Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells . In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile ; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures .Beside the effects on various levels of gene expression (reviewed in reference 1), an increase in temperature may also cause quite dramatic rearrangements in the cytoplasm . In our recent communication (6) we described two proteins from Drosophila melanogaster Kc cells (mol wt 46,000 and 40,000) that appear to change their cellular distribution after a brief heat shock. Here we report on further characterization of these proteins . To this end, monoclonal antibodies have been prepared against the 46,000 mol wt protein and used to determine its location in the cytoplasm of Kc cells and to analyse the relationship between the two proteins from Drosophila and to cross-reacting proteins from hamster cells . Indirect immunofluorescence studies led us to the conclusion that these two proteins are contained in a heat-labile cytoskeleton in Drosophila.Three different cytoskeletal structures have been described and characterized by immunofluorescence microscopy : microfilaments, microtubules, and intermediate-sized (10-nm) filaments (for review see reference 11 and 38) . Our 46,000 mot wt Drosophila protein cannot be actin, because Drosophila actins have molecular weights of 44,000 (36) and do not comigrate with the two proteins described here. Drosophila tubulins do not differ in size from tubulins in other organisms and have molecular weights of 54,000 and 52,000 (14) . MATERIALS AND METHODS Protein Extractions from Kc Cells and OrganismsD. melanogaster Kc cells were grown under constant stirring at a temperature of 25°C in D20 medium (5) and harvested at a density of 5-9x 106 cells/ml. Cells were heat-shocked by immersing the cultures in a 70°C water bath under continuous stirring until the temperature of the culture had reached 37°C. Cultures were shifted to a 37°C water bath and kept for an additional 5 min at elevated temperature . Cellsfrom 500-ml cultures were collected by centrifugation, washed once in 150 mM NaCl, 10 mM Tris/HCI, pH 7.5, 5 mM MgClz, 0.2 mM phenylmethylsulfonylfluoride (PMSF), and taken up in 20 ml of the same buffer. Cells were homogenized without detergent with a motor-driven teflon homogenizer until --50% of the cells were broken . Cell debris was rem...

After 5 minutes heat shock at 37 0C Drosophila melanogaster Kc-cell nuclear proteins were extracted with 0.4M NaCl and compared by SDS gel electrophoresis with extracts from cells grown at 250C. Two proteins (39 000 and 46 000) were only found in heat shock nuclei. Reconstitution with total Drosophila DNA or a DNA fragment from the heat inducible locus 87A/C covalently coupled to sepharose was performed. In the presence of calf thymus competitor DNA these proteins and also others of lower molecular weight showed preferential binding to the homologous DNA.