TOVIS · Cryo- Electron Microscopy of vitreous sections of biological cells and tissues
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2007-07-01 → 2009-06-30
- EU contribution
- €159,331
- Participants
- 1
- Scheme
- EIF
Lines connect the coordinator with its partners.
Results in brief
Final Activity Report Summary - TOVIS (Cryo- electron microscopy of vitreous sections of biological cells and tissues)
Cell-cell adhesion is important in binding the cells of normal tissue together thus maintaining tissue integrity. They also play key roles in embryonic development, skin and heart diseases, and cancer. The adhesions concerned, called desmosomes are small regions on cell surfaces where adhesion molecules, called cadherins, are concentrated and connected to the cell cytoskeleton through a set of proteins making up an electron-dense region called desmosomal plaque. Our objective was to resolve the molecular structure of the desmosome. Despite considerable efforts, the molecular organisation of desmosome has been obscure. The desmosome is a large macromolecular complex which makes it inaccessible to conventional structural determination and biochemical methods. Using cryo-electron tomography of vitreous sections from human epidermis we were able to visualise the three-dimensional (3D) molecular architecture of desmosomal cadherins at close to native conditions. The resulting 3D reconstructions showed a regular array of densities at app.70 Angstrom intervals along the midline with a curved shape resembling the X-ray structure of C-cadherin. 3D image processing of extracted sub-tomograms revealed the cadherin organisation. After fitting the C-cadherin atomic structure into the averaged sub-tomograms, we saw a periodic arrangement of a trans W-like and a cis V-like interaction corresponding to molecules from opposing and the same cell membrane, respectively. These results provide the first molecular model of cadherin organisation in vivo that is compatible with previous EM data and X-ray crystal structures of isolated cadherins. Furthermore, the resulting model of cadherin organisation explains existing two-dimensional data and yields insights into a possible mechanism of cadherin-based cell adhesion.
Data: CORDIS, © European Union
Project objective
During my postdoctoral research, I would like to investigate the macromolecular organization of synapses and desmosomes, in brain and skin tissue respectively, by combining cryo-sectioning (CS) and cryo-electron tomography (CET). This novel combination of CS with CET is currently the only approach which should allow the three-dimensional (3D) visualization of the organization of untreated (i.e. unfixed, unstained) tissue at macromolecular resolution.The 3D molecular architecture of the synaptic cleft will help to understand the synaptic transduction processes. Revealing the 3D macromolecular organisation of the desmosomes and their associated structures (e.g. intermediate filaments) will have a great impact on understanding the mechanism by which desmosomes differentiate, and help to understand desmosome-related diseases (e.g. Pemphigus). In order to accomplish these goals, I will expand my expertise in CS with training in CET and image processing to be able to identify and analyse macromolecules in a cellular context. Tomographic series of cryo-serial sections are going to be recorded in the electron microscope followed by computationally reconstructing several 3D tomograms. Subsequently, the tomograms, of these sections will be computationally assembled in to a large 3D image which will represent a significant portion of the cellular volume at app. 2 nm resolution.In the resulting reconstructions, I hope to reveal the molecular architecture of the desmosomes and synapses and thus we should be able to fit hi gh-resolution structures of adhesion proteins obtained by e.g. X-ray crystallography (e.g. Cadherin proteins in the desmosomes; neural cell adhesion molecules in the synaptic cleft). Finally I look forward to have strong interactions in the interdiscriplinary environment of EMBL, where I will also be able to supervise students and have knowledge-transfer in mutual directions. This should allow me to pave the way for a future leading position in science.
Original text from CORDIS.
Participants
- EUROPEAN MOLECULAR BIOLOGY LABORATORY · HEIDELBERGCoordinatorGermany
Links
Data: CORDIS, © European Union