Immunofluorescence studies on cartilage matrix synthesis. The synthesis of link protein, chondroitin sulfate proteoglycan monomer and type II collagen.
A comparison of the synthesis and deposition of fibrous type II collagen and the constituents of chondroitin sulfate proteoglycan (CSPG) aggregates, CSPG monomer and link protein, was made for chicken sternal chondrocytes in culture, using simultaneous double immunofluorescence and lectin localization. Chondrocytes deposited only CSPG constituents--and not type II collagen--into the extracellular matrix (ECM). Intracellular precursors of CSPG monomer were localized primarily in perinuclear regions, but were observed in other cytoplasmic vesicles as well. Link protein antibodies stained the same intracellular structures, but stained the perinuclear cytoplasm less intensely. In contrast, type II procollagen was distributed in vesicles throughout the cytoplasm and was clearly absent from the distinctive, CSPG precursor-containing vesicles. Fluorescence-labelled lectins were used to further identify intracellular membrane compartments. Wheat germ agglutinin (WGA) and Ricinus lectins (which recognize carbohydrates added in the Golgi) stained the perinuclear cytoplasm, while concanavalin A (conA) (which recognizes mannose-rich oligosaccharides added co-translationally) stained vesicles throughout the rest of the cytoplasm and not the perinuclear cytoplasm. The distinctive CSPG-containing vesicles were not stained with WGA or Ricinus agglutinins. Data presented elsewhere demonstrate that the vesicles do not react with monoclonal antibodies which recognize chondroitin sulfate (CS) or keratan sulfate (KS) determinants. Thus, we conclude that the vesicles accumulate CSPG precursors which have not been modified by Golgi-mediated processes. The data indicate that matrix molecules may be segregated selectively prior to transit through the Golgi complex. The co-distribution of link protein and CSPG monomer precursors in vesicles prior to further, Golgi-mediated modification may reflect an as yet undetermined function of these vesicles in the processing or assembly of CSPG.
D000818:Animals; D002356:Cartilage / Q000166:cytology*; D002478:Cells, Cultured; D002642:Chick Embryo; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D003094:Collagen / Q000096:biosynthesis*; D005109:Extracellular Matrix / Q000032:analysis*; D016326:Extracellular Matrix Proteins; D005455:Fluorescent Antibody Technique; D037102:Lectins / Q000032:analysis; D046911:Macromolecular Substances; D009844:Oligosaccharides / Q000032:analysis; D014176:Protein Biosynthesis; D011509:Proteoglycans / Q000096:biosynthesis*; D014018:Tissue Distribution; D000099249:Proteoglycan Link Protein
D011508:Chondroitin Sulfate Proteoglycans; D003094:Collagen; D016326:Extracellular Matrix Proteins; D037102:Lectins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein