The synthesis and secretion of collagen by cultured sea urchin micromeres.
Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.
Benson
S
S
Department of Biological Sciences, California State University, Hayward 94542.
Smith
L
L
Wilt
F
F
Shaw
R
R
D000818:Animals; D001757:Blastomeres / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D015870:Gene Expression; D008648:Mesoderm / Q000378:metabolism; D012617:Sea Urchins / Q000378:metabolism*
D003094:Collagen