title: Cortical F-actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells.
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.
authors.affiliation: 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.
authors.forename: Cristina J
authors.initials: CJ
authors.lastname: Torregrosa-Hetland
authors.forename: José
authors.initials: J
authors.lastname: Villanueva
authors.forename: Virginia
authors.initials: V
authors.lastname: Garcia-Martínez
authors.forename: Giovanna
authors.initials: G
authors.lastname: Expósito-Romero
authors.forename: Maria Del Mar
authors.initials: Mdel M
authors.lastname: Francés
authors.forename: Luis M
authors.initials: LM
authors.lastname: Gutiérrez
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D005089:Exocytosis / Q000235:genetics* / Q000502:physiology; D018613:Microscopy, Confocal; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D015220:Calcium Channels; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25