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

Krylyshkina O, Kaverina I, Kranewitter W, Steffen W, Alonso MC, Cross RA, Small JV - J. Cell Biol. (2002)

Bottom Line: Small. 1999.Cell Biol. 146:1033-1043).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Billrothsthstrasse 11, Salzburg 5020, Austria.

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.

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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).
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fig7: 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).

Mentions: In vitro experiments with the T93N heavy chain kinesin construct showed that this rat kinesin head mutant bound to microtubules at a stoichiometry of 1 head per microtubule heterodimer, and with a dissociation constant in the presence of ATP of <100 nM (unpublished data). In in vitro competition binding experiments, T93N heads competed off wild-type rat kinesin dimers with one T93N displacing 1 wt dimer. The microtubule-activated ATPase of T93N was 100-fold lower than that of wild-type rat kinesin-1. Because the activity of this construct was not limited to cell type, we injected it into both Xenopus and fish fibroblasts. At an injection concentration of 20 μM the T93N construct caused a collapse of mitochondria around the nucleus, as observed with the SUK-4 antibody (Fig. 5). Examples of experiments for cells labeled for vinculin and zyxin are shown for fish fibroblasts in Fig. 7 (Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1). As shown, injection of T93N kinesin at a concentration of 20 μM caused both cell depolarization and an increase in size of substrate adhesions. However, the effect was not as long lived as with the SUK-4 antibody, presumably due to degradation of the protein. Because of the different time of measurement, quantification of the changes in focal adhesion size and number are not included in Fig. 2. Quantification was made in this case for 17 image pairs of cells, as in Fig. 1. Injection of T93N kinesin induced a change in the number of focal adhesions from a range of 22–69 before injection to 5–31 1 h thereafter, and a corresponding shift in size from a range of 0.96–1.78 to 2.80–4.11 μm. These changes were similar to those observed with SUK-4–injected and nocodazole-treated cells (Fig. 2).


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

Krylyshkina O, Kaverina I, Kranewitter W, Steffen W, Alonso MC, Cross RA, Small JV - J. Cell Biol. (2002)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199234&req=5

fig7: 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).
Mentions: In vitro experiments with the T93N heavy chain kinesin construct showed that this rat kinesin head mutant bound to microtubules at a stoichiometry of 1 head per microtubule heterodimer, and with a dissociation constant in the presence of ATP of <100 nM (unpublished data). In in vitro competition binding experiments, T93N heads competed off wild-type rat kinesin dimers with one T93N displacing 1 wt dimer. The microtubule-activated ATPase of T93N was 100-fold lower than that of wild-type rat kinesin-1. Because the activity of this construct was not limited to cell type, we injected it into both Xenopus and fish fibroblasts. At an injection concentration of 20 μM the T93N construct caused a collapse of mitochondria around the nucleus, as observed with the SUK-4 antibody (Fig. 5). Examples of experiments for cells labeled for vinculin and zyxin are shown for fish fibroblasts in Fig. 7 (Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1). As shown, injection of T93N kinesin at a concentration of 20 μM caused both cell depolarization and an increase in size of substrate adhesions. However, the effect was not as long lived as with the SUK-4 antibody, presumably due to degradation of the protein. Because of the different time of measurement, quantification of the changes in focal adhesion size and number are not included in Fig. 2. Quantification was made in this case for 17 image pairs of cells, as in Fig. 1. Injection of T93N kinesin induced a change in the number of focal adhesions from a range of 22–69 before injection to 5–31 1 h thereafter, and a corresponding shift in size from a range of 0.96–1.78 to 2.80–4.11 μm. These changes were similar to those observed with SUK-4–injected and nocodazole-treated cells (Fig. 2).

Bottom Line: Small. 1999.Cell Biol. 146:1033-1043).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Billrothsthstrasse 11, Salzburg 5020, Austria.

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.

Show MeSH
Related in: MedlinePlus