Sanjeevi Sivasankar

Resolving cadherin interactions and binding cooperativity at the single-molecule level

The cadherin family of Ca2+-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell–cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell–cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.

Direct Measurements of Multiple Adhesive Alignments and Unbinding Trajectories between Cadherin Extracellular Domains

Direct measurements of the interactions between antiparallel, oriented monolayers of the complete extracellular region of C-cadherin demonstrate that, rather than binding in a single unique orientation, the cadherins adhere in three distinct alignments. The strongest adhesion is observed when the opposing extracellular fragments are completely interdigitated. A second adhesive alignment forms when the interdigitated proteins separate by 70 +/- 10 A. A third complex forms at a bilayer separation commensurate with the approximate overlap of cadherin extracellular domains 1 and 2 (CEC1-2). The locations of the energy minima are independent of both the surface density of bound cadherin and the stiffness of the force transducer. Using surface element integration, we show that two flat surfaces that interact through an oscillatory potential will exhibit discrete minima at the same locations in the force profile measured between hemicylinders covered with identical materials. The measured interaction profiles, therefore, reflect the relative separations at which the antiparallel proteins adhere, and are unaffected by the curvature of the underlying substrate. The successive formation and rupture of multiple protein contacts during detachment can explain the observed sluggish unbinding of cadherin monolayers. Velocity-distance profiles, obtained by quantitative video analysis of the unbinding trajectory, exhibit three velocity regimes, the transitions between which coincide with the positions of the adhesive minima. These findings suggest that cadherins undergo multiple stage unbinding, which may function to impede adhesive failure under force.

Ideal, catch, and slip bonds in cadherin adhesion

Classical cadherin cell-cell adhesion proteins play key morphogenetic roles during development and are essential for maintaining tissue integrity in multicellular organisms. Classical cadherins bind in two distinct conformations, X-dimer and strand-swap dimer; during cellular rearrangements, these adhesive states are exposed to mechanical stress. However, the molecular mechanisms by which cadherins resist tensile force and the pathway by which they convert between different conformations are unclear. Here, we use single molecule force measurements with an atomic force microscope (AFM) to show that E-cadherin, a prototypical classical cadherin, forms three types of adhesive bonds: catch bonds, which become longer lived in the presence of tensile force; slip bonds, which become shorter lived when pulled; and ideal bonds that are insensitive to mechanical stress. We show that X-dimers form catch bonds, whereas strand-swap dimers form slip bonds. Our data suggests that ideal bonds are formed as X-dimers convert to strand-swap binding. Catch, slip, and ideal bonds allow cadherins to withstand tensile force and tune the mechanical properties of adhesive junctions.

Mechanism of homophilic cadherin adhesion

Direct measurements of the distance-dependent forces between membrane-bound cadherins were used to test current models of homophilic cadherin interactions. The results reveal a complex binding mechanism in which the proteins adhere in multiple alignments that involve more than the amino-terminal domains.

Strain-Dependent Photoluminescence Behavior of CdSe/CdS Nanocrystals with Spherical, Linear, and Branched Topologies

The photoluminescence of CdSe/CdS core/shell quantum dots, nanorods, and tetrapods is investigated as a function of applied hydrostatic and non-hydrostatic pressure. The optoelectronic properties of all three nanocrystal morphologies are affected by strain. Furthermore, it is demonstrated that the unique morphology of seeded tetrapods is highly sensitive to non-isotropic stress environments. Seeded tetrapods can thereby serve as an optical strain gauge, capable of measuring forces on the order of nanonewtons. We anticipate that a nanocrystal strain gauge with optical readout will be useful for applications including sensitive optomechanical devices and biological force investigations.

Cadherin recognition and adhesion

Classical cadherins are the principle adhesive proteins at cohesive intercellular junctions, and are essential proteins for morphogenesis and tissue homeostasis. Because subtype-dependent differences in cadherin adhesion are at the heart of cadherin functions, several structural and biophysical approaches have been used to elucidate relationships between cadherin structures, biophysical properties of cadherin bonds, and cadherin-dependent cell functions. Some experimental approaches appeared to provide conflicting views of the cadherin binding mechanism. However, recent structural and biophysical data, as well as computer simulations generated new insights into classical cadherin binding that increasingly reconcile diverse experimental findings. This review summarizes these recent findings, and highlights both the consistencies and remaining challenges needed to generate a comprehensive model of cadherin interactions that is consistent with all available experimental data.

Resolving the molecular mechanism of cadherin catch bond formation.

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated.

Spatially Indirect Emission in a Luminescent Nanocrystal Molecule

Recent advances in the synthesis of multicomponent nanocrystals have enabled the design of nanocrystal molecules with unique photophysical behavior and functionality. Here we demonstrate a highly luminescent nanocrystal molecule, the CdSe/CdS core/shell tetrapod, which is designed to have weak vibronic coupling between excited states and thereby violates Kashas rule via emission from multiple excited levels. Using single particle photoluminescence spectroscopy, we show that in addition to the expected LUMO to HOMO radiative transition, a higher energy transition is allowed via spatially indirect recombination. The oscillator strength of this transition can be experimentally controlled, enabling control over carrier behavior and localization at the nanoscale.

Forces controlling protein interactions: theory and experiment☆

Abstract This work reviews both the theory and experimental measurements of the fundamental forces that control protein solution behavior. In addition to the Derjaguin–Landau–Verwey–Overbeek (DLVO) forces, we also discuss the relative importance of hydrodynamic, solvation, and lock-and-key interactions in controlling protein solution behavior. The more common computational methods used to calculate both electrostatic and van der Waals potentials are described. Particular attention is given to the differences between proteins and ideal colloidal particles, and the computational methods used to address those differences. In addition to theoretical investigations of protein interactions, the results of recent direct measurements of the forces governing protein interactions are reviewed. These experimental results provide not only measurements against which the theories can be tested, but also demonstrate directly the relative importance of both DLVO and non-classical DLVO forces in the control of protein behavior.

Different roles of cadherins in the assembly and structural integrity of the desmosome complex.

ABSTRACT Adhesion between cells is established by the formation of specialized intercellular junctional complexes, such as desmosomes. Desmosomes contain isoforms of two members of the cadherin superfamily of cell adhesion proteins, desmocollins (Dsc) and desmogleins (Dsg), but their combinatorial roles in desmosome assembly are not understood. To uncouple desmosome assembly from other cell–cell adhesion complexes, we used micro-patterned substrates of Dsc2aFc and/or Dsg2Fc and collagen IV; we show that Dsc2aFc, but not Dsg2Fc, was necessary and sufficient to recruit desmosome-specific desmoplakin into desmosome puncta and produce strong adhesive binding. Single-molecule force spectroscopy showed that monomeric Dsc2a, but not Dsg2, formed Ca2+-dependent homophilic bonds, and that Dsg2 formed Ca2+-independent heterophilic bonds with Dsc2a. A W2A mutation in Dsc2a inhibited Ca2+-dependent homophilic binding, similar to classical cadherins, and Dsc2aW2A, but not Dsg2W2A, was excluded from desmosomes in MDCK cells. These results indicate that Dsc2a, but not Dsg2, is required for desmosome assembly through homophilic Ca2+- and W2-dependent binding, and that Dsg2 might be involved later in regulating a switch to Ca2+-independent adhesion in mature desmosomes.