Transcription of the c-fos gene is known to be induced transiently by many types of cellular stimuli in various cultured cell lines; however, several authors have reported that the c-fos gene is constitutively transcribed in lymphoid cells. We detected, in fact, abundant transcripts which hybridized with a v-fos DNA probe in nuclear run-off transcripts and poly(A)⁺ RNA of both quiescent mouse splenic lymphocytes and unstimulated monocytic tumor cell lines. However, human c-fos cDNA did not hybridize with most of these transcripts. No signal was detected by v-fos probe in nuclear run-off transcripts of 3T3 fibroblasts, and c-fos was inducible in both the 3T3 cells and the monocytic tumor cell lines. In quiescent lymphocytes, only the 0.3 kb HincII-PvuII portion of v-foss DNA, which contains a repeat of CAAAA, hybridized with these transcripts; neither other parts of v-fos nor human c-fos DNAs did. These results suggest that a significant portion of the previously reported 'constitutive' transcripts detected by v-fos DNA in lymphocytes and monocytes are not transcripts of c-fos but of other sequences which are specifically expressed in lymphoid cells and have homology with the 0.3 kb HincII-PvuII fragment of v-fos.

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Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings. Also, we found that synapsin I, in the axonal and dendritic growth cones does not necessarily associate with vesicles, but rather with fuzzy filamentous structures in the cytoplasm. In search of the structure of synapsin I in vivo, we employed the quick-freeze, deep-etch technique after immunogold labeling. Synapsin I seems to thereby connect synaptic vesicles or anchor them to cytoskeletons in presynaptic endings.

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We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4°C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide.
Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the micro filaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl₂, but they were similar to those formed in the presence of 2 mM MgCl₂, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.

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An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of micro filament-stabilizing solution squirted from a syringe. The cell membranesleft on the coverslip were immediately stained with tetramethylrhodamine- conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membraneand cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dotlike structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.

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Three-dimensional cellular structures formed by MCF-7human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7cells from S-type to M-type does not occur in cells in volved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.

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Two monoclonal antibodies against a-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeasterjaponicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunobloting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16: 239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

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A pollen grain in Tradescantia reflexa consists of two cells, the generative and the vegetative cells, the generative cell being surrounded completely by the vegetative cell. The generative cell has many lobes or surface invaginations. A complicated network of rER extends throughout the entire vegetative cytoplasm, forming a system of channels made up by the cisternae of rER. Lipid granules are surrounded by ER. Branches of the rER enter all the concavities of the invaginations and attach to the plasma membranesat the bottoms of the invaginations. In the generative cell, no reserve substances, such as lipids, are seen. There is little ER, mitochondria are few in number, and Golgi bodies seem to be less active within this type of cell. Bundles of microtubules run parallel to the long axis of the generative cell. No microtubules or micro filaments can be detected at or near the bottoms of concavities, either on the generative or the vegetative side. ERis the sole cell element that bears a positional relationship to the invaginations. It appears, therefore, that rER is intimately involved in the shaping of the invaginations. This is the first report that a cell element other than microtubules and microfilaments can be involved in the formation of the outer shape of a cell. The possibility that materials from decomposed lipid droplets are transported through the rER to the generative cell is also discussed.

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tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G₀ mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G₀ and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34°C. However, most of these clones grew slowly at 40°C, producing many floating dead cells, and some clones were killed at 40°C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40°C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G₀ phase and are metabolically imbalanced at 40°C during self-stimulation from the G₀ to S phase. We propose that a drug which exclusively block, G₀-G₁ transition would be cytocidal to transformed cells but cytostatic to normal cells.

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Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetiri and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.

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