GFP tagged Hsp60 used to investigate the translocation of Hsp60 in response to mitochondrial stress in HeLa cells
Short, M. E. (2013). GFP tagged Hsp60 used to investigate the translocation of Hsp60 in response to mitochondrial stress in HeLa cells (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/7934
Permanent Research Commons link: http://hdl.handle.net/10289/7934
Heat shock protein 60 (Hsp60) is a mitochondrial stress protein that is elevated in response to mitochondrial impairment. It is involved in protein folding and, more recently, it has been identified as a signalling molecule. It has been found elevated in patients suffering from diabetes, atherosclerosis and cardiomyopathy. In order to investigate the translocation of Hsp60 from mitochondria we employed RG224428, a plasmid housing genes for a human Hsp60 connected to a green fluorescent protein (GFP) bound at the C-terminus. The plasmid was cloned inside DH5α E. coli (DH5α) and purified using an endotoxin-free plasmid extraction kit. Using endotoxin-free DNA we transfected the human cervical cancer cell line HeLa with the Hsp60-GFP DNA. It has been found that endotoxin (or otherwise known as lipopolysaccharide) can affect Hsp60 expression in mammalian cells, so by using endotoxin-free plasmid DNA we could induce controlled stress treatments without the unwanted effects the DH5α endotoxin would exhibit. Propidium iodide staining found the plasmid posed very low-to-no toxicity on HeLa cells. Mitochondrial staining using Mitotracker CMX Ros showed that the Hsp60-GFP was being taken up by mitochondria almost exclusively. High-glucose and sodium azide were used to stress the transfected HeLa cells and the translocation of the Hsp60-GFP signal was recorded using an Olympus FV1000 laser scanning confocal microscope. We found that the Hsp60-GFP colocalises with mitochondria to a high degree. We also found that high-glucose can be linked to the translocation of Hsp60-GFP out of mitochondria. The results here support published data that suggest higher glucose concentrations may play a role in the translocation of Hsp60 out of mitochondria and into extracellular fluids. We have also found that the use of fluorescent-confocal microscopy may provide a useful way to detect the movement of the biomarker Hsp60 under stress and therapeutic treatments.
University of Waikato
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