Pac1/LIS1 stabilizes an uninhibited conformation of dynein to coordinate its localization and activity

Abstract

Dynein is a microtubule motor that transports many different cargos in various cell types and contexts. How dynein is regulated to perform these activities with spatial and temporal precision remains unclear. Human dynein is regulated by autoinhibition, whereby intermolecular contacts limit motor activity. Whether this mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its role in regulating dynein function remain unclear. Here, we use a combination of negative stain electron microscopy, single-molecule assays, genetic, and cell biological techniques to show that autoinhibition is conserved in budding yeast, and plays a key role in coordinating in vivo dynein function. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast), plays an important role in regulating dynein autoinhibition. Our studies demonstrate that, rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate dynein localization and activity in cells.

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Fig. 1: The yeast dynein complex adopts an autoinhibited phi-particle conformation.
Fig. 2: Disrupting phi-particle contact points extends single-molecule run lengths.
Fig. 3: The autoinhibited conformation restricts cellular localization of dynein and its interaction with dynactin.
Fig. 4: Preventing dynein autoinhibition enables Pac1/LIS1-independent localization and function.
Fig. 5: Pac1 promotes the release of the autoinhibited conformation of dynein.
Fig. 6: Reducing Pac1–microtubule binding minimizes the Pac1-mediated reduction of dynein velocity.

Data availability

All of the yeast strains, datasets and raw video files that were generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank S. Reck-Peterson for the 8His-ZZ-Pac1-SNAP-expressing yeast strain, and members of the Markus and DeLuca laboratories for discussions. Electron microscopy was performed at the University of Colorado, Boulder EM Services Core Facility in the MCDB Department, with the technical assistance of facility staff. This work utilized the RMACC Summit supercomputer, which is supported by the National Science Foundation (awards ACI-1532235 and ACI-1532236), the University of Colorado Boulder and Colorado State University. The RMACC Summit supercomputer is a joint effort of the University of Colorado Boulder and Colorado State University. We also thank E. Osborne-Nishimura, D. King and S. Bowerman for their assistance with using software on Summit. This research was funded by the NIH/NIGMS (GM118492, to S.M.M.).

Author information

Affiliations

Authors

Contributions

M.G.M. and S.M.M. designed the study. M.G.M. performed and analysed most of the assays, with support from S.M.M. and J.M.G. Electron microscopy was performed by Garry Morgan at the University of Colorado Boulder Electron Microscopy facility. Single-particle analysis was performed by S.M.M. with assistance from the EM facility. Reagents were generated by S.M.M., M.G.M. and J.M.G.

Corresponding author

Correspondence to Steven M. Markus.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Representative raw EM image and quantitation of conformational states.

a, Representative EM image of negative stained yeast dynein complex (red arrow, phi particle conformation; green arrow, open, uninhibited conformation; yellow arrow, ambiguous). b, Quantitation of indicated conformational states from raw images (n = 435 particles).

Extended Data Fig. 2 Fluorescence intensity analysis of native and overexpressed single molecules of dynein.

Histogram of fluorescence intensity values for single molecules of motile dyneins, as indicated, along with accompanying Gaussian fits and modeled parameters (determined using the model-based clustering algorithm Mclust66). The percentages reflect the relative proportion of molecules that fall within each component (i.e., for mean 1, and mean 2). The two mean values for each likely represent single-labeled (mean 1) and dual-labeled (mean 2) dynein dimers, respectively. Red outlined region in panel D (“aggregates”), delineate particles with ~3-fold higher fluorescence intensity values than the single labeled complexes. Source data

Extended Data Fig. 3 D2868K mutation has no effect on GST-dyneinMOTOR motility.

a, Schematic of the plasmid used to produce GST-dyneinMOTOR (wild-type and D2868K mutant). Restriction digest with ApaI (cuts within URA3 gene) targets the plasmid for homologous recombination into the ura3-1 locus as depicted. b, Cartoon representation of the minimal GST-dimerized dynein motor domain (amino acids 1219-4092 of the dynein heavy chain, Dyn1). (c and d) Plots depicting mean values (left) and all values (right) for velocity (c) and run length (d) of wild-type and D2868K GST-dyneinMOTOR, along with the standard error (n = 217 and 238 motors for each). Source data

Extended Data Fig. 4 Uninhibited dynein mutant exhibits only moderate increase in microtubule landing activity.

a, Plots depicting relative microtubule landing rate of full-length wild-type (WT) and D2868K (DK) dynein, as measured from single molecule motility experiments (mean ± standard deviation; n = 554 wild-type motors from 1532 µm of microtubules, and 553 D2868K motors from 1177 µm of microtubules; 3 independent experiments were quantitated for each). Diamonds represent mean normalized values obtained from each independent replicate experiment. Briefly, equivalent concentrations of full length wild-type or D2868K dynein were added to imaging chambers (after taking into account relative differences in labeling efficiencies, as determined from fluorescent scans of protein gels), and the number of moving motors were quantitated. Statistical significance was determined using a two-tailed Welch’s t test. (b and c) Representative gel (b; Sypro Ruby-stained) and quantitation (c) of microtubule co-sedimentation assay with full-length wild-type (WT) and D2868K (DK) dynein done in the absence and presence of ATP (mean ± standard deviation; n = 2 independent experiments; diamonds represent values obtained from each replicate). Relative microtubule binding was determined by measuring background-corrected band intensities of each, and subtracting any non-specific microtubule-independent pelleting (as determined from experiment performed in the absence of microtubules). Source data

Extended Data Fig. 5 Synthetic interactions between dynein mutants and Kar9.

(ac) Serial dilutions of cells with indicated genotype were plated on rich media (YPA supplemented with 2% glucose) and grown at 30 °C for 2-4 days (a, extended incubation of plates shown in Fig. 4e were grown for 4 days; all others were incubated for 2 days). Note the severe growth defects in dyn1HL3 kar9∆ cells (in panel c), suggesting that dyneinHL3 is not active in cells. Note that similar results were obtained from 2 independent replicates.

Extended Data Fig. 6 Pac1-microtubule binding behavior, and its contribution to dynein activity.

(a and b) Representative images (a) and intensity scatter plots (b; bars depict mean ± standard deviation) of microtubule-bound Pac1 in different buffers. Pac1-SNAP647 diluted in motility buffer (50 nM, dimer concentration) with indicated salts was introduced into a chambers with coverglass-adhered microtubules, and images were acquired (yellow and magenta circles represent data acquired from each independent experiments; n = 38, 49, 41, 49, and 45 microtubules that span 911 µm, 1074 µm, 1077 µm, 906 µm, 1017 µm in length for each condition, left to right). (c) Pac1-microtubule binding is reduced after enzymatic removal of the unstructured tubulin carboxy-terminal tails (see Methods; similar results were obtained from 2 independent experiments). (dg) Addition of cell extracts reduces Pac1-microtubule binding, and attenuates Pac1-mediated dynein velocity reduction. Representative fluorescence images of Pac1-SNAP647 on microtubules (d; Pac1-SNAP647 shown as a heat map) and scatter plots depicting intensity values (e; bars depict mean ± standard deviation; n= 48 and 47 microtubules that span 890 µm and 841 µm in length for each condition, left to right; similar results were obtained from 2 independent experiments). (f and g) Plots depicting the motility properties for GST-dyneinMOTOR in the absence and presence of 25 nM Pac1 (dimer concentration) in low ionic strength buffer (50 mM potassium acetate) in the presence of cell extracts (0.96 mg/ml final; 275 and 258 motors, left to right, from 2 independent experiments were quantitated). Note the small Pac1-mediated GST-dyneinMOTOR velocity reduction in the presence of cell extracts (22.1%, compared to 69.5% in the absence of extracts). (h) Additional representative kymograph of GST-dyneinMOTOR comigrating with Pac1 in buffer with 150 mM KCl (see Fig. 6d and Extended Data Fig. 7E – G for quantitation and statistics). Note the diffusive behavior of Pac1 on microtubules. Scale bars in panels a, c and d, 4 µm. Source data

Extended Data Fig. 7 Assessment of dynein-Pac1 stoichiometry, structural analysis of the Pac1-dynein-microtubule complex, and additional motility plots.

(a and b) Representative kymographs (a) and quantitation (b) of dynein–Pac1/2×Pac1 complex motility (in motility buffer supplemented with 120 mM potassium acetate; n = 870 dynein molecules from 3 independent experiments; mean values with standard error are shown, along all datapoints for middle and right plots; similar results were obtained from each replicate). Statistical significance was determined by calculating Z scores (left; ***, p < 0.0001), using a two-tailed Mann-Whitney test (middle; p = 0.6068), or with a two-tailed Welch’s t test (right; p = 0.6581). Note the estimated fraction of dynein-2xPac1 complexes (5.7%; see main text) is less than what would be expected if there was no cooperativity for Pac1-dynein binding (i.e., the product of the probabilities of two single, independent binding events, 10.8%). (c and d) Structural and cartoon model of a microtubule and Pac1-bound dynein monomer (generated with pdbs 4RH736, 3J1T67, 5VH928, and 3J6G68). Note the close proximity of Pac1 to the microtubule surface, the latter of which is lacking the unstructured E-hooks. (d) Cryo-EM data reveals the dynein-microtubule angle varies due to a hinge point within the MTBD, and can be much steeper than that shown in panel A69,70 (Θ ≥ 15-20°, with average = 55°). Cartoons depict range of angles sampled by dynein on microtubules, and thus the distances between Pac1 and the microtubule. (e - g) Non-normalized plots of mean values (e and f) and all data points (g; see Fig. 6d for n values) showing the relationship between Pac1-mediated dynein velocity reduction and Pac1-microtubule binding (for panels b left, e, and f, diamonds represent mean values obtained from each independent replicate experiment; for panel g, mean values and standard deviations are depicted with red lines). Source data

Supplementary information

Reporting Summary

Supplementary Table

A list of yeast strains used throughout the study.

Supplementary Video 1

Three-dimensional classification of the full-length yeast dynein phi particle reveals similarities to human dynein. Video of the 3D class average of the autoinhibited yeast dynein conformation. The high-resolution cryo-EM structure of the human dynein phi particle (PDB 5NVU1) was manually docked into the EM density map. Note that the atomic structures of the two tail domains have been slightly rotated with respect to the motor domains to better fit the 3D model, and that the atomic structures of both TcTEX and Robl have been eliminated due to their absence from the yeast dynein complex.

Supplementary Video 2

Uninhibited dynein mutants can bypass the need for Pac1 to move the mitotic spindle. The mitotic spindle (GFP–Tub1) is observed in pac1∆kar9∆ cells expressing either wild-type (top), K1475E (middle) or D2868K (bottom) dynein. Each frame is a maximum-intensity projection of a 2.4 μm z stack of confocal images collected at intervals of 10 s. The appearance of green circles in the top-left corner of each video corresponds to frames in which dynein-mediated spindle movements are apparent. Note the presence of two spindles in the wild-type cell. Quantification and n values are provided in Fig. 4f.

Supplementary Video 3

Proposed mechanism for autoinhibition restricting dynein processivity. In combination with the tail-dependence of the autoinhibited conformation, our observation of 2D classes with apparent tail–tail contacts — but not motor–motor contacts (Fig. 1, classes vii and viii) — indicates that these contacts may initiate the transition to the autoinhibited state. Subsequent to adopting the autoinhibited conformation, we hypothesize that dynein dissociates from the microtubule.

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Marzo, M.G., Griswold, J.M. & Markus, S.M. Pac1/LIS1 stabilizes an uninhibited conformation of dynein to coordinate its localization and activity. Nat Cell Biol 22, 559–569 (2020). https://doi.org/10.1038/s41556-020-0492-1

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