Analysis of Cortical Flow Models In Vivo

Abstract

Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow. INTRODUCTIONCortical flow is the process whereby material in the cell cortex (the outer ϳ1-5 m of the cell) is translocated parallel to the plane of the plasma membrane (Bray and White, 1988). Although cortical flow is observed in a variety of cellular motility processes, it is especially striking during cytokinesis, when cortical F-actin (Cao and Wang, 1990), F-actin-binding proteins (Sanger et al., 1994), myosin-2 (DeBiasio et al., 1996, cortical organelles (Scott, 1960;Hird and White, 1993), and cell surface proteins (Koppel et al., 1982;Wang et al., 1994) all converge on the site of the incipient furrow. The extent of cortical flow varies in different systems and under different conditions. Cortical flow can occur over the entire surface of cells or embryos (Hird and White, 1993) or it may be restricted to a defined region of the cell (Wang et al., 1994; Fishkind and Wang, 1996). In either case, the net result is the same, with cortical F-actin, cortical organelles, and cell surface proteins converging on a particular area of the cortex.Cortical flow is thought to be important during cytokinesis because evidenc...

Leading Tip Drives Soma Translocation via Forward F-Actin Flow during Neuronal Migration

“…Furthermore, we observed a significant difference of myosin II activity between the leading tip and the soma in migrating neurons, indicating higher actomyosin contractility at the leading tip, as reflected also by the higher dynamics of the GC-like tip structure in migrating neurons. Since cortical actomyosin network is known to flow toward the region with higher cortical tension in animal cells (White and Borisy, 1983;Bray and White, 1988;Benink et al, 2000;Munro et al, 2004), we propose that the front-to-rear difference in actomyosin activity is responsible for driving the forward F-actin flow along the leading process, which in turn pulls the soma forward. We observed that soma translocation was accelerated and impeded when myosin II activity was elevated and inhibited at the front, respectively, whereas these manipulations at the rear end of the migrating neuron resulted in opposite effects.…”

“…Moreover, pharmacological disruption of microtubules increases F-actin-mediated contractility in many different cell types, including PC12 cells (Dennerll et al, 1988), fibroblasts (Danowski, 1989), retinal photoreceptor cells (Madreperla and Adler, 1989), and cardiac muscle cells (Tsutsui et al, 1993). In Xenopus oocytes, cortical F-actin flow could be suppressed by microtubule polymerization and enhanced by microtubule depolymerization (Canman and Bement, 1997;Benink et al, 2000). In addition, disrupting microtubules in chicken embryonic fibroblast resulted in increased phosphorylation of MLC (Kolodney and Elson, 1995), an effect that may help to fuel the F-actin flow.…”

“…Since the cortical F-actin is known to flow toward the region of the higher actomyosin contractility (White and Borisy, 1983;Bray and White, 1988;Benink et al, 2000;Munro et al, 2004), we compared the actomyosin activity in migrating versus nonmigrating neurons by immunostaining of p-MLC(serine-19), which marks the activated form of myosin II. Neurons were first assayed for their migratory state with time-lapse video microscopy, fixed immediately and immunostained for p-MLC.…”

“…Moreover, how the direction of F-actin flow in migrating neuron is determined remains unclear. Since cortical F-actin is known to flow toward the region with higher myosin contractility in the cell (White and Borisy, 1983;Bray and White, 1988;Benink et al, 2000;Munro et al, 2004), a forward F-actin flow in the leading process suggests a higher myosin II activity at the distal rather than the proximal leading process. Overall, it remains unclear how the myosin II-dependent driving force is generated and exerted on the soma during neuronal migration.…”

Polarisation des oeufs et des embryons : principes communs

“…Les phénomènes contrôlant les réorganisations corticales et cytoplasmiques restent mystérieux. L'analyse des flux corticaux a toutefois été amorcée sur des oeufs de xénope artificiellement activés ainsi que sur leurs extraits cytoplasmiques [80]. Ces études mettent en évidence le rôle essentiel des microtubules dans le démarrage et l'orientation des flux corticaux dont la force motrice est le réseau d'acto-myosine cortical.…”