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J. Biol. Chem., Vol. 279, Issue 26, 27450-27457, June 25, 2004

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New Roles of Myosin II during Vesicle Transport and Fusion in Chromaffin Cells^*

Patricia Ñeco, Daniel Giner¶, Salvador Viniegra, Ricardo Borges||, Alvaro Villarroel**, and Luis M. Gutiérrez

From the Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, E-03550 Alicante, Spain, the ^||Unidad de Farmacología, Facultad de Medicina Universidad de la Laguna, E-38071 Tenerife, Spain, and the ^**Instituto Cajal, CSIC, E28002 Madrid, Spain

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K⁺ or BaCl₂, as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.

Received for publication, October 20, 2003 , and in revised form, March 16, 2004.

^* This work was supported in part by Spanish Ministry of Science and Technology Grant BMC2002-00845 and Generalitat Valenciana Grant GRUPOS03/040. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a fellowship from the Generalitat Valenciana.

^¶ Recipient of a fellowship from the Spanish Ministry of Science and Technology.

To whom correspondence should be addressed: Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain. Tel.: 34-96-5919563; Fax: 34-96-5919484; E-mail: luisguti{at}umh.es.

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