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Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-layering at the lens equator occurred on days 1 and 2, but then reorganised into two discrete layers by day 3. E- and N-cadherin expression preceded cell polarity being re-established during the first week. Aquaporin 0 (AQP0) was first detected in the elongated cells at the lens equator at day 7. Cells at the capsulotomy site, however, behaved very differently expressing the epithelial mesenchymal transition (EMT) markers fibronectin and alpha-smooth muscle actin (SMA) from day 3 onwards. The physical interaction between the apical surfaces of the anterior and posterior LECs from day 3 after surgery preceded cell elongation. In the human BIL sample fibre cell formation was confirmed by both histological and proteome analyses, but the cellular response is less ordered and variable culminating in Soemmerring's ring (SR) formation and sometimes Elschnig's pearls. This we evidence for lenses from a single patient. No bow region or recognisable epithelial-fibre cell interface (EFI) was evident and consequently the fibre cells were disorganised. We conclude that lens cells require spatial and cellular cues to initiate, sustain and produce an optically functional tissue in addition to capsule integrity and the EFI., (Copyright © 2021. Published by Elsevier Ltd.)

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca 2+ . The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca 2+ concentrations., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

How the lens ages successfully is a lesson in biological adaption and the emergent properties of its complement of cells and proteins. This living tissue contains some of the oldest proteins in our bodies and yet they remain functional for decades, despite exposure to UV light, to reactive oxygen species and all the other hazards to protein function. This remarkable feat is achieved by a shrewd investment in very stable proteins as lens crystallins, by providing a reservoir of ATP-independent protein chaperones unequalled by any other tissue and by an oxidation-resistant environment. In addition, glutathione, a free radical scavenger, is present in mM concentrations and the plasma membranes contain oxidation-resistant sphingolipids what compromises lens function as it ages? In this review, we examine the role of small molecules in the prevention or causation of cataracts, including those associated with diet, metabolic pathways and drug therapy (steroids)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/μm2; equator: 8.2 ± 1.9 particles/μm2; posterior pole: 4 ± 0.9 particles/μm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/μm2; equator: 5 ± 2 particles/μm2; posterior pole: 1.9 ± 0.7 particles/μm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The lens epithelium possesses an apical junctional complex (AJC) comprising adherens and tight junctions (AJs and TJs) and yet several key structural components and associated regulatory proteins have not been identified or localised in these cells. Here we determine the subcellular distribution of the archetypal TJ markers (ZO-1, claudin-1, and occludin) and TJ-associated cell polarity proteins (aPKC, Par3 and Par6beta) with AJ markers, E- and N-cadherin. As seen in other polarised epithelia, all these markers were located by confocal immunofluorescence microscopy to the apical ends of the lateral plasma membranes of bovine lens epithelial cells at sites of cell-cell interaction. Using immunoelectron microscopy, we show that ZO-1 concentrated at "kissing points" between neighbouring cells and these data, when taken in the context of our confocal immunofluorescence microscopy and blotting data, suggest the presence of TJs within the AJC. Likewise, immunogold labelling for E-cadherin identified AJs within these AJCs. We also report aPKC immunogold labelling localised to the AJC. These data show that the AJC of lens epithelial cells are a composite of TJs and AJs.

The differentiation of epithelial cells in the vertebrate lens involves a series of changes that includes the degradation of all intracellular organelles and a dramatic elongation of the cells. The latter is accompanied by a substantial remodelling of the cytoskeleton and changes in the distribution of the actin, microtubule and intermediate filament cytoskeletons during lens cell differentiation have been well documented. There have, however, been no studies of microtubule organizing centres (MTOCs) and specifically centrosomes during lens cell differentiation. We have investigated the fate of the centrosomal MTOCs during cellular differentiation in the bovine lens using gamma-tubulin, ninein, centrin 2 and centrin 3 as markers. Our studies show that these markers oscillate between a clear centrosome-based association in epithelial cells and a defocused cluster in lens fibre cells. Our data further reveal a transient loss of signal for the typical centrosomal marker gamma-tubulin as the lens epithelial cells begin to differentiate into lens fibre cells. This marker apparently disappears in the most distal epithelial cells at the lens equator, only to reappear in early lens fibre cells. The changes in gamma-tubulin distribution are mirrored by the other centrosomal markers, centrins 2 and 3 and ninein that also show a similar transient loss of their signals and subsequent clustering at the apical ends of differentiating fibre cells. The transient loss of staining for these centrosomal markers in the most posterior epithelial cells is a distinctive feature that precedes lens cell elongation. The dramatic reorganization of MTOC markers coincides with gap junction reorganization as seen by the loss of connexin 43 (alpha1-connexin) in these lens epithelial cells suggesting that these events mark a significant change preceding subsequent cell elongation and differentiation into fibre cells.

Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.

Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

A missense mutation in one of the three lens connexins, alpha8-connexin, has been recently shown to be the genetic basis of the zonular pulverant lens cataract. This connexin had been considered to be expressed only in lens fibre cells. The present studies show that alpha8-connexin is also expressed in the lens epithelial cell layer. For this study, the distribution of gap junctions in the adult bovine lens has been investigated by confocal immunofluorescence microscopy using antibodies against alpha8-connexin (MP70) and alpha1-connexin (Cx43). In addition to the anticipated localisation of alpha8-connexin to the broad faces of lens fibre cells as reported in other species, alpha8-connexin was also found colocalized with alpha1-connexin at plaques in the lateral epithelial-epithelial plasma membranes of the bovine lens. These data suggest that mixed alpha8-connexin/alpha1-connexin plaques are between epithelial cells at their apico-lateral plasma membranes, rather than between epithelial and fibre cells. Indeed, freeze fracture analyses of the epithelial-fibre cell interface failed to reveal gap junctions connecting the epithelium and the underlying fibre cells. Importantly, microdissection and subsequent immunoblotting of lens epithelium samples confirmed the immunolocalisation results. The data suggest mature mammalian lens epithelial cells could form either heteromeric, heterotypic and/or mixed homomeric-homotypic gap junctional complexes with unique physiological properties, an important point when considering the role of epithelial cell connexins in cataractogenesis., (Copyright 1999 Academic Press.)

A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.