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 kinesin (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. Here, the microtubule axis is defined as the x-direction, and the direction perpendicular to the microtubule is defined as the z-direction. 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 (L(t)) 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 stall duration Δt (violet region) was defined based on the angular fluctuation and the rate of relative increase in NS’s length (Methods), where the red regions represent the attachment durations decided based on the angular fluctuations. 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 86 stall events (left). The right panel shows the distribution of stall forces for each KIF1A molecule in which six or more stall events were observed. c Lstall as a function of stall duration Δt with (n=105) and without (n=86) PEG. The correlation coefficient (R) between Lstall and Δt is shown in the figure (left). The right panel presents the magnified view of the blue rectangle in the left panel, and clearly indicates that R is small. d Comparison of Lstall and Δt with and without PEG (Mann–Whitney U test, p = 0.4718 for Lstall, p = 0.0616 for Δt). 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 of NS extension are shown for homodimers and WT-mutant heterodimers of P305L, V8M, and A255V (b,c,d,e,f,g). The black lines (traces) represent the average over 10 frames. The lighter-colored regions in the graph represent the attachment durations, while the darker-colored regions indicate the stall durations. The identification of these durations is described in the Methods section.

Comparison of Lstall and Δt among KAND mutants.

Lstall (a) and Δt (b) for homodimers and heterodimers of P305L, V8M and A255V, compared with WT.