Abstract
Filamin A (FLNA), an actin cross-linking protein, acts as a mechanosensor and mechanotransducer by exposing the cryptic binding site on repeat 21 (R21) to interact with integrin. Here, we investigated if any other biological molecule interacts with the cryptic binding site. Using proteomics and an in silico screening for a FLNA-binding motif, we identified and characterized a protein termed fimbacin (filamin mechanobinding actin cross-linking protein), encoded in the LUZP1 gene, as a novel FLNA-binding partner. Fimbacin does not interact with canonical full-length FLNA, but the exposure of a cryptic integrin-binding site of FLNA R21 enables fimbacin to interact. We have identified two FLNA binding sites on fimbacin and determined critical amino acid residues for the interaction. We also found that fimbacin itself is a new actin cross-linking protein and mapped the actin-binding site on amino acid residues 400-500. Fimbacin oligomerizes (estimated as an octamer on size exclusion chromatography) through the amino-terminal domain that is predicted to be a coiled-coil to cross-link actin filaments. When expressed, fimbacin localized to actin stress fibers in tissue culture cells. Although the interaction with FLNA is not necessary for fimbacin to colocalize with F-actin, fluorescent recovery after photobleaching (FRAP) revealed that their interaction stabilizes fimbacin on the actin cytoskeleton and that inhibition of Rho-kinase, an upstream activator of myosin II, also decreases the interaction presumably due to a loss of internal mechanical stress. Taken together, these data identify fimbacin as a new actin cross-linking protein that interacts with the FLNA mechanosensing domain R21.