Identifying the role of specific motifs in the lens fiber cell specific intermediate filament phakosin

PURPOSE: Phakosin and filensin are lens fiber cell-specific intermediate filament (IF) proteins. Unlike every other cytoplasmic IF protein, they assemble into a beaded filament (BF) rather than an IF. Why the lens fiber cell requires two unique IF proteins and why and how they assemble into a structure other than an IF are unknown. In this report we test specific motifs/domains in phakosin to identify changes that that have adapted phakosin to lens-specific structure and function. METHODS: Phakosin shows the highest level of sequence identity to K18, whose natural assembly partner is K8. We therefore exchanged conserved keratin motifs between phakosin and K18 to determine whether phakosin's divergent motifs could redirect the assembly of chimeric K18 and K8. Modified proteins were bacterially expressed and purified. Assembly competence was assessed by electron microscopy. RESULTS: Substitution of the phakosin helix initiation motif (HIM) into K18 does not alter assembly with K8, establishing that the radical divergence in phakosin HIM is not by itself the mechanism by which IF assembly is redirected to BF assembly. Unexpectedly, K18 bearing phakosin HIM resulted in normal IF assembly, despite the presence of an otherwise disease-causing R-C substitution, and two helix-disrupting glycines. This disproves the widely held belief that mutation of the R is catastrophic to IF assembly. Additional data are presented that suggest normal IF assembly is dependent on sequence-specific interactions between the IF head domain and the HIM. CONCLUSIONS: In the lens fiber cell, two members of the IF family have evolved to produce BFs instead of IFs, a change that presumably adapts the IF to a fiber cell-specific function. The authors establish here that the most striking divergence seen in phakosin is not, as hypothesized, the cause of this altered assembly outcome. The authors further establish that the HIM of IFs is far more tolerant of mutations, such as those that cause some corneal dystrophies and Alexander disease, than previously hypothesized and that normal assembly involves sequence-specific interactions between the head domain and the HIM.     

Characterization of a mutation in the lens-specific CP49 in the 129 strain of mouse

PURPOSE: The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies. METHODS: The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp. RESULTS: The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity. CONCLUSIONS: The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.     

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells

PURPOSE. To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS. Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS. The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS. Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.     

The Mr 115 kd fiber cell-specific protein is a component of the lens cytoskeleton

Electron microscope level immunocytochemistry was used to localize a lens fiber cell-specific protein with an Mr of 115 kd. Affinity-purified polyclonal antibodies were utilized on sections of detergent-extracted, acrylic-embedded lens cortical fiber cells. Monoclonal antibodies were utilized for pre-embeddment labelling of a subcellular fraction of lens fiber cells generated by homogenization, and high-speed centrifugation. The results indicate that the Mr 115 kd antigen is a component of the lens fiber cell cytoskeleton, specifically the beaded filament (BF), a cytoskeletal element thought to be unique to the differentiated lens fiber cell.     

Kinetics of cyclic AMP-dependent accumulation of novel intermediate filament proteins in cultured chick lens cells.

PURPOSE: To refine the parameters affecting the accumulation of cytoskeletal markers of lens fiber terminal differentiation. METHODS: Primary cultures of chick lens annular pad cells were treated with a lipid soluble cyclic AMP analog under various culture conditions. The accumulation of beaded filament proteins, unique markers of lens fiber terminal differentiation, was quantified with an ELISA assay. The incorporation of beaded filament proteins into macromolecular structures was followed with immunofluorescence microscopy. RESULTS: In a time- and dose-dependent manner, beaded filament protein levels were increased in cyclic nucleotide treated cells. The addition of serum to treated cells caused a further dose-dependent increase in beaded filament protein levels. The continuous presence of cyclic nucleotides for maximal beaded filament protein accumulation was also established. At the light microscopic level, cyclic nucleotide treatment produced much more extensive multilayering of cells and lentoid formation. Macromolecular structures containing beaded filament proteins also increased in both abundance and complexity after cyclic nucleotide treatment and were restricted to the multilayers/lentoids. CONCLUSIONS: These results indicate that multiple mechanisms (including cyclic AMP, serum factors, and the degree of cell-cell interactions) affect the accumulation of beaded filament proteins during the normal differentiation of lens fibers.     

The C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and CP49

PURPOSE: Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens. METHODS: AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections. RESULTS: The proteomics approach used to identify affinity-purified proteins revealed the lens-specific intermediate filament proteins filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that filensin and AQP0 were present in the same membrane compartments. CONCLUSIONS: These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization.     

Differentiation of chick lens epithelial cells: involvement of the epidermal growth factor receptor and endogenous ligand

PURPOSE: To characterize the constitutively activated epidermal growth factor receptor in a lens epithelial cell population experiencing initial stages of lens fiber formation, the chick lens annular pad. METHODS: Phosphotyrosine levels of the receptor were examined with western blot analysis and immunoprecipitation after ligand stimulation. Endogenous receptor ligands were immunologically identified in whole cell lysates of freshly isolated cells. The expression of lens fiber-specific differentiation marker proteins was examined with western blot analysis and enzyme-linked immunosorbent assay (ELISA) in short-term primary cultures of annular pad cells exposed to ligand. RESULTS: The major phosphotyrosine-containing protein in annular pad cells comigrated with the epidermal growth factor receptor and increased its phosphotyrosine content after epidermal growth factor treatment. Both time- and dose-dependent responses were noted. The constitutive activation of the receptor was determined in the presence of phosphatase inhibitors. Endogenous transforming growth factor-alpha, but not epidermal growth factor, was detected in freshly isolated cells. Transforming growth factor-alpha (TGF-alpha) treatment produced greater increases in receptor phosphotyrosine levels than equimolar levels of epidermal growth factor. Finally, TGF-alpha treatment induced increased expression of the beaded filament protein filensin when compared with control cells. Filensin expression was increased further when cells were costimulated with TGF-alpha and cAMP analogs. CONCLUSIONS: At least in the postnatal lens, endogenous TGF-alpha may affect overall growth patterns by modulating differentiation-specific protein expression. Furthermore, signaling pathways elicited by TGF-alpha and cAMP analogs converge to cooperatively enhance lens fiber differentiation.     

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality,but does not induce cataract

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 spontaneous mutation affects programmed cell death during development of the rat eye

We have discovered a spontaneous mutation in the Sprague-Dawley rat with a novel eye phenotype that we have named Nuc1. The Nuc1 mutation behaves as a single semi-dominant locus with an intermediate phenotype in the heterozygotes. Heterozygotes exhibit nuclear cataracts. Homozygous Nuc1 rats are fully viable and have microphthalmia, retinal abnormalities and disruption of lens structure shortly before birth. The homozygous mutant shows no obvious pathology outside of the eye, indicating that the mutation is highly eye specific in its effects. An unusual feature of the mutation is that it prevents the normal programmed loss of nuclei from lens fiber cells, but does not affect the loss of other organelles. TUNEL, light, and electron microscopic studies show normal intact nuclei in lens fibers, in contrast to many other models with degenerate nuclei and unlike normal lenses where no such nuclei remain. The beaded filament protein, filensin, is down-regulated in fibers of Nuc1, while heat shock cognate 70 is up-regulated. Homozygous retinas are thicker than normal, and TUNEL labeling indicates roughly half the number of apoptotic cells compared to a wild-type retina. The transient layer of Chievitz persists in adult Nuc1 retina, indicative of delayed development. Hence, Nuc1 is a novel mutation that could be an eye-specific regulator of apoptosis.     

Lens intermediate filaments

The ocular lens assembles two separate intermediate filament systems sequentially with differentiation. Canonical 8-11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific beaded filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed.     

Immunochemical characterization of a Mr 115 lens fiber cell-specific extrinsic membrane protein

Monoclonal and polyclonal antibodies have been produced against a lens fiber cell extrinsic membrane protein, with a relative molecular weight of approximately 115 kd. Enzyme Linked Immunosorbent Assays (ELISA) of retina, ciliary body-iris, liver, and skeletal muscle, utilizing these antibodies, suggest that the antigen is unique to the lens. Immunocytochemistry indicates that the antigen is present only in the differentiated fiber cell, and is absent from the lens epithelium. Further, immunocytochemical reactivity is predominantly associated with the fiber cell plasma membrane. However, sequential extraction of fiber cell homogenate, followed by quantitative, competitive ELISA analysis, indicates that most of the antigen is recovered in the neutral buffer extract. ELISA analysis using monoclonal antibodies indicates that an analogous antigen is present in human and rabbit lenses. On the basis of these results we characterize this antigen as a conserved extrinsic membrane protein, which is unique to the differentiated lens fiber cell. The relationship of this antigen to a previously described Mr 95 beaded filament-associated protein is discussed.     

Immunologic conservation of the fiber cell beaded filament

Lenses were obtained from the eyes of four different classes of Chordates, including Mammalia (rat, mouse, cow, human), Aves (chicken), Amphibia (tiger salamander), and Osteichthyes (steelhead), as well as from one Mollusca (squid). Buffer soluble, urea soluble and urea insoluble fractions were prepared from each, and probed by western blot analysis for the presence of the lens fiber cell 115 and 49 kD beaded filament proteins. Application of both polyclonal and monoclonal antibodies revealed that an immunologic homologue to the bovine fiber cell 115 kD protein is present in all examples of Chordates tested, and that this homologue possessed properties very similar to those of its bovine counterpart. Both monoclonal and polyclonal antibodies revealed an immunologically cross-reactive homologue in squid as well, but suggested that the squid protein had a native molecular weight of closer to 70-80 kD. A monoclonal antibody to the bovine 49 kD beaded filament protein was successful at identifying an immunologic homologue to this protein in mouse, chicken, and tiger salamander. Ultrastructural analysis of rat, human, and fish lenses showed that a beaded filament was present in these lenses, which was indistinguishable from that seen in the bovine lens. In the squid a filamentous, beaded structure was observed, but it differed from that seen in the bovine lens. We conclude from the data presented that the beaded filament, and its constituent proteins, are well-conserved. This data should facilitate the identification of lens cytoskeletal proteins and structure in a wide range of animal models, and establish that probes for these proteins may be of broad applicability.     

Unexpected variation in unique features of the lens-specific type I cytokeratin CP49.

PURPOSE: CP49 is a fiber cell-specific type I cytokeratin, but its function as part of the fiber cell-beaded filament remains unknown. To provide a rational basis for mutational studies that would contribute to an elucidation of function, the study was designed to define elements of CP49s that are highly conserved, discriminate conserved features from species-specific variations, and identify where CP49s have diverged from consensus type I features in their adaptation to selective pressures in the lens. METHODS: The primary sequence and gene structure of CP49 from a third vertebrate order was determined from a combination of cDNA and genomic sequencing. Protein product was characterized by SDS-PAGE and Western blot analysis. Consensus features and phylogenetic relationships were identified by multiple alignment. Coiled-coil analysis was conducted to define central rod domains. RESULTS: Trout CP49 is unique among CP49s in having a 39-amino-acid tail domain and shows both unique sequence and allelic variation at the LNDR motif. Comparison of consensus sequences identified unprecedented divergence between CP49s and other type I cytokeratins, including a shortened central rod domain that is conserved among CP49s, but distinct from type I cytokeratins. CONCLUSIONS: The considerable differences that have emerged between the consensus features of the type I cytokeratins and the CP49s suggest that the beaded filament serves a significantly different function from intermediate filaments in other epithelia and that type I cytokeratins may have limited utility as a model for studies on lens beaded filaments. These differences, in concert with consensus features identified among CP49s, suggest sites that are probably critical to CP49 function in the lens fiber cell.     

Differential protein expression in lens epithelial whole-mounts and lens epithelial cell cultures

PURPOSE: Lens fibergenesis is a problem in several types of cataract and in the posterior capsular opacification following cataract surgery. To correct improper fiber differentiation or to prevent unwanted growth on the posterior capsule following cataract surgery requires a thorough understanding of normal and abnormal fiber formation. To this end, studies were initiated to characterize fiber differentiation in the bovine lens and in lens epithelial cell cultures. METHODS: Indirect immunofluorescence and immunoblot analysis were employed to study the expression of vimentin, beta-crystallin, gamma-crystallin, filensin, aquaporin 0 and the Na, K-ATPase catalytic subunit isoforms (alpha1, alpha2, alpha3) in bovine lens epithelium whole-mounts as well as lens epithelial cell cultures propagated in medium containing 10% bovine serum or in medium supplemented with bovine serum concentrations < or =4%. RESULTS: Three distinct cell types were observed in the bovine lens epithelium. The cells of the central zone were identified by a polarized distribution of two distinct Na, K-ATPase catalytic subunit isoforms, alpha1 to the apical (fiber side) and alpha3 to the basal (aqueous humor side) membranes. Lateral to the polarized central zone, was the germinative zone of cells, best characterized by perinuclear vimentin basket-like structures and the loss of polarized Na, K-ATPase catalytic subunit isoforms. Lateral to the germinative zone were the cells of the transition zone (meridinal rows) where expression of the lens specific proteins beta-crystallin, gamma-crystallin, filensin and aquaporin 0 as well as the lens fiber-, adipocyte- and brain glia-specific Na, K-ATPase catalytic subunit, alpha2 are expressed. The cultured cells propagated in medium supplemented with 10% serum bore no resemblance to any of the cells of the bovine lens epithelium whole-mounts. The cells propagated in the medium supplemented with the lower bovine serum levels resembled the differentiating fibers of the transition zone of the bovine lens epithelium whole-mounts as well as superficial cortical fibers. CONCLUSIONS: Since the low-serum lens epithelial cell cultures bear a remarkable resemblance to early differentiating fibers, they are reasonable models for the study of early fiber differentiation or prevention of differentiation. The culture conditions employed do not yield the polarized cells of the central zone. Nor has the function of these polarized cells in lens fluid, nutrient and ion homeostasis been determined.     

Lens actin: purification and localization

Actin was purified from the chick lens using DEAE-52 column chromatography followed by hydroxylapatite chromatography. The antibody produced against the purified actin cross-reacted specifically with lens actin from other species in addition to smooth and skeletal muscle actin and labelled the stress bundles of cultured fibroblasts. Actin was localized, using immunological methods, primarily to the plasma membrane of the epithelial and fiber cells of the chick and human lens. Actin filaments were also identified by HMM S-1 labeling in bovine cortical fiber cells. Using this procedure, the actin filaments were found throughout the fiber cell but were mainly concentrated near the plasma membrane and in cell processes. They formed a population distinct from the beaded filaments. The initial DEAE-52 column chromatography was also useful in the initial purification of lens fiber cell intermediate filament protein and two species of beta-crystallins.