Two Phases of Actin Polymerization Display Different Dependencies on PI(3,4,5)P₃Accumulation and Have Unique Roles during Chemotaxis

Abstract

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten- cells. Surprisingly, the fast phase of PI(3,4,5)P3 accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis.

Novel Mechanism of PTEN Regulation by Its Phosphatidylinositol 4,5-Bisphosphate Binding Motif Is Critical for Chemotaxis

“…In a gradient of cAMP, these PTEN mutants are normally located at the rear of chemotaxing cells. Similarly, PTEN shows normal distribution in the presence of the PI3K inhibitor LY294002 (ϳ100 M), which almost completely inhibits PI(3,4,5)P 3 production and the redistribution of the PH domain derived from Crac upon cAMP stimulation in Dictyostelium cells (39). These results clearly indicate that PTEN localization is regulated by a signaling pathway distinct from PI(3,4,5)P 3 -mediated mechanisms.…”

S-adenosylhomocysteine hydrolase is localized at the front of chemotaxing cells, suggesting a role for transmethylation during migration

“…The time course of actin polymerization after addition of 1 M cAMP to differentiated cells (3 ϫ 10 7 per ml) in 10 mM phosphate buffer (pH 6.2), 2 mM MgSO 4 , and 3 mM caffeine was determined as described (51). Cells were fixed and stained in buffer containing 3.7% formaldehyde, 0.15 Triton X-100, 250 nM TRITC-phalloidin, 20 mM potassium phosphate, 10 mM Pipes, 5 mM EGTA, and 2 mM MgCl 2 , pH 6.8.…”

Moving Forward: Mechanisms of Chemoattractant Gradient Sensing

“…In amoebae exposed to a uniform stimulus of cAMP, these responses are transient and biphasic. A rapid initial response peaks by ~10-15 s after stimulation and is terminated after ~30 s. This is followed by a second, slower phase of activity that peaks at 1-2 min and then slowly subsides (5). During this time, PH domains localize to discrete patches on the plasma membrane, where they initiate random pseudopod formation (5,35).…”

“…A rapid initial response peaks by ~10-15 s after stimulation and is terminated after ~30 s. This is followed by a second, slower phase of activity that peaks at 1-2 min and then slowly subsides (5). During this time, PH domains localize to discrete patches on the plasma membrane, where they initiate random pseudopod formation (5,35). In a gradient, however, PI3K is stably recruited from the cytosol to a defined region of plasma membrane that labels the front of the cell along with PH domain proteins (FIGURE 2).…”

“…Disruption of the two most active PI3K isoforms in Dictyostelium has only a partial effect on chemotaxis (5,11). The cells move slower and deviate more frequently from the straight-line path to the chemoattractant source but nevertheless move toward chemoattractants and are able to aggregate when plated on nonnutrient agar.…”