V8M and Y89D mutant motors show delayed transport of membrane-bound cargo in cells. (A) Schematic of the peroxisome dispersion assay. A kinesin motor fused to monomeric mNG and an FRB domain (KIF1A(1–393)-mNG-FRB) is coexpressed in COS-7 cells with a peroxisome-targeting sequence (PEX3) fused to mRFP and an FKBP domain (PEX3-mRFP-FKBP). Addition of rapamycin (+Rap) causes heterodimerization of the FRB and FKBP domains and recruitment of motors to the peroxisome membrane. Recruitment of active motors drives cargo dispersion to the cell periphery. (B) Representative images of peroxisome dispersion before (−Rap) and 10 min after (10 min Rap) recruitment of WT or mutant motors to the peroxisome surface. Blue lines indicate the nucleus and periphery of each cell. Blue arrowheads indicate peroxisomes. Scale bar, 10 µm. Percentages in the upper right corner indicate the percentage of cells with the indicated dispersion phenotype: black, clustered peroxisomes; dark gray, partially dispersed peroxisomes; light gray, diffusely dispersed peroxisomes; white, peripherally dispersed peroxisomes. (C) Qualitative analysis of peroxisome dispersion. Cells were scored as clustered (black), partially dispersed (dark gray), diffusely dispersed (light gray), or peripherally dispersed (white). The phenotypes of n ≥ 43 cells across three experiments were combined into a stacked bar plot for each construct at each time point.