PRCD is essential for high-fidelity photoreceptor disc formation.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties.

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. Mouse lenses contain Tpm3.5 (also known as TM5NM5), a previously unstudied isoform encoded by Tpm3, which is associated with F-actin on lens fiber cell membranes. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and α-actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience, leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that a Tpm protein is essential for cell biomechanical stability in a load-bearing non-muscle tissue, and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivoThis article has an associated First Person interview with the first author of the paper.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice.

The wavy square array junctions are composed of truncated aquaporin-0 (AQP0) proteins typically distributed in the deep cortical and nuclear fibers in wild-type lenses. These junctions may help maintain the narrowed extracellular spaces between fiber cells to minimize light scattering. Herein, we investigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1-22 months were used. By light microscopy examinations, cav1-KO lenses at age 1-18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 µm deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 µm in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses.

Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse.

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1-16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 µm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

The tectonic complex regulates membrane protein composition in the photoreceptor cilium.

The primary cilium is a signaling organelle with a unique membrane composition maintained by a diffusional barrier residing at the transition zone. Many transition zone proteins, such as the tectonic complex, are linked to preserving ciliary composition but the mechanism remains unknown. To understand tectonic's role, we generate a photoreceptor-specific Tctn1 knockout mouse. Loss of Tctn1 results in the absence of the entire tectonic complex and associated MKS proteins yet has minimal effects on the transition zone structure of rod photoreceptors. We find that the protein composition of the photoreceptor cilium is disrupted as non-resident membrane proteins accumulate in the cilium over time, ultimately resulting in photoreceptor degeneration. We further show that fluorescent rhodopsin moves faster through the transition zone in photoreceptors lacking tectonic, which suggests that the tectonic complex acts as a physical barrier to slow down membrane protein diffusion in the photoreceptor transition zone to ensure proper removal of non-resident membrane proteins.

Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens.

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 µm 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/µm2 membrane) which were approximately two times greater than that of the cholesterol-rich gap junctions (i.e., 188 FCCs/µm2 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.

Gap junction remodeling associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens.

PURPOSE: To investigate the structural remodeling in gap junctions associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens. We also evaluated the hypothesis that the cleavage of the COOH-terminus of chick Cx50 (formerly Cx45.6) during fiber cell maturation would affect the gap junction remodeling. METHODS: Freeze-fracture TEM and filipin cytochemistry were applied to visualize structural remodeling of gap junction connexons associated with cholesterol redistribution during fiber cell maturation in adult leghorn chickens (42-62 weeks old). Freeze-fracture immunogold labeling (FRIL) was used to label the specific Cx50 COOH-terminus antibody in various structural configurations of gap junctions. RESULTS: Cortical fiber cells of the adult lenses contained three subtypes of cholesterol-containing gap junctions (i.e., cholesterol-rich, cholesterol-intermediate, and cholesterol-poor or -free) in both outer and inner cortical zones. Quantitative studies showed that approximately 81% of gap junctions in the outer cortex were cholesterol-rich gap junctions whereas approximately 78% of gap junctions in the inner cortex were cholesterol-free ones. Interestingly, all cholesterol-rich gap junctions in the outer cortex displayed loosely-packed connexons whereas cholesterol-free gap junctions in the deep zone exhibited tightly, hexagonal crystalline-arranged connexons. Also, while the percentage of membrane area specialized as gap junctions in the outer cortex was measured approximately 5 times higher than that of the inner cortex, the connexon density of the crystalline-packed gap junctions in the inner cortex was about 2 times higher than that of the loosely-packed ones in the outer cortex. Furthermore, FRIL demonstrated that while the Cx50 COOH-terminus antibody was labeled in all loosely-packed gap junctions examined in the outer cortex, little to no immunogold labeling was seen in the crystalline-packed connexons in the inner cortex. CONCLUSIONS: Gap junctions undergo significant structural remodeling during fiber cell maturation in the adult chicken lens. The cholesterol-rich gap junctions with loosely-packed connexons in the young outer cortical fibers are transformed into cholesterol-free ones with crystalline-packed connexons in the mature inner fibers. In addition, the loss of the COOH-terminus of Cx50 seems to contribute equally to the transformation of the loosely-packed connexons to the crystalline-packed connexons during fiber cell maturation. This transformation causes a significant increase in the connexon density in crystalline gap junctions. As a result, it compensates considerably for the large decrease in the percentage of membrane area specialized as gap junctions in the mature inner fibers in the adult chicken lens.

Age-related changes in eye lens biomechanics, morphology, refractive index and transparency.

Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.

Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens.

PURPOSE: To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the embryonic chicken lens. The possible mechanism by which cholesterol is removed from gap junction plaques is also investigated. METHODS: Filipin cytochemistry in conjunction with freeze-fracture TEM was used to visualize cholesterol, as represented by filipin-cholesterol complexes (FCCs) in gap junction plaques. Quantitative analysis on the heterogeneous distribution of cholesterol in gap junction plaques was conducted from outer and inner cortical regions. A novel technique combining filipin cytochemistry with freeze-fracture replica immunogold labeling (FRIL) was used to label Cx45.6 and Cx56 antibodies in cholesterol-containing gap junctions. Filipin cytochemistry and freeze-fracture TEM and thin-section TEM were used to examine the appearance and nature of the cholesterol-containing vesicular structures associated with gap junction plaques. RESULTS: Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL studies confirmed that cholesterol-containing junctions were gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were often observed in the gap junction plaques. They appear to be involved in the removal of cholesterol from gap junction plaques through endocytosis. CONCLUSIONS: Gap junctions in the young fibers are enriched with cholesterol because they are assembled in the unique cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner mature fibers during fiber cell maturation. A distinct endocytotic process appears to be involved in removing cholesterol from the cholesterol-containing gap junctions, and it may play a major role in the transformation of cholesterol-rich gap junctions into cholesterol-free ones during fiber cell maturation.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

PURPOSE: To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein. METHODS: We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers. RESULTS: F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, ß2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, ß2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers. CONCLUSIONS: These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a ß2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens.

PURPOSE: Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. METHODS: The loss of AQP0 in AQP0(-/-) lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0(-/-) lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. RESULTS: Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 µm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. CONCLUSIONS: This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC-induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF-α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.