Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration
Fig 5
Gβ regulates coupling between individual actin oscillators.
(A) Method of analysis. Cells expressing LimE-GFP were imaged by confocal microscopy in the plane where cells make contact with the coverslip. At each timepoint, the cell periphery was divided computationally into 36 sectors. For each sector, the intensity of LimE-GFP was quantified over time. The graph shows the trace for a single sector. (B) The temporal order of actin regulators at peripheral actin foci. The indicated actin reporters showed pulsatile behavior at the cell’s periphery. We measured their appearance, relative to LimE, in the same sectors in double-labeled cells. Oscillators from several cells (HSPC-300 n = 9; ABD n = 11; Arp2 n = 4; Coronin n = 4) were analyzed (plotted are means +/- SEM). An example for a dual color sequence of LimE and Coronin oscillations is shown in S10 Fig. Raw data can be found in S1 Data. (C) Traces for all 36 sectors of one control cell in which Gβ was not sequestered. Individual sectors oscillate, but the overall average does not. (D) Traces for all 36 sectors of one cell in which Gβ sequestration was induced by treatment with 1 μM rapamycin. Individual sectors oscillate, and so does the overall average. (E) Analysis of oscillation parameters. Each point represents the average of all 36 sectors of one cell. Amplitude and period of oscillations are similar in unsequestered (Gβ+), Gβ-sequestered (Gβ-sequ.), and Gβ-null cells. In contrast, synchrony of oscillations is increased in acutely sequestered cells. Raw data can be found in S1 Data. Further data are presented as supplements: a histogram of oscillation periods of individual sectors for one cell for each condition is shown in S9 Fig. To extract and compare phase information, we used the Hilbert transform, as shown in S11 Fig. A histogram of phase distributions for one cell each is shown in S12 Fig. Membership of individual oscillating sectors with the phase locked consensus is fluid as shown in S13 Fig.