FH2-dependent Localization of FHOD Formins in the Sarcomere

Abstract

Formins are a class of actin nucleating factors containing a highly-conserved FH2 domain, which binds actin. There are 15 mammalian formin proteins in seven sub-families that have been found to nucleate, cap, sever, bundle, and polymerize linear actin filaments. An expression analysis of all 15 human formins across 22 different cell and tissue types showed high levels of expression for one formin sub-family in striated muscle: FHOD. While FHOD1 is highly expressed across many cell and tissue types, FHOD3 is only found at comparatively high levels in striated muscle cells. The mature structure of skeletal muscle is a very recognizable periodic repetition of filaments that allows muscles to contract. The sarcomere is the smallest contractile unit of skeletal muscle, with the two main filaments, the thick filament containing myosin, and the thin filament containing actin moving back and forth via the interaction of myosin heads with the thin filament, which allows muscles to contract. While the molecular functioning and the mature structure of skeletal muscle is well understood, the exact mechanism by which the sarcomere is assembled, and specifically, how the actin-core of the thin filament is formed and incorporated into the thin filament remains unknown. The high levels of expression of FHOD formins in striated muscle, combined with their ability to interact with filamentous actin warranted a closer look at FHOD1 and FHOD3 in the sarcomere. In this study we found that FHOD1 and FHOD3 have distinct sarcomeric localizations in C2C12 cells. FHOD1 localizes to the barbed end of the actin filament at the Z-disk and FHOD3 localizes to the pointed end of the actin filament near the center of the sarcomere. Full-length cDNA constructs for FHOD1 and FHOD3 were introduced into skeletal muscle cells, and we were able to recreate the endogenous localization of FHOD3 with the exogenous cDNA. The FHOD1 cDNA localized not to the barbed end of the actin filament, as endogenous FHOD1 does, but instead to the pointed end of the actin filament, where endogenous and exogenous FHOD3 were found. We hypothesized that FHOD binding to actin is dependent upon two highly conserved actin-binding sites in the FH2 domain. Mutations of the actin-binding residues in the FH2 domain impaired the actin-binding ability of both exogenous FHOD1 and FHOD3 and showed that the localization of FHOD1 and FHOD3 in the sarcomere is actin-binding dependent.