This sandwich placement will be based in the lab of Richard Treisman.
Project background and description
Actin is the principal structural component of cytoskeleton structures. Its regulated polymerisation is essential for adhesion cell adhesion and motility, and most actin-binding proteins control actin filament assembly, disassembly, and crosslinking. In contrast, unpolymerised actin (G-actin) controls the activity of RPEL-family proteins. The MRTFs are transcriptional regulators. Their nuclear accumulation and targeting to DNA is inhibited by G-actin, which competes for MRTF binding with importins and with their DNA targeting cofactor SRF. Our recent data indicates that RPEL-bound actin regulates MRTF-SRF interaction by making additional interactions with MRTF’s SRF-binding region.
Puzzlingly, expression of certain actin mutants which stabilise F-actin – S14C, G15S and V159N – activates rather than inhibits the MRTFs. These mutations affect the structure of the actin ATP-binding pocket and its interaction with the actin C-terminal helix. The mechanism by which they promote MRTF activation remains unexplained: at least G15S and V159N retain the ability to bind the MRTFs, but they induce rather than inhibit nuclear accumulation, and presumably do not inhibit SRF interaction. The project will establish how these mutations affect actin’s interaction with MRTF and with SRF.
The project will involve purification and characterisation of the three mutant actins. Recombinant actins will be produced using an established baculovirus system together with derivatives containing the R62D mutation, which prevents their polymerisation. Recombinant MRTF and SRF protein derivatives are available in the lab.
Biochemical and biophysical methods will be used to compare interaction of the mutant actins with the MRTF RPEL motif peptides (Fluorescence polarisation assays), with the intact MRTF RPEL domain, including the SRF-interacting sequences (Octet biolayer interferometry, HDX-MS), and with SRF (DNA pulldown assay). Wildtype and individual mutants’ R62D derivatives will cocrystallised with RPEL peptides. All these assays are established in the lab.
Candidate background
The post holder should embody and demonstrate the Crick ethos and ways of working: bold, open and collegial. The candidate must be registered at a UK Higher Education Institution, studying in the UK and must have completed a minimum of two years’ undergraduate study in a relevant discipline, and on track to receive a final degree grade of 2:1 or 1. In addition, they should be able demonstrate the following experience and key competencies:
- This project will suit those studying biochemistry, molecular biology, and structural biology, with interests in gene expression and cell biology.
- Good knowledge in relevant scientific area(s)
- Good written and spoken communication skills
- Ability to work independently and also capable of interacting within a group
References
1. Vartiainen, M.K., Guettler, S., Larijani, B. and Treisman, R. (2007)
Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.
Science 316: 1749-1752. PubMed abstract
2. Mouilleron, S., Langer, C.A., Guettler, S., McDonald, N.Q. and Treisman, R. (2011)
Structure of a pentavalent G-actin•MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator.
Science Signaling 4: ra40. PubMed abstract
