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The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.

Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2005)

Bottom Line: Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2).Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2.The dynamic remodeling of this ternary complex controls cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunlogy, Centre Medical Universitaire, 1211 Geneva 4, Switzerland.

ABSTRACT
During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

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FRAP of activated integrins. FRAP analysis of β3 integrin dynamics within stationary or sliding peripheral high-intensity focal contacts and de novo formed dot- or streaklike integrin clusters in B16F1 cells. The analysis was performed in stably transfected EGFP-tagged WT (β3-wt), D723A (β3-D723A), or N305T (β3-N305T) cells. FRAP sequences of peripheral focal contacts in control cells (β3-wt; A), Mn2+-treated cells (β3-wt/Mn2+; B), and cells expressing D723A-activated β3 integrin (β3-D723A; C; Videos 1–3). FRAP sequences of de novo formed dot- (D) and streaklike (E) low-intensity integrin clusters in Mn2+-treated cells (Video 4). FRAP sequences of stationary peripheral focal contacts in cells expressing N305T-activated β3 integrin (β3-N305T; F; Video 5). Note the twofold expanded time range in F. FRAP sequence of WT β3 integrins in sliding focal contacts of Mn2+-stimulated cells (β3-wt/Mn2+; G; Video 6). Arrowheads in G indicate the position of the inner edge of the sliding contact before bleaching. (H) Comparison of the FRAP curves of peripheral focal contacts in WT (β3-wt, diamonds) or by mutationally activated β3-EGFP integrins (β3-D723A, squares; β3-N305T, open triangles), respectively. (I) Comparison of FRAP curves of control (diamonds) or Mn2+-treated (open triangles) peripheral high intensity focal contacts, as well as Mn2+-induced de novo, dotlike, low-intensity integrin clusters (squares). Superimposed on this graph is the increase in integrin fluorescence at the inner edge of bleached, inward-sliding focal contacts (filled triangles). Error bars correspond to the standard deviation of three independent experiments with each comprising at least three cells. Bars: (A–C) 7.4 μm; (D and E) 5.4 μm; (F) 8 μm; (G) 10.7 μm.
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fig5: FRAP of activated integrins. FRAP analysis of β3 integrin dynamics within stationary or sliding peripheral high-intensity focal contacts and de novo formed dot- or streaklike integrin clusters in B16F1 cells. The analysis was performed in stably transfected EGFP-tagged WT (β3-wt), D723A (β3-D723A), or N305T (β3-N305T) cells. FRAP sequences of peripheral focal contacts in control cells (β3-wt; A), Mn2+-treated cells (β3-wt/Mn2+; B), and cells expressing D723A-activated β3 integrin (β3-D723A; C; Videos 1–3). FRAP sequences of de novo formed dot- (D) and streaklike (E) low-intensity integrin clusters in Mn2+-treated cells (Video 4). FRAP sequences of stationary peripheral focal contacts in cells expressing N305T-activated β3 integrin (β3-N305T; F; Video 5). Note the twofold expanded time range in F. FRAP sequence of WT β3 integrins in sliding focal contacts of Mn2+-stimulated cells (β3-wt/Mn2+; G; Video 6). Arrowheads in G indicate the position of the inner edge of the sliding contact before bleaching. (H) Comparison of the FRAP curves of peripheral focal contacts in WT (β3-wt, diamonds) or by mutationally activated β3-EGFP integrins (β3-D723A, squares; β3-N305T, open triangles), respectively. (I) Comparison of FRAP curves of control (diamonds) or Mn2+-treated (open triangles) peripheral high intensity focal contacts, as well as Mn2+-induced de novo, dotlike, low-intensity integrin clusters (squares). Superimposed on this graph is the increase in integrin fluorescence at the inner edge of bleached, inward-sliding focal contacts (filled triangles). Error bars correspond to the standard deviation of three independent experiments with each comprising at least three cells. Bars: (A–C) 7.4 μm; (D and E) 5.4 μm; (F) 8 μm; (G) 10.7 μm.

Mentions: In migrating cells, stationary focal complexes at the front of the cell exhibit slow integrin exchange rates, whereas sliding and stationary focal contacts at the rear of the cell demonstrate rapid integrin exchange rates (Ballestrem et al., 2001). To determine a possible link between integrin exchange rates and the integrin affinity switch, we tested whether blocking αvβ3 integrin in high-affinity states modifies the dynamics of integrin exchange in stress fiber–linked peripheral focal contacts exhibiting high integrin fluorescence intensities (Ballestrem et al., 2001). We performed FRAP experiments with WT-, D723A-, N305T-, or Mn2+-activated β3-EGFP integrin–transfected cells (White and Stelzer, 1999; Ballestrem et al., 2001). Integrin activation by the intracellular mutation D723A or treatment with Mn2+ did not affect integrin exchange rates in focal contacts (Fig. 5, A–C, H, and I; and Videos 1–3, available at http://www.jcb.org/cgi/content/full/jcb.200503017/DC1). In contrast, integrin activation by the ectodomain mutation N305T resulted in a fivefold slower integrin exchange rate (Fig. 5, F and H; and Video 5). These data suggest that the rate-limiting step in the integrin exchange in peripheral focal contacts is determined by the flexibility of the integrin ectodomain. It further suggests that the facilitated interactions of the cytoplasmic tail of integrins, with its intracellular adaptors, do not influence the rate of integrin exchange in focal contacts.


The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells.

Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B - J. Cell Biol. (2005)

FRAP of activated integrins. FRAP analysis of β3 integrin dynamics within stationary or sliding peripheral high-intensity focal contacts and de novo formed dot- or streaklike integrin clusters in B16F1 cells. The analysis was performed in stably transfected EGFP-tagged WT (β3-wt), D723A (β3-D723A), or N305T (β3-N305T) cells. FRAP sequences of peripheral focal contacts in control cells (β3-wt; A), Mn2+-treated cells (β3-wt/Mn2+; B), and cells expressing D723A-activated β3 integrin (β3-D723A; C; Videos 1–3). FRAP sequences of de novo formed dot- (D) and streaklike (E) low-intensity integrin clusters in Mn2+-treated cells (Video 4). FRAP sequences of stationary peripheral focal contacts in cells expressing N305T-activated β3 integrin (β3-N305T; F; Video 5). Note the twofold expanded time range in F. FRAP sequence of WT β3 integrins in sliding focal contacts of Mn2+-stimulated cells (β3-wt/Mn2+; G; Video 6). Arrowheads in G indicate the position of the inner edge of the sliding contact before bleaching. (H) Comparison of the FRAP curves of peripheral focal contacts in WT (β3-wt, diamonds) or by mutationally activated β3-EGFP integrins (β3-D723A, squares; β3-N305T, open triangles), respectively. (I) Comparison of FRAP curves of control (diamonds) or Mn2+-treated (open triangles) peripheral high intensity focal contacts, as well as Mn2+-induced de novo, dotlike, low-intensity integrin clusters (squares). Superimposed on this graph is the increase in integrin fluorescence at the inner edge of bleached, inward-sliding focal contacts (filled triangles). Error bars correspond to the standard deviation of three independent experiments with each comprising at least three cells. Bars: (A–C) 7.4 μm; (D and E) 5.4 μm; (F) 8 μm; (G) 10.7 μm.
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fig5: FRAP of activated integrins. FRAP analysis of β3 integrin dynamics within stationary or sliding peripheral high-intensity focal contacts and de novo formed dot- or streaklike integrin clusters in B16F1 cells. The analysis was performed in stably transfected EGFP-tagged WT (β3-wt), D723A (β3-D723A), or N305T (β3-N305T) cells. FRAP sequences of peripheral focal contacts in control cells (β3-wt; A), Mn2+-treated cells (β3-wt/Mn2+; B), and cells expressing D723A-activated β3 integrin (β3-D723A; C; Videos 1–3). FRAP sequences of de novo formed dot- (D) and streaklike (E) low-intensity integrin clusters in Mn2+-treated cells (Video 4). FRAP sequences of stationary peripheral focal contacts in cells expressing N305T-activated β3 integrin (β3-N305T; F; Video 5). Note the twofold expanded time range in F. FRAP sequence of WT β3 integrins in sliding focal contacts of Mn2+-stimulated cells (β3-wt/Mn2+; G; Video 6). Arrowheads in G indicate the position of the inner edge of the sliding contact before bleaching. (H) Comparison of the FRAP curves of peripheral focal contacts in WT (β3-wt, diamonds) or by mutationally activated β3-EGFP integrins (β3-D723A, squares; β3-N305T, open triangles), respectively. (I) Comparison of FRAP curves of control (diamonds) or Mn2+-treated (open triangles) peripheral high intensity focal contacts, as well as Mn2+-induced de novo, dotlike, low-intensity integrin clusters (squares). Superimposed on this graph is the increase in integrin fluorescence at the inner edge of bleached, inward-sliding focal contacts (filled triangles). Error bars correspond to the standard deviation of three independent experiments with each comprising at least three cells. Bars: (A–C) 7.4 μm; (D and E) 5.4 μm; (F) 8 μm; (G) 10.7 μm.
Mentions: In migrating cells, stationary focal complexes at the front of the cell exhibit slow integrin exchange rates, whereas sliding and stationary focal contacts at the rear of the cell demonstrate rapid integrin exchange rates (Ballestrem et al., 2001). To determine a possible link between integrin exchange rates and the integrin affinity switch, we tested whether blocking αvβ3 integrin in high-affinity states modifies the dynamics of integrin exchange in stress fiber–linked peripheral focal contacts exhibiting high integrin fluorescence intensities (Ballestrem et al., 2001). We performed FRAP experiments with WT-, D723A-, N305T-, or Mn2+-activated β3-EGFP integrin–transfected cells (White and Stelzer, 1999; Ballestrem et al., 2001). Integrin activation by the intracellular mutation D723A or treatment with Mn2+ did not affect integrin exchange rates in focal contacts (Fig. 5, A–C, H, and I; and Videos 1–3, available at http://www.jcb.org/cgi/content/full/jcb.200503017/DC1). In contrast, integrin activation by the ectodomain mutation N305T resulted in a fivefold slower integrin exchange rate (Fig. 5, F and H; and Video 5). These data suggest that the rate-limiting step in the integrin exchange in peripheral focal contacts is determined by the flexibility of the integrin ectodomain. It further suggests that the facilitated interactions of the cytoplasmic tail of integrins, with its intracellular adaptors, do not influence the rate of integrin exchange in focal contacts.

Bottom Line: Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2).Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2.The dynamic remodeling of this ternary complex controls cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunlogy, Centre Medical Universitaire, 1211 Geneva 4, Switzerland.

ABSTRACT
During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

Show MeSH
Related in: MedlinePlus