Fusion between Phagosomes, Early and Late Endosomes: A Role for Actin in Fusion between Late, but Not Early Endocytic Organelles

Abstract

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 membranebound myosins, to facilitate fusion. INTRODUCTIONActin is essential for many cellular processes and dynamic polymerization/depolymerization of actin filaments is a critical property of all eukaryotic cells (Mitchison and Cramer, 1996;Carlier, 1998;Machesky and Insall, 1999;Small et al., 1999;Amann and Pollard, 2000). Much of the F-actin assembled in cells is intimately associated with membranes, especially the plasma membrane (Tilney, 1976;Hoglund et al., 1980;Lindberg et al., 1981;Carraway and Carraway, 1989;Dickinson and Purich, 2002). The first links between F-actin and membranes were reported by Tilney and Cardell (1970) who demonstrated the role of plasma membranes in actin nucleation, and Orci et al. (1972) who showed that cytochalasin can either stimulate or inhibit exocytic fusion, depending on the conditions. In addition to exocytosis (Bernstein et al., 1998;Lang et al., 2000;Bader et al., 2002), actin is now known to be involved in many other trafficking events, including phagocytosis (Swanson et al., 1999; Desjardin and Griffiths, 2003) and transcytosis (Durrbach et al., 2000). In the endocytic pathway, actin and myosins are essential for two different transport steps: clathrin-dependent internalization from the plasma membrane and transport to lysosomes (Durrbach et al., 1996;Riezman et al., 1996;Buss et al., 2001). More recently, actin has also been shown to be directly involved in homotypic fusion between yeast vacuoles (see DISCUSSION).Our interest in actin emerged from our analysis of an in vitro phagosome-endocytic organelle fusion assay that used latex bead phagosomes (LBP) and early endosomes (EE) and late endocytic org...

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).…”

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum

“…4). We hypothesize that fusion between the FV and the cytostome requires the nucleation of new actin microfilaments to stabilize this event, as was proposed in phagolysosome fusion and exocytic vesicle fusion with the plasma membrane (Kjeken et al, 2004;Sokac and Bement, 2006). Thus, JAS may be inhibiting step 6 (Fig.…”

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.…”