ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

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 (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.

Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

“…An actin-remodeling requirement has been implicated in several membrane fusion systems (Stamnes, 2002;Eitzen, 2003), but the precise role of actin during fusion is still a matter of debate. Current data clearly support roles for F-actin polymerization (Jahraus et al, 2001;Kjeken et al, 2004), depolymerization (Vitale et al, 1991;Muallem et al, 1995), or both (Bernstein et al, 1998;Eitzen et al, 2002). Signaling pathways exist on membranes of isolated organelles that control actin assembly (Defacque et al, 2000;Eitzen et al, 2002).…”

Annexin A8 Regulates Late Endosome Organization and Function

“…Pharmacological disruption of the actin cytoskeleton impairs receptor degradation in several cellular models (van Deurs et al, 1995;Durrbach et al, 1996) and relocalizes late endosomes to the cell periphery. Perinuclear aggregation and both fusion of late endosomes with each other or with lysosomes, critically depend on actin (van Deurs et al, 1995;Jahraus et al, 2001;Kjeken et al, 2004). Likewise, homotypic vacuole fusion in yeast that is analogous to mammalian late endosomal/lysosomal fusion requires actin (Eitzen et al, 2002).…”

“…Likewise, homotypic vacuole fusion in yeast that is analogous to mammalian late endosomal/lysosomal fusion requires actin (Eitzen et al, 2002). Further emphasizing a link to the actin cytoskeleton, late endosomes/lysosomes can nucleate F-actin on their membranes (Taunton et al, 2000;Jahraus et al, 2001;Kjeken et al, 2004), thereby presumably assembling actin filaments to facilitate fusion with neighboring organelles. Based on our results, we propose that annexin A8 present on the limiting membrane of late endosomes might link late endosomes to the actin cytoskeleton through either direct interaction or through the organization of specific membrane/ actin attachment sites.…”

“…Altering annexin A8 levels did not change the overall appearance of the actin cytoskeleton (data not shown), arguing against a general role of the protein in the organization of the cellular actin network. Because annexin A8 specifically affects properties of late endosomes and because many observations link the actin cytoskeleton to late endosomal distribution, morphology, trafficking, and fusion (Jahraus et al, 2001;Eitzen et al, 2002;Kjeken et al, 2004), we next analyzed whether annexin A8 regulates the association of actin with late endosomes.…”

The unconventional myosin-VIIa associates with lysosomes

“…This process is initiated by the sequential fusion of endosomes, lysosomes, and melanosomes with the ROS containing phagosomes, creating the so-called phagolysosome (reviewed in Vieira et al 2002). Actin may facilitate fusion between endocytic compartments and existing phagosomes, as judged by studies that depolymerize actin (Jahraus et al 2001). A class I myosin has been implicated in membrane trafficking occurring between endosomes and lysosomes (Raposo et al 1999), so it was proposed that myosin-VIIa may play a similar role in organelle function in RPE.…”