Structural Proteomics to spatially resolve interactions with mechanosensitive synaptic scaffold proteins

About the Project

Over the past decades, tremendous progress has been made in unveiling how synaptic plasticity wires and rewires neural circuits, forming the basis of learning and memory. Yet, we lack sufficient understanding of the underlying mechanisms through which molecules compute and store information in synapses. The project will combine state-of-the art technologies in a unique way to map the talin interactome during cell signalling and LTP. The supervisory team are world leading experts in the structural mechanobiology of talin, proteomics and cell adhesion biology and cell culture.

Over the past decades, tremendous progress has been made in unveiling how synaptic plasticity wires and rewires neural circuits, forming the basis of learning and memory. Yet, we lack sufficient understanding of the underlying mechanisms through which molecules compute and store information in synapses. Non-neuronal cells form focal adhesions (FAs), transmitting mechanical force through the actin cytoskeleton to the extracellular matrix (ECM). In FAs, proteins such as talin alter their structure in response to these forces, thereby rewiring the activity of intracellular signalling pathways. We hypothesize that mechanosensing is the key missing link mediating signalling in synapses, thereby encoding 'mechano-memories.' We propose that these are built on talin’s ability to undergo an elaborate sequence of structural transitions, which form the nexus for biochemical computations that regulate synaptic plasticity and strength.

This project is based on our exciting preliminary data of the essential role of talin in synaptic functions; i) talin contains 13 force-dependent binary switch domains that cells can open using their motor proteins, ii) talin is under tension in synapses, iii) CRISPR deletion of talin from neurons prevents long-term potentiation (LTP) and iv) mutations in the talin gene are a previously unrecognized cause of childhood epilepsy (collaboration with Boston Children’s Hospital). Our research has shown that key to talin’s capacity to orchestrate enzymatic processes is its ability to change its dimensions by >10-fold as its switch domains unfold. As a result, the enzymes decorating talin can get spatially resolved over the entire length of the synapse from 60 nm when at rest to greater than 500 nm. This leads to our central hypothesis that the talin interactome changes during LTP.

Crosslinking mass spectrometry is a powerful technology for identifying protein-protein interactions in highly complex samples. This technique employs small, reactive reagents to form covalent bonds between amino acids that are in close proximity within the protein’s native state. Following reduction, alkylation, and digestion, the resulting crosslinked peptides—two peptides bound together by the crosslinking reagent—are analysed by mass spectrometry to reveal peptide identities and the specific location of the crosslink. This process provides not only interaction details but also valuable structural information, allowing precise mapping of interaction interfaces between proteins – precisely the information relevant for this project.

The project will combine state-of-the art technologies in a unique way to map the talin interactome during cell signalling and LTP. The supervisory team are world leading experts in the structural mechanobiology of talin, proteomics and cell adhesion biology and cell culture. Our neuroscience collaborators in Finland, have provided us with neuronal cell extracts taken at different stages of LTP.

The student will be embedded in the Biochemistry, Cell and Systems Biology department at the University of Liverpool, where they will train in protein expression and purification, biochemistry and proteomics-based approaches. This interdisciplinary approach will provide them with an excellent well rounded scientific training. The supervisory team have an excellent track record of PhD student success, both in them graduating in 4 years but also in publishing papers and going on to future careers.

We will 1) optimize the structural proteomics assay with talin fragments and interactions in vitro first where the positions of binding sites are known. 2) Once the structural proteomics-based approaches are optimised for working on talin, the student will use them to map the talin interactome at sub-nanometre resolution in cell culture. Finally, the methodology will be applied in neuronal cells, to identify precisely i) what ligands are bound to talin and ii) where on talin each ligand is bound, during synaptic function and during LTP.

Supervisors:

Prof Ben Goult - b.t.goult@liverpool.ac.uk

Prof Richard Scheltema - Richard.Scheltema@liverpool.ac.uk

Dr Paul Atherton - Paul.Atherton@liverpool.ac.uk

Prof Martin Humphries - Martin.Humphries@manchester.ac.uk

Funding Notes

This project is open to UK and international applicants with their own funding. Funding should cover course fees, living expenses and research expenses (bench fees).

References

1. Kang M, Otani Y, Guo Y, Yan J, Goult BT* and Howe A*. (2024) The focal adhesion protein talin is a mechanically-gated A-kinase anchoring protein (AKAP). PNAS 121(13):e2314947121 PMID: 38513099
2. Gallego-Paez LM, Edwards WJS, Chanduri M, Guo Y, Koorman T, Lee C-Y, Grexa N, Derksen P, Yan J, Schwartz MA, Mauer J and Goult BT* (2023) TLN1 contains a cancer-associated cassette exon that alters talin-1 mechanosensitivity. J. Cell Biol 222(5):e202209010 PMID: 36880935
3. Barnett SFH and Goult BT* (2022) The MeshCODE to scale—visualising synaptic binary information. Front. Cell. Neurosci. 16:1014629 PMID: 36467609
4. Gough RE, Jones MC, Zacharchenko T, Le S, Yu M, Jacquemet G, Muench SP, Yan J., Humphries JD, Jørgensen C, Humphries MJ and Goult BT*. (2021) Talin mechanosensitivity is modulated by a direct interaction with cyclin dependent kinase-1. J Biol Chem 297(1):100837 PMID: 34118235
5. Klykov O, Steigenberger B, Pektaş S, Fasci D, Heck AJR and Scheltema RA*. (2018) Efficient and robust proteome-wide approaches for cross-linking mass spectrometry. Nature Protocols. 13(12):2964-2990 PMID: 30446747