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Vol. 15, Issue 1, 345-358, January 2004
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* European Molecular Biology Laboratory, D-69117 Heidelberg, Germany;
Central Electron Microscopy Facility, Universität Ulm, D-89069 Ulm, Germany; and
|| Institute of Cell Biology and Biosystems Technology, University of Rostock, D-18051 Rostock, Germany
Submitted May 27, 2003;
Revised August 25, 2003;
Accepted August 26, 2003
Monitoring Editor: Jean Gruenberg
Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.
Abbreviations used: BSA, bovine serum albumin; EE, early endosome(s); FESEM, field emission scanning electron microscope; HB, homogenization buffer; HRP, horseradish peroxidase; LatA, latrunculin A; LE, late endosome(s); LEO, late endocytic organelle(s); LBP, latex bead phagosome(s); PNS, membranes endocytic organelles within a crude postnuclear supernatant PMEE, 35 mM PIPES-KOH, 5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, pH 7.4; T
4, thymosin 4.
These authors contributed equally to this study.
Corresponding author. E-mail address: kjeken{at}bio.uio.no.
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