James L. Lessard

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin.

SummaryMyofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against α- and γ-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the “sarcomeric” proteins α-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of “sarcomeric” proteins in regard to myofibrillogenesis.

Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin.

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the α- and γ-isoforms of actin☆

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Tropomyosin isoforms from the γ gene differing at the C‐terminus are spatially and developmentally regulated in the brain

Tropomyosin is an actin‐binding protein responsible for stabilizing the actin microfilament system in the cytoskeleton of nonmuscle cells and is involved in processes such as growth, differentiation, and polarity of neuronal cells. From the γ gene, at least 11 different isoforms have been described, with three different C‐terminal exons used (9a, 9c, 9d). The precise roles that the different isoforms play are unknown. To examine the localization and hence determine the function of these isoforms in developing mouse, specific antibodies to exons 9a and 9c were made. These were used with previously developed 9d and N‐terminal 1b antibodies on Western blots and immunohistochemical analysis of mouse brains. We were able to show that all three C‐termini are used in the brain. 9c isoforms are highly enriched in brain and neural cells, and we also detected significant amounts of 9a‐containing isoforms in brain. γ gene activity is relatively constant in the brain, but the choice of C‐terminus is developmentally regulated. A more detailed study of the brain revealed regional expression differences. The hippocampus, cerebellum, and cortex were analyzed in depth and revealed that different isoforms could be sorted into different neuronal compartments, which change with development for 9d. Furthermore, a comparison with a homologous exon to 9c from the α‐tropomyosin gene showed that expression from these exons is related to the maturational state of the neuron, even though both are sorted differently intracellularly. These data suggest that the large numbers of tropomyosin isoforms are likely to have specific roles in microfilament dynamics and neural cell function.

A solid-phase assay for antiactin antibody and actin using protein-A

Abstract A method has been developed for the detection of an immunocomplex between an agaroselinked antigen and its antibody using 125 I-labeled protein-A. This procedure has been used to follow the appearance of antiactin antibody in rabbits injected with 200 μg of sodium dodecyl sulfate (SDS)-denatured and electrophoretically purified murine skeletal muscle actin on Days 0 and 20. Significant immunoglobulin G (IgG) binding to agarose-actin was detected at about Day 50 and the titer was maximum at about Day 100. The antibody binding reaction is not affected by bovine serum albumin, ovalbumin, or a bacterial extract, but can be blocked by soluble actin. Murine skeletal muscle actin (0.1 mg/ml) prevents about 80–90% of the binding, with 50% inhibition observed at about 10 μg/ml. Using an SDS extract of neuroblastoma cells, only about 60–70% inhibition was obtained, suggesting that the nonmuscle actin in this preparation does not react with all the antibodies which bind to muscle actin. The specificity and general usefulness of this assay are also suggested by the fact that antiactin antibodies do not bind to agarose-BSA, agarose-ovalbumin, or agarose-uterine myosin, while antibodies to the two latter proteins bind preferentially to their respective antigens. Thus, this method can potentially be used to assay any antigen immunologically active in the solid phase, the antibody to that antigen, and also crossreacting antigens.

Expression of Actin Isoforms in Developing Rat Intestinal Epithelium

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.

Fetal expression of muscle-specific isoactins in multiple organs of the Wistar-Kyoto rat.

SummaryActin, a cytoskeletal and contractile protein, is expressed in six different isoforms that exhibit striking specificity. No studies have considered the muscle-specific actin expression in multiple organ systems in the intact fetus. Using a monoclonal antibody (B4) which reacts specifically with the isoactins of the smooth and skeletal muscle our immunohistochemical study examined whole fetal body sections to follow the development of actin expression throughout the last third of gestation in the Wistar-Kyoto rat. B4 staining was exclusively localized to muscle, confirming its high specificity and its usefulness for studying the ontogeny of muscle-specific isoactins. At 15 days of gestation, B4 staining was detected in the heart, the thoracic aorta and the skeletal muscle of the chest wall. The distribution and intensity of staining in the heart were initially higher than in the aorta or skeletal muscle and remained unchanged throughout the remainder of gestation, suggesting that the maturation of cardiac actin expression is well developed, although not fully completed before birth. Expression of muscle-specific actins in skeletal muscle was age-dependent and correlated with the maturational changes of muscle cell precursors. B4 staining in the fetal kidney was not apparent until day 20 of gestation and was localized to the inner cortical vessels. in association with the most mature nephrons, suggesting a centrifugal maturation of the intrarenal vasculature. The intensity of B4 staining in most tissues including bronchi, bowel, diaphragm, chest wall muscle and peripheral and pulmonary arteries increased by the end of gestation.

APPEARANCE OF c-AMP DEPENDENT ACTIN PHOSPHORYLATION IN RAT LUNG AND TYPE II EPITHELIAL CELLS

c-AMP enhances surfactant release from Type II epithelial cells, presumably by activation of protein kinases (PK) and increased protein phosphorylation. cAMP dependent (dep) phosphorylation of endogenous proteins was therefore assessed in rat lung during development and in adult Type II epithelial cells. Protein Mr=43,000 was the major substrate of c-AMP-PK in cytosol from postnatal lung and in purified adult Type II cells. Evidence that [32P]43,000 is the cytoskeletal protein, actin, includes migration on 2-D SDS-PAGE and phospho-peptide mapping. [32P]Serine was the only phosphoamino acid detected. Phosphorylation was reversible and entirely cAMP dep, EC50=5×10−7 M. cAMP dep 32P-actin was barely detectable until 21d gestation and increased 25-fold during the perinatal period. cAMP dep protein kinase activity did not correlate with developmental increase in 32P-actin. cAMP-PK was higher in fetal than adult preprations, p<.01. Lung actin content did not change with age. Addition of purified actin but not cAMP-PK to fetal cytosol enhanced 32P-actin. Actin is a major endogenous cytosolic substrate of c-AMP-PK in Type II epithelial cells and in postnatal lung. Actin phosphorylation is developmentally regulated in association with other aspects of lung maturation. Mechanisms that might account for the developmental changes in lung 32p-actin include 1) availability of actin to serve as substrate of c-AMP- PK, 2) new actin forms, or 3) ontogenic appearance of specific c-AMP dependent actin kinase activity.

Metabolic and Structural Requirements for Concanavalin A Capping in Phagocytic Cells

Summary Colchicine induced Con A capping in polymorphonuclear leukocytes, peritoneal monocytes, and alveolar macrophages, which was prevented in the polymorphonuclear leukocyte and peritoneal monocyte by cytochalasin B. Isoelectrophoresis of whole cell homogenates and purified cytoplasmic actin revealed β- and γ-actin to be present in similar amounts in all three cell types. Alveolar macrophages failed to cap in the presence of 1 mM potassium chloride whereas polymorphonuclear leukocytes and peritoneal monocytes maintained the capping response. In contrast, alveolar macrophages continued to cap during treatment with 5 mM 2-deox-yglucose abeit at lower levels than control; whereas in polymorphonuclear leukocytes and peritoneal monocytes, capping was substantially attenuated. These studies demonstrate the differing metabolic requirements for capping in phagocytic cells and suggest that different topographical relationships of actin-microfilaments to the plasma membrane might exist in the alveolar macro-phages compared to the phagocytic cells dependent on anaerobic metabolism.