Kinesin light chains: why so many?

运动蛋白-1是一种微管马达，它在细胞内移动许多货物，这种运输对生命和健康至关重要。它对神经元至关重要，神经元依靠驱动蛋白-1将诸如分泌囊泡、线粒体、蛋白质和mrna等货物从神经细胞体运送到可能超过1米远的轴突尖端。Kinesin-1是两个运动亚基（KHCs）和两个相同的轻链多肽（KLCs）的四聚体。在脊椎动物中有4个KLC基因，在线虫C. elegans中有2个，不同的KLC蛋白高度保守，除了它们的c端，它们也受到进一步的交替剪接。因此，KLC品种提供了一种机制，指导电机到它的许多不同的货物。目前尚不清楚含有单一KLC异构体的激酶蛋白-1四聚体是如何组装的，但一种可能性是每个KLC异构体的mRNA被包装成单独的、不同的mRNA颗粒（mRNPs），这些mRNA颗粒也含有KHC mRNA。本项目将深入分析激酶1的功能和组装，以及许多不同的KLCs的作用。我们将使用培养的人类细胞（非神经元细胞和类似神经元的SH-SY-5Y）和遗传上易于处理的模式生物秀丽隐杆线虫。广泛的细胞和分子生物学，生物化学和遗传技术将被使用，包括CRISPR/Cas9。我们将使用晶格光片显微镜对活细胞和蠕虫中的动态事件进行成像，这是一种先进的成像技术，仅在英国5家机构中可用。我们将:1。使用单分子FISH测试KHC和KLC mRNA的定位是否在激酶1本身的合成中起作用，并评估活细胞中的mRNA动态。2. 研究kinesin通过运输MARS复合体（一种氨基酰基-tRNA合成酶复合体，催化氨基酸与其相应的tRNA结合）来确保蛋白质合成所需tRNA的供应，从而支持整个神经元的蛋白质合成的假设。3. 确定激酶在定位神经寻路分子NAV1（秀丽隐杆线虫中的Unc-53）中所起的作用，NAV1是一种微管+端结合蛋白。4. 研究KLCs 1和KLCs 2在秀丽隐杆线虫中的功能：它们是否具有不同的或共同的作用？现有的KLC1和KLC2突变体将用于研究体内运动蛋白-1转运体的运动性。通过使用CRISPR/Cas9标记内源性蛋白来评估KLCs 1和KLCs 2的表达谱和分布。该项目与DTP主题“世界一流的基础生物科学”相一致，并解决了BBSRC的战略“理解生命的规则”，因为它使用了广泛的复杂的实验方法来研究至关重要的微管马达-激酶-1的功能。它将利用bbsrc资助的晶格光片显微镜，这是全国仅有的5台显微镜之一。这将确保学生获得尖端显微镜和分析方面的专业知识。该项目还将涉及为动力学蛋白研究产生许多新工具。因此，该项目与“变革性技术”目标以及培养在定量、综合和数据密集型生物科学方面具有多学科专业知识的人才直接相关。对秀丽隐杆线虫中KLC功能的分析也将产生与“健康的生物科学和综合理解”相关的数据，包括正常生理、早期发育和整个生命周期。它可以归入BBSRC研究分类“技术与方法发展”或“分子、细胞和工业生物技术”。

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'.