Mandato CA , Bement WM .

GM52932-04A1 NIGMS NIH HHS , R01 GM052932 NIGMS NIH HHS

	Figure 1. Wound-induced actomyosin arrays are contractile. All times are in min:sec. (A) Images from a 4D video of oocyte injected with TR-phalloidin and wounded with imaging laser to produce square wound. Wound rounds up during closure. See Video 1. (B) Images from 4D video of oocyte injected with AX-phalloidin and wounded twice with nitrogen pump laser to generate oval wound. Long axis of wound closes faster than short axis until both are same length. (C) Quantification of oval wound closure. Regardless of probe used, long axes shrink faster than short axes. (D) Fluorescent, color-coded images obtained from 4D videos of oocytes wounded after injection with OG-actin and rewounded either outside the actin ring (left), or on (right) the actin ring. The last image of the 4D video before rewounding is shown in red; the first image taken after rewounding is shown in green. The linear wound made outside the closing ring with a laser Z-scan (white line) is limited in its lateral spread (arrows), whereas the wound made to the ring itself results in the springing open of the ring and extensive lateral spreading (arrows). (E) Fluorescence images from a 4D video of an oocyte injected with AX-phalloidin and TR and then subjected to a “cauterizing” wound (see text). The 00:00 time point is shown in green and red to reveal both the F-actin (green) and the presence of the cauterized region (red square on inside of wound). Subsequent time points show only the phalloidin signal. Closing actin ring breaks on right side of cauterized square and free edges (arrowheads) recoil away from break. See Video 2. The supplemental videos are available at http://www.jcb.org/content/vol154/issue4.
	Figure 2. A vortex of cortical flow at the wound border. (A) Images from 4D video of wound closure in oocyte injected with AX-phalloidin. Stable F-actin flows from surrounding cortex to wound edge, creating dark halo of F-actin depletion (white line) that spreads from the original wound edge. See Video 3. (B) Image from a 4D video of wound closure in an oocyte injected with AX-phalloidin. Stable F-actin around wound edge is in cables oriented parallel to wound border, whereas peripheral F-actin forms cables oriented perpendicular to wound border (arrows). (C) Images representing boxed area in B. The perpendicular actin cables (arrowheads) flow toward wound edge. See Video 4. (D) Projection of 14, 1 min frames from a 4D video of F-actin particle moving to wound border (W). Over time, F-actin (arrowhead) moves increasing distances toward the wound. (E) Correlative plot of F-actin cable velocity versus distance from the wound. The fluorescence micrograph is taken from the 4D video used to generate the instantaneous velocity measurements shown in the graph; X-axis of the graph corresponds to the area from the micrograph where the measurements were made. Instantaneous velocity measurements show that F-actin more than ∼28 μm away from the wound moves slowly; in regions close to wound, rate of movement increases sharply until plateau near wound edge. The threshold distance is correlated with the transitional area between low and high cortical F-actin (gray line). (F) Kymograph of AX-phalloidin–labeled actin motility at the wound edge. Horizontal arrow is 90 s; vertical arrow is 10 μm. Acceleration of F-actin as it approaches wound (W) is revealed by progressively greater distances moved over time by individual actin spots (arrowhead). The supplemental videos are available at http://www.jcb.org/content/vol154/issue4.
	Figure 3. A zone of highly dynamic actin forms around wounds. (A) Images from a 4D video of oocyte injected with OG-actin and wounded. Actin is concentrated in an ∼6-μm wide zone around wound (black line) that broadens over time and is the site of actin comet formation (arrows). Zone is flanked by dark halo of F-actin depletion (white line). Radial actin cables run perpendicular to the wound border (arrowheads) and disappear into zone of intense actin accumulation. See Video 5. (B) Fluorescence intensity scans from 4D video images of wounds made in an OG-actin–injected oocyte (note that imaging was started much sooner than in Fig. 3 A). At the start (00:00), peaks of F-actin signal (arrows) are evident next to wound (W) even though little depletion of signal is seen in flanking regions. However, at later time points, increasingly prominent signal “troughs” (arrowheads) flanking the peaks are evident. (C) Images from 4D video of oocyte injected with OG-actin and wounded. Wound edge is shown. Actin-rich “fingers” (arrowhead) extend from zone of high actin signal and vanish within 1 min. Fingers often appear to contact each other (double arrowheads). An F-actin comet (arrow) swims lazily through wound area. See Video 6. (D) Images from 4D video showing comets form in or near zone of high actin density (Z). Comets typically taper at one (arrowhead) or both (arrows) ends. See Video 7. (E) Histogram showing relationship between comet position and the zone of high actin intensity. Individual comets that moved more than two frames were followed in 10 experiments. The supplemental videos are available at http://www.jcb.org/content/vol154/issue4.
	Figure 4. The contractile ring is contained within the zone of actin polymerization. (A) Double label images from 4D videos made of oocytes injected with OG-actin and either TR-phalloidin (top) or TMR–myosin 2 (bottom). The TR-phalloidin and TMR–myosin 2 (red) act as markers for the contractile array of F-actin, which forms a tight band (black bar) within the broader zone of polymerization revealed by the green OG-actin (white bar). (B) Quantification of zone and contractile ring width from 4D videos made of oocytes injected with both OG-actin and TR-phalloidin. Zone is ∼5 times broader than the contractile ring and increases in width over time. Results are mean ± SEM from three independent experiments. (C) Fluorescence images of F-actin distribution in wounded oocytes stained with phalloidin after fixation. A 15° tilt of an image of a 4-μm stack reveals cables of F-actin parallel to the wound border (arrows) as well as fingers of F-actin extending into the wound (arrowheads). An en face view of the periphery of the wound (periphery) made from a 1.2-μm stack reveals cables of F-actin running perpendicular to the wound border (arrowheads). An en face view made from an 8-μm stack of wound fixed late in healing process (late) reveals F-actin fingers contacting each other across wound (arrows) and apparently pulling the sides of wound together as revealed by indentations in the F-actin array around the wound (arrowheads). (D) Images from a 4D video of an oocyte wound in the late stages of healing. Fingers of F-actin (arrows) contact each other across the wound, shorten over time, and pull edges of wound together, causing indentations actin array around wound (arrow heads). In region of finger contact in middle of the wound, F-actin staining becomes progressively brighter (double arrowheads). See Video 8. The supplemental video is available at http://www.jcb.org/content/vol154/issue4.
	Figure 5. Myosin 2 accumulates around wounds with little obvious flow. Time shown is in min:sec. (A) Images from 4D video of oocyte injected with TMR–myosin 2 and wounded. Bright foci of myosin 2 (arrowheads) accumulate around wound border and eventually fuse into tight, continuous ring (arrow). See Video 9. (B) Fluorescence intensity scans from 4D video images of wounds made in TMR-myosin 2–injected oocyte. At start of imaging (00:00), peaks of myosin 2 signal (arrows) are evident flanking wound (W) even though no depletion of signal is seen in the flanking regions. Even at later time points, the signal trough (arrowhead) flanking signal peaks are quite limited relative to those seen with actin probes (compare to Fig. 3 B above). (C) Confocal, double-labeled image (D) of a wound edge stained for F-actin (FA) and myosin 2 (M2) with a corresponding signal intensity plot, where the red line is F-actin signal and the blue line is myosin 2 signal. Quantification of signal from region 1 (beyond the dark halo of depletion) versus region 2 (in the dark halo) shows that the degree of F-actin depletion in the dark halo is significantly greater than that of myosin 2 (results are mean ± SEM; asterisk indicates P < 0.05; n = 20 wounds from 3 experiments). (D) Kymograph of TMR-myosin motility at wound edge. Horizontal arrow is 45 s; vertical arrow is 10 μm. Some myosin 2 foci move toward the wound at the same rate as the edge moves forward (double arrowheads); some move backward (arrowheads) and some new foci appear during the course of imaging (asterisks). Few foci show acceleration toward the wound edge seen for F-actin (compare to Fig. 2 F). (E) Vector plot diagram of myosin 2 foci movement from Video 10. Each point is separated by 15 s; some foci move steadily toward wound (W), some move intermittently, and some move parallel to wound edge. Asterisks represent areas where foci appeared during the course of imaging. See Video 10. The supplemental videos are available at http://www.jcb.org/content/vol154/issue4.
	Figure 6. Uncoupling actin assembly from cortical contraction and flow. (A) Images from 4D videos made of oocytes injected with OG-actin and then wounded. After 10:00 min, control wound (control) has rounded up and begun closure and shows typical F-actin distribution, including flow-dependent formation of a dark halo around the wound. In contrast, when flow is blocked by treatment with 100 ug/ml WGA or injection of 4 mg/ml NEM-S1, rounding and closure are perturbed and the dark halo fails to appear. Nevertheless, a zone of actin polymerization still forms around wounds. However, at later time points, the zone becomes destabilized and disappears from around wound (NEM-S1, 20:00). (B) Quantification of cortical flow inhibition by 4 mg/ml NEM-S1 and 100 μg/ml WGA. Cortical flow rates were calculated from 4D videos made from oocytes subjected to the indicated treatments. Results are mean ± SEM of three independent experiments; asterisk indicates P < 0.001. (C) Immunoblot (top) analysis of Xenopus egg extracts shows that antibodies to Arp3 (α-Arp3) and profilin (α-Prof) recognize proteins from Xenopus egg extracts of 50 and 15 kD, respectively (arrows). Bars indicate molecular weight markers of 211, 128, 84, 42, 32, and 17 kD (Arp3 blot) and 38, 29, 18, and 9 kD (profilin blot). In both absence (control) and presence (cyto-B) of 40 μM cytochalasin, profilin and Arp3 accumulate around wounds.
	Figure 7. Uncoupling myosin assembly from cortical contraction and cortical flow. (A) Images from 4D videos made from oocytes injected with TMR–myosin 2 and wounded. After 10 minutes, the control wound (control) has rounded up, commenced closure, and assembled a smooth, continuous ring of myosin 2. In contrast, the wound from oocyte treated with 100 μg/ml WGA was not rounded up nor closed, yet myosin 2 accumulated around the wound border in an irregular, discontinuous array. (B) Images from oocyte injected with TMR–myosin 2 and then wounded after treatment with 40 μm cytochalasin B. Bright foci of myosin 2 accumulate over time around wound border, but fail to coalesce into a continuous ring. (C) High magnification images from a 4D video of oocyte injected with TMR–myosin 2 and wounded after cytochalasin treatment. At the start of imaging (00:00), myosin 2 is visible as faint band. Bright foci of myosin appear adjacent to this band (arrowheads), and band is eventually surrounded by a ∼5–10-μm zone of myosin 2 foci (20:00). Most individual myosin 2 foci (e.g., arrow) grow progressively larger over time in the absence of any apparent transport. See Video 11. (D) Triple-label, confocal fluorescence analysis showing F-actin (green), TMR–myosin 2 (red), and endogenous myosin 2A (blue) in oocytes wounded after pretreatment with cytochalasin. TMR–myosin 2 (TMR-M2) and endogenous myosin 2A (XM2A) are codistributed around the wound. In some cases, they overlap with F-actin which remains after cytochalasin treatment (arrow), but in other cases they do not (arrowhead). The supplemental video is available at http://www.jcb.org/content/vol154/issue4.
	Figure 8. Breakage of the contractile ring destabilizes the zone of actin polymerization. Double-label images from 4D video of oocyte injected with OG-actin and TMR–myosin 2 and then subjected to cauterization wounding. See Video 12. Hatched line indicates burnt area of cytoplasm. Initially actin is present around wound as typical zone of polymerization. 6 min after start of imaging cortical flow has commenced, as revealed by development of dark halo around wound. The contractile ring is visible as a tight red-yellow ring within broader green zone of polymerization. The ring encounters edge of cauterized cytoplasm (arrow), stops movement, and begins to stretch. The zone of actin polymerization is broader in this region (arrowheads). By 10 min the contractile ring has broken, two recoiling free edges are visible (arrows) and zone widens and dims locally near break. At later time points (14:30) the edge of the spreading zone (arrowhead) is only barely visible near the original break site, whereas the spreading itself (double arrowheads) is apparently following the retreating free edges of the broken contractile ring (arrows). By 21 min the zone is almost gone near the original break point, but by 30 min, a new zone has assembled (arrowheads). The supplemental video is available at http://www.jcb.org/content/vol154/issue4.

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