Characterising novel mitotic microtubule associated proteins in the early Drosophila embryo

Abstract

Cell division is a fundamental biological process driven by the formation of a microtubule (MT) based mitotic spindle, ensuring the accurate segregation of chromosomes. MT length, nucleation and dynamics are all determined by microtubule associated proteins (MAPs). Following on from previous work carried out by the Hughes et al., (2008) this investigation has applied quantitative comparative proteomics to cycling and mitotic Drosophila embryo extracts prepared via MT cosedimentation assay, in order to compare their MAP complements as they progress though the cell cycle or whilst fixed at the metaphase-anaphase transition. We have demonstrated that many MAPs known for their roles in cell division increase their association with MTs during mitosis, and in addition our approach has identified a number of protein classes not previously characterised as a MAP, binding to MTs during mitosis. One of these protein classes was the Replication Factor C complex (RFC). The RFC complex is most well-known for its role as the sliding clamp loader in DNA replication, however it is now presenting as MAPs with a potential mitotic function. By combining techniques from biochemistry, fluorescence microscopy and further proteomic analysis we have been able to begin to investigate the localisation and functions of this complex during mitosis. Thus far we have been able to biochemically show that the RFC complex is a true MT binding protein and that all three alternative RFC complexes, as well as the archetypal complex, are present in mitotic embryo extracts following immunoprecipitation of RFC3. We have also shown via fluorescence imaging that the RFC complex presents a weak localisation to the mitotic spindle. Application of these techniques has also led to further investigation into the known MAPs, Asp and DTACC, for which we have identified novel protein interactors and investigated localisation during the process of Drosophila embryonic spindle self-assembly.