Nuclear proteins in Drosophila melanogaster cells after heat shock and their binding to homologous DNA

Falkner, F G; Biessmann, Harald

doi:10.1093/nar/8.5.943

Cited by 12 publications

(5 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We recently described two proteins (mol wt 46,000 and 40,000) that appear to change their cellular distribution after a 5-min heat shock of D. melanogaster Kc cells (6) . To determine the localization of these proteins within Kc cells, we fractionated cells into nuclear, mitochondrial, microsomal, and soluble cytoplasmic components using differential centrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…Cell debris was removed by centrifugation at 10,000 g for 15 min, and this postmitochondrial supernate subjected to a 100,000 gcentrifugation in a SW56 rotor (Beckman Instruments, Inc., Fullerton, Calif.) for 5 h. The pellet is referred to as microsomal fraction whereas the supernate represents the soluble fraction . Nuclei were prepared and extracted with buffered 0.4 M NaCl as described recently (6).…”

Section: Methodsmentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

“…FIGURE6 (a) SDS-polyacrylamide gel electrophoresis of total BHK proteins (lane 2), and protein fractions of various stages of vimentin purification from these cells (33) (lanes 3, 4, 5) . Proteins after final PBS-EDTA washes (lane 3), protein after DNase I treatment (lane 4), proteins extracted from the perinuclear "caps" after colcemid treatment of BHK cells (lane 5) .…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Falkner¹,

Saumweber²,

Biessmann³

1981

The Journal of Cell Biology

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Falkner¹,

Saumweber²,

Biessmann³

1981

The Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…A possible explanation for the reported association ofthe small hsps with chromatin and nuclear matrix also comes in part from studies of the 46-kDa IFP. Falkner and Biessmann (33) found that this protein is present in nuclear extracts from heat-shocked cells and will bind to DNA. Thus, any disruption of nuclei (e.g., isolation of chromatin, salt extraction, DNase treatment, or, perhaps, severe heat shock) may allow the IFPs to enter the nucleus, where they can stick to chromosomes and other nuclear structures.…”

mentioning

confidence: 99%

Small heat shock proteins of Drosophila associate with the cytoskeleton.

Leicht¹,

Biessmann²,

Palter³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vnmentin-like intermediate filament proteins (EFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mehanis. Indirect Immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe-indeed, lethal-shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the some perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.The heat shock response is a ubiquitous cellular response to physiological stress characterized by the rapid induction of a small number of heat shock proteins (hsps; reviewed in refs. 1-3). The extreme conservation of the response and of the hsps themselves suggests that these proteins carry out essential functions. However, aside from their role in the development of thermotolerance-the ability to withstand a second heat shock at an otherwise lethal temperature (4-10)-little is known about their function(s). Thus far, the most progress has been made for hsp70. In yeast, replacement of several members of the hsp7O gene family with mutant genes indicates a role for hsp70 in growth at high temperatures (11). Likewise, mutants in dnaK (the Escherichia coli counterpart ofhsp70) are temperature-sensitive for growth (12) and are unable to recover normal protein synthesis following heat shock (13). hsp70 appears to play a similar role in Drosophila (14, 15) and apparently carries out this function within the nucleus (16).As a means of addressing the function of the low molecular weight family of hsps, several investigators have examined the intracellular distribution of these proteins. Several reports have argued for an association with chromatin and nucleoli (8,17). Yet, others appear to rule out association with chromatin and suggest that these proteins are part of the nuclear matrix (18,19 (20)(21)(22)(23). This cofractionation appears to be due to the collapse of the IFP network against the nucleus within 5 min of heat shock (20-23). In order to assess the contribution of the collapse of the IF cytoskeleton on the localization of the small hsps, we have reinvestigated the intracellular distribution of the small hsps ofDrosophila by biochemical fractionation and indirect immunofluorescence. The data best support association of these proteins with the IF cytoskeleton. For heat shock, cells were incubated at 370C for 15 min prior to harvest. Cells were rinsed with Echalier's medium, concentrated to 1 ml, and labeled with 50 ,uCi (1 Ci = 37 GBq) of [35S]methionine (Amersham) at 250C or 37TC for 1 hr. T...

show abstract

The Effect of Steroid Hormones on Gene Transcription

Anderson

1984

Biological Regulation and Development

View full text Add to dashboard Cite

Nuclear proteins in Drosophila melanogaster cells after heat shock and their binding to homologous DNA

Cited by 12 publications

References 39 publications

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

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

Small heat shock proteins of Drosophila associate with the cytoskeleton.

The Effect of Steroid Hormones on Gene Transcription

Contact Info

Product

Resources

About