Hook Adaptors Induce Unidirectional Processive Motility by Enhancing the Dynein-Dynactin Interaction

Abstract

Cytoplasmic dynein drives the majority of minus end-directed vesicular and organelle motility in the cell. However, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Recent work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. Of particular interest, studies in fungal systems have implicated Hook proteins in the regulation of microtubule motors. Here we investigate the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. We used optogenetic approaches to specifically recruit Hook proteins to organelles and observed rapid transport of peroxisomes to the perinuclear region of the cell. This rapid and efficient translocation of peroxisomes to microtubule minus ends indicates that mammalian Hook proteins activate dynein rather than kinesin motors. Biochemical studies indicate that Hook proteins interact with both dynein and dynactin, stabilizing the formation of a supramolecular complex. Complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of end-binding (EB) proteins but cannot bind directly to microtubules. Single-molecule motility assays using total internal reflection fluorescence microscopy indicate that both Hook1 and Hook3 effectively activate cytoplasmic dynein, inducing longer run lengths and higher velocities than the previously characterized dynein activator bicaudal D2 (BICD2). Together, these results suggest that dynein adaptors can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking.

Keywords: cytoskeleton; dynein; intracellular trafficking; kinesin; microtubule.

FIGURE 1.

Hook proteins redistribute peroxisomes to the perinuclear region in an optogenetic assay. A, schematic of inducible the dimerization assay and corresponding constructs. B, using a photoactivatable dimerization system (cTMP-Htag dimerizer) (48), motors/adaptors (-mCh-DHFR tagged) were recruited to peroxisomes (PEX3-Halo-GFP-labeled) by 405-nm light, and the resulting motility was observed by live cell confocal microscopy. Scale bars = 10 μm. Arrows indicate peroxisome clustering after recruitment. C, overlay of pre- and post-dimerization images of peroxisomes.

FIGURE 2.

Hook proteins differentially redistribute peroxisomes to the MTOC. A, dimerization assay in fixed cells stained with γ-tubulin antibody. Images are maximum projections of confocal z stacks. Scale bars = 10 μm. White arrows point to γ-tubulin stained MTOC, and yellow arrows point to peroxisome clusters. Cell outlines were determined from corresponding X-mCH-DHFR images (data not shown). B, distribution of peroxisomes from MTOC measured in a fixed time point dimerization assay (analyzed using Cell Profiler (47)). The endosomally linked adaptor Hook1 tightly clusters peroxisomes to the MTOC compared with Hook3 and BICD2. Cells analyzed per condition: K560, n = 36; p150^Glued, n = 19; BICD2, n = 26; Hk1, n = 23; Hk3, n = 32. Error bars show standard error based on number of cells.

FIGURE 3.

Mammalian Hook proteins interact with the dynein-dynactin complex. A, Western blot showing IP of endogenous p150^Glued (subunit of dynactin) and DIC from mouse brain lysates, with anti-myc used as a mouse IgG (Ms IgG) control. IP of p150^Glued shows interaction with Hook1, whereas disruption of the dynein-dynactin complex in IP with anti-DIC shows loss of this interaction (n = 3). B, Western blots showing IP of endogenous p150^Glued from COS7 cells expressing Halo-Hook1, Halo-Hook3, and Halo-BICD2 (1–572), with the HaloTag expressed as a negative control. C, graph of the DIC- to-p150^Glued IP ratio from the experiments in B (n = 4). The ratio of DIC to p150^Glued IP for the control condition (HaloTag only) was normalized to 1, and all other conditions are shown as a -fold change from the control. Error bars show standard error.

FIGURE 4.

Hook proteins bind microtubules indirectly. A and C, MT binding assays were performed using cell lysates from HA-Hook1 transfected COS7 cells (A) and recombinant purified Hook1 dimer (1–443 aa) and Hook3 (1–210 aa-GCN4) (1 μm) (C). MT binding assays were performed by mixing equal amounts of protein to increasing amounts of Taxol- or GMPCPP-stabilized MTs. Supernatant (S) and pellets (P) were analyzed by SDS-PAGE gels and Western blotting, with the HA tag and p150^Glued antibodies as noted. The gels in C are Coomassie-stained SDS-PAGE gels. Hook1 from cell lysates co-sediments with MTs, but purified Hook1 and Hook3 constructs do not pellet with MTs, suggesting indirect binding. Endogenous and purified p150^Glued (1–210 aa-Htg construct) were used as controls. GMPCPP-stabilized MT binding assay gels are not shown. B, binding assay quantification of A. Error bars show standard error. Cell lysates experiments, n = 5; purified experiments, n = 2–3.

FIGURE 5.

Hook proteins lack conserved regions for MT binding. A, sequence alignment based on the secondary structure for Hook1 and EB3. The coloring is based on the BLOSUM62 score. Magenta boxes indicate MT interaction regions in EB3 (residues within 6 Å of the tubulin surface in the PDB 3JAK structure (34)). Arrows indicate residues that ablate microtubule association in EB1 when mutated and are not conserved in Hook proteins (35). B, comparison of N-terminal mouse Hook1 (PDB code 1WIX) and EB3 (PDB code 3JAK (34)) structures with predicted microtubule interactions sites highlighted in magenta. Numbers correspond to boxed regions in the alignment (A). C-ter, C terminus; N-ter, N terminus.

FIGURE 6.

Pulldown of Halo-Hook1 with the dynein-dynactin complex requires the N-terminal region. A, conserved domains and predicted coiled-coil regions in Hook1. MTBD, putative microtubule binding domain; CBD, cargo binding domain. B, Western blot showing pulldown (PD) of Halo-Hook1 constructs with endogenous dynein-dynactin from mouse brain lysates. DHC, dynein heavy chain. Pulldown of the Hook1 full-length (FL) and Hook1 (1–554 aa) constructs shows interaction with dynein-dynactin, whereas pulldown of Hook1 (171–728 aa, 171-E) shows loss of interaction with the dynein-dynactin complex. NT, non-transfected control. n = 3. C, graphs of DIC or p150^Glued to Hook1 (Hk) ratio from experiments in B (n = 3). Error bars show standard error.

FIGURE 7.

Hook proteins display high velocities and long run lengths. A, example time series of particles moving to the minus end of microtubules (polarity is marked for Hk1 and Hk3; plus end shown in green). Scale bars = 2 μm. FL, full-length. B, maximum projections of Halo-Hook1 (full-length) expressed in cells under mock or dynein heavy chain (DHC) siRNA conditions and imaged in the TIRF assay. Scale bars = 5 μm. C, Western blot of mock and dynein heavy chain siRNA knockdown lysates used for TIRF assays. D, track displacement and velocity distributions for particles tracked with the ImageJ plugin TrackMate. Data were fitted with a custom maximum likelihood estimation program (38) and plotted as probability density functions with 95% confidence interval bootstrapping. (BICD2, n = 242; Hk1, n = 90; Hk3, n = 84). E, table of motility parameters based on fits from data in D. F, percent of events with a mean velocity of more than 1 μm/s. G, per-track standard deviation of instantaneous velocity. Data are plotted as a box plot with Tukey whiskers.

FIGURE 8.

C-terminally truncated Hook proteins display similar motility as full-length proteins. A, conserved domains and predicted coiled-coil regions in Hook1 and truncated constructs below with their corresponding motility in TIRF assays (+, motility; −, no observable motility). MTBD, putative microtubule binding domain; CBD, cargo binding domain. B, track displacement and velocity distributions for particles tracked with the ImageJ plugin TrackMate. Data were fitted with a custom maximum likelihood estimation program (38) and plotted as probability density functions with 95% confidence interval bootstrapping (Hk1 (1–554 aa), n = 107; Hk3 (1–552 aa), n = 156. C, percent of events with a mean velocity of more than 1 μm/s. D, per-track standard deviation of instantaneous velocity. Data are plotted as a box plot with Tukey whiskers. BICD2 data are repeated from Fig. 7 for comparison.