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Lis1 activates dynein motility by modulating its pairing with dynactin

Nature Cell Biology volume 22, pages 570–578 (2020)Cite this article

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Abstract

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.

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Fig. 1: Lis1 increases the stepping rate of dynein–dynactin.
Fig. 2: Lis1 increases the force production of dynein–dynactin.
Fig. 3: Lis1 does not affect the force generation and velocity of single dynein complexed to dynactin and a cargo adaptor.
Fig. 4: Lis1 favours the recruitment of two dyneins to dynactin.
Fig. 5: Lis1 binding decreases the velocity of dynein–dynactin.
Fig. 6: Lis1 promotes assembly of the dynein transport machinery.

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Data availability

All data that support the conclusions are available from the authors on request. Source data for Figs. 1–6 and Extended Data Figs. 1–7 are presented with the paper.

Code availability

Codes used in this paper are available from the corresponding author upon request.

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Acknowledgements

We thank the members of the Yildiz laboratory for helpful discussions and V. Madan (Medical Research Council) for sharing unpublished results. This work was funded by grants from the National Institutes of Health (GM094522) and National Science Foundation (MCB-1055017 and MCB-1617028) to A.Y., the Medical Research Council (MC_U105178790) to S.L.B. and the Deutsche Forschungsgemeinschaft research fellowship (BA5802/1–1) to J.B.

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  1. Mohamed M. Elshenawy

    Present address: Quantum Biosystems, Menlo Park, CA, USA

Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, CA, USA

    Mohamed M. Elshenawy & Ahmet Yildiz

  2. Biophysics Graduate Group, University of California at Berkeley, Berkeley, CA, USA

    Emre Kusakci, Sara Volz & Ahmet Yildiz

  3. MRC Laboratory of Molecular Biology, Division of Cell Biology, Cambridge, UK

    Janina Baumbach & Simon L. Bullock

  4. Physics Department, University of California at Berkeley, Berkeley, CA, USA

    Ahmet Yildiz

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Contributions

M.M.E., J.B., S.L.B. and A.Y. conceived the study and designed the experiments. M.M.E. purified dynein, dynactin and cargo adaptors. J.B. purified Lis1 proteins. M.M.E. and E.K. labelled the proteins with DNA and fluorescent dyes and performed the single-molecule motility experiments. M.M.E. and E.K. performed fluorescent tracking assays. M.M.E., S.V. and E.K. performed optical-trapping assays. M.M.E., S.L.B. and A.Y. wrote the manuscript, and all authors read and edited the manuscript.

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Correspondence to Ahmet Yildiz.

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Extended data

Extended Data Fig. 1 Lis1 increases the velocity of complexes assembled with wtDyn.

a, Assembly of wtDDB and wtDDR. b, Velocity distribution of wtDDB and wtDDR complexes assembled in the presence and absence of 600 nM Lis1. The line and whiskers represent the mean and SD, respectively. From left to right, n = 106, 72, 75, and 81, and mean values are 538, 718, 924, 1113 nm s-1 (three independent experiments). p-values are calculated from a two-tailed t-test. c, Velocity distribution of complexes assembled with wtDyn and mtDyn in the absence of Lis1. The line and whiskers represent the mean and SD, respectively. From left to right, n = 106, 132, 75, and 307, and mean values are 538, 652, 924, 1155 nm s−1 (three independent experiments). p-values are calculated from a two-tailed t-test. d, The percentage of processive wtDDB complexes that are dual-labeled when an equimolar mixture of TMR- and LD650-dynein motors were assembled with dynactin and BicD2N in the absence of Lis1 (mean ± SEM, n = 246 and 178 from left to right). Error bars represent SE calculated from multinomial distribution and the p-value is calculated from the two-tailed z-test. 

Source data

Extended Data Fig. 2 Step analysis of mtDDB in the presence and absence of Lis1.

a, Additional examples of mtDDB stepping in the presence and absence of 600 nM Lis1. b, The average size of steps taken in forward (µf), backward, (µb), and both (µcum) directions along the longitudinal axis of the MT. Error bars are SEM. In a and b, six independent experiments were performed per condition. c, Stepping rates estimated from the exponential fit in Fig. 1f. Error bars are SE of the fit. In b and c, p values are calculated from a two-tailed t-test; sample size (n) distribution of data are provided in Fig. 1f. 

Source data

Extended Data Fig. 3 Lis1 does not increase the stall duration of dynein bound to dynactin and a cargo adaptor.

a, Inverse cumulative distribution of stall durations in the absence and presence of 600 nM Lis1. Solid curves represent fitting to a two-exponential decay (decay time ± SE). b, Mean stall times of mtDDB and mtDDR in absence and presence of 600 nM Lis1 (± SEM). p values are calculated from a two-tailed t-test. In a and b, n = 53, 27, 50, and 39 from left to right, four independent experiments per condition. 

Source data

Extended Data Fig. 4 Lis1 does not affect stall time and stepping rate of single dynein bound to dynactin.

a, Distribution of dwell times between consecutive steps along the longitudinal axis of the MT. A fit to an exponential decay reveals the decay rate (rate ± SE, n = 734 for mtDTR-Lis1 and 724 for mtDTR+Lis1). b, Inverse cumulative distribution of stall durations of mtDTR in the presence and absence of 600 nM Lis1. Solid curves represent fitting to a two-exponential decay (decay time ± SE, n = 118 for mtDTR-Lis1 and 100 for mtDTR+Lis1, three independent experiments). 

Source data

Extended Data Fig. 5 Lis1 does not stimulate the recruitment of dynein tail to dynactin.

Representative kymographs show the motility of LD650-Dyn and TMR-DynLT assembled with BicD2N or BicDR1 in the presence and absence of 600 nM Lis1. White arrows point to complexes that contain both LD650-mtDyn and TMR-DynLT (three independent experiments were performed per condition).

Extended Data Fig. 6 Additional examples of binding events of Lis1 to mtDDB and mtDTR during processive movement.

a, Schematic depiction of mtDDB complex assembled in the presence of TMR-Lis1. b, Representative kymographs show binding of Lis1 to motile mtDDB complexes assembled by mixing 1 nM LD650-mtDDB and 75 nM TMR-Lis1 and immediately recording motility with free proteins in solution (see methods). White arrows represent colocalization of LD650-Dyn (red) and Lis1-TMR (cyan). c, Velocity distribution of mtDDB complexes not bound to Lis1 moves faster than complexes that are bound to Lis1 during single-molecule motility. The line and whiskers represent the mean and SD, respectively. From left to right, n = 270 and 117 and mean values are 921 and 813 nm s-1. In b and c, three independent experiments were performed per condition. The p-value is calculated from a two-tailed t-test. d, Rare events of dynamic binding of Lis1 to dynein as mtDDB walks along an MT assembled in the presence of 50 nM Lis1. White arrows represent the colocalization of LD650-Dyn (red) and TMR-Lis1 (cyan). In the top kymograph, Lis1 initially diffuses on an MT and then binds to mtDDB during processive movement. Lis1 binding reduces the velocity of the complex. In the middle kymograph, dissociation of Lis1 during mtDDB motility increases the velocity. In the bottom kymograph, a diffusing Lis1 initially binds and later dissociates from mtDDB, without affecting the velocity of the complex (four independent experiments). e, Additional kymographs show single- and dual Lis1 binding to motile mtDTR complexes assembled in the presence of 50 nM Lis1. Red arrows represent the colocalization of Atto488-DynLT (green) and Cy5-Lis1 (red). White arrows represent the colocalization of Atto488-DynLT (green) with both Cy5-Lis1 (red), and TMR-Lis1 (cyan). Three independent experiments were performed per condition. 

Source data

Extended Data Fig. 7 At limiting dynein concentration, Lis1 recruits single dynein to dynactin and BicD2N.

a, Schematic depiction of wtDDB assembly using 5 nM LD650-wtDyn and TMR-wtDyn in the absence and presence of 600 nM Lis1. b, Fraction of processive and static/diffusive wtDDB complexes on MTs (mean ± SEM, n = 59, 788, 303 and 984 from left to right, three independent experiments). 

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Motility of single DDBs along MTs at 1 mM ATP. LD650-mtDyn was assembled with dynactin and BicD2N and motility along surface-attached MTs in the absence and presence of Lis1 was imaged under TIRF illumination. Scale bar is 5 µm. Stopwatch shows time in seconds. Four independent experiments were performed for each condition.

Supplementary Video 2

Motility of single DDRs along MTs at 1 mM ATP. LD650-mtDyn was assembled with dynactin and BicDR1 and motility along surface-attached MTs in the absence and presence of Lis1 was imaged under TIRF illumination. Scale bar is 5 µm. Stopwatch shows time in seconds. Four independent experiments were performed for each condition.

Supplementary Video 3

Motility of DDB-kinesin co-localizers. LD650-mtDyn was assembled with dynactin and BicD2N. LD650-DDB (red) and TMR-kinesin (cyan) were tethered using a DNA scaffold and motility along surface-attached MTs in the absence and presence of 600 nM Lis1 was imaged with two-color TIRF illumination. Colocalizers are denoted by white arrows. Scale bar is 5 µm. Stopwatch shows time in seconds. Three independent experiments were performed for each condition.

Supplementary Video 4

Motility of single DTRs along MTs at 1 mM ATP. LD650-mtDyn (red) and TMR-DynLT (cyan) were mixed in the presence of dynactin and BicDR1. Motility along surface-attached MTs in the absence and presence of 600 nM Lis1 was imaged with two-color TIRF illumination. Colocalizers that move along a single MT are denoted by white arrows. Scale bar is 5 µm. Stopwatch shows time in seconds. Three independent experiments were performed for each condition.

Supplementary Video 5

Recruitment of two dyneins to dynactin by BicD2N and BicDR1. LD650-mtDyn (red) and TMR-mtDyn (cyan) were mixed with dynactin and a cargo adaptor in the absence and presence of 600 nM Lis1. Motility of DDB and DDR complexes along surface-attached MTs were imaged with two-color TIRF illumination. Colocalizers along a single MT are denoted by white arrows. Scale bar is 5 µm. Stopwatch shows time in seconds. Four independent experiments were performed for each condition.

Supplementary Video 6

Colocalization of two Lis1 dimers to DTR. Atto488-DynLT (green), TMR-Lis1 (blue), and LD650-Lis1 (red) were mixed with dynactin and BicDR1. Motility along surface-attached MTs was imaged with three-color TIRF illumination. Atto488, TMR, LD650, and overlaid frames are separately shown for ease of visualization. Colocalizers are denoted by white arrows. Scale bar is 3 µm. Stopwatch shows time in seconds. Three independent experiments were performed for each condition.

Supplementary Video 7

Motility of single DDBs along MTs at limiting dynein concentration. 5 nM of LD650-mtDyn or LD650-wtDyn was mixed with dynactin and BicD2N in the absence and presence of 600 nM Lis1. Motility along surface-attached MTs was imaged under TIRF illumination. Scale bar is 5 µm. Stopwatch shows time in seconds. Three independent experiments were performed for each condition.

Source data

Source Data Fig. 1

Statistical source data.

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Elshenawy, M.M., Kusakci, E., Volz, S. et al. Lis1 activates dynein motility by modulating its pairing with dynactin. Nat Cell Biol 22, 570–578 (2020). https://doi.org/10.1038/s41556-020-0501-4

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