Figures and data
Experimental design for the stall force measurement of KIF1A using a NS.
a Schematic of the domain structure of full-length KIF1A and the recombinant construct used in the experiments. To stabilize KIF1A dimers, which do not form stably without cargo-binding domains, a leucine zipper was incorporated, and SNAP-tags were added at the N-termini to enable chemical coupling to the NS. b Schematic of the chemical modifications required for coupling the NS to KIF1A (Methods). c An inert KIF5B is anchored to the microtubule, and the NS extends as a KIF1A moves toward the plus end. The NS is uniformly labeled with Cy3 fluorophores, allowing force to be calculated from its extension. The micrographs depict the NS in the retracted and extended states. d Force–extension relationship of the NS, showing nonlinear elastic behavior, calibrated by acoustic force spectroscopy (AFS)21 and fitted with an exponential function (Methods).
Length estimation using DNA calibration rods.
a The fluorescence intensity of the DNA calibration rod was theoretically modeled as a superposition of Gaussian functions with variance σ2 aligned along a straight line, and an artificial image was generated accordingly. b The σ value of the Gaussian function used in the model was fitted to match the point spread function of Cy3 fluorescent dye (black line), resulting in a value of 90.65 nm. c Artificial image generated by placing 116 two-dimensional Gaussian functions (top). When 116 Gaussian functions were superimposed, they appeared as a single bright spot with an elliptical shape. The image (top) was fitted by the Gaussian fitting method (equation (1)) (middle) and the chain fitting method (equation (2)) (bottom). d Estimated length plotted against the true length of the model (L) using the Gaussian fitting method (equation (1)) (dark colors) and the chain fitting method (equation (2)) (bright colors), respectively. The dotted line represents the linear equation y = x. The chain fitting model provides estimates the true value of L in the simulation. e Fluorescence micrographs of the DNA calibration rods21 obtained in real experiments, with lengths of 398 nm, 501 nm, 599 nm, and 658 nm. The scale bars indicate 2 µm. f Estimated length plotted against the true length of the rods using the Gaussian fitting method (equation (1)) (dark colors) and the chain fitting method (equation (2)) (bright colors), respectively. The dotted line represents the linear equation y = x. The chain fitting method provides estimates closer to the true value of L.
Stall force measurement of wild type KIF1A homodimers using NSs.
a Time course of NS extension in the case of wild-type KIF1A. The black line (trace) represents the average over 10 frames. As illustrated in the schematic in Fig. 1c, the NS is stretched (micrograph, top) as a KIF1A moves toward the plus end of the microtubule. When the load reaches the maximum force that the KIF1A can generate, a stall is observed, followed by the detachment of the KIF1A from the microtubule. The NS then returns to its original retracted state (micrograph, bottom). The attachment duration Δt (red region) was defined as the period during which the angular fluctuation reaches a minimum (Methods). For each stall event, the histogram of NS extension exhibits a bimodal Gaussian distribution, with the higher peak corresponding to the stall length Lstall (left panel). b Histogram of Lstall values calculated from 81 stall events. c Lstall as a function of attachment duration Δt with (n=81) and without (n=65) PEG. d Comparison of Lstall with and without PEG (p=0.55). n.s., not significant (p ≥0.05). The green bars indicate the median values along with the first and third quartiles.
Stall force of KIF1A(1-393) estimated from the average value of Lstall.
The stall force values were calculated from the mean values of Lstall by using the force-extention relationship of the NS (Fig. 1d). The error of Lstall value represents the standard deviation (SD).
Stall force measurement of KAND mutants KIF1A homodimers and heterodimers using NSs.
a Schematic of KIF1A domain structure showing the functions affected by the P305L, V8M, and A255V mutations6. Representative traces are shown for homodimers and WT-mutant heterodimers of P305L, V8M, and A255V (b,c,d,e,f,g,h,i,j). The black lines (traces) represent the average over 10 frames. The comparison reveals mutation-specific differences in stall behavior (d,g,j). p=5.8× 10-35 for WT/WT vs. P305L/P305L, p=0.018 for WT/WT vs. P305L/WT, p=1.3×10-5 for P305L/P305L vs. P305L/WT. p=7.5×10-13 for WT/WT vs. V8M/V8M, p=6.7×10-12 for WT/WT vs V8M/WT, p=0.063 for V8M/V8M vs. V8M/WT. p=9.1×10-7 for WT/WT vs. A255V/A255V, p=1.4×10-6 for WT/WT vs. A255V/WT, p=1.0 for A255V/A255V vs. A255V/WT. n.s., not significant (p ≥0.05). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.
Comparison of Δt between homodimers and heterodimers for each KAND mutant.
Δt values for the cases of P305L, V8M and A255V. p=0.50 for P305L/P305L vs. P305L/WT. p=0.017 for V8M/V8M vs. V8M/WT. p=0.31 for A255V/A255V vs. A255V/WT. n.s., not significant (p ≥0.05). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.
