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(Investigative Ophthalmology and Visual Science. 2003;44:5252-5258.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0224
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Targeted Deletion of the Lens Fiber Cell–Specific Intermediate Filament Protein Filensin

Azita Alizadeh,1 John Clark,2 Teri Seeberger,2 John Hess,1 Tom Blankenship,1 and Paul G. FitzGerald1

1From the Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, California; and the 2Department of Biological Structure, University of Washington, Seattle, Washington.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the function of the lens fiber cell–specific cytoskeletal protein, filensin, in lens biology.

METHODS. Targeted genomic deletion was used to delete exon 1 and the transcriptional start site of the filensin gene. Resultant chimeric animals were bred to homozygosity for the mutant allele. These animals were outbred to mice bearing the wild-type CP49 alleles to eliminate the mutant CP49 gene carried by the 129 strain of mice. Animals homozygous for the mutated filensin gene and wild-type CP49 gene were compared with wild-type and heterozygous animals by Northern and Western blot analyses, light and electron microscopy, and slit lamp microscopy.

RESULTS. Disruption of the filensin gene successfully blocked production of filensin mRNA, reduced levels of filensin’s assembly partner CP49, and prevented the assembly of beaded filaments. Despite the absence of beaded filaments, lenses did not show obvious changes in fetal development, nor in the differentiation of epithelial cells into mature fiber cells, as judged by light microscopic analysis. Filensin knockouts began to show evidence of light-scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a reduction in filensin transcript and moderate light-scattering at 5 months.

CONCLUSIONS. The lens fiber cell–specific intermediate filament protein filensin is essential for beaded filament assembly. However, although beaded filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of filensin and the beaded filament affects optical clarity has yet to be defined.

Lens fiber cells express two fiber cell–specific proteins, CP49 and filensin, both of which are unusually divergent members of the intermediate filament (IF) family of proteins.1 2 3 4 5 6 Both proteins have been localized to a cytoskeletal structure referred to as the beaded filament.7 8 9 10 These proteins are expressed shortly after differentiation is initiated, but do not accumulate to maximum levels until the fiber cell has fully elongated.11

The role of the beaded filament in lens biology has not been defined, but two lines of evidence suggest that the beaded filament is a requirement for sustained optical clarity: (1) mutations in human CP49 have been implicated in two separate families who have autosomal dominant inherited cataract. These individuals are born with clear lenses, but opacities develop when they are children or young adults12 13 ; and (2) targeted deletion of CP49 expression in mice results in a subtle opacification that worsens over time.14 15 It is probably critical to note that in both cases the opacification is not evident at birth but progresses in severity with age.

More than 50 different IF proteins have been defined, resulting in a very large body of literature documenting the impact of mutations and deletions of these proteins. Although the details of the phenotypes vary in each case, there is a general trend in the literature that suggests that IF proteins commonly provide structural support and durability to the differentiated phenotype.16 17 18 19 Thus, the observations that beaded filament perturbation results in a phenotype that worsens with age is consistent with a role for beaded filaments in stabilizing the phenotype and enhancing resistance to accumulated stresses.

We sought to define the mechanism by which the beaded filament contributes to the lens’ ability to maintain optical clarity during aging. Toward this end, we created a loss-of-function mutant in which the expression of filensin was negated.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Use
Animal use protocols were approved by the University of California, Davis, (UCD) campus veterinarian and animal use committee and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Filensin Targeting Vector and Generation of Knockout Mice
Targeted inactivation of the mouse filensin gene was performed by standard methods using electroporation of embryonic stem (ES) cells, selection of positive recombinants, and generation of mice by fusion of the ES cells with mouse blastulas and implantation into female mice.

A bacterial artificial chromosome (BAC) clone containing a large region of the mouse filensin gene isolated from mouse 129/SVJ library was purchased from Incyte Genomics (St. Louis, MO). From this clone, several restriction fragments from the promoter, exon 1 and exon 2 regions, were subcloned into plasmid vectors. From these clones, a 5-kb BamHI fragment containing most of intron 1, exon 2, and a portion of intron 2 was isolated and subcloned into the BamHI site of plasmid pKO scrambler NTKV-1901 (Stratagene, La Jolla, CA). Into this construct, a Klenow blunt-ended, 2-kb EclI-StuI fragment was subcloned into the HpaI site. The resultant plasmid contained a 2-kb small arm of homology to the mouse filensin promoter region and a 5-kb large arm of homology to intron 1, exon 2, and a small region of intron 2. Replacement of the endogenous filensin sequences with sequences in the targeting vector by homologous recombination was designed to delete the endogenous filensin exon 1, the transcription initiation site, and several potential promoter elements.20

This plasmid was linearized with NotI, and 25 µg was electroporated into 4 x 107 GK129 embryonic stem cells derived from 129/OLa mice.21 Cells were selected by double selection with the neomycin analogue G418 and the nucleoside analogue 2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl-5-iodouracil (FIAU).22 Approximately 470 resistant clones were chosen and grown and the DNA prepared.

Genomic DNA from the resistant ES clones was screened for homologous recombination between the targeting vector and endogenous filensin sequences by PCR. A homologous recombination event would bring neo coding sequences into the filensin gene, eliminating exon 1 and the transcriptional start signals. A pair of PCR primers were designed so that only a homologous recombination event would produce an amplified product. Primer 1 (PKO m filensin KO down 2 (5' CTT GTC ATG GTC TGA GTG AGC CAT TCC A) anneals to sequences 60 bases upstream from the small arm of homology. Primer 2 (PKO 2100 up (3' GAG CTA GAG GTA CCC TAG AAA GCT TCC) anneals to sequences within the targeting vector, at the 3' end of the neo cassette. Although the filensin promoter region is difficult to amplify, using a commercial PCR system (Expand High Fidelity PCR; Roche Diagnostics, Indianapolis, IN), we were able to develop conditions that reproducibly and vigorously amplified this region. As designed, a homologous recombination event would be detected by production of a ~2.3-kb PCR product. Genomic DNA from five 96-well plates was screened and six positives were identified. These six positives were screened with a second PCR reaction (m115 2.6 R1 T7 out: 3' GAA AGC AGT TGC TGT CCG AAA GCA CC) and (m115 I2 dn: 5' GAA CGG CTT AAC AAG GTG AGC AG) to confirm that the homologous recombination event occurred with the large arm of homology. One positive clone was found to have an abnormal structure at the 3' end of the large arm of homology and was not considered further.

Five positive ES cells identified were expanded and reconfirmed by PCR, and three of the clones (4C2, 2G1, and 2C1) were used by the UCD Targeted Genomics Laboratory for production of chimeras. High-percentage agouti male chimeric mice were bred to female C57/B6 mice. F1 offspring from chimeric x C57/B6 parents were screened by PCR for the CP115 exon 1 sequence using the primers m filensin amino acids 12–19 up, 3' CTG GGC GCG CTC GTA GCG CTC CTG, and m filensin promoter 2050, 5' CGG AAC AAA GAG GTC CTT GCC CGA TG, and for the neomycin gene using the primers PKO neo gene, up, 801, 3' TGC CCT GAA TGA ACT GCA GGA CGA, and PKO neo gene, dn, 287, 5' GCC GCC AAG CTC TTC AGC AAT ATC.

Primers were developed to permit discrimination between wild-type and mutant CP49129 alleles. Filensin F1 mice were screened for the presence of a C57/B6 CP49 gene (CP49B6 allele), and outbred to eliminate the mutant CP49 allele.

Northern Blot Analysis
Total RNA was isolated from 4-week-old litters from wild-type, heterozygous, and knockout mice, using a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture.23 Total RNA was quantified by spectroscopy. Approximately 10 µg of total RNA was electrophoresed in formaldehyde-agarose gel and transferred to nylon membrane (Immobilon-Ny+; Millipore, Bedford, MA) according to standard procedures.24 After UV cross-linking, the blots were probed with 32P-labeled cDNAs for CP49 (full-length cDNA probe), or filensin (partial cDNA probe consisting of 500 bp from the filensin rod domain), washed, exposed, and developed according to standard procedures. Each sample was run in duplicate. Signal density was determined with a phosphorescence imager (Storm Phosphorimager and ImageQuant software; Amersham Biosciences, Sunnyvale, CA). The values determined for the {gamma}S signal in the wild-type, heterozygous, and knockout lanes were used to normalize the signals for the filensin and CP49 blots.

SDS-PAGE and Western Blot Analysis
Whole lenses were decapsulated and solubilized in SDS-PAGE sample buffer containing 2% SDS, 5 mM ß-mercaptoethanol, and 5% glycerol in 50 mM Tris (pH 8.0). Samples were electrophoresed on 12.5% polyacrylamide gels and either stained with Coomassie blue or transferred electrophoretically to nylon membrane (Immobilon P; Millipore) for Western blot analysis. After the reaction was blocked in 5% normal goat serum, 2% powdered milk, and 0.1% Tween 20 in Tris-buffered saline for 20 minutes, samples were probed with antisera raised against either recombinant human filensin or mouse CP49, diluted 1:1000 in blocker. Visualization was achieved by second-stage labeling with goat anti-rabbit antibody conjugated to alkaline phosphatase, developed with 5-bromo-4-chloro-3-indoyl phosphate (BCIP). Quantitation was conducted on computer (ImageQuant; Amersham Biosciences), using an average derived from three lanes per data point.

Light Microscopy
Whole eyes were removed and an aperture cut through the sclera at the site of the optic nerve head. Eyes were immersed in fixative (2% formaldehyde, 2.5% glutaraldehyde in phosphate buffer), at 37°C for a minimum of 4 hours followed by room temperature overnight. Tissues were dehydrated through immersion in progressively increasing concentrations of ethanol, and equilibrated in 100% ethanol before immersion in glycol methacrylate (Technovit 7100; Heraeus Kulzer, Wehrheim, Germany). Subsequent embedding followed the manufacturer’s directions, except that infiltration times were extended. Sections 1 to 2 µm thick were cut and stained with either toluidine blue or DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate).25

In Situ Examination of Lenses
Unanesthetized mice were examined with a slit lamp ophthalmoscope (FS-2; Nikon, Tokyo, Japan). Mouse eyes were dilated with a 1:1 mixture of 1% tropicamide (Alcon, Fort Worth, TX) and 10% phenylephrine hydrochloride (Akorn, Abita Springs, LA). The angle of the slit lamp was approximately 40, and the slit width was approximately 0.2 mm. Examinations were recorded by digital video (Optura Pi; Canon, Tokyo, Japan). Still images were captured (Premiere; San Diego, CA) and processed (Photoshop; Adobe).

Electron Microscopy and Immunocytochemistry
Enucleated eyes were immersed in optimal cutting temperature (OCT) compound and frozen on dry ice. Sections 150 µm thick were cut on a cryostat (Leica, Deerfield, IL, in the "Trim" mode) and immediately placed in 25 mL of phosphate-buffered saline, with 5 mM EDTA containing a commercial anti-protease preparation (Complete Mini; Roche, Indianapolis, IN). Samples were very gently agitated on a rotary shaker, set at approximately 10 rpm. Buffer was replaced after 30 minutes. After 1 hour of total incubation, the supernatant was removed, replaced with PBS, and fixed as described earlier. To confirm the identity of beaded filaments, the wild-type lens slices were incubated in antibody to rabbit anti-filensin, diluted 1:250 in blocker (5% normal goat serum in Tris-buffered saline-Tween). After 2 hours, the medium was replaced five times with blocker over the course of 1 hour and incubated in goat anti-rabbit colloidal gold (BioCell, Rancho Dominquez, CA) for 1 hour. Slices were washed as just described. All samples were rinsed in PBS and immersed in PBS containing 1% tannic acid in PBS. After extensive washing with water, the samples were immersed in 1% osmium tetroxide for 1 hour, washed in water, and immersed in 1% uranyl acetate in water for 1 hour. Samples were dehydrated through 100% ethanol and then propylene oxide and embedded in mounting medium (PolyBed 812; Polysciences, Warrington, PA) for thin sectioning.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Gene Targeting
The structure of the wild-type and disrupted filensin alleles, the location of PCR probes used for screening stem cells and for characterizing the resulting mice, and the PCR analyses are presented in Figure 1 . The targeting vector was designed to replace promoter elements, the transcriptional start site, and exon 1. Quinlan et al., reported that the 129 strain of mice carry a mutation in the CP49 gene (Quinlan R, et al. IOVS 2001;44:ARVO Abstract 4695). We have characterized that mutation and defined PCR primers to permit its identification by PCR.26 To eliminate the 129 CP49 mutation from the filensin knockout line, we bred the filensin knockout mice against littermates bearing the wild-type CP49 alleles, ultimately obtaining mice that were homozygous for both the disrupted filensin allele and the wild-type CP49 allele. We established identity between the C57BL6 CP49 sequence and the Swiss Webster sequence, as previously described.5 All data reported herein were derived from animals bearing this wild-type CP49 genotype.



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  		FIGURE 1. (a) The structures of the wild-type filensin allele, the targeting vector, and the mutant filensin allele are shown. Arrows: Location of PCR primers used in screening ES cells and resultant animals. (b) Ethidium bromide–stained agarose gel showing PCR results from the screening of wild-type (lanes 2, 5), heterozygous (lanes 3, 6), and knockout (lanes 4, 7) animals. Molecular weight standards are shown (lanes 1, 8).

 
To establish that gene targeting effectively eliminates transcription of the filensin mRNA, Northern blot analyses were performed on lens RNA from both wild-type and filensin knockout animals. The results, presented in Figure 2a , show that the filensin transcript was not detectable in animals that were homozygous for the disrupted allele. Animals heterozygous for the disrupted allele showed 68% of the wild-type levels of filensin mRNA. Northern blot analysis was also used to determine whether the disruption of filensin transcription alters transcription of the CP49 gene. A comparison of the filensin knockout, heterozygous, and wild-type animals is shown in Figure 2b . Levels of CP49 transcript were elevated slightly in the heterozygote, and to 153% of wild-type levels in the knockout.



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  		FIGURE 2. Northern blot analysis (top) of RNA isolated from filensin wild-type (lane 1), heterozygous (lane 2), and knockout (lane 3) lenses. {gamma}S-crystallin was used as an internal control for mRNA levels. Filensin was clearly absent from the knockout lens (lane 3). Densitometry was used to assess {gamma}S signal in each of the genotypes and the data were then used to normalize densitometry data from the filensin and CP49 scans (bottom). Signal is therefore presented as a percentage of wild-type, normalized for RNA loading, as determined by densitometry of the {gamma}S signals for that genotype.

 
Western Blot Analysis
Western blot analysis was used to confirm the absence of filensin in animals homozygous for the mutated filensin allele and to assess the levels of filensin’s assembly partner CP49. Figure 3 compares nine littermates from a heterozygote–heterozygote breeding. Genotypes were established by PCR and are indicated at the top of the figure. Lanes 2, 5, and 8 are homozygous null (-/-) animals. No obvious differences were seen in the total protein profiles when the wild-type, heterozygous, and knockout animals were compared (Fig. 3 , top, Coomassie blue–stained gel). However, the levels of filensin, CP49, and vimentin were too low to be identified in total lens preparations such as these. The absence of filensin was clearly evident in Western blot analysis (Fig. 3b , lanes 2, 5, and 8). Densitometry analysis of the Western blots suggests that filensin levels in the heterozygote were slightly less than 80% of the wild-type levels.



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  		FIGURE 3. SDS-PAGE, Western blot analysis, and densitometry of an example of a litter of nine animals resulting from heterozygote–heterozygote breeding. PCR was used to determine the genotype for each animal. (a) Coomassie blue–stained gel showing total lens proteins for each animal. No apparent differences were seen in the total protein profile of wild-type, heterozygous, and knockout lenses, because filensin, CP49, and vimentin were not evident in total lens gel profiles. (b–d) Western blots with (b) anti-filensin, (c) anti-CP49, and (d) anti-vimentin. (b) Filensin was not detectable in the lenses from knockout animals and appeared to be slightly reduced in the lenses from heterozygotes. (c) CP49 levels were reduced in the filensin knockout lenses. Also, the CP49 breakdown patterns were notably different between the knockout and the other two genotypes. (d) Vimentin levels were unchanged by targeted deletion of filensin. (e) Western blots were analyzed by densitometry to assess the extent of protein changes. Three lanes for each genotype were averaged and normalized to the wild-type signal. For CP49, two different calculations were made: one that compared just the parent CP49 and a second that compared the parent molecule plus the labeled breakdown products. For vimentin, only signal from the parent molecule was assessed.

 
In the Western blot probed with anti-CP49, it was evident in the knockout animals that the absence of filensin resulted in reduction of the parent CP49 molecule (Fig. 3c , lanes 2, 5, and 8). In heterozygous animals (lanes 3, 4, and 9), there was a dramatic loss of the CP49 breakdown products. Densitometry conducted on several litters confirmed the reduction in levels of the parent molecule in the knockout to approximately 60% of wild-type levels, a somewhat surprising result in the face of increased CP49 mRNA in the knockouts. Heterozygotes showed a slight reduction in CP49 levels. If the CP49 breakdown products are included in the calculation for CP49, the difference between the heterozygote and knockout increases sharply. Identical samples were also probed for vimentin (Fig. 3d) , a type III IF protein also expressed in lens epithelium and differentiating fiber cells. Comparison of vimentin levels in the three genotypes showed only minor differences, suggesting that absence of filensin did not affect vimentin levels to any significant degree.

In Situ Examination of Lenses
Slit lamp microscopy was performed on the lenses of living homozygous null, heterozygous, and wild-type animals (Fig. 4) . Eleven animals were examined, ranging in age from 10 weeks to 6 months. Subtle opacification of the lenses of homozygous null animals appeared at approximately 10 weeks of age and worsened progressively in older animals. Six-month-old homozygous null animals had a cataract of approximately stage 3 on a scale of 1 to 6. Lenses of wild-type animals were clear. Heterozygous lenses showed an intermediate phenotype with slight opacification in the older animals.



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  		FIGURE 4. Slit lamp views of living wild-type (a, 5.2 months), heterozygous (b, 5.3 months), and homozygous null (c, 5.8 months) 5- to 6-month-old CP115 null mice. In the slit lamp view, the thin bright line on the right (anterior) is caused by light-scattering from the cornea. The dark band to the left (posterior) of the cornea is the aqueous chamber. The lens capsule and epithelium appear as the next thin light band. In wild-type animals, the remainder of the lens is clear (a). CP115 null mice show layered rings of opacity (c), with the greatest scattering in the nucleus of the lens. Heterozygous animals have much less opacity (b) than the homozygous null animals.

 
Histology
To determine whether the absence of beaded filaments results in changes in the fetal development and normal differentiation of the lens fiber cells, histologic examination was conducted on embryonic and postnatal lenses. A comparison of filensin knockout and wild-type lenses is shown in Figure 5 . Some sections were stained conventionally with toluidine blue (Figs. 5a 5b) . These revealed no obvious differences in the lenses between wild-type and knockout lenses, using conventional staining. To obtain a clearer view of cell boundaries, similar sections were stained with DiI, which labels membranes with a fluorochrome (Fig. 5c 5d) . Again, there appeared to be no differences between the wild-type and knockout lenses in the lens epithelium and in the differentiating fiber cells. Thus, the process of fiber cell differentiation appeared to unfold normally, even in the absence of beaded filaments, at least as judged by light microscopy.



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  		FIGURE 5. Glycol methacrylate sections of wild-type (a, c) and knockout (b, d) lenses, stained with toluidine blue (a, b) and DiI (c, d). Overall lens shape, size, and distribution of fiber cell nuclei were indistinguishable between the wild-type and knockout lenses, although differences were seen, because of the plane of the sections. Bar, 30 µm.

 
In the deeper cortical and nuclear fibers, however, DiI staining revealed more irregularity in fiber cell shape and packing. This may have been greater in the knockout lenses, a finding that is reasonable to expect. However, we note that there was much variability in this finding and that wild-type lenses often showed the same loss of organization and cell shape as occurred in the knockouts. Because the penetration of fixative into the lens interior is very slow, taking hours to days depending on the size of the lens, we are concerned that artifactual changes in the inner fiber cells may contribute to this variability, a variability compounded by the dramatic impact that subtle changes in the plane of section can make in the appearance of these cells and by the different sizes of lenses within litters. Until the variable of fixation time can be eliminated, we are reluctant to reach conclusions on possible changes in the architecture of the inner lens. These efforts are in progress.

Electron Microscopy
To explore how the absence of filensin impacted the beaded filament, we examined extracted, thick, frozen sections by electron microscopy (Fig. 6) . This approach reduces crystallin levels at the surface of the cut section, so that the underlying cytoskeleton can be visualized. Beaded filaments were structurally evident (Fig. 6a , inset, arrow), and their identification was confirmed by immunogold labeling with antibodies to filensin (Fig. 6a , inset). It is worth noting that residual crystallins were cross-linked by fixation to any remaining surface, such as plasma membranes or 10-nm IFs. The latter is particularly troublesome, as it creates the impression of a filament that is beaded. Labeled 10-nm IFs can be distinguished structurally on the basis of a thicker and more rigid core filament, but also by immunochemical methods.



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  		FIGURE 6. Frozen, extracted sections of wild-type (a) and knockout (b) lenses. (a) The abundance of beaded filaments is evident in the lower magnification view of the wild-type lenses, seen as filamentous material spanning the fiber cell cytoplasm. Inset, arrowhead: specific examples of beaded filaments at higher magnification, confirmed by immunogold labeling with anti-filensin antiserum. (b) Knockout lenses show a dramatically reduced presence of cytoplasmic filaments, as evidenced in the lower magnification overview of a region of lens comparable to that in (a). Inset, arrowhead: higher magnification view, revealing the presence of occasional 10-nm IFs. Scale bar: (a, b) 2 µm; (a, b, insets) 0.15 µm.

 
Figure 6b shows a lower magnification overview of extracted, frozen, thick sections of filensin knockout lenses. Although 10-nm IFs were evident (Fig. 6b , inset), beaded filaments were not identifiable, confirming the essential role of filensin in the architecture of beaded filaments.

A comparison of the fiber cells in the wild-type (Fig. 6a) and knockout (6b) lenses showed a much greater degree of irregularity in the shape of the knockout fiber cells. This is an artifact associated with the mechanical transfer of these thin, structurally flimsy sections. A similar degree of irregularity was generated in wild-type lenses, and very regular shape and packing were seen in knockout lenses. Images portraying the degree of variability were intentionally selected to show the variation that occurs and to caution against overinterpretation of such images.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Filensin and CP49, two members of the large IF family of proteins, are expressed only in the differentiating lens fiber cells. Both proteins are highly divergent from the other 50 or so members of this family in both primary sequence and predicted secondary structure.4 5 6 27 28 29 These proteins also appear to be unique among IFs, in that they are localized to the beaded filament and not to 10-nm IFs. However, CP49 and filensin have been shown in vitro to assemble into 10 nm IFs.30 31 Presumably, these variations from the rest of the IF family serve some function unique to the fiber cell, but that function has not yet been defined.

The data presented herein establish that neither filensin nor the beaded filament is required for normal lens development or for normal fiber cell differentiation, as judged by light microscopy. In situ examination of filensin knockout lenses showed a slight degree of light-scatter at 10 weeks that worsened with age. Similar results were seen in the CP49 knockout,14 suggesting that the beaded filament may not be necessary to achieve clarity but is certainly essential to maintain it. These results parallel findings in humans that suggest that mutations in human CP49 may cause inherited autosomal dominant cataract, but one that occurs several years after birth.12 13 Collectively, these findings suggest that the beaded filament may confer on the lens a capacity for increased resistance to changes that result in opacification, suggesting that such knockouts constitute a valuable model for the study of age-dependent opacification.

The absence of CP49 (FitzGerald P, unpublished data, 2003) and filensin each result in the absence of beaded filaments, suggesting that the resultant phenotypes should be identical. However, there appear to be subtle differences in these phenotypes. The degree of light-scattering in the filensin knockouts appeared to be greater, and at an earlier age, than in the CP49 knockouts. Also, the filensin heterozygotes showed a slight increase in light scatter. What accounts for such differences is unclear, but they could be related to absolute levels of unmated CP49 or filensin, or differences in the degree of light scatter caused by these insoluble proteins. Alternatively, filensin may interact with more than one molecule in the fiber cell cytoplasm. Thus, the absence of filensin may disrupt its association with CP49 and also with other processes by which the beaded filament is integrated into the fiber cell cytoplasmic function. We speculate also that the absence of a partner protein may account for the differences in the pattern of breakdown products seen in the CP49 Western blot, arising from different mechanisms of protein processing. Although intact beaded filaments appear to be degraded by calcium-activated proteases, it is reasonable to speculate that unpartnered, insoluble CP49 may be recognized as a misfolded protein and targeted to the proteasome pathway.

It was clear from electron microscopy that even in the presence of significant levels of CP49, beaded filaments were not present in the fiber cells of filensin knockout animals. This confirms the essential role of filensin in the assembly of the beaded filament, a result that has not been obtained before.

Flavoring all speculation on beaded filament function is the fact that the beaded filaments, and indeed essentially all other cytoskeletal proteins, are largely degraded in most cells of the adult lens inner cortex and nucleus.8 11 32 33 34 35 36 37 Thus, because opacification appears in these animals with age, it might reasonably be predicted that changes would be found in lens nuclear cells. However, these proteins were identified in intact form only in the younger cells, nearer the lens surface, suggesting that they may function only in the limited population of younger cells. Alternatively, what we view as degradation may represent further posttranslational processing into molecules that remain functional contributors to lens biology. This remains to be established.

Exactly how the beaded filament contributes to the maintenance of optical clarity remains undefined. The IF literature suggests that IFs contribute to the stabilization of the differentiated phenotype, conferring added capacity to resist mechanical stresses. In light of the exceptional degree of structural differentiation in the fiber cell, such a hypothesis seems reasonable, and predicts a change or alteration in fiber cell structure. In the present study the normal fiber cell differentiation appeared to proceed routinely, evidenced by normal fiber cell shape and elongation in histologic sections.

Whether changes occur in older fiber cells of the inner cortex and nucleus is technically more difficult to establish with any confidence. Fixatives penetrate very slowly into lenses, and in large lenses can take days. The time frame for adequate fixation of the interior of mouse lenses has not been established in a controlled manner, and much caution must therefore be used in interpreting the structure of the lens interior. That the gradient of fixation in lenses is the same as the gradient of fiber cell age is of particular concern when deciding whether to attribute changes in fiber cells to decreased resistance to aging or to other time-dependent factors: Are the differences due to age of the cell or to delayed fixation? Indeed, histologic differences between wild-type and knockout lenses may not be real differences, but differences that emerge after death because of differences in their susceptibility to the insult of delayed fixation. Thus, there is sufficient doubt about the status of these inner cells at the time of fixation to warrant some caution in the interpretation of results.


   Footnotes
 
Supported by Grants R01 EY08747 (PGF), P30 EY12576 (University of California, Davis), and R01 EY04542 (JC) from the National Eye Institute and the Office of Research, School of Medicine, University of California, Davis.

Submitted for publication March 4, 2003; revised July 1, 2003; accepted July 30, 2003.

Disclosure: A. Alizadeh, None; J. Clark, None; T. Seeberger, None; J. Hess, None; T. Blankenship, None; P.G. FitzGerald, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Paul G. FitzGerald, Department of Cell Biology and Human Anatomy, School of Medicine, University of California, 1 Shields Avenue, Davis, CA 95616; pgfitzgerald{at}ucdavis.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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