CP49 and filensin intermediate filaments are essential for formation of cold cataract

Mice and formation of cold cataract

Mouse care and breeding were performed according to the Animal Care and Use Committee approved animal protocol (University of California Berkeley, Berkeley, CA) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three mouse strains, C57BL/ 6J wild-type, 129 wild-type [12], and filensin gene knockout [15] on the C57BL/6J background, were used. Enucleated eyeballs were immediately collected from mice euthanized by CO₂ inhalation and were dissected in 4 °C cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) to prepare the 4 °C treated lens samples or in PBS at room temperature to prepare the 25 °C and 37 °C treated lens samples. The dissected lenses were immediately transferred into fresh PBS at 4 °C, 25 °C, and 37 °C, respectively, for imaging under a Leica MZ16 (Leica, Wetzlar, Germany) dissecting microscope with a digital camera. The whole procedure including dissecting and imaging took about 10–15 min. A transparent lens developed a full cold cataract within less than 3 min after immersion in 4 °C PBS, and a cold cataract disappeared within less than 3 min after immersion in 37 °C PBS. We also made a small-interval temperature controller to sequentially change temperatures from 4 °C to 25 °C by using the thermoelectric cooler Peltier 12V CL-C067 (Heke Tech, Shenzhen, China) with a plate (40 × 40 × 3.87 mm) equipped with copper tubes for the circulating solution and a fan to dissipate heat in air. This temperature controller utilizes a feedback signal that forms a closed-loop control refrigeration system to detect, change, or maintain the temperature of the PBS in the plate.

Statistical analysis

Statistical significance was determined with one-way analysis of variance (ANOVA) with Tukey’s post-hoc test, using GraphPad Prism software (San Diego, CA). P values less than or equal to 0.01, 0.001, and 0.0001 were considered statistically significant.

Lens vibratome section and immunohistochemistry

For the examination of 4 °C induced cold cataract, fresh lenses were dissected in 4 °C PBS and fixed in 4% paraformaldehyde/PBS solution at 4 °C for overnight. For the 25 °C or 37 °C treated lens samples, fresh lenses were dissected in PBS at room temperature and then transferred into 25 °C or 37 °C 4% paraformaldehyde/PBS solution for 3–5 h fixation. Fixed lenses were washed with PBS three times before being embedded in 4% low-melting agarose gel and then were superglued to an aluminum mounting block as in the following procedures: A drop of 4% low melted agarose (about 35 µl) was added on the surface of the aluminum mounting block. A wedge was cut in the solidified agarose gel to hold a lens, which was oriented for cutting either anterior-posterior (A/P; along the optical axis) or cross-sectional (along the lens equator) planes. Then 0.4 µl of superglue was added to the wedge (not attached to the lens) to glue the agarose gel with the oriented lens on the aluminum mounting block. The entire lens was further covered with agarose. After the agarose gel solidified, the block with the agarose embedded lens was mounted on a Leica VT1000 S Vibratome, and lens sections with 100 μm thickness were cut. Lens sections around the lens equator were collected for immunostaining. The lens vibratome sections were first incubated with a blocking solution containing 3% wt/vol bovine serum albumin (BSA; Gemini Bio-products, West Sacramento, CA), 3% vol/vol normal goat serum (Vector Laboratories, Inc., Burlingame, CA), and 0.3% vol/vol Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 2 h, followed by incubation with rabbit polyclonal antibodies against CP49 or filensin (generated in Paul Fitzgerald’s laboratory at the University of California at Davis) at 4 °C for overnight. After washing three times in PBS, the sections were incubated in rhodamine-labeled secondary antibodies and fluorescein (FITC)-phalloidin (Vector Laboratories, Inc.) for 2 h at room temperature. Then the lens sections were washed three times in PBS and were mounted in Vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Fluorescent images were collected using a Zeiss LSM 700 confocal microscope. ImageJ software was used to quantify the intensity and distribution of the immunostained CP49 and filensin protein signals in different areas in regions in lens zones I, II, and III. The signal intensity ratios between the cellular boundary and cytosol were analyzed in selected areas in the three zones. Immunostaining analysis for each antibody was performed in lens vibratome sections from at least three different mice including female and male mice for each genotype.

Immunostaining of single lens fiber cells

Fixed lenses with capsule were cracked open from the lens center along the A/P axis to produce three or four pieces of lens mass by using forceps with a fine sharp tip (Student Dumont #5 Forceps, Fine Science Tools, Foster City, CA). The lens pieces were fixed in a drop of 4% paraformaldehyde/PBS solution for 5 min and then washed with PBS drops three times under the dissecting microscope. Lens pieces from the lens cortex to the lens core were used for whole mount staining with rabbit polyclonal anti-filensin antibody on parafilm paper in a small humidified chamber. The same immunostaining procedure for the lens vibratome sections was used. To maximize the dissociation of single lens fibers, fine forceps were used to tease apart the lens pieces into small bundles before they were mounted in Vectashield mounting medium with DAPI. Low-magnification images of whole mount lens pieces were used to determine the location of single fibers from the lens surface, and high-magnification images of single fibers that partly detached from the surface of bundled fibers in different zones were collected under a Zeiss LSM 700 confocal microscope. Filensin protein staining images of single fibers in representative regions of zones I, II, and III were analyzed for the intensity plot of the single fiber cell surface by using ImageJ software.

Differences in formation of cold cataract in B6 wild-type, 129 wild-type, and B6 filensin KO mouse lenses

We examined transparency or opacity in enucleated fresh lenses from P14 B6 wild-type (B6WT), 129 wild-type (129WT), and B6 filensin KO mice at three representative temperatures, 37 °C, 25 °C, and 4 °C (Figure 1). The B6WT lenses, immersed in cold PBS in vitro, showed typical cold cataracts, as opacity occurred in the lens nucleus at 25 °C, and larger opacity that expanded toward the cortex appeared at 4 °C, while the lens periphery remained clear (Figure 1A). Unexpectedly, the 129WT and B6 filensin KO lenses had no opacity at 25 °C but showed similar opacity with reduced light scattering in the lens core at 4 °C (Figure 1B,C). More than three male and three female 129WT and B6 filensin KO mice were examined, and we observed the identical cold cataract phenomenon. There were no noticeable differences in the formation of cold cataract among lenses from more than three different male and female mice. No phenotypic difference was observed between the two lenses of a single mouse from these mouse lines.

Quantitative densitometry scanning measurements of these lens images at the equatorial plane further revealed that the light scattering distribution of the B6WT cold cataract (Figure 1D) obviously differed from those of the 129WT and B6 filensin KO cold cataracts which showed gradually reduced opacity toward the lens core at 4 °C (Figure 1E,F). The size of the P14 lenses of different mice was similar with a diameter about 1,735 ± 50.00 μm (n = 9). The diameters of the 4 °C cold cataracts were 1,279 ± 25.00 μm for B6WT, 1,264 ± 21.00 μm for 129WT, and 1,276 ± 26.00 μm for B6 filensin KO, about 73% of the whole lens diameter. However, the diameter of the 25 °C cold cataracts in the B6WT mice, 939 ± 27.0 μm, was about 27% smaller than that of the 4 °C cold cataracts (Figure 1G).

The formation of cold cataracts was completely reversible in all mouse lenses. Lens opacity disappeared within minutes when the cold cataractous lenses were immersed in the 37 °C PBS solution. We further assessed the recovery of P14 B6WT and B6 filensin KO lens transparency during a continuous warm-up course from 4 °C to 25 °C in PBS and then 37 °C (Appendix 1).

Based on the diameters of the cold cataracts at 4 °C and 25 °C, we proposed a schematic cold cataract model by arbitrarily dividing the lens into three zones (I, II, and III). Zone I represents the transparent part of the lens cortex that never develops cold cataracts, zone II covers the expanded area of the cold cataract at 4 °C, and zone III covers the lens core region where 25 °C induced cold cataract occurs. The B6WT cold cataract seemed to be more opaque than the 129WT and B6 filensin KO cold cataracts. The 129WT mouse strain is known to contain a CP49 gene deletion. The results suggested that CP49 and filensin are important for the formation of cold cataracts. To understand the roles of CP49 and filensin in the formation of cold cataracts, we performed immunostaining to determine the distributions of the CP49 and filensin proteins in selective areas of zones I, II, and III in lenses treated with different temperatures.

Formation of cold cataract correlates with accumulations of CP49 and filensin proteins in interior fiber cell boundaries

Lens intermediate filaments composed by CP49 and filensin play essential roles in maintaining the cell shape of the lens fiber and regulating cell morphogenesis in the maturation of fiber cells. We characterized the distribution of the CP49 and filensin proteins in the inner fibers of the B6WT lenses (zone I to zone III) under the treatment of different temperatures. Confocal images of the lens cross-sectional view revealed CP49 immunostaining in lens fibers from the periphery to the core. Uniform CP49 signals appeared in the zone I, II, and III lens fibers in the 37 °C treated samples as well as in zone I fibers of the 25 °C and 4 °C treated lens samples (Figure 2). However, evenly segregated CP49 signals appeared in zone II interior fibers, but unevenly segregated CP49 signals occurred in the zone III fibers of the lens core in the 25 °C treated samples. Moreover, irregularly aggregated or accumulated CP49 signals often appeared along the fiber cell boundaries in the zone II and zone III fibers of the 4 °C treated lens samples (Figure 2A–D). Quantitative measurements of the CP49 signal intensity ratios between the fiber cell boundaries versus the cytosol in zones I, II, and III of these samples are shown in Table 1. These data suggest that increased CP49 accumulative signals near fiber cell boundaries were statistically significant in zone II and zone III of the 4 °C treated lens samples and in zone III of the 25 °C treated lens samples, which were correlated with the appearance of cold cataracts (Figure 2E–G).

Immunohistochemical analysis was performed to examine the filensin distributions in B6WT lens vibratome sections along the anterior-posterior axis. Similar to the CP49 protein distributions, the filensin signals were uniformly distributed in all fibers of the 37 °C treated lens samples but became unevenly accumulated near the fiber cell boundaries in zone III of the 25 °C treated lens samples, as well as in the zone II and zone III fibers of the 4 °C treated lens samples (Figure 3A–D). Quantitative measurements of the filensin signal intensity ratios between the fiber cell boundaries versus the cytosol in zones I, II, and III of these samples are shown in Table 2. These data suggest that the accumulated filensin signals near the fiber cell boundaries were statistically significant in zone II and zone III of the 4 °C treated lens samples and in zone III of the 25 °C treated lens samples, which were correlated with the appearance of cold cataracts (Figure 3E–G). Thus, the results suggest that uneven accumulations of lens beaded filaments, consisting of CP49 and filensin proteins near fiber cell boundaries, were correlated with the formation of cold cataract in lens inner fibers in zone II and zone III.

We also performed immunohistochemical analysis of filensin and CP49 in the 129WT lens vibratome sections. The confocal fluorescent images showed extremely low levels of the filensin protein in lens fibers at zones I, II, and III from the 37 °C, 25 °C, and 4 °C treated samples (Appendix 2) and the absence of the CP49 protein or background signal in all lens fibers (Appendix 3). Therefore, the cold cataracts in the 129WT lenses were independent of the beaded intermediate filaments in the interior lens fibers.

Uneven accumulations of filensin proteins detected in interior single fibers of cold cataract lens

To further characterize the changes in filensin protein distributions in cold temperature–induced cataractous lens, we performed double-labeling of anti-filensin antibody and FITC-phalloidin (F-actin) in single lens fiber cells at different zones of the 4 °C treated B6WT lens. Filensin staining signals were uniformly distributed in the single lens fiber cells of zone I (Figure 4A), unevenly distributed in the single lens fiber cells of zone II (Figure 4B), and predominately accumulated near fiber cell boundaries in single lens fiber cells of zone III (Figure 4C). Quantitative analysis further verified that uneven accumulations of filensin proteins were correlated with the formation of cold cataracts in zone II (Figure 4E) and zone III (Figure 4F). These single lens fiber cell results, consistent with the B6WT lens vibratome section data, revealed that beaded intermediate filaments are important in the formation of cold cataracts.