Figure 5.

Chemotaxis defects can be rescued with cAMP pulses. (A) Chemotaxis in a Dunn chamber. Each line is a vector whose direction is the direction of movement and whose length corresponds to cell velocity in a stationary cAMP gradient. The y-axis points in the direction of the gradient. The velocity unit is micrometers per minute. In each case, >50 cells were followed. Top left, behavior of wild-type cells without cAMP prepulsing. n = 30 cells. Average velocity, 3.8 μm/min. Top right, behavior of wild-type cells with cAMP prepulsing. n = 11 cells. Average velocity, 7.8 μm/min. Bottom left, behavior of icmA⁻ cells without cAMP prepulsing. Only 2 of 54 cells were able to move in the gradient. Average velocity, 1.4 μm/min. Bottom right, behavior of icmA⁻ cells after cAMP prepulsing. n = 27 cells. Average velocity, 6.0 μm/min. (B) Micropipette chemotaxis assay. Cells for assays in the bottom panel had been pulsed with 50 nM cAMP for 5 h. The micropipette tips were filled with 150 μM cAMP. (C) Motility under agar. Pulsed cells were plated in an under agar chemotaxis assay and monitored with Nomarski optics. (D) Actin organization. Wild-type and mutant cells were starved with shaking for 6 h, plated on glass coverslips for 1 h, and then fixed and stained with rhodamine-conjugated phalloidin. (E) Myosin II localization in cells migrating under agar. Bars, 10 μm. See Supplemental Material.