Expression and Subcellular Localization of Mammalian Formin Fhod3 in the Embryonic and Adult Heart

Abstract

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.

Mammalian formin Fhod3 plays an essential role in cardiogenesis by organizing myofibrillogenesis

“…AB078608) (Kanaya et al, 2005) under the control of the α-myosin heavy chain promoter, a generous gift from Dr. Jeffery Robbins (Cincinnati Children's Hospital Medical Center) (Gulick et al, 1991), were generated on a C57BL/6 background, as previously described (Kan-o et al, 2012); expression level of exogenous Fhod3 in the transgenic mice was ten-fold more than that of endogenous Fhod3. Transgenic mice expressing a mutant Fhod3 carrying the I1127A substitution (Taniguchi et al, 2009) under the control of the α-myosin heavy chain promoter were also generated on a C57BL/6 background.…”

The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response

“…The muscle-specific FHOD3 has been implicated in myogenesis. Fhod3 mutant mice display strong defects in myofibril maturation resulting in embryonic lethality (Iskratsch et al, 2010;Kan-O et al, 2012a;Kan-O et al, 2012b;Taniguchi et al, 2009). This muscle-specific function of FHOD isoforms seems to be evolutionarily conserved and C. elegans lacking the Fhod3-related gene shows prominent cytoskeletal organization defects in muscle cells (Mi-Mi et al, 2012).…”

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

“…In muscle cells, the specific mechanisms for actin nucleation and maintenance in sarcomeres were poorly understood (8). Recent studies have uncovered actin nucleation activities of the nebulin-N-WASP complex (9) and of formin proteins FHOD3 (10)(11)(12), mDia2, DAAM, FMNL1, and FMNL2 (13,14) in sarcomeres. In particular, leiomodin (Lmod) has been identified as a class of potent tandem-G-actin-binding nucleators in muscle cells (15,16); Lmod1 is found in smooth muscle of many human tissues, and Lmod2 and Lmod3 are found in cardiac and skeletal muscle (17).…”