Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

The 27,000 daltons stress proteins are phosphorylated by protein kinase C during the tumor promoter-mediated growth inhibition of human mammary carcinoma cells

Phorbol-12-myristate-13-acetate (PMA) inhibited growth of human mammary carcinoma cell lines and increased mainly the phosphorylation of two cytosolic phosphoproteins (pp) of 27 kD with isoelectric points of 5.5 (pp27a) and 5.0 (pp27b). The time course of pp27 phosphorylation closely paralleled the rapid PMA-induced subcellular redistribution of protein kinase C (PKC) activity and its subsequent down regulation. Addition of phospholipase C and fetal calf serum to intact cells or purified PKC to a cell free system enhanced the phosphorylation of both pp27 suggesting that the two polypeptides are specific substrates for PKC. Exposure of human mammary carcinoma cells to stress inducers such as arsenite or cadmium increased the 32P incorporation of both pp27 to an extent comparable to PMA. The increased phosphorus content following stress was rather due to a higher rate of synthesis of both pp27 than to a higher phosphorylation state of these polypeptides as determined by [3H]-leucine labeling. These results indicate that the major substrates of PKC, phosphorylated during the PMA-induced growth inhibition of human mammary carcinoma cells, are members of the stress protein family, suggesting a new possible function for these proteins.     

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.     

Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes

The subcellular distribution of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) in epithelial and fibroblastic cells was determined by indirect immunofluorescence microscopy. In interphase cells both regulatory (R II) and catalytic (C) subunits were concentrated in a perinuclear area. By comparison of the R II distribution with the location of a bona fide Golgi membrane constituent, this area was identified as the Golgi complex. The cytochemical localization of R II was confirmed by subcellular fractionation. In addition, cAMP-dPK II was associated with microtubule-organizing centers, in particular with mitotic spindle poles. These distributions of cAMP-dPK II probably represent important factors in mediating the effects of cAMP on basic cellular activities ranging from secretion and proliferation to cell shape and motility.     

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Comparison of M-line and other myofibril components during reversible phorbol ester treatment

The events occurring during phorbol ester mediated destruction of myofibrils in differentiated muscle cells were followed at the fluorescence and electron microscope levels using antibodies which bind troponin-T, a newly discovered 185 000 dalton M-line protein called myomesin and muscle type creatine kinase. The following series of events is proposed. Within one day of phorbol ester treatment, Z-bands and thin filaments, including troponin-T, are absent from many myofibrils resulting in the rapid loss of longitudinal and lateral alignment. A-bands become randomly oriented and clustered into ever smaller compartments within the rounding, myosac-like, multinucleated cells until after 3 days of treatment they too disappear. The M-line proteins are always present in existing A-bands. These results suggest that the Z-band and associated structures are responsible for the maintenance of alignment and the lateral register of myofibrils, whereas the M-line is responsible for the structural integrity of the A-band. When phorbol ester is removed, the cells revert to a myotube morphology and within 2 to 3 days are filled with myofibrils. A comparison of the appearance of troponin-T and the 185 000 dalton myomesin in the recovery period to their appearance during normal myofibrillogenesis reveals that these proteins are more temporally co-ordinated during myofibrillogenesis than in the phorbol ester experimental system.     

Visualization of freeze-dried and shadowed myosin molecules immobilized on electron microscopic films

The mica replication technique first described by Hall [5] has produced myosin molecules which were heterogeneous in appearance in terms of shadowing, decoration, contrast and background. Therefore, an alternative technique for the visualization of myosin molecules was developed: Myosin molecules are sprayed directly onto glow discharged or silicium-monoxide coated carbon filmed grids, omitting glycerol. After washing several times with distilled water, rapid freezing, and freeze-drying, the immobilized myosin molecules are visualized by shadow-casting at low temperature and at varying angles. After backing with carbon the "in situ" shadowed molecules are observed in the electron microscope. This technique has several advantages over the standard method in that it yields more reproducible results. It is potentially useful for investigating interactions of myosin binding proteins with myosin and for visualizing unshadowed myosin in the STEM.