The In vitro reconstituted the lens-specific intermediate filament with filensin and phakinin replicates the genotype-phenotype correlation for cataracts

Abstract

The intermediate filaments (IFs) family is one of the cyto-skeletons that regulate cell shape, size, stiffness, and movement. Unlike other cytoskeletons such as actin and tubulin, alpha-helical linear proteins of the IFs family constitute filament formation, which can be classified in six types depending on their assembly mechanisms. Filensin and phakinin, which are classified into the type-VI IFs, are only expressed in lens fiber cells and are composed of the lens-specific IFs. However, it is unclear how filensin and phakinin constitute filaments and have an impact on lens properties. Here, we studied the in vitro molecular assembly of human filensin and phakinin to identify the structural and functional relationships. We reconstituted the co-assembled filaments with human recombinant filensin and phakinin and determined its stoichiometry as the ratio of one-to-one. Filensin and phakinin filaments interacted with alpha-crystallin and assembled to make a beeded structure detected by the sedimentation assay and TEM. Moreover, the cataract disease mutant phakinin E233del caused short filaments and reduced resistance against heat and shear stress. We further showed that the alpha-helical rod domains in each protein are involved in the interaction between two proteins, and the intrinsically disordered head and tail domains regulates filament extension. Overall, we determined the molecular interaction in the lens-IFs, which confers the crystalline lens with stability against physical stress. These results suggest that the impaired integrity of the IF can lead to age-related diseases like cataracts and presbyopia.