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Int J Biochem Cell Biol. 2013 Mar;45(3):583-92. doi: 10.1016/j.biocel.2012.11.021. Epub 2012 Dec 7.

Cortical F-actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells.

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1
Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, Sant Joan d'Alacant, Carretera Nacional 332 s/n, 03550 Alicante, Spain.

Abstract

It has been proposed recently that the F-actin cytoskeleton organizes the relative disposition of the SNARE proteins and calcium channels that form part of the secretory machinery in chromaffin cells, a neurosecretory model. To test this idea, we used confocal microscopy do determine if DsRed-SNAP-25 microdomains, which define the final sites of exocytosis along with syntaxin-1, preferentially remain in contact with F-actin cortical structures labelled by lifeact-EGFP. A quantitative analysis showed that in cells over-expressing these constructs there is a preferential colocalization, rather than a random distribution of SNAP-25 patches. To analyze the possible interactions between these proteins, we designed FRET experiments and tested whether treatment with agents that affect F-actin mobility would modify SNAP-25 movement. The significant FRET efficiencies detected suggest that direct molecular interactions occur, whereas dynamic experiments using TIRFM revealed that attenuation of cortical F-actin movement clearly diminishes the mobility of SNAP-25 clusters. Taken together, these data can be explained by a model that associates components of the secretory machinery to the F-actin cortex through flexible links.

PMID:
23220175
DOI:
10.1016/j.biocel.2012.11.021
[Indexed for MEDLINE]
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