HSP70 kinetics study by continuous observation of HSP-GFP fusion protein expression on a perfusion heating stage.
The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.
Wang
Sihong
S
Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.
Xie
Weijun
W
Rylander
Marissa Nichole
MN
Tucker
Philip W
PW
Aggarwal
Shanti
S
Diller
Kenneth R
KR
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006361:Heating / Q000379:methods; D007700:Kinetics; D008657:Metabolic Clearance Rate; D008856:Microscopy, Fluorescence / Q000379:methods*; D010477:Perfusion; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure; D013696:Temperature
D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins