ATP-dependent membrane assembly of F-actin facilitates membrane fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macr ophage cytosol (Jahraus et al., 1998). Here, we show that different reagent s affecting the actin cytoskeleton can either inhibit or stimulate this fus ion process. Because the membranes of purified phagosomes can assemble F-ac tin de novo from pure actin with ATP (Defacque et al., 2000a), we focused h ere on the ability of membranes to nucleate actin in the presence of J774 c ytosolic extracts. For this, we used F-actin sedimentation, pyrene actin as says, and torsional rheometry, a biophysical approach that could provide ki netic information on actin polymerization and gel formation. We make two ma jor conclusions. First, under our standard in vitro conditions (4 mg/ml cyt osol and 1 mM ATP), the presence of membranes actively catalyzed the assemb ly of cytosolic F-actin, which assembled into highly viscoelastic gels. A m odel is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP de pletion than under high-ATP conditions, even in the absence of membranes; w e discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.