The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to 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 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.

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...

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P 2 -binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P 2 , and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P 2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P 2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P 2 -binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4-and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. INTRODUCTIONA significant fraction of the de novo nucleation of actin in cells occurs on the cytoplasmic surface of eukaryotic cell membranes, especially the plasma membrane (Tilney, 1976;Carraway and Carraway, 1989;Small et al., 1995;Mitchison and Cramer, 1996), and a role for phosphoinositides in this elusive process has been widely discussed (Divecha and Irvine, 1995;Martin, 1998;Caroni, 2001). However, the precise function of these lipids is still not clear and is likely to be quite complicated. In several cellular systems that show rapid actin assembly in response to extracellular ligands, synthesis of phosphoinositides, especially phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], and in some cases phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ], coincides precisely with the transient burst of actin assembly (Eberle et al., 1990;Dobos et al., 1992;Apgar, 1995;Hartwig et al., 1995;Gachet et al., 1997). In addition, overexpression of phosphatidylinositol-4-phosphate [PI(4)P] 5-kinase in cells leads to a significant polymerization of actin (Shibasaki et al., 1997). However, in other systems, the synthesis of PI(4,5)P 2 as well as PI(3,4,5)P 3 coincides more with actin depolymerization, after a transient assembly of F-actin (Apgar, 1995;Gratacap et al., 1998).One important clue to the functions of phosphoinositides in actin assembly/disassembly is that these lipids can bind in vitro to an increasing number of actin-binding proteins (ABPs). Interestingly, two different...

Vitamin D and retinoids cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic U937 leukemia cells. In the present work, we investigated the role of TGF-beta as an endogenous mediator of this process. We found that the TGF-beta1 precursor began to accumulate in cell culture supernatants soon after the addition of 1alpha,25 dihydroxyvitamin D3 (VD) and retinoids. We used neutralizing antibodies (AbTGF-beta) and antisense oligonucleotide (AS Oligo) to inhibit its possible effects. Our data demonstrated that AbTGF-beta partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to the chemotactic peptide fMLP, and lysozyme secretion. AS Oligo was also inhibitory, and the effects of AS Oligo and AbTGF-beta were cumulative. Cell growth inhibition induced by VD and retinoids was completely reversed, and differentiation was reduced by about 75% when both inhibitors were associated. Time course experiments based on the delayed addition of AbTGF-beta and AS Oligo showed that TGF-beta1 was required for cell differentiation 24 h after the addition of inducers. Studies on TGF-beta receptors revealed that, while the expression of type II receptor was stable, the level of type I TGF-beta receptor mRNA and the expression of the protein began to decline early during the differentiation process. As a whole, these results support the notion that an autocrine TGF-beta pathway, activated by VD and retinoids in U937 cells, is involved in the early steps of the process leading to cell growth arrest and differentiation.