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Vol. 12, Issue 1, 155-170, January 2001
*European Molecular Biology Laboratory, Heidelberg, Germany;
§Physik Department E22 Technische Universität
München, James Franck Strasse, 85747 Garching, Germany;
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 (Jahraus et al.,
1998). 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 (Defacque et al.,
2000a), 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.
Max-Planck-Institut für Medizinische Forschung c/o
Max-Planck-Institut für Zell Biologie, Ladenburg, Germany
Present addresses: Laboratory
of Molecular Biology, Wageningen Agricultural University, Dreijenlaan
3, 6703 HA Wageningen, The Netherlands
¶
Present address: Observatorie Océanologique
de Banyuls, Laboratoire Arajo, 3P44, 66651 Sur Mer, France.
@
Corresponding author. European Molecular Biology
Laboratory, Meyerhoffstrasse 1, Postfach 102209, 69012 Heidelberg,
Germany. Fax: 0049 6221 387306. Tel: 0049 6221 387508. E-mail:
griffiths{at}embl-heidelberg.de.
These authors contributed equally to this study.
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