Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation.
Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to its actin-based motility. In Listeria-infected PtK2 cells Akt-pleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C delta (PLC delta)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P3 by expression of Akt-PH-GFP and available PtdIns(4,5)P2 by expression of PLC delta-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC delta-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P3 production, indicating a central role for PI 3-kinases in Listeria intracellular actin-based motility and filopod formation.
Sidhu
Gurjit
G
Department of Medicine, Division of Infectious Diseases, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Li
Wei
W
Laryngakis
Nicholas
N
Bishai
Ellen
E
Balla
Tamas
T
Southwick
Frederick
F
D000199:Actins / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004173:Dipodomys; D004285:Dogs; D007527:Isoenzymes / Q000502:physiology; D008089:Listeria monocytogenes / Q000502:physiology*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology; D018129:Phosphatidylinositol Phosphates / Q000502:physiology; D054803:Phospholipase C delta; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D010738:Type C Phospholipases / Q000502:physiology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002867:Chromones; D007527:Isoenzymes; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D054803:Phospholipase C delta; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D013844:Thiazoles; D053778:Thiazolidines; D010738:Type C Phospholipases; C060974:phosphatidylinositol 3,4,5-triphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C037067:latrunculin A