16. Touching Adhesions
Two lines of evidence indicate that microtubule tips approach focal adhesions at a range close enough for the precise exhange of molecular signals. The first evidence comes from the use of evanescent wave microscopy (Toomre and Mannstein, 2001).
If a beam of light illuminates a transparent, reflecting surface at the angle of total internal reflection, an evanescent wave penetrates above the surface to a depth of around 100-200nm. With living cells growing on a glass coverslip, the illumination is then restricted to the dorsal surface, which includes the substrate adhesion sites.
Evanescent wave microscopy of cells labelled with GFP-tubulin shows that microtubules dip down into the evanescent wave as they grow towards the cell periphery (Fig. 16-1).
Figure 16-1. Evanescent wave microscopy of a fibroblast expressing GFP-tubulin. By this technique, the sample is illuminated with exciting light in a layer only 100-200nm above the substrate. The appearance of microtubules only at the cell periphery indicates that microtubules dip down towards the dorsal cell surface in these peripheral regions. Note that some microtubules follow identical tracks. (from Krylyshkina et al., 2003).
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Double labeling of cells for an adhesion component (zyxin) and microtubules shows that the dipping down of microtubules correlates with their targeting of adhesion sites (Fig. 16-2).
Figure 16-2. Evanescent wave microscopy of a fibroblast expressing GFP-tubulin and Ds-Red zyxin and imaged simultaneously in the green and red fluorescent channels. Both pseudo colour and a black and white superimposition of images are shown. Where microtubules dip down towards the dorsal cell surface, they target adhesion sites. (from Krylyshkina et al., 2003).
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The intensity of the evanescent wave decreases exponentially away from the substrate and the intensity of fluorescence of the microtubule tips gives a measure of their depth of penetration into the evanescent wave. Calculations show that the microtubules approach the dorsal cell surface within a range of 50nm. We return later to this phenomenon in the context of the complex of proteins found at microtubule tips.
A second indication of an intimate cross-talk between microtubules and focal adhesions is indicated by the ability of adhesion sites to capture depolymerising microtubules and to temporarily stabilise them. This is illustrated in the video of Fig. 16-3. In this experiment, the cell was exposed during the sequence to nocodazole to depolymerise microtubules. Following addition of the drug (at the point of loss of focus), microtubules start to shorten. One of them (T) is subsequently captured during shortening at an adhesion site (V) and stops depolymerising there for several minutes (real time) before finally shrinking into the cell body (Kaverina et al., 1998).
Figure 16-3. Adhesion sites can capture microtubules. Video shows the peripheral region of a fibroblast that was co-injected with cy-3 tubulin and rhodamine-vinculin. During the video, nocodazole was added to the medium to initiate the depolymerisation of microtubules (addition at point of defocus). A shrinking microtubule (T) is captuterd at a focal adhesion (V) and temporarily stabilised at the adhesion against depolymerisation. (from Kaverina et al., 1998).
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