Microtubule targeting of substrate contacts promotes their relaxation and dissociation

We recently showed that substrate contact sites in living fibroblasts are s pecifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Sm all. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microt ubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fi broblasts in which microtubules and contact sites were simultaneously visua lized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein. For cells allowed to spread in the presence of nocodazole the turnover of c ontacts was retarded, as compared with controls and adhesions that were ret ained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell f ront and larger adhesions, corresponding to focal adhesions, at the retract ing flanks and rear. At retracting edges, multiple microtubule contact targ eting preceded contact release and cell edge retraction. The same effect co uld be observed in spread cells, in which microtubules were allowed to re-a ssemble after local disassembly by the application of nocodazole to one cel l edge, At the protruding front of polarized cells, focal complexes were al so targeted and as a result remained either unchanged in size or, more rare ly, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusi on stimulated by the injection of constitutively active Rac, peripheral foc al complexes became abnormally enlarged. We further found that the local ap plication of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as obs erved after microtubule targeting. Our data are consistent with a mechanism whereby microtubules deliver local ized doses of relaxing signals to contact sites to retard or reverse their development, We propose that it is via this route that microtubules exert t heir well-established control on cell polarity.