Nancy S. Rafferty

Cytoplasmic filaments in the crystalline lens of various species: functional correlations.

Abstract The distribution of cytoplasmic filaments in lenses of five species was studied with the electron microscope. Two distinct patterns emerged. One pattern, in which filaments are grouped in characteristic bundles around the nucleus, in processes, and throughout the subcortical cytoplasm of epithelial cells, is typical of spherical, non-accommodating lenses of mice and rats. The second pattern is associated with anteriorly-flattened, accommodating lenses of infant human, squirrel and frog. In these, filaments are scattered in epithelial cells, but are accumulated on either side of the plasma membrane junction between epithelial cells and lens fibers. They are especially dense on the lens fiber side of the junction, and form a lattice associated with the lens fiber plasma membrane. The lattice is less extensive along the sides of lens fibers not in contact with epithelial cells. In spherical lenses the epithelial-fiber lattice is greatly reduced. Filaments in both types of lenses ranged in diameter between 5 and 11 nm. The filaments are thought to be a mixture of thin and intermediate filaments. It is hypothesized that the role of cytoplasmic filaments in lens, depending on the pattern present, is either to structurally support a spherical shape, or to provide a contractile force or elasticity to return the flattened anterior surface to the accommodated state in conjunction with the elasticity of the lens capsule.

An electron-microscope study of adult mouse lens: some ultrastructural specializations.

The ultrastructure of three regions of adult mouse lens was studied with the transmission electron microscope. Epithelial cells of the central and preequatorial peripheral regions are similar and contain organelles which characterize moderately metabolically active cells. These cells also contain large quantities of 50 A diameter filaments located primarily around the nucleus and in cytoplasmic processes, but which, in cells of older mice, are arrayed in bundles which intersect in a herringbone pattern. The only specialized cell junctions observed are probable gap junctions, but ordinary cell contacts between epithelial cells are nearly twice as wide as contacts between lens fibers, and cell-to-fiber contacts are intermediate. The difference in thickness of cell contacts is due to a narrowed intercellular space and not to plasma membrane thickness. This specialization may be related to physical requirements for optimal light transmission.

Comparative study of actin filament patterns in lens epithelial cells. Are these determined by the mechanisms of lens accommodation

Actin filament patterns in lens epithelia from animals of various taxonomic groups were studied using rhodamine phalloidin fluorescence microscopy of epithelial whole mounts and transmission electron microscopy of tangential sections. The results were compared with the accommodative mechanism operating in each case as reported in the literature. Lenses that accommodate by deformation of the anterior surface, in squirrel, chipmunk, rabbit, monkey and human, showed polygonal arrays (PAs) at the apical end of the epithelial cells. Lenses that translate as a whole, in shark, bony fish and frog, showed stress fibers (SFs) at the basal or apical end of the cells. No specialized actin pattern was seen in turtle and bird, which have lenses that are squeezed into an anterior lenticonus; cat, where the lens is translated forward; or rat, cow and most mice, which have no defined accommodation. In exception, certain strains of laboratory mice did show sequestered actin bundles (SABs) and/or PAs. Based on our findings, we conclude that PAs, which resemble geodesic domes, do not take an active part in near-point accommodation; but like SFs, may serve to resist overextension by internal pressure of the fiber mass or by zonular tension.

Immunocytochemical evidence for an actin-myosin system in lens epithelial cells

Since filamentous actin had been shown earlier to exist in lens epithelial and fiber cells, we inquired whether this could represent a contractile system with myosin and other actin-associated proteins. We resolved this question in freshly removed or organ-cultured rabbit and squirrel lens epithelial whole mounts using immunocytochemical techniques and by immunoblots of extracts separated by electrophoresis. In the former, methods were developed using long fixation times and long incubation in primary antibodies and biotinylated second antibodies visualized by streptavidin immunofluorescence and by diaminobenzidine peroxidase. Myosin was found to be localized along the filamentous rays and at central vertices of polygonal arrays situated at the apices of epithelial cells. It was not clear whether myosin and actin occurred together along the same or adjacent filaments in a bundle. Tubulin and vimentin were found deeper in the cells and were not aligned with actin and myosin filaments. Control lens epithelia treated similarly except for deletion of the primary antibodies showed no staining. As positive controls, pieces of glycerinated sartorius muscle exhibited characteristic cross-banded patterns of actin and myosin when incubated with the same reagents used on the lens epithelium. Denatured extracts of rabbit lens epithelium and of cortical fiber cells separated by electrophoresis and transferred to nitrocellulose paper, stained specifically with the same myosin and tubulin antibodies used in the immunocytochemistry experiments. The molecular weight profile of the myosin polypeptide indicated that lens tissue has myosin II. We conclude that a contractile system exists in lens epithelial and cortical fiber cells, although the function is not understood at this time. We conjecture that the system may act to stabilize lens shape by providing contractile tone.

The effects of near-UV radiation on elasmobranch lens cytoskeletal actin

The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringers medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Polygonal arrays of microfilaments in epithelial cells of the intact lens

Polygonal arrays of microfilaments have been discovered to line the inner apical plasma membrane of anterior epithelial cells of the intact rabbit lens. When tangential sections are studied with the electron microscope, the polygonal arrays are seen to consist of central vertices interconnected by rays of filaments. The rays near the cell periphery insert into the lateral plasma membrane. The vertices are spaced about 1 micron apart and appear to be attached to the apical plasma membrane. The polygonal arrays have little depth as judged by stereo-pairs and are incorporated within the dense band of microfilaments seen in cross-section at the epithelio-fiber junction. The diameter of the filaments and their similarity to actin-containing polygonal arrays described by other investigators in cultured cells suggest that these structures contain actin in lens epithelial cells. The function of the polygonal arrays in relation to maintenance of lens shape or to changes in lens shape in accommodation is discussed.

A scanning electron microscopic study of lens fibers in healing mouse lens

Abstract Anterior cortical lens fibers of young, normal and injured mouse lenses were studied with the scanning electron microscope. The lenses were injured by a transcorneal needle penetration. Following injury the specimens were examined at intervals of 17 and 24 hr, 3, 4, 8, and 11 days. In all injured lenses studied, there appeared a localized cortical lens fiber response to the injury. Adjacent to the wound area, the fibers appeared flattened and attenuated into interdigitating fiber processes. This response had begun at 17 hr post-injury, seemingly peaked at 4 days, and showed a reversion to normal morphology at 8 days. We feel that this is indicative of a plastic healing response to injury rather than a cataractogenic process. Examination of the nuclear and suture areas of control lenses showed a similarly complex morphology which we feel indicates that lens fibers have an inherent capability for morphological change and healing.

Immunogold-EM localization of actin and vimentin filaments in relation to polygonal arrays in lens epithelium in situ

An indirect immunogold technique for transmission electron microscopy was used for localizing two cytoskeletal proteins, actin and vimentin, in the epithelium of freshly removed rabbit lens, especially in relation to the polygonal array structures located at the apices of the epithelial cells. Antibody specificity was determined on semi-pure chicken breast muscle actin and bovine lens vimentin using Western blotting of these proteins and extracts of rabbit lens epithelium separated by SDS-PAGE. Whole lenses of rabbits were lightly fixed in glutaraldehyde and embedded in LR White resin. Tangential sections were taken at 70 to 80 nm and at 0.25 micron and used for single-labeling, and double-labeling with antibodies raised in different hosts and treated with appropriate second antibodies conjugated with non-overlapping sizes of gold particles. Routine and stereomicroscopy were used to analyze gold-label patterns. The study shows that the rays of the polygons project deeply into the cell from the vertices lying on the inner apical membrane. Actin is located on the filaments of rays, but vimentin is not associated with the polygons at the level in the cell that we studied. Vimentin filaments are found in deeper regions of the epithelial cell. Stereopairs were useful in differentiating where the gold-label was located and in fact, this technique demonstrated that most of the label is on the surface of sections where the filaments are exposed.

Development of actin polygonal arrays in rabbit lens epithelial cells

In searching for a clue to the role of actin filament bundles organized into polygonal arrays, or geodomes, in lens epithelial cells, we examined several physical events occurring in the young rabbit lens which may initiate their formation. We used NZW rabbits between the ages of 24 days gestation and 50 days postnatal. Data were obtained from TEM, SEM and fluorescence microscopy. Parameters measured were lens weights, apical surface areas of cells in epithelial whole mounts, epithelial cell thickness, and timing of eyelid opening, breakdown of the tunica vasculosa lentis (TVL) and formation of the ciliary zonules; these findings were correlated with the first signs of development of the arrays. Polygonal arrays formed slowly beginning at one to two days after birth, and with advancing time these thickened and made more numerous connections with the lateral plasma membranes. Development of the arrays was not correlated with onset of vision or disappearance of the TVL or a sudden increase in cell area, since these events occur postnatally at about 9-10 days, nor with the development of zonular fibers since these are already in place at 24 days of gestation. Only lens weights showed a dramatic increase between 24 days gestation and birth. It is surmised that the expanding lens mass may be involved in some way in signaling the organization of actin filaments into geodomes.

An ultrastructural study of fixation artifacts in lens epithelium

The conditions providing for optimal preservation of the ultrastructure of the lens epithelium of embryonic and young chicks were sought, especially with regard to avoiding fixation artifacts and to enhancing the lens cytoskeleton. The optimal fixative/buffer solution for these purposes was found to consist of 2% glutaraldehyde in 0.05M phosphate buffer containing 0.2% tannic acid and 0.002% CaCl2, pH 7.2, whose total osmolarity is about 340 mOsm, and postfixation in 1% osmium. As the osmolarity was increased by use of 0.075M or 0.1M phosphate buffer, intercellular spaces and myelin-like figures appeared along the cell membranes of the epithelial cells and superficial cortical fibers. As the osmolarity was decreased, using 0.02M phosphate buffer, plasma membranes became flaccid and interrupted, nuclei underwent severe shape changes, and the cytoplasm became electron-lucent. Delays of 30 minutes or one hour before fixation caused swelling of cytoplasmic organelles and the nuclear envelope. Primary fixation in osmium resulted in interrupted cell membranes and in swollen organelles. Many of these artifacts seen in the superficial chick lens, produced merely by manipulating the tonicity or the time of fixation after death, have been previously attributed in the literature to cataractous or aging changes. Based on the present findings caution in interpreting electron micrographs of pathologic changes in lens is urged.