PI(4,5)P2-dependent microdomain assemblies capture microtubules to promote and control leading edge motility

Abstract

The lipid second messenger PI(4,5)P2 modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P2-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P2 directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility. The accumulation of PI(4,5)P2 at rafts, together with Cdc42, promotes patch assembly through N-WASP. The patches exhibit locally regulated PI(4,5)P2 turnover and reduced diffusion-mediated exchange with their environment. Patches capture microtubules (MTs) through patch IQGAP1, to stabilize MTs at the leading edge. Captured MTs in turn deliver PKA to patches to promote patch clustering through further PI(4,5)P2 accumulation in response to cAMP. Patch clustering restricts, spatially confines, and polarizes protrusive motility. Thus, PI(4,5)P2-dependent raft-rich patches enhance local signaling for motility, and their assembly into clusters is regulated through captured MTs and PKA, coupling local regulation of motility to cell polarity, and organization.

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…[21][22][23] In agreement with previous observations, 34 we observed that such probe almost exclusively localizes at the plasma membrane where it distributes in discrete microdomains partially colocalizing with lipid rafts (R.G., unpublished data, December 2005). The interaction of NK cells with target cells was followed by a rapid consumption of PIP2 in the area of cytolytic synapse, whereas in contact-free membrane areas or in noncytolytic interactions we did not observe major PIP2 variations, thus ruling out rapid membrane turnover as the process responsible for the localized loss of membrane-associated GFP-PH probe.…”

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

“…Using two chemically distinct dyes to uniformly stain the plasma membrane, we find that the puncta and patches are areas containing increased membrane surface area (folds and microvilli), consistent with two other reports (Colarusso and Spring, 2002;van Rheenen and Jalink, 2002) and are not sites of PIP 2 and/or lipid raft enrichment. Our observations, however, may be cell-type specific, because other groups using similar membrane dyes have concluded that PLC␦ 1 PH domain and mGAP43 accumulation in localized regions of the plasma membrane is not caused by locally increased membrane area but instead is attributed to rafts (Huang et al, 2004;Golub and Caroni, 2005). Recently, PKC␣ has been demonstrated to associate with detergent-insoluble fractions in a Ca 2ϩ -dependent manner attributed to raft binding (Rucci et al, 2005).…”

“…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).…”

“…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). To resolve these possibilities, we first used the lipophilic membrane dyes DiD and FM4-64 to provide uniform membrane staining in cells expressing mGAP43-YFP (Raucher et al, 2000;van Rheenen and Jalink, 2002;Golub and Caroni, 2005). Cells expressing low levels of mGAP43-YFP were treated with DiD at 37°C for 5 min before being cooled to 4°C to slow the movement of dye to interior cell membranes.…”

Wiskott‐Aldrich syndrome protein (WASp) and N‐WASp are involved in the regulation of NK‐cell migration upon NKG2D activation

“…Thus, different pathways regulate cytoskeletal rearrangement and may compensate for the WASp defect. In addition to actin dynamics, Cdc42/WASp pathway regulates accumulation of raft patches at the cell surface in different cell lines [44]. These domains act as microtubule stabilizers through IQGAP1 association and promote sustained directional motility.…”