Fig 1.
Schematic molecular model of hypothesized signaling events linking Ca2+ and PIP3 signaling at the leading edge of polarized leukocytes.
The model [8,14,20] proposes that Ca2+ signals can stimulate PIP3 production by (a) driving Ca2+-PKC binding to membrane where it is activated by DAG (produced by Ca2+-PLC, not pictured). The resulting Ca2+-DAG-activated PKC (b) phosphorylates MARCKS and displaces it from sequestered PIP2 [20,26]. Following MARCKS displacement, the increased density of free PIP2 on the membrane surface provides additional target lipid binding sites that (c) recruit additional active PI3K molecules to the membrane surface, thereby (d) increasing net PI3K lipid kinase activity and PIP3 production. The increased PIP3 levels in turn (e) drive increased recruitment of PIP3- specific PH domain proteins, including PDK1, to the membrane surface.
Fig 2.
Strategy employed to quantify modulator-triggered changes in the leading edge area, or in key leading edge signaling reactions.
Representative pair of RAW 264.7 cells treated with activator (ATP) in the top two rows and inhibitor (Go6976) in the bottom two rows, showing the masks used to quantify leading edge area and activity changes. (A) Leading edge area changes were determined by first outlining the leading edge region using the freehand selection tool in FIJI, while excluding the bulk of the cell body. The mask baseline (yellow line) was added at the base of the actively ruffling leading edge membrane prior to modulator addition. Changing leading edge area subsequent to addition of modulator, or modulator vehicle, was measured relative to that baseline at time = 0. (B) Leading edge activity changes were quantified by measuring sensor fluorescence (XFP sensor or CKAR or CellMask) within a defined boundary, depicted by the yellow outline enclosing a portion of leading edge membrane and adjacent cytoplasm. As the timecourse progressed, the mask was manually moved in order to remain proximal to the leading edge boundary. Additional details in Methods.
Fig 3.
Steady-state changes in leading edge area following modulator addition.
Polarized, actively ruffling RAW macrophages expressing the indicated activity sensor were imaged at specific times following addition of the indicated modulators (green = activator, red = inhibitor, black = carrier medium control). Open bars represent the initial leading edge area, normalized to 1.0, immediately after modulator addition. Filled bars represent the fold change as the leading edge area approaches a new steady state size approximately 5 min after modulator addition (t = 4.5 to 5.5 min (see Methods)). As expected for functional leading edge signaling in all sensor backgrounds, activators trigger significant leading edge expansion, inhibitors trigger significant leading edge contraction, and controls have no significant effect. 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 the initial area at t = 0 (one, two or three asterisks indicate p < 0.05, p < 0.01, or p < 0.001, respectively). Image analysis described in Fig 2A and Methods.
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.
Fig 5.
Timecourses of leading edge area changes (A) and leading edge activity changes (B-E) following modulator addition. Timecourses were measured for same polarized, actively ruffling RAW macrophages imaged in Figs 2 and 3 (see those figure legends for additional details) following addition of the indicated modulators (green = activator, red = inhibitor, open = carrier medium control). (A) The area change data indicate that the leading edge area expansion triggered by activators is slower, and appears to exhibit biphasic kinetics with a lag phase, compared to the more monophasic contraction triggered by inhibitors. (B-D) The most rapid leading edge activity changes are observed for the inhibitor-triggered decreased in PIP3 density sensed by AKTPH-mRFP, while the slowest change is observed for the attractant PDGF-triggered dissociation of MARCKSp-mRFP from the leading edge membrane. Error bars represent standard errors of the mean for 15–35 cells measured in at least 4 independent experiments.