The structure of desmosomes and desmosomal cadherins

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

We are investigating the molecular mechanism that binds cells in tissues so strongly together and makes tissues so tough.
Strong adhesion is especially important in tissues that are constantly subjected to physical stress such as cardiac muscle and the outer layer of skin, the epidermis. Failure of adhesion can result in heart failure or extremely unpleasant, potentially lethal skin disease.
To cope with everyday wear and tear normal cells must vary their adhesion. In order to repair a wound, epidermal cell must weaken their adhesion so that they can rearrange themselves and spread over the wound surface.
We believe that strong adhesion is mediated by minute structures called desmosomes that are like little Velcro patches on the cells. Our hypothesis is that desmosomes have a special property that distinguishes them from other cell adhesions and Velcro. Once they have formed initial weak adhesion between cells they can lock themselves into a strongly adhesive state in which they remain unless the tissue is wounded when they revert to the weaker state.
We will use electron microscopy and X-ray crystallography to investigate how desmosomes are able to do this.
Our results may suggest novel disease treatments and ways to accelerate wound healing.

Technical Summary

The cells of tissues subject to mechanical stress, e.g. epidermis and cardiac muscle, are bound together by adhesive intercellular junctions known as desmosomes. Defects in desmosomes cause the potentially lethal human diseases epidermolysis bullosa, pemphigus and arrhythmogenic right ventricular cardiomyopathy. We recently proposed the concept of hyper-adhesion to explain how desmosomes mediate strong adhesion. Tissue desmosomes are hyper-adhesive apparently because they have an organised, quasi-crystalline arrangement of the extracellular (EC) domains of their adhesion molecules, the desmosomal cadherins (DCs). Hyper-adhesive desmosomes resist disruption by chelating agents. Hyper-adhesiveness is lost in wounded cell sheets, accompanied by loss of organisation and acquisition of sensitivity to chelating agents. Loss of hyper-adhesiveness appears to be mediated by protein kinase C alpha. Our recent work shows that the DCs desmocollin and desmoglein exhibit homophilic, isoform-specific adhesive binding that may be fundamental to desmosome structural organisation. Our hypothesis is that the organised arrangement of the DC EC domains relates to an organised molecular arrangement within the plaque and that modulation of the latter by PKCa causes concomitant disorganisation of the former and loss of hyper-adhesiveness. To provide evidence for this hypothesis we will investigate the structure of the plaque of isolated bovine nasal epidermal desmosomes by immunogold-labelling and electron microscopy (EM), and by atomic force microscopy (AFM), and determine the crystal structure of the desmocollin and desmoglein EC domains. Isolated desmosomes will be adsorbed to EM grids, labelled with antibodies to desmosomal plaque components and gold particles distributions analysed by multivariate analysis to map the 2D arrangement of plaque molecules. Desmosomal plaque architecture will be analysed by AFM including dissection of the plaque by application of increasing force via the tip stylus. AFM examination of freeze fracture replicas of desmosomes will be used to analyse structure within the membrane and intercellular space. Isolated desmosomes will be treated with PKCa and examined by EM and AFM to characterise configurational changes in the plaque, and will be used to study phosphorylation of plaque components and calcium binding. The structure of the EC domains of desmocollin and desmoglein will be determined by X-ray crystallography to reveal features involved in homophilic binding and hyper-adhesion. The effect of calcium concentration on the configuration of DC EC domains will be determined by small angle X-ray solution scattering and AFM.

Publications

10 25 50
 
Description Proof of Pricipal Awards, University of Manchester Intellectual Property/University of Manchester Itellectual Property Ltd
Amount £87,000 (GBP)
Organisation University of Manchester 
Department Intellectual Property
Sector Academic/University
Country United Kingdom
Start 06/2010 
End 05/2011
 
Title Novel method for immunolabelling of desmosomes 
Description Isolated desmosomes are adsorbed onto coated em grids and, without fixation, directly labelled with immuno-gold antibody. This will enable novel studies on the structure of the desmosomal plaque 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact None yet - a manuscript is currently in preparation 
 
Description Adhesion of skin to transdermal sugical implants 
Organisation University College London
Department Mechanical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Design of experiments, provision of antibodies and recombinant proteins
Impact Not yet
Start Year 2010
 
Description Down-regulation of desmosomal adhesion 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Institute of Medical Biology
Country Singapore 
Sector Academic/University 
PI Contribution Proposal and joint supervision of graduate student research project.
Collaborator Contribution Joint supervision of project
Impact Reseach is progressing well and should result in publication as well as application for new funding.
Start Year 2014
 
Description Glycosylation of desmosomal cadherins 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of material for anlysis. Interpretation of data. Testing consequent hypotheses
Collaborator Contribution Mass spectromic anlysis of material supplied
Impact Prelimianry data obtained showing that detailed analysis of glycosylation patterns can proceed, providing outcomes that can be tested in cells. The collaboration involves interactions between chemists, structural biologists and cell biologists.
Start Year 2015
 
Description Laser surface texturing of Ti-6Al-4V for improving biocompatibility 
Organisation University of Manchester
Department Faculty of Engineering and Physical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Designing and supervising experiments to assess the adhesion of osteoblasts to the laser-treated surface if titanium alloy with a view to the production of surgical implants.
Collaborator Contribution Additional collaboration
Impact A surface modification that produces excellent cell adhesion has been produced and has given rise to a further collaboration involving implants into experimental animals. Multi-disciplinary - biology and aerospace
Start Year 2007
 
Description Mouse model for pemphigus vulgaris 
Organisation University of Bern
Department Vetsuisse Faculty
Country Switzerland 
Sector Academic/University 
PI Contribution Electron microscopy of mouse epidermis after injection of pathogenic antibody
Collaborator Contribution A paper has been submitted and further collaboration is in prospect
Impact A manuscript has been submitted to the Journal of Investigative Dermatology.
Start Year 2009
 
Description Role of keratins in desmsomal adhesion 
Organisation University of Leipzig
Department TRM Leipzig & Biologie
Country Germany 
Sector Academic/University 
PI Contribution supply of materials and intellectual input
Collaborator Contribution experimental work and financial support
Impact none so far
Start Year 2013
 
Description Studies on wound healing 
Organisation King Saud University
Department Department of Rehabilitation Medicine
Country Saudi Arabia 
Sector Academic/University 
PI Contribution I am supervising research projects on desmosomes, stem cells and nitric oxide in wound healing. The experiments are being carried out at the University of Mansoura in Egypt, and are being funded buy King Saud University
Impact None yet
Start Year 2010
 
Description Testing of surgical implants in sheep 
Organisation University College London
Department Mechanical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Planning of experiments involving implantation of laser-treated titanium alloy into sheep
Collaborator Contribution It has led to a further collaboration
Impact None yet
Start Year 2010
 
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact Lectures to school biology society

Much interest from pupils
Year(s) Of Engagement Activity 2007,2008
 
Description Seminar at local grammar school 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact 50 pupil attended research-type seminar, which provoked many excellent questions

None
Year(s) Of Engagement Activity 2008,2009