Supervisors: Prof Mark C Field, Prof Eric Schirmer
Project Description:
Self-assembly of larger protein networks is a central feature of replicating systems from viral capsids to the cytoskeleton that gives cells structure and polarity. One important example is the nuclear lamina, a subset of the cytoskeleton responsible for nuclear structural integrity, controlling the demarcation between active and inactive chromatin and the developmental control of gene expression programs. The mammalian lamina is comprised of lamins, 60kDa coiled-coil proteins that assemble into a network that maintains nuclear structure and stabilizes the genome, yet with significant flexibility to support transcription and DNA replication. Lamins are also a potential model for driving self-association of synthetic polymers due to their biological and biochemical properties. Lamins differ from other filament systems such as actin, tubulin and spectrins in being the most flexible while also being the most resistant to sheer and tensile force, so that understanding their assembly and strength properties could be used to model novel synthetic polymers.
One way to understand these properties is to compare the wide range of lamins that share these physical properties, yet have significantly diverged in sequence over evolution. As lamins are the ancestral intermediate filaments, searches have identified nuclear lamin homologs in organisms for which cytoplasmic intermediate filaments have not been found such as dictostylium and by us in trypanosomes. We, and collaborators, recently demonstrated the presence of multiple lamina systems across the eukaryotes. This will enable us to test what features are essential to achieve and support these functions through comparative analysis.
Our chosen system is the trypanosome lamina, comprised of two interacting coiled-coil proteins NUP-1 and NUP-2 (1,2,3). Both have high molecular weights, an extended configuration, and are mutually dependent for localisation. The principle aims of our project are:
1. Creation of NUP-1 and NUP-2 mutants with epitope tags and deletions. Year one.
2. Isolate nuclear envelopes from a select set of mutant trypanosomes and image the lamina using epitope tag/immunogold labelling to determine the arrangement of these proteins in vivo. Year two.
3. Reconstitute NUP-1 and NUP-2 complexes from recombinant fragments and image the complexes using EM/rotary shadowing. Year three.
4. Perform crosslinking mass spectrometry with GFP-tagged domains of NUP-1 and NUP-2 to gain structural insights. Year three.
5. Analyse the ability of NUP-1 and NUP-2 fragments to interact. Year four.
The training environment in Dundee and Edinburgh is excellent, and provision is made for teaching of informatics, presentation, and communication skills together with a rigorous mentoring.
References:
1. DuBois, K.N., Alsford, S., Holden, J.M., Buisson, J., Swiderski, M., Bart, J.M., Ratushny, A.T., Wan, Y., Bastin, P., Barry, J.D., Navarro, M., Horn, D., Aitchison, J.D., Rout, M.P., and Field, M.C., (2012) ‘NUP-1 is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.’ PLoS Biology 10 e1001287
2. Obado, S., Brillantes, B., Uryu, K., Zhang, W-Z., Ketaren, N.E., Chait, B.T. Field, M.C., and Rout, M.P. (2016) 'Interactome mapping reveals the evolutionary history of the nuclear pore complex.' PLoS Biology 14 e1002365
To apply for this project, please go to this link.
