Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.

Abstract

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...

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

“…1) reveal three classes: (i) the bulk of mRNAs, which are nontranslated during heat shock but are preserved (Fig. 1H); (ii) a few control mRNAs whose translation continues during heat shock, e.g., mRNAs cod- (18,57 (14,17,22,34,(64)(65)(66) (17,34). The close location is impressively illusosition of prosomes (1) and pre-HSGs (2) trated by a freeze fracture image of chicken cells showing .…”

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm

“…The preparation of the affinity-purified polyclonal antiserum against the kinesin-II 115 K subunit is described in Wedaman et al [1996]. An anticentrosome monoclonal antibody raised against Drosophila intermediate filaments which cross reacts with sea urchin centrosomes [see Falkner et al, 1981;Schatten et al, 1987] …”

Functions of intermediate filaments

“…GFAP is expressed in glial cells, primarily within the central nervous system, whereas the neurofilament proteins are expressed exclusively in neurons [see Traub, 19851. 'Bartnik et al, 1985;'Bartnik et al, 1986;3Biessmann et al, 1982;4Bilbant, 1980;'Coggeshall, 1967;7Eagles et al, 1981;'Falkner et al, 1981;'Gilbert, 1975;"Harris and Shaw, 1984;"Kensler et al, 1979;"Lasek et al, 1979;''Phillips et al, 1983;I5Pumplin and Muller, 1983;16Walter and Biessman, 1984;I7Zackroff and Goldman, 1980; '*Kauri and Eckert, 1986;"Jeffery and Meier, 1983;20White and Jeffery, 1987;21Foelix and Hauser, 1979;22Venuti and Jeffery, 1988;23Boyle and Ernst, 1989;Z4Weber et al, 1988;25White et al, 1988.…”