Motor recruitment to the filaments with the rapalog system and repositioning of the vimentin network in U2OS cells. (A) Fluorescence images of COS-7 cells expressing Vim-mCh-FKBP. The overexpressed vimentin is visualized by mCherry fluorescence, while total vimentin intensity is detected via anti-vimentin immunostaining. The dashed box indicates the region of the cell enlarged in the zoom image. Images were captured using Airyscan microscopy. (B and C) Fluorescence images of COS-7 cells co-transfected with Vim-mCh-FKBP and either FRB-GFP-GCN4-ppKin14 (B) or HA-KIF5A-FRB (C), with or without 5-min rapalog treatment. The top panels show untreated cells, while the bottom panels display rapalog-treated cells. FRB-GFP-GCN4-ppKin14 and HA-KIF5A-FRB are detected using anti-GFP and anti-HA antibodies respectively. Images were captured using Airyscan microscopy. (D and E) Representative fluorescence images of U2OS cells co-transfected with Vim-mCh-FKBP and FRB fusions of the indicated motors, with or without 1 h of rapalog treatment. Transfected cells are outlined with dashed lines, and non-transfected cells serve as controls. Anti-vimentin and anti-HA antibodies detect total vimentin and HA-KIF5A-FRB, respectively.