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

除细菌外，所有生物的细胞都含有被称为微管的细丝，它作为物质从一个区域运输到另一个区域的轨道。这种重要的交通是由沿着微管“行走”的蛋白质携带的，它们就像微型马达一样，移动着许多不同的货物。微管运动蛋白有两个家族，运动蛋白和动力蛋白，大多数运动蛋白将货物从细胞中心运送到细胞周围。这些马达和它们携带的货物对神经细胞的健康和大脑功能绝对至关重要，因为它们的突变会导致或促成肌肉萎缩症、阿尔茨海默病、亨廷顿病、痉挛性截瘫和精神分裂症等疾病。它们的功能对身体的所有细胞都至关重要，而不仅仅是神经元。运动蛋白-1运输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：KIF5亚基提供运动活性，而运动蛋白轻链（KLCs）与货物蛋白结合并控制运动蛋白的活性。KLC有几种不同的类型，所以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，KLC1基因被选择性地剪接，产生至少17种不同的蛋白质（同种异构体），这些蛋白质只在一端（它们的c端）的氨基酸序列不同。我们的功能研究表明，KLC1B和KLC1D两种同工异构体各自控制不同膜类型的运动，这支持了KLC变异对货物选择至关重要的观点。KLC1剪接在生理上是重要的，因为某些剪接形式的水平变化与阿尔茨海默病和精神分裂症有关。总之，很明显，KLCs是驱动蛋白-1货运结合和随后激活的核心。然而，我们并不完全了解不同的klcs -特别是KLC1亚型-在激酶-1功能中的重要性。作为第一步，我们必须确定它们结合的细胞货物，以及将它们招募到这些货物的特定蛋白质。为了做到这一点，我们使用了一种叫做BioID的技术，将生物素分子添加到特定KLC异构体附近的任何蛋白质上。然后用质谱法分离和鉴定生物素化蛋白。我们发现KLC1D，而不是KLC2或3，与三种参与内吞作用的蛋白非常接近。这种途径是细胞从细胞外吸收物质（例如营养物质和生长因子）的途径。KLC1D的两个近邻，SNX1和CCDC22，参与分类应该从其中回收的材料进行降解。第三种是BIRC6，在细胞分裂过程中细胞分裂成两个细胞需要BIRC6。该项目的一个主要目标是测试含有KLC1D异构体的激酶1如何在早期内体中促进蛋白质分选，重点关注需要SNX1和CCDC22的两条途径。我们还将研究激酶在细胞分裂中的作用。为了做到这一点，我们将制作一个新的激酶工具箱，它将允许我们快速地从细胞中移除KLC1，或者用缺乏运动结构域的非活性版本替换细胞上的激酶。我们将使用光学显微镜和生化分析来监测破坏运动蛋白功能后内吞作用的变化。我们还将确定SNX1， CCDC22和BIRC6是否可以直接与KLC1D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KLC1异构体是否确实使用三种额外的KLC1剪接形式将激酶-1靶向到特定的细胞器。总的来说，这个项目将为了解KLC1剪接如何影响激酶的功能和与特定产物的关联提供重要的见解。

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.