The finding of LIS1 at the leading edge of the migrating cells in the current paper identifies a new site for dynein and LIS1 colocalization

The finding of LIS1 at the leading edge of the migrating cells in the current paper identifies a new site for dynein and LIS1 colocalization. conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin made up of structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant unfavorable dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement. Keywords: microtubule; lissencephaly; motor protein; lamellipodia Introduction Cytoplasmic dynein is usually a molecular motor associated with diverse subcellular structures, such as membranous organelles and kinetochores, which it transports or pulls toward the minus end of microtubules. Two lines of recent evidence have implicated dynein in a new and potentially general role in directed LIN28 inhibitor LI71 cell movement. First, dynein interacts with the LIS1 protein (Faulkner et al., 2000; Sasaki et al., 2000; Smith et al., 2000) mutations in which cause lissencephaly, a disease resulting from incomplete migration of neural progenitor cells from the ventricular zone during early brain development (Reiner et al., 1993). A second line of evidence implicating dynein itself in cell movement has come from analysis of cytoskeleton reorganization in wounded monolayer cultures (Etienne-Manneville and Hall, 2001; Palazzo et al., 2001), a system which provides an excellent means for controlling the timing and direction of cell movement. An early step in wound healing involves the reorientation of the centrosome, the major microtubule organizing structure, to a position ahead of the nucleus and toward the leading cell edge (Gundersen and Bulinski, 1988). Microtubules located at the front of the cells also become preferentially stabilized, and the cells migrate to close the wound. Injection of antidynein antibody or overexpression of the dynactin subunit dynamitin each interfered with centrosome reorientation (Etienne-Manneville and Hall, 2001; Palazzo et al., 2001) without affecting microtubule stabilization or organization (Palazzo et al., 2001). These data implicated dynein in the reorientation process. However, the mechanism by which dynein contributed to this behavior, and a possible direct role in cell translocation were not assessed. We now report LIN28 inhibitor LI71 that dynein, its associated regulatory complex dynactin, and LIS1 are enriched at the leading LIN28 inhibitor LI71 cell edge in wounded NIH3T3 fibroblast monolayers during MTOC reorientation and subsequent Ebf1 cell migration. Inhibition of dynein, dynactin, and LIS1 interfere not only with reoriention of the microtubule network, but also with persistent directed cell migration as well. Results and discussion Localization of dynein and its related protein during wound curing To monitor the behavior of cytoplasmic dynein during cell migration, we performed immunofluorescence microscopy using antibodies to dynein or its accessories proteins. We recognized a stunning enrichment of dynein in the leading edge from the cell monolayer as the cells migrated to close the wound (Fig. 1, aCc, arrows; Fig. 2 j; Fig. 3 b). Dynactin, which includes been implicated in dynein focusing on (Echeverri et al., 1996) and processivity (Ruler and Schroer, 2000) was also enriched at these websites (Fig. 1, dCf, arrows), where it colocalized with dynein (Fig. 1, gCi, arrows). In lots of cells, dynein and dynactin had been enriched at areas toward which microtubules had been aimed (Fig. 1, aCf). Both punctate and diffuse staining had been observed. The second option pattern could possibly be noticed even in areas including few or no microtubules (Fig. 1, jCl, arrowheads). It had been often seen in parts of lamellipodial protrusion as judged by the current presence of membrane ruffles visualized by phase-contrast microscopy (Fig. 2, aCc arrows; and Fig. S1 A, offered by http://www.jcb.org/cgi/content/full/jcb.200310097/DC1). The staining didn’t overlap using the ruffles precisely. Furthermore, in lots of cells, dynein and dynactin had been enriched in accordance with the membrane marker Compact disc44 (Fig. 2 mCo; Fig. S1, C and B; Perschl et al., 1995), and industry leading staining was obviously noticed by confocal (Fig. S1 D) and total inner representation fluorescence microscopy (TIRF; discover Fig. 3). Open up in.