Immunofluorescent Visualization of 100 Å Filaments in Different Cultured Chick Embryo Cell Types

Abstract. Experiments are described supporting the proposition that the assembly of stress fibers in nonmuscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membraneassociated 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 cellsubstrate 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, a-actin, and sarcomeric ot-actinin (s-ot-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 ot-actinin-containing structures invariably are positive for the sarcomeric isoform, o~-actin and related sarcomeric proteins; they lack non-sot-actinin, -y-actin, and caldesmon; (g) in fibroblasts all ot-actinin-containing structures are positive for the non-sarcomeric isoform, -y-actin, and related nonsarcomeric 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. CONSIDERABLE information has accumulated regarding the molecular composition and possible functions of the submembranous complexes into which stress fibers insert (4,5,11,26,34,46). In cell-cell junctions of the adherens type the distal tips of the microfilament bundles insert into submembranous F-actin/o~-actinin/vinculin complexes that often involve additional membrane-associated molecules such as A-CAM (31, 60), radixin (58) etc. A somewhat different class of submembranous complexes characterizes the termini of stress fibers where they insert into the cell-substrate junctions of spread, nontranslocating cultured ceils. These F-actin/ot-actinin/vinculin complexes, known as adhesion plaques (APs),t are also positive for talin (11), paxillin (59), integrin (17), and other proteins. APs are also sites for a Ca2 § protease (5) and a1. Abbreviations used in this paper: AJ, adherens junction; AP, adhesion plaque; MHC, myosin heavy chain; s, sarcomeric; SAP, subsarcolemmal adhesion plaque.protein kinase C (35) suggesting they may be involved in transmembrane signaling. In both the adherens junction (AJs) and the APs, ct-actinin appears to play a bifunctional role. Distally, facing the inner surface of the c...

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“…The polyclonal anti-desmin has been described; it does not stain vimentin either in cells or blots (7). Three antibodies to ~-actinin are used.…”

Section: Antibodiesmentioning

confidence: 99%

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

DiLullo

Schultheiss

et al. 1992

The Journal of Cell Biology

115

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“…Unless otherwise noted, the IgG fraction of a rabbit antiserum against chicken gizzard desmin and a guinea-pig antiserum against chicken muscle myosin heavy chain were used for immunohistochemical localization of desmin and myosin, respectively. The rabbit desmin antiserum [2,12] was kindly provided by Dr. H. Holtzer (University of Pennsylvania, Philadelphia). The IgG fraction of this desmin antiserum was further purified in our laboratory using protein A-Sepharose and was shown to react only with the desmin band by polyacrylamide gel electrophoresis and immunoblot analysis [35].…”

Section: Antibody Stainingmentioning

confidence: 99%

“…Desmin is the major subunit of muscle-type intermediate filaments and is expressed by skeletal, cardiac and most types of smooth muscle cells in both embryonic and adult tissues [2,19,21,28]. In the skeletal-muscle lineage, desmin expression has been thought to be specific to postmitotic myoblasts and myotubes.…”

Section: Introductionmentioning

confidence: 99%

“…In the skeletal-muscle lineage, desmin expression has been thought to be specific to postmitotic myoblasts and myotubes. This conclusion was based on studies of primary myogenic cultures employing immunofluorescence [2,3,16], protein analysis by gel electrophoresis [16], or mRNA analysis [5]. Other cell culture studies have demonstrated that during chicken myogenesis the expression of desmin precedes that of other musclespecific proteins [11], and it has been suggested that replicating myoblasts can express desmin.…”

Section: Introductionmentioning

confidence: 99%

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Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle

Yablonka–Reuveni

Nameroff

1990

Differentiation

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Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [ 3 H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [ 3 H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [ 3 H]thymidine and were desminpositive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosinnegative, [ 3 H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.

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“…Furthermore, vimentin is highly conserved in phylogenesis as suggested by the cross reaction of antibodies produced against mammalian vimentin with the corresponding protein in birds and amphibians (Bennett et al 1978 ;Szaro & Gainer, 1988 ;Zamora & Mutin, 1988 ;Bodega et al 1994). Thus the knowledge of phylogenetic and ontogenetic relations between the different cell types belonging to the glial cell lineage might be facilitated by comparative studies on glial cells in the CNS of species which are members of different vertebrate classes.…”

mentioning

confidence: 99%

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of adult Podarcis sicula (Squamata, Lacertidae)

Lazzari

Franceschini

2001

Journal of Anatomy

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The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula. GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star-shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.

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Immunofluorescent Visualization of 100 Å Filaments in Different Cultured Chick Embryo Cell Types

Cited by 79 publications

References 43 publications

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

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

Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of adult Podarcis sicula (Squamata, Lacertidae)

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