Cryo Electron Microscopy of Desmosome Assemblies

Project completed 2013.

Professor Richard Palmer, School of Physics
Professor Michael Overduin, Institute of Cancer and Genomic Sciences
Dr Martin Chidgey, Institute of Cancer and Genomic Sciences
Professor Roy Johnston, School of Chemistry

Desmosomes are intercellular junctions of epithelia and cardiac muscle. They are essential for the maintenance of tissue integrity; loss of desmosomal adhesion can have devastating consequences for human health causing life-threatening diseases of the skin and heart. This project aims to develop and deploy a novel cryo electron microscopy technique to image the structure of the head domain of desmoplakin, and perhaps later other desmosomal proteins or complexes.

Frozen-hydrated biological samples are successfully used in several laboratories to study the ultrastructure of cellular organelles and their molecular components in a native state. Three-dimensional reconstructions of these structures can be made using electron tomography (ET), where many projection images at different tilt angles are computationally combined and compared with structural models of the proteins. Alignment of the images from a tilt series is one of the crucial steps in ET.

Alignment of the images involves (a) cross-correlation of collected images and (b) alignment using fiducial markers added to specimens. Conventional markers are colloidal gold particles (aqueous gold particle suspensions can be added to particulate samples before freezing). However, it is not always possible to generate a randomly distributed and/or highly concentrated solution of markers without seriously affecting the properties of the sample, and colloidal particles also have a tendency to form aggregates. This project will therefore apply soft-landed, size-selected gold clusters as alternative, high-resolution fiducial markers.

Link to ethesis: