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J. Biol. Chem., Vol. 276, Issue 38, 35652-35659, September 21, 2001

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Real-time Visualization of Processive Myosin 5a-mediated Vesicle Movement in Living Astrocytes^*,

Stanley J. Stachelek, Richard A. Tuft, Lawrence M. Lifschitz, Deborah M. Leonard, Alan P. Farwell^§, and Jack L. Leonard^¶

From the  Departments of Cellular and Molecular Physiology and ^§ Medicine, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green florescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

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