Vesicle motion and fusion are altered in chromaffin cells with increased SNARE cluster dynamics.
The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a Delta9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 Delta9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 Delta9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population.
López
Inmaculada
I
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, Sant Joan d'Alacant, Alicante 03550, Spain, Spain.
Ortiz
Jose A
JA
Villanueva
José
J
Torres
Vanesa
V
Torregrosa-Hetland
Cristina J
CJ
del Mar Francés
Maria
M
Viniegra
Salvador
S
Gutiérrez
Luis M
LM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D005089:Exocytosis; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D011336:Probability; D011485:Protein Binding; D050600:SNARE Proteins / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*
D050600:SNARE Proteins; D002118:Calcium