Micropipette manipulation measurements quantified the pre-steady state binding kinetics between cell pairs mediated by Xenopus cleavage stage cadherin. The time-dependence of the intercellular binding probability exhibits a fast forming, low probability binding state, which transitions to a slower forming, high probability state. The biphasic kinetics are independent of the cytoplasmic region, but the transition to the high probability state requires the third extracellular domain EC3. Deleting either EC3 or EC3-5, or substituting Trp 2 for Ala reduces the binding curves to a simple, monophasic rise in binding probability to a limiting plateau, as predicted for a single site binding mechanism. The two stage cadherin binding process reported here directly parallels previous biophysical studies, and confirms that the cadherin ectodomain governs the initial intercellular adhesion dynamics.The cadherin family of adhesion proteins mediates cell-cell interactions in all solid tissues (1). These calcium-dependent cell surface glycoproteins are critical for morphogenesis and for directing the segregation of cells into distinct tissues during development. In addition to their mechanical role as adhesion molecules, they are also signaling proteins that influence cytoskeletal reorganization, cell migration, and proliferation through interactions with other cadherins and possibly with other cell surface receptors.Classical cadherins are the most extensively studied of the cadherin superfamily. The proteins comprise an extracellular region, and single-pass transmembrane domain, and a cytoplasmic domain (2). The extracellular region embeds the adhesive and selectivity functions of the protein. It folds into five structurally homologous extracellular (EC) 4 domains numbered 1-5 from the N-terminal domain (3). The cytoplasmic domain mediates signaling through interactions with catenins (1).Several approaches have been used to investigate cadherin recognition, binding, and signal transduction. Sequence exchange and cell aggregation studies mapped the specificitydetermining region to the first extracellular domain EC1 (4). For this reason, this domain has been the focus of the majority of mechanistic studies of cadherin adhesion and binding specificity. In the crystal structure of the soluble N-terminal domain (EC1) of neural cadherin, the Trp 2 (W2) residue was docked in a hydrophobic pocket of the adjacent EC1 domain (5). This reciprocal Trp 2 exchange is referred to as a "strand dimer." The structure of the ectodomain of Xenopus cleavage stage cadherin (C-cadherin) similarly exhibited this Trp 2 exchange, but between anti-parallel EC1 domains (3). Electron tomography images of desmosomal cadherins in mouse epidermis also suggested that similar interactions form in tissue, although the images contain a wide variety of other configurations and possible interactions (6). Studies showing that W2A and W2G mutations eliminate cell adhesion, also suggest that the docked Trp 2 side chain forms the sole adhesive interface (7, 8). Other...

Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

Single-molecule biomechanical measurements, such as the force to unfold a protein domain or the lifetime of a receptor-ligand bond, are inherently stochastic, thereby requiring a large number of data for statistical analysis. Sequentially repeated tests are generally used to obtain a data ensemble, implicitly assuming that the test sequence consists of independent and identically distributed (i.i.d.) random variables, i.e., a Bernoulli sequence. We tested this assumption by using data from the micropipette adhesion frequency assay that generates sequences of two random outcomes: adhesion and no adhesion. Analysis of distributions of consecutive adhesion events revealed violation of the i.i.d. assumption, depending on the receptor-ligand systems studied. These include Markov sequences with positive (T cell receptor interacting with antigen peptide bound to a major histocompatibility complex) or negative (homotypic interaction between C-cadherins) feedbacks, where adhesion probability in the next test was increased or decreased, respectively, by adhesion in the immediate past test. These molecular interactions mediate cell adhesion and cell signaling. The ability to ''remember'' the previous adhesion event may represent a mechanism by which the cell regulates adhesion and signaling.adhesion frequency assay ͉ Markov sequence ͉ single-molecule mechanics ͉ Bernoulli sequence B iomechanical studies of protein, DNA, and RNA at the level of single molecules provide insights that complement information obtained from conventional measurements on ensembles of large numbers of molecules (1). These experiments employ ultrasensitive force techniques, for example, atomic force microscopy (2) and the biomembrane force probe technique (3), to mechanically characterize a single molecule that physically links the force sensor to a sample surface. Fig. 1 [and supporting information (SI) Movie 1] illustrate a simple experiment: the micropipette adhesion frequency assay (4). A human red blood cell (RBC) pressurized by micropipette aspiration is used as an adhesion sensor to test interactions between ligands coated on the RBC membrane and receptors expressed on a second cell (Fig. 1 A). The receptor-expressing cell is put into contact with the RBC for a given duration (Fig. 1B) and then retracted. If adhesion results, retraction will stretch the RBC (Fig. 1C), otherwise the RBC will smoothly return to its initial shape (Fig. 1D). When adhesion does occur, additional quantities can be measured by using the RBC picoforce transducer or any other ultrasensitive force technique, including rupture force (3), adhesion lifetime (2), molecular elasticity (5), protein unfolding (6), and protein refolding (7).Single-molecule biomechanical measurements are inherently stochastic because molecular events (e.g., unfolding of a protein domain or unbinding of a receptor-ligand bond) are determined not only by the weak, noncovalent interactions (within a single molecule or between two interacting molecules) but also by thermal excitations from the envi...

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.

SummaryThis study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherindependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the twodimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting.Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than merely controlling cell-cell cohesion.