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Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein

Nature Cell Biology volume 16, pages 1118–1124 (2014)Cite this article

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Abstract

Cytoplasmic dynein is a two-headed microtubule-based motor responsible for diverse intracellular movements, including minus-end-directed transport of organelles1,2,3. The motility of cargo transporters is regulated according to the presence or absence of cargo4,5,6; however, it remains unclear how cytoplasmic dynein achieves such regulation. Here, using a recombinant and native dynein complex in vitro, we show that lone, single dynein molecules are in an autoinhibited state, in which the two motor heads are stacked together. In this state, dynein moves diffusively along a microtubule with only a small bias towards the minus end of the microtubule. When the two heads were physically separated by a rigid rod, the movement of dynein molecules became directed and processive. Furthermore, assembly of multiple dynein molecules on a single cargo enabled them to move unidirectionally and generate force cooperatively. We thus propose a mechanism of autonomous on–off switching of cargo transport, in which single dynein molecules in the cell are autoinhibited through intramolecular head–head stacking and become active when they assemble as a team on a cargo.

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Figure 1: Motile properties of single full-length human cytoplasmic dynein.
Figure 2: Diffusive behaviour of dynein is related to the compact conformation in the ADP•Pi state.
Figure 3: Stacking of two motor heads leads to the diffusive, autoinhibited state.
Figure 4: Collective movement of multiple full-length dynein molecules.

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Acknowledgements

We thank K. H. Bui, T. Ishikawa and H. Sakakibara for technical instruction in electron microscopy, K. Sutoh for critical comments on the manuscript, K. Kawaguchi for helpful discussion, K. Yamamoto for early work on DNA nanotubes, M. Amino and Y. Watari for technical assistance, K. Shibata for constructing the monomeric yeast dynein construct and Y. Utsumi for the chicken α-actinin plasmid. This work was supported by a Grant-in-Aid for the Japan Society for the Promotion of Science (JSPS) fellowship from JSPS, a JSPS Grant-in-Aid for Scientific Research (B), a JSPS Grant-in-Aid for Scientific Research (C) and a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan

    Takayuki Torisawa, Muneyoshi Ichikawa, Kei Saito & Yoko Y. Toyoshima

  2. Advanced ICT Research Institute, National Institute of Information and Communications Technology, Kobe, Hyogo 651-2492, Japan

    Takayuki Torisawa, Akane Furuta, Kazuhiro Oiwa, Hiroaki Kojima & Ken’ya Furuta

  3. CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0076, Japan

    Takayuki Torisawa & Kazuhiro Oiwa

  4. Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan

    Kazuhiro Oiwa

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Contributions

T.T., Y.Y.T. and K.F. designed research, T.T., M.I., A.F., K.S. and K.F. performed experiments and T.T., M.I., K.O., H.K., Y.Y.T. and K.F. analysed data and wrote the manuscript.

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Correspondence to Yoko Y. Toyoshima or Ken’ya Furuta.

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Integrated supplementary information

Supplementary Figure 1 Properties of full-length cytoplasmic dynein.

(a) Average gliding velocities of human full-length dynein (determined by single-Gaussian fitting) versus ATP concentration. The plots and error bars represent the means and SDs obtained from the Gaussian fitting, respectively. The gliding velocities were fitted to the following equation: V = (Vmax × [ATP])/(Km, ATP + [ATP]), in which V is velocity at a given ATP concentration [ATP],Vmax is a maximum velocity under saturated ATP concentration, and Km, ATP is ATP concentration required for half maximum velocity. The number of gliding microtubules (MTs) scored is 113, 136, 130, 165, 161, 165, 148, 145 and 103 for 10, 50, 100, 200, 400, 500, 1,000, 2,000 and 5,000 μM ATP, respectively. The experiment was conducted three times. The apparent discrepancy between the average velocity of MT gliding (∼1,100 nm s−1) and that of linked dynein molecules on MTs (∼200 nm s−1;Supplementary Table 5) could be explained by the difference in the number of motors involved in the movement and the geometric arrangement of dynein and the MT, as previously discussed1. (b) Histogram of the maximum force generated by single full-length dynein molecules in the presence of 1 mM ATP. The histogram was fitted by a single Gaussian function. The number of force production analysed was 580 from eight independent experiments. (c) Processivity (red) and force (black) versus trap stiffness measured for single human full-length cytoplasmic dynein. The small force should be actually generated through a processive stepping of dynein because a weaker optical trap gave rise to longer runs within the trap. The number of traces used to calculate the average maximum force and displacement for each trap stiffness was 50. Note that the experiment was conducted independently of the experiment for b using different optical trap apparatus. Error bars represent SD of triplicates. (d) Mean square displacement (MSD) analysis of the movement of single full-length dynein molecules in the presence of 1 mM ATP. The number of traces analysed was 330 from four independent experiments The traces used in this figure are identical to those in Fig. 1e. (e) Histogram of segmental velocity measured at a time interval of 1.0 s (grey bar). The segmental velocities were calculated from the displacement between the two frames separated by a time interval of 1.0 s. The histogram is identical to that shown in Fig. 1e when the horizontal axis is multiplied by 1 s. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. The data were fitted to the sum of two Gaussian distributions (black line), which correspond to pauses (blue line) and biased diffusion (dashed red line). The equation was y = A × exp(−(x − m1)2/2σ12) + B × exp(−(x − m2)2/2σ22), where m1 and m2 are the mean values, and σ1 and σ2 is the standard deviation (m1 = −3.0, m2 = 30, σ1 = 24 and σ2 = 207). The SD of the stationary component (24 nm s−1) is similar to that obtained from immobilized dynein molecules on MTs (∼20 nm s−1). The stationary component can be interpreted as the molecules that stuck to the MT or glass surface, or as the stationary state in the diffusive movement. (f) Intensity distribution of full-length human dynein molecules labelled with Alexa Fluor 488 (n = 411 from a single experiment). The two Gaussian distributions in the histograms indicate that we were observing single molecules of full-length dynein dimer. The histograms are fitted by the following equation: y = A × exp(−(x − m1)2/2σ2) + B × exp(−(x − m2)2/2σ2), where m1 and m2 are the mean values and σ is the standard deviation (m1 = 6.8, m2 = 10.7 and σ = 2.1). (g) Kymographs depicting the diffusive movement of single full-length human and porcine dynein molecules carrying a quantum dot (QD) in the presence of 1 mM ATP. The experiment was repeated three times. (h) Mean square displacement (MSD) of QD–mammalian dynein versus time interval (n = 72 and 65 for human and porcine dynein, respectively). The diffusion coefficients of human and porcine dyneins were 21 ± 0.3 × 103 and 22 ± 1.0 × 103 nm2 s−1, respectively. The experiment was repeated three times (i) Distance travelled by full-length human dynein in the presence of 1 mM ATP (n = 330). The distance between the appearance and disappearance of a fluorescent spot on the MTs was measured. The distance toward the direction of dynein displacement (MT minus end) is shown as a positive value. The bias of the average travel distance indicates the minus-end directionality of a single dynein molecule. The histogram was well fitted by a single Gaussian function (mean = 100 nm, SD = 461.5 nm and R2 value = 0.9796). The traces used in this figure are identical to those in Fig. 1e. (j) Steady-state MT-activated ATPase activities of full-length dynein. The ATPase activity is expressed per dynein motor domain, and the MT concentration is expressed per tubulin heterodimer. The experiment was conducted three times. The number of measurement was 15. The plots show a hyperbolic dependence with kcat value of 3.9 ± 0.4 s−1. The Km, MT value is 10.5 ± 2.3 μM. (k) Kymographs showing the behaviour of single full-length human dynein molecules at different ionic strengths in the presence of 1 mM ATP. As the concentration of potassium acetate was increased, the duration of dynein on MTs decreased, suggesting that the electrostatic interactions between dynein and the MT are responsible for the diffusive movement. The experiment was repeated three times. (l) Kymographs depicting the behaviour of single full-length human dynein molecules on subtilisin-digested MTs in the presence of 1 mM ATP (see Supplementary Methods). The digestion of the negatively charged C-terminal region of tubulin by subtilisin reduced the duration and diffusive component of dynein on MTs, similarly to the case at higher ionic strengths shown in k, suggesting that the diffusion was mediated by the highly charged C terminus of tubulin. The experiment was repeated three times (m) Example representation of how the velocity was determined by the analysis carried out in previous studies. The trajectories of the dynein movement were manually divided into segments and classified into either plus-end- or minus-end–directed movement. The processive segments that moved over 200 nm were defined as a run (indicated by yellow lines for example). The plus (+) and minus (−) symbols refer to the polarity of the MT. (n) Histogram of the full-length dynein velocity in the presence of 1 mM ATP. The histogram was compiled from the velocity of each segment that was determined as described in m. Plus and minus values of the velocity represent forward (the direction of dynein displacement) and backward (the opposite direction) movements, respectively. The histogram was fitted to the sum of two Gaussian functions. The mean ± s.d. and the number of segments analysed are shown at the top of the graph. The experiment was repeated three times.

Supplementary Figure 2 Compact conformation of full-length human cytoplasmic dynein.

(a) MSD analysis of the movement of single dynein molecules under different nucleotide conditions. The number of analysed traces is 223, 229, 278 and 231 for apo, AMPPNP, ATP•Vi and ADP, respectively. The experiments at ATP•Vi and other conditions were repeated four and three times, respectively. (b) Typical negative-stain EM images of dynein molecules in a stacked-head conformation on MTs in the presence of 1 mM ATP and 1 mM Vi. The yellow circles indicate the dynein molecules in a stacked-head conformation bound to the MTs. (c) Gallery of dynein molecules in a stacked-head conformation on MTs. The arrowheads indicate the motor domain of dynein. (d) Representative class average of the head region of dynein molecules in a stacked-head conformation and the corresponding snapshot of the crystal structure (PDB: 3VKH). This view (top view) shows the dynein particle that is rotated 90 degrees from the view of Fig. 2e around the axis of the dynein tail. The number of averaged particle is 124. (e) Class-average images of the side view (#1–5, upper row) and the top view (#6–8, lower row) of the dynein head domains in the ADP•Vi state. The number of images used in each class is indicated at the bottom of the image. The asterisks denote the representative class-average images that are shown in Fig. 2e and Supplementary Fig. 2d. EM images showed that a major fraction of the stacked conformation comprises two motor domains with their C-terminal sides facing each other; however, further study is required to determine the stacking surface. (f) Representative class average of the stalk region showing two stalks crossing each other. The mask was applied to most of the head domains. The number of images used is indicated at the bottom of the image.

Supplementary Figure 3 Properties of artificially dimerised dynein constructs.

(a) Negative-stain EM images of the GST-D425 dynein particle in which the two heads are stacked together. The gallery represents the typical images of the side view (upper row) and top view (lower row), respectively. (b) Negative-stain EM image of the GST-D425 dynein in which the two heads are separated. (c) Fraction of GST-D425 particles in a stacked-head conformation in the presence of ATP and Vi, or ADP. The bars represent the mean and SE of bootstrapped data sets. The graph clearly shows the nucleotidedependent conformational change of GST-D425. The experiment was repeated three times. (d) Segmental velocity histograms of single molecules of GST-D425, D417, D405 and D382 measured at a time interval of 1.0 s. The segmental velocity was calculated from all traces obtained for each construct. The experiment was conducted three times each. (e) Kymographs showing the behaviour of tail truncated dimers on MTs in the presence of ATP and Vi. GST-D425 showed diffusive movement on MTs, whereas GST-D417, D405 and D382 only showed transient binding events. (f) Structure of homodimeric rod domain of chicken α-actinin (270–740 a.a. fragment) used in this study. The C termini of the rod domains are indicated by the letter ‘C’. The distance between two C termini was measured in the cryo-EM structure (PDB: 1SJJ) using a modelling software (RasMol). (g) Segmental velocity histograms of single molecules of ACTN-D382, ACTN-14GS-D382 and GST-D425-GFP measured at a time interval of 1.0 s. The segmental velocity was calculated from all traces obtained for each construct. The experiments were conducted three times each. (h) Kymographs showing the movement of DNA–dynein complex in the presence of 1 mM ATP. The experiments were conducted three times. The statistics are summarised in Table 1. (i–k) Distribution of maximum forces generated by single molecules of tail-truncated dyneins and time series showing the typical forces in the presence of 1 mM ATP. The grey lines show the raw trace acquired at 11 kHz. The black lines represent the 25-Hz median filtered trace. The experiments were repeated two (GST-D405, n = 230), four (GST-D417, n = 172) and four (GST-D425, n = 210) times. The distributions were fitted by a single Gaussian function. The mean and the standard deviation were shown.

Supplementary Figure 4 Properties of dynein teams and comparison between human and yeast dyneins.

(a) Kymograph showing the movement of QDs driven by multiple molecules of full-length human dynein in the presence of 1 mM ATP. (b) Velocity distributions of the moving portion selected from the traces of QD–dynein movements. The velocity was derived from linear-fitting of each trace (1 mM ATP). The directed movements of QD–dynein were selected by visual inspection, unlike the case of Fig. 4c. The movement that moved for over 200 nm was measured. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. The experiments were repeated three times. (c) Kymographs depicting the movement of 2-, 4- and 8-dynein teams in the presence of 1 mM ATP. Figure 4b shows all traces analysed for each construct. (d) Velocity distribution of the moving portion selected from the movement of two coupled full-length dynein molecules. The velocity was derived from linear-fitting of each trace (1 mM ATP, n = 471 from three independent experiments). The directed movements were selected by visual inspection, unlike the case of Fig. 4c. The processive segment that moved over 200 nm was analysed. This histogram can be compared with the histograms in b. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. (e) Comparison of the segmental velocities among different dynein complexes measured at a time interval of 1.0 s. The vertical axis is expressed as density so that the shape of the three distributions can be directly compared. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. The number of traces is 330, 375, 128 and 127 for single, 2-, 4- and 8-dynein teams, respectively. The experiment with single dyneins and other constructs was repeated four and three times, respectively. (f) MSD analysis of the movement of different dynein teams in the presence of 1 mM ATP. The traces of single dynein molecules used in this figure are identical to those in Supplementary Fig. 1d. The traces of 2-, 4- and 8-dynein teams are identical to those in e. (g) Comparison of MSD between single and two coupled dynein molecules in the presence of 1 mM ATP. The traces used are identical to those in f. Each plot represents mean ± s.e.m. The MSD plots were fitted by MSD(t) = vdrift2t2 + 2Dt + δ(vdrift, a drift velocity; D, a diffusion coefficient; δ, a constant). The diffusive nature in the inhibited state would allow dynein to explore the MT in search of cargo with low ATP consumption. The thermally driven 1D diffusion covers shorter distances (<0.5 μm) more rapidly than directed movement2. (h) More examples of typical forces generated by multiple full-length dynein molecules. The grey line shows the raw trace acquired at 11 kHz. The black line represents the 25-Hz median filtered trace. (i) Cluster mode analysis of the force distribution of multiple full-length dynein (grey bar, the histogram is identical to that shown in Fig. 4f). Each Gaussian distribution (solid line) represents the predicted Gaussian mode. In this case, the best model according to the algorithm was an unequal variance model with 4 clusters. The algorithm provides parameter estimation without any prior knowledge about the number of clusters. The values at the top of Gaussian distributions denote the means of them. (j) Comparison of duration in the strongly bound state (ADP or apo state) between human and yeast monomeric dyneins at no load. The cumulative plots of durations were fitted by a one-phase exponential decay model. The decay constants (± s.e.m. of fitting) are shown. The experiment was conducted three times each.

Supplementary Figure 5 Gel analysis of cytoplasmic dynein constructs.

(a) SDS-PAGE analysis (4–15%) of recombinant full-length human and native porcine dyneins. Both the human and porcine dynein heavy chain were co-purified with endogenous intermediate chains (ICs), light intermediate chains (LICs) and light chains (LCs), but without the dynactin complex. The protein bands were visualised by Stain-Free technology (Bio-Rad). (b) SDS-PAGE analysis (4–15%) of the GST-D425 dynein. The image shows the absence of subcomponents in the GST-D425 dynein. The protein band was visualised by Stain-Free technology (Bio-Rad). (c) SDS-PAGE analysis of two coupled motor domains of dynein on DNA scaffolds with the spacing of 3 nm, 17 nm and 32 nm. SDS-PAGE were performed on a 4% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue G250-based reagent (24590, Thermo Scientific). (d) SDS-PAGE analysis of two coupled full-length dynein molecules. SDS-PAGE was performed on a 5% polyacrylamide gel. (e) Gel shift assay of Alexa Fluor 647-labelled DNA origami nanotubes carrying 4 and 8 dynein molecules. Electrophoresis was performed on a 0.5% agarose gel. The DNA bands were visualised by epifluorescence imaging with red LED excitation (ChemiDoc MP, Bio-Rad).

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Diffusive movement of single full-length dynein molecules (3 × real-time).

The movements were observed in the presence of 1 mM ATP. Green lines indicate the position of the MT. Bars represent 3 μm. (AVI 831 kb)

Directed movement of single ACTN-D382 dynein molecules (10 × real-time).

The movements were observed in the presence of 1 mM ATP. Bars represent 3 μm. (AVI 926 kb)

Directed movement of single GST-D425-GFP dynein molecules (10 × real-time).

The movements were observed in the presence of 1 mM ATP. Bars represent 3 μm. (AVI 483 kb)

Directed movement of two coupled full-length dynein molecules (10 × real-time).

The movements were observed in the presence of 1 mM ATP. Bars represent 3 μm. (AVI 464 kb)

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Torisawa, T., Ichikawa, M., Furuta, A. et al. Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein. Nat Cell Biol 16, 1118–1124 (2014). https://doi.org/10.1038/ncb3048

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