LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes

Abstract

Cytoplasmic dynein-1 is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions that range from retrograde axonal transport to mitotic spindle assembly^1,2. Activated dynein complexes consist of one or two dynein dimers, the dynactin complex and an 'activating adaptor', and they show faster velocity when two dynein dimers are present^3-6. Little is known about the assembly process of this massive ~4 MDa complex. Here, using purified recombinant human proteins, we uncover a role for the dynein-binding protein LIS1 in promoting the formation of activated dynein-dynactin complexes that contain two dynein dimers. Complexes activated by proteins representing three families of activating adaptors-BicD2, Hook3 and Ninl-all show enhanced motile properties in the presence of LIS1. Activated dynein complexes do not require sustained LIS1 binding for fast velocity. Using cryo-electron microscopy, we show that human LIS1 binds to dynein at two sites on the motor domain of dynein. Our research suggests that LIS1 binding at these sites functions in multiple stages of assembling the motile dynein-dynactin-activating adaptor complex.

Extended Data Fig. 1. Effect of Lis1 on the motility and microtubule binding of activated dynein complexes.

a. SDS-PAGE gel stained with Sypro Red of human dynein, dynactin and the activating adaptors BicD2-S (aa 25–398), BicD2-L (aa 1–598), Hook3 (aa 1–552), and Ninl (aa 1–702) used here. The dynein heavy chain was tagged with the SNAP tag, the dynactin subunit p62 with the HaloTag, and each activating adaptor with the HaloTag. The dynein light chains are too small to be seen on this low percentage gel. SDS-PAGE gels were run after all protein purifications. b. Example microscopy images for microtubule binding density data in the absence (white circles) or presence (black circles) of 300 nM Lis1 presented in Figure 1 d and e. Microtubules in magenta and dynein or activating adaptor foci in green. Scale bars are 10 μm. c. Example kymographs of dynein/ dynactin/ activating adaptor complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1. Scale bars are 10 μm (x) and 20 sec (y). d. Percent processive runs of dynein/ dynactin/ activating adaptor complexes in standard motility buffer in the absence (white circles) or presence (black circles) of 300 nM Lis1. Statistical analysis was performed on data pooled from all replicates with a chi-squared test. e. Immunoblots of cell lysates from human U2OS cells co-transfected with PEX3-mEmerald-FKBP and BicD2-S-V5-FRB constructs, as well as either scramble siRNA or Lis1 siRNA 1 or 2. Blots were performed for each biorep with similar results. f. Peroxisome velocity in human U2OS cells with scrambled or Lis1 siRNA pool knockdown. The median and interquartile range are shown. At least 7 peroxisome motility events were measured per cell. g. Immunoblots of cell lysates from human U2OS cells co-transfected with PEX3-mEmerald-FKBP and BicD2-S-V5-FRB constructs and scramble or Lis1 siRNA pool. Two bio-replicates (1 and 2) are shown. An anti-V5 antibody detects BicD2-S-V5-FRB, an anti-Lis1 antibody assesses the efficiency of Lis1 knockdown, and an anti-actin antibody serves as a loading control for immunoblots shown in e and g. Statistical data and unprocessed gel and blot images are available as source data for Extended Data Figure 1.

Extended Data Fig. 2. Characterization of the dynein binding interface and dimerization domain of Lis1.

a. Example SEC-MALS traces with Lis1 dimer (orange), dynein monomer (grey), and dynein monomer with Lis1 dimer (black). The intensity of the UV signal (solid line) and the molecular weight fit (dashed line) are shown. Dimeric Halo-tagged-Lis1 is expected to be 161.4 kDa and monomeric dynein is expected to be 380.4 kDa. In this experiment we observe Halo-tagged-Lis1 to be 157.6 kDa, monomeric dynein to be 489.5 kDa and the Lis1-dynein complex to be 700.1 kDa. The high apparent molecular weight of monomeric dynein may be due to a self-association species that appears as a shoulder in the UV trace. The experiment was repeated in triplicate yielding similar results, giving a stoichiometry of 1.2 +/− 0.3 Lis1 dimers per dynein monomer. Based on this data we cannot rule out that some dynein monomers are bound to two Lis1 dimers (which has been reported to occur), but our data suggest that most dynein monomers bind a single Lis1 dimer, and that Lis1 does not tether two dynein monomers. b. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes with increasing concentrations of Lis1. The median and interquartile range are shown. c. Single-molecule velocity of dynein/ dynactin/ Hook3 complexes in the presence of a higher ionic strength buffer in the absence (white circles) or presence (black circles) of 300 nM Lis1 or Lis1–5A. The data in the presence and absence of WT Lis1 was also presented in Fig. 1h. The median and interquartile range are shown. d. Example kymographs of dynein/ dynactin/ Hook3 complexes in a higher ionic strength buffer in the absence or presence of 300 nM Lis1 or Lis1–5A. Scale bars are 10 μm (x) and 20 sec (y). Data is quantified in Extended Data Fig. 2C. e. Example SEC-MALS trace of Lis1ΔN (orange). The intensity of the UV signal (solid line) and the molecular weight fit (dashed line) are shown. Monomeric Halo-tagged-Lis1ΔN is expected to be 71.5kDa. In this experiment we observe Halo-tagged-Lis1ΔN to have a monomer peak at 72.0 kDa and a dimer peak at 141.2 kDa. The experiment was repeated in triplicate yielding similar results. Statistical data is available as source data for Extended Data Figure 2.

Extended Data Fig. 3. Quantification of the velocity of one-color and two-color activated dynein complexes in the presence of absence of Lis1.

a. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1 with colocalized dynein (two color) or without observed colocalization (one color). The median and interquartile range are shown. Statistical data is available as source data for Extended Data Figure 3.

Extended Data Fig. 4. Characterization of Lis1 binding to microtubules and activated dynein complexes.

a. Example microscopy images for imaging 50 nM TMR-Lis1 (magenta in merge) in the presence of microtubules (green in merge). Lis1 does not colocalize with microtubules. Scale bars are 10 μm. The experiment was repeated in triplicate yielding similar results. b. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes in the absence (white circles) or presence (black circles) of 50 nM TMR-Lis1 or TMR-Lis1–5A. The median and interquartile range are shown. c. Representative kymographs of Alexa647-dynein/ dynactin/ BicD2-S complexes in the presence of 50 nM TMR-Lis1–5A. Scale bars are 10 μm (x) and 20 sec (y). Statistical data is available as source data for Extended Data Figure 4.

Fig. 1.. Lis1 increases microtubule binding and velocity of activated dynein complexes.

a. Current model for dynein activation. Dynein is autoinhibited in the Phi conformation, opens, and then adopts a parallel conformation in the activated dynein complex, which can contain two dynein dimers (A and B). b. Schematic of the AAA+ ATPase dynein heavy chain. The two Lis1 binding sites, “Site_ring” and “Site_stalk” are shown. Microtubule binding domain (MTBD). c. Activating adaptor constructs used in this study. Dashed lines highlight the regions that were truncated. d, e. Binding density (mean ± s.e.m.) of full-length recombinant human dynein with its associated intermediate, light intermediate and light chains (d) or dynein/ dynactin/ activating adaptor complexes (e) on microtubules in the absence (white circle) or presence (black circle) of 300 nM Lis1. Data was normalized to a density of 1.0 in the absence of Lis1. f. Single-molecule velocity of dynein/ dynactin/ activating adaptor complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1. The median and interquartile range are shown. g. Single-molecule velocity of dynein/ dynactin/ Hook3 complexes in a higher ionic strength buffer (67.5 mM compared to 37.5 mM in our standard buffer) in the absence (white circle) or presence (black circle) of 300 nM Lis1. The median and interquartile range are shown. h. Percent processive runs of dynein/ dynactin/ Hook3 complexes in standard and higher (grey panel) ionic strength motility buffer in the absence (white circles) or presence (black circles) of 300 nM Lis1. Statistical analysis was performed on data pooled from all replicates with a chi-squared test. i. Peroxisome relocation assay. The peroxisomal protein, Pex3, is fused to mEmerald and FKBP and BicD2-S is fused to FRB. Rapalog induces the association of FKBP and FRB. j. Peroxisome velocity in human U2OS cells with scrambled or Lis1 siRNA knockdown with two independent siRNAs. The median and interquartile range are shown. Statistical data is available as source data for Fig. 1.

Fig. 2.. Human Lis1 binds the human dynein motor domain at AAA3/4 and the stalk.

a. 2D class averages of human dynein monomers bound to human Lis1 dimers in the presence of ATP-vanadate. b. Best-matching projections of a model combining human dynein-2 bound to ATP-vanadate (PDB: 4RH7) with homology models of human Lis1 at the locations where Lis1 binds to yeast dynein in the presence of ATP-vanadate (PDB: 5VLJ). The two Lis1’s (“Site_ring” and “Site_stalk”) identified in yeast dynein, dynein’s AAA ring, stalk and buttress are labeled. c. Projections of 4RH7 alone in the same orientations as those shown in (b). d. Homology model of human Lis1 (from SWISS-MODEL) showing the five residues mutated to alanine in “Lis1–5A”. e. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes in the absence (white circles) or presence (black circles) of Lis1 or Lis1–5A. The median and interquartile range are shown. f. Percent processive runs of dynein/ dynactin/ Hook3 complexes in a higher ionic strength buffer in the absence (white circles) or presence (black circles) of 300 nM Lis1 or Lis1–5A. Data in the presence of 300 nM Lis1 was also presented in Fig. 1h. Statistical analysis was performed on data pooled from all replicates with a chi-squared test. g. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1 dimer or 600 nM Lis1ΔN. Since Lis1ΔN is largely monomeric (Extended data Figure 2e), 300 nM Lis1 dimer is roughly equivalent to 600 nM Lis1ΔN. The median and interquartile range are shown. Statistical data is available as source data for Fig. 2.

Fig. 3.. Lis1 recruits a second dynein dimer to dynein/ dynactin/ BicD2-S complexes.

a. Schematic of the dynein/ dynactin complex formation assay. b, c. Percent dynein bound to BicD2-S-coupled beads (mean ± s.e.m.) in the presence (b) or absence (c) of dynactin and in the absence (white circle) or presence (black circle) of 150 nM Lis1. d. Schematic depicting the maximum probability of forming various dynein/ dynactin/ BicD2-S complexes containing two dynein dimers. The maximum probability of colocalization was 45% (grey dashed line shown in f and g) given our labeling efficiency (see Methods). e. Representative kymographs showing the colocalization of TMR- and Alexa647-labeled dynein in moving dynein/ dynactin/ BicD2-S complexes in the presence of 300 nM Lis1. Each channel is shown separately (left and middle panels) and the merged TMR- and Alexa647-channels in pseudocolor (right panel). Scale bars are 10 μm (x) and 20 sec (y). Data is quantified in Fig. 3f. f. Percent two-color dynein/ dynactin/ BicD2-S runs in the absence (white circle) or presence (black circle) of 300 nM Lis1. Statistical analysis was performed using a chi-squared test. Statistical data is available as source data for Fig. 3.

Fig. 4.. Lis1 is not required to sustain fast velocity of activated dynein complexes.

a. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes in the absence (white circle) or presence (black circle) of 50 nM TMR-Lis1. The median and interquartile range are shown. b. Single-molecule velocity of Alexa647-dynein/ dynactin/ BicD2-S complexes in the presence of 50 nM TMR-Lis1 either co-migrating with TMR-Lis1 (yes) or not (no). The median and interquartile range are shown. c. Representative kymographs of the Alexa647-dynein and TMR-Lis1 channels (left and middle panels) and the merged images in pseudocolor (right panel). Arrowheads indicate colocalized runs. Scale bars are 10 μm (x) and 20 sec (y). Data is quantified in Fig. 4b. d. Kymographs showing examples of dynein’s velocity changing upon loss of the TMR-Lis1 signal. The Alexa647-dynein and TMR-Lis1 channels are shown (left and middle panels) and the merged images in pseudocolor (right panel). Arrowheads indicate instances of velocity change. Four such events were observed. Scale bars are 10 μm (x) and 20 sec (y). Data is quantified in Fig. 4b. Statistical data is available as source data for Fig. 4.

Fig. 5.. Lis1 preferentially binds to Open dynein and enhances the formation of complexes containing two Open dynein dimers.

a. One of the dynein protomers in the Phi conformation (PDB: 5NVU) was aligned to the structure of yeast dynein (AAA3-Walker B) bound to Lis1 in the presence of ATP-vanadate (PDB: 5VLJ). The inset shows the cryo-EM map for the yeast structure with Lis1 docked at Site_ring and highlights the steric incompatibility between the Phi conformation and binding of Lis1 at this site. b. Determination of the binding affinity of Lis1 for wild-type (WT) dynein (blue, K_d = 144 nM ± 25) and Open dynein (green, K_d = 80 nM ± 8.1). c. Percent (mean ± s.e.m.) of WT dynein (blue) and Open dynein (green) bound to BicD2-S conjugated to beads in the absence (white circles) or presence (black circles) of 150 nM Lis1. Data with WT dynein in the presence and absence of Lis1 is also presented in Fig. 3b. Statistical analysis was performed using a two-tailed unpaired t test with Bonferroni corrected significance levels for two comparisons. d. Single-molecule velocity of dynein/ dynactin/ BicD2-S complexes with WT dynein (blue) and Open dynein (green) in the absence (white circles) or presence (black circles) of 300 nM Lis1. The median and interquartile range are shown. Data with WT dynein with and without Lis1 was also presented in Fig. 1f. e. Percent two-color colocalized runs with activated dynein complexes with Open dynein in the absence (white circle) or presence (black circle) of 300 nM Lis1. Statistical analysis was performed using a chi-squared test. The labeling efficiency for both TMR- and Alexa-647 dynein in this experiment was 100%. f. Model for the roles of Lis1 in forming activated dynein complexes. Statistical data is available as source data for Fig. 5.