Cytoskeletal rearrangements in adult rat cardiomyocytes in culture.
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Animals↗
Biomedical subjects
H M Eppenberger
Publications and source records attributed to H M Eppenberger.
Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.
Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mib-CK was isolated in a two-step isolation procedure starting with 1 litre of isopropyl beta-D-thiogalactopyranoside-induced E. coli culture, whereas previous attempts to express other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mib-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mib-CK expression. Recombinant Mib-CK displayed an identical N-terminal amino acid sequence, identical Km for phosphocreatine and Vmax. values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mib-CK had the same molecular mass as native chicken Mib-CK in m.s. analysis, indicating that post-translational modification of the enzyme in chicken tissue does not occur. As judged by gel-permeation chromatography and electron microscopy, recombinant enzyme formed predominantly octameric oligomers with the same overall structure as the chicken heart enzyme. Furthermore, the enzymes isolated from both sources formed protein crystals of space group P42(1)2, when grown in the absence of ATP, with one Mi-CK octamer per asymmetric unit. The indistinguishable X-ray-diffraction patterns indicate identical structures for the native and recombinant proteins.
Animals↗
Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis.
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Adenosine Triphosphate↗
In situ compartmentation of creatine kinase in intact sarcomeric muscle: the acto-myosin overlap zone as a molecular sieve.
Creatine kinase isoenzymes (CK = ATP: creatine N-phosphoryl transferase, EC 2.7.3.2) were localized in situ in cryosections of intact sarcomeric muscle by immunocytochemical staining. Similar to cardiac muscle, spermatozoa and photoreceptor cells, mitochondrial-type CK (Mi-CK) localization in skeletal muscle was also restricted to mitochondria. Besides the well-documented localization of muscle-type (M-CK) at the M-line and at the sarcoplasmic reticulum, surprisingly, most of the sarcoplasmic M-CK was also highly compartmentalized and was mainly confined to the I-band. The localization of M-CK at the I-band coincided with that of adenylate kinase and aldolase. In intact muscle, the diffusion equilibrium decisively favours occupancy by all three enzymes of the I-band, with the acto-myosin overlap region of the A-band acting as a molecular sieve, excluding to a large extent all three enzymes from the acto-myosin overlap region. This indicates that in intact muscle, this region of the A-band may be less accessible in vivo to soluble, sarcoplasmic enzymes than thought before. If muscle were permeabilized by chemical skinning before fixation, I-band CK, as well as aldolase and adenylate kinase, were solubilized and disappeared from the myofibrils, but the fraction of M-CK which was specifically associated with the M-line remained bound to the myofibrils. Implications of these findings are discussed with respect to the functional coupling of I-band-CK with glycolysis, to the formation of large multienzyme complexes of glycolytic enzymes with CK and to the supply of energy for muscle contraction in general.
Actins↗
Fast myosin light chain expression in chicken muscles studied by in situ hybridization.
We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.
Animals↗
Urination frequency and cystic pressure resistance after fractionated whole or partial irradiation of the rabbit urinary bladder.
Urination frequency and cystic pressure resistance have been used as end-points to assess x-ray-induced changes of bladder function. Whole or half bladders of adult male rabbits were irradiated, caudally or cranially. The absorbed dose was 33 Gy, 36 Gy or 39 Gy, given in 5 daily fractions. Animals which received a whole bladder dose of 39 Gy or 36 Gy showed increased urination frequency and enhanced bladder pressure resistance during the whole follow-up time of 100 weeks, compared with the sham-irradiated controls. At half bladder irradiation, only the highest doses (39 Gy to the cranial part of the bladder and 39 Gy or 36 Gy to the caudal part) gave rise to a slight increase in frequency at about 20 weeks after exposure.
Animals↗
Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.
Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.
Actins↗
Interaction of mitochondrial creatine kinase with model membranes. A monolayer study.
The interaction of mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi-CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi-CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligomeric form of Mi-CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi-CK was able to interact equally well with both inner and outer mitochondrial membranes.
Animals↗
Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers.
Mitochondrial creatine kinase isolated from chicken cardiac muscle was crystallized by vapor diffusion techniques. Depending on the growth conditions, fine needles and platelets as well as large single crystals appeared after a few days. Large crystals were shown to diffract to at least 3.2 A resolution (Schnyder, T., Winkler, H., Gross, H., Sargent, D., Eppenberger, H. M., and Wallimann, T. (1990) Biophys J. 57, 420 and thus are suited for a detailed X-ray analysis in the future. The relatively high density of single crystals measured by a linear organic solvent density gradient indicates a tight packing of mitochondrial creatine kinase molecules within the crystals. Microcrystals, however, were subjected to electron optical examination either after prefixation with glutaraldehyde followed by conventional negative staining or by freeze-fracturing crystals in mother liquor and heavy metal replication with platinum/carbon. In both cases the crystals exhibited a square lattice with parameters of a = b = 139 A and a = b = 132 A in negatively stained and replicated crystals, respectively. No other lattice parameters were found, suggesting that these microcrystals represent a quasi-cubic three-dimensional lattice, which is in accordance with the finding that the building blocks of the crystals are the cube-like octamers described (Schnyder, T., Engel, A., Lustig, A., and Wallimann, T. (1988) J. Biol. Chem. 263, 16954-16962). Digital image processing applied to electron micrographs of crystals clearly revealed the arrangement of mitochondrial creatine kinase octamers in the crystal lattice as well as the subdivision of the octamer into its subdomains at a resolution of 23 A.
Animals↗
Heading of the 20th European Muscle Club meeting in Oxford in the fall 1991.
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Europe↗
Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.
The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.
Animals↗
Adult rat cardiomyocytes cultured in creatine-deficient medium display large mitochondria with paracrystalline inclusions, enriched for creatine kinase.
In adult regenerating cardiomyocytes in culture, in contrast to fetal cells, mitochondrial creatine kinase (Mi-CK) was expressed. In the same cell, two populations of mitochondria, differing in shape, in distribution within the cell and in content of Mi-CK, could be distinguished. Immunofluorescence studies using antibodies against Mi-CK revealed a characteristic staining pattern for the two types of mitochondria: giant, mostly cylindrically shaped, and, as shown by confocal laser light microscopy, randomly distributed mitochondria exhibited a strong signal for Mi-CK, whereas small, "normal" mitochondria, localized in rows between myofibrils, gave a much weaker signal. Transmission EM of the giant mitochondria demonstrated paracrystalline inclusions located between cristae membranes. Immunogold labeling with anti-Mi-CK antibodies revealed a specific decoration of these inclusions for Mi-CK. Addition of 20 mM creatine, the substrate of Mi-CK, to the essentially creatine-free culture medium caused the disappearance of the giant cylindrically shaped mitochondria as well as of the paracrystalline inclusions, accompanied by an increase of the intracellular level of total creatine. Replacement of creatine in the medium by the creatine analogue and competitor beta-guanidinopropionic acid caused the reappearance of the enlarged mitochondria. It is believed that the accumulation of Mi-CK within the paracrystalline inclusions, similar to those observed in certain myopathies, represents a compensatory effect of the cardiomyocytes to cope with a metabolic stress situation caused by low intracellular total creatine levels.
Animals↗
Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina.
Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.
Animals↗
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.
Animals↗
Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle.
Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.
Animals↗
Mitochondrial creatine kinase from chicken brain. Purification, biophysical characterization, and generation of heterodimeric and heterooctameric molecules with subunits of other creatine kinase isoenzymes.
In a recent study it has been shown that mitochondrial creatine kinase from chicken brain (Mia-CK) and heart (Mib-CK) are two distinct isoenzymes differing in ten out of the thirty N-terminal amino acids (Hossle, J.P., Schlegel, J., Wegmann, G., Wyss, M., Böhlen, P., Eppenberger, H.M., Wallimann, T., and Perriard J.C. (1988) Biochem. Biophys. Res. Commun. 151, 408-416). The present article describes the purification and biophysical characterization of the mitochondrial creatine kinase isoenzyme from chicken brain (Mia-CK). Gel permeation chromatography, direct mass measurements of individual molecules by scanning transmission electron microscopy, and analytical ultracentrifugation confirmed the existence of two different oligomeric forms, dimeric and octameric Mia-CK, with molecular masses of 85 kDa and 306-352 kDa and with sedimentation constants of 4.9-5.3 and 11.6-12.0 S, respectively. In addition, it was tested if Mia- and Mib-CK can form heterodimeric and heterooctameric molecules with subunits of other CK isoenzymes. By denaturation in urea or guanidine hydrochloride and subsequent renaturation, MiaMib-CK and surprisingly also MiaM-CK heterodimers could be generated. In contrast, no heterodimers were obtained between Mib- and M- or B-CK. Furthermore, reoctamerization of a mixture of Mia- and Mib-CK homodimers led to the formation of MiaMib-CK heterooctamers. In these heterooctamers, the Mia- and Mib-CK homodimers remained the fundamental building blocks. No subunit exchange between adjacent dimers within the heterooctamer could be observed even after storage for 3 months at 4 degrees C. The relevance of these data on the structural organization of the Mi-CK octamer and on the physiological aspects of tissue-specific isoenzyme expression are discussed.
Amino Acid Sequence↗
Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo.
In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.
Animals↗
Functional studies with the octameric and dimeric form of mitochondrial creatine kinase. Differential pH-dependent association of the two oligomeric forms with the inner mitochondrial membrane.
Phosphate extraction of mitochondrial creatine kinase (Mi-CK, EC 2.7.3.2) from freshly isolated intact mitochondria of chicken cardiac muscle, after short swelling in hypotonic medium, yielded more than 90% of octameric and only small amounts of dimeric Mi-CK as judged by fast protein liquid chromatography-gel permeation analysis of the supernatants immediately after extraction of the enzyme. In extraction buffer, octameric Mi-CK displayed a tendency to dissociate, albeit at a slow rate with a half-life of approximately 3-5 days, into stable dimers. Experiments with purified Mi-CK octamers or dimers, or defined mixtures thereof, incubated under identical conditions with Mi-CK-depleted mitoplasts revealed that both oligomeric forms of Mi-CK can rebind to mitoplasts. However, the association of Mi-CK was strongly pH-dependent and, in addition, octameric and dimeric Mi-CK showed different pH dependences of rebinding. Therefore, it was possible under certain pH conditions to rebind either both oligomeric forms or selectively the octamers only. Furthermore, evidence is presented that Mi-CK dimers partially form octamers upon rebinding to the inner membrane. The differential association of the two oligomeric Mi-CK forms with the inner mitochondrial membrane together with the dynamic equilibrium between octameric and dimeric Mi-CK (Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, H.M., and Wallimann, T. (1988) J. Biol. Chem., 263, 16942-16953) suggest that both oligomeric forms are physiologically relevant. A change in the octamer to dimer ratio may influence the association behavior of Mi-CK in general and thus modulate mitochondrial energy flux as discussed in the phosphoryl creatine circuit model (Wallimann, T., Schnyder, T., Schlegel, J., Wyss, M., Wegmann, G., Rossi, A.-M., Hemmer, W., Eppenberger, H.M., and Quest, A.F.G. (1989) Prog. Clin. Biol. Res. 315, 159-176.
Animals↗