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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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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.
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fig4: 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.

Mentions: The result of a block in dynein-associated transport on substrate adhesion sites in Xenopus fibroblasts is illustrated in Fig. 4 . Fig. 4, A and B, shows a control cell that was injected only with vinculin and imaged immediately after injection and 3 h later. The typical result obtained with cells that were additionally injected with the m74–2 antibody is shown in Fig. 4, C and D. The size and distribution of adhesion sites was essentially unchanged over the 3-h period after antibody injection. This was confirmed by measurements (Fig. 2) of a total of 1,000–1,500 adhesion sites in corresponding regions in 26 cell pairs of the type shown in Fig. 4. Further, time-lapse recording of control and m74–2-injected cells over a period of 90 min revealed no detectable differences in adhesion site dynamics in the shorter term. Cell polarization and the ability for directional movement also seemed to be unaffected (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1). Essentially, the same result was obtained with cells transfected with green fluorescent protein (GFP)*dynamitin: the translocation of rhodamine-labeled lysosomes to the cell periphery was readily apparent in all overexpressing cells, but no major change in the size and distribution of substrate adhesion sites or in the cell shape and motility could be detected (unpublished data).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: 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.
Mentions: The result of a block in dynein-associated transport on substrate adhesion sites in Xenopus fibroblasts is illustrated in Fig. 4 . Fig. 4, A and B, shows a control cell that was injected only with vinculin and imaged immediately after injection and 3 h later. The typical result obtained with cells that were additionally injected with the m74–2 antibody is shown in Fig. 4, C and D. The size and distribution of adhesion sites was essentially unchanged over the 3-h period after antibody injection. This was confirmed by measurements (Fig. 2) of a total of 1,000–1,500 adhesion sites in corresponding regions in 26 cell pairs of the type shown in Fig. 4. Further, time-lapse recording of control and m74–2-injected cells over a period of 90 min revealed no detectable differences in adhesion site dynamics in the shorter term. Cell polarization and the ability for directional movement also seemed to be unaffected (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200105051/DC1). Essentially, the same result was obtained with cells transfected with green fluorescent protein (GFP)*dynamitin: the translocation of rhodamine-labeled lysosomes to the cell periphery was readily apparent in all overexpressing cells, but no major change in the size and distribution of substrate adhesion sites or in the cell shape and motility could be detected (unpublished data).

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