A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages
Fig 4
Steady state changes in leading edge activity sensors following modulator addition.
The same polarized, actively ruffling RAW macrophages imaged in Fig 3 were also monitored for leading edge signaling activities as detected by the indicated activity sensor at specific times following addition of the indicated modulators (green = activator, red = inhibitor, black = carrier medium control). At each timepoint, the fluorescence signal of the sensor was measured to quantify the leading edge activity it monitors. Open bars represent the initial leading edge activity, normalized to 1.0, immediately after modulator addition. Filled bars represent the fold change as the activity approaches a new steady state level approximately 5 min after modulator addition (t = 4.5 to 5.5 min (see Methods)). The findings indicate that activators significantly increase (and inhibitors significantly decrease) the leading edge PIP3 density sensed by AKTPH-mRFP, and the leading edge PKC activity sensed by CKAR. In contrast, the opposite significant changes are observed for MARCKS binding to the leading edge membrane sensed by MARCKSp-mRFP. No significant changes in leading edge membrane binding were observed for the MARCKSp-SA4-mRFP sensor that lacks the Ser residues required for phosphoregulation by PKC. Error bars represent standard errors of the mean for 15–35 cells measured in at least 4 independent experiments. Asterisks indicate significance of each change from t = 0 (one, two or three asterisks indicate p < 0.05, p < 0.01, or p < 0.001, respectively). Image analysis described in Fig 2B and Methods.