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© The Rockefeller University Press, 0021-9525/2000//519 $5.00
The Journal of Cell Biology, Volume 148, Number 3, , 2000 519-530

Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp

^a Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
^b Lawrence Berkeley National Laboratory, Berkeley, California 94720
^c Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720
^d Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195
Harvard Medical School, Department of Cell Biology, 240 Longwood Ave., Boston, MA 02115.(617) 432-3702(617) 432-3804

jack_taunton{at}hms.harvard.edu

We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA), and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

Key Words: actin assembly • fertilization • Wiskott-Aldrich • phorbol ester • Rho-GDI

© 2000 The Rockefeller University Press

Abbreviations used in this paper: BIM-I, bisindolylmaleimide I; GDI, guanine-nucleotide dissociation inhibitor; GFP, green fluorescent protein; PKC, protein kinase C; PNS, postnuclear supernatant.

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