Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy
Author:
Pfister Gerald1, Stroh Cordula M.2, Perschinka Hannes1, Kind Michaela3, Knoflach Michael3, Hinterdorfer Peter2, Wick Georg3
Affiliation:
1. Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria 2. Institute for Biophysics, University of Linz, A-4040 Linz, Austria 3. Institute of Pathophysiology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria
Abstract
The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59±2 pN, which correlated nicely to the 51±1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.
Publisher
The Company of Biologists
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