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First published online 22 March 2005
doi: 10.1242/jcs.02292

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

Gerald Pfister^2,*, Cordula M. Stroh^1,*, Hannes Perschinka², Michaela Kind³, Michael Knoflach³, Peter Hinterdorfer^1, and Georg Wick³

¹ Institute for Biophysics, University of Linz, A-4040 Linz, Austria
² Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria
³ Institute of Pathophysiology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria

View larger version (27K): [in a new window]   Fig. 4. Flow cytometric analysis of unstressed (left panels) and heat-stressed (right panels) living HUVECs. (a) Dot plots of forward scatter (cell size) against side scatter (surface granularity) showing similar size and granularity in control and heat-stressed cells. (b) Dot plots of forward scatter (cell size) against FITC fluorescence intensity (FITC FLI) showing a significant increase of cells with HSP60 surface expression from 1.1% in the control cells to 9.5% in heat-stressed cells. Cells have been incubated with the FITC labelled monoclonal antibody II-13 specific for human HSP60. (c) Histogram plots of cell counts against propidium iodide intensity showing full cell membrane integrity in control cells and heat-stressed cells. The full membrane integrity is indicated by the equal high percentage (100%) of propidium iodide negative cells in both unstressed and stressed HUVECs.

View larger version (102K): [in a new window]   Fig. 1. Production of HSP60 in the mitochondria of HUVECs fixed with ice-cold acetone/methanol. The nuclei appear red after staining with the fluorescent DNA dye TOPRO-3 and excitation at 543 nm. HSP60 appears green after labelling with a FITC-conjugated monoclonal anti-HSP60 antibody and excitation at 488 nm. (a) Unstressed control cells. (b) Cells exposed to heat stress (42°C, 30 minutes). (c) Detailed view of mitochondrial localization of HSP60 in heat stressed cells.

View larger version (79K): [in a new window]   Fig. 2. CLSM images of HSP60 on the surface of living HUVECs after incubation with the FITC-conjugated monoclonal anti-HSP60 antibody II-13. All images are focused on the top of the cells. (a) Heat-stressed (42°C, 30 minutes) cell showing HSP60 on the cell surface (green regions marked by arrows). The insert shows another heat stressed cell expressing HSP60 on the cell surface. (b) Unstressed control cells with almost no surface expression of HSP60.

View larger version (71K): [in a new window]   Fig. 3. Image series of a three dimensional reconstruction of 25 single zx-profiles of a living heat-stressed HUVEC. Surface HSP60 is presented as a thermo-false-colour image. The image series is from a video (see Movie 1 in supplementary material).

View larger version (14K): [in a new window]   Fig. 5. Force spectroscopy of AbII-13-coated tip on HUVECs. (a) Schematic representation of a force-distance cycle carried out to measure specific molecular forces. (b) The force-distance cycle on HUVECs with an AbII-13-coated AFM tip shows specific interaction in the retrace (jump at 75 nm). The specific interaction is blocked using free AbII-13 in solution (inset). (c) The probability density function (pdf) is constructed from an ensemble of forces, gives the distribution of unbinding forces fu. (d) Binding probabilities of AbII-13-coated tips on non-stressed cells (NC), heat-shocked cells (HS) and on heat shocked cells blocked with free AbII-13 in solution (Block), proving the specificity of the measured molecular interactions.

View larger version (37K): [in a new window]   Fig. 6. Force spectroscopy of AbII-13-coated tip on a protein layer of isolated HSP60. HSP60 was electrostatically adsorbed onto mica. (a) Topography image of HSP60 on mica. Single proteins of about 3 nm in height and small aggregates cover the surface of the 500 x 500 nm large area almost completely. The false colour bar at the bottom of the image (0 to 5 nm, from dark to bright) indicates the heights of the objects. (b) Probability density function (pdf) of specific molecular forces giving the distribution of unbinding forces fu. (c) Binding probabilities of AbII-13-coated tips on isolated HSP60 without (left) and with free AbII-13 in solution (Block), proving the specificity of the measured molecular intersections.