Dissecting subsecond cadherin bound states reveals an efficient way for cells to achieve ultrafast probing of their environment.

Cells continuously probe their environment with membrane receptors, achieving subsecond adaptation of their behaviour [Diez, G., Gerisch, G., Anderson, K., Müller-Taubenberger, A. and Bretschneider, T. (2006) Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc. Natl. Acad. Sci. USA 102, 7601-7606, Shamri, R., Grabovsky, V., Gauguet, J.M., Feigelson, S., Manevich, E., Kolanus, W., Robinson, M.K., Staunton, D.E., von Andrian, U.H. and Alon, R. (2005) Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat. Immunol. 6, 497-606, Jiang, G., Huang, A.H., Cai, Y., Tanase, M. and Sheetz, M.P. (2006) Rigidity sensing at the leading edge through alpha(V)beta(3) integrins and RPTPalpha. Biophys. J. 90, 1804-2006]. Recently, several receptors, including cadherins, were found to bind ligands with a lifetime of order of one second. Here we show at the single molecule level that homotypic C-cadherin association involves transient intermediates lasting less than a few tens of milliseconds. Further, these intermediates transitionned towards more stable states with a kinetic rate displaying exponential decrease with piconewton forces. These features enable cells to detect ligands or measure surrounding mechanical behaviour within a fraction of a second, much more rapidly than was previously thought.

Mechanism and dynamics of cadherin adhesion.

Cadherins are essential cell adhesion molecules involved in tissue morphogenesis and the maintenance of tissue architecture in adults. The adhesion and selectivity functions of cadherins are located in their extracellular regions. Biophysical studies show that the adhesive activity is not confined to a single interface. Instead, multiple cadherin domains contribute to binding. By contrast, the specificity-determining site maps to the N-terminal domains, which adhere by the reciprocal binding of Trp2 residues from opposing proteins. Structural cooperativity can transmit the effects of subtle structural changes or ligand binding over large distances in the protein. Increasingly, studies show that differential cadherin-mediated adhesion, rather than exclusive homophilic binding between identical cadherins, direct cell segregation and the organization of tissue interfaces during morphogenesis. Force measurements quantified both kinetic and strength differences between different classical cadherins that may underlie cell sorting behavior. Despite the complex adhesion mechanisms and differences in binding properties, cadherin-mediated cell adhesion is also regulated by many other biochemical processes. Elucidating the mechanisms by which cadherins organize cell junctions and tissue architecture requires not only quantitative, mechanistic investigations of cadherin function but also investigations of the biochemical and cellular processes that can modulate those functions.

Engineered protein a for the orientational control of immobilized proteins.

This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A. The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports. The fragment was expressed at high yield in E. coli and purified to homogeneity. When selectively immobilized to histidine binding matrices, the protein A fragment exhibits high affinity for soluble IgG. We further demonstrate from adsorption isotherms that the receptor exhibits a homogeneous, high affinity population at densities where steric crowding between large ligands does not affect the apparent receptor affinity. This engineered receptor is appropriate for a range of applications including sensor design or those using immobilized Fc-tagged proteins.