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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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Contact targeting and retraction after recovery from local application of nocodazole. Cell shown was cotransfected with zyxin-EGFP and tubulin-EGFP and treated on one edge with 50 μg/ml nocodazole applied through a microneedle to locally depolymerize microtubules. Video sequences show recovery after removal of needle at time 0′00″. Arrowheads indicate sequential targeting events at peripheral contact sites and asterisks the positions of contacts that had already been targeted. Time is in minutes and seconds. Bar, 5 μM. Video available at http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1
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Figure 6: Contact targeting and retraction after recovery from local application of nocodazole. Cell shown was cotransfected with zyxin-EGFP and tubulin-EGFP and treated on one edge with 50 μg/ml nocodazole applied through a microneedle to locally depolymerize microtubules. Video sequences show recovery after removal of needle at time 0′00″. Arrowheads indicate sequential targeting events at peripheral contact sites and asterisks the positions of contacts that had already been targeted. Time is in minutes and seconds. Bar, 5 μM. Video available at http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1

Mentions: Video 8: Fig. 6. http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1


Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

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

Contact targeting and retraction after recovery from local application of nocodazole. Cell shown was cotransfected with zyxin-EGFP and tubulin-EGFP and treated on one edge with 50 μg/ml nocodazole applied through a microneedle to locally depolymerize microtubules. Video sequences show recovery after removal of needle at time 0′00″. Arrowheads indicate sequential targeting events at peripheral contact sites and asterisks the positions of contacts that had already been targeted. Time is in minutes and seconds. Bar, 5 μM. Video available at http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Contact targeting and retraction after recovery from local application of nocodazole. Cell shown was cotransfected with zyxin-EGFP and tubulin-EGFP and treated on one edge with 50 μg/ml nocodazole applied through a microneedle to locally depolymerize microtubules. Video sequences show recovery after removal of needle at time 0′00″. Arrowheads indicate sequential targeting events at peripheral contact sites and asterisks the positions of contacts that had already been targeted. Time is in minutes and seconds. Bar, 5 μM. Video available at http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1
Mentions: Video 8: Fig. 6. http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1

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