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Originally published as MBC in Press, 10.1091/mbc.E02-09-0626 on February 21, 2003

Vol. 14, Issue 5, 2016-2028, May 2003

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Dynamin Regulates Focal Exocytosis in Phagocytosing Macrophages

Anke Di, Deborah J. Nelson, Vytautas Bindokas, Mary E. Brown, Frances Libunao, and H. Clive Palfrey *

Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637

Submitted September 26, 2002; Revised December 16, 2002; Accepted January 30, 2003
Monitoring Editor: Vivek Malhotra

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase{bullet}amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein{bullet}dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E02-09-0626. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-09-0626.

Online version of this article contains video material for some figures. Online version available at www.molbiolcell.org.

* Corresponding author. E-mail address: hpalfrey{at}midway.uchicago.edu.




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