Cell cycle synchrony in the developing chicken lens epithelium.

Synchronous oscillations of DNA synthesis and histone 2B mRNA expression occur during normal development of 13- to 16-day-old embryonic chicken lens epithelium. At least four cycles were observed with peak values of DNA synthesis and histone 2B mRNA 5 to 10 times greater than baseline values. Fourier analysis of DNA synthesis identified a statistically significant oscillatory period of 18 hr, the approximate length of the cell cycle at this age. Minor components of 7-9 and 12 hr were also identified in the data sets. Lenses labeled with 3H-thymidine and analyzed by autoradiography at 13.8 days of embryogenesis revealed more than twice the number of labeled nuclei at this time than in lenses labeled 9 hr later; histone 2B mRNA followed this same pattern. These findings demonstrate that a significant population of cells is synchronized with respect to the cell cycle in the developing lens epithelium in ovo. The temporal pattern of mitosis may be the basis of the fiber cell architecture and consequent lens transparency.

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.

Calpactin I in the differentiating embryonic chicken lens: mRNA levels and protein distribution.

Calpactin I, one of the EDTA-extractable proteins of the lens membrane, binds phospholipid and actin in a calcium-dependent manner. It is also known substrate of the pp60arc kinase. Analysis of embryonic chicken lens RNA with a bovine calpactin I-specific cDNA probe revealed the presence of a approximately 1.8 Kb calpactin mRNA in the lens cells. Six-day embryonic chicken lenses were microdissected into central epithelium, equatorial epithelium, and fiber cells. Total cytoplasmic RNA was isolated from these samples and calpactin I mRNA levels were determined by the polymerase chain reaction (PCR) following reverse transcription (RT). Quantitative PCR indicates that the calpactin I mRNA levels in the equatorial epithelium are greater than in the central epithelium by a factor of 12.7 +/- 2.7. Calpactin I mRNA in fiber cells is an additional 3.5 +/- 1.5 times greater than in the equatorial epithelium. Whole mounts of embryonic chicken lens epithelia and histological sections of whole lenses were also examined with an antibody directed against chicken calpactin I. Calpactin I was predominantly localized in a punctate distribution in equatorial epithelial cells and near the plasma membrane of elongate fiber cells. The elevated levels of calpactin I mRNA observed in the equatorial epithelium and fiber cells and the immunological localization of the protein suggest a possible role of calpactin I in the elongation of fiber cells during lens differentiation.

The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading.

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture.

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.