Research Article

Biochemistry and Chemical Biology

Stall force measurement of the kinesin-3 motor KIF1A using a programmable DNA origami nanospring

The Institute for Solid State Physics, The University of Tokyo, Japan
Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, The University of Tokyo, Japan
Systems Biochemistry in Pathology and Regeneration, Yamaguchi University Graduate School of Medicine, Japan
Graduate School of Frontier Bioscience, The University of Osaka, Japan
Advanced ICT Research Institute, National Institute of Information and Communications Technology, Japan
Immunology Frontier Research Center (IFReC), The University of Osaka, Japan

Mar 25, 2026

https://doi.org/10.7554/eLife.108477.3

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Figures
Videos
Tables
Additional files

6 figures, 2 videos, 2 tables and 8 additional files

Figures

Figure 1 with 2 supplements

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Experimental design for the stall force measurement of KIF1A using an 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) (Matsubara et al., 2023) and fitted with an exponential function (Methods).

Figure 1—source data 1 Excel file containing force–extension measurements of the nanospring.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig1-data1-v1.xlsx
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Figure 1—figure supplement 1

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Purification of recombinant SNAP-tagged KIF1A and KIF5B proteins.

(A) Size-exclusion chromatogram for SNAP-tagged wild-type or mutant KIF1A dimerized motors (1–393-LZ). Elution positions of prep contaminants (void.) are noted. Dimerized KIF1A motor domain was shown by arrowheads. Below: Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) of the column elution fractions. (B) Size-exclusion SDS–PAGE NGC chromatogram showing elution profiles of KIF5B (G234A).

Figure 1—figure supplement 2

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Purification of recombinant SNAP-tagged KIF1A heterodimers.

Results of size-exclusion chromatogram for heterodimers composed of wild-type and mutant KIF1A. Elution positions of prep contaminants (void.) and heterodimerized KIF1A motor domain are noted. Below: Coomassie-stained SDS–PAGE of the column elution fractions showing dimerized KIF1A.

Figure 2

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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 $σ^{2}$ 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 of the true value of $L$ in the simulation. Note that we generated 30 artificial videos, each consisting of 300 frames. (e) Fluorescence micrographs of the DNA calibration rods (Matsubara et al., 2023) obtained in real experiments, with lengths of 398, 501, 599, and 658 nm. The scale bars indicate $2 μ m$ . (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. Note that approximately 30 videos were recorded, each containing about 300 frames for each DNA calibration rod.

Figure 2—source data 1 Excel file containing estimated lengths of fluorescence images of DNA rods for simulations and experiments.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig2-data1-v1.xlsx
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Figure 3 with 3 supplements

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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 Figure 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 $∆ t$ (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 $L_{s t a l l}$ (left panel). (b) Histogram of $L_{s t a l l}$ 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) $L_{s t a l l}$ as a function of stall duration $∆ t$ with (n = 105) and without (n = 86) PEG. The correlation coefficient ( $R$ ) between $L_{s t a l l}$ and $∆ t$ 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 $L_{s t a l l}$ and $∆ t$ with and without PEG (Mann–Whitney U test, p = 0.4718 for $L_{s t a l l}$ , p = 0.0616 for $∆ t$ ). n.s., not significant (p ≥ 0.05). The green bars indicate the median values along with the first and third quartiles.

Figure 3—source code 1 Time-course analysis of single-molecule videos.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig3-code1-v1.zip
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Figure 3—source data 1 Excel file containing the time course, $L_{s t a l l}$ and $∆ t$ from the stall force experiment of wild-type KIF1A.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig3-data1-v1.xlsx
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Figure 3—figure supplement 1

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Time course of a nanospring (NS) extension at a recording rate of 100 fps for wild-type KIF1A homodimers.

No significant difference in $L_{s t a l l}$ was observed between recordings at 33 fps (Figure 3, main text) and 100 fps. 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.

Figure 3—figure supplement 2

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Estimation of NS extensions using the Gaussian fitting method (Equation 1, main text).

The conversion from $σ_{l o n g}$ to $L_{m}^{G}$ was performed using the relation $L_{m}^{G} = 3.67 \times σ_{l o n g} - 224$ (Methods, main text). The $L_{s t a l l}$ estimated by the Gaussian fitting method was 534 ± 58 (SD) nm.

Figure 3—figure supplement 3

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Cumulative distributions of stall duration ( $∆ t$ ) in the case of the KIF1A(WT).

(a) When $∆ t \leq 20$ s, the data were fitted using $1 - e^{- k t}$ with $k = 0.17$ . (b) When fitting over a wide range of $∆ t$ , the data were fitted using $A k_{1} e^{- k_{1} t} + (1 - A) {k_{2} e}^{- k_{2} t}$ with $k_{1} = 0.26$ , $k_{2} = 0.062$ and $A = 0.44$ .

Figure 4 with 2 supplements

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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 mutations (Budaitis et al., 2021). Representative traces of NS extension are shown for homodimers and WT-mutant heterodimers of P305L, V8M, and A255V (**b–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.

Figure 4—source data 1 Excel file containing the time course data from the stall force experiment of KIF1A mutants.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig4-data1-v1.xlsx
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Figure 4—figure supplement 1

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Displacement (calculated from the extensions of NSs) for KIF1A KAND mutants.

Approximately 10 stall events were overlaid. V8M and A255V are slightly slower than WT, whereas P305L is significantly slower.

Figure 4—figure supplement 2

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Histograms of $L_{s t a l l}$ for KIF1A KAND mutants.

The time courses of the NS extensions are presented in Figure 4 of the main text. The mean values of $L_{s t a l l}$ are listed in Table 1 of the main text. Stall events: n = 58 for P305L/P305L, n = 41 for P305L/WT, n = 95 for V8M/V8M, n = 61 for V8M/WT, n = 83 for A255V/A255V, and n = 40 for A255V/WT.

Figure 5 with 1 supplement

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Comparison of $L_{s t a l l}$ and $∆ t$ among KAND mutants.

$L_{s t a l l}$ (a) and $Δ t$ (b) for homodimers and heterodimers of P305L, V8M, and A255V, compared with WT.

Figure 5—source data 1 Excel file containing $L_{s t a l l}$ and $Δ t$ from the stall force experiment of KIF1A mutants.: https://cdn.elifesciences.org/articles/108477/elife-108477-fig5-data1-v1.xlsx
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Figure 5—figure supplement 1

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Comparison between stall force and KAND severity scores.

These scores are cited from the previous study [Boyle et al., *HGG Advance* 2, 100026 (2021)].

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Videos

Video 1

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posterframe for video — Wild-type KIF1A pulling a nanospring.

The video is shown at 10× speed.

Video 2

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Tables

Table 1

Stall force of KIF1A(1–393) estimated from the average value of $L_{s t a l l}$ .

The stall force values were calculated from the mean values of $L_{s t a l l}$ by using the force–extension relationship of the NS (Figure 1d). The error of $L_{s t a l l}$ value represents the standard deviation (SD).

	Stall force (pN)	Detachment force (pN)	Termination force (pN)
WT/WT	4.7 (n = 86) ( $L_{s t a l l} = 562 \pm 48$ nm)	2.7^* 2.2^†	4 ^‡ 6 ^§
P305L/P305L	0.2 (n = 58) ( $L_{s t a l l} = 375 \pm 49$ nm)	0.7^†	-
P305L/WT	0.3 (n = 41) ( $L_{s t a l l} = 402 \pm 54$ nm)	-	-
V8M/V8M	2.2 (n = 95) ( $L_{s t a l l} = 516 \pm 57$ nm)	1.9*	-
V8M/WT	1.0 (n = 61) ( $L_{s t a l l} = 469 \pm 27$ nm)	-	-
A255V/A255V	3.0 (n = 83) ( $L_{s t a l l} = 535 \pm 46$ nm)	-	-
A255V/WT	2.5 (n = 40) ( $L_{s t a l l} = 524 \pm 26$ nm)	-	-

Detachment force and termination force measured by using optical tweezers.
*

Reported in reference (Budaitis et al., 2021).
†

Reported in reference (Lam et al., 2021).
‡

Reported in reference (Pyrpassopoulos et al., 2023) for the single-bead assay.
§

Reported in reference (Pyrpassopoulos et al., 2023) for the three-bead assay.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Recombinant DNA reagent	Plasmid pSN672: pET21a(+) human KIF1A(1–393)-LZ-6His	Anazawa et al., 2022	RRID:Addgene_177362	Template for SNAP-tag insertion
Recombinant DNA reagent	Plasmid pSN643: pET28a(+) human KIF1A(1–393)-LZ-mScarlet-I-Strep-tagII	Anazawa et al., 2022	—	Template for SNAP-tag insertion
Recombinant DNA reagent	Plasmid pSNAP-tag (T7)-2 vector	New England Biolabs	#N9181S	Template for SNAP-tag cDNA amplification
Recombinant DNA reagent	Plasmid pET21a(+) human KIF1A(1–393)-LZ-SNAP-6His	This study	—	Motor domain construct for force measurement
Recombinant DNA reagent	Plasmid pET28a(+) human KIF1A(1–393)-LZ-SNAP-Strep-tagII	This study	—	Used for heterodimer preparation
Recombinant DNA reagent	Plasmid pET21a(+) human KIF1A(1–393, P305L)-LZ-SNAP-6His	This study	—	KIF1A mutant construct associated with KAND individuals
Recombinant DNA reagent	Plasmid pET21a(+) human KIF1A(1–393, V8M)-LZ-SNAP-6His	This study	—	KIF1A mutant construct associated with KAND individuals
Recombinant DNA reagent	Plasmid pET21a(+) human KIF1A(1–393, A255V)-LZ-SNAP-6His	This study	—	KIF1A mutant construct associated with KAND individuals
Recombinant DNA reagent	Plasmid pYS05: KIF5B(1–560,G234A)::RA::SNAP::6His	Shimamoto et al., 2015	—	Inactive kinesin-5B mutant; gift from Dr. Yuta Shimamoto
Strain, strain background (Escherichia coli)	BL21(DE3)	Novagen	#69450
Peptide, recombinant protein	Tubulin (porcine brain)	Tokyo Shibaura Organ	—	Purified in-house
Peptide, recombinant protein	NeutrAvidin	Thermo Scientific	31000	DNA calibration rod immobilization
Peptide, recombinant protein	Biotinylated BSA	Thermo Scientific	29130	Surface coating
Chemical compound, drug	IPTG	Sigma-Aldrich	—	Protein expression inducer
Chemical compound, drug	Taxol	FUJIFILM Wako	163–28163	Microtubule stabilization
Chemical compound, drug	Casein	Sigma-Aldrich	C5890-500G	Surface blocking
Chemical compound, drug	BG-GLA-NHS	New England Biolabs	S9151S	SNAP-tag labeling
Chemical compound, drug	d-Desthiobiotin	Sigma-Aldrich	D1411	Strep-tag elution
Chemical compound, drug	K10–PEG5K	Alamanda Polymers	mPEG5K-b-PLKC10	DNA origami surface passivation
Sequence-based reagent	p8064 (DNA scaffold)	Tilibit Nanosystems	—	DNA origami scaffold
Sequence-based reagent	Core and handle staples (oligonucleotides)	Integrated DNA Technologies	—	Sequences in Tables S2–S7
Sequence-based reagent	DNA nanospring (DNA nanostructure)	This study	—	Designed with caDNAno
Sequence-based reagent	DNA calibration rod (DNA nanostructure)	Matsubara et al., 2023	—	Used for force calibration
Software, algorithm	caDNAno	Douglas et al., 2009	—	DNA origami design
Software, algorithm	High Speed Recording	Hamamatsu Photonics	—	Video acquisition
Software, algorithm	Python	Python Software Foundation	RRID:SCR_008394	Version 3.11.3
Software, algorithm	Chain fitting analysis	This study	—	Custom Python code using scipy.optimize

Additional files

Supplementary file 1 Plasmid list. Listed below are the plasmids used in this study.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp1-v1.docx
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Supplementary file 2 Handle staples and anti-handles used to link KIF1A and inert KIF5B at the end of NSs. In the sequences of staples handle 32A and 32B, italicized regions indicate single-stranded DNA (ssDNA) handle sequences.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp2-v1.docx
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Supplementary file 3 Core staples and handle staples for Cy3 to build the NS. Sequences in italics indicate the handle site.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp3-v1.docx
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Supplementary file 4 Antihandle carrying Cy3 to label the NS.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp4-v1.docx
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Supplementary file 5 Core staples to build the DNA calibration rod.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp5-v1.docx
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Supplementary file 6 Handle staples for the DNA calibration rod. Sequences in italics indicate the handle site.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp6-v1.docx
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Supplementary file 7 Antihandles for DNA calibration rod.: https://cdn.elifesciences.org/articles/108477/elife-108477-supp7-v1.docx
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MDAR checklist: https://cdn.elifesciences.org/articles/108477/elife-108477-mdarchecklist1-v1.pdf
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Nobumichi Takamatsu
Hiroko Furumoto
Takayuki Ariga
Mitsuhiro Iwaki
Kumiko Hayashi

(2026)

Stall force measurement of the kinesin-3 motor KIF1A using a programmable DNA origami nanospring

eLife 14:RP108477.

https://doi.org/10.7554/eLife.108477.3

Figures

Experimental design for the stall force measurement of KIF1A using an NS.

Figure 1—source data 1

Purification of recombinant SNAP-tagged KIF1A and KIF5B proteins.

Purification of recombinant SNAP-tagged KIF1A heterodimers.

Length estimation using DNA calibration rods.

Figure 2—source data 1

Stall force measurement of wild-type KIF1A homodimers using NSs.

Figure 3—source code 1

Figure 3—source data 1

Time course of a nanospring (NS) extension at a recording rate of 100 fps for wild-type KIF1A homodimers.

Estimation of NS extensions using the Gaussian fitting method (Equation 1, main text).

Cumulative distributions of stall duration ( $∆ t$ ) in the case of the KIF1A(WT).

Stall force measurement of KAND mutants KIF1A homodimers and heterodimers using NSs.

Figure 4—source data 1

Displacement (calculated from the extensions of NSs) for KIF1A KAND mutants.

Histograms of $L_{s t a l l}$ for KIF1A KAND mutants.

Comparison of $L_{s t a l l}$ and $∆ t$ among KAND mutants.

Figure 5—source data 1

Comparison between stall force and KAND severity scores.

Videos

Wild-type KIF1A pulling a nanospring.

The P305L mutant pulling a nanospring.

Tables

Stall force of KIF1A(1–393) estimated from the average value of $L_{s t a l l}$ .

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

MDAR checklist

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Experimental design for the stall force measurement of KIF1A using an NS.

Figure 1—source data 1

Purification of recombinant SNAP-tagged KIF1A and KIF5B proteins.

Purification of recombinant SNAP-tagged KIF1A heterodimers.

Length estimation using DNA calibration rods.

Figure 2—source data 1

Stall force measurement of wild-type KIF1A homodimers using NSs.

Figure 3—source code 1

Figure 3—source data 1

Time course of a nanospring (NS) extension at a recording rate of 100 fps for wild-type KIF1A homodimers.

Estimation of NS extensions using the Gaussian fitting method (Equation 1, main text).

Cumulative distributions of stall duration (∆t) in the case of the KIF1A(WT).

Stall force measurement of KAND mutants KIF1A homodimers and heterodimers using NSs.

Figure 4—source data 1

Displacement (calculated from the extensions of NSs) for KIF1A KAND mutants.

Histograms of Lstall for KIF1A KAND mutants.

Comparison of Lstall and ∆t among KAND mutants.

Figure 5—source data 1

Comparison between stall force and KAND severity scores.

Wild-type KIF1A pulling a nanospring.

The P305L mutant pulling a nanospring.

Stall force of KIF1A(1–393) estimated from the average value of Lstall.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

Supplementary file 6

Supplementary file 7

MDAR checklist

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Cumulative distributions of stall duration ( $∆ t$ ) in the case of the KIF1A(WT).

Histograms of $L_{s t a l l}$ for KIF1A KAND mutants.

Comparison of $L_{s t a l l}$ and $∆ t$ among KAND mutants.

Stall force of KIF1A(1–393) estimated from the average value of $L_{s t a l l}$ .