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. 2022 Jul 7;119(28):e2120193119. doi: 10.1073/pnas.2120193119

Fig. 4.

Fig. 4.

Single-molecule analysis of Kif4A-GFP movement in the presence of K401-clip-647 or Kif4A-mCherry. (A–F) Kymographs obtained from time-lapse image sequence acquired in examining microtubule interaction of (A) 45 pM Kif4A-GFP (scale bar: x = 2 µm; y = 6 s), (B) 1,000 pM K401-clip-647 (scale bar: x = 5 µm; y = 11 s), (C) 300 pM Kif4A-mCherry (scale bar: x = 2 µm; y = 7 s), (D) 45 pM Kif4A-GFP + 1,000 pM K401-clip-647 (dimer 15 to 28% labeled) (scale bar: x = 3 µm; y = 10 s), (E) 45 pM Kif4A-GFP + 180 pM K401-clip-647 (dimer 58 to 82% labeled) (scale bar: x = 1.5 µm; y = 10 s), and (F) 45 pM Kif4A-GFP + 300 pM Kif4A-mCherry (scale bar: x = 1 µm; y = 2 s). (G and H) Two methods of quantitative analysis of the colocalization of the GFP with either K401-clip-647 or Kif4A-mCherry from the two color experiments from D–F (Materials and Methods). The kymograph schematic and bar graph show percentage of colocalization of (G) clip-647/mCherry pixels with GFP tracks (Kif4A-GFP + K401-clip-647 [dimer = 15 to 28% labeled], mean 6 ± 2%, n = 11; Kif4A-GFP + K401-clip-647 [dimer = 58 to 82% labeled], 7 ± 2%, n = 32; Kif4A-GFP + Kif4A-mCherry, 5 ± 1%, n = 50) and (H) clip-647/mCherry tracks (greater than 5 pixels) with GFP tracks (Kif4A-GFP + K401-clip-647 [dimer = 15 to 28% labeled], mean 1 ± 1%, n = 24; Kif4A-GFP + K401-clip-647 [dimer = 58 to 82% labeled], 3 ± 2%, n = 20; Kif4A-GFP + Kif4A-mCherry, 5 ± 2%, n = 51). The kymograph overlay schematic shows GFP (green lines), clip-647/mCherry (red lines), and the overlap between GFP and clip-647/mCherry (yellow lines). The measured events are indicated by the black arrows. The error is the SEM.