Collagen synthesis in chick-embryo frontal bone and 3T3 fibroblasts from mice was measured by incorporation in vitro of ['4C]proline into collagenasedigestible material. About 15-25% of the collagen synthesized by the frontal bone in 60 min, and 60% of that synthesized by the fibroblasts in 2 hr, was found to be soluble in the culture medium. The microtubular disruptive drugs colchicine and vinblastine, at 10 MM, inhibited collagen secretion in both systems almost completely. Formation of collagen hydroxyproline from proline was not inhibited by these drugs. Cytochalasin B, which impairs microfilament function, had no effect on collagen secretion. Our results support the theory that collagen is transported in vesicles to the cell membrane, where it is secreted. This conclusion is based on the similarity of the collagen-secreting system to other systems in which the movement of secretory vesicles or storage granules is inhibited by microtubule disruption.It has been known for some time that colchicine and vinblastine can bind to mitotic spindle protein and arrest mitosis; more recently, it has been shown that these alkaloids can dissociate the microtubule structure into its subunits (1). By the use of these agents, it has also been demonstrated that microtubules are involved in the secretion of thyroid hormone by mouse thyroid glands (2), of insulin by islets of Langerhans from the rat (3), of histamine by rat mast cells (4), and are involved in intracellular transport of amine granules by nerve cells (5), and melanin granules in melanocytes in frog skin (6).In order to better understand the mechanism by which collagen is transported from the intracellular site where it is synthesized to the extracellular matrix, we
MethodsFrontal bones were removed from 15-day-old chick embryos and cleaned of adhering tissues; individual bones were incubated in 0.5 ml of Eagle's minimal essential medium plus 0.25 mM sodium ascorbate in an atmosphere of 5% C02-95% air. After 30 min of incubation in the presence or absence of 10 uM colchicine, 10 AuM vinblastine, or 10 ,ug/ml of cytochalasin B, 1 MACi of L-[14C]proline was added and the incubation was extended for an additional 60 min. The bones were removed, homogenized in 1 ml of 0.05 M Tris-HCl (pH 7.6) in a stainless-steel mortar, and then sonicated for 20 sec with a Branson sonifier (3 amp). Protein was prepared from the sonicate and digested (7) with purified, protease-free bacterial collagenase. In this procedure, radioactivity released by collagenase and remaining in the supernatant after addition of 5% trichloroacetic acid-0.25% tannic acid is a measure of the collagen synthesized by the bone, while noncollagen protein is precipitated. Carrier protein (1 mg of chick-embryo protein) was added to the medium and this also was analyzed for Mouse fibroblasts (Balb 3T3) were obtained from Dr. E. Scolnick and cultivated at 5 X 105 cells per plate in Eagle's minimal essential medium containing 10% fetal-calf serum, 25 mM tricine buffer (pH 7.4), and 13 mM bicarbonate i...