Several studies have shown that the depolymerisation of microtubules in fibroblasts leads to an increase in contractility of the actin cytoskeleton (Small et al., 2002). This is shown most vividly by cultivating cells on a suitably flexible substrate: after microtubule depolymerisation the substrate is pulled into creases (Danowski, 1989).
As we have already seen, an increase in contractility in the actin cytoskeleton leads to an increased bundling of actin filaments and to the growth of focal adhesions. This is what is also observed in cells treated with microtubule inhibitors. This figure shows the same cell before and after treatment with nocodazole for 3h. After treatment, the cell is no longer polar and there are fewer and larger focal adhesions:
Change in cell polarity and adhesion patterns in response to microtubule disruption. A Xenopus fibroblast was transfected with GFP-zyxin and imaged before (A) and after (B) treatment with 2.5µM nocodazole for 3h. Note the loss of cell polarity and an increase in size of focal adhesions, in response to nocodazole. The increase in focal adhesion size is diagnostic of an increase in contractility in the actin cytoskeleton. (From Krylyshkina et al., 2002)
The amplification of contractility by microtubule disruption is confirmed by the observation that myosin inhibitors prevent the augmentation of focal adhesions by nocodazole (Bershadsky et al., 1996).
These findings suggest (see also Bershadsky et al, 1996) that the microtubule cytoskeleton mediates a suppression of contractility in the actin cytoskeleton.
We have proposed that microtubules provide a “fibre delivery system” that allows this supression of contractility to be directed precisely to selected focal adhesions. The delivery of highly localised “relaxing” signals to single adhesion sites would then retard their growth or promote their disassembly.
Circumstantial evidence in support of this idea comes from experiments in which cells were challenged locally with inhibitors of myosin contractility. Filamentous myosin II, which generates contractility in the actin cytoskeleton, is activated by phosphorylation of the myosin regulatory light chain in the neck of the molecule. This phosphorylation is mediated by the myosin light chain kinase (MLCK) and can be blocked by inhibitors of MLCK, such as ML-7. When ML-7 is applied locally to a cell edge, the retraction of the edge is induced, mimicking that seen at the flanks of a migrating cell:
A local inhibition of contractility induces the release of substrate adhesions. A fish fibroblast was injected with rhodamine-vinculin to mark adhesion sites and then exposed on one edge to the myosin relaxant-ML-7– through a micropipette. (From Kaverina et al., 2000)
This effect is due to local relaxation of myosin at the cell periphery, as shown using cells injected with fluorescent myosin. The contractile activity in different regions of a cell are reflected by the change in spacing of myosin assemblies. As seen in the figure below, myosin assemblies do not decrease their spacing at the cell edge exposed to ML-7, but they become concentrated further away from the edge in the body of the cytoskeleton. Thus, this local relaxation at the periphery suffices to release adhesions, but does not block the overal contractility in the actin bundles that drives retraction of the cell edge.
Experiment as in the previous figure, but with a cell injected with rhodamine-labelled smooth muscle myosin. A reduction in spacing between the myosin „spots“ gives a qualitative measure of contractility. The local application of the myosin inhibitor causes cell edge retraction, whereby the retraction is due to adhesion release together with contraction of the medial zones of the actin filament bundles. At the cell edge, no reduction in myosin spacing is seen, consistent with a local relaxation, which promotes adhesion release. (From Kaverina et al., 2000)
- Bershadsky, A., Chausovsky, A., Becker, E., Lyubimova, A., Geiger, B. (1996). Involvement of microtubules in the control of adhesion-dependent signal transduction. Curr. Biol. 10, 1279 — 1289.
- Danowski, B., A. (1989). Fibroblast contractility and actin organization are stimulated by microtubule inhibitors. J. Cell. Science. 93, 255 — 266.
- 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.
- Krylyshkina, O., Kaverina, I., Kranewitter, W., Steffen, W., Alonso, M. C., Cross, R. A., Small, J. V. (2002). Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1. J. Cell Biol. 156, 349 — 359.
- Small, J. V., Geiger, B., Kaverina, I., Bershadsky, A. (2002). How do microtubules guide migrating cells? Nat. Rev. Mol. Cell Biol. 3, 957 – 64.