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The Journal of Neuroscience, September 26, 2007, 27(39):10636-10645; doi:10.1523/JNEUROSCI.1228-07.2007

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Cellular/Molecular
Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

Claire Desnos,^1 * Sébastien Huet,^1 * Isabelle Fanget,¹ Catherine Chapuis,¹ Caroline Böttiger,¹ Victor Racine,² Jean-Baptiste Sibarita,² Jean-Pierre Henry,¹ and François Darchen¹

¹Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1929, Université Paris 7 Denis Diderot, 75005 Paris, France, and ²Institut Curie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, 75248 Paris Cedex 05, France

Correspondence should be addressed to Dr. François Darchen, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1929, 13 rue Pierre et Marie Curie, 75005 Paris, France. Email: francois.darchen{at}ibpc.fr

Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, D_xy, was observed. Almost immobile SGs (D_xy < 5 x 10^–4 µm² · s^–1) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a D_xy below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a D_xy below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.

Key words: myosin V; actin; exocytosis; docking; secretory vesicle; TIRFM

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Received March 19, 2007; revised Aug. 16, 2007; accepted Aug. 16, 2007.

Correspondence should be addressed to Dr. François Darchen, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1929, 13 rue Pierre et Marie Curie, 75005 Paris, France. Email: francois.darchen{at}ibpc.fr

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