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Figure 1. Endogenous and exogenous rho GTPase activity in resting and wounded oocytes. (A) Confocal images of plasma membrane (PM) fluorescence of oocytes injected with a GBD probe, GST, or eGFP, and subsequently injected with water or GTP-γ-S. Bars, 10 μm. (B) Quantification of PM fluorescence signal in oocytes containing the indicated probes and injected with water or GTP-γ-S (n = 30; results are shown as means ± SEM; asterisks indicate P < 0.001). (C) Quantification of background fluorescence intensity in resting oocytes containing the indicated probes and toxin B, C3 exotransferase (C3), or the specified construct, compared with controls containing the probes alone (n ≥ 3; results are shown as means ± SEM; asterisks indicate P < 0.001). (D) Confocal images show lack of GST (green) localization around a wound relative to F-actin array (red). Bar, 10 μm. (E) Confocal images show ringlike localization of GST-rGBD (green) on interior of F-actin array (red). Bar, 10 μm. (F) Confocal images show ringlike colocalization of eGFP-wGBD (green) with F-actin array (red). Bar, 10 μm. (G) Confocal image shows zone of GST-rGBD (red) encircled by that of eGFP-wGBD (green). Bar, 10 μm.
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Figure 2. Rho GTPase activity zone dynamics. (A) Montage shows that zone of RhoA activity (eGFP-rGBD; green) appears as broad zone before focusing into a tight ring on actin array interior (red), whereas Cdc42 activity (eGFP-wGBD; green) appears and is maintained as a narrow zone in the middle of the actin array during wound healing (time in seconds; see Fig. S1). Bars, 15 μm. (B) Montage shows that RhoA (eGFP-rGBD; green) and Cdc42 (mRFP-wGBD; red) segregate into discrete zones during wound healing (time in seconds). Bar, 10 μm. (C) Montage shows that RhoA zone intensity (rGBD) is greatly reduced in the presence of C3 exotransferase (C3), toxin B, or constitutively active Rho (CARho), compared with control (time in seconds). Bars, 10 μm. (D) Montage shows that Cdc42 zone intensity of activity (wGBD) is greatly reduced in the presence of dominant negative Cdc42 (DNCdc42), toxin B, or constitutively active Cdc42 (CACdc42), compared with control (time in seconds). Bars, 10 μm. Videos available at http://www.jcb.org/cgi/content/full/jcb.200411109/DC1.
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Figure 3. External calcium is required for local activation of RhoA and Cdc42. (A) Montage shows suppression of RhoA (eGFP-rGBD) and Cdc42 (mRFP-wGBD) activity zones at 90 s after wounding in calcium-free medium. Bars, 10 μm. (B) Quantification of GBD zone fluorescence intensity in the absence of external calcium, relative to controls, over time (n = 11; results are shown as means ± SEM; asterisks indicate P < 0.001).
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Figure 4. F-actin and rho GTPase zones. (A) Montage shows RhoA (rGBD) or Cdc42 (wGBD) activity zones (green) relative to actin (red) in controls and cytochalasin D–treated cells (Cyt D) at 90 s after wounding. Bars, 15 μm. (B) Montage shows that RhoA (rGBD; green) and Cdc42 (wGBD; red) activity zones appear and segregate in the presence of cytochalasin D during wound healing (time in seconds). Bar, 10 μm. (C) Quantification of GBD fluorescence intensity in resting oocytes in the presence of cytochalasin D, relative to controls (n = 5; results are shown as means ± SEM; asterisks indicate P < 0.05). (D) Quantification of GBD zone width over time in the presence of cytochalasin D, relative to controls (width refers to the distance from the outer edge of the zone to the inner edge; n = 7; results are shown as means ± SEM; asterisks indicate P < 0.05). (E) Montage shows activity zones of RhoA (rGBD) or Cdc42 (wGBD; green) relative to actin (red), in the presence of latrunculin B (Lat B), during wound healing (time in seconds). Bars, 10 μm. (F) Confocal image shows activity zones of RhoA (green) and Cdc42 (red) 60 s after wounding in the presence of latrunculin B. Bar, 10 μm.
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Figure 5. Microtubules and rho GTPase activation. (A) Montage shows the broadening of RhoA (rGBD) or Cdc42 (wGBD; green) activity relative to actin (red) in cells treated with nocodazole (noc; time in seconds). Bars, 10 μm. (B) Quantification of GBD zone width over time in the presence of nocodazole, relative to controls (n = 3; results are shown as means ± SEM; asterisks indicate P < 0.05). (C) Montage shows the broadening of RhoA (rGBD) or Cdc42 (wGBD; green) activity zones relative to actin (red) in cells treated with taxol (tax; arrowheads show rGBD signal on the outside of the actin array; time in seconds). Bars, 10 μm. (D) Quantification of GBD zone width over time in the presence of taxol, relative to controls (n = 3; results are shown as means ± SEM; asterisks indicate P < 0.05). (E) Montage shows the segregation of RhoA (rGBD; green) and Cdc42 (wGBD; red) activity zones at 60 s after wounding in control and nocodazole- and taxol-treated cells (arrowheads show gap between zones in taxol). Bar, 10 μm. (F) Quantification of the time needed for GBD zone segregation in the presence of nocodazole or taxol, compared with controls (n = 11; results are shown as means ± SEM; asterisks indicate P < 0.05). (G) Quantification of zone segregation, comparing signal ratio (red/green; mRFP-wGBD/eGFP-rGBD) in the red zone, of controls and oocytes treated with nocodazole or taxol (n = 9; results are shown as means ± SEM; asterisks indicate P < 0.05).
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Figure 6. Crosstalk and rho GTPase zones. (A) Montage shows a decrease in RhoA activity zone intensity (rGBD) in the presence of DNCdc42 and an increase in zone width in the presence of CACdc42 (time in seconds). Bars, 10 μm. (B) Quantification of rGBD zone fluorescence intensity over time in the presence of DNCdc42 relative to controls (n = 8; results are shown as means ± SEM; asterisks indicate P < 0.05). (C) Quantification of rGBD zone width over time in the presence of CACdc42, relative to controls (n = 11; results are shown as means ± SEM; asterisks indicate P < 0.05). (D) Montage shows an increase in Cdc42 activity zone intensity (wGBD) in the presence of C3 and an increase in zone width with both C3 and CARho (time in seconds). Bars, 10 μm. (E) Quantification of wGBD zone signal width over time in the presence of C3 or CARho, relative to controls (n ≥ 4; results are shown as means ± SEM; asterisks indicate P < 0.05). (F) Quantification of wGBD zone fluorescence intensity over time in the presence of C3, relative to controls (n = 8; results are shown as means ± SEM; asterisks indicate P < 0.05).
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Figure 7. Activity zones and myosin-2 light chain phosphorylation. (A) Confocal image shows phosphorylated myosin-2 regulatory light chain staining (P-MLC; green) circumscribed by Cdc42 activity (red) in a fixed cell. Bar, 25 μm. (B) Confocal images of fixed cells show that P-MLC staining is broadened in cells treated with CACdc42 and reduced with C3, compared with control, at 3 min after wounding. Bars, 25 μm.
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Figure 8. Contributions of rho GTPases to actin assembly and closure. (A) Montage shows varied appearance and dynamics of actin in cells treated with either C3, DNCdc42, CARho, or CACdc42, compared with control (arrowheads indicate the outer edge of the dark halo in control). Bars, 15 μm. (B) Montage of brightest point projections made from movies (available at http://www.jcb.org/cgi/content/full/jcb.200411109/DC1) shows that movement of individual actin filament clusters around wounds is greatly reduced in cells treated with C3, DNCdc42, or CARho, but increased with CACdc42, compared with control. Videos available at http://www.jcb.org/cgi/content/full/jcb.200411109/DC1.
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Figure 9. Summary, crosstalk, and working model for observed discrete localization of rho GTPase activity zones during wound healing. (A) Schematic shows observed concentric subcellular compartmentalization of RhoA and Cdc42 activity zones at wound border, summary of crosstalk, and their respective effects on actin dynamics. (B) Diagram shows working model for maintenance of wound-induced response. Upon wounding, a cytoplasmic activity module (inside box) is established in which activation of localized RhoA and Cdc42 leads to directed cortical contraction and flow, which cause accumulation of both actin and microtubules, which, in turn, further promotes zones of activity.
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