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FIGURE 1:. Multiple, distinct lipid domains form around single-cell wounds. (A) eGFP-PH (GRP1) detects PIP3 accumulation. (Aâ²) PIP3 peak enrichment occurs inside the active Cdc42 zone at 90 s. (B) eGFP-PH (PLCδ) detects PIP2 enrichment. (Bâ²) PIP2 peak enrichment is outside the active Cdc42 zone at 90 s. (C) eGFP-C2 (lactadherin) detects PS enrichment. (Câ²) PS peak enrichment occurs inside the active Cdc42 zone at 90 s. (D) eGFP-Spo20 detects a region of PA depletion (arrowheads) immediately after wounding, followed by enrichment. (Dâ²) The peak of PA signal is found inside the zone of active Cdc42 at 90 s. (E) eGFP-C1 (PKCη) detects a region of DAG enrichment. (Eâ²) The peak of DAG enrichment leads the peak of active Cdc42 at 90 s. (F) The peak of DAG enrichment leads the peak of PIP2. (G) The peak of PIP3 enrichment leads the peak of PIP2. (H) The peak PS signal leads the peak PA signal. (I) The peak DAG and PA signal are colocalized. (J) Schematic of lipid localization relative to the active Rho and Cdc42 zones. Scale bar, 20 μm. W, wound.
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FIGURE 2:. PA and DAG are generated at the wound edge coincident with Rho GTPase activation. (A) Recruitment times for each lipid were quantified. PA appeared at 16.8 s, and DAG at 18.5 s, whereas PIP3, PIP2, and PS accumulated at â¥32 s (PA, n = 10; DAG, n = 12; PIP3, n = 12; PIP2, n = 8; PS, n = 8; error bars, SEM). (B) The ratio of zone/background fluorescence intensity was calculated for each lipid at 90 s (PA, PIP3, n = 6; DAG, PIP2, PS, n = 5; error bars, SEM). (C) Cells injected with mCherry-rGBD and eGFP-Spo20 were wounded. A kymograph shows corecruitment of active Rho and PA to the wound edge. (Câ²) The intensity/time plotted from a vertical line drawn through the wound edge in C. (D) Cells injected with eGFP-rGBD and mRFP-C1 were wounded. A kymograph shows corecruitment of active Rho and DAG to the wound edge. (Dâ²) Intensity/time plotted from a vertical line drawn through the wound edge in D. (E) Brightest-point projections from movies show the contribution of cortical flow (indicated by lines perpendicular to the wound edge) to enrichment of each lipid. PA and DAG are less dependent on cortical flow than PIP3, PIP2, and PS. (F) Cells were treated with latrunculin A to abolish actin-mediated cortical flow, but PA and DAG still accumulated, as indicated by arrowheads. Scale bar, 10 μm (C, D); 20 μm (E, F).
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FIGURE 3:. DAG is generated via the PC-PLC/SMS pathway, whereas PA is generated via PLD and DAG kinase. (A) DAG is present at wounds in control cells but blocked by the PC-PLC/SMS inhibitor D609. (Aâ²) Quantification of wound DAG (***p < 0.005). (B) DAG is present at similar levels at wounds in control cells and those treated with FIPI, a PLD inhibitor. (Bâ²) Quantification of wound DAG (p > 0.05). (C) DAG at wounds is elevated in cells treated with the PIP2-PLC inhibitor U-73122. (Câ²) Quantification of wound DAG (***p < 0.005). (D) PA is decreased in cells treated with D609 relative to controls. (Dâ²) Quantification of wound PA (*p < 0.05). (E) PA wound signal is blocked in cells treated with FIPI. (Eâ²) Quantification of wound PA (***p < 0.005). (F) PA levels at wounds are decreased in cells treated with R59949, a DAG kinase inhibitor. (Fâ²) Quantification of wound PA (**p < 0.01). (G) Schematic of pathways leading to DAG and PA generation and corresponding inhibitors. Top and bottom whiskers represent maximum and minimum values, respectively. Scale bar, 20 μm.
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FIGURE 4:. Blocking generation of DAG, but not PA, inhibits Rho and Cdc42 activation. (A) Cells were injected with eGFP-rGBD and mRFP-wGBD to detect active Rho and active Cdc42, respectively. In cells treated with D609, Rho and Cdc42 activity is inhibited and wound healing stalls, whereas FIPI treatment does not inhibit GTPase activity or healing. (B) Intensity of Rho and Cdc42 zones from control and D609-treated cells was quantified at 60 s (Rho: control vs. D609, p < 0.01; Cdc42: control vs. D609, p < 0.005). (C) Intensity of Rho and Cdc42 zones from control and FIPI-treated cells was quantified at 60 s (Rho: control vs. FIPI, p >0.05; Cdc42: control vs. FIPI, p < 0.01). Top and bottom whiskers represent maximum and minimum values, respectively. Scale bar, 20 μm.
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FIGURE 5:. SPK601 inhibits PC-PLC, DAG production, and Rho and Cdc42 activation. (A) SPK601 (SPK) is as an effective inhibitor of PC-PLC in vitro as D609. Lipase activity of purified PC-PLC was assayed at the indicated concentrations of each inhibitor. (B) SPK601 suppresses DAG production around wounds. (Bâ²) Quantification of wound DAG (p < 0.005). (C) SPK601 suppresses PA production around wounds. (Câ²) Quantification of wound PA (p < 0.005). (D) SPK601 suppresses Rho and Cdc42 activation around wounds. Intensity of Rho and Cdc42 zones from control and SPK601-treated cells was quantified at 60 s (Rho: control vs. SPK601, p < 0.005; Cdc42: control vs. SPK601, p < 0.005). Top and bottom whiskers represent maximum and minimum values, respectively. Scale bar, 20 μm.
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FIGURE 6:. Dose-dependent suppression of healing and DAG production by SPK601 or D609. (A) Cells were treated with DMSO, the vehicle, or the indicated micromolar concentrations of SPK601 (S) or D609 (D) for 1 h, stab wounded with a glass needle, cultured overnight, and then assessed for survival. SPK601 and D609 both significantly suppressed healing at concentrations of 100 or 200 μM. (B) Quantification of wound DAG levels in cells treated as in A. Bars, mean ± SD; *p < 0.05; ***p < 0.005.
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FIGURE 7:. PKCβ and PKCη differentially regulate Rho and Cdc42 zones. (A) Cells injected with PKCβ -eGFP and PKCη-mCherry reveal localization of both PKCs to the wound edge, with PKCβ spanning a broader region than PKCη. (B) A line scan from the 90-s time point of A. (C) Cells were injected with eGFP-rGBD and mRFP-wGBD alone or with PKCβ, DN PKCβ, PKCη, DN PKCη, or DN PKCβ and PKCη. PKCβ up-regulates Rho and Cdc42 activity zones, whereas DN PKCβ down-regulates zones. PKCη inhibits Rho and Cdc42 activity zones, whereas DN PKCβ up-regulates Rho and Cdc42 activity on the cortex. Expression of DN PKCβ and full-length PKCη results in dramatic inhibition of both Rho and Cdc42 activity zones. (D) Quantification of Rho and Cdc42 zone intensity at 30 and 60 s with PKCβ expression (control, n = 6; PKCβ, n = 7). (E) Quantification of Rho and Cdc42 zone intensity at 60 s and background intensity with DN PKCβ expression (control, n = 16; PKCβ DN, n = 11). (F) Quantification of Rho and Cdc42 zone intensity at 30 and 60 s with PKCη expression (n = 14). (G) Quantification of Rho and Cdc42 zone intensity at 60 s and background intensity with DN PKCη expression (control, n = 16; PKCη DN, n = 11). (H) Quantification of Rho and Cdc42 zone intensity at 30 and 60 s with expression of DN PKCβ and full-length PKCη (control, n = 16, PKCβ DN/PKCη, n = 13). Green bars, Rho; red bars, Cdc42. Top and bottom whiskers represent maximum and minimum values, respectively. Scale bar, 20 μm. W, wound.
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