Experimental Design and Raw Data from Motor-DNA Tensiometers.

(A) Schematic of a motor-DNA tensiometer, consisting of a dsDNA (burgundy) connected on one end to a kinesin motor through a complimentary oligo (blue), and on the other end to the MT using biotin-avidin (tan and gray, respectively). A Qdot functionalized with GFP binding protein nanobodies is attached to the motor’s GFP tag and used to track motor position. (Not to scale; motor and Qdot are both ∼20 nm and DNA is ∼1 micron) (B) Predicted force extension curve for a worm-like chain 3009 bp dsDNA based on a 50 nm persistence length. (C) Representative kymographs of motor-DNA tensiometers for kinesins-1, -2 and -3. (D) Enlarged kymograph showing diffusion around the origin, ramp, and stall. (E) Example distance vs. time trace (kinesin-3), highlighting detached durations (red), ramps and stalls (black) where the motor has pulled the DNA taut, and transient slips during stall (red). (F-H) Representative distance vs. time plots for kinesin-1 (F), kinesin-2 (G) and kinesin-3 (H), corresponding to the kymographs in (C). Further examples are shown in Figure S3.

Tensiometer Stall Durations Indicate Catch-bond Behavior for Kinesin-1 and -2.

Tensiometer stall durations are plotted for A) kinesin-1 (blue), (B) kinesin-2 (purple), and (C) kinesin-3 (green). Unloaded run durations for each motor are plotted in gray. Distributions were fit with a single exponential function using MEMLET to generate time constants, representing the mean durations. (D) Comparison of unloaded and stall durations for the three motors, with error bars indicating 95% CI. Stall durations >20s were excluded from the fit (three events for kinesin-1 and two events for kinesin-2). Bi- exponential fits of all data including >20 s are shown in Figure S5.

During Ramps, Kinesin-3 Detaches More Readily Than Under Zero Load.

Unloaded, ramp, and stall duration parameters were estimated using a Markov process model, coupled with Bayesian inference methods. Curves show the posterior probability distributions of the duration parameters for (A) kinesin-1, (B) kinesin-2 and (C) kinesin-3. Bars below each peak indicate the 95% credible regions for the ramp (green), unloaded (gray) and stall (blue) duration parameters. Notably, the estimated ramp durations are larger, the same, and smaller than the unloaded run durations for kinesin-1, -2, and -3, respectively. For the unloaded and stall durations, this estimation method produces almost identical values as the maximum likelihood estimates in Figure 2 (values provided in Table S2).

Restart Kinetics for Kinesin-1, -2 and -3.

(A) Fraction of slip, fast rebinding, and slow rebinding events for each motor, with example kymographs for each (top; scale bars are 0.5 μm and 0.2 s). Solid colors indicate slips during stall, where the motor resumes a new ramp within a single frame (∼40 ms), crosshatching indicates rapid reattachment events (100 ms) following fall to baseline, and open bars indicate slow reattachment events with >100 ms fluctuations around baseline. (B) Kinesin-3 stall durations, with unloaded run times in gray, stall durations terminated by slips in light green, and stall durations terminated by falling to the baseline (ignoring slips) in dark green. Unloaded and stall durations (replotted from Figure 2) were fit with single exponential functions in MEMLET. Stall durations ignoring slips were fit with a bi-exponential by least squares (ρ1 = 2.01 s [95% CI: 1.52, 2.35 s], A1 = 0.66 s [0.54, 0.83 s], ρ2 = 11.0 s [9.18, 13.70], A2 = 0.33 [0.23, 0.48]). Weighted average of the two time constants is displayed in plot for comparison to other time constants. Similar results for kinesin-1 and -2 are shown in Figure S7. (C) Comparison of stall durations for kinesins -1, -2 and -3 with slips observed as stall terminations or ignored. (D-F) Distribution of restart times for each motor fit to a tri-exponential (least squares). Confidence intervals of parameters determined by bootstrapping with 1000 iterations are given in Table S3.

Chemomechanical Model of Proposed Catch-bond Mechanism.

(A) Diagram of kinesin chemomechanical cycle model consisting of strongly- and weakly-bound states that make up the stepping cycle, and slip and detached states that terminate runs and stalls. Note that two pathways of detachment from the slip state (and reattachment) are incorporated into the model, but only one pathway is shown for simplicity (see Supplementary Methods for details). (B) Table of rate constants used to simulate unloaded and stall durations and restarting times. All rate constants are derived from fits to experimental data, as described in Supplemental Methods. kS-W and kslip depended exponentially on load (𝑘(𝐹) = 𝑘!𝑒#$%) with 8 for kS-W of -2.7, -2.4, and -3.6 nm and 8 for kslip of 1.6, 1.3 and 2.7 nm for kinesin-1, -2 and -3, respectively; see also Figure S8A). (C-E) Experimental (symbols) and simulated (lines) unloaded and stall durations. 10,000 events were simulated for each condition and plotted with minimum cutoffs matching experiments. Kinesin-3 ramp durations were taken from parameter estimated in Figure 3. (F-H) Experimental (symbols) and simulated (lines) restart times.