Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens

Abstract

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2β2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

“…Lens fibers contain two specialized intermediate filament proteins, CP49 (phakinin) and filensin (24). Together, these proteins form obligate heteropolymers, termed beaded intermediate filaments, that are required for mechanical strength (27,36) and life-long transparency of the lens (1,36,73). Moreover, mutations in CP49 lead to autosomal-dominant cataracts in humans (15,16,44,92).…”

Inverse elastographic method for analyzing the ocular lens compression test

“…Our own measurement of 11.9 mN/mm at low extents of compression is similar to the measurements of 12.4 mN/mm by Gokhin et al at 6% compression. 25 Extrapolation indicates that, if our experiments had achieved 27% compression, the exponential sti®ness would have been 22.0 mN/mm whereas Gokhin et al measured 27.6 mN/mm. This gives con¯dence that the methods used to generate force-displacement data and subsequently estimate the extrinsic sti®ness of the lens are reliable.…”

“…The tangent to this exponential curve at various points may then be interpreted as the instantaneous sti®ness. 24,25 This approach was also considered here by¯tting all data with an exponential curve, then computing the The role of contact area is clari¯ed by re-plotting the measurements in terms of the Hertz displacement (i.e., displacement raised to the 3/2 power), resulting in nearly straight lines for the loading curves.…”

“…6 Previous methods for estimating lens sti®ness include spinning tests, [7][8][9] dynamic mechanical analysis, 10 indentation, 11,12 penetration, 13 Brillouin microscopy, [14][15][16][17][18][19] acoustic methods, 20,21 and compression. [22][23][24][25][26][27] The lens capsule has been mechanically characterized by in°ation testing 28,29 and uniaxial tensile testing. 30,31 However, no test has yet enabled a simultaneous mechanical analysis of both the lens and its capsule.…”

“…Recently, they have also been used as a model for quantifying the contribution of speci¯c proteins to lens mechanical properties by computing an extrinsic sti®ness parameter derived from lens compression. 25,27 However, the use of an extrinsic mechanical property is inherently confounded by altered lens size and shape 35,36 -a common side e®ect of genetic manipulation. 37 The intrinsic mechanical properties have been extracted from larger lenses (i.e., porcine and human) using spinning tests coupled with an inverse FE method.…”