Abstract
Scratch-wound assays are commonly used to study the ability of cells to polarize and migrate. In a previous study we showed that Golgi reorientation in response to a scratch wound is actin-dependent in NIH 3T3 cells but not in astrocytes. In this investigation, to study cell polarity and motility further, we used the polarization of the Golgi and microtubule organizing center (MTOC), as well as the ability of NIH 3T3 cells to migrate, in a scratch-wound assay. Unlike Golgi polarization, MTOC polarization was not dependent on actin, the Arp2/3 complex or Wiskott-Aldrich syndrome protein (WASP)-family proteins. By contrast, disruption of microtubules inhibited MTOC polarity, but not Golgi polarity. Migration was found to be dependent both on actin and microtubules. Expression of the formin-homology 2 (FH2) region of mDia1 inhibited Golgi polarization and migration but not MTOC polarization. Similarly, ST638, a Src inhibitor, inhibited Golgi polarization and migration but not MTOC polarization, whereas expression of the actin regulator IRSp53 only inhibited cell migration. Interestingly, the inhibition of cell migration by the mDia1 FH2 domain could be overcome by addition of Y27632, an inhibitor of ROCK (Rho-associated kinase). In fact, in the presence of ROCK inhibitor, cell migration was accelerated but polarization of both the Golgi and MTOC were inhibited. These data show that, in NIH 3T3 cells, different aspects of cell polarization and migration occur by different mechanisms, and both actin and microtubule networks are required. In addition, this study indicates that MTOC and Golgi polarization events are separately controlled.
Highlights
Microtubules (MTs) are essential for the polarization of many cell types (Etienne-Manneville and Hall, 2001; Goldman, 1971; Gundersen and Cook, 1999; Vasiliev et al, 1970)
We investigated the role of several proteins in the regulation of MT organizing center (MTOC) polarization, including regulators of both the actin and microtubule cytoskeletons
As we showed that the actin nucleator Arp2/3 complex is essential for Golgi polarization in wound-edge NIH 3T3 cells (Magdalena et al, 2003) and that MTOC polarization occurs in response to wounding, we tested whether Arp2/3 was necessary for MTOC polarization
Summary
Microtubules (MTs) are essential for the polarization of many cell types (Etienne-Manneville and Hall, 2001; Goldman, 1971; Gundersen and Cook, 1999; Vasiliev et al, 1970). Despite the central role of MTs in cell polarization, relatively little is known about the molecular mechanisms by which they exert their polarizing activity. There is a coordination of MT and actin cytoskeletons by mDia (Ishizaki et al, 2001). The mDia protein contains two forminhomology domains: FH1 and FH2. MDia exerts it effects on actin and MTs through its FH1 and FH2 regions, respectively (Ishizaki et al, 2001). MDia works in association with ROCK for the formation of stress fibers and focal adhesions (Nakano et al, 1999)
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