Structural Reorganization of Nuclear Proteins During Mitosis

Iowa State University, Ames IA

Investigators

Abstract

The long term objective of this study is to test the hypothesis that nuclear derived proteins form a spindle matrix during mitosis that is distinct from the microtubule spindle apparatus. The concept of a spindle matrix was proposed long ago based on theoretical considerations of the requirements for force production to help organize and stabilize the microtubule spindle during mitosis; however, molecular evidence corroborating its existence has been elusive. Dr. Johansen previously characterized a novel nuclear protein in Drosophila, Skeletor, the distribution of which exhibits many features consistent with it being part of a spindle matrix. Skeletor is localized to chromatin during interphase but reorganizes into a true fusiform spindle-structure during prophase at a time when tubulin is still excluded from the nucleus. However, Skeletor encodes a low-complexity protein with no obvious motifs, making it unlikely that Skeletor itself is a structural component of a spindle matrix but rather that it is a member of a multi-protein complex. In searching for other members of such a complex, Dr. Johansen recently identified a protein directly interacting with Skeletor that she named Chromator. Chromator contains a chromodomain and co-localizes with Skeletor on the chromosomes at interphase as well as to the Skeletor-defined spindle during metaphase. Reduction of Chromator levels reveals that Chromator is an essential gene affecting spindle function and chromosome segregation. Dr. Johansen will directly test the hypothesis that Chromator plays a functional role in the cell cycle, by analyzing the effects on cell cycle progression of reduced levels of Chromator using P-element mutations. She will use EMS-mutagenesis to generate a range of Chromator mutant alleles, including complete nulls, which will allow her to genetically dissect the functional requirements of Chromator in different contexts and which will help in identifying essential residues. The necessary and sufficient domains of Chromator will be identified by expressing mutated and deleted constructs of Chromator transgenically in null mutant flies. The hypothesis that Chromator is a member of a multi-protein complex will be further tested by using Chromator as a means to identify other components of the putative spindle matrix by immunoaffinity purification and pull-down assays. The functional analysis of Chromator and its associated proteins will provide new and important insights into the mechanisms behind the complex and highly choreographed process of chromosome segregation during cell division. This project will further provide the research basis for undergraduate and high school training on internships and honors projects within the laboratory. The PI will continue her efforts to motivate elementary and college students to consider research careers by both working directly with such students in the laboratory as well as by presenting general research talks at primarily undergraduate institutions and high schools that introduce students to the controversies and mysteries still to be explained in cell division.