Confocal microscope

To understand how living cells work, it is essential to visualise the processes that go on in them. We will use a specialised but powerful type of microscopy, confocal microscopy, to do this. Confocal microscopy yields very clear images of objects at a single level in the cell, by excluding light from other levels. Advances in technology make it easier to identify multiple structures and follow them in living cells. We will use this technology to study two processes in the fruitfly Drosophila / cell division, and communication between nerve cells (neurons). The correct segregation of duplicated chromosomes to daughter cells is essential for correct transmission of the genetic material. In the human egg, mistakes in chromosome segregation mean that offspring do not develop correctly because they have the wrong number of chromosomes. Such mistakes also predispose dividing cells to become tumour cells. Thus the mechanisms of chromosome segregation are important for critical aspects of medical science. The segregation of chromosomes into daughter cells requires a specialised structure called the spindle. Although first discovered over a century ago, we are only beginning to understand the intricacies of this complex molecular machine. The spindle is a bipolar structure, its two poles anchored in the two future daughter cells. It is built from microtubules, polymers along which replicated chromosomes move towards the two poles. Microtubules are dynamic: they grow and shrink, and tubulin monomers flow along their length. Their dynamics are regulated by many accessory proteins, including motor proteins that pull the chromosomes to the poles. At the spindle poles, microtubules interact with a body known as the centrosome. We will study how the centrosome is duplicated during the cell cycle. Chromosomes have a specialised component, the kinetochore, that interacts with the mitotic spindle. The kinetochore provides a platform for molecules that mediate chromosome attachment to the spindle and their transmission, along with molecules that monitor whether this process occurs correctly. Cell division is highly dynamic and proteins can flip from one state of activity to another by the addition of phosphate groups through the action of enzymes known as protein kinases. We are studying the roles of the protein kinases that orchestrate cell division. Finally, once chromosomes are segregated to daughter cells, the cell itself divides in a process known as cytokinesis. This requires changes in the spindle that regulate formation and constriction of a contractile circular ring-structure that is itself built of numerous molecular components. This work will study the interactions between the various components of the spindle and this contractile ring to bring about this process. Neuronal communication is fundamental to both normal brain function and neurological disease. Using confocal microscopy, we will study how neurons are connected. We will also study some of the ways in which they communicate. Communication depends on the ability to bud off a small area of a larger membrane to form a spherical structure (a vesicle), and then to traffic the vesicle to another location in the cell where it fuses with a target membrane. When an electrical signal reaches a nerve terminal, vesicles that contain neurotransmitter fuse with the cell membrane, releasing neurotransmitter that activates the next cell. The new surface membrane must be recovered by budding of vesicles into the cell interior, where they replenish the supply of synaptic vesicles. Budding also internalises signaling molecules bound to receptors on the cell surface, so that they can signal further, or be trafficked to where they are degraded. Using confocal microscopy, we will monitor receptors, vesicles, and the microtubules that they move along, to understand the mechanisms by which this traffic occurs, and how it contributes to neuronal signalling.

We will install a basic and an advanced confocal microscope for use in Drosophila cell biology. First, in the area of cell division, we will study the effect on the centriole duplication cycle of over-expressing the SAK/PLK4 kinase in cultured cells. The dependence of the dramatic over-replication of centrioles in Drosophila embryos over-expressing SAK/PLK4 upon the conserved SAS4 and SAS6 proteins will also be tested. This analysis requires optical sectioning of living and fixed whole embryos. The recruitment of spindle organising proteins to centrosomes will be followed dynamically as they mature on mitotic entry, and dependence of this process upon Polo kinase and the interacting Greatwall kinase will be investigated. We will tag microtubule-associated proteins that regulate spindle formation with fluorescent proteins to follow their dynamic behaviour in mitosis in wild-type and mutant tissues. Protein complexes that regulate cytokinesis will be tracked in living cells and recovery of fluorescence after photo-bleaching (FRAP) used to follow the recruitment of such protein complexes into the contractile ring. Novel proteins present in mitotic protein complexes, identified with the help of BBSRC Project Grant support, will be tagged with fluorescent proteins to facilitate study of their dynamic localisation in dividing cells. Second, we will also use the instruments for Drosophila neuronal cell biology. We will use molecular and genetic neuroanatomy to characterize neuronal connections relevant to learning, and provide a basis for functional analysis. We will use high resolution immunolabeling and multiple fluorescently tagged proteins in live cells to study how BMP signaling and receptor endocytosis is regulated and regulates the axonal and synaptic cytoskeleton, and how endocytic proteins contribute to synaptic vesicle traffic. The instruments will also be available to other investigators in the areas of cell division and Drosophila cell biology.