Cargo Recognition and Regulation of the Ncd Motor Protein

驱动蛋白和动力蛋白超家族的微管依赖性马达蛋白是有丝分裂和减数分裂纺锤体的重要组成部分，纺锤体是驱动染色体分离的复杂细胞机器。至少对于驱动蛋白，我们现在已经对这些马达如何沿着沿着运动有了相当好的理解，但是要完全理解给定马达在细胞中的作用以及蛋白质如何发挥其作用，还需要对每个马达回答更多的问题。这些问题包括：什么是发动机的货物和如何识别货物？非运动亚单位是否与运动亚单位相关？如果是，它们的作用是什么？ 电机的位置和功能如何调节？对于大多数驱动蛋白，答案可能是相互依赖的，但在回答这些问题的主要绊脚石是，货物的身份是未知的许多驱动蛋白。然而，某些驱动蛋白，包括C-末端，MKLP-1和bimC亚家族的成员，识别和运输一个众所周知的和充分表征的货物：另一个MT。研究这些被认为是驱动MT滑动的电机，应该有助于回答上述问题。 本项目的总体目标是了解果蝇C-末端Ncd马达在卵子发生的减数分裂和胚胎早期有丝分裂中的功能。Ncd可能是最具特征的C-末端驱动蛋白，但关于这种蛋白如何识别货物，无论是马达滑动还是简单地捆绑相邻的MT，天然Ncd的组成，以及它如何被调节以在细胞分裂期间的适当时间产生适当的力，还有很多东西有待了解。 有两个具体目标。第一个将检查Ncd尾与货物微管的ATP非依赖性结合。实验将确定重组Ncd是否能使MT相互滑动，哪些特定残基参与结合货物微管，以及二聚体Ncd尾部结构域如何与货物微管相互作用。第二个目标将检查天然Ncd的亚基组成并表征天然Ncd的活性。像大多数驱动蛋白一样，Ncd从未从天然来源中纯化出来，并且对该蛋白的工作知识是基于在细菌中表达的重组马达亚基。为了确定天然Ncd是否也包含非马达亚基，将使用离子交换色谱和微管亲和技术的组合来纯化天然马达。此外，天然Ncd的马达特性将被确定，以确定先前的工作与重组Ncd马达亚基是一个准确的反映天然Ncd活性。 为了实现这些目标，步行者博士将使用生物化学，生物物理和分子生物学方法的组合。对Ncd活性和功能的进一步了解将揭示C-末端驱动蛋白的机制，并最终揭示微管依赖性马达的基本特性和依赖于这些蛋白质的细胞过程。

Microtubule-dependent motor proteins of the kinesin and dynein superfamilies are essential components of mitotic and meiotic spindles, the complicated cellular machines that power chromosome segregation. For kinesins at least, there is now a fairly good understanding of how these motors move along microtubules, but to understand completely the role that a given motor plays in the cell and how the protein performs its role, additional questions need to be answered for each motor. These questions include: What is the motor's cargo and how is that cargo recognized? Are non-motor subunits associated with the motor subunits and if so, what is their role? How is the motor's location and function regulated? For most kinesins, the answers are likely to be interdependent, but a major stumbling block in answering these questions is that the identity of the cargo is unknown for many kinesins. However, certain kinesins, including members of the C-terminal, MKLP-1, and bimC subfamilies, recognize and transport a well-known and well-characterized cargo: another MT. Study of these motors, which are thought to drive MT sliding, should facilitate answering the above questions. The overall objective of this projectis to understand how the Drosophila C-terminal Ncd motor functions during the meiotic divisions of oogenesis and the early mitotic divisions of the embryo. Ncd is perhaps the best-characterized C-terminal kinesin, yet much remains to be learned about how this protein recognizes cargo, whether the motor slides or simply bundles adjacent MTs, the composition of native Ncd, and how it is regulated to produce appropriate forces at appropriate times during cell division. There are two specific objectives. The first will examine the ATP- independent binding of the Ncd tail to cargo microtubules. Experiments will determine if recombinant Ncd can slide MTs past each other, what specific residues are involved in binding cargo microtubules, and how dimeric Ncd tail domains interact with cargo microtubules.The second objective will examine the subunit composition of native Ncd and characterize the activity of native Ncd. Ncd, like majority of kinesins, has never been purified from native sources and working knowledge of the protein is based on recombinant motor subunits expressed in bacteria. To determine if native Ncd also contains non-motor subunits, a combination of ion exchange chromatography and microtubule affinity techniques will be used to purify the native motor. In addition, the motor properties of native Ncd will be determined to determine if previous work with recombinant Ncd motor subunits is an accurate reflection of native Ncd activity. To accomplish these objectives, Dr. Walker will use a combination of biochemical, biophysical and molecular biology approaches. Increased understanding of Ncd activity and function will shed light of the mechanisms of C-terminal kinesins, and ultimately on the fundamental properties of microtubule-dependent motors and the cellular processes dependent on these proteins.