Figure 10.
Figure 10. PIP3, F-actin structures, and blebs during re-orientation of wild-type and PhdA/CRAC double-null cells. (A) Re-orientation of a wild-type cell. PIP3 accumulates at the membrane adjacent to the cyclic-AMP micropipette within 5 s of the micropipette movement, and F-actin microspikes form at the same time. At 14 s after the move a substantial bleb forms from the region of the membrane with highest PIP3 accumulation (see Video 9). (B) Re-orientation of a PhdA−/CRAC− double-mutant cell. These cells are generally less elongated than the wild type, but can still turn by forming a new leading edge from their flank. Although a PIP3 patch and F-actin microspikes form normally, the cell does not turn with a bleb, but uses a pseudopod instead (see Video 10). Turning experiments, using a micropipette filled with cyclic-AMP, were performed as in Fig. 6 with wild-type and HM1589 cells expressing PH-CRAC-GFP and LifeAct-RFP, and starved for 5.5 h.

PIP3, F-actin structures, and blebs during re-orientation of wild-type and PhdA/CRAC double-null cells. (A) Re-orientation of a wild-type cell. PIP3 accumulates at the membrane adjacent to the cyclic-AMP micropipette within 5 s of the micropipette movement, and F-actin microspikes form at the same time. At 14 s after the move a substantial bleb forms from the region of the membrane with highest PIP3 accumulation (see Video 9). (B) Re-orientation of a PhdA−/CRAC− double-mutant cell. These cells are generally less elongated than the wild type, but can still turn by forming a new leading edge from their flank. Although a PIP3 patch and F-actin microspikes form normally, the cell does not turn with a bleb, but uses a pseudopod instead (see Video 10). Turning experiments, using a micropipette filled with cyclic-AMP, were performed as in Fig. 6 with wild-type and HM1589 cells expressing PH-CRAC-GFP and LifeAct-RFP, and starved for 5.5 h.

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