Mapping of the dynein-interacting domain of NuMA. (A) Schematic representation of NuMA, with its coil-coiled domain and the regions within its C terminus known to mediate interaction with LGN and microtubules. The NLS is also indicated. The truncated forms for NuMA used in the experiments are depicted on the bottom with their amino acid boundaries. The red asterisk on top of fragment 1–705 denotes interaction with dynein. N, N terminus; C, C terminus. (B–E) Untransfected control HeLa cell (B) or HeLa cells transfected with GFP-NuMA(1–705) (C), GFP-NuMA(706–1,410) (D), or GFP-NuMA(1,411–2,115) (E) and stained for the dynactin subunit p150^Glued. Note loss of p150^Glued from spindle poles in cells transfected with GFP-NuMA(1–705) and also from the cell membrane in cells transfected either with GFP-NuMA(1–705) (C) or GFP-NuMA(1,411–2,115) (E). 50 cells were scored for each condition. Arrows point to cortical p150^Glued. (F–H) Untransfected control HeLa cells (F) or HeLa cells transfected with GFP-NuMA(1–705) (G) or GFP-NuMA(706–1,410) (H) and then transferred to a coverslip with an L-shaped fibronectin micropattern, fixed, and stained with antibodies against GFP and the 58K Golgi marker. Note that the Golgi is dispersed upon transfection of GFP-NuMA(1–705). 30 cells were scored for each condition. (I) Extracts from mitotic cells expressing either GFP-NuMA(1–705) or GFP-NuMA(706–1,410) were immunoprecipitated with GFP-Trap, and the resulting blots were probed for p150^Glued, dynein intermediate chain (DIC), and GFP as indicated. Molecular mass is indicated in kilodaltons. IN, input (2% of total); IP, immunoprecipitate (20% of total).