Role of kinesin light chain 1 in binding to specific cargoes.

除细菌外，所有生物体的细胞都含有被称为微管的细丝，微管充当将物质从一个区域运输到另一个区域的轨道。这种重要的运输是由蛋白质进行的，蛋白质沿着沿着微管“行走”，充当移动许多不同货物的微小马达。微管马达蛋白有两个家族，驱动蛋白和动力蛋白，大多数驱动蛋白将货物从细胞中心运送到细胞周边。这些马达及其携带的物质对神经细胞的健康和大脑功能至关重要，因为它们的突变可能导致或促成肌肉萎缩症、阿尔茨海默氏病、亨廷顿氏病、痉挛性截瘫和精神分裂症等疾病。它们的功能在身体的所有细胞中都是至关重要的，而不仅仅是神经元。驱动蛋白1转运许多不同种类的货物，从膜细胞器到细胞骨架蛋白。它由两种类型的蛋白质亚基组成：KIF 5亚基提供运动活性，而驱动蛋白轻链（KLC）与货物蛋白结合并控制驱动蛋白的活性。KLC有几种不同的类型，因此一种可能性是每种货物都使用含有特定KLC的驱动蛋白-1。有四个KLC基因，其中KLC 1基因被选择性剪接以产生至少17种不同的蛋白质（同种型），这些蛋白质的氨基酸序列仅在一端（其C-末端）变化。我们的功能研究表明，两种亚型，KLC 1B和KLC 1D，各自控制不同膜类型的运动，支持KLC变异对货物选择至关重要的观点。KLC 1剪接在生理学上是重要的，因为某些剪接形式水平的变化与阿尔茨海默病和精神分裂症有关。总之，很明显，KLC对驱动蛋白-1货物结合和随后的激活是至关重要的。然而，我们并不完全了解不同的KLC，特别是KLC 1亚型的重要性，在驱动蛋白1的功能。作为第一步，我们必须确定它们所结合的细胞货物，以及将它们招募到这些货物中的特定蛋白质。为了开始这样做，我们使用了一种称为BioID的技术，该技术将生物素分子添加到特定KLC同种型附近的任何蛋白质上。然后使用质谱法分离和鉴定生物素化的蛋白质。我们发现，KLC 1D，而不是KLC 2或3，是在密切接近三个蛋白质参与内吞作用。这条途径是细胞从细胞外吸收物质（例如营养素和生长因子）的途径。KLC 1D的两个近邻，SNX 1和CCDC 22，参与了从要降解的材料中回收材料的分类。第三个是BIRC 6，它是细胞分裂后一分为二所必需的，这一过程称为胞质分裂。该项目的一个主要目标是测试含有KLC 1D同种型的驱动蛋白-1如何有助于早期内体的蛋白质分选，重点是需要SNX 1和CCDC 22的两个途径。我们也将研究驱动蛋白在胞质分裂中的作用。为了做到这一点，我们将制作一个新的驱动蛋白工具箱，使我们能够快速地从细胞中去除KLC 1，或者用一个缺乏运动域的非活性版本取代细胞上的驱动蛋白。我们将使用光学显微镜和生化分析来监测在破坏驱动蛋白功能后内吞作用发生了什么。我们还将确定SNX 1，CCDC 22和BIRC 6是否可以直接与KLC 1D结合，如果可以，则解剖这种结合所需的每种蛋白质的区域。最后，我们将使用BioID来测试KLC 1亚型是否确实将驱动蛋白-1靶向特定的细胞器，使用三种额外的KLC 1剪接形式。总的来说，这个项目将提供重要的洞察KLC 1剪接如何影响驱动蛋白功能和与特定货物的关联。

Cells of all organisms except bacteria contain filaments, called microtubules, that act as tracks for the transport of material from one region to another. This vital traffic is carried by proteins that 'walk' along microtubules, acting as minute motors that move many different cargoes. There are two families of microtubule motor proteins, the kinesins and dyneins, with most kinesins carrying cargo away from the cell centre towards the cell periphery. These motors and the cargoes they carry are absolutely vital for the health of nerve cells and for brain function, since mutations in them can cause or contribute to diseases such as muscular dystrophy, Alzheimer's disease, Huntington's disease, spastic paraplegia and schizophrenia. Their function is vital in all cells of the body, not just neurons.Kinesin-1 transports many different kinds of cargo, ranging from membrane organelles through to cytoskeletal proteins. It is made up of two types of protein subunits: the KIF5 subunits provide the motor activity, while the kinesin light chains (KLCs) bind to cargo proteins and also control kinesin's activity. The KLCs come in several different types, so one possibility is that each cargo uses kinesin-1 containing a particular KLC. There are four KLC genes, with the KLC1 gene being alternatively spliced to generate at least 17 different proteins (isoforms) that vary in amino acid sequence only at one end, their C-terminal. Our functional studies showed that two isoforms, KLC1B and KLC1D, each control the movement of different membrane types, supporting the idea that KLC variation is crucial for cargo selection. KLC1 splicing is important physiologically, as changes in the levels of certain splice forms have been linked to Alzheimer's disease and schizophrenia. Altogether, it is clear that KLCs are central to kinesin-1 cargo binding and subsequent activation. However, we do not fully understand the importance of different KLCs-and particularly KLC1 isoforms-in kinesin-1 function. As a first step, we must identify the cellular cargoes to which they bind, and the specific proteins that recruit them to those cargoes. To begin to do this, we have used a technique called BioID that adds a biotin molecule to any protein in the near neighbourhood of a specific KLC isoform. Biotinylated proteins are then isolated and identified using mass spectrometry. We found that KLC1D, but not KLC2 or 3, was in close proximity to three proteins involved in endocytosis. This pathway is the route by which cells take up material (nutrients and growth factors, for example) from outside the cell. Two of the KLC1D near-neighbours, SNX1 and CCDC22, are involved in sorting material that should be recycled from that to be degraded. The third, BIRC6, is needed for cells to divide into two after cell division in a process called cytokinesis. A major goal of this project is to test how kinesin-1 containing the KLC1D isoform contributes to protein sorting at the early endosome, focussing on the two pathways that require SNX1 and CCDC22. We will also investigate kinesin's role in cytokinesis. To do this, we will make a new kinesin tool-box that will allow us to remove KLC1 rapidly from cells, or replace the cell's kinesin on cargo with an inactive version that lacks the motor domains. We will use light microscopy and biochemical assays to monitor what happens to endocytosis after disrupting kinesin function. We will also determine if SNX1, CCDC22 and BIRC6 can bind directly to KLC1D, and if so, dissect the regions of each protein needed for this binding. Finally, we will use BioID to test if KLC1 isoform do indeed target kinesin-1 to specific organelles, using three additional KLC1 splice forms. Overall, this project will provide important insight into how KLC1 splicing affects kinesin function and association with specific cargoes.