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Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules

Nature Chemical Biology volume 20, pages 521–529 (2024)Cite this article

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Abstract

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1–stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

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Fig. 1: Lis1 binding reduces the velocity and increases the run time of dynein.
Fig. 2: Lis1 slows dynein motility in the absence of its interaction with microtubules.
Fig. 3: Lis1 does not affect the stall force of dynein.
Fig. 4: Lis1 reduces the asymmetry in the F–V behavior of dynein in the presence of ATP.
Fig. 5: Lis1 reduces the asymmetry in F–V of dynein in the absence of ATP.
Fig. 6: Mutagenesis of the Lis1-binding site on the dynein stalk recovers the asymmetry in F–V of dynein.

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Data availability

A reporting summary for this article is available as a Supplementary Information file. The main data supporting the findings of this study are available within the article and its Extended Data figures. Protocols that support the findings of this study can be found in Methods. Yeast strains and raw data will be made available by the corresponding authors upon request. Source data are provided with this paper.

Code availability

The custom code used to analyze experimental data is uploaded to the Yildiz Lab code repository (www.yildizlab.org/code_repository) and GitHub (https://github.com/Yildiz-Lab/YFIESTA).

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Acknowledgements

We thank S. Can, M. ElShenawy, L. S. Ferro, J. T. Canty and other members of the Yildiz and Reck-Peterson laboratories for helpful discussions, and S. M. Markus for sharing the yeast strain that expresses dyneinGal. This work was supported by grants from the National Institute of General Medical Sciences (GM094522, A.Y.), the National Science Foundation (MCB-1617028 and MCB-1055017, A.Y.) and the Fellowship of the Ministry of Education of the Turkish Republic (E.K.). S.L.R.-P. is a Howard Hughes Medical Institute Investigator and is also supported by the National Institutes of Health (1R35GM141825).

Author information

Authors and Affiliations

  1. Graduate Group in Biophysics, University of California Berkeley, Berkeley, CA, USA

    Emre Kusakci & Ahmet Yildiz

  2. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

    Zaw Min Htet, Yuanchang Zhao & Ahmet Yildiz

  3. Physics Department, University of California Berkeley, Berkeley, CA, USA

    Yuanchang Zhao & Ahmet Yildiz

  4. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

    John P. Gillies & Samara L. Reck-Peterson

  5. Department of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA

    Samara L. Reck-Peterson

  6. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Samara L. Reck-Peterson

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  1. Emre Kusakci
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Contributions

E.K., S.L.R.-P. and A.Y. conceived the study and designed the experiments. Z.M.H. generated the yeast constructs for dynein and Lis1. E.K. purified the proteins. E.K. and Y.Z. performed single-molecule experiments and analyzed the data. J.P.G. performed experiments on monomeric Lis1. E.K., S.L.R.-P. and A.Y. wrote the manuscript. All authors read and revised the manuscript.

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Correspondence to Ahmet Yildiz.

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Nature Chemical Biology thanks Richard McKenney and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Single molecule motility of dynein in the presence of Lis1.

a) Representative kymographs of TMR-dynein with increasing concentrations of unlabeled Lis1. b) (Left) 1-CDF of run time under different Lis1 concentrations. Fitting to a double exponential decay (solid curves) reveals the weighted average of run time in each condition. (Right) The weighted average of run time under increasing Lis1 concentrations (± s.e.; N = 861, 534, and 312 from left to right). c) (Left) 1-CDF of run length under different Lis1 concentrations. Solid curves represent a fit to a double exponential decay. (Right) The weighted average of run length in each condition (bar graphs, ± s.e.; N = 861, 534, and 312 from left to right). d) An example kymograph shows that the transient binding of Lis1 slows whereas the subsequent release of Lis1 restores the velocity (arrowheads). In b and c, P values were calculated by the two-tailed Kolmogorov-Smirnov test.

Source data

Extended Data Fig. 2 Lis1 interacts with the microtubule lattice through its dynein binding site in vitro.

a) Microtubule co-pelleting assay with Lis1WT and Lis15A (mean ±s.e.m., three replicates per condition). The solid curve represents a fit to a binding isotherm to determine KD (±s.e.; N.D.: not determined). The statistical analysis was performed using an extra sum-of-squares F test (p = 10−4). b) The subtilisin treatment of microtubules reduces the molecular weight of tubulin in a denaturing gel (three independent experiments). c) The structure of Lis1 bound to the AAA ring with the five residues mutated in Lis15A are shown as spheres and all lysine and arginine residues are in blue (Protein Data Bank: 7MGM15). d) Representative kymographs of dynein in the presence and absence of Lis15A. Assays were performed in 50 mM KAc. e) The velocity, run time, and run length of dynein in the presence and absence of Lis15A (N = 326, 534, and 336 from left to right). The center line and whiskers represent the mean and s.d., respectively. P values were calculated by a two-tailed t-test with Welch correction for velocity and by the two-tailed Kolmogorov-Smirnov test for run time and run length. f) Surface-immobilized microtubules were decorated by 100 nM TMR-Lis1 before and after removing unbound Lis1 in the flow chamber. The assay was performed in the absence of added salt to maximize the Lis1-microtubule interaction. Washing the chamber with buffer and introducing dynein reduces 92% of the Lis1 signal on microtubules (three independent experiments).

Source data

Extended Data Fig. 3 Motility of dyneinGal in the presence of unlabeled Lis1.

a) Representative kymographs of dyneinGal with increasing concentrations of unlabeled Lis1. Assays were performed in 1 mM ATP and 150 mM KAc. b) The velocity of dyneinGal under different Lis1 concentrations (mean ± s.e.m.; N = 611, 694, 230, 260, 251, and 852 from left to right; two biological replicates). c) 1-CDF of run time and run length of dyneinGal under different Lis1 concentrations. Fitting to a double exponential decay (solid curves) reveals the weighted average of run time and run length of the motor in each condition (bar graphs, ± s.e.; N = 611, 694, and 852 from left to right). d) Representative kymographs of dyneinGal in the presence or absence of 1,000 nM unlabeled Lis15A. e) The velocity, run time, and run length of dyneinGal in the presence or absence of 1,000 nM Lis15A (N = 610 and 209 from left to right). The center line and whiskers represent the mean and s.d., respectively. In c and e, P values were calculated by a two-tailed t-test with Welch correction for velocity and by the two-tailed Kolmogorov-Smirnov test for run time and run length.

Source data

Extended Data Fig. 4 Motility of dyneinGal in the presence of fluorescently-labeled Lis1.

a) Representative kymographs of two-color imaging of LD655-labeled dyneinGal and LD555-labeled Lis1 under different Lis1 concentrations. b) The velocity, run time, and run length distributions of dyneinGal under different concentrations of Lis1 (N = 611, 526, 168, 233, 175, 55, 69, 126, and 145 from left to right). c) The fraction of LD655-dyneinGal that colocalizes with LD555-Lis1 under different Lis1 concentrations. A fit to a binding isotherm function (solid curve, see Methods) reveals KD (±s.e.). d) The velocity distribution of dyneinGal in the absence of Lis1 compared to the motors that do not colocalize with Lis1 when fluorescently labeled Lis1 is present in the chamber (N = 611 and 233 from left to right). e) Representative kymographs show colocalization of 5 nM TMR-labeled and 5 nM Cy5-labeled Lis1 to unlabeled dyneinGal. f) Velocity, run length, and run time distributions of dyneinGal in the absence (light gray) and presence of Lis1 (N = 287, 233, 175, and 274 from left to right). DyneinGal motors that colocalize with 0, 1, or 2 colors of Lis1 were analyzed separately. In b, d, and f, the center line and whiskers represent the mean and s.d., respectively. P values were calculated by two-tailed t-tests with Welch correction for velocity and by the two-tailed Kolmogorov-Smirnov test for run time and run-length measurements.

Source data

Extended Data Fig. 5 The addition and removal of Lis1 whilst recording dynein motility.

a) A kymograph (left) and velocity (right) of dynein before and after Lis1 was flown into the chamber (red arrowhead and a dashed line, N = 44 and 42 from left to right). b) A kymograph of dyneinGal when Lis1 was flown into the chamber (red arrowhead and a dashed line). (Middle) The velocity of the complexes that were already walking on the microtubules during Lis1 addition moved at the same velocity after flowing Lis1 (N = 26 and 26 from left to right). Only the motors that do not colocalize with Lis1 were included in the analysis. (Right) The velocity of the complexes that land onto microtubules within 4 min after Lis1 addition was analyzed (N = 122 and 84 from left to right). The motors that colocalize with Lis1 walked slower than motors that do not colocalize with Lis1. c) A kymograph (left) and velocity (right) of dyneinGal before and after washing excess Lis1 from the flow chamber (red arrowhead and a dashed line). The velocity of the motors that colocalize (+Lis1) or do not colocalize (−Lis1) with Lis1 remained unaltered by removing excess Lis1 from the chamber (N = 9, 9, 22, 22 from left to right). In a, b, and c, assays were performed in 1 mM ATP and 50 mM KAc. The center line and whiskers represent the mean and s.d., respectively. P values were calculated by a two-tailed t-test with Welch correction.

Source data

Extended Data Fig. 6 Lis1 reduces the stall force of the tail-truncated dynein.

a) Representative trajectories of beads driven by tail-truncated dynein constructs dimerized with a Glutathione S-transferase (GST) tag (GFP-GST-Dyn331kDa and GFP-GST-Dyn314kDa) in the presence and absence of 300 nM Lis1. Assays were performed in 2 mM ATP. Red arrowheads represent the detachment of the motor from the microtubule followed by the snapping back of the bead to the trap center. b) Stall force histograms of Dyn331kDa and Dyn314kDa in the presence and absence of 300 nM Lis1 (mean ± s.e.m.; p = 2 × 10−7 for Dyn331kDa and 0.006 for Dyn314kDa, two-tailed t-test). c) Stall times of Dyn331kDa and Dyn314kDa in the presence and absence of 300 nM Lis1. Fitting to a double exponential decay (solid curves) reveals the weighted average of stall time (±s.e.).

Source data

Extended Data Fig. 7 Lis1 slows the motility but does not substantially affect the stall force of dyneinEQN.

a) Representative kymographs of dyneinEQN motility under different concentrations of unlabeled Lis1. b) The velocity of dyneinEQN motility under different concentrations of unlabeled Lis1 (mean ± s.e.m.; N = 659, 283, 482, 484, 487, 610, and 132 from left to right). The fit of the dyneinEQN velocity data (solid curve) reveals KD (±s.e., see Methods). c) A representative kymograph of a three-color imaging assay shows two Lis1s bind to the same dyneinEQN motor (white arrows). d) The velocities of dyneinEQN motors not colocalizing (-Lis1) or colocalizing (+Lis1) with Lis1 (N = 98 and 92 from left to right). The center line and whiskers represent the mean and s.d., respectively. The P-value was calculated by a two-tailed t-test with Welch correction. e) Representative trajectories of beads driven by dyneinEQN in the presence or absence of 900 nM Lis1 in a fixed trapping assay. Assays were performed in 2 mM ATP. Red arrowheads represent the detachment of the motor from the microtubule followed by the snapping back of the bead to the trap center. f) Stall force histograms of dyneinEQN in the presence and absence of 900 nM Lis1 (mean ± s.e.m.; p = 10−3, two-tailed t-test). g) Stall times of dyneinEQN in the presence and absence of 900 nM Lis1. Fitting to a double exponential decay (solid curves) reveals the weighted average of stall time (±s.e.).

Source data

Extended Data Fig. 8 The comparison of the F-V behavior of dynein and dyneinEQN in the presence and absence of Lis1.

(Left) F-V measurements of dynein and dyneinEQN in the absence of Lis1 (mean ±s.e.m., from left to right, N = 41, 38, 37, 861, 27, 52, 106, 28, 51, 60, 89, 115, 124, 62, 23, 38, 40 for dynein and N = 31, 66, 62, 48, 47, 26, 42, 39, 23, 45, 33 for dyneinEQN). (Right) F-V measurements of dynein in 300 nM Lis1 compared to F-V of dyneinEQN in 900 nM Lis1 (mean ±s.e.m., from left to right, N = 34, 52, 25, 312, 33, 42, 49, 37 62, 47 33 42, 36, 29, 18 for dynein; and N = 54, 36, 61, 31, 37, 17, 54, 14, 19, 38, 26 for dyneinEQN). Dynein velocity under assisting forces is lower than that of dyneinEQN in the presence of Lis1. Assays were performed in 2 mM ATP.

Source data

Supplementary information

Supplementary Information

Supplementary Table 1: The list of S. cerevisiae strains used in this study.

Reporting Summary

Supplementary Video 1

Colocalization of two Lis1s to the same dynein dimer: The three-color video shows GFP-dynein colocalized with both LD655-Lis1 and TMR-Lis1 as it moves along the microtubule (arrows). 5 nM final concentration of dynein is incubated on ice with 2 nM TMR-Lis1 and 2 nM LD655-Lis1 for 10 min. The videos were acquired on a time-shared TIRF microscope with a 108 nm effective pixel size and 0.3 s per frame per channel. The videos were analyzed using FIJI. The assay was performed in 1 mM ATP.

Supplementary Video 2

Lis1 binding pauses dynein motility and its unbinding restores dynein velocity: The two-color video shows the binding and unbinding of TMR-Lis1 to Cy5-dynein during processive motility. The red arrows show the position of dynein, whereas the cyan arrows show the Lis1 binding. Lis1 is introduced to the imaging chamber via a microfluidic system as dynein motors walk along the microtubule. The videos were acquired on a time-shared TIRF microscope with a 160 nm effective pixel size and 0.65 s per frame per channel. The videos were analyzed using FIJI. The assay was performed in 1 mM ATP.

Supplementary Video 3

Cy5-dynein moves along the microtubules in the absence of Lis1: Cy5-dynein walking along the microtubules in 1 mM ATP. The assay was performed in the absence of added salt or Lis1. The videos were acquired on a TIRF microscope with a 160 nm effective pixel size and 0.5 s per frame channel time resolution.

Supplementary Video 4

Cy5-dynein moves on a Lis1-decorated microtubule: The two-color video shows that Cy5-dynein motility is not slowed by the presence of Lis1 on the microtubule. 100 nM TMR-Lis1 is flowed into the chamber and allowed to decorate surface-immobilized microtubules in the absence of added salt. The chamber is washed twice with the stepping buffer to remove unbound Lis1 and 5 nM Cy5-dynein was added to the chamber in 1 mM ATP without added salt. The videos were collected on a TIRF microscope with a 160 nm effective pixel size and 0.65 s per frame per channel. The videos were analyzed using FIJI.

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Kusakci, E., Htet, Z.M., Zhao, Y. et al. Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules. Nat Chem Biol 20, 521–529 (2024). https://doi.org/10.1038/s41589-023-01464-6

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