The signaling mechanisms underlying cell polarity and chemotaxis

Abstract

Chemotaxis--the directed movement of cells in a gradient of chemoattractant--is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) used by a neutrophil to choose its direction of polarity and to crawl effectively in response to chemoattractant gradients. Recent technological advances are beginning to reveal many fascinating details of the intracellular signaling components that spatially direct the cytoskeleton of neutrophils and D. discoideum and the complementary mechanisms that make the cell's front distinct from its back.

Figure 1.

Examples of chemotaxis. (A) A human neutrophil chasing a Staphylococcus aureus microorganism on a blood film among red blood cells, notable for their dark color and principally spherical shape (imaged by David Rogers, courtesy of Thomas P. Stossel). Bar, 10 µm. Chemotaxis is also necessary for (B) D. discoideum to form multicellular aggregates during development (courtesy of M.J. Grimson and R.L. Blanton, Texas Tech University), and (C) for axons to find their way in the developing nervous system. Photo provided by Kathryn Tosney, University of Miami.

Figure 2.

(A–D) Polarization of a neutrophil in response to gradient of chemoattractant. Nomarski images of unpolarized neutrophil responding to a micropipette containing the chemoattractant fMLP (white circle) at (A) 5 s, (B) 30 s, (C) 81 s, and (D) 129 s of stimulation. Bar = 5 µm. (Figure is taken from Weiner et al. 1999, with permission.) Human neutrophils stimulated with fMLP show highly polarized morphology and asymmetric cytoskeletal assemblies. (E–G) Human neutrophils were stimulated by a uniform concentration of fMLP (100 nM) and fixed 2 min after stimulations. Fixed cells were stained for F-actin with rhodamine-phalloidin (E, red) and an antibody raised against activated myosin II (phosphorylated specifically at Ser19, p[19]-MLC) (F, green). These fluorescent images are merged with Nomarski image in (G). Bars, 10 µm.

Figure 3.

PI(3,4,5)P3 shows a polarized distribution during chemotaxis. GFP-PH-AKT was used as a probe for the PI3K lipid product PI(3,4,5)P3. (A) The probe is uniformly distributed inside the cytosol of unstimulated dHL-60 cells, but accumulates on the up-gradient face of cells exposed to a chemoattractant gradient, delivered by a micropipette containing fMLP (B). The white circle denotes the position of the micropipette. Bar, 10 µm. (C) 3T3 fibroblasts exposed to a gradient of PDGF (white circle). Bar, 20 µm. (D) D. discoideum exposed to gradient of cAMP (white circle). The arrow heads point to the site of GFP-PH-AKT accumulation. Bar, 10 µm. (A) and (B) are taken from Servant et al. (2000), and (C) and (D) are modified from Haugh et al. (2000) and Meili et al. (1999), respectively.

Figure 4.

Mechanisms for neutrophil polarization. (A) A model for cell polarization during chemotaxis. In this model, polarization is assumed to arise from the interplay between a local activator, capable of catalyzing its own production, and a global inhibitor. In the case of the formation of polarized clusters on the cell surface, a membrane-bound activator (green circles) recruits other activator molecules to proximal regions of the membrane via a positive feedback mechanism (red arrows). In addition, membrane-bound activator is assumed to trigger inhibitor molecules (red lines). The inhibitor molecules act in a long-range inhibitory fashion and inhibit activation elsewhere (blue lines). Yellow dots denote chemoattractants. Competition between the activator and inhibitor limits the size and number of the clusters. (B) Overview of the feedback mechanism during neutrophil polarization. Chemoattractants, such as fMLP, trigger signaling by activating their specific GPCRs (denoted as R) and Gi proteins at the surface of neutrophils, leading to release of the Gβγ subunit, which in turn activates PI3Kγ, resulting in PI(3,4,5)P3 accumulation. PI(3,4,5)P3 triggers the translocation of DOCK2, a specific GDP to GTP exchange factor for Rac and increases its activity. Activated DOCK2 in turn activates the Rac and Cdc42 that ultimately transmit signals to the actin polymerization machinery. PI(3,4.5)P3, Rac, and polymerized actin serve as signals in a positive feedback loop that consolidates the leading edge of the neutrophils, although how actin polymers (or Rac) promotes PI(3,4,5)P3 is still unclear. In addition, there are signals that can activate Rac independently of PI(3,4,5)P3 (Inoue and Meyer 2008). The biochemical nature of these signals remains to be defined (dotted lines).

Figure 5.

Distinct actin assemblies modulate sensitivity to attractant and self-organizing polarity of neutrophils. Chemoattractant binds to a GPCR (R), which in turn activates different trimeric G proteins to generate two divergent, opposing signaling pathways, which promote actin polymerization (frontness) and actin–myosin contraction (backness), respectively. Localized mechanochemical incompatibility of the two cytoskeletal responses, combined with the ability of each to damp signals that promote the other (dashed inhibitor lines), then gradually drive them to separate into distinct domains of the membrane. As a result, a morphologically distinct pseudopod, which is highly sensitive to attractant, demarcates itself from relatively insensitive membrane, enriched with myosin, at the back and sides.