Ca-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

Celica Biomedical Center, Proletarska 4, 1000 Ljubljana, Slovenia.
Experimental Cell Research (Impact Factor: 3.25). 12/2007; 313(18):3809-18. DOI: 10.1016/j.yexcr.2007.08.020
Source: PubMed


Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca(2+)-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca(2+) level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca(2+)-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.

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    • "This is in line with WNV vesicles, which have 100 nm in diameter until they start to fuse and become approximately the size of 500 nm [2]. A minor portion of DiD-TBEV particles (6% at 2 h p.i., 5% at 4 h p.i. and 20% at 18 h p.i.) was even comparable in apparent size to peptidergic and glutamatergic vesicles monitored in astrocytes (50–100 nm, [38]; [56]). The rest of TBEV-loaded vesicles were between 500 or 600 nm (diameter), which is in the range of late endosomes and lysosomes [54], [55]. "
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    ABSTRACT: Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention.
    PLoS ONE 01/2014; 9(1):e86219. DOI:10.1371/journal.pone.0086219 · 3.23 Impact Factor
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    • "Increases in [Ca 2þ ] i Decrease the Mobility of AQP4e Vesicles An increase in [Ca 2þ ] i differentially affects vesicle mobility in rat astrocytes and appears to be vesicle type specific (Potokar et al., 2007, 2008, 2010; Stenovec et al., 2007). The speed of AQP4e vesicles was unaffected by increases in [Ca 2þ ] i , as observed in vesicles on their way to the plasma membrane (Potokar et al., 2005, 2007). "
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    ABSTRACT: Aquaporin 4 (AQP4) is the predominant water channel in the brain, expressed mainly in astrocytes and involved in water transport in physiologic and pathologic conditions. Besides the classical isoforms M1 (a) and M23 (c), additional ones may be present at the plasma membrane, such as the recently described AQP4b, d, e, and f. Water permeability regulation by AQP4 isoforms may involve several processes, such as channel conformational changes, the extent and arrangement of channels at the plasma membrane, and the dynamics of channel trafficking to/from the plasma membrane. To test whether vesicular trafficking affects the abundance of AQP4 channel at the plasma membrane, we studied the subcellular localization of AQP4 in correlation with vesicle mobility of AQP4e, one of the newly discovered AQP4 isoforms. In cultured rat astrocytes, recombinant AQP4e acquired plasma membrane localization, which resembled that of the antibody labeled endogenous AQP4 localization. Under conditions mimicking reactivation of astrocytes (increase in cytosolic cAMP) and brain edema, an increase in the AQP4 plasma membrane localization was observed. The cytoskeleton remained unaffected with the exception of rearranged actin filaments in the model of reactive astrocytes and vimentin meshwork depolymerization in hypoosmotic conditions. AQP4e vesicle mobility correlated with changes in the plasma membrane localization of AQP4 in all stimulated conditions. Hypoosmotic stimulation triggered a transient reduction in AQP4e vesicle mobility mirrored by the transient changes in AQP4 plasma membrane localization. We suggest that regulation of AQP4 surface expression in pathologic conditions is associated with the mobility of AQP4-carrying vesicles.
    Glia 06/2013; 61(6). DOI:10.1002/glia.22485 · 6.03 Impact Factor
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    • "Next we used dextran labeling and time-lapse confocal imaging to study the mobility of late endosomes/lysosomes in living astrocytes. To determine whether vesicle mobility in IFN-γ-treated cells is dependent on cytosolic Ca2+ levels, we exposed the cells to 1 mM ATP to increase cytosolic [Ca2+[33,34]. Cells were imaged every 2 s for 30 s (15 frames) to determine the maximal displacement (MD) and the track length (TL) of vesicles. "
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    ABSTRACT: In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) 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. The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ 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-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation 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.
    Journal of Neuroinflammation 06/2012; 9(1):144. DOI:10.1186/1742-2094-9-144 · 5.41 Impact Factor
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