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D A Fischman

Publications and source records attributed to D A Fischman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Molecular cloning of chicken myosin-binding protein (MyBP) H (86-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type III motifs.

The complete nucleotide sequence of a cDNA clone encoding the chicken skeletal muscle myosin-binding protein H (MyBP-H), formerly termed 86-kDa protein, has been established and the predicted amino acid sequence compared with other proteins entered into the GenBank data base. The full-length cDNA of 2066 base pairs contains a single open reading frame of 1611 base pairs encoding a muscle-specific protein of 58,487 Da. The predicted molecular weight differs significantly from the relative mobility of 86-kDa protein in reducing sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The full-length protein expressed in Escherichia coli also exhibits an anomalously slow mobility in SDS-PAGE; this gel retardation is a property of the N-terminal 24 kDa of the protein which contain two extended motifs of alternating alanine and proline residues, resembling the N terminus of skeletal muscle myosin light chain 1 (Nabeshima, Y. I., Fujii-Kuriyama, Y., Muramatsu, M., and Ogata, K. (1984) Nature 308, 333-338). The C-terminal 40 kDa share 49.6% sequence identity and 17% conservative substitutions with chicken skeletal muscle MyBP-C (C-protein) (Einheber, S., and Fischman, D. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2157-2161). The protein contains four internal repeats of approximately 100 amino acids each, two of which bear significant resemblance to the C2 set of the immunoglobulin superfamily, and the other two are related to the type III fibronectin repeat. The arrangement of these repeats, -III-C2-III-C2-, is identical to that seen in the C-terminal 40-kDa section of MyBP-C. This repeat structure is implicated in myosin binding for the MyBP family. Finally, genomic Southern blots indicate that a single gene encodes fast skeletal muscle MyBP-H.

Amino Acid Sequence↗
PubMed1993

Human myosin-binding protein H (MyBP-H): complete primary sequence, genomic organization, and chromosomal localization.

Vertebrate striated muscle contains a set of myosin-associated proteins with discrete distributions in the A-band. Some of these proteins, including MyBP-H and MyBP-C, are characterized by a series of internal repeats (motifs) with homology to either the C2-set of the immunoglobulin superfamily or the fibronectin type III repeat. These repeats are predicted to be involved in protein-protein interactions within the myofibril. The cDNA sequence, the genomic organization, and the chromosomal localization of the human homologue of MyBP-H are presented. The 1.8-kb cDNA encodes a 52-kDa polypeptide containing two Ig-C2 and two type III repeats. The mRNA is expressed in a skeletal muscle-specific pattern. A 28-kb region of genomic sequence has been isolated that encompasses the 5' and 3' ends of the cDNA. This region includes 4.2 kb of upstream sequence with a potential promoter and 14 kb of downstream sequence containing the polyadenylation site. The chromosomal assignment was made by high resolution chromosomal in situ hybridization. This method maps the gene to chromosome 1q32.1. The repeat structure described previously in chicken MyBP-H and MyBP-C was also detected in human MyBP-H. The primary sequence of the C-terminal Ig-C2 motif and its predicted secondary structure have been extensively conserved in MyBP-H homologues and other members of the MyBP family. This Ig-C2 motif has been implicated in myosin binding.

Amino Acid Sequence↗
PubMed1993

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

Amino Acid Sequence↗
PubMed1992

Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardial morphogenesis between stages 17 and 27 (days 3-5) of egg incubation. To trace cells which give rise to the coronary arteries in vivo, we applied retroviral cell tagging procedures and analyzed clonal populations of vascular smooth muscle, endothelium, and connective tissue in the hearts of post-hatch chickens. Our data provide direct proof that (i) vascular smooth muscle progenitors begin to enter the heart at stage 17, substantially after the heart begins propulsive contractions; (ii) cardiac myocytes, vascular smooth muscle, perivascular fibroblasts, and coronary endothelial cells all derive from independent precursors when these cells migrate into the heart; (iii) endothelial cells of the coronary vessels have a different clonal origin than endothelial cells of the endocardium; (iv) coronary arteries form by the coalescence of discontinuous colonies (i.e., in situ vasculogenesis), each derived from a founder cell tagged at the time of retroviral injection (stages 17-18); and (v) coronary arteries contain discrete segments composed of "polyclones." These studies indicate the feasibility of gene targeting to coronary progenitors through the use of recombinant retroviruses.

Animals↗
PubMed1992

Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus: I. Formation of the ventricular myocardium.

Cells of the precardiac mesoderm (stages 4-6) and dividing myocytes of early hearts (stages 10-15) were tagged with a replication-incompetent retrovirus (CXL) (Mikawa et al., 1991b) encoding bacterial beta-galactosidase (beta-gal). Two protocols were used to infect the cardiogenic cells. (1) Small blocks (approximately 50 micron 2) of anterolateral mesoderm were dissected from gastrula-stage embryos (stages 4-6) and incubated in liquid medium containing the retrovirus. After removal of CXL, the tissues were dispersed into single-cell suspensions and pressure injected into the precardiac areas of recipient embryos (stages 4-6). Such embryos were then incubated in vitro at 37 degrees C for 2 days (New, 1968), and those embryos with beating hearts were fixed for X-gal histochemistry and paraffin serial sectioning. (2) CXL was pressure injected in ovo (embryonic stages 4-15) into cardiogenic tissues and the eggs subsequently returned to an incubator. At selected stages of development embryos or whole hearts were fixed, stained with X-gal, and serially sectioned after paraffin embedding. The first method showed that (1) cells of the precardiac mesoderm could be infected with the retrovirus, (2) the transplanted cells would differentiate into beating myocytes, and (3) beta-gal expression was sufficiently high to be detected histochemically. With the second procedure we could show that (1) beta-gal-tagged cells formed colonies in the myocardium, (2) the labeled cells were exclusively myocytes, (3) the number of cells per colony increased with increasing age of embryonic development, (4) the size of colonies was larger in the left than the right ventricle, (5) many of the colonies were transmural, i.e., they extended from epicardial to endocardial layers of the myocardium and generally exhibited a cone or funnel-shape with the base of the cone nearest the epicardium, (6) the orientation of myocytes within each colony changed at different layers of the myocardium, and (7) the cones contained both beta-gal+ and beta-gal- myocytes. DNA labeling studies with [3H]thymidine indicated that cardiogenic cells divided every 16-18 hr during the first week of development and that the CXL-labeled cells divided indistinguishably from unlabeled myocytes. Based on these observations a model for the growth of the myocardium is presented.

Animals↗
PubMed1992

Expression of sarcomeric myosin in the presumptive myocardium of chicken embryos occurs within six hours of myocyte commitment.

The distribution of sarcomeric myosin heavy chain (MyHC) has been examined immunocytochemically in the presumptive myocardial cells of chicken embryos (stages 6-10) prior to the onset of the heart beat. Embryos were stained with monoclonal antibody MF20, a reagent which recognizes all chicken sarcomeric MyHCs (Bader et al., 1982), and then examined both in whole mount by immunofluorescence and in semithin, plastic-embedded sections following immunoperoxidase labeling. We observed that myosin could be detected as early as stage 7 (0-2 pairs of somites) in 29% of the 31 embryos examined, and by stage 8 (4 pairs of somites) more than 80% of the embryos were MF20+. Every embryo with 5 pairs of somites (stage 8+) labeled strongly with MF20. Labeling was first detected at stage 7 to 7+ as a diffuse fluorescent signal within pleomorphic cells of the splanchnic mesoderm located in two crescent-shaped regions bordering each side of the anterior intestinal portal (AIP). With progressive development, the two crescent-shaped regions merged at the apex of the AIP, and as the two heart tubes began fusion at stage 9, the MyHC+ regions extended cranially and medially. By somite stages 9-10, the myosin-positive cells completely encircled the heart tube. From stages 7 to 9 the myosin signal had no sarcomeric distribution; i.e., there were no MyHC striations nor periodic repeats evident in the presumptive myocytes until late stage 9 and stage 10. Semithin sections revealed that myosin was first distributed in apical regions of the myocytes, adjacent to the pericardial coelom. The implications of these findings for myocyte determination, differentiation and morphogenesis are discussed.

Animals↗
PubMed1992

Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus. III: Polyclonal origin of adjacent ventricular myocytes.

Replication-incompetent variants of the avian spleen necrosis virus (SNV) encoding cytoplasmic or nuclear-directed beta-galactosidase (beta-gal) have been used to trace the clonal growth of myocytes during left ventricular free-wall formation. Tubular-stage hearts were infected with a mixed suspension of both retroviruses and, after hatching, the progeny of marked cells in the ventricular wall were examined by X-gal histochemistry. When a small number of virions was introduced individual blue patches contained myocytes with only one label type (cytoplasmic or nuclear). These results confirmed our previous conclusion that each cluster or patch represents a single clone (Mikawa et al., 1992, Dev. Dynamics, 193:11-23). Each of these clones formed a clone-shaped patch which often extended through the entire thickness of the ventricular myocardium, but typically each patch was heterogeneous, containing a mixture of labeled and unlabeled cells. We then asked whether the two populations of myocytes in each patch were clonally related or generated from more than one progenitor. When hearts were infected with high titer viral suspensions many patches were observed in which cytoplasmic-tagged myocytes were intermingled with nuclear-tagged myocytes. Thus, the cone-shaped myocyte patches in the ventricular wall are polyclones derived from separate progenitors in the precardiac mesoderm. This finding led us to examine the separation of clonally related ventricular myocytes in the developing hearts. Embryos were infected with retroviral suspensions at varying stages of development and the resulting colonies examined after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Myosin heavy chain composition of single fibres and their origins and distribution in developing fascicles of sheep tibialis cranialis muscles.

The myosin heavy chain (MHC) composition of single muscle fibres in developing sheep tibialis cranialis muscles was examined immunohistochemically with monoclonal antibodies to MHC isozymes. Data were collected with conventional microscopy and computerized image analysis from embryonic day (E) 76 to postnatal day (PN) 20, and from adult animals. At E76, 23% of the young myofibres stained for slow-twitch MHC. The number of these fibres considerably exceeded the number of primary and secondary myotubes. By E100, smaller fibres, negative for slow-twitch MHC, encircled each fibre from the initial population to form rosettes. A second population of small fibres appeared in the unoccupied spaces between rosettes. Small fibres, whether belonging to rosettes or not, did not initially express slow-twitch MHC, expressing mainly neonatal myosin instead. These small fibres then diverged into three separate groups. In the first group most fibres transiently expressed adult fast myosin (maximal at E110-E120), but in the adult expressed slow myosin. This transformation to the slow MHC phenotype commenced at E110, was nearing completion by 20 postnatal days, and was responsible for approximately 60% of the adult slow twitch fibre population. In the other two groups expression of adult fast MHC was maintained, and in the adult they accounted for 14% (IIa MHC) and 17% (IIb MHC) of the total fibre numbers. We conclude that muscle fibre formation in this large muscle involves at least three generations of myotube. Secondary myotubes are generated on a framework of primary myotubes and both populations differentiate into the young myofibres which we observed at E76 to form rosettes. Tertiary myotubes, in turn, appear in the spaces between rosettes and along the borders of fascicles, using the outer fibres of rosettes as scaffolds.

Animals↗
PubMed1992

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes.

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.

Actinin↗
PubMed1992

cDNA cloning and sequence comparisons of human and chicken muscle C-protein and 86kD protein.

Thick filaments in vertebrate striated muscles are composed of myosin heavy chain (MHC) and myosin light chains (MLCs) plus at least eight other proteins: C-protein, 86kD protein (birds) or H-protein (mammals), M-protein, myomesin, titin, MM-creatine kinase, skelemin, and AMP-deaminase. Except for CPK and AMP deaminase, none have well defined functions. Analysis of cDNA clones encoding chicken C-protein and 86kD protein has revealed a high degree of shared amino acid identity, particularly in the C-terminal 40kD. To identify functionally significant regions, the human counterpart of each protein was cloned, sequenced and analysed. Two human C-protein cDNAs were isolated with significant homology to chicken fast C-protein. Clone H75, with 69% identity to chicken fast C-protein, shows the same pattern of hybridization as the chicken fast C-protein in chicken muscles. The other clone, H8 with 60% identity, shows a pattern of hybridization in chicken muscles which is consistent with the expression of chicken slow C-protein. The human 86kD protein shares 66% DNA sequence identity with the chicken 86kD protein. Assuming that essential sequences would be conserved during evolution, we compared the chicken and human proteins using PALIGN. Chicken and human fast C-proteins possess 66% peptide identity over their deduced length plus 10% conservative substitutions. Human slow C-protein and chicken fast C-protein share 44% peptide sequence identity, plus 16% conservative substitutions. Chicken and human 86kD proteins are also very similar: 54% peptide identity plus 20% conservative substitutions. This high degree of sequence identity between chicken and human C- and 86kD proteins suggests selective pressure on the primary sequence. Recent primary sequence analyses of projectin and mini-titins from Drosophila, twitchin from C. elegans, C-protein, smMLCK, 86kD protein, and M-protein from the chicken, titin from the rabbit, and skelemin from the mouse reveals that all these proteins possess multiple internal repeats of approximately 100 amino acids. These repeating domains are of two types: one is homologous to the internal repeats which define the C-2 subset of the immunoglobulin superfamily, the other is related to the fibronectin type III repeat. Both human C-proteins possess comparable internal repeats and preliminary evidence suggests the presence of the same repeats in human 86kD. This duality of repeat structure is found in many extracellular proteins and is typified by the N-CAMs.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Molecular analysis of protein assembly in muscle development.

The challenge presented by myofibril assembly in striated muscle is to understand the molecular mechanisms by which its protein components are arranged at each level of organization. Recent advances in the genetics and cell biology of muscle development have shown that in vivo assembly of the myofilaments requires a complex array of structural and associated proteins and that organization of whole sarcomeres occurs initially at the cell membrane. These studies have been complemented by in vitro analyses of the renaturation, polymerization, and three-dimensional structure of the purified proteins.

Actins↗
PubMed1991

Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity.

The incorporation of actin into myofibrils has been examined in a cell-free system [Bouché et al.: Journal of Cell Biology 107:587-596, 1988; Goldfine et al.: Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S-methionine (35S-actin) or purified from muscle and labeled with fluorescein-5-isothiocyanate (FITC-actin). Myofibrils were incubated with either 35S-actin or FITC-actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC-actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z-band regions and less so in I-bands. No fluorescence was detected in non-overlap regions of A-bands. Confocal microscopic analysis of these myofibrils indicated that FITC-actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC-actin showed a variety of non-physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I-buffer). However, when ATP was added to I-buffer, FITC-actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I-buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both alpha- and beta-actins exhibited incorporation into the myofibrils but there was a three-fold greater incorporation of the alpha isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.

Actins↗
PubMed1991

Visualization of myosin exchange between synthetic thick filaments.

Exchange of myosin molecules between synthetic thick filaments was examined by fluorescence energy transfer and visualized by electron microscopy using streptavidin-gold to detect exchanged biotinylated myosin molecules. N-hydroxysuccinimido-biotin (NHS-biotin) was covalently linked to purified adult chicken pectoralis myosin to obtain assembly-competent biotinylated myosin molecules. Two distinct classes of synthetic filaments, distinguishable by length, were prepared. Biotinylated filaments (575 +/- 100 nm) were assembled by a quick dilution (QD) method and unlabelled filaments (1025 +/- 250 nm) were obtained by a sequential dilution (SD). The two filament population maintained their distinct length distributions even when mixed. To measure exchange, biotinylated short (QD) filaments were combined with unlabelled long (SD) filaments at a 1:5 ratio, sampled at varying times and the entry of biotinylated myosin into the previously unlabelled long filaments visualized by the addition of streptavidin-gold. The number of gold particles per micron was examined for fully biotinylated short filaments (less than 700 nm), unlabelled long filaments (greater than 900 nm), and exchanged filaments. Equivalent binding of streptavidin-gold to the two filament types was detected by 60 min suggesting randomization of biotinylated monomers by this time. The precise location of streptavidin-gold sites on the long filaments was also measured. Although labeling was detected along the full length of the filaments, at the earliest time points (5 min) filament ends contained twice the number of gold particles as the filament centers. Approximately equivalent labeling along the entire length of the filaments was observed by 60 min. These results provide additional support for our earlier report of extensive myosin exchange between synthetic thick filaments and show that extensive exchange takes place rapidly along the full length of synthetic thick filaments.

Actin Cytoskeleton↗
PubMed1991

Cell-free incorporation of newly synthesized myosin subunits into thick myofilaments.

Although a substantial literature exists on the in vitro polymerization of purified myosin, little is known about native thick filament assembly, remodeling or turnover. We have recently described a cell-free system (Bouche et al., 1988) to examine the interactions between thick filaments and soluble, newly synthesized myofibrillar proteins. In the present manuscript we describe our studies on myosin heavy (MHC) and light chain (LC) incorporation into myofibrils or native and synthetic thick filaments. 35S-labeled myofibrillar proteins or myosin subunits were synthesized in a reticulocyte lysate translation system after which myofibrils or myofilaments were added and incubated with these proteins in the lysate. The added filaments were then sedimented and analyzed by SDS-PAGE and fluorography to establish which of the labeled protein subunits were co-pelleted. Operationally, this co-sedimentation of labeled proteins with myofilaments has been termed 'protein incorporation'. We observed that newly synthesized MHC, LCs 1, 2 and 3 all incorporated into the thick filaments. However, the quantity and specificity of LC incorporation depended upon the structure or composition of the filaments. LCs 1 and 3 were preferentially incorporated into myofibrils and native thick filaments, whereas LC2 was selectively taken up by synthetic filaments prepared from purified myosin. These results suggest that soluble MHCs and LCs interact independently with myofilaments. This hypothesis is supported by the observation that selective removal of soluble MHCs, or of a single LC, did not alter the incorporation of the remaining myosin subunits. Similarly, MHCs synthesized in the absence of LCs also incorporated into myofilaments or myofibrils. We propose that myosin subunits are capable of independent incorporation into and exchange from myofilaments.

Actin Cytoskeleton↗
PubMed1991

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species.

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.

Animals↗
PubMed1990

Polarized release of enveloped viruses in the embryonic chick heart: demonstration of epithelial polarity in the presumptive myocardium.

The presumptive myocardium of the embryonic vertebrate heart is composed of cells which exhibit the morphology of a cuboidal epithelium. To examine the functional polarity of these developing myocytes, embryonic chick hearts (Hamburger-Hamilton stages 10-13) were infected with either influenza virus (FLU) or vesicular stomatitis virus (VSV). These viruses have been shown to sort vectorially to either apical (FLU) or basolateral (VSV) membrane surfaces in monolayers of polarized kidney (MDCK) cells. Our results demonstrate that these viruses bud with comparable polarity from differentiating myocytes. However, there appear to be stage-dependent differences in the polarized budding of the two viruses: restricted basolateral release of VSV is present before or shortly after the formation of the heart tube, whereas polarized budding of FLU is established later in development. These results are discussed in terms of plasma membrane organization during the early stages of cardiac development.

Animals↗
PubMed1990

Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily.

C-protein is a thick filament-associated protein located in the crossbridge region of vertebrate striated muscle A bands. Its function is unknown. To improve our understanding of its primary structure, we undertook the molecular cloning of C-protein mRNA. We describe the isolation and characterization of a cDNA clone, lambda C-86, that encodes approximately 80% of the fast isoform of C-protein in the chicken. Sequence analysis of the insert revealed that C-protein, although an intracellular, nonmembrane-associated protein, is a member of the immunoglobulin superfamily. Like several cell surface adhesion molecules that belong to this superfamily, C-protein contains sequence motifs that resemble immunoglobulin domains and fibronectin type III repeats. Computer searches using the C-protein sequence also lead to the identification of related domains in chicken smooth muscle myosin light chain kinase that have not been reported previously.

Amino Acid Sequence↗