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Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

Kaverina I, Krylyshkina O, Small JV - J. Cell Biol. (1999)

Bottom Line: The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge.At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria.

ABSTRACT
We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein. For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting. Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.

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Focal adhesions are dissociated during recovery from nocodazole. Control image shows fish fibroblast cotransfected with EGFP-tubulin and EGFP-zyxin pretreated with and replated in 2.5 μg/ml nocodazole (NOC) for 1 h. Subsequent frames show recovery after nocodazole washout. Time is given in minutes and seconds. Arrowheads indicate some of the adhesion sites that were dissociated during microtubule reassembly. Bar, 10 μm.
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Figure 2: Focal adhesions are dissociated during recovery from nocodazole. Control image shows fish fibroblast cotransfected with EGFP-tubulin and EGFP-zyxin pretreated with and replated in 2.5 μg/ml nocodazole (NOC) for 1 h. Subsequent frames show recovery after nocodazole washout. Time is given in minutes and seconds. Arrowheads indicate some of the adhesion sites that were dissociated during microtubule reassembly. Bar, 10 μm.

Mentions: An influence of microtubules on substrate contact turnover could also be observed during the reassembly of microtubules in cells that had first been allowed to spread to their maximum extent in the presence of nocodazole. In this case (Fig. 2) cells were used that had been transiently transfected with both EGFP-zyxin and EGFP-tubulin. As seen in Fig. 2, the recovery of the microtubule network was associated with the disassembly of a large proportion of contacts in the perinuclear region of the cell. Out of 61 nonperipheral contacts, 29 disappeared during microtubule recovery and 32 were retained, but even these became smaller or less intense. Video sequences from a further 3 cells showed similar results.


Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

Kaverina I, Krylyshkina O, Small JV - J. Cell Biol. (1999)

Focal adhesions are dissociated during recovery from nocodazole. Control image shows fish fibroblast cotransfected with EGFP-tubulin and EGFP-zyxin pretreated with and replated in 2.5 μg/ml nocodazole (NOC) for 1 h. Subsequent frames show recovery after nocodazole washout. Time is given in minutes and seconds. Arrowheads indicate some of the adhesion sites that were dissociated during microtubule reassembly. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169483&req=5

Figure 2: Focal adhesions are dissociated during recovery from nocodazole. Control image shows fish fibroblast cotransfected with EGFP-tubulin and EGFP-zyxin pretreated with and replated in 2.5 μg/ml nocodazole (NOC) for 1 h. Subsequent frames show recovery after nocodazole washout. Time is given in minutes and seconds. Arrowheads indicate some of the adhesion sites that were dissociated during microtubule reassembly. Bar, 10 μm.
Mentions: An influence of microtubules on substrate contact turnover could also be observed during the reassembly of microtubules in cells that had first been allowed to spread to their maximum extent in the presence of nocodazole. In this case (Fig. 2) cells were used that had been transiently transfected with both EGFP-zyxin and EGFP-tubulin. As seen in Fig. 2, the recovery of the microtubule network was associated with the disassembly of a large proportion of contacts in the perinuclear region of the cell. Out of 61 nonperipheral contacts, 29 disappeared during microtubule recovery and 32 were retained, but even these became smaller or less intense. Video sequences from a further 3 cells showed similar results.

Bottom Line: The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge.At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, A-5020 Salzburg, Austria.

ABSTRACT
We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein. For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting. Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.

Show MeSH
Related in: MedlinePlus