Using data automatically acquired by microinterferometry from large numbers of chick fibroblasts, we have detected rapid microfluctuations in the rates of protrusion and retraction of the cell margin which were strongly suppressed by colcemid, nocodazole and taxol. Fluctuations in the rate of retraction of the margin were about twice as powerful as fluctuations in the rate of protrusion. High-frequency fluctuations were also apparent in the cell track and in measures of cell spreading, shape and speed. These rapid fluctuations were also all suppressed by colcemid and nocodazole, sometimes by doses insufficient to disrupt the majority of microtubules. Taxol on the other hand did not suppress fluctuations in direction of the cell track nor fluctuations in the spreading of the cells but it was very effective at suppressing variations in protrusion and retraction and in cell speed and shape. We discovered that much slower, larger-scale variations in protrusion, retraction, spreading, shape and speed resulted from the accumulation of these rapid, microtubule-dependent fluctuations of the cell margin. These large-scale variations in cell behaviour were also suppressed by the same drug treatments that were effective in suppressing the corresponding high-frequency fluctuations. We speculate that a function of microtubules is to enhance the fibroblast's responses to its environment by causing microfluctuations of the cell's margin which give rise to large-scale variations in cell behaviour.
We have used a fluorescent derivative of kinesin, AF-kinesin (kinesin conjugated with 5-(iodoacetamido)fluorescein), to investigate the binding site of kinesin on microtubules and to compare this site with that to which tau binds. Microtubules saturated with tau will bind AF-kinesin in the presence of the ATP analogue, 5′-[beta,gamma-imino]triphosphate (AdoPP[NH]P). This shows that there are distinct binding sites for the two proteins. Further evidence comes from digestion studies where taxol-stabilised microtubules were treated with subtilisin, resulting in the cleavage of C-terminal residues from both the alpha- and beta-tubulin subunits. These treated microtubules can no longer bind tau, but are able to bind AF-kinesin in the presence of AdoPP[NH]P. Finally, AF-kinesin will support the gliding of subtilisin-digested microtubules in the presence of ATP at rates comparable to those obtained with non-digested microtubules. These results show directly that the binding site for kinesin is outside the C-terminal region of tubulin that is removed by subtilisin and is distinct from the binding site of tau.