Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy

Gerald Pfister,Cordula M Stroh,Peter Hinterdorfer,Michael Knoflach,Hannes Perschinka,Georg Wick,Michaela Kind

doi:10.1242/jcs.02292

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.

Highlights

Heat shock proteins (HSP) are ubiquitous and structurally highly conserved molecules
Owing to the strong homology between microbial and human HSP60 an immune reaction against infectious microbes may lead to a cross-reactive autoimmune response against the native human HSP60, which is increasingly produced in arterial endothelial cells (ECs) stressed by classical atherosclerosis risk factors (Mayr et al, 1999; Wick and Xu, 1999; Wick et al, 2001; Wick et al, 2004)
A spacer molecule, used to bind the ligand to the Atomic force microscopy (AFM) tip, enhances the mobility of the ligand, thereby increasing its chance to bind to the cognitive receptor attached to the surface (Hinterdorfer et al, 1996; Hinterdorfer et al, 1998). It helps to discriminate between specific and nonspecific interactions. Since this method works with receptors embedded in membranes of live and fixed cells (Lehenkari and Horton, 1999) we aimed to prove the presence of HSP60 on the cell surface of stressed ECs and to measure the interaction strength between HSP60 and a monoclonal antibody (AbII-13) directed against human HSP60, which was coupled to the AFM tip

Summary

Introduction

Heat shock proteins (HSP) are ubiquitous and structurally highly conserved molecules. They have important physiological functions in folding and intracellular transport of newly synthesized proteins and in preventing their aggregation and misfolding. Owing to the strong homology between microbial and human HSP60 an immune reaction against infectious microbes may lead to a cross-reactive autoimmune response against the native human HSP60, which is increasingly produced in arterial endothelial cells (ECs) stressed by classical atherosclerosis risk factors (Mayr et al, 1999; Wick and Xu, 1999; Wick et al, 2001; Wick et al, 2004). The atherogenic importance of the stress-induced production of HSP60 in ECs provides the rational basis for our analysis of HSP60 on the surface of ECs after stress exposition

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