Ted W. Huiatt

Purified desmin from adult mammalian skeletal muscle: A peptide mapping comparison with desmins from adult mammalian and avian smooth muscle

Abstract Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca 2+ -activated muscle protease, trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.

Molecular Characteristics of the Novel Intermediate Filament Protein Paranemin SEQUENCE REVEALS EAP-300 AND IFAPa-400 ARE HIGHLY HOMOLOGOUS TO PARANEMIN

Paranemin was initially found to copurify with the intermediate filament (IF) proteins vimentin and desmin from embryonic chick skeletal muscle and was described as an IF-associated protein (IFAP). We have purified paranemin from embryonic chick skeletal muscle, prepared antibodies, and demonstrated that they label at the Z-lines of both adult avian and porcine cardiac and skeletal muscle myofibrils. We determined the cDNA sequence of paranemin by immunoscreening a λgt22A cDNA library from embryonic chick skeletal muscle. Northern blot analysis revealed a single transcript of 5.3 kilobases, which is much smaller than predicted from the size of paranemin (280 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The derived amino acid sequence of paranemin (1,606 residues; 178,161 kDa) contains the conserved IF rod domain (308 amino acids), which has highest homology to the rod domains of nestin and tanabin. Thus, paranemin is an IF protein rather than an IFAP. Sequence analysis also revealed that the partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, are almost identical to regions of the cDNA sequence of paranemin. The complete paranemin cDNA was expressed in a cell line (SW13) with, and without, detectable cytoplasmic IFs. Antibody labeling of these cells suggests that paranemin does not form IFs by itself, but rather is incorporated into heteropolymeric IFs with vimentin.

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture☆

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named alpha-synemin and beta-synemin, with the larger alpha-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller beta-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within beta-synemin is within the C-terminal 332 amino acid region (SNbetaTII) at the end of the long tail domain. Transient expression of SNbetaTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.

Muscle Intermediate Filament Proteins

Publisher Summary Intermediate filaments (IFs) appear to function as mechanical integrators of cellular space and provide the overall cytoskeletal integrity and strength, as well as the organization, necessary for supporting contraction. The five muscle cell IF proteins include desmin, vimentin, synemin, paranemin (avian)/nestin (mammalian ortholog), and syncoilin. This chapter provides some background information and methods on the purification of intermediate filament proteins from muscle, expression of muscle intermediate filament proteins and domains, and muscle intermediate filament protein interactions. The chapter discusses some pitfalls, such as the Bradford protein assay available commercially as the Bio–Rad Protein Assay that gives unreliable results with desmin, and recommends the use of the Folin-Lowry or BCA (Pierce) protein assays for measuring the concentration of desmin. Syncoilin is an intriguing muscle IF protein; that it has not yet been shown to form heteropolymeric IFs may put it in a functional class of its own. Although, some progress is being made in identifying proteins within other cytoskeletal structures with which muscle IF proteins interact, it is likely, or at least possible, that many of the most important ones have not yet been identified.

Integrin expression in developing smooth muscle cells.

We studied the specific expression patterns and distributions of α1 and β1 integrin subunits, the major cell adhesion receptors in smooth muscle, in developing smooth muscle cells from 16-, 18-, and 20-day embryonic gizzards and from 1- and 7-day post hatch chick gizzards by SDS-PAGE, immunoblotting, and immunoelectron microscopy. Antibodies raised against α1 and β1 integrins isolated from avian gizzards were used as probes. Gels and blots showed that the amount of α1 and β1 integrins increased as age increased, with major increases at 1 and 7 days post hatch. Image analysis of immunoelectron micrographs demonstrated that statistically significant labeling increases occurred between embryonic Days 16 and 18, between embryonic Day 20 and 1 day post hatch, and between 1 day and 7 days post hatch. Immunolabeling with both anti-α1 and anti-β1 integrin was prominent at membrane-associated dense plaques (MADPs) and at filament anchoring regions at cell ends. This indicates that α1 and β1 integrin expression coincides temporally with the intracellular proliferation and reorientation of myofilaments. The similarity in distribution patterns of α1 and β1 integrins during development suggests that the two integrin subunits are synchronously expressed during development and do not appear sequentially.

Regulatory Role of Arginine-Specific Mono(ADP-Ribosyl)Transferase in Muscle Cells

Earlier we demonstrated that meta-iodobenzylguanidine (MIBG), a specific inhibitor of arginine mono-ADP-ribosylation blocks proliferation and differentiation of chick skeletal myogenic cells in culture (Exp. Cell Res., 1992, 201:33-42). Membrane fractions from 4-day, myotube cultures of embryonic chick muscle cells were incubated with 32P-NAD+. Several proteins were labeled, but labeling of two hands of about 53 and 36 kDa appeared to be due to arginyl ADP-ribosylation. Immunoprecipitation with D3 monoclonal antibody to the intermediate filament protein desmin, SDS-PAGE and autoradiography demonstrated that the 53 kDa band contained desmin, and that this desmin is ADP-ribosylated by the endogenous arginine-specific mono(ADP-ribosyl)transferase (Exp. Cell Res., 1996, in press). Desmin is the muscle-specific intermediate filament protein, and it appears to be one of the first muscle-specific proteins expressed during terminal myogenic differentiation. We have examined whether desmin can be ADP-ribosylated in muscle cells by use of polyclonal antibodies for ADP-ribosylated arginyl residues. We have found that soluble desmin is present in 5-6 day myogenic cell cultures and that this desmin contains ADP-ribose, demonstrating that desmin is ADP-ribosylated in skeletal muscle cells. We also found that purified avian desmin contains antigenic material that reacts with these antibodies. In both cases, NaCl had no effect on the reactivity, but NH2OH did, which is consistent with an arginine-ADPR linkage. In summary, these results suggest that ADP-ribosylation is an important regulatory mechanism in differentiating muscle cells, and that the intermediate filament protein desmin is an important substrate for modification in muscle cells.

Disparate responses of cultured skeletal muscle cells and growing chicks to tripeptide aldehyde protease inhibitors and an in vivo interaction with ethanol

Abstract The objective of this study was to determine the effects of leupeptin and two leupeptin analogs (calpain inhibitors I and II) on protein turnover in embryonic chick skeletal muscle cell cultures and on growth of chicks. For in vitro studies, inhibitors (70 μg/mL) were added to the medium of cell cultures for 24 hr and proteolysis was determined by measuring the release of natural leucine into medium containing all leucine as [5,5,5- 2 H]leucine. Compared with controls, leupeptin and calpain inhibitors I and II decreased proteolysis 17.0%, 55.4%, and 60.6%, respectively. For in vivo studies, 7-day-old male White Leghorn chicks were given the inhibitors by daily intraperitoneal injection for 7 days. In experiment 1, inhibitors were administered in a 25% ethanol:75% saline carrier at doses of 2.0 or 20.0 mg/kg body weight. Calpain inhibitors I and II had no effect on lean tissue gain or feed efficiency. Leupeptin-injected chicks gained 51.1 ± 2.1 g and control chicks gained 43.2 ± 2.1 g ( P P P