Self-assembly of large protein networks is a central feature of many 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 cytoskeletal system responsible for nuclear structural integrity, controlling the demarcation between active and inactive chromatin and the developmental control of gene expression programs. The lamina in mammalian cells is an intermediate filament assembly comprised of lamins, 60kDa coiled-coil proteins that assemble in a precise and hierarchical manner to build a network that maintains nuclear structure and tethers and stabilises a subset of the genome, yet with significant flexibility to support transcription and DNA replication. In most multicellular organisms and all vertebrates the lamina is disassembled and reassembled with each mitosis, largely driven by post-translational modifications. Thus the dynamic interplay of such modification with its ability to self-associate and re-establish genome organisation is of considerable biological interest. Lamins are also of interest as a potential model for driving self-association of synthetic polymers due to their biological and biochemical properties. Lamins and intermediate filaments differ from other filament systems such as actin and tubulin in being both more flexible and resistant to sheer and tensile force; 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. We, and collaborators, recently demonstrated the presence of multiple lamina systems across the eukaryotes, revealing that distinct solutions to solving the many problems of structural support of nuclear functions have arisen, enabling us to now test what features are essential to achieve and support these functions through comparative analysis. The presence of several self-assembling lamina systems offers the potential to understand the structural principles by which coiled-coil protein networks assemble, and the general and specific features that differentiate these networks.
Our chosen system is the trypanosome lamina, recently characterised as minimally comprised of two interacting coiled-coil proteins NUP-1 and NUP- 2 (1,2). Both have high molecular weights, an extended configuration, and exhibit a mutual dependence in terms of localisation. We have available within our laboratories the ability to map protein-protein interactions, to visualise networks in vivo by in situ epitope tagging/mutation and advanced ultrastructural methods for visualisation of both isolated nuclear envelopes plus recombinant reconstitution of NUP-1/NUP-2 systems in vitro. Much of the structural analysis will also require computational modelling to facilitate reconstructing the lamina structure. The principle aims of our project are:
1. Creation of a set of NUP-1 and NUP-2 mutants with epitope tags and deletions suitable for expression both in trypanosomes and heterologous expression systems. Year one.
2. Map the interactions and positions of NUP-1 and NUP-2 with respect to each other, additional nuclear markers (specifically the nuclear pore complex and telomeres), and genome regions and assess the impact of NUP mutations on nuclear structure. Years one and two.
3. 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.
4. Reconstitute NUP-1 and NUP-2 complexes from recombinant fragments and image the complexes using EM/rotary shadowing. Year three.
5. Perform crosslinking mass spectrometry with GFP-tagged domains of NUP-1 and NUP-2 to gain structural insights. Year three.
6. Analyse the ability of NUP-1 and NUP-2 fragments to interact with each other and with additional components. Year four.
This integrated set of investigations will provide an excellent basis for graduate research, connecting the student to excellent science in the UK and US, and an exceptional platform from which to build a career. The environment in Dundee and Edinburgh is excellent, and provision is made for teaching of informatics, communication skills together with a rigorous mentoring program designed to support to the student. In combination with the connections with the several laboratories studying the lamina, the multidisciplinary nature of understanding gene expression, self-assembly and the structure of biopolymers we believe offers a state of the art opportunity.
1. Maishman, L., Obado, S., Alsford, S., Bart, J.M., Navarro, M., Horn, D., Ratushny, Aitchison, J.D., Chait, B.T., Rout, M.P., and Field, M.C., (2015) 'Codependence between Trypanosoma nuclear lamina components in nuclear stability and control of gene expression.' Nucleic Acids Research (in press)
2. 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
3. Makarov, A.A., Rizzotto, A., Meinke, P., Schirmer, E.C., (2016) Purification of lamins and soluble fragments of NETs. Methods Enzymol. 569 79-100.