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Understanding microtubule-actin crosstalk in neuronal cell migration

Primary Supervisor: Professor Andrew Fry

Secondary Supervisor: Professor Ian Forsythe

PhD project title: Understanding microtubule-actin crosstalk in neuronal cell migration

University of Registration: University of Leicester

Project outline:

Summary

This BBSRC-funded 4-year PhD studentship will address the mechanism of action of a microtubule-associated protein, EML4, in promoting microtubule-actin crosstalk and neuronal cell migration [1]. Exciting new evidence discovered in our lab has suggested a role for EML4 in regulating cell migration by increasing crosstalk between microtubules and the actin network. However, the mechanisms for this crosstalk are unknown. By addressing these mechanisms, this project will sit at the interface between fundamental cell biology and neuroscience and will use cutting edge experimental techniques to explore EML4 function in microtubule-actin crosstalk with a focus on training in a range of subcellular imaging technologies.

Background

EML4 is a microtubule-associated protein that is conserved across multicellular eukaryotes. In humans, it is a member of a family of six proteins, EML1 to EML6, that to date remain relatively poorly characterized. These proteins are highly expressed in the nervous system of developing embryos suggesting a role in neurogenesis [2]. Indeed, inherited mutations in EML1 lead to a developmental brain disorder.

We have been studying EML4 regulation through the cell cycle and discovered that it is less tightly associated with microtubules in mitosis thereby allowing a highly dynamic microtubule-based mitotic spindle to assemble and promote chromosome congression and segregation [3]. We have also been studying the oncogenic mechanism of the EML4-ALK fusion protein that is frequently observed in lung cancer. Excitingly, we found that EML4-ALK fusion proteins can cause accelerated cell migration and invasion that may drive the high rates of metastasis typical of this cancer and have identified a kinase-dependent pathway that could be targeted with new therapeutics. Preliminary evidence suggests that this involves modulation of both the microtubule and actin cytoskeleton networks.

Intriguingly, wild-type EML4 also drives increased cell migration and, considering its expression in neuronal cells, we propose that the physiological role of EML4 is to regulate microtubule-actin crosstalk in neuronal cells. We believe that this is necessary for neuronal cell migration and, ultimately, normal brain development.

Hypothesis and Aims

Our hypothesis is that the EML4 microtubule-associated protein regulates neuronal cell migration through as yet unknown mechanisms that depend on microtubule-actin crosstalk. The aim of this PhD project is to test this hypothesis and explore potential molecular mechanisms through which EML4 exerts its function in neurons.

Experimental Objectives and Methods

  1. Determine the expression and localization of EML4 in neuronal cell lines and rodent hippocampal neurons using a bioinformatics approach, as well as Western blot, qPCR, confocal and super-resolution microscopy.
  2. Examine the functional consequences of removing EML4 on the microtubule and actin networks by imaging following RNAi and CRISPR-Cas9-mediated gene editing. Quantitative analysis of migration and neurite outgrowth will also be analysed.
  3. Search for novel interacting partners of EML4 in neuronal cells by proteomic analysis and follow up candidate regulators of microtubule-actin crosstalk by identifying regions of interaction and colocalization in neuronal cells.

Impact of Project

The immediate impact of this project will be new mechanistic insights into how EML4 regulates cell migration. The longer term impact is that it will help us understand the processes that underlie diseases associated with brain development and degeneration, including recovery from brain injuries, dementia, and mental health disorders. Furthermore, as indicated from our preliminary work, it may reveal new approaches to treat cancers associated with EML4-ALK fusion proteins.

References:

  1. Dogterom & Koenderink (2019) Nature Reviews Molecular Cell Biology 20, 38-55.
  2. Houtman et al. (2007) Neuroscience 144, 1373-82.
  3. Adib et al. (2019) Science Signaling 12, eaaw2939.

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing

Techniques that will be undertaken during the project:

  • Bioinformatics
  • Cell and neuronal tissue culture
  • Protein expression analysis
  • Functional assays using siRNA depletion and CRISPR-mediated gene editing
  • Multi-modal quantitative imaging, incl. super-resolution, confocal, live-cell
  • Proteomic analysis

Contact: Professor Andrew Fry, University of Leicester