Development of an in vivo bio imaging system to facilitate real-time cytoskeleton studies

The development and use of fluorescence microscopy technologies has allowed significant breakthroughs to be made in our understanding of the fundamental processes within a cell. This includes the ability to observe the actin and microtubule cytoskeletons, which are the extremely dynamic polymer based intracellular structures responsible for the internal organisation within a cell and allow the cell to respond rapidly to changes in both the intra- and extra- cellular environments. The actin cytoskeleton plays an important role in a plethora of conserved cellular processes within cells. These include the transport of molecules to distinct cellular locations. This is facilitated by myosin motor proteins, which move along actin filaments to deliver cargoes to specific cellular locations. The speed at which the myosins move within the cell (up to 10 um/sec) can make visualising their movements in vivo extremely challenging. Over recent years this lab has been using the fission yeast model system to study the motility and function of the class V myosins, Myo52. This has led to the development of strains and techniques to visualise this myosin's movement in vivo, and the identification of a number of cargoes for these motor proteins (Cell Motil Cytoskeleton 2006 63: 149; J Cell Sci 2007 120: 4093; J. Cell Sci. 2009 122: In press; J. Cell Sci. 2009 122: In press.). However, limitations in current off the shelf bio-imaging equipment have made it difficult / impossible to examine whether the speed of the motor is affected by interactions with: (a) different cargoes (e.g. through protein folding / regulating post-translational modifications / mass increase); or (b) different types of actin polymer, where the presence of different actin binding proteins may regulate myosin movement. This hypothesis is supported by our finding that Myo52 movements fall into at least two populations. This project sets out to (i) Develop an imaging system to facilitate automated Fluorescence Resonance Energy Transfer (FRET) based system biology screens to define proteins which interact with either of the fission yeast class V myosins, and (ii) To simultaneously track the position and dynamic behaviour of the actin track, the myosin motor and its cargo, to examine how their interactions affect motility and whether cargo and track affect the velocity of these molecular motors in vivo. (i) The FRET based screen will be developed using known interacting and non-interacting proteins to optimise screen conditions. The student will optimise the light path and write software scripts to allow automated screening using multiwell plates. Then using a fission yeast YFP fusion expression vector library, the student will look for FRET interactions with Myo52-CFP, in a system biology based fluorescence screen. This screen can then be applied to define interacting proteins for each cytoskeleton component studied within this lab. Each positive result will subsequently be confirmed by yeast 2-hybrid and biochemical techniques. (ii) We will develop a bioimaging system capable of following movements throughout the entire cell using 3 wavelengths (track motor and cargo) in the sub-second time-scale. Previous work in this lab has defined specific protein cargoes of Myo52. The student will generate yeast strains in which Myo52, actin filaments and cargoes are tagged with different combinations of CFP, GFP and mCherry fluorophores, and define which one allows optimum visualisation in combination with optimum functionality. This will introduce the student to the rigours of careful controls and careful experimental procedures of a repetitive nature. Using proprietary technologies developed by Cairn, the student will develop the imaging system to allow simultaneous observation of the three wavelengths or multiple z layers. The student will highlight which aspects of the technology can be improved on, and make developments which can be applied to future imaging systems.