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运动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。至少对于运动蛋白来说，现在对这些马达如何沿着微管运动已经有了相当好的理解，但是要完全理解一个给定马达在细胞中扮演的角色以及蛋白质如何发挥其作用，还需要回答每个马达的其他问题。这些问题包括：汽车的货物是什么？如何识别这些货物？非运动亚基与运动亚基有关联吗？如果有，它们的作用是什么？电机的位置和功能是如何调节的？对于大多数动力蛋白来说，答案可能是相互依赖的，但回答这些问题的一个主要障碍是，许多动力蛋白的货物身份是未知的。然而，某些驱动蛋白，包括c端，MKLP-1和bimC亚家族的成员，识别和运输一种众所周知的和具有良好特征的货物：另一种MT。研究这些被认为驱动MT滑动的马达，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端Ncd运动如何在卵发生的减数分裂和胚胎的早期有丝分裂中起作用。Ncd可能是最具特征的c端运动蛋白，但关于这种蛋白质如何识别货物，马达是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的适当时间产生适当的力，还有很多有待研究。有两个具体目标。第一项研究将检查Ncd尾部与货物微管的ATP非依赖性结合。实验将确定重组Ncd是否可以使mt相互滑动，结合货物微管涉及哪些特定残基，以及二聚体Ncd尾部结构域如何与货物微管相互作用。第二个目标将检查原生非传染性疾病的亚基组成和表征原生非传染性疾病的活性。Ncd，像大多数运动蛋白一样，从未从天然来源中纯化过，并且对该蛋白的工作知识是基于在细菌中表达的重组运动亚基。为了确定天然Ncd是否也含有非运动亚基，将使用离子交换色谱和微管亲和技术的组合来纯化天然运动。此外，将确定天然Ncd的运动特性，以确定先前对重组Ncd运动亚基的研究是否准确反映了天然Ncd的活性。为了实现这些目标，沃克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有助于揭示c端运动蛋白的机制，并最终揭示微管依赖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.