Dissection of kinetochore structure and function in Drosophila

The correct segregation of duplicated chromosomes to daughter cells is a fundamental part of biology as it ensures the correct transmission of the genetic material. When this process does not occur correctly in the development of the human egg, it leads to offspring that do not develop correctly because they have the wrong number of chromosomes. Mistakes in chromosome segregation in the dividing cells of our bodies can predispose these cells to turn into tumour cells. This is because loss of one chromosome carrying a wild-type copy of a tumour suppressor gene can leave only its counterpart carrying a mis-functioning gene. Thus an understanding of the mechanisms that regulate chromosome segregation are important for our understanding of these critical aspects of medical science. Chromosomes have a specialised component, the kinetochore, that interacts with microtubules of the mitotic spindle, the molecular machine required for chromosomes to be directed into the two daughter cells. The kinetochore provides a platform for the molecules that mediate chromosome attachment to microtubules and their transmission together with molecules that monitor whether this process occurs correctly. Here we propose to study how a kinetochore is built in the fruit fly, a model organism that has been used to study chromosome inheritance for almost 100 years. An understanding of how a kinetochore is constructed will help us to understand its function. The kinetochores of yeasts comprise over 70 proteins and those of animal cells could contain more. It is thought that kinetochores from all species comprise four main sub-complexes. Our pilot studies have confirmed the identity of 3 proteins that are members of two of these sub-complexes. These gives a 'way in' to the study of the organisation and function of the kinetochore in this model system using a combination of genetic and biochemical approaches. We will study the effects of disrupting these two kinetochore sub-complexes by a process that allows us to deplete these proteins from cultured cells and by studying their mutants in the developing fly. For both types of study we will use time-lapse microscopy to follow spindle microtubules tagged with a protein that makes them fluoresce green and chromosomes that carry a protein tagged with another protein that fluoresces red. This will allow us to follow the defects in the behaviour of this complex molecular machine. We will also examine the consequences of disrupting one of the sub-complexes for the assembly of the rest of the kinetochore. In so doing we will examine not only the recruitment of the core components of the kinetochore into the final structure but also the molecules that regulate chromosomes attachment and those that are individual motors within the machine. We will adapt cultured fruitfly cells so that they make kinetochore components that have been 'tagged' in such a way that they can be fished out from the complex mix of proteins present in the cell. This also fishes out the proteins that they bind to. We can identify such proteins in an instrument that analyses the precise mass to charge ratio of breakdown products from the proteins. This gives a characteristic signature that can be recognised in the genetic blueprint of the fruit fly. Having identified the components of the kinetochore we will be able to study the way in which they interact and how this affects function. As protein-protein interactions can be influenced by enzymes that place negatively charged phosphate groups on proteins, we will study the effects of these enzymes upon the protein interactions within the complexes of proteins that make up the kinetochore.

We will disrupt the MIND and NDC80 kinetochore sub-complexes of Drosophila and study effects on attachment of chromosomes to spindle microtubules, chromosome congression and, if applicable, anaphase separation. This will be carried out on both fixed preparations and on living cells by time lapse microscopy. Down-regulation will be achieved in cultured cells by RNAi with the 3 genes we have identified. In 2 cases we will also examine cells of the whole organism in specific mutants. We will also build constructs to express hairpin RNAs under control of the bipartite GAL4-UAS system in specific tissues and developmental stages. In each of these situations we will follow recruitment of other core kinetochore components, molecules that interact with microtubules, and spindle assembly checkpoint (SAC) molecules. We will determine how this might be regulated by kinetochore associated mitotic protein kinases and phosphatases using kinase or phophatase mutants, RNAi and chemical inhibitors of kinase function. We will express kinetochore components tagged in such a way to facilitate their purification from S2 cells together with interacting proteins by affinity chromatography. Interacting co-purifying proteins will be identified by mass spectrometry and the functions of novel putative kinetochore proteins assessed by RNAi. We will examine proteins that show changes in migration on 2D electrophoresis following treatment of cells with RNAi or chemical inhibitors of protein kinases. For Polo substrates we will also assess the status of the 3F3/2 phosphoepitope. We will also isolate and phosphorylate kinetochore sub-complexes in vitro with purified kinases. We will determine the phosphorylation sites of substrates in anticipation of making mutant genes encoding these proteins that cannot be phosphorylated or that are phosphomimics. In the longer term we will study the effects of such mutations on the assembly of the kinetochore and its function.