Craig A. Mandato

Why do we wound Xenopus oocytes to study contractile systems?

Cellular wounding triggers two complementary repair mechanisms. The first is the calcium-dependent fusion of membrane vesicles at the damaged area (Steinhardt et aa., 1994; Terasaki et al., 1997). This fusion event takes place within seconds and provides a temporary barrier (a membrane patch) that prevents the inrush of ions which would ultimately kill the cell (McNeil et al., 2000). This response has been documented in cultured mammalian cultured cells (Miyake and McNeil, 1995), sea urchin eggs and embryos (Steinhardt et al., 1994; Bi et al., 1995; Terasaki et al., 1997; McNeil et al., 2000).

The second repair mechanism, described in detail for amphibian oocytes, entails the assembly and subsequent contraction of a circumferential array of F-actin and myosin-2 (Bement et al., 1999). This mechanism begins within 30 seconds to a minute of the initial breach in the plasma membrane. The role of this contraction event is to reestablish a functional cortex. That is, the patch membrane lacks the various transmembrane proteins and cytoskeletal linkages found in the normal plasma membrane and cortex. Thus, as the contractile ring closes, the wounded area is replenished with functional plasma membrane and cortex from the surrounding area.

Movie 1A [.mov]

Oocytes are injected with the a fluorescent probes, mounted, wounded with either the krypton/argon confocal imaging laser or with a high-energy nitrogen dye pulse laser and imaged via confocal microscopy. This is a 4D movie of oocyte injected with Texas Red phalloidin and wounded with imaging laser to produce square wound. The wound is contractile -- it rounds up during closure.

High magnification schematic illustrating the spatial relationship between the contractile actomyosin ring and the zone of de novo actin and myosin-2 polymerization.

Movie 3A Copy [.mov] A 4D movie of oocyte injected with Oregon-green G-actin and wounded. Actin is concentrated in ~6 umm wide zone around wound that broadens over time and is site of actin comet formation. Zone is flanked by dark halo of F-actin depletion. Radial actin cables run perpendicular to the wound border and disappear into zone of intense actin accumulation.

Quicktime 2B [.mov] A 4D movie of wound closure in an oocyte injected with Alexa-488 Phalloidin. Stable F-actin around wound edge is in cables oriented parallel to wound border while peripheral F-actin forms cables oriented perpendicular to wound border.

Movie 3 [.mov] Double-labeled low magnification 4D movie of oocyte injected with Oregon green-taxol (green) and Alexa-568-phalloidin (red) and wounded reveal that microtubules move toward the wound association with F-actin and buckle upon reaching wound edge.

Acttube movie [.mov] Higher magnification movie showing cotransport of F-actin (red) and microtubules (green). Lengthwise interactions between microtubules and F-actin are observed as microtubules move toward wound. Microtubules bend and break at the wound border.