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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.)

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 lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.

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.

Although cataract is a characteristic feature of myotonic dystrophy type 1 (DM1), little is known of the underlying mechanisms. We generated four lens epithelial cell lines derived from DM1 cataracts and two from age-matched, non-DM cataracts. Small-pool PCR revealed typical large triplet repeat expansions in the DM1 cells. Furthermore, real-time PCR analysis showed reduced SIX5 expression and increased expression of the Ca(2+)-activated K(+) channel SK3 in the DM1 cells. These cells also exhibited longer population doubling times which did not arise through reduced proliferation, but rather increased cell death as shown by increased release of lactate dehydrogenase (LDH). Using (86)Rb(+) as a tracer for K(+), we found no difference in the resting K(+) influx or efflux kinetics. In all cases, the ouabain sensitive component of the influx contributed approximately 50% of the total. However, stimulating internal Ca(2+) by exposure to ionomycin not only caused greater stimulation of K(+) ((86)Rb) efflux in the DM1 cells but also induced a higher rate of cell death (LDH assay). Since both the hyper-stimulation of K(+) efflux and cell death were reduced by the highly specific SK inhibitor apamin, we suggest that increased expression of SK3 has a critical role in the increased Ca(2+)-induced fragility in DM1 cells. The present data, therefore, both help explain the lower epithelial cell density previously observed in DM1 cataracts and provide general insights into mechanisms underlying the fragility of other DM1-affected tissues.

Purpose: The expression of the Sigma 1 receptor and the ability of receptor antagonists to inhibit growth and induce pigment formation were investigated in human lens epithelial cells., Methods: Capsular bags were formed for experimental purposes by performing sham cataract operations on donor lenses. The resultant bags were cultured in Eagle's minimum essential medium (EMEM) alone or supplemented with the Sigma receptor antagonists rimcazole (3 microM) and BD1047 (10 microM). Cell growth was monitored by phase microscopy. Tyrosine incorporation was quantified by culturing in the presence of 14-C tyrosine for 24 hours. At the end of the culture period, some bags were fixed in 4% paraformaldehyde for electron microscopy, and others were plunged into liquid nitrogen for later immunoblot and PCR analyses. Protein levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) were quantified by Western blot analysis. The presence of pigment granules within epithelial cells were monitored by phase and electron microscopy techniques., Results: The Sigma-1 receptor was expressed in native human lens cells and in cultured capsular bag cells. The Sigma receptor antagonists BD1047 and rimcazole inhibited lens cell growth and, surprisingly, lens cells accumulated pigment granules in the presence of the antagonists. The antagonists raised preexisting levels of TYR and TYRP1, whereas there was no change in TYRP2., Conclusions: The human lens normally expresses components of the melanin synthesis pathway, and this suggests a possible origin for the pigment granules that have been observed under certain conditions in the human lens. Exposure of lens cells to Sigma receptor antagonists leads to growth inhibition and pigment granule production.

The transcriptional status of cells can be deduced from the staining pattern of various nuclear markers such as the Cajal body, nucleolus and nuclear speckles. In this study we have used these markers to correlate transcriptional status with cell differentiation in the lens. As a closed system with no cell loss and with each stage being spatially preserved, it is particularly well suited to such studies. To confirm that the nuclear markers in lens cells follow the same trends as in other cells, primary bovine lens epithelial cells were cultured and then treated with actinomycin D to inhibit transcription. This reduced the Cajal body markers to one or two foci per nucleus and the nucleoli became compacted as revealed by fibrillarin staining. The nuclear speckles, containing snRNPs (e.g. Sm) and the splicing factor, SC35, also became larger and more numerous while the signal for trimethylguanine (TMG) decreased suggesting a role hierarchy for the various speckle factors during transcriptional shutdown. The signal for survival of motor neurones gene product (SMN) also decreased at this point. In the lens epithelium, postmitotic cells near the equatorial region had one or two Cajal bodies per nucleus, indicating these cells had only basal levels of transcription. Sm was also present as large foci in these cells. Interestingly, both the speckles and Cajal bodies were NuMA-positive in these post-mitotic cells. At the epithelial-fibre cell transition, Cajal body number increased, while their size decreased indicative of increased transcriptional activity. Fibrillarin adopted the open floret pattern indicating increased transcriptional activity. The nuclear speckles adopted a more diffuse nucleoplasmic pattern, although some spots were still observed. All NuMA colocalisation with the Cajal bodies and nuclear speckles was lost at this stage of lens cell differentiation. Transcriptional shutdown occurs at a later stage in fibre cell differentiation, prior to programmed nuclear destruction. In the lens, both the Cajal bodies and nuclear speckles again became NuMA-positive, although separate NuMA spots were also formed during transcriptional shutdown. These data suggest the nuclear matrix is important in the concentration of Cajal body and speckle components into large, distinct spots in transcriptionally inactive nuclei and also suggest a new role for NuMA in post-mitotic cells to assist in these sub-nuclear reorganisations.

Discusses the use of fluorescently tagged keratin from a transfected enhanced green fluorescent protein construct to observe keratin intermediate filament networks in living cultured epithelial cells. Details on the inhibitory effect of nocodazole on keratin particle movement; Detection of particulate keratin structures in untransferred epithelial cells; Analysis of th substructure of cells.

Cell-to-cell contact and polarisation of epithelial cells involve a major reorganisation of the microtubules and centrosomal components. The radial microtubule organisation is lost and an apico-basal array develops that is no longer anchored at the centrosome. This involves not only the relocation of microtubules but also of centrosomal anchoring proteins to apical non-centrosomal sites. The relocation of microtubule minus-end-anchoring proteins such as ninein to the apical sites is likely to be essential for the assembly and stabilisation of the apico-basal arrays in polarised epithelial cells. In this study, we establish that ninein is highly dynamic and that, in epithelial cells, it is present not only at the centrosome but also in the cytoplasm as distinct speckles. Live-cell imaging reveals that GFP-ninein speckles are released from the centrosome and move in a microtubule-dependent manner within the cytoplasm and thus establishes that epithelial cells possess the mechanical means for relocation of ninein to non-centrosomal anchoring sites. We also provide evidence for the deployment of ninein speckles to apical anchoring sites during epithelial differentiation in both an in situ tissue and an in vitro culture system. In addition, the findings suggest that the non-centrosomal microtubule anchoring sites associate with adherens junctions in polarised epithelial cells. [ABSTRACT FROM AUTHOR]