Demonstration of different patterns of microtubule organization in Physarum polycephalum myxamoebae and plasmodia using immunofluorescence microscopy

We have used anti-tubulin antibodies and immunofluorescence microscopy to determine the overall distribution of microtubules during interphase and mitosis in both the myxamoebae and plasmodia of the slime mold Physarum polycephalum. We have paralleled these observations with electron microscopy of the same stages. The myxamoebae possess a network of cytoplasmic microtubules whilst the coenocytic plasmodium does not possess any cytoplasmic microtubules--at either interphase or mitosis. In plasmodia microtubules are, however, elaborated by an intranuclear microtubule organizing centre (MTOC) during prophase of mitosis and these microtubules proceed to form part of the mitotic spindle. There is little difference in the overall distribution and arrangement of microtubules during division of either the myxamoebal or plasmodial nuclei. These findings are discussed in relation to the synthesis of tubulin during the plasmodial cell cycle and the rearrangements of the nuclear envelope during mitosis.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells.

A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4 x 10(5) cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 microgram DNA or 5 micrograms protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

A purified cellular extract accelerates the cell cycle in Physarum polycephalum

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells

Summary A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4×10⁵ cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling, with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 μg DNA or 5 μg protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous, for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Halophilic Bacteria Susceptibility to Peracetic Acid Vapor and Ethylene Oxide

Extremely to slightly halophilic bacteria were tested for susceptibility to two sterilizing agents, peracetic acid (PAA) and ethylene oxide (ETO). PAA susceptibility was explored by two methods: an agar plate (constant pH of 7.2) and a filter strip (constant incubation period of 37 days); 100% susceptibility was obtained by both methods. The dosage (0.5 ml/min) was applied to a filter pad in a petri dish cover. Glove box experiments with ETO (input 1.5 lb. [ca. 680.4 g]/24 hr, the only constant) yielded 100% susceptibility for all halophiles tested. These experiments demonstrated the efficacy of two lethal agents for extreme halophiles, PAA and ETO. Variation in pH did not affect susceptibility.     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

Chinese hamster ovary cell mitosis and its response to ionizing radiation: a morphological analysis of the living cell

Repeated microscopic observations of exponentially growing Chinese hamster ovary cells were made and the times and mitotic stages were recorded in control and irradiated cultures at 37 degrees C. As determined by autoradiography, the time from the end of S phase to early prophase (the G2 phase) was 46 min, to breakdown of the nuclear envelope was 91 min, and to restoration of the nuclear envelope was 116 min. The time spent in morphologically distinguishable phases of mitosis and the effects of 0.5, 1.0, 1.5, 2.0, and 4.0 Gy of gamma or X radiation on cells at each phase were determined. Affected cells were found to be delayed without or with reversion to an earlier mitotic stage before recovering and advancing through mitosis. Cells were timed in the five steps comprising delay with reversion: inertia, cessation I, regression, cessation II, and reprogression. No cells treated in late prophase, i.e., within 8-10 min of nuclear envelope breakdown, were delayed by the doses used; therefore the critical or transition point must be situated in middle prophase. Cells irradiated in this stage were not delayed by 0.5 or 1.0 Gy, but suffered a dose-dependent delay with or without reversion after 1.5, 2.0, and 4.0 Gy. Cells irradiated in early prophase and very late interphase responded similarly, but a greater percentage of the latter reverted.     

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and mice were killed at various intervals after an intravenous injection of 3H-thymidine to time the acquisition of label by the various phases of mitosis. The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G1 and G2 stages were 6.8 and 1.0 hr, respectively. From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurrence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr. Ten minutes after an intravenous injection of 3H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase. We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G2. Also, nuclei are in telophase during the early half of G1 but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G1 into early and late periods is practical.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Three-dimensional culture of bovine chondrocytes in rotating-wall vessels

Summary The Rotating-Wall Vessel (RWV) was used to culture chondrocytes for 36 d to observe the influence of low-shear and quiescent culture conditions allowing three-dimensional freedom on growth, differentiation, and extracellular matrix formation. Chondrocytes were freshly isolated from bovine cartilage and placed into the RWV with Cytodex-3 microcarriers. Nonadherent petri dishes were initiated with microcarriers as representative of standard culture conditions. In the RWV, large three-dimensional aggregates (5–7 mm) were formed in suspension. In addition, a large sheet of matrix adhered to the oxygenator core and vessel endcaps. Petri dish culture resulted in the formation of sheets of chondrocytes with no matrix production. Immunocytochemical analyses on histologic sections of tissue obtained from the RWV and the petri dish controls were performed with antibodies against fibronectin, collagen II, chondroitin-4-sulfate, chondroitin-6-sulfate, and vimentin. Results demonstrated increased signal in the RWV material while the petri dishes demonstrated a slight decrease in signal. In addition, differentiated chondrocytes were observed in sections of RWV material through 36 d, while few were observed in the sections of petri dish material. These results indicate that the unique conditions provided by the RWV afford access to cellular processes that signify the initiation of differentiation as well as production of normal matrix material.     

The intranuclear microtubule organizing centre in the plasmodium of the myxomycete Physarum polycephalum

Physarum possesses two different microtubule cytoskeletons. In amoebae, cytoplasmic and mitotic microtubules are nucleated by a typical centrosome. In contrast, it has been reported that plasmodia have an intranuclear spindle organizing centre (SPOC) devoid of centrioles. We present genetic evidence suggesting that the SPOC located in the centrosome is very similar to the intranuclear plasmodial SPOC. The immunostaining properties of a new monoclonal antibody against Physarum centrosome has been used to compare these different MTOCs. Moreover, a dense plasmodial microtubule network was present in interphase plasmodia and absent in plasmodia undergoing mitosis. MTOCs responsible for the nucleation of the cytoplasmic microtubule network and intranuclear SPOCs were located in two different compartments of the plasmodium.     

Plasmodium falciparum gametocyte formation in vitro: its stimulation by phorbol diesters and by 8-bromo cyclic adenosine monophosphate

Phorbol 12-myristate 13-acetate increased the number of gametocytes by 50 to 100% in well or petri dish cultures of the HB-3 clone of Plasmodium falciparum. Phorbol dibutyrate had a similar effect. The optimal concentration for each of these agents was 20 ng/ml or approximately 30 nM. No effect of forskolin was found, other than a general inhibition of growth at concentrations over 10 microM. An inhibitor of phosphodiesterase, 8-bromo cyclic adenosine monophosphate (at concentrations of 0.1 and 1.0 microM) also significantly increased the number of gametocytes formed by this clone.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Cryopreservation of mouse embryos at -196 degrees C by vitrification

Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U. PMSG and 5 I.U. hCG given 48 hr apart. Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS). Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2. After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish. After 3 washings in culture medium the embryos were kept in CO2 incubator for further development. In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture. Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).     

SYNTHESIS OF RNA IN MAMMALIAN CELLS DURING MITOSIS AND INTERPHASE

Chinese hamster cells in the mitotic and G₁ phases of the growth cycle were incubated for 30 or 60 min in suspension tissue culture and pulse-labeled with tritiated uridine. After appropriate chases, washes, and extractions, it was found that all incorporation into the nucleic acid may be accounted for by those cells in interphase. An average of 410 counts was found for incorporation into the cell population (approximately 2.0 x 10⁵ cells) of which over 80% of the cells was initially in mitosis. The increasing number of cells leaving mitosis and entering interphase during the 30 min incubation was theoretically able to account for 470 counts. In addition, short-pulse labeling experiments have shown a consistent linear relationship between the percentage of cells in division and the incorporation of the isotope, which strongly suggests that, if 100% of the cells were in mitosis, the counts would be essentially zero. Thus, the entire label may be attributed to those cells in interphase where portions of the chromosomal material are known to be already extended.