In the background of the cover image of the November 15 issue of Biophysical Journal, we show a composite immunofluorescence image of cells stained for the desmosomal proteins desmoglein 2 (cyan) and desmoplakin (pink). In the foreground, we show an artistic representation of enhanced green fluorescent protein (EGFP). EGFP is shown as a rainbow-colored ribbon enclosed in a translucent bubble representing the solvent-excluded surface area of the protein. We show a laser beam exciting the chromophore (lime green) in the center of the structure.
Despite recent advances in traditional structural techniques, elucidating the architecture of large, membrane-bound protein assemblies remains incredibly challenging. Cell-cell junctions such as the desmosome are perfect examples of this challenge; desmosomes are embedded in two plasma membranes, are bound to two cytoskeletons, and are heterogenous in both size and composition. To overcome these challenges, we used excitation-resolved fluorescence polarization microscopy (FPM) to study the architecture of the proteins responsible for desmosomal adhesion, the desmosomal cadherins. By engineering a series of cadherin-EGFP chimeras, we were able to study desmosome architecture in a near-native setting. The cover image was designed to reflect the difference in scale between the underlying physical basis of FPM and the biological questions we set out to answer. On the one hand, fluorescence is a process that occurs at the atomic scale; the probability of the EGFP chromophore absorbing a photon from the laser beam depends on its orientation relative to the orientation of the photon’s electric field. On the other hand, desmosomes are large, geometrically complex protein assemblies responsible for the mechanical integrity of entire tissues; each of the fluorescent puncta in the image represents a single desmosome containing hundreds of individual proteins. In our research, we exploited the foundational principles of fluorescence to study the nanoscale geometry of desmosomal proteins. Importantly, despite the diffraction limit imposed by traditional optical imaging, we were able to resolve architectural differences between different domains in individual cadherins.
Desmosomes are required for all vertebrate life, and dysfunction of their constituent proteins is associated with severe disease of the heart and skin. Many of these diseases are linked to single-point mutations in the cadherins, for which no governing disease-causing mechanisms have been established. By using our methodology, it should now be possible to pinpoint the link between mutations in the cadherins and their effect on desmosomal adhesion, which will aid in understanding the molecular mechanisms of diseases associated with desmosome dysfunction.
The desmosome is only one example of a biological assembly that is difficult to study by using traditional methods. The strategies used here could easily be extended to study the organization of other large, membrane-bound complexes.
Readers may view recent research from our laboratory on our laboratory website: http://www.mattheyseslab.com.
- William F. Dean and Alexa L. Mattheyses