Abstract
Purpose :
Filensin and phakosin are two divergent intermediate filament proteins expressed only in the lens. They assemble into the Beaded Filament (BF), a fiber cell-specific cytoskeletal element. Flensin undergoes a well-conserved cleavage event early in differentiation, that yields two major fragments. Mass Spectroscopy suggests that the cleavage occurs at a caspase motif. We used the CRISPR gene editing approach to generate mutants to gain insight into the role of this caspase site in murine BF biology.
Methods :
Two mutant strains of mice were generated: 1) a “deletion mutant” with the caspase site deleted, and 2) a truncation mutant with a stop codon immediately after the caspase site. Lenses were analyzed by western blotting, transmission and scanning electron microscopy, and immunofluorescence. Monoclonal antibodies were generated to track the fate of different regions of the filensin protein.
Results :
The truncated filensin accumulated in fiber cells, though its subcellular distribution was altered. No beaded filaments were found by TEM. The truncation mutant showed a loss of the highly-ordered alignment of fiber cells in the deeper cortex, as seen in the filensin and phakosin KOs. In contrast, beaded filaments still formed in the caspase site deletion mutant. Western blotting showed that the deletion mutant still underwent progressive cleavage events, but the pattern of fragments differed from that seen in the WT, and fiber cell stacking remained normal. Subcellular distribution of filensin also appeared normal.
Conclusions :
1) The filensin tail domain appears to be is essential for BF assembly in situ (TEM). 2) The truncation mutant confirms that the tail domain appears to be required for the BF association with the plasma membrane usually seen in younger fiber cells (IHC). 3) The 433-caspase cleavage site/event is not required for filament assembly, correct packing of fiber cells, or association of the BF with the plasma membrane of younger fiber cells (TEM, IHC). 4) The 433 caspase cleavage site does not appear to be required for the translocation of immunoreactivity from membrane-associated to cytoplasmic (IHC). 5) In contrast to the bfsp1 and bfsp2 KO mice, the absence of filament assembly in the tail-less mutant does not appear to result in removal of the unassembled filensin or phakosin (WB, IHC).
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.