Kinesin light chains: why so many?

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Kinesin-1 is a microtubule motor that moves many cargoes within cells, and this transport is crucial for life and health. It is vital in neurons, which rely on kinesin-1 to carry cargo such as secretory vesicles, mitochondria, proteins and mRNAs from the nerve cell body to the axon tip, which may be more than 1 metre away. Kinesin-1 is a tetramer of two motor subunits (KHCs) and two identical light chain polypeptides (KLCs). There are four KLC genes in vertebrates, and two in the nematode worm, C. elegans, and the different KLC proteins are highly conserved except at their C-termini, which are also subject to further alternate splicing. KLC variety therefore provides a mechanism for directing the motor to its many different cargoes. It is not known how kinesin-1 tetramers containing a single KLC isoform are assembled, but one possibility is that the mRNAs for each KLC isoform are packaged into separate, distinct mRNA granules (mRNPs) that also contain KHC mRNAs. This project will provide an in-depth analysis of kinesin-1 function and assembly, and the role of the many different KLCs. We will use cultured human cells (non-neuronal and the neuron-like SH-SY-5Y) and the genetically tractable model organism, C. elegans. A wide range of cell and molecular biological, biochemical and genetic techniques will be used, including CRISPR/Cas9. We will image dynamic events in living cells and worms using lattice lightsheet microscopy, an advanced imaging technique that is available in only 5 UK Institutions. We will:1. Test if the localisation of KHC and KLC mRNAs plays a part in the synthesis of kinesin-1 itself, using single molecule FISH, and assessing mRNA dynamics in live cells. 2. Investigate the hypothesis that kinesin supports protein synthesis throughout the neuron by transporting the MARS complex (the aminoacyl-tRNA synthetase complex that catalyses the attachment of amino acids to their corresponding tRNAs) to ensure a supply of tRNA for protein synthesis. 3. Determine the role of kinesin in localising the essential for neuronal pathfinding molecule NAV1 (Unc-53 in C. elegans), a microtubule plus end binding protein. 4. Investigate the function of KLCs 1 and 2 in C. elegans: do they have distinct or shared roles? Existing KLC1 and KLC2 mutants will be used to investigate the motility of kinesin-1 cargoes in vivo. The expression profile and distribution of KLCs 1 and 2 will be assessed by tagging the endogenous protein using CRISPR/Cas9.This project aligns with the DTP theme World Class Underpinning Biosciences, and addresses the BBSRC strategy "Understanding the Rules of Life", because it is using a wide range of sophisticated experimental approaches to investigate the function of the fundamentally important microtubule motor, kinesin-1. It will take advantage of the BBSRC-funded lattice lightsheet microscope, one of only 5 in the country. This will ensure the student gains expertise in cutting-edge microscopy and analysis. The project will also involve generating many new tools for kinesin research. The project is therefore directly relevant to the "Transformative Technologies" goal, and the development of people with multidisciplinary expertise in quantitative, integrative and data-intensive bioscience. The analysis of KLC function in C. elegans will also generate data relevant to the "Bioscience for and integrated understanding of health", in terms of normal physiology, early development and across the lifespan. It could sit under the BBSRC Research Classification 'Technologies & methodological development' or 'Molecules, cells and industrial biotechnology'.