Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein

Abstract

Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150^Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150^Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150^Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

Fig. 1. Deletion of the intermediate chain N-terminus has no effect on dynein complex integrity or function.

a Cartoon depicting the yeast and metazoan dynein complexes with (right) and without (left) the activating cargo adapter and dynactin. Inset depicts heavy chain- (HC) and light chain (LC)-bound intermediate chain (IC) with N-terminus (ICN) highlighted. Studies suggest that the ICN is an interaction site for both coiled-coil 1b (CC1b) of p150 (Nip100 in yeast) and Ndel1 (Ndl1 in yeast). b Schematics of ICs from budding yeast (Pac11) and humans (Dync1I2/IC2C) with domains indicated. SAH, single alpha-helix. c Analytical size exclusion chromatography of wild-type (WT) and dynein^∆ICN complexes purified from yeast and human cells, along with coomassie-stained gel and immunoblots of human dynein depicting the presence of accessory chains. Data from five independent replicates. d Mass photometric analysis of purified human dynein complexes. e Negative stain electron micrographs reveal intact dynein complexes from yeast (2D class averages shown; n = 2; scale bar, 10 nm) and human (raw images shown; n = 2; scale bars, 50 nm). f Representative kymographs, and plots (mean ± SD, along with mean values from individual replicates for single molecule data, with circles representing all data points for gliding velocity; mean run length values were determined by fitting raw data to one phase decay) depicting motility parameters for purified yeast (using single molecule assays; n = 183/177/174 WT, and 183/177/208 ∆ICN motors from 3 independent replicates; P values were calculated using a Mann–Whitney test) and human (via microtubule-gliding assays, n = 20/20 microtubules for WT and dynein^∆ICN from 2 replicates; P value was calculated using a two-tailed t-test) dynein reveal similar motility parameters between WT and dynein^∆ICN complexes. g Kymograph depicting two-color single molecule motility assay in which yeast dynein HC (HaloTag^JFX49-Dyn1) and LC (Dyn2-S6^LD650) are visualized together. Plot (mean ± SD, along with all data points) depicts fluorescence intensity values for single molecules of Dyn2 bound to either WT or dynein^∆ICN, indicating the mutant binds to the same number of LCs as the WT complex (n = 101/100 and 101/100 Dyn2 foci from WT and dynein^∆ICN motors, respectively, from 2 independent replicates, represented by different shades of blue and orange). P value was calculated using a Mann–Whitney test.

Fig. 2. The dynein intermediate chain N-terminus is required for in-cell dynein function and DDA assembly.

a Immunofluorescence images of Flp-In^TM T-REx^TM 293 cells inducibly expressing either WT or IC2C^∆ICN (n = 3). b Plots (mean ± SD, along with mean values for individual replicates) depicting fractions of cells in mitosis (left), or those with abnormal spindles (right) for uninduced cells, or those induced to express WT or IC2C^∆ICN (n = 717/16/25 uninduced WT cells, 802/50/27 induced WT cells, 1243/29/27 uninduced IC2C^∆ICN cells, and 1150/45/33 induced IC2C^∆ICN cells, from 3 independent replicates; P values were calculated using a two-tailed t-test). c Representative images of cells expressing fluorescent α-tubulin and plot (weighted mean ± weighted standard error of proportion, along with values from individual replicates) depicting fractions of cells with mispositioned anaphase spindles (n = 53/56/49 WT cells, 38/56/61 dyn1∆ cells, and 49/66/69 pac11^∆ICN cells, from 3 independent replicates). Two-tailed P values were calculated from Z scores. d Immunoblots and plot (mean ± SD, n = 2) depicting relative degree of dynein-dynactin-Hook3 (DDH) assembly as a consequence of mutation or addition of factors. Quantification of the relative combined band intensities for p150/p135 in the presence of dynein, dynactin, Hook3, and LIS1 is shown. P value: two-tailed t-test. e Fluorescence images of cells expressing mTurquoise2-Tub1, Jnm1-3mCherry (homolog of human p50), and either WT or dynein^∆ICN−3GFP (arrows, plus end foci; arrowhead, cortical focus; n = 2). f Plot depicting degree of colocalization for indicated foci in WT or Pac11^∆ICN cells (mean ± SD; n = 221/193 foci in WT cells, and 57/38 foci in pac11^∆ICN cells from 2 independent replicates). g Plots (weighted mean ± weighted standard error of proportion, along with mean values from individual replicates) depicting fractions of cells exhibiting indicated foci in WT or pac11^∆ICN cells (Dyn1: n = 266/265 WT cells and 208/200 pac11^∆ICN cells from 2 independent replicates; Jnm1: n = 266/265 WT cells and 208/200 pac11^∆ICN cells from 2 independent replicates). Two-tailed P values were calculated from Z scores. h Schematics of human and yeast p150/Nip100 with domains indicated (CAP-Gly cytoskeleton-associated protein, glycine-rich, CC coiled-coil, ICD inter coiled domain). i Images and quantitation depicting degree to which Nip100^CC1 binds WT and dynein^∆ICN (mean ± SD, along with mean values from individual replicates; n = 20 microtubules for each, from 2 independent replicates).

Fig. 3. ICN-bound Ndel1/Ndl1 recruits LIS1/Pac1 to promote in-cell dynein localization.

a Schematics of human Ndel1 and its budding yeast homolog Ndl1 with coiled-coil domains (CC) indicated. b Representative images and quantitation (mean ± SD, along with mean values from individual replicates) depicting role of ICN in dynein-Ndl1 binding (n = 30 microtubules for each, from 3 independent replicates). P value: unpaired two-tailed Welch’s t-test. Fluorescence images and quantitation (mean ± SD; dashed line indicates relative dynein-Pac1/LIS1 binding in the absence of Ndl1/Ndel1) depicting relative dynein-Pac1 (c) or LIS1 (d) binding. Binding was determined from relative intensity values for microtubule-associated Pac1 or LIS1 with respect to dynein (for c, 20 nM Pac1 and ~15 nM dynein were used; for d, 20 nM dynein and 60 nM LIS1 were used). Note that buffer conditions were used such that neither Pac1, LIS1, Ndl1, nor Ndel1 was recruited to microtubules in the absence of dynein. Thus, the degree of microtubule localization for Pac1, LIS1, Ndl1 and Ndel1 is directly proportional to the extent of their dynein binding (c: n = 10/10/10 microtubules for WT, and 10/10/10 microtubules for dynein^∆ICN, from 3 independent replicates; d: n = 53/50, 52/45, 56/56, 56/56, 52/72, 56/61 microtubules for WT, and 52/56, 58/53, 51/59, 54/71, 52/52, 55/57 microtubules for dynein^∆ICN, mean ± SD from 2 independent replicates; scale bars, 5 µm). e, f Plots (mean ± SD, as well as all data points for intensity values) depicting the extent of dynein localization in cells with and without Ndl1 (e: n = 105/102 NDL1 and 104/101 ndl1∆ cells, and 95/95 and 41/42 foci from NDL1 and ndl1∆ cells, respectively, all from 2 independent replicates), or with and without overexpressed Ndl1 (f: n = 113/100 uninduced, and 102/100 induced cells, and 74/52 and 103/106 foci from uninduced and induced cells, respectively, all from 2 independent replicates). P values were calculated using a Mann–Whitney test (for intensity values), or by calculating Z scores (two-tailed). For cells in panel f, which were engineered to possess a GAL1 promoter upstream of the NDL1 locus, Ndl1 overexpression was controlled by the exclusion or inclusion of galactose in the media for 3 hours immediately prior to imaging.

Fig. 4. Dynein and Ndel1/Ndl1 compete for binding to LIS1/Pac1.

a, b Alphafold2-Multimer models of 2 Ndel1^CC:2 LIS1^WD40:2 ICN (a) or 2 Ndl1^CC:2 Pac1^WD40:2 ICN (b) complexes (also see Supplementary Fig. S3). Insets highlight residues on each protein mutated in this study or others^,,. c, d Mass photometric analysis of individual proteins or mixtures thereof. Proteins were mixed together at a 1:1 molar ratio. Fits of mean mass values for each species, and relative fraction of particles with indicated mass are shown. Although the majority of species were comprised of 1:1 complexes, a minor but reproducible population of 2 Pac1:1 Ndl1 were apparent in all experiments with yeast proteins only (also see Supplementary Fig. S3e, f). e Mass photometry of the human GST-dimerized dynein motor domain (depicted in cartoon schematic) with and without LIS1 and Ndel1^CC. Proteins were mixed together at a 1:1 molar ratio. f Representative fluorescence images and quantitation depicting relative binding between microtubule-bound dynein-dynactin-BicD2 complexes (DDB) and LIS1 ± Ndel1^CC in the presence of AMPPNP (mean ± SD, n = 88/71 microtubules for chambers without Ndel1^CC, and 57/68 microtubules for those with Ndel1^CC, from 2 independent replicates; P value calculated using a one-way ANOVA; scale bar, 5 µm). g, h Plots (mean ± SD, as well as all data points for intensity values) depicting the extent of dynein^MOTOR localization in cells with and without Ndl1 (g; n = 66/63 NDL1 and 64/63 ndl1∆ cells, and 87/82 and 115/107 foci from NDL1 and ndl1∆ cells, respectively, all from 2 independent replicates), or with and without overexpressed Ndl1 (h; n = 61/62 uninduced, and 62/63 induced cells, and 109/113 and 92/78 foci from uninduced and induced cells, respectively, all from 2 independent replicates). P values were calculated using a Mann–Whitney test, or by calculating Z scores (two-tailed). For cells in panel h, which were engineered to possess a GAL1 promoter upstream of the NDL1 locus, Ndl1 overexpression was controlled by the exclusion or inclusion of galactose in the media for 3 h immediately prior to imaging. Cartoon schematic in h depicts the proposed manner by which Ndl1 competes Pac1 away from dynein^MOTOR.

Fig. 5. Excess p150^CC1 and Ndel1 competitively inhibit DDA assembly but do not perturb pre-assembled complexes.

a The fraction of cells with a mispositioned spindle are plotted (weighted mean ± weighted standard error of proportion, along with values from individual replicates; n = 53/64/44 WT + galactose cells, 26/62/31 GAL1p:NIP100^CC1 + glucose cells, and 50/60/43 GAL1p:NIP100^CC1 + galactose cells, all from 3 independent replicates). Two-tailed P values were generated by calculating Z scores. b Representative fluorescence images of Nip100^CC1-overexpressing cells (after growth in galactose-containing media) depicting localization of Nip100^CC1 within cells (arrows, plus end foci; arrowheads, SPB foci). c Cartoon schematic depicting experimental strategy to assess DDA complex formation in the absence or presence of indicated recombinant proteins, added either prior to addition of Hook3 to lysates, or 60 min thereafter. d–f Immunoblots and plots depicting relative degree of DDH assembly as a consequence of addition of indicated wild-type factors (d and e), or indicated mutant Ndel1^CC (f). g Representative kymographs from two-color movies depicting motility of 30 nM DDH complexes (assembled as indicated in panel c) in the presence of indicated concentration of fluorescent p150^CC1 or Ndel1. Note the low frequency of comigration of p150^CC1 and Ndel1 with DDH. h Plot (mean ± SD, along with values from independent replicates) depicting frequency of comigration of p150^CC1 or Ndel1 with DDH complexes (n = 646/837/698/539/542/243, 512/1020/449/646/670/558, 775/664/978/486/412/516, 2188/860/770/328/537/349 processively migrating DDH particles for 30 nM and 150 nM p150^CC1, 30 nM and 150 nM Ndel1, respectively). P values were calculated using a one-way ANOVA.

Fig. 6. Model for dynein activation.

1 Dynein stochastically switches between the autoinhibited phi and open states. 2 Ndel1 binding to the ICN SAH, which binds equally well to the phi and open states (see Supplementary Fig. 4a), recruits LIS1 to dynein. 3 An unknown mechanism leads to LIS1 unbinding from Ndel1, and subsequently binding to open dynein, thus stabilizing this conformation. 4 Interactions between H2 of the ICN and p150^CC1b (refs. ^,) competitively inhibit ICN-Ndel1 binding, initiate dynein-dynactin binding (independent of a cargo adapter), and potentially relieves dynactin autoinhibition (the latter of which is due to interactions between p150^CC1 and the pointed end complex). 5 Binding of a cargo adapter to the adapter-independent dynein-dynactin complex, which requires ICN-p150 binding, leads to assembly of the active dynein-dynactin-adapter (DDA) complex. Insets show AF2 models of human ICN with either Ndel1 or p150^CC1. Note that data suggests that the ICN-p150 interaction involves both the SAH and H2 of ICN, but only the AF2 predicted H2-p150 interaction is shown here. 6 Coincident with microtubule binding, LIS1 dissociates, and the active DDA complex processively transports cargoes along microtubules.