Beaded Filaments (BFs) are a structurally and biochemically filamentous cytoskeletal structure found in lens fiber cells. In 40 years, neither the BF nor the two proteins required for assembly of the BF (BF Proteins CP49 and filensin), have been demonstrated in cells other than differentiated lens fiber cells. In Preliminary Data, we show that BF proteins are expressed in lens epithelium, but also in a subset of biopsied human cancers. Moreover, BF protein expression in lens epithelium is not in a filamentous structure, but instead in a large vermiform structure, which is only expressed after 5 weeks of age. As cells near the bow region where differentiation is triggered, the structure disappears. These data: 1) are the first to report (to our knowledge) the expression of BF proteins outside of the fiber cell, and outside of the lens 2) suggest that the lens epithelium may assemble a novel organelle/inclusion, and 3) that developmental switches are still being flipped in the lens epithelium at 5 weeks of age. Thus, these observations suggest a previously unknown function for the BF Proteins, both in the lens, and in non-lens tissue, and in both normal and pathologic biology. We have no idea what this function is, and invoke this R21 to begin the process of establishing this/these function(s). Our lab has worked on Beaded Filaments and Intermediate Filaments for the past 25 years. In doing so we have accumulated a combination of BF/IF tools, resources, and experience in lens biology, which uniquely situates us to make progress in revealing this "non-canonical" BF protein function. Because of the availability of these tools (CP49 knockout, filensin knockout, and vimentin-knockout animals, a range of antibodies to BF proteins, vimentin, and several other lens proteins, cDNA constructs, etc) and because of the limited availability of rare human tumor material, we focus these initial efforts on lens. There are, of course, a vast number of questions that can be asked about a novel structure, but the R21 limitations constrain the scope of inquiry. For that reason we are proposing questions at three levels, the molecular, cellular and tissue levels, to cast the widest net possible. The questions asked are guided by our knowledge of lens, intermediate filaments, beaded filaments, and the literature on cellular organelles and inclusions. Just as our previous work started with a fiber cell-specific cytoskeletal structure (the BF) and grew into a general attack on the structure of Intermediate Filaments, we hope that these studies will transcend from lens epithelial-specific to the broader field of cell biology.
In preliminary data we demonstrate that two proteins, previously thought to exist only in the fiber cells of the ocular lens, are also expressed in lens epithelium and in a subset of human cancers. In the lens epithelium they appear to assemble into a novel organelle, but only after 6 weeks of age. These observations suggest a previously unknown function for these proteins, both in the lens, and in non-lens tissue, and in both normal and pathologic biology, but what that function may be is unknown. This proposal begins the process of identifying that function.