Purpose: Ball-and-sockets and protrusions are specialized interlocking membrane domains between lens fibers of all species studied. Ball-and-sockets and protrusions are similar in their shape, size, and surface morphology, and are traditionally believed to play a key role in maintaining fiber-to-fiber stability. Here, we evaluate the hypothesis that ball-and-sockets and protrusions possess important structural and functional differences during fiber cell differentiation and maturation.Methods: Intact lenses of leghorn chickens (E7 days to P62 weeks old) and rhesus monkeys (1.5-20 years old) were studied with SEM, freeze-fracture TEM, freeze-fracture immunogold labeling (FRIL), and filipin cytochemistry for membrane cholesterol detection.Results: SEM showed that ball-and-sockets were distributed along the long and short sides of hexagonal fiber cells, whereas protrusions were located along the cell corners, from superficial to deep cortical regions in both chicken and monkey lenses. Importantly, by freeze-fracture TEM, we discovered the selective association of gap junctions with all ball-and-sockets examined, but not with protrusions, in both species. In the embryonic chicken lens (E18), the abundant distribution of ball-and-socket gap junctions was regularly found in an approximate zone extending at least 300 mu m deep from the equatorial surface of the superficial cortical fibers. Many ball-and-socket gap junctions often protruded deeply into neighboring cells. However, in the mature fibers of monkey lenses, several ball-and-sockets exhibited only partial occupancy of gap junctions with disorganized connexons, possibly due to degradation of gap junctions during fiber maturation and aging. FRIL analysis confirmed that both connexin46 (Cx46) and connexin50 (Cx50) antibodies specifically labeled ball-and-socket gap junctions, but not protrusions. Furthermore, filipin cytochemistry revealed that the ball-and-socket gap junctions contained different amounts of cholesterol (i.e., cholesterol-rich versus cholesterol-free) as seen with the filipin-cholesterol-complexes (FCC) in different cortical regions during maturation. In contrast, the protrusions contained consistently high cholesterol amounts (i.e., 402 FCCs/mu m(2) membrane) which were approximately two times greater than that of the cholesterol-rich gap junctions (i.e., 188 FCCs/mu m(2) membrane) found in ball-and-sockets.Conclusions: Gap junctions are regularly associated with all ball-and-sockets examined in metabolically active young cortical fibers, but not with protrusions, in both chicken and monkey lenses. Since these unique gap junctions often protrude deeply into neighboring cells to increase membrane surface areas, they may significantly facilitate cell-to-cell communication between young cortical fiber cells. In particular, the large number of ball-and-socket gap junctions found near the equatorial region may effectively facilitate the flow of outward current toward the equatorial surface for internal circulation of ions in the lens. In contrast, a consistent distribution of high concentrations of cholesterol in protrusions would make the protrusion membrane less deformable and would be more suitable for maintaining fiber-to-fiber stability during visual accommodation. Thus, the ball-and-sockets and protrusions are two structurally and functionally distinct membrane domains in the lens.

This study shows that caveolae are present in lens epithelia of rabbit and guinea pig under normal conditions. Caveolae are unique lipid membrane microdomains observed in many cell types. They are believed to play crucial roles in a variety of basic physiological functions including signal transduction, lipid and transcellular transport. Using TEM, immunocytochemistry and immunoblotting we show for the first time the existence of caveolae and the co-localization of their signature marker integral proteins, caveolin-1 and caveolin-2, in the intact lens of rabbit and guinea pig. Thin-section TEM shows that among several species studied, lens epithelia of rabbit and guinea pig exhibited a large number of caveolae. The caveolae were pear shaped, approximately 70nm in diameter, and were found frequently along the lateral membranes of epithelial cells in the intact lens. In the intact cortical fibers, only a small number of caveolae was seen in the superficial cells. In cultured lens epithelial cells, however, caveolae were observed along all membrane surfaces, but were more abundant at the apical membrane of the cells. Immunofluorescence and immunoblot analyses confirmed the presence of caveolin-1 and caveolin-2 in the lens epithelium. In addition, caveolin-1 and caveolin-2 co-exist in the lens epithelium of both rabbit and guinea pig. HRP tracer study demonstrated that caveolae could carry out endocytosis, suggesting their involvement in molecular transport. Cultured rabbit lens epithelial cells (line N/N1003A) were used to examine the response of caveolae to methyl-β-cyclodextrin (MBCD), a specific cholesterol-depleting drug. The lens epithelial cells were incubated in freshly prepared MEM medium plus 8% rabbit serum containing 10mm MBCD for 0 (control), 15, 30 or 60min. Controls for MBCD treatment were cultured in MEM plus 8% rabbit serum. MBCD treatment for 30min revealed that depletion of cholesterol abolished the majority of caveolae in cultured lens epithelial cells. This result strongly suggests that caveolae are cholesterol-rich lipid rafts that are likely to play important roles in the lens.

Zonulae adherens and associated actin bundles (ZA/AB) are believed to play a major role in epithelial folding and invagination during morphogenesis of neural tube and other vesicular structures. The lens morphogenesis is associated with the formation of the lens vesicle in which ZA/AB would be needed during the formation process. However, the existence of ZA/AB in the lens has never been established. In this study we report for the first time the existence of ZA/AB in both lens epithelium and fiber cells during embryonic development of chicken lens from E4 to E20. Light microscopy revealed contacts between the lens epithelium and primary fiber cells, and between the lens epithelium and secondary fiber cells at E4 and E11, respectively. Thin-section electron microscopy consistently revealed ZA/AB near both the apical ends of lens epithelial cells and primary fiber cells at E4. This arrangement manifests as a parallel pair of belt-like ZA/AB along the epithelium–fiber interface. In semi-tangential sections, a continuous belt-like ZA/AB was also evidenced in individual epithelial cells and fiber cells. Furthermore, the same ZA/AB arrangement was observed near both the apical ends of epithelial cells and secondary fiber cells at E11. Besides ZA/AB, macular-type fasciae adherens were distributed regularly between epithelial cells, between primary fibers, between secondary fibers, and between epithelium and both primary and secondary fibers. Immunofluorescence strongly and preferentially labeled N-cadherin at both the apical ends of lens epithelium and primary or secondary fibers at the corresponding ages, suggesting a direct association with the zonulae adherens. Also punctate N-cadherin labeling was commonly seen along various regions of primary and secondary fiber cells at different ages, and to a larger extent in the mature fibers of older lenses. This study suggests that: (1) ZA/AB located at the apices of lens epithelial cells may play a crucial role in the early stages of lens morphogenesis (e.g. lens vesicle formation); (2) ZA/AB of primary and secondary fiber cells originate from the epithelial cells during their elongation and differentiation; (3) owing to the restricted distribution of ZA/AB, abundant fasciae adherens are needed to maintain the structural stability of the epithelium and fiber cells during development and maturation; and (4) N-cadherin is the principle adhesion protein for both the zonulae adherens and fasciae adherens in the lens.

A unique association between actin filament bundles and gap junctions in cortical fiber cells of human and monkey lenses was studied with thin-section electron microscopy and immunocytochemistry. Thin-section electron microscopy showed that distinct layers of filament bundles (∼ 55 nm thick) were consistently associated with fiber gap junctions (∼ 16 nm thick) from intermediate to deep cortical regions in both species studied. The filament bundle was composed of 6-8 nm microfilaments which lay along both cytoplasmic surfaces of the junction. Fluorescence microscopy revealed a patchy pattern of F-actin labeling along the fiber cell membranes in the intermediate and deep cortical regions of the lens. The size and distribution pattern of F-actin labeling appear to correlate well with those of filament bundles/gap junctions seen in thin-section electron microscopy. By immunoelectron microscopy, the anti-actin antibody was shown to be localized to filament bundles/gap junctions in the intermediate cortical fibers of human lens, indicating that filament bundles are F-actin in nature. The identical filament bundle/gap junction association was not found in other species examined, including rodent, bird and fish, by the same procedure, suggesting that an association between actin bundles and gap junctions has a special functional role in the primate lens. It is proposed that gap junction-associated actin bundles may provide added structural stability for the primate lens.

A rapid, peripheral disease model utilizing the Bunyavirus, Caraparu, was established in mice for the evaluation of antiviral therapy with immunomodulators. 4-6-week-old B6C3F1 female mice, inoculated intraperitoneally with virus, developed coagulative liver necrosis and died between 4-6 days after infection. This Caraparu disease model was relatively resistant to treatment with immunomodulators, such as ABMP, Ampligen, alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta). However, a significant increase in median survival time (MST) was consistently observed upon treatment with gamma-interferon (IFN-gamma). The nucleoside analog - ribavirin - was highly effective against Caraparu virus in repeated treatment schedules begun on either day - 1, day 0, or day + 1 of infection. Ribavirin gave little protection when initiation of treatment was delayed until day + 2. However, combined treatment with IFN-gamma, starting on day 0 and ribavirin starting on day + 2. significantly reduced mortality.

Electron microscopy confirms the presence of a high concentration of glycogen particles in the lens nuclear region of birds of flying habit such as the ring-neck dove and pigeon. This observation is consistent with Raman spectroscopy. The glycogen particles in the dove lens, which are approximately 35 nm in diameter, are classified as beta type particles. Although this type has been previously characterized by high rates of glycogen turnover in other tissues, its localization in the lens nucleus indicates that it may serve a structural function rather than as a storage depot of carbohydrate in the lens. In a comparative electron microscopy study, glycogen particles were not observed in the chicken lens.