Dr Mark Prescott
Room 217, Bld 13D, Clayton
Phone: +61 3 9905 3724
Fax: +61 3 9542 7199
The laboratory has a strong research focus on the development and application of novel GFP-like proteins. We use a range of biophysical techniques including X-crystallography and spectroscopy to investigate how the structure of these intriguing proteins determines the wide range of optical characteristics, one of which is their colour. GFP technology is an indispensible tool for use in molecular cell biology research.
Indeed, the 2008 Nobel Prize for Chemistry was awarded for work of others on this family of proteins. We engineer proteins to develop fluorescent biosensors suitable for monitoring events in living cells and organisms. One biosensor we have developed, ‘Rosella' (named for the colourful bird) changes colour with pH and is used to follow delivery of cellular material and compartments (eg. mitochondria) to the acidic lumen of the lysosome in a process called autophagy.
Green fluorescent protein (GFP) is just one member of a large super-family. In addition to the many different coloured proteins that together span the entire visible spectrum others have novel and intriguing properties. For example some are intensely coloured but non-fluorescent whilst others change colour when exposed to light of a particular wavelength (photoswitching) or a particular environment (e.g. pH).
These properties arise from differences in the way the side-chains of amino acids interact with the chromophore buried in the protein. We use a wide range of biophysical techniques to investigate how these interactions alter how different proteins respond to light.
We use GFP technology to investigate the molecular mechanism of autophagy. Autophagy is a process whereby cells degrade or turnover their internal components. It takes place in all eukaryotic cells and is important for cellular homoeostasis, differentiation and development. Autophagy is implicated in a growing list of disease processes including cancer and Parkinson's Disease.We use yeast as a biological model to understand the complex regulation of the different types of autophagy. For example, we are investigating the mechanism and regulation of the mitochondrial turnover by autophagy or mitophagy. In this process damaged mitochondria are delivered by one of several mitophagy pathways to the acidic lumen of the vacuole (lysosome in mammalian cells) for degradation. It is important to identify those genes involved in the process and how they are regulated. We use high-throughput imaging screens of yeast cells expressing Rosella to interrogate various gene libraries and experimental conditions. You can watch an example of how we use fluorescent the protein biosensor Rosella at http://www.jove.com/video/2779/a-fluorescence-microscopy-assay-for-monitoring-mitophagy-yeast
Potential Project Areas
- Investigating the mechanisms and regulation of mitophagy
- Developing new fluorescent biosensors
- Investigating the structure and function of novel GFP-like proteins
In our laboratory you will be trained in a wide range of techniques including the following:
- Cloning and mutagenesis
- Protein engineering, expression and purification
- SDS-PAGE and Western blots
- Yeast molecular biology
Yeast is an important model organism studied by a number of research groups in the Monash Yeast Collective - see http://med.monash.edu.au/biochem/monash-yeast-collective-mainpage.html
- Absorbance and fluorescence spectroscopy
- X-ray crystallography
- High-throughput imaging screens
- Analytic gel filtration and analytical ultracentrifugation
- Confocal laser scanning microscopy and wide-field fluorescence microscopy
- Fluorescence live cell imaging
- Advanced fluorescence imaging techniques: lifetime imaging and FRET imaging
Some relevant publications from our laboratory
- ‘Ultramarine, a chromoprotein acceptor for Förster resonance energy transfer' Anne Pettikiriarachchi, Lan Gong, Matthew A. Perugini, Rodney J. Devenish and Mark Prescott PloS One (2012) 7 (7):e41028.
- ‘HcRed, a genetically encoded fluorescent binary cross-linking agent for cross-linking of mitochondrial ATP synthase in Saccharomyces cerevisiae'.Lan Gong, Georg Ramm, Rodney J. Devenish and Mark Prescott (2012) PLoS One 7, (4) e35095.
- "Autophagy in human embryonic stem cells". Tra, T., Gong, L., Kao, L.P., Li, X.L., Grandela, C., Devenish, R.J., Wolvetang, E. & Prescott M. (2011) PLoS One. 6 (doi:10.1371/journal.pone.0027485).
- "Autophagy as a macrophage response to bacterial infection" Lan Gong, Rodney J. Devenish and Mark Prescott (2012) IUBMB Life doi: 10.1002/iub.1070
- "Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines". Cullinane, M., Gong, L., Li, X., Lazar-Adler, N., Tra, T., Wolvetang, E., Prescott, M. Boyce, J.D., Devenish, R.J. and Adler, B. (2008) Autophagy. 4, 744-53.
- "A Fluorescence Microscopy Assay for Monitoring Mitophagy or Nucleophagy in the Yeast Saccharomyces cerevisiae" Mijaljica D, Prescott M and Devenish RJ Journal Vis Exp (2011) Mijaljica, D., Prescott, M. & Devenish, R.J.
- "Hungry for Power: Elimination of mitochondria by mitophagy", May, A., Prescott, M. Australian Biochemist (2011), 42 (2), Australia, pp. 4-7.
- "Receptor protein complexes are in control of autophagy" Dalibor Mijaljica, Taras Nazarko, John Brumell, Wei-Pang Huang, Masaaki Komatsu, Mark Prescott, Anne Simonsen, Ai Yamamoto, Hong Zhang, Daniel Klionsky and Rodney Devenish. (2012) Autophagy. Accepted July 3rd 2012.
- "The 2.2 a crystal structure of a pocilloporin pigment reveals a non-planar chromophore conformation". Prescott, M., Ling, M., Beddoe, T., Oakley, A.J., Dove, S., Hoegh-Guldberg, O., Devenish, R.J. & Rossjohn, J. (2003) Structure 11, 275-284.
- "The 2.0 Å crystal structure of eqFP611, a far-red fluorescent protein from the sea anemone Entacmaea quadricolor." Petersen, J., Wilmann, P.G., Beddoe, T, Aaron J. Oakley, A.J. Devenish, R.J. Prescott, M. & Rossjohn, J. (2005) J. Biol Chem. 278, 44626-31.
- "The 2.1 A crystal structure of the far-red fluorescent protein HcRed: inherent conformational flexibility of the chromophore" Wilmann, P.G., Petersen, J., Pettikiriarachichi, A., Buckle, A. M., Smith, S. C., Olsen, S., Devenish, R.J., Prescott, M. & Rossjohn, J. (2005) J. Mol. Biol. 349, 223-37.
- "Variations on the GFP chromophore: A polypeptide fragmentation within the chromophore revealed in the 2.1 Å crystal structure of a non-fluorescent chromoprotein from Anemonia sulcata" Wilmann, P.G., Petersen, J., Devenish, R.J., Prescott, M. & Rossjohn, J. (2005) J. Biol. Chem. 280, 2401-2404.