The idea that the spatial regulation of contractility in a cell has a primary impact on cell polarity is supported by experiments with fibroblasts made apolar and immobile by the depolymerisation of microtubules. When such cells are exposed to inhibitors of contractility on one edge, the retraction induced is accompanied by protrusion on the opposite edge (next movie). Continued exposure of inhibitor to the retracting edge leads to the development of polarity and directional locomotion.
Asymmetric relaxation can restore polarisation and motility to cells lacking microtubules. Two examples are shown of cells transfected with GFP-zyxin that were treated and maintained in nocodazole to disassemble microtubules. After nocodazole treatment, the cells become symmetric and develop large focal adhesions at their periphery. The video shows the result of exposing the cells on one edge with the myosin relaxant, ML-7, through a micropipette. Upper panels show fluorescence images and lower panels the phase contrast images. Note that the release of adhesions at one edge, induced by local relaxation, is followed by protrusion at the opposite edge. (From Kaverina et al., 2000)
One conclusion from this experiment is that microtubules influence cell polarity by inducing an asymmetry in contractility in the actin cytoskeleton. As already suggested from the preceding experiments, this effect on contractility is mediated by the interaction of microtubules with the sites of linkage of contractile actin bundles with the substrate, namely with the focal adhesions.
It is worth comparing the characteristics of focal adhesion turnover in cells lacking microtubules and forced to move with myosin inhibitors (previous movie), with that in normal cells moving spontaneously (next movie). Note that in the control cell, focal adhesions formed behind the cell front dissolve in the wake of the cell body. Not so in the cells lacking microtubules; here they persist as the cell body moves over them. We conclude that in normally moving cells, microtubules play a role in disassembling the older adhesions behind the cell front, thus promoting turnover of the cytoskeleton to facilitate further protrusion.
The turnover of adhesion sites in a motile fish fibroblast, with an intact microtubule network. The cell was transfected with GFP-zyxin. Adhesion sites formed behind the cell front dissolve before the cell body reaches them. This contrasts with cells lacking microtubules that are forced to move as shown in the first movie.
- Kaverina, I., Krylyshkina, O., Gimona, M., Beningo, K., Wang, Y. L., Small, J. V. (2000). Enforced polarisation and locomotion of fibroblasts lacking microtubules. Curr Biol. 10, 739 — 742.