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Ca-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

Celica Biomedical Center, Proletarska 4, 1000 Ljubljana, Slovenia.
Experimental Cell Research (Impact Factor: 3.37). 12/2007; 313(18):3809-18. DOI: 10.1016/j.yexcr.2007.08.020
Source: PubMed

ABSTRACT 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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    • "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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    Glia 06/2013; 61(6). DOI:10.1002/glia.22485 · 6.03 Impact Factor
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    • "To test that the vesicle mobility recorded in cultured astrocytes also occurs in vivo, vesicle dynamics was studied in brain slices in which cell-to-cell contacts are preserved and tissue architecture is closer to the one present in the brain (Potokar et al., 2009). The mobility of specifically labelled recycling glutamatergic and peptidergic vesicles (immunopositive for VGLUT1 or ANP respectively) was similar to that in cultured astrocytes (Stenovec et al., 2007; Potokar et al., 2008). "
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    ASN Neuro 02/2012; 4(2). DOI:10.1042/AN20110061 · 4.44 Impact Factor
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    • "TIRFM were estimated to be ∼40 nm based on comparison to 40 nm diameter fluorescent beads ( Cali et al . 2008 ; Marchaland et al . 2008 ) . Glutamatergic vesicles labelled by capturing an extracellular antibody against VGLUT1 in a Ca 2+ - dependent manner while recycling with the plasma membrane are electron - lucent and have diameters of ∼50 nm ( Stenovec et al . 2007 ) . Vesicles found within gliosomes , subcellular components of astrocytic processes isolated from brain by fractionation ( Nakamura et al . 1993 ) , express synaptobrevin 2 and VGLUT1 and measure ∼30 nm in diameter ( Stigliani et al . 2006 ) ."
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    The Journal of Physiology 07/2011; 589(Pt 17):4271-300. DOI:10.1113/jphysiol.2011.210435 · 4.54 Impact Factor
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