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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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Compact integrin with and without THD roughly segregate into separate classes. Panels are images of 50 × 50-nm fields. Images of nanodiscs containing compact integrins from sample prepared in the presence or absence of THD were grouped together, and the pooled images were then classified and averaged iteratively. 20 classes were generated during the iterative process (parent classes) and at the final step, each class was split to two subclasses (daughter subclasses) to verify the internal consistency of the class averages. Each bracket contains two daughter subclasses from a parent class. Little or no difference between the daughter subclasses indicated robust alignment and classification by the iterative procedure. Within each class, the percentage of images from samples containing either integrin nanodiscs in the presence or absence of THD are depicted in a pie chart. Each resulting class average in which the integrin structure was clearly defined was greatly enriched in images from the integrin nanodiscs alone or from the THD-containing integrin nanodisc population. For example, the first row of images shows classes dominated by images from the integrin alone sample (up to 89%). In contrast, the second and third row are classes dominated by images from the sample containing added THD (up to 91%). In the classes in which the integrin structure is not clearly resolved, both samples were more evenly represented (fourth row). 8 classes of empty nanodiscs were omitted from this figure.
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fig8: Compact integrin with and without THD roughly segregate into separate classes. Panels are images of 50 × 50-nm fields. Images of nanodiscs containing compact integrins from sample prepared in the presence or absence of THD were grouped together, and the pooled images were then classified and averaged iteratively. 20 classes were generated during the iterative process (parent classes) and at the final step, each class was split to two subclasses (daughter subclasses) to verify the internal consistency of the class averages. Each bracket contains two daughter subclasses from a parent class. Little or no difference between the daughter subclasses indicated robust alignment and classification by the iterative procedure. Within each class, the percentage of images from samples containing either integrin nanodiscs in the presence or absence of THD are depicted in a pie chart. Each resulting class average in which the integrin structure was clearly defined was greatly enriched in images from the integrin nanodiscs alone or from the THD-containing integrin nanodisc population. For example, the first row of images shows classes dominated by images from the integrin alone sample (up to 89%). In contrast, the second and third row are classes dominated by images from the sample containing added THD (up to 91%). In the classes in which the integrin structure is not clearly resolved, both samples were more evenly represented (fourth row). 8 classes of empty nanodiscs were omitted from this figure.

Mentions: In the presence of THD, the compact integrin densities appeared slightly less compact than when THD is absent (Fig. 7, A and B). We verified this observation by pooling images of compact integrins in the presence or absence of THD, and subjecting this mixed pool to image analysis of alignment and classification. The slightly less compact forms were strongly associated with the presence of THD (Fig. 8). This result suggests that THD may also induce a more subtle difference in conformation.


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)

Compact integrin with and without THD roughly segregate into separate classes. Panels are images of 50 × 50-nm fields. Images of nanodiscs containing compact integrins from sample prepared in the presence or absence of THD were grouped together, and the pooled images were then classified and averaged iteratively. 20 classes were generated during the iterative process (parent classes) and at the final step, each class was split to two subclasses (daughter subclasses) to verify the internal consistency of the class averages. Each bracket contains two daughter subclasses from a parent class. Little or no difference between the daughter subclasses indicated robust alignment and classification by the iterative procedure. Within each class, the percentage of images from samples containing either integrin nanodiscs in the presence or absence of THD are depicted in a pie chart. Each resulting class average in which the integrin structure was clearly defined was greatly enriched in images from the integrin nanodiscs alone or from the THD-containing integrin nanodisc population. For example, the first row of images shows classes dominated by images from the integrin alone sample (up to 89%). In contrast, the second and third row are classes dominated by images from the sample containing added THD (up to 91%). In the classes in which the integrin structure is not clearly resolved, both samples were more evenly represented (fourth row). 8 classes of empty nanodiscs were omitted from this figure.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Compact integrin with and without THD roughly segregate into separate classes. Panels are images of 50 × 50-nm fields. Images of nanodiscs containing compact integrins from sample prepared in the presence or absence of THD were grouped together, and the pooled images were then classified and averaged iteratively. 20 classes were generated during the iterative process (parent classes) and at the final step, each class was split to two subclasses (daughter subclasses) to verify the internal consistency of the class averages. Each bracket contains two daughter subclasses from a parent class. Little or no difference between the daughter subclasses indicated robust alignment and classification by the iterative procedure. Within each class, the percentage of images from samples containing either integrin nanodiscs in the presence or absence of THD are depicted in a pie chart. Each resulting class average in which the integrin structure was clearly defined was greatly enriched in images from the integrin nanodiscs alone or from the THD-containing integrin nanodisc population. For example, the first row of images shows classes dominated by images from the integrin alone sample (up to 89%). In contrast, the second and third row are classes dominated by images from the sample containing added THD (up to 91%). In the classes in which the integrin structure is not clearly resolved, both samples were more evenly represented (fourth row). 8 classes of empty nanodiscs were omitted from this figure.
Mentions: In the presence of THD, the compact integrin densities appeared slightly less compact than when THD is absent (Fig. 7, A and B). We verified this observation by pooling images of compact integrins in the presence or absence of THD, and subjecting this mixed pool to image analysis of alignment and classification. The slightly less compact forms were strongly associated with the presence of THD (Fig. 8). This result suggests that THD may also induce a more subtle difference in conformation.

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