Figure 7. MAP7D3 binds to KIF5B more tightly than MAP7 and promotes its activity in vitro. (A) Schemes of MAP7 family proteins and KIF5B constructs used in this figure. (B) SEC-MALS was used to determine the oligomerization state of indicated proteins in solution. Lines depict the relative UV-absorbance measured at 280 nm plotted against the elution volume. The molecular mass determinations by multi-angle light scattering are depicted by dashed lines. (C and D) ITC of MAP7D3 (C) or MAP7 (D) against KIF5B. Top: Enthalpograms of the respective titrations. Bottom: integrated heat change (black dots) and the associated fitted curve (red line in C). Controls are shown in Fig. S4 (E–G). (E) Kymographs of K560-GFP on dynamic MTs in the presence of full-length (FL) MAP7D3 or MAP7D3-Ct (both purified from E. coli). (F) Quantifications of MAP7D3-FL or -Ct intensities on dynamic MTs using images acquired under identical conditions on a TIRF microscope. n = 40, n = 38, and n = 42 MTs from two independent experiments. Representative images are shown in Fig. S5 A. (G) Quantification of landing frequency per MT and corrected for MT length, time of acquisition, and kinesin concentration. n = 167, 36, 36, and 31 MTs from two independent experiments. (H) Histograms showing kinesin run lengths fitted to a single exponential decay (red) with the indicated rate constants (tau) as a measure of mean run length; n = 241, 271, and 209 kinesin runs from two independent experiments. The associated bar plots are shown in Fig. S5 C. (I) Gaussian fits of kinesin velocities. Histograms are shown in Fig. S5 B. (J) Kymographs of single-molecule FRAP experiments on K560-GFP motors and MAP7D3-Ct. Photobleaching with a 561-nm red laser was performed at time points indicated by red lightning bolts. Fluorescent recovery is indicated by arrowheads. (K) Cumulative frequency distribution plot of mCherry-MAP7D3-Ct recovery after photobleaching (black dots) fitted to an exponential decay (red line) with the indicated decay constant (tau); n = 79 from three independent experiments.
Figure 7.

MAP7D3 binds to KIF5B more tightly than MAP7 and promotes its activity in vitro. (A) Schemes of MAP7 family proteins and KIF5B constructs used in this figure. (B) SEC-MALS was used to determine the oligomerization state of indicated proteins in solution. Lines depict the relative UV-absorbance measured at 280 nm plotted against the elution volume. The molecular mass determinations by multi-angle light scattering are depicted by dashed lines. (C and D) ITC of MAP7D3 (C) or MAP7 (D) against KIF5B. Top: Enthalpograms of the respective titrations. Bottom: integrated heat change (black dots) and the associated fitted curve (red line in C). Controls are shown in Fig. S4 (E–G). (E) Kymographs of K560-GFP on dynamic MTs in the presence of full-length (FL) MAP7D3 or MAP7D3-Ct (both purified from E. coli). (F) Quantifications of MAP7D3-FL or -Ct intensities on dynamic MTs using images acquired under identical conditions on a TIRF microscope. n = 40, n = 38, and n = 42 MTs from two independent experiments. Representative images are shown in Fig. S5 A. (G) Quantification of landing frequency per MT and corrected for MT length, time of acquisition, and kinesin concentration. n = 167, 36, 36, and 31 MTs from two independent experiments. (H) Histograms showing kinesin run lengths fitted to a single exponential decay (red) with the indicated rate constants (tau) as a measure of mean run length; n = 241, 271, and 209 kinesin runs from two independent experiments. The associated bar plots are shown in Fig. S5 C. (I) Gaussian fits of kinesin velocities. Histograms are shown in Fig. S5 B. (J) Kymographs of single-molecule FRAP experiments on K560-GFP motors and MAP7D3-Ct. Photobleaching with a 561-nm red laser was performed at time points indicated by red lightning bolts. Fluorescent recovery is indicated by arrowheads. (K) Cumulative frequency distribution plot of mCherry-MAP7D3-Ct recovery after photobleaching (black dots) fitted to an exponential decay (red line) with the indicated decay constant (tau); n = 79 from three independent experiments.

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