Periplakin Interactions with Lens Intermediate and Beaded Filaments

The vertebrate lens consists predominantly of elongated fiber cells assembled with a high degree of precision and long-range order. Covering the anterior surface of the lens, and representing a tiny fraction of the total lens mass, is a single layer of flattened cuboidal lens epithelial cells. Fiber cells arise by differentiation of lens epithelial cells, a process that represents a remarkable transformation of the epithelial cell, approximately 20 μm in diameter, into a fiber cell that measures 2 × 6 × 10,000 μm, depending on the species. As each generation of epithelial cells undergoes this differentiation process, a new layer of fiber cells is added to the lens, giving rise to a series of concentric shells arranged chronologically by “birth date.” This differentiation and addition of new layers goes on throughout life, albeit more slowly in the adult organism. Because older lens cells do not die, the lens contains a full spectrum of cells ranging from those at the center, created in the embryonic period, to those at the surface that have just recently differentiated. This process is all the more remarkable in that a fiber cell will dismantle all its membranous organelles shortly after completing the elongation process, a process that occurs approximately 50 fiber cells from the lens equatorial surface.1–6 The other thousand or more fiber cells lack organelles and, therefore, the repair and renewal processes associated with them. Despite this, these nonrenewing fiber cells last the life of the organism, which can span several decades. As with some other tissue types, such as epidermis, lens cells undergo a differentiation-dependent switch in the expression of intermediate filament (IF) proteins.7–9 The lens epithelium predominantly expresses vimentin, a well-conserved type 3 IF protein common to many mesenchymal tissues.10–14 However, in contrast to epidermis, which switches from one keratin pair to another, the lens fiber cells express an entirely novel, fiber cell–specific pair of IF proteins, CP49 and filensin.15–20 These are considered the most divergent IF proteins yet described and are often assigned to their own class because of the low levels of identity with existing classes.18–22 Moreover, CP49 and filensin are not found in canonical 11-nm IFs but rather in the structurally unique beaded filament (BF).15,16,23 Targeted genomic deletion of either BF protein in the mouse eliminates the assembly of the BF in the lens fiber cell.24–27 The resultant change in phenotype is subtle, showing a slight light scatter in the lens that is detectable at approximately 3 weeks of age but that grows progressively worse over the lifespan of the animal. Electron microscopy of the knockout lens suggests that the elaborate differentiation of fiber cells proceeds in a manner not identifiably different from that of the wild type. This phenotype gradually is lost, as is the exquisite long-range stacking of fiber cells, suggesting that the BF is not required to achieve the differentiated phenotype but is required to maintain cellular and tissue-level phenotypes with aging.27,28 Thus, despite the very high degree of divergence in the primary sequence, in the predicted secondary structure and in the filament that is ultimately assembled, the BF remains true to its roots as an IF protein in that it serves to provide mechanical support and to resist changes in the differentiated phenotype.29–34 IF function is clearly dependent on integration with other cellular structures and other cytoskeletal elements, in part through IF-associated proteins (IFAPs). As a result, mutations in IFAPs would be predicted to have the potential to result in cataracts that emulate the absence of an IF by breaking the link that connects IFs to other cell structures. The plakins are a major class of IFAP that have been identified as crucial to IF function.35–38 To date, seven plakins have been identified, desmoplakin, plectin, BPAG-1, envoplakin, periplakin, epiplakin, and MACF-1.37 With the exception of epiplakin, they share a common structure: a central coiled-coil domain, flanked by globular domains at the N- and C termini.35,37,39,40 The IF-binding domain has been localized to the C terminus, whereas the N-terminus mediates binding to cell adhesion sites at the plasma membrane or other cytoskeletal networks.41–45 Previous reports have identified periplakin as a constituent of lens fiber cells.46 Periplakin has been shown to bind to IF proteins, specifically vimentin and K8. The binding is mediated by periplakin's C-terminal Linker domain, which targets rod domain 2 on the IF protein. In this report, we examine the relationships formed between periplakin and lens IF and BF proteins. We were particularly interested in establishing whether the high degree of sequence and structural divergence of the BF proteins mandated the emergence of a novel IFAP adapted to BF structure or whether the BF could make use of established IFAPs.