Nano Biomechanics Lab

Mechanical insights into the physiological functions of intercellular adhesion molecules

Epithelial cells that line the airway tract, gastrointestinal tract and renal tract are attached to one another through intercellular adhesion molecules or proteins (Figure 1). The intercellular adhesion protein complex consists of a variety of proteins that contribute to distinct physiological functions. While proteins at the adherens junctions (AJ) like nectins and e-cadherins are important for initiating and maintaining cell adhesion, the proteins localizing at the tight junctions (TJs) act as gates to regulate solute flux across these monolayers (Figure 2). Furthermore, some of these proteins also act as receptors for several viruses as well as bacterial toxins. Our primary objective is to understand the adhesion kinetics of the interactions mediated by different adhesion molecules. We hope that this would provide a better perspective not only on how these proteins organize and function but also how we can modulate these interactions for improving drug delivery.

Single molecule force spectroscopy using the atomic force microscope was used to analyse the kinetic properties and adhesion strength mediated by various intercellular adhesion proteins. This method makes use of soft flexible cantilevers functionalized with the protein of interest (in our case TJ and AJ proteins like GST-claudin-1, Fc-nectin-1 and Fc-JAM-A) to probe either cells or functionalized surfaces (Figure 3). The underlying principle is to probe how the interacting molecules behave under an externally applied force (stressed state) and then extrapolating the results to see how they behave in the absence of a force (unstressed state). The behaviour of the molecular interactions in the unstressed state can give insight into the biophysical nature of the molecular interactions being probed.

Our results show that proteins localizing at the TJs that regulate paracellular diffusion of solutes (e.g. claudin-1and JAM-A) show weak and short-lived interactions. Such unstable and dynamic interactions would facilitate breaking and resealing of TJ strands to regulate the paracellular diffusion of solutes. It is also seen that interactions of the TJ protein JAM-A with the reovirus attachment protein σ1 is much more stable as compared to the homophilic JAM-A interactions. It is likely that stable interactions of the viral protein with JAM-A help the virus in entering the cell. Furthermore, adhesion kinetics parameters of the interactions involving the AJ protein nectin-1 strongly support their role in initiating cell adhesion. Interactions mediated by nectins are weak but kinetically more stable and form much more rapidly than e-cadherins. Hence, nectin mediated interactions are important for the initial stages of cell adhesion. Once the adhesion is established, e-cadherin are recruited to the adhesion sites to strengthen it.

In summary, we showed that single molecular force spectroscopy can be a versatile technique to investigate the biophysical characteristics of molecular interactions. The study of molecular interactions involving cell adhesion molecules will provide us with a better understanding of their role in physiological functions as well as pathological roles.

This work was supported by the Biomedical Research Council (BMRC) from the Agency for Science, Technology & Research (A*STAR), Singapore. Their funding support is gratefully acknowledged.

Figure 1. Schematic showing epithelial cells forming barriers which regulate the passage of solutes and fluids between various body compartments e.g. gastro intestinal tract, respiratory tract, etc. The intercellular adhesion proteins (such as TJs and AJs) are responsible for stabilizing the adhesion and regulating the paracellular pathway (from K. Matter and M. S. Balda, Nature Reviews, Molecular Cell Biology, March 2003).

Figure 2. Schematics showing composition and organization of adherens junctions (AJs) and tight junctions (TJs) . JAM: Junction Adhesion Molecule (from J. Miyoshi, Y. Takai, Advanced Drug Delivery Reviews, 2005)

Figure 3. Schematic of the atomic force microscopy (AFM) experimental set up. Intercellular adhesion molecules Claudins, Nectin-1and JAM-A were linked to the AFM tip to probe their interactions at the level of single molecule. The arrows indicate the direction of pulling in the AFM experiment. JAM: Junction Adhesion Molecule.

References

Vedula, S R K, T S Lim, W Hunziker, C T Lim, Mechanistic Insights into Physiological Functions of Cell Adhesion Proteins Using Single Molecule Force Spectroscopy, Molecular & Cellular Biomechanics, 2008. (in press)
Vedula, S R K, T S Lim, E Kirchner, K M Guglielmi, T S Dermody, T Stehle, W Hunziker, C T Lim, A Comparative Molecular Force Spectroscopy Study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma-1, Journal of Molecular Recognition, 2008. (in press)
Vedula, S R K, T S Lim, P J Kausalya, B Lane, G Rajagopal, W. Hunziker, C T Lim, Quantifying forces mediated by integral tight junction proteins in cell-cell adhesion, Experimental Mechanics, 2008. (in press)
Lim, T S, S R K Vedula, J Kausalya, W Hunziker, C T Lim, Single Molecular Level Study of Claudin-1 Mediated Adhesion, Langmuir, 24 (2008):490-495.
Vedula, S R K, T S Lim, H Shi, J P Kausalya, B Lane, G Rajagopal, W Hunziker, C T Lim, Molecular force spectroscopy of homophilic nectin-1 interactions. Biochemical and Biophysical Research Communications, 362, 4 (2007): 886-892.
Lim, CT, S R K Vedula, T S Lim, P J Kausalya, R Gunaretnam, W Hunziker, Molecular interactions of tight junction proteins in cell-cell interaction. Journal of Biomechanics, 39, Supplement 1 (2006): S241.
Vedula, S R K, T S Lim, P J Kausalya, W Hunziker, G Rajagopal, C T Lim, Biophysical approaches for studying the integrity and function of tight junctions, Molecular & Cellular Biomechanics, 2, 3 (2005): 105-124.

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