Crystallin Synthesis Research Articles

Macromolecular events during differentiation of the chicken lens: Paper from the symposium ȁlens proteins and related subjects,ȁ Ghent, Belgium, June, 1967

The synthesis of lens proteins during differentiation of the chicken lens was studied with antisera to adult lens and to fractions isolated by polyacrylamide gel electrophoresis. δ-Crystallin (FISC) was first detected by immunoelectrophoresis at 60 hr. Lens placode cells showed immunofluorescence at 52 hr, mainly in their basal parts. Autoradiography after exposure to [3H]thymidine showed that their nuclei undergo interkinetic migration. During interkinesis they are located in the basal parts of the cells, but for mitosis the cells contract to the lumen. Following division the daughter cells again elongate and the nuclei return to the retinal side. Thus it is not surprising that δ-crystallin first appears here. At 57 hr the reaction had spread throughout the posterior wall, and at 3 days the epithelium was positive. At 8 days the latter reacted progressively weaker but the fibers remained positive. In 5-week-old chicks and in adults the cortex was negative while the center still showed strong fluorescence. α-Crystallin was detectable by immunoelectrophoresis on the fourth day of incubation. It was first demonstrated with immunofluorescence in centrally located lens fibers at 3ȁ3½ days. At 8 days the epithelium became positive and the fibers lost some fluorescence. This continued until in 5-week-old chicks the lens core was negative. The β-crystallins of the adult chicken consist of 8 or 9 immunologically related proteins. At 7 days the first component was seen immunoelectrophoretically. The pattern became more complicated until it was comparable at 17 days of embryonic life with that of the adult. Fluorecence was first observed in 28-somite embryos in the same location as δ-crystallin. At 3½ days the epithelium began to react and at 4 days the complete lens was positive. The reaction disappeared from the nucleus at a faster rate than that for α-crystallin. γ-Crystallins were not found. Fluorescence was never seen outside the lens. We conclude that crystallin synthesis is restricted to the lens itself and first occurs after placode formation has taken place. The most plausible explanation is that lens differentiation is dependent on differential gene activity at the level of the lens cell nuclei.

Stabilization of mRNA templates in bovine lens epithelial cells

Through the use of actinomycin D, it has been shown that the half-life of α-, β- and γ-crystallin mRNA in bovine lens epithelium increases with lens age. Crystallin synthesis in epithelial cells from embryonic lenses was potently inhibited by actinomycin D; the synthesis of these same proteins in adult lens epithelial cells was not affected. In both cases, all RNA synthesis was completely turned off by the antibiotic. Since the mitotic index of epithelial cells in embryonic lenses is much greater than that of the adult epithelial cells, our results indicate that stabilization of the protein-synthesizing templates found in adult epithelial cells may be related to the decreased replicative activity of these cells.

The changing cellular localization of α-crystallin in the lens of the chicken embryo, studied by immunofluorescence

The appearance and distribution of α-crystallin in chicken embryos and in animals after hatching were studied with the indirect fluorescent antibody method. α-Crystallin was isolated by two-dimensional polyacrylamide gel electrophoresis. Antisera were prepared against the purified fraction and against total chicken lens. They were controlled for their specificity in Ouchterlony plates and immunoelectrophoresis. The first fluorescence with total lens antiserum was observed in the morphologically most advanced cells of the lens placode of 23-somite embryos (50–53 hours of incubation). α-Crystallin antisera did not react at this time, indicating that another structural protein was synthesized before α-crystallin appeared. With further development of the lens rudiment the fluorescence gradually spread to more peripheral parts, and at 3 to 3/12 days the entire lens reacted with total lens antiserum. At this time α-crystallin could first be detected in and around the nuclei of a few centrally located fibers. Gradually more peripheral fibers became involved, then the equatorial zone, and finally the anterior epithelium. While some cells reacted very strongly, others were only weakly fluorescent or even remained negative; there resulted a mosaiclike staining pattern. At 8 days of embryonic life all epithelial cells were positive for α-crystallin and the reaction in the fibers became progressively weaker. In the adult lens the center of the lens was negative, the epithelial cells and equatorial zone, with the exception of a few completely negative cells, were strongly positive and the superficial fibers showed an intermediate reaction. The fluorescence was always confined to the lens. It is concluded that lens placode formation is independent from crystallin synthesis. Furthermore, the production of crystallins is restricted to the lens rudiment and lens and does not take place in the optic cup and its derivates or in other ectodermal tissues. α-Crystallin appears relatively late in development and cannot be considered as an essential protein for lens formation.