Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

Abstract

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...

Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

“…However, in contrast to PI(4)P, PI(4,5)P 2 was not required for PLF, in line with a direct effector function for PI(4)P. This does, of course, not exclude a role of PI(4,5)P 2 in late phagosome maturation in vivo. For example, conversion of PI(4)P to PI(4,5)P 2 on phagosomes is required for polymerization of F-actin (21), which facilitates their fusion with lysosomes (42). This possible in vivo role of PI(4,5)P 2 would not be reconstituted in our cell-free PLF assay, which does not require actin polymerization (2).…”

“…Conversely, early subreactions of phagosome maturation (i.e., fusion of early phagosomes with early endosomes or late endosomes) did not require PI(4)P (this study). Both, PI(4)P and PI(4,5)P 2 have occasionally been detected in phagolysosomes (21) and lysosomes (19,20), yet specific functions have only been attributed to PI(4,5)P 2 . Even in other cellular processes, PI(4)P was long thought to be merely the precursor of PI(4,5)P 2 (41) and here, too, phagosomal PI(4)P was converted to PI(4,5)P 2 under fusion conditions.…”

“…Phagolysosomes and Lysosomes Contain PI(4)P and PI(3)P. PI(4)P is enriched in the Golgi complex and endoplasmic reticulum (18) and PI(3)P in early endosomes, yet both lipids have occasionally been observed in late endosomes (19,20) and phagosomes (21,22). Because PLF required free PI(4)P and PI(3)P, we tested using a reverse-phase high performance liquid chromatographymass spectrometry (RP-HPLC-MS) approach (23) whether our LBP and lysosome preparations do contain these PIPs: LBPs or lysosomes were incubated under fusion assay conditions and lipids were extracted, deacylated, and analyzed for monophosphorylated PIPs.…”

Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell Phagosomes

“…Likewise, flashing does not depend on the molecular features of the internalized object. Inhibition of PI-3 kinase does not block comet tail formation (Rozelle et al, 2000) nor in vitro assembly of actin on bead phagosomes (Defacque et al, 2002). Similarly, inhibition of PI 3-kinase had no effect on flashing.…”

Cholesterol-Binding Peptides and Phagocytosis

“…For example, Hope & Pike, 1996 showed that polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low-density Triton-insoluble membrane fraction that contains caveolin; this fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P 2 . Defacque et al, 2002 suggested that PI(4,5)P 2 may exist in raftlike microdomains on latex bead phagosomes after isolation. On activation of cells with agonists or addition of ATP to the in vitro actin assay, PIPs are rapidly synthesized and may aggregate laterally into larger raft domains.…”