Disrupted microtubule dynamics are a key mechanism underlying the selective vulnerability of neurons across many different motor neuron diseases. Microtubules facilitate the long range transport of cargo along axons and stabilise the endoplasmic reticulum and mitochondria distribution along neurons. This project will investigate the microtubule based molecular causes of Hereditary Spastic Paraplegia type 8 (HSP8), a motor neuron disease originating from point mutations in the protein Strumpellin. You will address this question using a wide range of techniques including super-resolution live cell microscopy of neurons; cell biological analysis of the signalling pathways and in vitro reconstitution of microtubule dynamics.
The WASH complex –a pentameric complex including Strumpellin- polymerises actin networks on intracellular organelles like endosomes and the centrosome. It was thought to be primarily required for retrograde transport of selected receptors to the plasma membrane (MacDonald et al. 2018). However, recently we and others have identified major functions for the WASH complex in regulating microtubule dynamics throughout the cell cycle (Farina et al. 2019; Inoue et al. 2019). Depletion of WASH complex member expression leads to increased microtubule intensity and acetylation in cells.
Neuronal microtubule dynamics are especially tightly regulated to allow for effective axonal transport and stability of neuronal morphology. We have evidence that the HSP8 causing point mutations in Strumpellin alter neuronal microtubule dynamics and affect neuronal fitness. This provides us with the testable hypothesis that HSP8 mutations lead to motor neuron degeneration through deregulated microtubule dynamics.
Detailed Experimental Approach to test this hypothesis:
1.) Define changes in neuronal microtubule dynamics. Fibroblasts harbouring the heterozygous HSP8 causing Strumpellin point mutations have been generated using CRISPR/Cas9 knock-ins. HSP8 mutant fibroblasts will be transformed into neuronal like cells and investigated for their microtubule dynamics/modifications and changes in neurite dynamics and growth. Parallel studies can be carried out in primary motor neuron cultures from inducible conditional Strumpellin knockout mice.
2.) We will use the aforementioned cell types to investigate the underlying molecular mechanism. We have preliminary evidence that points towards Strumpellin being implicated in a microtubule stabilising signaling loop on endosomal membranes. You will investigate this signalling loop using loss of function and domain mutants to decipher the required steps.
3.) In vitro reconstitution of mechanism. We will investigate the effect of wildtype and HSP8 mutation harbouring Strumpellin/WASH complex on microtubule dynamics using in vitro reconstitution assays. By using purified tubulin to carry out TIRF microscopy assays, the rates of microtubule polymerisation, catastrophe and nucleation will be quantified. They will be characterised both in the presence of whole cell lysates with and without WASH/Strumpellin mutations. This will characterise the molecular phenotype the WASH complex has on microtubule stability, differentiating whether this is due to the WASH complex directly and/or its actin polymerisation ability.
This is a collaborative project of the Zech and Twelvetrees labs. You will primarily be based in Liverpool but also learn techniques and carry out work in Alison Twelvetrees lab in Sheffield. More information about all our research interest can be found here:
Tobias Zech lab: https://tzech6.wixsite.com/zechlab
Alison Twelvetrees lab: https://www.twelvetreeslab.co.uk
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/
MacDonald E, Brown L, Selvais A, Liu H, Waring T, Newman D, Bithell J, Grimes D, Urbé S, Clague MJ, Zech T. HRS-WASH axis governs actin-mediated endosomal recycling and cell invasion. J Cell Biol. 2018 Jul 2;217(7):2549-2564.
Farina F, Ramkumar N, Brown L, Samandar Eweis D, Anstatt J, Waring T, Bithell J, Scita G, Thery M, Blanchoin L, Zech T, Baum B. Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis. EMBO J. 2019 Jun 3;38(11).
Inoue D, Obino D, Pineau J, Farina F, Gaillard J, Guerin C, Blanchoin L, Lennon-Duménil AM, Théry M. Actin filaments regulate microtubule growth at the centrosome. EMBO J. 2019 Jun 3;38(11).