A signal transduction pathway involved in microtubule‐mediated cell polarization

Since Keith Porter first observed subcellular structures under the electronic microscope half a century ago, cell biologists around the world have been challenged by the fundamental question of how cells establish and maintain an asymmetrical distribution of organelles. It is evident now that microtubules (MTs) play an essential role in maintaining the distribution of cellular organelles, because the breakdown of MTs by MT antagonists results in the disruption of the normal distribution of mitochondria (1), endoplasmic reticulum (ER) (2), lysosomes and endosomes (3), Golgi (4), and vimentin intermediate filaments (IFs) (5). MTs have been shown to be dynamic structures turning over with a half-life of 5–10 min in proliferating cells in culture (6, 7). This raises the question of how such dynamic structures as MTs can contribute to cellular organization, which would appear to require at least some structural stability. Kirschner and Mitchison (8) hypothesized that MT dynamics may allow MTs to efficiently search the 3-dimensional space of the cell and, in response to environmental cues, dynamic MTs may become locally stabilized. Indeed, in polarized and differentiating cells, a subset of MTs are much more stable than the bulk, dynamic MTs, and, consistent with a role for MTs in organizing cellular organelles, the stable MTs are usually oriented in the axis of polarization or differentiation (reviewed in ref 9). One of the questions we have been addressing in our laboratory is how cells distinguish stabilized MTs from dynamic MTs. One clue to this question is that when MTs are stabilized in vivo, the tubulin comprising the MTs is modified by one or more posttranslational modifications (reviewed in ref 9). One of the best characterized of these modifications is detyrosination, which involves the removal of the carboxyl-terminal tyrosine residue from a-tubulin by tubulin carboxypeptidase (10), which preferentially utilizes polymeric tubulin as a substrate (11). Thus, only stabilized MTs that have persisted long enough have a high level of detyrosinated tubulin, and detyrosination thus serves as a marker for stable MTs. Detyrosination generates a-tubulin with a glutamate residue at the carboxyl terminus. (The two forms of tubulin are called Tyr and Glu tubulin after their respective carboxyl termini.) Detyrosination is reversible and Glu tubulin can be retyrosinated by tubulin tyrosine ligase (12). This enzyme is active only on soluble tubulin and is responsible for keeping the monomer pool of tubulin completely tyrosinated in vivo (13). As a result of the properties of tubulin carboxypeptidase and tubulin tyrosine ligase, and the dynamics of cellular MTs, cells have a mixture of dynamic MTs enriched in Tyr tubulin (Tyr MTs), stable MTs enriched in Glu tubulin (Glu MTs), and subunit tubulin comprised of only Tyr tubulin. We can distinguish the two forms of MTs by using antibodies that specifically react with Glu and Tyr tubulin (14). The biological system we use in most of our studies is an in vitro wound healing model. Fibroblasts are grown to confluency, and narrow strips of cells are then scraped off the substratum. The cells at the wound edge are initially unpolarized, but, in response to wounding, they become polarized with their long axis perpendicular to the wound and with an active leading edge at the portion of the cell facing the cell-free area. As a result of this polarization, the cells are able to move directionally into the wound. One response of wound-edge cells to wounding is the stabilization of a subset of MTs that are oriented toward the wound (15, 16). The stabilization of the MTs oriented toward the wound edge requires a serum factor (17), which we subsequently identified as the mitogenic lipid lysophosphatidic acid (LPA) (18). LPA or serum is capable of generating stabilized MTs in as little as 5 min after the addition to serum-starved cells (18). This suggests that MTs are poised to respond rapidly to extracellular cues and can function on a time scale that is consistent with many dynamic cell behaviors. In our initial effort to identify intracellular factors that mediate the LPA-induced stabilization of MTs, we found that activated Rho (a Ras-related, small GTPase) is both necessary and sufficient to mediate the selective stabilization of MTs at the wound edge (18). Activated Rho did not alter the parameters of the