Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures

Abstract

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.

Membrane Ruffling Requires Coordination between Type Iα Phosphatidylinositol Phosphate Kinase and Rac Signaling

“…2C). PIP 2 has also been detected at membrane ruffles with a phosphatidylinositol 4,5-bisphosphate-specific PH domain of PLC␦ (36,42). These data show that endogenous PIPKI␣ localizes to membrane ruffles and correlates with the localized synthesis of PIP 2 .…”

“…To verify that PIP 2 was produced at sites of actin remodeling, a GFP-PLC␦ PH domain was used to detect local changes in PIP 2 (36,44). The GFP-PLC␦ PH domain expression vector was coexpressed with PIPKI␣ by microinjection.…”

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…In principle, the observed puncta and patches could represent large clusters of lipids and proteins containing high local densities of PIP 2 on the cytoplasmic surface of the plasma membrane, such as clusters of lipid rafts (Laux et al, 2000;Raucher et al, 2000;Tall et al, 2000;Brown et al, 2001;Kwik et al, 2003;Huang et al, 2004;Golub and Caroni, 2005). Alternatively, the puncta and patches could represent geometric features arising from high local densities of membrane surface area (Colarusso and Spring, 2002;van Rheenen and Jalink, 2002).…”

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…Thus, by regulating the association of PLC-δ1 to membranes, the PH domain plays a key role in governing the activity of this enzyme. Moreso, the PH domain plays a key role in localizing it to substrate-containing membranes in cells [35].…”