that the upregulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.
COBISS.SI-ID: 3272561
Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurospherecultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on theactivity of gamma-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin. STEM Cells2012;30:2320-2329.
COBISS.SI-ID: 30181849
In the brain, astrocytes signal to the neighboring cells by the release of chemical messengers (gliotransmitters) via regulated exocytosis. Recent studies uncovered a potential role of signaling lipids in modulation of exocytosis. Hence, we investigated whether sphingosine and the structural analog fingolimod/FTY720, a recently introduced therapeutic for multiple sclerosis, affect (i) intracellular vesicle mobility and (ii) vesicle cargo discharge from cultured rat astrocytes. Distinct types of vesicles, peptidergic, glutamatergic, and endosomes/lysosomes, were fluorescently prelabeled by cell transfection with plasmids encoding atrial natriuretic peptide tagged with mutant green fluorescent protein and vesicular glutamate transporter tagged with enhanced green fluorescent protein or by LysoTracker staining, respectively. The confocal and total internal reflection fluorescence microscopies were used to monitor vesicle mobility in the cytoplasm and near the basal plasma membrane, respectively. Sphingosine and FTY720, but not the membrane impermeable lipid analogs, dose-dependently attenuated vesicle mobility in the subcellular regions studied, and significantly inhibited stimulated exocytotic peptide and glutamate release. We conclude that in astrocytes, cell permeable sphingosine-like lipids affect regulated exocytosis by attenuating vesicle mobility, thereby preventing effective vesicle access/interaction with the plasma membrane docking/release sites.
COBISS.SI-ID: 30039513