Steven Bassnett

Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

αA- and αB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of αA- and αB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of αA- and αB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of αA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of αB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor α. To further study the relative protective activities of αA- and αB-crystallins, we created a cell line derived from αA−/−αB−/− double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line αAαBKO1 derived from αA/αB double knockout cells was transfected with αA- or αB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either αA-crystallin or αB-crystallin were isolated. The ability of αA- or αB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of αA-crystallin was two to three-fold higher than that of αB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of αA- and αB-crystallin. Since the expression of αA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.

On the mechanism of organelle degradation in the vertebrate lens.

The programmed elimination of cytoplasmic organelles occurs during terminal differentiation of erythrocytes, keratinocytes and lens fiber cells. In each case, the process is relatively well understood phenomenologically, but the underlying molecular mechanisms have been surprisingly slow to emerge. This brief review considers the particular case of the lens where, in addition to their specialized physiological roles, organelles represent potential sources of light scattering. The article describes how the elimination of organelles from lens cells located on the visual axis contributes to the transparency of lens tissue. Classic anatomical studies of lens organelle degradation are discussed, along with more contemporary work utilizing confocal microscopy and other imaging modalities. Finally, recent data on the biochemistry of organelle degradation are reviewed. Several review articles on lens organelle degradation are available [Wride, M.A., 1996. Cellular and molecular features of lens differentiation: a review of recent advances. Differentiation 61, 77-93; Wride, M.A., 2000. Minireview: apoptosis as seen through a lens. Apoptosis 5, 203-209; Bassnett, S., 2002. Lens organelle degradation. Exp. Eye Res. 74, 1-6; Dahm, R., 2004. Dying to see. Sci. Am. 291, 82-89] and readers are directed to these for a comprehensive discussion of the earlier literature on this topic.

Regulation of tissue oxygen levels in the mammalian lens

Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long‐term preservation of  lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (Q̇O2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on Q̇O2 and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2‐photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, PO2 gradients extended beyond the boundaries of the mitochondria‐containing cell layer, indicating the presence of non‐mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion–consumption model. Typical values were 3 × 10−5 cm2 s−1 for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in Q̇O2 and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen‐rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.

The role of MIP in lens fiber cell membrane transport

Abstract. MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 μm/sec whereas normal fiber cell membrane pH2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 μm/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

The Molecular Chaperone αA-Crystallin Enhances Lens Epithelial Cell Growth and Resistance to UVA Stress

αA-Crystallin (αA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of αA, but in differentiated fiber cells, αA is the most abundant soluble protein. The lenses of αA-knock-out mice develop opacities at an early age, implying a critical role for αA in the maintenance of fiber cell transparency. However, the function of α-crystallin in the lens epithelium is unknown. To investigate the physiological function of αA in lens epithelial cells, we used the following two systems: αA knock-out (αA(−/−)) mouse lens epithelial cells and human lens epithelial cells that overexpress αA. The growth rate of αA(−/−) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of αA(−/−) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the αA(−/−) cultures was increased 40-fold over wild type. In cells lacking αA, UVA exposure modified F-actin, but actin was protected in cells expressing αA. Stably transfected cell lines overexpressing human αA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoαA. Cells overexpressing αA were resistant to UVA stress, as determined by clonogenic survival. αA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of αA was not associated with its relocalization to the nucleus. These results indicate that αA has important cellular functions in the lens over and above its well characterized role in refraction.

Role of the Executioner Caspases during Lens Development

The notion that the cell death machinery is utilized during lens organelle degradation is supported by the observation that well characterized apoptotic substrates are cleaved during this process. Here, we test directly the role of executioner caspases (caspase-3, -6, and -7) in fiber cell differentiation. The distribution of mRNA, protein, and enzymatic activity for each caspase was determined in the mouse lens. Transcripts for all three executioner caspases were identified in lens fiber cells by real time RT-PCR, although only caspase-6 and -7 proteins were detected subsequently by Western blot analysis. Endogenous proteolytic activity was noted for caspase-3 but not caspase-6 or -7. We tested the role of executioner caspases in organelle degradation by examining lenses from mice deficient in each caspase. Knock-out lenses appeared grossly normal with the exception of caspase-3-/- lenses, which exhibited marked cataracts at the anterior lens pole. The distribution of lens organelles was mapped by confocal microscopy. There was no significant difference in the size of the lens organelle-free zone (OFZ)1 between wild-type and knock-out lenses. In response to treatment with staurosporine, caspase-3 and -6 (but not caspase-7) enzymatic activities were induced. We generated double knock-out animals to examine the phenotype of lenses deficient in both caspase-3 and -6. Histological examination of such lenses indicated the presence of a properly formed OFZ. Thus, no single executioner caspase (nor a combination of caspase-3 and -6) is required for organelle loss, although caspase-3 activity may be required for other aspects of lens transparency.

The stratified syncytium of the vertebrate lens.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein.

Aquaporin‐0 (AQP0) is the major intrinsic protein of lens fiber cells and the founder member of the water channel gene family. Here we show that disruption of the AQP0 gene by an early transposon (ETn) element results in expression of a chimeric protein, comprised of ∼75% AQP0 and ~25% ETn long terminal repeat (LTR) sequence, in the cataract Fraser (CatFr) mouse lens. Immunoblot analysis showed that mutant AQP0‐LTR was similar in mass to wild‐type AQP0. However, immunofluorescence microscopy revealed that AQP0‐LTR was localized to intracellular membranes rather than to plasma membranes of lens fiber cells. Heterozygous CatFr lenses were similar in size to wild‐type but displayed abnormal regions of translucence and light scattering. Scanning electron microscopy further revealed that mature fiber cells within the core of the heterozygous Cat Fr lens failed to stratify into uniform, concentric growth shells, suggesting that the AQP0 water channel facilitates the development of the unique cellular architecture of the crystalline lens.—Shiels, A., Mackay, D., Bassnett, S., Al‐Ghoul, K., Kuszak, J. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0‐LTR protein. FASEB J. 14, 2207–2212 (2000)

Morphometric analysis of fibre cell growth in the developing chicken lens.

The optical characteristics of any lens are determined by its internal composition, size and shape. In the lens of the eye, the macroscopic form of the tissue reflects the arrangement and behaviour of its component cells. In the current study, we quantified changes in the morphology and organization of chicken lens fibre cells during embryonic development. Lens radii, fibre cell length, shape, cross-sectional aspect ratio, cross-sectional area, cross-sectional perimeter, and cell packing organization were measured from confocal and transmission electron micrographs using computer assisted image analysis. Derived values for cell surface area and volume were also calculated. Because of the radial symmetry of the avian lens, we were able to employ a novel coordinate system to track the fate of identified cohorts of cells at successive developmental stages. This allowed kinetic information, such as the rate of increase in length or volume, to be derived. By sampling identified cell populations (i.e. those located at a specific point on the lens radius) at regular intervals it was possible, for the first time, to reconstruct the life history of fibre cells buried within the cellular conglomerate of the lens. The measurements indicated that a surprising degree of structural remodeling occurs during fibre cell elongation and continues after extant cells have been buried by waves of newly differentiated fibres. Even in the anucleated cells of the lens core, the size and surface topology of the cells were altered continually during development. However, some aspects of fibre cell organization were established early in development and did not vary thereafter. For example, the packing arrangement of cells in the adult lens was traced to a cellular template established on the tenth day of embryonic development.

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.