Two phases of actin polymerization display different dependencies on PI(3,4,5)P3 accumulation 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.

Figure 1.

Assessment of actin dynamics in wild-type (WT) cells after a uniform cAMP stimulus. (A) Typical biphasic profile of actin polymerization assay from wild-type cells. All values were normalized to the amount of F-actin at time 0, which was taken just before addition of 1 μM cAMP. Typical cell shapes corresponding to time points are illustrated. (B) PH_CRAC-RFP and Coronin-GFP coexpressed WT cells were used to simultaneously monitor the localization of PI(3,4,5)P₃ and new F-actin in a living cell. Fluorescence images were captured at the indicated times after addition of 1 μM cAMP. (See video 1 in supplementary data.)

Figure 2.

Monitoring PH_CRAC-GFP translocation in wild-type (WT) and pten^- cells. (A) PH_CRAC-GFP expressed in WT and pten^- cells was visualized by fluorescence microscopy. Images were captured at the indicated times after addition of 1 μM cAMP. When present, LY294002 was added 15 min before experiments. (See videos 2a-2d in supplementary data.) (B) PH_CRAC-GFP translocation assay in WT and pten^- cells. Amount of membrane associated PH_CRAC-GFP in lysates prepared at times after addition of 1 μM cAMP was determined by immunoblot. Top: a typical immunoblot. Bottom: normalize data from two to four experiments.

Figure 3.

Actin polymerization assay in wild-type (WT), pten^-, and pi3k1^-/2^- cells in the presence or absence of LY294002. Five-hour stage WT cells (A), pten^- cells (B), and pi3k1^-/2^- cells (C) were treated with 30 μM LY for 15 min before addition of 1 μM cAMP. Data shown are average of at least three independent experiments.

Figure 4.

Chemotaxis to a micropipette filled with 10 μM cAMP. Fluorescent microscopic images of wild-type (WT) and pten^- cells expressing PH_CRAC-GFP in the presence or absence of 30 uM LY were taken at 10-s interval for 10 min. Frames demonstrate typical cell shape and PH_CRAC-GFP localization for each condition. White spot indicates the location of the micropipette. (See videos 4a-4d in supplementary data.) Average speed and chemotactic index were calculated from at least three independent experiments. Distribution of the speed (presented as pixels covered in 10 min) and chemotactic index of cells were also calculated and plotted.

Figure 5.

Comparison of cell morphology and biphasic actin response at different stages of development. (A) Wild-type (WT) cells chemotaxing in cAMP gradients. Typical examples of cells at 5- and 7-h stage are shown. (B) Actin polymerization assay in 5- and 7-h stage WT cells. Data shown are average of five independent experiments.

Figure 6

(facing page). Analysis of wild-type (WT) cells at the 5-h stage in the absence and presence of LY and at the 7-h stage switching directions after micropipette repositioning. (A) Fluorescent images of cells switching direction in response to new cAMP gradient. Relative times between frames are indicated on images. (See videos 6a-6c in supplementary data.) (B) Quantification of the “delay time” required for switching direction. Time between frames was 10 s. The time required for the leading edge to display sustained movement for two continuous frames toward the micropipette was determined. Number of events were binned in 20-s intervals and plotted as a histogram. One direction switch was considered one event. Five independent experiments (22 cells, 98 events) were quantified for 5-h stage WT cells. Seven independent experiments were quantified for both 30 μM LY-treated WT cells (22 cells, 41 events) and 7-h stage WT cells (30 cells, 63 events). Three independent experiments were quantified for both pten^- (11 cells and 79 events) and pten^- plus LY (19 cells and 31 events).