Developing yeast single cell microinjection technology for the study of protein

The ability to maintain protein quality throughout the lifetime of a cell is crucial to the maintenance of longevity and healthy ageing. Loss of the ability to correctly manage protein quality control systems leads to the production of protein aggregates which can prove to be toxic and which are often linked to disease. The production of protein aggregates is complex and can result from mutation, a variety of cell stresses such as oxidative and heat stress, mitochondrial dysfunction, loss of chaperone activity or defects in process that can clear mis-folded protein products such as autophagy. To unravel the complexities that underlie protein quality control and its downstream effects many researchers turn to genetically tractable model organisms such as the budding yeast S. cerevisiae. Within this studentship we will conduct research that will facilitate the development of new technologies designed to improve and accelerate research into protein aggregation via collaboration with Singer Instruments. The project design offers a wide range of training within academic and industrial settings under an umbrella project that fits within the BBSRC strategic area of "Lifelong Health and Wellbeing".
Project Overview:
The student will focus upon the disease related aggregate forming protein superoxide dismutase 1 (SOD1). Mutations in SOD1 lead to familial Amyotrophic Lateral Sclerosis (ALS, Motor Neuron Disease) for which there is no known cure and for which the cause of cell death is currently unknown. The Sod1 protein is evolutionary conserved and mutant forms that are associated with ALS can be expressed in yeast leading to toxicity (our unpublished data). We are therefore able to utilise yeast as convenient platform to determine the nature of mutant Sod1 toxicity. The student will produce and purify mutant forms of Sod1 that are most commonly found in ALS patients using yeast expression systems and standard biochemical techniques. Purified Sod1 will then be induced to aggregate in vitro and microinjected into yeast cells using new technology being developed at Singer Instruments. This approach offers the unique advantage of being able to control the size of aggregates and concentration delivered to the cell at specific stages of the cell cycle. Following injection the health of cells will be monitored using a variety of assays that are well developed within our lab such as respiratory rate, ROS levels, viability and redox potential within specific compartments. Expression of mutant Sod1 and its toxicity will also be achieved by standard gene expression techniques. We will then micro-inject Sod1 aggregates into yeast cells lacking genes that control specific functions known to be involved in protein homeostasis and organelle function. This medium throughput will allow us to obtain new insights into the mode of Sod1 aggregate mediated toxicity. It will also provide a means to test exciting new microinjection technology that has tremendous potential and which will have many applications.
Training:
The students will receive the benefits of training opportunities in both the academic and industrial applicant labs. Within the academic environment the student will receive training in molecular biology techniques, live cell imaging, biochemical assays, protein purification and flow cytometry amongst others. At Singer Instruments the student will develop a different set of key skills that centre upon customised robotics, analytical software development and IT, quality control procedures and industrial standard lab protocols. The package represents an exciting opportunity that underpins cutting edge technology development and scientific research.