Cargo Recognition and Regulation of the Ncd Motor Protein

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

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.