Abstract
Dynactin is the longest known cytoplasmic dynein regulator, with roles in dynein recruitment to subcellular cargo and in stimulating processive dynein movement. The latter function was thought to involve the N-terminal microtubule-binding region of the major dynactin polypeptide p150Glued, although recent results disputed this. To understand how dynactin regulates dynein we generated recombinant fragments of the N-terminal half of p150Glued. We find that the dynein-binding coiled-coil α-helical domain CC1B is sufficient to stimulate dynein processivity, which it accomplishes by increasing average dynein step size and forward-step frequency, while decreasing lateral stepping and microtubule detachment. In contrast, the immediate upstream coiled-coil domain, CC1A, activates a surprising diffusive dynein state. CC1A interacts physically with CC1B and interferes with its effect on dynein processivity. We also identify a role for the N-terminal portion of p150Glued in coordinating these activities. Our results reveal an unexpected form of long-range allosteric control of dynein motor function by internal p150Glued sequences, and evidence for p150Glued autoregulation.
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Acknowledgements
We thank R. McKenney for help in initiating this project and A. Baffet for helpful comments on the manuscript. Support by GM102347 to R.B.V. and GM070676 to S.P.G.
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R.B.V., S.P.G., S.J.W. and S.K.T. designed experiments; S.K.T., S.J.W. and C.C. performed the experiments; P.A. provided kinesin; R.B.V., S.P.G., S.K.T., C.C. and S.J.W. wrote the paper.
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Supplementary Figure 3 Temperature dependent melting and reannealing of p150Glued CC1 and CC1B fragments.
Molar ellipticity of CC1 and CC1B at 222 nm was monitored under increasing and decreasing temperature. Unfolding was reversible. Tm values were 23.5 °C for CC1B and 33 °C for CC1. (One independent experiment)
Supplementary Figure 4 Additional examples of force traces and MSD curves.
(A) Video trace showing measurement of single dynein force followed by its run-length. Blue star marks force production, red arrow indicates turning off of optical trap, and blue arrow shows end of runlength (B) and (C): Force traces from dynein alone and with P150. Experiments were done at a bead binding fraction of 30%. (D): The MSD curve for beads with P150 alone (30% binding fraction) diffusing along microtubules. (E) The MSD curve for single-molecule dynein beads (30% bf) with CC1A, diffusing along MTs. (F, G): MSD curves for dynein (again 30% bf) with P150 (E) and P135 (F). The blue curves reflect the diffusing beads, whereas the red curves reflect the processively moving beads.
Supplementary Figure 5 Measurement of force distribution for diffusive beads.
(A) (left):Force trace of free bead, held in trap. The quadrant photo diode (QPD) signal was obtained at 4 kHz, with trap stiffness of 1.5 pN/100nm. (right):histogram of detected forces due to thermal motion for the free bead (B) Force trace for bead attached to microtubule by a single p150 1-555 without dynein. (C) Higher temporal resolution image of (B). (D) Distribution of displacements (from fits in (B)) from many traces (475 force events). (E, F, and G) (775 force events) Same as (A, B and C) for, dynein with p150 1-555. (H, I and J (518 force events)) Same as (A, B and C) for dynein with p135-CC1. In all cases, the QPD signal was averaged to 1 kHz and analysed using Kerssemakers’ step detection algorithm, with waiting time restricted to ≥ 50 ms. While (D) is described by a single Gaussian (no additional force production), (G) and (J) each require the sum of three Gaussian peaks. The small peaks indicate the presence of some force. We interpret these small forces as likely reflecting transient binding/release events by dynein (see Supplementary Discussion).
Supplementary Figure 6 Characterization of step detection.
Step sizes were measured with an optical trap and an acousto optic deflector (AOD) force feedback system on single-motor (30% binding fraction) beads. The bead was maintained at the trap center by AOD feedback every 40 nm (blue arrows in A). (A) A trace with detected steps (in red) for single kinesin moving at a velocity similar to dynein (500 nm s−1). (B, F, H and J) Simulated tracks with detected steps for kinesin (309 steps), dynein with 8nm steps only, dynein (number of steps = 659) and CC1B-dynein (428 steps). (C, D, G, I and K) Step size distributions determined from experiment (blue star, real tracks such as in Fig. 3a, b) and simulated tracks with real noise (red circles) for single-molecule kinesin with plus end directed 8 nm steps only, kinesin involving back-steps, dynein with minus end directed 8 nm steps only and for single dynein with and without CC1B (405 steps used in K). Purple open circles are the residual, indicating difference between distributions. (E) Integrated residual from (C) and (D) is smaller when back-steps are for kinesin are included. (L) Normalized step probability for dynein with and without CC1B. (M) Steps were detected from video tracking traces (48 traces) of moving beads coated with dynein or dynein/CC1B (30 frames/sec) without force feedback. Step distributions are in qualitative agreement with step detection from AODs.
Supplementary Figure 7 Characterization of lateral motion.
Beads with adsorbed kinesin, dynein, or dynein plus the p150Glued CC1B fragment were analyzed for bead motion perpendicular to the microtubule long axis (Y- bead position). (A) Example traces of Y-bead position vs. time for kinesin at saturating ATP. (B) Detected steps (red lines) from (A). (C) Gaussian distribution of detected steps (25 processive bead). (D) Lateral step size distribution of kinesin in the presence of AMP-PNP (20 beads checked). Distributions in (C) and (D) are similar indicating that kinesin does not take lateral steps. (E) Lateral step size distributions of dynein without ATP (black) or dynein (blue), and dynein with CC1B (red) with saturating ATP (428 lateral steps). (F and G) are example traces of Y-FLOP of bead for dynein and CC1B-dynein.
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Tripathy, S., Weil, S., Chen, C. et al. Autoregulatory mechanism for dynactin control of processive and diffusive dynein transport. Nat Cell Biol 16, 1192–1201 (2014). https://doi.org/10.1038/ncb3063
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DOI: https://doi.org/10.1038/ncb3063
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