Enhanced Processivity and Collective Force Production of Kinesins at Low Radial Forces

Reviewer #1 (Public review):

Summary:

The manuscript by Hensley and Yildez studies the mechanical behavior of kinesin under conditions where the z-component of the applied force is minimized. This is accomplished by tethering the kinesin to the trapped bead with a long double-stranded DNA segment as opposed to directly binding the kinesin to the large bead. It complements several recent studies that have used different approaches to looking at the mechanical properties of kinesin under low z-force loads. The study shows that much of the mechanical information gleaned from the traditional "one bead" with attached kinesin approach was probably profoundly influenced by the direction of the applied force. The authors speculate that when moving small vesicle cargos (particularly membrane-bound ones), the direction of resisting force on the motor has much less of a z-component than might be experienced if the motor were moving large organelles like mitochondria.

Strengths:

The approach is sound and provides an alternative method to examine the mechanics of kinesin under conditions where the z-component of the force is lessened. The data show that kinesin has very different mechanical properties compared to those extensively reported using the "single-bead" assay, where the molecule is directly coupled to a large bead, which is then trapped.

Weaknesses:

My primary concern is that in some of the studies, there are not enough data points to be totally convincing. This is particularly apparent in the low z-force condition of Figure 1C and in Figure 2B.

The substoichiometric binding of kinesins to multivalent DNA complicates the interpretation of the data.

Reviewer #2 (Public review):

This short report by Hensley and Yildiz explores kinesin-1 motility under more physiological load geometries than previous studies. Large Z-direction (or radial) forces are a consequence of certain optical trap experimental geometries, and likely do not occur in the cell. Use of a long DNA tether between the motor and the bead can alleviate Z-component forces. The authors perform three experiments. In the first, they use two assay geometries - one with kinesin attached directly to a bead and the other with kinesin attached via a 2 kbp DNA tether - with a constant-position trap to determine that reducing the Z component of force leads to a difference in stall time but not stall force. In the second, they use the same two assay geometries with a constant-force trap to replicate the asymmetric slip bond of kinesin-1; reducing the Z component of force leads to a small but uniform change in the run lengths and detachment rates under hindering forces but not assisting forces. In the third, they connect two or three kinesin molecules to each DNA, and measure a stronger scaling in stall force and time when the Z component of force is reduced. They conclude that kinesin-1 is a more robust motor than previously envisaged, where much of its weakness came from the application of axial force. If forces are instead along the direction of transport, kinesin can hold on longer and work well in teams. The experiments are rigorous, and the data quality is very high. There is little to critique or discuss. The improved dataset will be useful for modeling and understanding multi-motor transport. The conclusions complement other recent works that used different approaches to low-Z component kinesin force spectroscopy, and provide strong value to the kinesin field.

Major comments:

(1) Kinesin-1 is covalently bound to a DNA oligo, which then attaches to the DNA chassis by hybridization. This oligo is 21 nt with a relatively low GC%. At what force does this oligo unhybridize? Can the authors verify that their stall force measurements are not cut short by the oligo detaching from the chassis?

(2) Figure 1, a justification or explanation should be provided for why events lower than 1.5 pN were excluded. It appears arbitrary.

(3) Figure 2b, is the difference in velocity statistically significant?

(4) The number of measurements for each experimental datapoint in the corresponding figure caption should be provided. SEM is used without, but N is not reported in the caption.

Reviewer #3 (Public review):

Summary:

Hensley et al. present an important study into the force-detachment behaviour of kinesin-1, the most well-characterised motor protein. One of the key techniques used to characterise kinesins is in vitro optical trapping of purified proteins, which has provided remarkable insights into the biochemical and mechanical mechanisms of motor proteins under single- and multi-motor conditions. This study presents an adapted (from Urbanska et al.) methodological approach of DNA-tethering kinesin-1 to a bead, both under single- and multi-motor conditions, which is then trapped to characterise the run length, processivity, and stall behaviour under unloaded and loaded (both assisting and hindering) conditions. The new approach reduces the vertical or z-force and thus provides insights into the role of horizontal or x-forces acting on the motor. Based on their method of imposing dominant horizontal forces on the motor and their data, they conclude that kinesin-1 exhibits a higher asymmetry in its force-detachment kinetics, is less slippery, and exhibits slip-bond behaviour, particularly under hindering loads. Under assisting loads, similar slip-bond kinetics ensue, but detachment from the microtubule is far more sensitive. To demonstrate the implications of their method and data, they conduct a multi-motor assay and show that multiple kinesin-1 motors can generate significantly higher forces, almost proportional to motor number. Overall, this is important work, and the data are compelling.

Strengths:

The method of DNA-tethered motor trapping is effective in reducing vertical forces and can be easily optimised for other motors and protein characterisation. The major strength of the paper is characterising kinesin-1 under low z-forces, which is likely to reflect the physiological scenario. They report that kinesin-1 is more robust and less prone to premature detachment. The motors exhibit higher stall rates and times. Under hindering and assisting loads, kinesin-1 detachment is more asymmetric and sensitive, and with low z-force shows that slip-behaviour kinetics prevail. Another achievement of this paper is the demonstration of the multi-motor kinesin-1 assay using their low-z force method, showing that multiple kinesin-1 motors are capable of generating higher forces (up to 15 pN, and nearly proportional to motor number), thus opening an avenue to study multiple motor coordination.

Weaknesses:

The method of DNA-tethered motor trapping to enable low z-force is not entirely novel, but adapted from Urbanska (2021) for use in conventional optical trapping laboratories without reliance on microfluidics. However, I appreciate that they have fully established it here to share with the community. The authors could strengthen their methods section by being transparent about protein weight, protein labelling, and DNA ladders shown in the supplementary information. What organism is the protein from? Presumably human, but this should be specified in the methods. While the figures show beautiful data and exemplary traces, the total number of molecules analysed or events is not consistently reported. Overall, certain methodological details should be made sufficient for reproducibility.

The major limitation the study presents is overarching generalisability, starting with the title. I recommend that the title be specific to kinesin-1. The study uses two constructs: a truncated K560 for conventional high-force assays, and full-length Kif5b for the low z-force method. However, for the multi-motor assay, the authors use K560 with the rationale of preventing autoinhibition due to binding with DNA, but that would also have limited characterisation in the single-molecule assay. Overall, the data generated are clear, high-quality, and exciting in the low z-force conditions. But why have they not compared or validated their findings with the truncated construct K560? This is especially important in the force-feedback experiments and in comparison with Andreasson et al. and Carter et al., who use Drosophila kinesin-1. Could kinesin-1 across organisms exhibit different force-detachment kinetics? It is quite possible. Similarly, the authors test backward slipping of Kif5b and K560 and measure dwell times in multi-motor assays. Why not detail the backward slippage kinetics of Kif5b and any step-size impact under low z-forces? For instance, with the traces they already have, the authors could determine slip times, distances, and frequency in horizontal force experiments. Overall, the manuscript could be strengthened by analysing both constructs more fully.

Appraisal and impact:

This study contributes to important and debated evidence on kinesin-1 force-detachment kinetics. The authors conclude that kinesin-1 exhibits a slip-bond interaction with the microtubule under increasing forces, while other recent studies (Noell et al. and Kuo et al.), which also use low z-force setups, conclude catch-bond behaviour under hindering loads. I find the results not fully aligned with their interpretation. The first comparison of low z-forces in their setup with Noell et al. (2024), based on stall times, does not hold, because it is an apples-to-oranges comparison. Their data show a stall time constant of 2.52 s, which is comparable to the 3 s reported by Noell et al., but the comparison is made with a weighted average of 1.49 s. The authors do report that detachment rates are lower in low z-force conditions under unloaded scenarios. So, to completely rule out catch-bond-like behaviour is unfair. That said, their data quality is good and does show that higher hindering forces lead to higher detachment rates. However, on closer inspection, the range of 0-5 pN shows either a decrease or no change in detachment rate, which suggests that under a hindering force threshold, catch-bond-like or ideal-bond-like behaviour is possible, followed by slip-bond behaviour, which is amazing resolution. Under assisting loads, the slip-bond character is consistent, as expected. Overall, the study contributes to an important discussion in the biophysical community and is needed, but requires cautious framing, particularly without evidence of motor trapping in a high microtubule-affinity state rather than genuine bond strengthening.