Eileen Gibney

Cellular adhesion to collagen

Abstract BALB/3T3 cells were released from tissue culture plates with EGTA, and their rates of attachment to collagen gels polymerized on Millipore filters; were measured. Cell attachment in serum-free medium was 20–50% of that which occurred in medium containing 10% fetal calf serum (FCS). Cell attachment to gels pretreated with FCS and assayed in serum-free medium was identical with that of gels in FCS-containing medium. Thus, it seems there are two separate mechanisms of attachment to collagen; one involving direct attachment and a second mediated by a serum component(s) which binds to collagen.

Ferritoid, a Tissue-specific Nuclear Transport Protein for Ferritin in Corneal Epithelial Cells

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ∼50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

Type II collagen in the early embryonic chick cornea and vitreous: Immunoradiochemical evidence

Abstract The relative proportion of collagen types I and II was examined in early chick corneas and vitrous bodies by radioimmunoprecipitation. Embryos (4–5 days of incubation) were labelled in ovo with 3H-proline. After an additional 1–2 days, corneas and vitreous bodies were removed and the collagen was extracted and treated by limited pepsinization. Then, the labelled collagens were reacted with affinity purified antibodies against collagen types I and II, and the immune complexes precipitated with protein A-containing Staphylococcus aureus. In some cases, the precipitated collagen was further analysed by SDS-polyacrylamide gel electrophoresis. The results for the vitreous showed that greater than 90% of the labelled material precipitated by the antibodies was in type II. In the corneal samples, on the other hand, both types of antibodies precipitated appreciable labelled collagen with somewhat less than half being type II (30–46%). These results are in agreement with our earlier biochemical observations on the relative content of type II collagen in these extracellular matrix-rich structures from embryonic avian eyes.

Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody

The thermal stability of the helical domain of intracellular and matrix-associated type X collagen was examined in situ within the hypertrophic region of embryonic chick vertebral cartilages. For this we employed indirect immunofluorescence histochemistry of unfixed tissue sections reacted at progressively higher temperatures (Linsenmayer et al., J cell biol 99 (1984) 1405) with a conformation-dependent monoclonal antibody (X-AC9) (Schmid & Linsenmayer, J cell biol 100 (1985) 598). The hypertrophic chondrocytes which had most recently initiated synthesis of type X did not immediately secrete it, but instead retained it intracellularly within cytoplasmic organelles. This allowed for clear visualization of the intracellular type X. Within the pool of intracellular type X collagen, the epitope recognized by the antibody was stable up to 55 degrees C, but was destroyed at 60 degrees C. This is 5-10 degrees C higher than the thermal stability of the epitope when the molecule is in neutral solution (as determined by competition ELISA). The matrix-associated type X collagen is stable at least to 65-67.5 degrees C. We conclude that in situ the stability of the collagen helix in its normal intracellular environment is considerably greater than might be predicted from measurements made on molecules in solution.

Complete primary structure of the chicken α1(V) collagen chain

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Avian Epidermis Expresses Collagen Types I and II but not Type IX in a Restricted Spatial Pattern during Feather Morphogenesis

Type I1 collagen, once thought to be exclusively a mesenchymally derived, “cartilagespecific” molecule, is also a component of a number of widely diverse, epithelially derived matrices of avian embryos. These include ectodermal derivatives such as the primary corneal stroma,’.’ neuroepithelial derivatives such as the primary vitreous:’’ and a variety of other ~ites.4.~ Since these various type I1 collagen-containing matrices differ structurally and in their developmental fates, they likely differ in their composition as well. One possible source of variation among these matrices could be the presence or absence of other “cartilage-specific ” molecules that might be coexpressed with type I1 collagen, such as type IX collagen. WeZz6 and others’ have observed that type IX collagen is present, along with type 11, in some of these matrices but not in others. Recently we were surprised to observe6 in developing skin a transitory subepidermal deposition of type I1 collagen that is restricted to the interpapillary region of feather buds, being absent within the buds themselves. This suggests an involvement of the molecule in feather morphogenesis. The subepidermal matrix of interpapillary feather-forming skin is an example of the deposition of type 11 collagen without type IX. By immunofluorescence histochemistry, type I1 collagen (FIG. la) was restricted to the subepithelial matrix between the feather buds and was absent at the roots of, and within, the feather buds themselves. Type IX collagen (FIG. 1 b) was absent. Type I collagen (FIG. lc), however, was abundant within the feather buds, in the subepidermal matrix between them, and in the deeper tissues as well. The negative control was Type X collagen (FIG. Id). To examine the source and regulation of these subepidermal collagens, we performed in situ hybridizations employing 32P-labeled cDNA probes for the collagens.