Limits...
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.

Show MeSH

Related in: MedlinePlus

The 1E12 antigen is located intracellularly and codistributes with SMαA in aortic cells cultured from explants on a planar glass  substrate. The cells are double labeled with mAbs to SMαA and 1E12. (a, c, and e) Images of cells examined with simultaneous excitation in both fluorescein and rhodamine wavelengths. (b, d, and f) The same fields of cells under fluorescein only excitation, which denotes those cells that are 1E12 positive. (a and b) Low magnification views of aortic cells migrating from explanted tissue. The majority  of cells in these fields are labeled with SMαA (arrowhead, red cells). A subset of cells are double labeled with SMαA and 1E12 (arrow,  yellow cells). In this field the majority of cells are motile, spindle-shaped cells and, as such, do not show extensive stress fiber arrays, as  shown in c and d. (c and d) The explanted aortic cells have migrated onto the planar glass and have formed extensive microfilament  (stress fiber) arrays. As above, all the cells in this field were positive for SMαA, while a subset were positive for both SMαA and 1E12.  (e and f) A high magnification view of the embryonic aortic cells shows the 1E12 antigen codistributed with actin microfilament bundles.  As shown in f, 1E12 labels the entire length of the actin stress fibers in these cells. The faint staining in the other cells is due to optical  bleed-through from the rhodamine channel. Bars: (a and b) 50 μm; (c and d) 50 μm; (e and f) 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139835&req=5

Figure 1: The 1E12 antigen is located intracellularly and codistributes with SMαA in aortic cells cultured from explants on a planar glass substrate. The cells are double labeled with mAbs to SMαA and 1E12. (a, c, and e) Images of cells examined with simultaneous excitation in both fluorescein and rhodamine wavelengths. (b, d, and f) The same fields of cells under fluorescein only excitation, which denotes those cells that are 1E12 positive. (a and b) Low magnification views of aortic cells migrating from explanted tissue. The majority of cells in these fields are labeled with SMαA (arrowhead, red cells). A subset of cells are double labeled with SMαA and 1E12 (arrow, yellow cells). In this field the majority of cells are motile, spindle-shaped cells and, as such, do not show extensive stress fiber arrays, as shown in c and d. (c and d) The explanted aortic cells have migrated onto the planar glass and have formed extensive microfilament (stress fiber) arrays. As above, all the cells in this field were positive for SMαA, while a subset were positive for both SMαA and 1E12. (e and f) A high magnification view of the embryonic aortic cells shows the 1E12 antigen codistributed with actin microfilament bundles. As shown in f, 1E12 labels the entire length of the actin stress fibers in these cells. The faint staining in the other cells is due to optical bleed-through from the rhodamine channel. Bars: (a and b) 50 μm; (c and d) 50 μm; (e and f) 10 μm.

Mentions: The images in Fig. 1 show double immunofluorescence images of cultured embryonic aortic wall cells. Fig. 1, a and b, shows low magnification views depicting a field of cells that had recently migrated from the explanted aortic tissue. All cells were reactive with the SMαA mab, whereas far fewer cells immunostain with both the SMαA antibody and 1E12. The images in Fig. 1, a, c, and e, show an optical field in which wide-band excitation wavelengths suitable for both fluorescein and rhodamine were used. The redorange fluorescence depicts cells immunoreactive for SMαA, while the yellow staining designates cells immunopositive for both SMαA and 1E12. The green fluorescence in Fig. 1, b, d, and e, designates only those cells immunoreactive with 1E12, when the same optical fields are illuminated with a narrow band-pass fluorescein filter set.


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)

The 1E12 antigen is located intracellularly and codistributes with SMαA in aortic cells cultured from explants on a planar glass  substrate. The cells are double labeled with mAbs to SMαA and 1E12. (a, c, and e) Images of cells examined with simultaneous excitation in both fluorescein and rhodamine wavelengths. (b, d, and f) The same fields of cells under fluorescein only excitation, which denotes those cells that are 1E12 positive. (a and b) Low magnification views of aortic cells migrating from explanted tissue. The majority  of cells in these fields are labeled with SMαA (arrowhead, red cells). A subset of cells are double labeled with SMαA and 1E12 (arrow,  yellow cells). In this field the majority of cells are motile, spindle-shaped cells and, as such, do not show extensive stress fiber arrays, as  shown in c and d. (c and d) The explanted aortic cells have migrated onto the planar glass and have formed extensive microfilament  (stress fiber) arrays. As above, all the cells in this field were positive for SMαA, while a subset were positive for both SMαA and 1E12.  (e and f) A high magnification view of the embryonic aortic cells shows the 1E12 antigen codistributed with actin microfilament bundles.  As shown in f, 1E12 labels the entire length of the actin stress fibers in these cells. The faint staining in the other cells is due to optical  bleed-through from the rhodamine channel. Bars: (a and b) 50 μm; (c and d) 50 μm; (e and f) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The 1E12 antigen is located intracellularly and codistributes with SMαA in aortic cells cultured from explants on a planar glass substrate. The cells are double labeled with mAbs to SMαA and 1E12. (a, c, and e) Images of cells examined with simultaneous excitation in both fluorescein and rhodamine wavelengths. (b, d, and f) The same fields of cells under fluorescein only excitation, which denotes those cells that are 1E12 positive. (a and b) Low magnification views of aortic cells migrating from explanted tissue. The majority of cells in these fields are labeled with SMαA (arrowhead, red cells). A subset of cells are double labeled with SMαA and 1E12 (arrow, yellow cells). In this field the majority of cells are motile, spindle-shaped cells and, as such, do not show extensive stress fiber arrays, as shown in c and d. (c and d) The explanted aortic cells have migrated onto the planar glass and have formed extensive microfilament (stress fiber) arrays. As above, all the cells in this field were positive for SMαA, while a subset were positive for both SMαA and 1E12. (e and f) A high magnification view of the embryonic aortic cells shows the 1E12 antigen codistributed with actin microfilament bundles. As shown in f, 1E12 labels the entire length of the actin stress fibers in these cells. The faint staining in the other cells is due to optical bleed-through from the rhodamine channel. Bars: (a and b) 50 μm; (c and d) 50 μm; (e and f) 10 μm.
Mentions: The images in Fig. 1 show double immunofluorescence images of cultured embryonic aortic wall cells. Fig. 1, a and b, shows low magnification views depicting a field of cells that had recently migrated from the explanted aortic tissue. All cells were reactive with the SMαA mab, whereas far fewer cells immunostain with both the SMαA antibody and 1E12. The images in Fig. 1, a, c, and e, show an optical field in which wide-band excitation wavelengths suitable for both fluorescein and rhodamine were used. The redorange fluorescence depicts cells immunoreactive for SMαA, while the yellow staining designates cells immunopositive for both SMαA and 1E12. The green fluorescence in Fig. 1, b, d, and e, designates only those cells immunoreactive with 1E12, when the same optical fields are illuminated with a narrow band-pass fluorescein filter set.

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
Related in: MedlinePlus