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© The Rockefeller University Press,
0021-9525/1997//925 $5.00
The Journal of Cell Biology, Volume 137, Number 4,
, 1997 925-937
Article |
Identification of a Novel Marker for Primordial Smooth Muscle and Its Differential Expression Pattern in Contractile vs Noncontractile Cells
Department of Cell Biology and the Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston 29425; Invitrogen Corporation, San Diego, California 92121; || Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010; ¶ Department of Chemistry, University of Virginia, Charlottesville 22901; and ** Department of Pathology, University of Virginia, Charlottesville 22908
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
-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 -actinin. Our results suggest that the 1E12 antigen is a member of the -actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.
1. Abbreviations used in this paper: ECM, extracellular matrix; LSCM, laser scanning confocal microscopy; NMC, nonmuscle cell; PVDF, polyvinyldifluoride; SM
A, smooth muscle -actinin; SMC, smooth muscle cell; VSMC, vascular smooth muscle cell. Rocco Falchetto was supported by a fellowship from the European Molecular Biology Organization. This work was supported by awards from the National Institutes of Health (NIH) (R01HL45348 and R01HL57645) and the March of Dimes Birth Defects Foundation (FY950453) to C.D. Little. J.E. Hungerford was supported in part by an NIH training grant (T32HL07284-14).
Please address all correspondence to Dr. Charles D. Little, Department of Cell Biology, Medical University of South Carolina, BSB Room 626, 171 Ashley Avenue, Charleston, SC 29425. Tel.: (803) 792-9030. Fax: (803) 792-0664. e-mail: Charles_Little{at}smtpgw.musc.edu
J.E. Hungerford's current address is Department of Cell Biology, Box 439, University of Virginia, Charlottesville, VA 22908.
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