Synchronization of drosophila cells in culture

Summary We synchronized Drosophila cell lines (Schneider's line 2 and K_c) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S=14 to 16 hr; G₂=6 to 8 hr; M=0.4 hr) were similar to those of K_c cells. This work was performed under the auspices of the U.S. Energy Research and Development Administration. A.R. was supported by an NIH post-doctoral fellowship, No. CA01060.     

Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Intermediate-sized filaments in Drosophila tissue culture cells

In using a monoclonal antibody against a major cytoplasmic protein of 46,000 mol wt, we have characterized an intermediate-sized (10 nm) filamentous cytoskeleton in Drosophila melanogaster tissue culture cells. Indirect immunofluorescence, immunoelectron microscopy, and protein blotting show that this cytoskeleton exhibits features typical of the vertebrate vimentin cytoskeleton, including the diameter and appearance of filaments, sensitivity to 10(-6) M colcemid, and insolubility in buffers containing 1% Triton X-100. The antibody cross- reacts with vimentin and desmin from baby hamster kidney cells and stains a vimentin cytoskeleton in the vertebrate Chinese hamster ovary cell line. We, therefore, conclude that the 46,000-mol wt Drosophila protein is homologous to vertebrate vimentin. Three minor, higher- molecular-weight polypeptides are also detected in the Drosophila cells that react with the antibody. At least two of these are members of a family of proteins with properties resembling those of the 46,000-mol wt intermediate filament protein.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.     

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.     

Circular extrachromosomal copia-like transposable elements in Drosophila tissue culture cells

We find that in the circular extrachromosomal DNA from Drosophila tissue culture cells the transposable elements copia, 412, 297, and mdg 1 are present in variable amounts. There is no detectable circular DNA homologous to B104 . From the relationship between the intra- and extrachromosomal forms it appears that the amount of different circular elements is not related to the amount of the respective chromosomal elements.     

Pole cells of Drosophila melanogaster in culture. Normal metabolism, ultrastructure, and functional capabilities.

The metabolism, ultrastructure, and function of mass-isolated pole cells were examined during short-term culture in vitro. In addition to demonstrating that these cells functioned normally in culture, a number of new features of embryonic pole cells were discovered. Cell populations isolated from Renografin density gradients were incubated in medium containing tritiated valine, uridine, or thymidine. Although pole cells incorporated similar amounts of valine into protein as other embryonic cells throughout the first 6 hr in culture, they began to synthesize RNA only after 2 hr in culture. Approximately 30% of the pole cells synthesized DNA in vitro and this synthetic activity occurred largely during the first hour of culture. An ultrastructural analysis of colcemid-treated cells showed that 10% of the pole cells divide shortly after placement in culture. During pole cell culture in vitro, polar granules and nuclear bodies fragment and disperse so that they are eventually not detected in these cells. These changes also occur during pole cell development in vivo. Finally, we have obtained 25 to 33% germ line mosaicism among the fertile adults which were derived from embryos receiving transplantation of isolated pole cells before and after culture in vitro. These results demonstrate that these cells are able to follow their normal developmental program in vitro and are able to give rise to functional germ cells in vivo.     

Changes in nuclear proteins induced by heat shock in Drosophila culture cells

Nuclear proteins of normal and heat-shocked Drosophila cells were analysed by two-dimensional electrophoresis. The computerized processing of the gels allowed us to detect 6 proteins strongly induced by the heat treatment, but which were different from the usually described heat-shock proteins. The possible role of these proteins in genetic regulation is discussed, as is the value of this type of approach for the study of other genetic regulation phenomena.     

Heat-shock proteins are associated with hnRNA in Drosophila melanogaster tissue culture cells

Ribonucleoprotein complexes of Drosophila melanogaster Kc tissue culture cells grown at 24°C or heat-shocked at 37°C were cross-linked in vivo by u.v. irradiation. Cross-linked heterogeneous nuclear ribonucleoprotein (hnRNP) complexes were fractionated by oligo(dT)-cellulose chromatography and CsCI density centrifugation. The hnRNP complexes of both 24°C and 37°C culture cells possess buoyant densities in CsCI between = 1.38 g/cm^-3 and 1.43 g/cm^-3. The ³⁵S-labelled proteins bound to the hnRNA of 37°C culture cells correspond in mol. wt. to the so-called heat-shock proteins of 70 K, 68 K, 27 K, 26 K, 23 K and 22 K. The 70 K and 68 K proteins are also present in hnRNP complexes of 24°C culture cells. In addition, several other Drosophila hnRNPs of 140 K, 56 K, 45 K, 43 K, 38 K, 37 K and 34 K, whose synthesis is strongly repressed under heat-shock conditions, could be identified. The results demonstrate that the so-called heat-shock proteins possess a function as RNPs.     

Network-Specific Synchronization of Electrical Slow-Wave Oscillations Regulates Sleep Drive in Drosophila

1. Download : Download high-res image (224KB)
2. Download : Download full-size image

     

Drosophila grim induces apoptosis in mammalian cells.

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.