Phosphoinositides regulate membrane-dependent actin assembly by latex bead phagosomes.
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 (). 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.
Defacque
Hélène
H
European Molecular Biology Laboratory, 69012 Heidelberg, Germany.
Bos
Evelyne
E
Garvalov
Boyan
B
Barret
Cécile
C
Roy
Christian
C
Mangeat
Paul
P
Shin
Hye-Won
HW
Rybin
Vladimir
V
Griffiths
Gareth
G
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007840:Latex / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007840:Latex; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D010738:Type C Phospholipases; D000099149:Ezrin