Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1

Abstract

Recent studies have shown that the targeting of substrate adhesions by microtubules promotes adhesion site disassembly (Kaverina, I., O. Krylyshkina, and J.V. Small. 1999. J. Cell Biol. 146:1033-1043). It was accordingly suggested that microtubules serve to convey a signal to adhesion sites to modulate their turnover. Because microtubule motors would be the most likely candidates for effecting signal transmission, we have investigated the consequence of blocking microtubule motor activity on adhesion site dynamics. Using a function-blocking antibody as well as dynamitin overexpression, we found that a block in dynein-cargo interaction induced no change in adhesion site dynamics in Xenopus fibroblasts. In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Blockage of kinesin activity had no influence on either the ability of microtubules to target substrate adhesions or on microtubule polymerisation dynamics. We conclude that conventional kinesin is not required for the guidance of microtubules into substrate adhesions, but is required for the focal delivery of a component(s) that retards their growth or promotes their disassembly.

Figure 1.

Enlargement of focal adhesions following microtubule disassembly. Figure shows the same Xenopus fibroblast injected with TAMRA-vinculin, before (A) and after (B) treatment for 3 h with 2,5 μM nocodazole. (Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1.)

Figure 2.

Quantification of adhesion site size in control, nocodazole-treated and antibody-injected cells, as indicated. The data on adhesion site length was collected from image pairs of cells as in Fig. 1 (26 pairs for m74–2; 24 pairs for SUK-4 and 23 pairs for nocodazole), recorded immediately after injections (or beginning of treatment) and 3 h later. Note the closely similar increase in contact site size and decrease in number for nocodazole and kinesin inhibition with the SUK-4 antibody.

Figure 3.

Inactivation of dynein by the m74–2 intermediate chain antibody, as monitored by the redistribution of lysosomes to the cell periphery. Lysosomes were loaded with rhodamine-dextran. (A and C) Paired phase contrast (A) and fluorescence (C) images of Xenopus fibroblast taken just after antibody injection. (B and D) The same cell 24 min later (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1).

Figure 4.

Dynein inhibition has no effect on focal adhesions. Cells in A and C were injected with TAMRA vinculin to visualize adhesion sites. Cell in C was subsequently injected with the m74–2 anti-dynein antibody. After 3 h, the control (B) and antibody-injected cell (D) showed no enhancement of focal adhesions.

Figure 5.

Control of block in kinesin motor activity by SUK-4 antibody. Figure shows living Xenopus fibroblasts in which mitochondria were marked with Rhodamine-123. The upper cell was injected with SUK-4 plus TAMRA dextran (diffuse background label in cytoplasm). Mitochondria are collapsed around the nucleus in the injected cell.

Figure 6.

Induction and elongation of focal adhesions following inhibition of kinesin by the SUK-4 antibody. Two Xenopus fibroblasts are shown with adhesion sites marked with TAMRA vinculin (A and B) or GFP-zyxin (C and D). A and C show the cells just before injection of the SUK-4 antibody (0 min). B and D show the same cells respectively 3 h and 1 h later (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1).

Figure 7.

Enlargement and elongation of focal adhesions following inhibition of kinesin by the rigor kinesin T93N. Two CAR fibroblasts are shown with adhesion sites marked with TAMRA vinculin (A and B) or GFP-zyxin (C and D). A and C show the cells just before injection of T93N kinesin (0 min). B and D show the same cells respectively 90 min and 1 h later (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1).

Figure 8.

Microtubules distribution in Xenopus fibroblasts is unaffected by kinesin inhibition. Cells were preinjected with Cy-3–tubulin (control, middle) and additionally injected with 20 μM T93N (left) or SUK-4 antibody (right).

Figure 9.

Microtubule targeting of substrate adhesions in Xenopus fibroblasts. Figure shows selected video frames from peripheral region of Xenopus fibroblasts coinjected with Cy-3–tubulin and TAMRA vinculin (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1). Arrows indicate targeting events. Time is given in minutes and seconds.

Figure 10.

Kinesin inhibition does not block the targeting of substrate adhesions by microtubules. Figure shows video frames from a SUK-4 injected Xenopus fibroblast that was preinjected with Cy-3-tubulin and TAMRA-vinculin. Arrows indicate targeting events. Time is in minutes and seconds.