Genetic evidence that formins function within the nucleus

The murine limb deformity (ld) locus encodes a set of proteins, termed formins, that are required for embryonic limb and kidney development. Previous studies had indicated that these proteins are located in the nucleus and cytoplasm and have biochemical properties consistent with an action within the nucleus. To test the notion that nuclear localization is crucial for formin function, we carried out molecular and biochemical studies on three ld alleles. We show that two transgene induced alleles, ld(TgHd) and ld(TgBri), generate similar COOH truncated formins that lack the terminal 110 amino acids, while a third allele, ld(In2), generates a less extensively truncated formin that lacks the terminal 42 amino acids. Using subcellular fractionation analysis, we find that wild-type formin is detected in both nuclear and cytosolic fractions; in contrast, the truncated formins encoded by ld(TgHd) and ld(TgBri) are strictly cytosolic. The less extensively truncated ld(In2) formin shows a similar, but less complete, localization defect. Consistent with this weaker cellular phenotype, hind limbs from ld(In2) mice have milder skeletal defects than those of ld(TgBri) mice. These observations define a small region in the carboxyl terminus that is required for nuclear localization and suggest that nuclear localization plays a role in formin action.