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Demonstration of catch bonds between an integrin and its ligand.

Kong F, García AJ, Mould AP, Humphries MJ, Zhu C - J. Cell Biol. (2009)

Bottom Line: Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules.How force regulates integrin-ligand dissociation is unclear.Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range.

View Article: PubMed Central - PubMed

Affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.

ABSTRACT
Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

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Lifetimes of FN–α5β1 bonds. (A–C) Plots of lifetime versus force of α5β1-Fc–functionalized Petri dish dissociating from FNIII7–10-coated cantilever tips (circles) in Ca2+/Mg2+ (A), Mg2+/EGTA (B), and Mn2+ (C). (D) A qualitatively similar plot (triangles) of mα5β1 reconstituted into lipid bilayer dissociating from FNIII7–10-coated cantilever tips in Mg2+/EGTA confirmed the catch bond observation. Also plotted in A–C is the lifetime versus force curve of Fc dissociating from GG-7 (squares). For B and C, the black and gray curves overlap at forces >30 pN, indicating that measured lifetimes beyond 30 pN were caused by Fc–GG-7 dissociation instead of FNIII7–10–α5β1-Fc dissociation. Also shown in C is a lifetime versus force plot (diamonds) of α5β1-Fc–functionalized Petri dish dissociating from BSA-coated cantilever tips measured in Mn2+. (E) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII7–10 and α5β1-Fc or between α5β1-Fc and GG-7. (F) Schematic of the molecular arrangement for experiments that measured the capturing strength of the Fc–GG-7 interaction. Error bars indicate mean ± SEM.
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fig3: Lifetimes of FN–α5β1 bonds. (A–C) Plots of lifetime versus force of α5β1-Fc–functionalized Petri dish dissociating from FNIII7–10-coated cantilever tips (circles) in Ca2+/Mg2+ (A), Mg2+/EGTA (B), and Mn2+ (C). (D) A qualitatively similar plot (triangles) of mα5β1 reconstituted into lipid bilayer dissociating from FNIII7–10-coated cantilever tips in Mg2+/EGTA confirmed the catch bond observation. Also plotted in A–C is the lifetime versus force curve of Fc dissociating from GG-7 (squares). For B and C, the black and gray curves overlap at forces >30 pN, indicating that measured lifetimes beyond 30 pN were caused by Fc–GG-7 dissociation instead of FNIII7–10–α5β1-Fc dissociation. Also shown in C is a lifetime versus force plot (diamonds) of α5β1-Fc–functionalized Petri dish dissociating from BSA-coated cantilever tips measured in Mn2+. (E) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII7–10 and α5β1-Fc or between α5β1-Fc and GG-7. (F) Schematic of the molecular arrangement for experiments that measured the capturing strength of the Fc–GG-7 interaction. Error bars indicate mean ± SEM.

Mentions: Mechanical regulation of FN–α5β1 dissociation was quantified by the force dependence of mean lifetimes of mostly single FNIII7–10–α5β1 bonds measured in each of the three cation conditions (Fig. 3, A–C, circles). As force increased, lifetime first decreased to a minimum, then increased to a maximum, and decreased again, exhibiting a triphasic transition from slip bonds to catch bonds and then to slip bonds again. The first slip bond regimen was most clearly observed in Ca2+/Mg2+ (Fig. 3 A) but became less pronounced in Mg2+/EGTA (Fig. 3 B) and Mn2+ (Fig. 3 C). The much less frequent nonspecific binding between BSA-coated cantilevers and polystyrene Petri dishes functionalized with α5β1-Fc contributed negligibly to these lifetime versus force curves, as most of these events were ruptured at forces <20 pN (Fig. S2 A), and those that survived the ramping had very short lifetimes (Fig. 3 C, diamonds).


Demonstration of catch bonds between an integrin and its ligand.

Kong F, García AJ, Mould AP, Humphries MJ, Zhu C - J. Cell Biol. (2009)

Lifetimes of FN–α5β1 bonds. (A–C) Plots of lifetime versus force of α5β1-Fc–functionalized Petri dish dissociating from FNIII7–10-coated cantilever tips (circles) in Ca2+/Mg2+ (A), Mg2+/EGTA (B), and Mn2+ (C). (D) A qualitatively similar plot (triangles) of mα5β1 reconstituted into lipid bilayer dissociating from FNIII7–10-coated cantilever tips in Mg2+/EGTA confirmed the catch bond observation. Also plotted in A–C is the lifetime versus force curve of Fc dissociating from GG-7 (squares). For B and C, the black and gray curves overlap at forces >30 pN, indicating that measured lifetimes beyond 30 pN were caused by Fc–GG-7 dissociation instead of FNIII7–10–α5β1-Fc dissociation. Also shown in C is a lifetime versus force plot (diamonds) of α5β1-Fc–functionalized Petri dish dissociating from BSA-coated cantilever tips measured in Mn2+. (E) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII7–10 and α5β1-Fc or between α5β1-Fc and GG-7. (F) Schematic of the molecular arrangement for experiments that measured the capturing strength of the Fc–GG-7 interaction. Error bars indicate mean ± SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC2712956&req=5

fig3: Lifetimes of FN–α5β1 bonds. (A–C) Plots of lifetime versus force of α5β1-Fc–functionalized Petri dish dissociating from FNIII7–10-coated cantilever tips (circles) in Ca2+/Mg2+ (A), Mg2+/EGTA (B), and Mn2+ (C). (D) A qualitatively similar plot (triangles) of mα5β1 reconstituted into lipid bilayer dissociating from FNIII7–10-coated cantilever tips in Mg2+/EGTA confirmed the catch bond observation. Also plotted in A–C is the lifetime versus force curve of Fc dissociating from GG-7 (squares). For B and C, the black and gray curves overlap at forces >30 pN, indicating that measured lifetimes beyond 30 pN were caused by Fc–GG-7 dissociation instead of FNIII7–10–α5β1-Fc dissociation. Also shown in C is a lifetime versus force plot (diamonds) of α5β1-Fc–functionalized Petri dish dissociating from BSA-coated cantilever tips measured in Mn2+. (E) Schematic of the molecular arrangement indicating possible dissociation loci between FNIII7–10 and α5β1-Fc or between α5β1-Fc and GG-7. (F) Schematic of the molecular arrangement for experiments that measured the capturing strength of the Fc–GG-7 interaction. Error bars indicate mean ± SEM.
Mentions: Mechanical regulation of FN–α5β1 dissociation was quantified by the force dependence of mean lifetimes of mostly single FNIII7–10–α5β1 bonds measured in each of the three cation conditions (Fig. 3, A–C, circles). As force increased, lifetime first decreased to a minimum, then increased to a maximum, and decreased again, exhibiting a triphasic transition from slip bonds to catch bonds and then to slip bonds again. The first slip bond regimen was most clearly observed in Ca2+/Mg2+ (Fig. 3 A) but became less pronounced in Mg2+/EGTA (Fig. 3 B) and Mn2+ (Fig. 3 C). The much less frequent nonspecific binding between BSA-coated cantilevers and polystyrene Petri dishes functionalized with α5β1-Fc contributed negligibly to these lifetime versus force curves, as most of these events were ruptured at forces <20 pN (Fig. S2 A), and those that survived the ramping had very short lifetimes (Fig. 3 C, diamonds).

Bottom Line: Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules.How force regulates integrin-ligand dissociation is unclear.Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range.

View Article: PubMed Central - PubMed

Affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.

ABSTRACT
Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

Show MeSH
Related in: MedlinePlus