Cellular dynein–dynactin motility induced by WT and mutant BICD2. (A) Schematic of the cell-based peroxisome motility assay. U2OS cells were cotransfected with a plasmid expressing GFP–FRB with a PEX3 localization sequence, targeting the protein to peroxisomes, and an mCherry-BICD2–FKBP construct. After addition of rapamycin, mCherry-BICD2–FKBP is recruited to peroxisomes. If in an active state, the BICD2 recruits dynein–dynactin, which transport the peroxisome along microtubules toward the centrosome. (B) Representative epifluorescence images of the assay for both BICD2_1–594 and BICD2_FL. Perixosomes are shown in green, the white signal represents the far-red cell membrane marker, CellMask, and blue is DAPI. In the absence of rapamycin, peroxisomes decorated with GFP–FRB are distributed throughout the cytoplasm (columns 1 and 3). 1 h after rapamycin addition, peroxisomes in cells expressing BICD2_1–594 are heavily clustered at the cell center (column 2), whereas those expressing BICD2_FL exhibit less clustering (column 4). Bar, 10 µm. (C) Example image of an analyzed cell for both BICD2_1–594 and BICD2_FL. The white outline depicts the cell boundary; the inner green line indicates the area that contains 90% of fluorescence (see Materials and methods). Bar, 10 µm. (D and E) Quantification of the cell clustering data. The percentage of the cell area in which 90% of total fluorescence was contained is used as a measure for the degree of clustering (see Materials and methods). Lower values reflect a higher degree of clustering; mean and SD from n = 3 independent experiments (each experiment measuring a minimum of 25 cells for each construct); *, P ≤ 0.05.