Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells

Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit-erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady-state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation.     

Subcellular distribution of the enzymes of the malate-aspartate shuttle in rat heart and effect of experimental cardiac hypertrophy

The effect of experimental cardiac hypertrophy on the enzymes of the malate - aspartate shuttle aspartate aminotransferase (AAT) and malate dehydrogenase (MDH) was studied. ( l ) Aortic constriction in adult rats resulted in 25% cardiac hypertrophy in 2 1/2-3 weeks. Total DNA (mg per heart) did not change. ( 2 ) The proportions of mitochondrial and cytosolic isozymes of AAT and MDH did not change as a result of cardiac h y p e r t r o p h y . About two-thirds of each enzyme occurred in the mitochondrial form and one-third in the cytosolic form. ( 3 ) Total AAT in hypertrophic hearts, in enzyme units per mg DNA, increased by 24% compared to AAT content in the hearts of sham-operated animals . Total MDH did not change. SoIubilized protein increased by 20%. Normal hearts contained 10 times more enzyme units of MDH than of AAT. (4) Cardiac growth stimulation induced in newborn rats did not result in specific changes of either enzyme. It is suggested that true cardiac hypertrophy acts as a specific stimulus for the possibly rate-limiting enzyme AAT of the shuttle.     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

Collagen polysomes: Site of hydroxylation of proline residues

The biosynthesis of collagen on polysomes has been studied by using a newly devised method for obtaining polysomes in high yield from stationary-phase mouse fibroblast (line 3T6; Goldberg &, Green, 1967). These polysomes were completely disaggregated to monosomes by brief exposure to ribonuclease and they lost most of their radioactivity to the top of the sucrose gradients as a result of a 30-minute chase with unlabeled proline. After a ten-minute pulse with [³H]proline, nascent collagen peptides could be identified in these polysomes on sucrose gradients. Most of the proline residues susceptible to hydroxylation by collagen proline hydroxylase were found, in most cases, to be already hydroxylated in these nascent peptides. The nascent nature of these peptides was confirmed by the observation that treatment of the polysomes with RNase transferred the radioactive collagen peptides to the monosome area and these peptides could subsequently be removed to the soluble material at the top of the gradient upon treatment with puromycin. These findings therefore, show clearly that the hydroxylation of proline residues is occurring, in vivo under normal conditions, on nascent collagen chains. In no case was the degree of hydroxylation of the released collagen chains higher than that on the nascent collagen peptides. It seems likely, therefore, that the major site of proline hydroxylation is the nascent collagen peptide.     

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.     

Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments

Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.