HEAT-SHOCK PROTEIN HSP90 AND ITS ASSOCIATION WITH THE CYTOSKELETON - A MORPHOLOGICAL-STUDY

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M Cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell reparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5) M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque. However, after combined treatment of those preparations with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine, the filamentous structures double labeled with anti-HSP90 and anti-cytokeratins antibodies appeared to be similarly reorganized, showing comparable rearrangement characteristics, the later suggesting a possible association of HSP90 with keratin cytoskeleton. It is important to note that there is no in vitro cross-reaction of anti-HSP90 antibodies with any of the studied cytoskeletal proteins in Western blotting analysis.