Sabyasachi Rakshit

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.

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.

Resonance Energy Transfer from β-Cyclodextrin-Capped ZnO:MgO Nanocrystals to Included Nile Red Guest Molecules in Aqueous Media

Core-shell ZnO:MgO nanocrystals have been synthesized by a sequential preparative procedure and capped with carboxymethyl beta-cyclodextrin (CMCD) cavities, thereby rendering the surface of the nanocrystals hydrophilic and the particles water-soluble. The water-soluble CMCD-capped ZnO:MgO nanocrystals emit strongly in the visible region (450-680 nm) on excitation by UV radiation and are stable over extended periods and over a range of pH values. The integrity of the cyclodextrin cavities is preserved on capping and retains their capability for complexation of hydrophobic species in aqueous solutions. Here we report the use of the water-soluble cyclodextrin-capped ZnO:MgO nanocrystals as energy donors for fluorescence resonance energy transfer studies. The organic dye Nile Red has been included within the anchored cyclodextrin cavities to form a noncovalent CMCD ZnO:MgO-Nile Red assembly in aqueous solution. Significant Nile Red fluorescence at 640 nm is observed on band gap excitation of the ZnO:MgO in the UV, indicating efficient resonance energy transfer (RET) from the nanocrystals to the included dye. The number of acceptor molecules interacting with a single donor in the CMCD ZnO:MgO-Nile Red assembly may be altered by controlling the filling up of the anchored cavities by Nile Red, leading to a variation in the efficiency of resonance energy transfer. The donor-acceptor distance was estimated from the efficiency measurements. The Nile Red emission following RET shows a pronounced thermochromic shift, suggesting the possible use of the CMCD ZnO:MgO-Nile Red assembly as thermometers in aqueous solutions.

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.

Cross-linking of a charged polysaccharide using polyions as electrostatic staples

Charged polysaccharides play an essential role in biology and also serve as a promising class of biomaterials. We demonstrate, at the single molecule level, a simple method to cross-link anionic polysaccharides using polyions as electrostatic staples; positive charges on the polyion bridge opposite charges on the polysaccharide and trap its conformation. Cross-linking is detected by measuring step-like conformational transitions when the polysaccharides are stretched using an Atomic Force Microscope (AFM). The probability of cross-linking can be tuned by either titrating or screening the polysaccharide charges; this probability is measured from the width of transitions in the AFM force–extension curves. Polyion mediated cross-linking can be used to reversibly fold polysaccharides and engineer carbohydrates as a stimuli-responsive material that responds to changes in pH and ionic environment.

Resolving Desmosomal Cadherin Interactions at the Single Molecule Level

Desmocollin and Desmoglein are essential Ca2+ dependent cell adhesion proteins that mediate the integrity and functional organization of tissues in multi-cellular organisms; however, the molecular interactions that mediate their binding are not understood. It is currently believed that desmosomal cadherin cis-dimers are required to mediate adhesion, evidence for the role of cis-dimers is lacking. Furthermore, it is unclear if desmosomal cadherins interact via homophilic or heterophilic binding and the molecular mechanism by which desmosomal cadherins enhance their bond-strength. To resolve these questions we used Dynamic Force Spectroscopy with an Atomic Force Microscope to measure the homophilic and heterophilic binding of desmosomal cadherin monomers and cis-dimers at the single molecule level. Our measurements showed that desmosomal cadherin monomers alone mediate Ca2+ dependent adhesion, cis-dimers are not essential for adhesion. Furthermore, while desmocollin and desmoglein participate in Ca2+ dependent heterophilic binding, we could not measure Ca2+ dependent homophilic interactions between these cadherins. Dynamic Force Spectroscopy revealed that desmosomal cadherin interactions occur via a double barrier interaction potential; at low loading rates the outer barrier serves as the dominant impedance to unbinding while at higher loading rates the inner barrier serves as the primary kinetic barrier. These experiments resolve the molecular details of desmosomal cadherin binding and suggest a mechanism by which these essential cell adhesion molecules enhance bond strength and lifetime in the presence of force.