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Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

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This figure shows an amniotic SMC double labeled with SMαA (a) and 1E12 (b). These images were obtained by LSCM. Each  image is a series of 10 1-μm optical sections through the long axis of the cell projected into a single image plane. (a) In these contractioncompetent cells, SMαA expression is observed in fine, filamentous “whorls” surrounding the nucleus (arrow). Retraction fibers, which  are present at the surface of the coverslip, also are labeled with SMαA (arrowhead). (b) 1E12 expression is distributed in the same area  as SMαA (arrow) with the cell. However, 1E12 expression appears in a punctate and granular pattern. Additionally, the 1E12 antigen is  distributed to discrete patches of the cell (arrowhead), reminiscent of cell–substrate contact sites. As a means of visualizing the morphology of the amniotic SMC shown in a and b, digital image processing software was used to render the actin immunofluorescence pattern  as a surface relief object. From this topographical representation, it is clear that the actin microfilaments extend from the cell body as  long processes (arrowheads), which are reminiscent of retraction fibers. 1E12 immunolabeling was not present in these structures. Bar,  10 μm.
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Figure 2: This figure shows an amniotic SMC double labeled with SMαA (a) and 1E12 (b). These images were obtained by LSCM. Each image is a series of 10 1-μm optical sections through the long axis of the cell projected into a single image plane. (a) In these contractioncompetent cells, SMαA expression is observed in fine, filamentous “whorls” surrounding the nucleus (arrow). Retraction fibers, which are present at the surface of the coverslip, also are labeled with SMαA (arrowhead). (b) 1E12 expression is distributed in the same area as SMαA (arrow) with the cell. However, 1E12 expression appears in a punctate and granular pattern. Additionally, the 1E12 antigen is distributed to discrete patches of the cell (arrowhead), reminiscent of cell–substrate contact sites. As a means of visualizing the morphology of the amniotic SMC shown in a and b, digital image processing software was used to render the actin immunofluorescence pattern as a surface relief object. From this topographical representation, it is clear that the actin microfilaments extend from the cell body as long processes (arrowheads), which are reminiscent of retraction fibers. 1E12 immunolabeling was not present in these structures. Bar, 10 μm.

Mentions: When individual cells from contraction-competent cultures were examined at high magnification using laser scanning confocal microscopy (LSCM), the two fluorescent antigen patterns were not superimposable. This was determined by projecting a series of 10 1-μm optical sections into a single virtual image plane. Instead of microfilament bundles, the SMαA expression pattern resembled fine filamentous whorls surrounding a nonlabeled nuclear compartment (Fig. 2 a). Also, fine retraction fibers extending from the cell periphery, at the level of the glass coverslip, immunostained with the SMαA mAb. These structures are clearly shown by the digitally generated, surface relief image of the cell shown in Fig. 2 c. It is readily apparent from this and similar images that these contraction-competent cells do not form the typical stress fiber morphology of cells grown on planar substrates in the presence of serum.


Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

Hungerford JE, Hoeffler JP, Bowers CW, Dahm LM, Falchetto R, Shabanowitz J, Hunt DF, Little CD - J. Cell Biol. (1997)

This figure shows an amniotic SMC double labeled with SMαA (a) and 1E12 (b). These images were obtained by LSCM. Each  image is a series of 10 1-μm optical sections through the long axis of the cell projected into a single image plane. (a) In these contractioncompetent cells, SMαA expression is observed in fine, filamentous “whorls” surrounding the nucleus (arrow). Retraction fibers, which  are present at the surface of the coverslip, also are labeled with SMαA (arrowhead). (b) 1E12 expression is distributed in the same area  as SMαA (arrow) with the cell. However, 1E12 expression appears in a punctate and granular pattern. Additionally, the 1E12 antigen is  distributed to discrete patches of the cell (arrowhead), reminiscent of cell–substrate contact sites. As a means of visualizing the morphology of the amniotic SMC shown in a and b, digital image processing software was used to render the actin immunofluorescence pattern  as a surface relief object. From this topographical representation, it is clear that the actin microfilaments extend from the cell body as  long processes (arrowheads), which are reminiscent of retraction fibers. 1E12 immunolabeling was not present in these structures. Bar,  10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139835&req=5

Figure 2: This figure shows an amniotic SMC double labeled with SMαA (a) and 1E12 (b). These images were obtained by LSCM. Each image is a series of 10 1-μm optical sections through the long axis of the cell projected into a single image plane. (a) In these contractioncompetent cells, SMαA expression is observed in fine, filamentous “whorls” surrounding the nucleus (arrow). Retraction fibers, which are present at the surface of the coverslip, also are labeled with SMαA (arrowhead). (b) 1E12 expression is distributed in the same area as SMαA (arrow) with the cell. However, 1E12 expression appears in a punctate and granular pattern. Additionally, the 1E12 antigen is distributed to discrete patches of the cell (arrowhead), reminiscent of cell–substrate contact sites. As a means of visualizing the morphology of the amniotic SMC shown in a and b, digital image processing software was used to render the actin immunofluorescence pattern as a surface relief object. From this topographical representation, it is clear that the actin microfilaments extend from the cell body as long processes (arrowheads), which are reminiscent of retraction fibers. 1E12 immunolabeling was not present in these structures. Bar, 10 μm.
Mentions: When individual cells from contraction-competent cultures were examined at high magnification using laser scanning confocal microscopy (LSCM), the two fluorescent antigen patterns were not superimposable. This was determined by projecting a series of 10 1-μm optical sections into a single virtual image plane. Instead of microfilament bundles, the SMαA expression pattern resembled fine filamentous whorls surrounding a nonlabeled nuclear compartment (Fig. 2 a). Also, fine retraction fibers extending from the cell periphery, at the level of the glass coverslip, immunostained with the SMαA mAb. These structures are clearly shown by the digitally generated, surface relief image of the cell shown in Fig. 2 c. It is readily apparent from this and similar images that these contraction-competent cells do not form the typical stress fiber morphology of cells grown on planar substrates in the presence of serum.

Bottom Line: Biol. 178:375-392).Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin.Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.

ABSTRACT
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Show MeSH