Neutrophil polarization: Spatiotemporal dynamics of RhoA activity support a self-organizing mechanism

Abstract

Chemoattractants like fMet-Leu-Phe (fMLP) induce neutrophils to polarize with phosphatidylinositol 3,4,5-trisphosphate (PIP3) and protrusive F-actin at the front and actomyosin contraction at the sides and back. RhoA and its downstream effector, myosin II, mediate the ''backness'' response, which locally inhibits the ''frontness'' response and constrains its location to one part of the cell. In living HL-60 cells, we used a fluorescent PIP3 probe or a single-chain FRET biosensor for RhoA-GTP to assess spatial distribution of frontness or backness responses, respectively, during the first 3 min after exposure to a uniform concentration of fMLP. Increased PIP3 signal or RhoA activity initially localized randomly about the cell's periphery but progressively redistributed to the front or to the back and sides, respectively. Cells rendered unable to mount the frontness response (by inhibiting actin polymerization or G i, a trimeric G protein) responded to a micropipette source of attractant by localizing RhoA activity at the up-gradient edge. We infer that protrusive F-actin, induced by the frontness response, constrains the spatial distribution of backness by locally reducing activation of RhoA, thereby reducing its active form at the front. Mutual incompatibility of frontness and backness is responsible for self-organization of neutrophil polarity.actin cytoskeleton ͉ cell polarity ͉ Rho GTPases

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

“…Our data indicate that endogenous PIPKI␥ was enriched toward the rear of the cell ( Figure 1C) and did not colocalize with actin at the cell front. Previous studies have shown that the active form of RhoA is enriched in the rear of neutrophil-like HL-60 cells (Xu et al, 2003;Wong et al, 2006). Using GST-rhotekin as a probe for RhoA-GTP, we were able to detect enrichment of active RhoA toward the cell rear of primary neutrophils, similar to the staining observed for PIPKI␥ ( Figure 1D).…”

“…For example, leading edge components such as Rac, Cdc42, and PI3K are thought to promote the frontness response by triggering accumulation of actin and the phosphoinositide PtdIns(3,4,5)P 3 at the leading edge (Meili et al, 1999;Weiner et al, 1999;Servant et al, 2000;Van Keymeulen et al, 2006). In contrast, components of the cell rear such as Rho and ROCK are thought to promote backness by the generation of actomyosin-based contraction and regulation of rear release (Niggli, 1999;Eddy et al, 2000;Xu et al, 2003;Wong et al, 2006). Accordingly, spatial restriction of PIPKI␥661 to the cell rear suggests that PIPKI␥661 plays a role in backness signaling at the trailing edge during neutrophil chemotaxis.…”

“…HL-60 cells represent a widely used model system to study neutrophil chemotaxis (Servant et al, 2000;Weiner et al, 2002;Gomez-Mouton et al, 2004;Van Keymeulen et al, 2006;Wong et al, 2006), because this promyelocytic leukemia cell line differentiates into a neutrophil-like state when cultured with 1.25% DMSO (Collins et al, 1978) and becomes responsive to chemoattractants (Collins et al, 1979;Gallagher et al, 1979;Hauert et al, 2002). Wild-type and kinase-dead GFP-PIPKI␥661 were stably expressed in undifferentiated HL-60 cells.…”

ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization

“…In some migrating cell types, such as neutrophils and amoeba cells, pathways based on phosphoinositide 3-kinases and members of Rho family proteins control formation of the cell front and rear (reviewed in Dormann and Weijer, 2006;Kimmel and Firtel, 2004;Niggli, 2003;Ridley et al, 2003). In these cell types, feedback loops between pathways ensure that the cell front and rear form coordinately (Charest and Firtel, 2006;Schneider and Haugh, 2006;Van Keymeulen et al, 2006;Wong et al, 2006;Xu et al, 2003). However, in other migrating cell types, such as fibroblasts, mathematical models do not invoke feedback loops (Schneider and Haugh, 2005) and coordination is poorly understood in these cells.…”

“…However, all migrating cell types tested have the capacity to polarize in the absence of an externally applied gradient of chemoattractant. This infers that a significant contribution to establishing the cell front and rear must come from self-organization and selfamplification of important molecules within the cell Onsum et al, 2006;Verkhovsky et al, 1999b;Wong et al, 2006;Xu et al, 2003). In this regard, it is not obvious whether the cell front or rear should be expected to form first, which has implications for overall mechanism.…”