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Recreation of the terminal events in physiological integrin activation.

Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA, Sligar SG, Taylor KA, Ginsberg MH - J. Cell Biol. (2010)

Bottom Line: Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3.Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins.Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

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THD activates integrins in nanodiscs. (A) THD increases the binding of PAC1 to captured integrin nanodiscs. The inset shows the configuration of the assay, which is described in the Materials and methods section. The activation indices were calculated as (L − L0) / (Lmax − L0) where L = luminescence, L0 = luminescence in presence of 1 µM eptifibatide, and Lmax = luminescence in the presence of anti-LIBS6 activating anti-β3 antibody. (B) Mutations of THD reduce its capacity to activate integrins in nanodiscs. The increase in activation is calculated as in Fig. 3 B. A mutation that reduces membrane binding (K322D) or one that blocks binding to the integrin-β3 tail (L325R) reduced capacity of THD to activate αIIbβ3 in nanodiscs. (C) EM images of negatively stained fibrin and integrin nanodiscs alone. Relatively few integrin nanodiscs are bound to the fibrin. Bar, 100 nm. (D) EM images of negatively stained fibrin and integrin nanodiscs in the presence of 5 µM THD showing an increase in the number of bound integrin nanodiscs when THD is present. The top insets show enlarged views of integrin nanodiscs bound to the fibrin. Single fibrin-bound integrin nanodiscs are clearly visible, indicating that the increase in binding is due to increased monomer affinity rather than clustering. The bottom insets show enlarged views of unbound integrin nanodiscs. Both compact and extended conformations are visible. Bar, 100 nm. (E) The local concentrations of nanodiscs were estimated from EM images by manual enumeration of nanodiscs/µm2. The number of bound integrin nanodiscs per 10-nm length of fibrin strand was plotted for each concentration. The presence of THD increased the number of bound integrin nanodiscs. Error bars = SE (n = 4) for each concentration.
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fig6: THD activates integrins in nanodiscs. (A) THD increases the binding of PAC1 to captured integrin nanodiscs. The inset shows the configuration of the assay, which is described in the Materials and methods section. The activation indices were calculated as (L − L0) / (Lmax − L0) where L = luminescence, L0 = luminescence in presence of 1 µM eptifibatide, and Lmax = luminescence in the presence of anti-LIBS6 activating anti-β3 antibody. (B) Mutations of THD reduce its capacity to activate integrins in nanodiscs. The increase in activation is calculated as in Fig. 3 B. A mutation that reduces membrane binding (K322D) or one that blocks binding to the integrin-β3 tail (L325R) reduced capacity of THD to activate αIIbβ3 in nanodiscs. (C) EM images of negatively stained fibrin and integrin nanodiscs alone. Relatively few integrin nanodiscs are bound to the fibrin. Bar, 100 nm. (D) EM images of negatively stained fibrin and integrin nanodiscs in the presence of 5 µM THD showing an increase in the number of bound integrin nanodiscs when THD is present. The top insets show enlarged views of integrin nanodiscs bound to the fibrin. Single fibrin-bound integrin nanodiscs are clearly visible, indicating that the increase in binding is due to increased monomer affinity rather than clustering. The bottom insets show enlarged views of unbound integrin nanodiscs. Both compact and extended conformations are visible. Bar, 100 nm. (E) The local concentrations of nanodiscs were estimated from EM images by manual enumeration of nanodiscs/µm2. The number of bound integrin nanodiscs per 10-nm length of fibrin strand was plotted for each concentration. The presence of THD increased the number of bound integrin nanodiscs. Error bars = SE (n = 4) for each concentration.

Mentions: We captured the αIIbβ3 nanodiscs with an anti-β3 antibody and assessed their activation state by PAC1 binding (Fig. 6 A, top). We reasoned that because integrin nanodiscs are immobilized before addition of THD and ligands, neither THD nor ligand can induce further integrin clustering. THD induced a concentration-dependent increase in PAC1 binding (Fig. 6 A). To confirm that the increase in integrin activation is a result of physiologically relevant THD–β3 interaction, we compared the effect of THD, THD(L325R), a mutation that disrupts THD-β3 membrane proximal interaction site, and THD(K322D), a mutation that reduced THD–membrane interaction (Fig. 4 D) (Wegener et al., 2007). Both mutations reduced the capacity of THD to activate (Fig. 6 B). Thus, THD activates integrin αIIbβ3 in nanodiscs under conditions which disfavor clustering.


Recreation of the terminal events in physiological integrin activation.

Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA, Sligar SG, Taylor KA, Ginsberg MH - J. Cell Biol. (2010)

THD activates integrins in nanodiscs. (A) THD increases the binding of PAC1 to captured integrin nanodiscs. The inset shows the configuration of the assay, which is described in the Materials and methods section. The activation indices were calculated as (L − L0) / (Lmax − L0) where L = luminescence, L0 = luminescence in presence of 1 µM eptifibatide, and Lmax = luminescence in the presence of anti-LIBS6 activating anti-β3 antibody. (B) Mutations of THD reduce its capacity to activate integrins in nanodiscs. The increase in activation is calculated as in Fig. 3 B. A mutation that reduces membrane binding (K322D) or one that blocks binding to the integrin-β3 tail (L325R) reduced capacity of THD to activate αIIbβ3 in nanodiscs. (C) EM images of negatively stained fibrin and integrin nanodiscs alone. Relatively few integrin nanodiscs are bound to the fibrin. Bar, 100 nm. (D) EM images of negatively stained fibrin and integrin nanodiscs in the presence of 5 µM THD showing an increase in the number of bound integrin nanodiscs when THD is present. The top insets show enlarged views of integrin nanodiscs bound to the fibrin. Single fibrin-bound integrin nanodiscs are clearly visible, indicating that the increase in binding is due to increased monomer affinity rather than clustering. The bottom insets show enlarged views of unbound integrin nanodiscs. Both compact and extended conformations are visible. Bar, 100 nm. (E) The local concentrations of nanodiscs were estimated from EM images by manual enumeration of nanodiscs/µm2. The number of bound integrin nanodiscs per 10-nm length of fibrin strand was plotted for each concentration. The presence of THD increased the number of bound integrin nanodiscs. Error bars = SE (n = 4) for each concentration.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812850&req=5

fig6: THD activates integrins in nanodiscs. (A) THD increases the binding of PAC1 to captured integrin nanodiscs. The inset shows the configuration of the assay, which is described in the Materials and methods section. The activation indices were calculated as (L − L0) / (Lmax − L0) where L = luminescence, L0 = luminescence in presence of 1 µM eptifibatide, and Lmax = luminescence in the presence of anti-LIBS6 activating anti-β3 antibody. (B) Mutations of THD reduce its capacity to activate integrins in nanodiscs. The increase in activation is calculated as in Fig. 3 B. A mutation that reduces membrane binding (K322D) or one that blocks binding to the integrin-β3 tail (L325R) reduced capacity of THD to activate αIIbβ3 in nanodiscs. (C) EM images of negatively stained fibrin and integrin nanodiscs alone. Relatively few integrin nanodiscs are bound to the fibrin. Bar, 100 nm. (D) EM images of negatively stained fibrin and integrin nanodiscs in the presence of 5 µM THD showing an increase in the number of bound integrin nanodiscs when THD is present. The top insets show enlarged views of integrin nanodiscs bound to the fibrin. Single fibrin-bound integrin nanodiscs are clearly visible, indicating that the increase in binding is due to increased monomer affinity rather than clustering. The bottom insets show enlarged views of unbound integrin nanodiscs. Both compact and extended conformations are visible. Bar, 100 nm. (E) The local concentrations of nanodiscs were estimated from EM images by manual enumeration of nanodiscs/µm2. The number of bound integrin nanodiscs per 10-nm length of fibrin strand was plotted for each concentration. The presence of THD increased the number of bound integrin nanodiscs. Error bars = SE (n = 4) for each concentration.
Mentions: We captured the αIIbβ3 nanodiscs with an anti-β3 antibody and assessed their activation state by PAC1 binding (Fig. 6 A, top). We reasoned that because integrin nanodiscs are immobilized before addition of THD and ligands, neither THD nor ligand can induce further integrin clustering. THD induced a concentration-dependent increase in PAC1 binding (Fig. 6 A). To confirm that the increase in integrin activation is a result of physiologically relevant THD–β3 interaction, we compared the effect of THD, THD(L325R), a mutation that disrupts THD-β3 membrane proximal interaction site, and THD(K322D), a mutation that reduced THD–membrane interaction (Fig. 4 D) (Wegener et al., 2007). Both mutations reduced the capacity of THD to activate (Fig. 6 B). Thus, THD activates integrin αIIbβ3 in nanodiscs under conditions which disfavor clustering.

Bottom Line: Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3.Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins.Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

Show MeSH
Related in: MedlinePlus