Abstract:
:We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.
journal_name
Mol Biol Celljournal_title
Molecular biology of the cellauthors
Jahraus A,Egeberg M,Hinner B,Habermann A,Sackman E,Pralle A,Faulstich H,Rybin V,Defacque H,Griffiths Gdoi
10.1091/mbc.12.1.155keywords:
subject
Has Abstractpub_date
2001-01-01 00:00:00pages
155-70issue
1eissn
1059-1524issn
1939-4586journal_volume
12pub_type
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