Kinesin light chains: why so many?

Kinesin-1是一种微管马达，可以在细胞内移动许多货物，这种运输对生命和健康至关重要。这在神经元中至关重要，神经元依靠Kinesin-1将分泌囊泡、线粒体、蛋白质和mRNAs等货物从神经细胞体运送到轴突尖端，轴突尖端可能超过1米远。Kinesin-1是由两个运动亚基(KHC)和两个相同的轻链多肽(KLCs)组成的四聚体。在脊椎动物中有四个KLC基因，在线虫中有两个，不同的KLC蛋白高度保守，除了它们的C-末端，这也受到进一步的交替剪接。因此，KLC的多样性提供了一种机制，可以将马达引导到许多不同的货物上。目前尚不清楚含有单一KLC亚型的Kinesin-1四聚体是如何组装的，但一种可能性是每个KLC亚型的mRNAs被包装成单独的、不同的mRNPs，这些mRNPs也包含KHC mRNAs。本项目将深入分析Kinesin-1的功能和组装，以及许多不同KLCs的作用。我们将使用培养的人类细胞(非神经细胞和类似神经元的SH-SY-5Y)和遗传易驯化的模式生物秀丽线虫。将使用广泛的细胞和分子生物学、生化和遗传技术，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，只有5个英国机构可以使用。我们将：1.利用单分子FISH技术测试KHC和KLC mRNAs的定位是否在Kinesin-1本身的合成中发挥作用，并评估活细胞中的mRNA动力学。2.研究Kinesin通过运输MARS复合体(催化氨基酸与其对应的tRNA结合的氨基酰-tRNA合成酶复合体)来确保蛋白质合成所需的tRNA供应，从而支持整个神经元的蛋白质合成。3.确定Kinesin在定位神经元通路必需分子NAV1(线虫中的UNC-53)中的作用，NAV1是一种微管+末端结合蛋白。4.研究KLCS1和KLCS2在线虫中的功能：它们是具有不同的角色还是共享的角色？现有的KLC1和KLC2突变体将用于研究Kinesin-1货物在体内的运动性。KLCS1和KLCS2的表达谱和分布将通过使用CRISPR/Cas9标记内源蛋白来评估。该项目与DTP主题世界级支撑生物科学相一致，并解决了BBSRC战略“了解生命规则”，因为它使用了广泛的复杂实验方法来研究根本重要的微管发动机-Kinesin-1的功能。它将利用BBSRC资助的晶格光片显微镜，这是该国仅有的5台之一。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Kinesin研究开发许多新工具。因此，该项目直接关系到“变革性技术”的目标，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长的人。对线虫KLC功能的分析还将从正常生理、早期发育和整个寿命方面产生与“生物科学和对健康的综合理解”相关的数据。它可以被归入BBSRC研究分类“技术和方法发展”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'.