Fig. 7. N-terminal domain of BICD2 co-precipitates with dynein and disrupts microtubule minus-end-directed organelle distribution. (A) Overview of GFP–BICD2 deletion constructs. COS-1 or HeLa cells were transfected with the indicated GFP–BICD2 fusion constructs, fixed and stained with Golgi- or endosome-specific antibodies, to analyse whether the transfected construct was targeted to the Golgi (+ indicates Golgi accumulation of the GFP fusion construct and – indicates absence of such accumulation) and whether organelle distribution was affected by the transfection (+ indicates a disrupted Golgi and endosome distribution, and – indicates a normal Golgi and endosome organization). (B and C) Organelle disruption by GFP–BICD2^1-271 overexpression. In (B), fragmentation of the Golgi complex is scored, as detected with anti-p115 antibodies (B2). In (C), a redistribution of endosomes to the periphery of the cell is scored, as detected by staining with antibodies against the transferrin receptor (C2). Organelle disruption is a robust phenotype; when present it occurs in all transfected cells, while neighbouring, non-transfected cells show a normal, compact perinuclear Golgi staining (B2) and prominent juxtanuclear accumulation of endosomes (indicated by arrowheads in C2). Bars: 10 μm. (D) Lysates of COS-1 cells, transfected with the indicated GFP proteins, were immunoprecipitated with monoclonal anti-GFP antibodies, and precipitated proteins were analysed by western blotting with polyclonal anti-GFP antibodies or with anti-DIC antibodies.