Molecular mechanism of dynein-dynactin complex assembly by LIS1

Abstract

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

Fig. 1. JIP3 is an autoinhibited activating adaptor for dynein motility in vitro.

(A) Schematic representation of BICDR1 (also known as BICDL1), HOOK3, Rab11FIP3 and JIP3. The dotted lines illustrate the short coiled coil of JIP3 as compared to known dynein activating adaptors. (B) Kymographs of TMR-dynein-dynactin-LIS1 in the presence of HOOK3^1-522, JIP3^FL, JIP3^1-582, JIP3^1-560 and JIP3^1-185. (C) Quantification of the number of processive events/μm microtubule/minute with the mean ± S.D. plotted. The total number of events analyzed were 1364 (HOOK3^1-522), 95 (JIP3^FL), 348 (JIP3^1-582), 537 (JIP3^1-560) and 513 (JIP3^1-185). (D) An AlphaFold2 prediction of two copies of JIP3 (residues 1-600) showing an interaction between JIP3^helix (residues 570-582) and the RH1 domain. The JIP3^helix contains the same FFxxL motif as the DLIC^helix (residues 427-439 of the DLIC2 isoform) (bottom left), which has been shown to bind the RH1 domain (28) (bottom right). (E) Coomassie Blue-stained SDS-PAGE gel of purified recombinant protein mixtures prior to the addition of glutathione agarose resin (left) and of proteins eluted from glutathione agarose resin after GST pull-down (right). The RH1 domain construct was composed of JIP3^1-108 and JIP3^helix construct was composed of GST-JIP3^563-586. The asterisks denote the bands corresponding to degradation products. (F) Kymograph of TMR-dynein-dynactin-LIS1 in the presence of JIP3^1-582mut (left). Quantification of the number of processive events/μm microtubule/minute with the mean ± S.D. plotted. The total number of events analyzed were 568. Statistical significance in comparison with JIP3^1-582 and JIP3^1-560 is depicted. The in vitro motility assays were performed with three technical replicates and statistical significance was determined using ANOVA with Tukey’s multiple comparison.

Fig. 2. Cryo-EM structure of dynein-dynactin-JIP3-LIS1 on microtubules.

(A), (B) Composite density map of dynein-dynactin bound to JIP3 and LIS1 overlaid on 13 protofilament microtubule. The zoom-in (grey circle) shows the locally refined map of dynein-A1 bound to p150, DIC-N and LIS1. Schematic representation of the complex is shown on the bottom left.

Fig. 3. JIP3 interactions with dynein-dynactin.

(A) Overall organization of JIP3 bound to dynein-dynactin. The unresolved regions which are predominantly unstructured are depicted as a dotted line. (B) Trajectory of JIP3 along the dynein heavy chain tails is shown using a top view of the complex. Dynactin segments are removed to aid visualization. (C) The N-terminal RH1 domain (yellow) has extra density on either side which is explained by the DLIC C-terminal helix (blue). The AlphaFold2 prediction of this interaction is displayed as cartoon inside the density. (D) JIP3 is sandwiched between heavy chains of dynein-B1 and dynein-A2. JIP3 contains an HBS1 which interacts with the dynein heavy chains using glutamine residues followed by an acidic patch. (E) JIP3 interaction with the dynein-A2 is shown. (F) Multiple sequence alignment of the JIP3 HBS1 from different organisms is shown. The residues involved in interactions with dynein are highly conserved. Sequences of HBS1 from BICDR1 and HOOK3 are shown for comparison. (G) JIP3 segments bound at the dynactin pointed end are shown. The JIP3 Spindly motif binds to the p25 subunit of dynactin whereas the LZII and RH2 foldback on each other and are bound along the p25 and p62 subunits (left). Spindly motif residues bound at the p25 subunit are shown on the right.

Fig. 4. Organization of p150 and dynein intermediate chain.

(A) Domain architecture of p150 (top) and its organization in the autoinhibited conformation is shown. Domain architecture of dynein intermediate chain and the interaction sites for dynein light chains are depicted (bottom). (B) Cartoon representation of p150 (green), dynein intermediate chain (magenta), dynein light chains (ROBL (pink), LC8 (brown) and TCTEX1 (golden)) and LIS1 (coral) bound to dynein-A1 (blue; depicted as surface). Unresolved segments are depicted as dotted or solid lines. (C) Schematic of interaction sites on p150-CC1B (top) and cryo-EM density of dynein-A1 motor domain bound by CC1B and LIS1 (bottom). CC1B is further bound by DIC-N region 1 (residues 1-32). (D) The regions of CC1B involved in binding the dynein motor domain, DIC^1-32 and LIS1-N are mapped on the model of the autoinhibited p150. The open state of p150 solved in this study is shown on the right for comparison. The area covered by the two DIC^1-32 helices, LIS1-N as well as the dynein AAA2 and AAA3 domains when bound to CC1B are shown using dotted lines.

Fig. 5. LIS1 stabilizes the pre-powerstroke state of dynein-A.

(A) (i) Surface representation of LIS1 bound dynein-A and microtubule bound dynein-B. Dynein conformation and linker position is shown on the right. (B) Cryo-EM density of dynein-A and -B motor domains where the different subdomains and bound proteins are highlighted. (C) Regions involved in interacting with the ICD and CC1B segments of p150 are mapped (in yellow) onto the heavy chain of dynein-A (top) and dynein-B (bottom). (D) Surface representation of dynein-A motor domains with bound LIS1-WD40 domains (left). Interaction between dynein-A1 and A2 motor domains is shown (right) and the residues at the A1-A2 interface interacting with LIS1 are mapped (colored in coral).

Fig. 6. LIS1-N binding to p150 is important for dynein activation.

(A) A schematic of the mitochondrial relocation assay is shown. (B) Representative images showing the distribution of mitochondria (magenta) in LIS1 siRNA-treated HeLa GFP-BICD2N-MTS cells electroporated with different SNAP-tagged LIS1 proteins. Scale bars represent 10 μm. (C) Quantification of mitochondrial spread (μm) in LIS1 knockdown HeLa GFP-BICD2N-MTS cells electroporated with different LIS1 proteins. Data are plotted from 3 biological replicates. The total cells quantified were 3922 (blank), 3830 (LIS1-SNAP), 2541 (WD40-SNAP) and 3655 (GCN4-WD40-SNAP). (D) Kymographs of TMR-dynein-dynactin-JIP3^1-560 in the presence of (from left) blank, LIS1, GCN4-WD40, LIS1^mut and GCN4-WD40+LIS1-N. Cartoons depicting the LIS1 construct used are shown above each kymograph. (E) Quantification of the number of processive events per μm microtubule per minute with the mean ± S.D. plotted. The total number of movements analyzed were 74 (blank), 1592 (LIS1), 352 (GCN4-WD40), 116 (LIS1^mut), and 225 (GCN4-WD40 + LIS1-N). Data are plotted from 3 technical replicates. (F) Schematic summarizing the effect on dynein assembly when LIS1-N and WD40 domains are present on the same molecule (left). Kymographs of TMR-dynein-dynactin-JIP3^1-560 in the (from left) absence of LIS1, with LIS1, with FRB-WD40+LIS1-N-FKBP without and with rapalog are depicted (right). (G) Quantification of the number of processive events per μm microtubule per minute with the mean ± S.D. plotted. The total number of movements analyzed were 283 (blank), 675 (LIS1), 388 (FRB-WD40+LIS1-N-FKBP) and 665 (FRB-WD40+LIS1-N-FKBP with rapalog). Data are plotted from 3 technical replicates. Statistical significance was determined using ANOVA with Tukey’s multiple comparison.