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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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Schematic illustration of substrate contact dynamics in a moving fibroblast. (Top) Substrate contacts are initiated in the protruding and ruffling lamellipodium (Lam, ruf). Two classes of primary contacts are depicted: punctate focal complexes (fc) and linear contacts associated with some microspike bundles (ms/c). Their formation is associated with the activation of Rac and Cdc42, respectively. Each type of primary contact can develop into a precursor of a focal adhesion (pFA). Further abbreviations: iFA, intermediate focal adhesion in the body of the cell; tFA, focal adhesion at a trailing cell edge. (Bottom) Four types of contact sites with different strengths of anchorage to the substrate (anchors) are depicted and correspond to those in the upper diagram. All sites rely on contractility for their maintenance, indicated by different levels of myosin II–dependent tension (T and t). For precursor (pFA) and mature focal adhesions (iFA, tFA), myosin activation depends on Rho-kinase. The contractility of focal complexes is Rho-kinase independent. Microtubules (MT) interface with contact sites and modulate their turnover by locally inhibiting contractility. The relaxing dose is controlled by the total number and frequency of microtubule targeting events. Focal complexes may or may not be targeted by microtubules.
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Figure 9: Schematic illustration of substrate contact dynamics in a moving fibroblast. (Top) Substrate contacts are initiated in the protruding and ruffling lamellipodium (Lam, ruf). Two classes of primary contacts are depicted: punctate focal complexes (fc) and linear contacts associated with some microspike bundles (ms/c). Their formation is associated with the activation of Rac and Cdc42, respectively. Each type of primary contact can develop into a precursor of a focal adhesion (pFA). Further abbreviations: iFA, intermediate focal adhesion in the body of the cell; tFA, focal adhesion at a trailing cell edge. (Bottom) Four types of contact sites with different strengths of anchorage to the substrate (anchors) are depicted and correspond to those in the upper diagram. All sites rely on contractility for their maintenance, indicated by different levels of myosin II–dependent tension (T and t). For precursor (pFA) and mature focal adhesions (iFA, tFA), myosin activation depends on Rho-kinase. The contractility of focal complexes is Rho-kinase independent. Microtubules (MT) interface with contact sites and modulate their turnover by locally inhibiting contractility. The relaxing dose is controlled by the total number and frequency of microtubule targeting events. Focal complexes may or may not be targeted by microtubules.

Mentions: As we have seen, the retraction of an adhesion site at a trailing cell edge may be preceded by multiple targeting events. These findings are generally supported by the statistical characterization of regional microtubule dynamics by Wadsworth 1999; her studies showed that microtubule ends close to nonmotile edges exhibited shorter excursions and frequent catastrophes, whereas longer excursions and fewer catastrophes occurred in protruding regions. The shorter and more frequent excursions would correspond to what we observe at cell edges destined for retraction. We conclude that a single targeting event serves to provide a quantum relaxation dose and that further doses, as required, must be supplied by freshly charged microtubule ends in subsequent targeting forays. By modulating the number of targeting events, differential relaxation effects may then be exerted on contact assemblies in different regions of a cell. In this way, microtubule signaling at contact sites could modulate the substrate contact pattern of a cell and thereby determine its polarity (Fig. 9).


Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

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

Schematic illustration of substrate contact dynamics in a moving fibroblast. (Top) Substrate contacts are initiated in the protruding and ruffling lamellipodium (Lam, ruf). Two classes of primary contacts are depicted: punctate focal complexes (fc) and linear contacts associated with some microspike bundles (ms/c). Their formation is associated with the activation of Rac and Cdc42, respectively. Each type of primary contact can develop into a precursor of a focal adhesion (pFA). Further abbreviations: iFA, intermediate focal adhesion in the body of the cell; tFA, focal adhesion at a trailing cell edge. (Bottom) Four types of contact sites with different strengths of anchorage to the substrate (anchors) are depicted and correspond to those in the upper diagram. All sites rely on contractility for their maintenance, indicated by different levels of myosin II–dependent tension (T and t). For precursor (pFA) and mature focal adhesions (iFA, tFA), myosin activation depends on Rho-kinase. The contractility of focal complexes is Rho-kinase independent. Microtubules (MT) interface with contact sites and modulate their turnover by locally inhibiting contractility. The relaxing dose is controlled by the total number and frequency of microtubule targeting events. Focal complexes may or may not be targeted by microtubules.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169483&req=5

Figure 9: Schematic illustration of substrate contact dynamics in a moving fibroblast. (Top) Substrate contacts are initiated in the protruding and ruffling lamellipodium (Lam, ruf). Two classes of primary contacts are depicted: punctate focal complexes (fc) and linear contacts associated with some microspike bundles (ms/c). Their formation is associated with the activation of Rac and Cdc42, respectively. Each type of primary contact can develop into a precursor of a focal adhesion (pFA). Further abbreviations: iFA, intermediate focal adhesion in the body of the cell; tFA, focal adhesion at a trailing cell edge. (Bottom) Four types of contact sites with different strengths of anchorage to the substrate (anchors) are depicted and correspond to those in the upper diagram. All sites rely on contractility for their maintenance, indicated by different levels of myosin II–dependent tension (T and t). For precursor (pFA) and mature focal adhesions (iFA, tFA), myosin activation depends on Rho-kinase. The contractility of focal complexes is Rho-kinase independent. Microtubules (MT) interface with contact sites and modulate their turnover by locally inhibiting contractility. The relaxing dose is controlled by the total number and frequency of microtubule targeting events. Focal complexes may or may not be targeted by microtubules.
Mentions: As we have seen, the retraction of an adhesion site at a trailing cell edge may be preceded by multiple targeting events. These findings are generally supported by the statistical characterization of regional microtubule dynamics by Wadsworth 1999; her studies showed that microtubule ends close to nonmotile edges exhibited shorter excursions and frequent catastrophes, whereas longer excursions and fewer catastrophes occurred in protruding regions. The shorter and more frequent excursions would correspond to what we observe at cell edges destined for retraction. We conclude that a single targeting event serves to provide a quantum relaxation dose and that further doses, as required, must be supplied by freshly charged microtubule ends in subsequent targeting forays. By modulating the number of targeting events, differential relaxation effects may then be exerted on contact assemblies in different regions of a cell. In this way, microtubule signaling at contact sites could modulate the substrate contact pattern of a cell and thereby determine its polarity (Fig. 9).

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