Fignl1 directly binds the dynein/dynactin motor adaptor bicd1 in a protein complex including KIF1Bβ and dynactin 1. (A) Schematic representation of Ms_fignl1, Ms_bicd1, and their respective binding domains identified through a yeast two-hybrid screen (Y2H; black arrows). Specific domains are shown as boxes. Numbers indicate amino acids that delineate domain frontiers. AAA, ATPase domain; CC1, CC2, and CC3, coil-coiled domains. (B) COS-7 cells transfected with GFP-Ms_bicd1 and Dr_Fignl1-HA, and immunolabeled with HA and GFP antibodies. Dashed line delimits the transfected cell. Bottom panels represent higher magnifications of boxed regions in corresponding upper panels. Arrowheads indicate colocalization between HA-Fignl1 and GFP-bicd1 onto cytoplasmic vesicles. Scale bars, 10 µm. (C) Fignl1 associates with bicd1. Co-IP of Fignl1 isoforms and bicd1 from protein extracts of COS-7 cells transfected with GFP-Ms_bicd1 or GFP, Ms_ HA-fignl1, Dr_Fignl1-HA, or Dr_Fignl1Δ1-113-HA. (D) KIF1Bβ associates with bicd1. Co-IP of KIF1Bβ and bicd1 from protein extracts of COS-7 cells transfected with YFP-Hu_KIF1Bβ and Flag-Ms_bicd1. (E and F) Fignl1 is part of a protein complex including KIF1Bβ and the dynein adaptors bicd1 (E) and dynactin 1 (F). (E) Left-hand well: Co-IP of exogenous Fignl1 and bicd1 with endogenous kif1b from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA and GFP-Ms_bicd1. Middle and right-hand wells: Co-IP of exogenous Fignl1, bicd1, and KIF1Bβ from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA, Flag-Ms_bicd1, and YFP-Hu_KIF1Bβ or GFP. Immunoprecipitations were performed with GFP-trap (C–F), KIF1B (E), or HA (F) antibodies. Immunoprecipitated and coimmunoprecipitated proteins were revealed by Western blot using anti-tag or KIF1B and dynactin 1 antibodies or by Ponceau staining. IP, immunoprecipitation; WB, Western blot.