Fig. 4

Loss-of-function of the myosin phosphatase complex causes a liverless phenotype and the mypt1^sq181 mutation impairs the formation of the myosin phosphatase complex by compromising the binding of Mypt1 to PP1c. (A) Genetic complementation test between hi2653 and sq181 (sq181 x hi2653). (B,C) Both mypt1-MO and PP1c-MO morphants were either liverless or exhibited a small liver (arrow). (D) FLAG-tagged zebrafish and mouse wild-type (w) and M³⁶-mutant (m) Mypt1^1-305 were co-expressed with HA-tagged mouse PP1c in COS-7 cells, and were co-immunoprecipitated using an anti-FLAG antibody. PP1c was detected with an anti-HA antibody and Mypt1 with an antibody against human MYPT1 (PPP1R12A - HUGO). Cells transfected with HA-PP1c alone were used as the negative control (-); w, wild-type Mypt1^1-305; m, M³⁶-Mypt1^1-305; zb, zebrafish; mus, mouse. (E) Immunofluorescence microscopy of Hela cells transfected with FLAG-tagged zebrafish wild-type Mypt1^1-305 (Mypt1) or mutant M³⁶-Mypt1^1-305 (M³⁶-Mypt1). Cells containing Mypt1 or M³⁶-Mypt1 were detected with an anti-FLAG antibody (arrows) (left panels). Stress fibers were stained with TRITC-labeled phalloidin (right panels). Note that cells lacking Mypt1 or M³⁶-Mypt1 (marked with ^*) contained significant amounts of stress fibers, as did cells transfected with M³⁶-Mypt1^1-305 (arrows, lower right panel).