Axons are slender, cable-like projections of neurons which electrically wire our nervous system and bodies. They are the longest cellular processes and this unique architecture has to be maintained for an organismâs lifetime. Notably, axons contain dense bundles of parallel microtubules (MTs) which form the structural backbones as well as the tracks for long-distance transport between cell bodies and synaptic endings. We lose about 50% of axons towards high age, and this is often accompanied by disorganisation of MT bundles in axon swellings. My host group proposes the model of âlocal homeostasisâ to explain how MT bundles are maintained. The model proposes that (a) MT bundles are highly dynamic with constant turnover to prevent senescence caused by wear and tear; (b) in the axonal environment the dynamic MTs tend to go off-track into disorganisation; (c) different mechanisms mediated by MT regulators âtameâ MTs into axonal bundles. The aim of my project was to identify further molecular mechanisms contributing to MT bundle formation and maintenance, focussing on candidate MT regulators which are all linked to neurodegeneration. These proteins were (1) Futsch/MAP1B (expected to act as a MT cross-linker, potentially in conjunction with the spectraplakin Short stop/Shot), (2) Spastin (expected to sustain MT turnover through its MT severing activity), and (3) the motor protein kinesin-1/Kinesin heavy chain (Khc) (expected to be involved in the even distribution of newly forming MTs through its MT sliding activity). For my studies, I capitalised on the versatile Drosophila primary neurons. I used combinatorial genetics and pharmacological approaches, combined with informative subcellular readouts including MT organisation, axon length, mitochondrial shape and distribution, presynaptic proteins distribution and endoplasmic reticulum. Detailed studies of all three regulators failed to confirm the above proposed working hypotheses, with loss of Futsch, of Spastin or of the Khc-driven MT sliding function failing to generate any MT disorganisation even in long-term studies or when challenged in different ways. Nevertheless, my work on all three candidates produced important new data and concepts. First, my results shifted the focus from Futsch to Shot, in particular its plakin-repeat region (PRR; specific to one isoform abundant in neurons). My work provided first analysis on a new CRISPR-mediated shotïPRR allele lacking the PRR, which causes MT disorganisation, providing indication of the importance of PRR in MT bundling. Second, my results shifted the focus from Spastin to Katanin 60, another MT severer which I found to cause strong MT disorganisation when functionally depleted; future work will show whether it performs the roles I proposed for Spastin. Third, my main focus lay on Khc which causes strong MT disorganisation when depleted. However, this was not caused by absence of Khc-mediated MT sliding, but linked to its roles in cargo/vesicle transport (absence of the Kinesin light chain/Klc linker) and mitochondrial dynamics (absence of Miro or Milton linkers). My experiments aiming to explain how these sub-functions of Khc relate to MT bundle organisation led into new ways of thinking about kinesin-1-linked neurodegeneration. So far, the best supported model involves oxidative stress downstream of mitochondrial dysfunction. The second model involves force imbalance caused by loss of specific motor proteins from specific MT bundle subtracts; it is experimentally less well supported, but consistent with my experimental results so far. Although my experiments failed to support my original hypotheses for roles of Futsch, Spastin and Khc within the model of local axon homeostasis, the work I present opens up new concepts for thinking about axonal cell biology and provides new working hypotheses, candidate molecules and molecular tools readily available for future studies.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Viki Allan (Supervisor) & Andreas Prokop (Supervisor)|
Roles of microtubule-binding proteins during axon growth and maintenance
Liew, Y. T. (Author). 1 Aug 2018
Student thesis: Phd