Overexpression of rigor constructs of KIF5B, KIF3A, and KIF3B influences cell-surface exposure of MT1-MMP. Confocal micrographs of macrophages expressing MT1-MMP–mCherry transfected with (A-C) GFP control or GFP- or YFP-fused P-loop mutant constructs of kinesins: (D-F) KIF5B T92N, (G-I) KIF3A T107N, (J-L) KIF3B T103N, and (M-O) KIF1C K103A. Total MT1-MMP–mCherry signal shown in (A,D,G,J,M; red), GFP signal in (B,E,H,K,N; green). Cells were fixed, but not permeabilized, and stained with primary anti-mCherry antibody and secondary Cy5-conjugated antibody to label MT1-MMP–mCherry on the cell surface (C,F,I,L,O; white). Note reduction of MT1-MMP–mCherry at the cell surface upon overexpression of KIF5B, KIF3A, and KIF3B mutants, but not in case of the KIF1C mutant. White bar indicates 10 μm for all images of the same row. (P) Fluorescence intensities of surface-localized MT1-MMP–mCherry, based on Cy5 fluorescence. Bars indicate mean values ± SD. Fluorescence intensity for the control (A-C) was set to 100%. *P < .05, **P < .01 compared with control. For all values, 3 × 30 cells were evaluated. For specific values, see supplemental Table 1.