GFP tagged Hsp60 used to investigate the translocation of Hsp60 in response to mitochondrial stress in HeLa cells

Abstract

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.