Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.
Tall
E G
EG
Department of Physiology and Biophysics, State University of New York at Stony Brook, 11794, USA.
Spector
I
I
Pentyala
S N
SN
Bitter
I
I
Rebecchi
M J
MJ
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D007150:Immunohistochemistry; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins