Supplementary Figure S1

Transcription

1 Supplementary Figure S1 Supplementary Figure S1 Subcellular localization of Rab6a and LIS1 in the DRG neurons. (a) Exogenous expression of EGFP-Rab6a(wild-type) in the DRG neurons by the Neon transfection system (Life Technologies, CA, USA)(upper panels). Endogenous expression of Rab6a in the DRG neurons was demonstrated by immunocytochemistry using a specific antibody for Rab6 (lower panels). In both cases, Rab6a was highly expressed in the soma and tips of the neurite process. (b) Comparison of subcellular localization between LIS1 and Rab6a in the DRG neurons, which display similar localization at the periphery. The DRGs were permeabilized by 8 µm digitonin to remove the soluble cytosolic fraction. Scale bars, 10 µm. mdrg neurons; mouse dorsal root ganglion neurons.

2 Supplementary Figure S2 Supplementary Figure S2 Subcellular localization of Rab6a and DIC1. EGFP-DIC1 and TMR-HaloTag-Rab6a(Q72L) (upper panels), and EGFP-DIC1 and TMR-HaloTag-Rab6a(T27N) (lower panels) localization patterns at the periphery of DRG neurons are shown. Scale bars, 5 µm. DIC; dynein intermediate chain, DRG neuron; dorsal root ganglion neurons.

3 Supplementary Figure S3 Supplementary Figure S3 Quantitation of cis-interaction between dynein and Rab6a(Q72L) by FCCS at the periphery of DRG neurons. The inset is a schematic diagram demonstrating fluorescent-labeled proteins used with each experiment. Auto- and cross-correlation curves of fluorescent-labeled proteins were shown for EGFP and TMR-HaloTag (a), EGFP-DIC1 and TMR-HaloTag-Rab6a(T27N) (b), EGFP-DIC1 and TMR-HaloTag-Rab6a(Q72L) (c), and EGFP-DIC1 and TMR-HaloTag-LIS1 (d). FCCS; fluorescence cross-correlation spectroscopy, DRG neurons; dorsal root ganglion neurons.

4 Supplementary Figure S4 Supplementary Figure S4 Analysis of the single molecule movements labeled with Q-dots. (a) TIRF microscopy of 605Q-dot-dynein (green) are shown. Dynein displayed unidirectional translocation on microtubules (green arrow head). Scale bar, 2 µm. (b) A representative kymograph of 605Q-dot-dynein moving along a microtubule. Vertical scale bar, 2 µm; Horizontal scale bar, 1.0 sec. (c) Histogram of velocities of single native dynein molecules moving along microtubules. The histogram was fitted to a Gaussian curve (N = 121). (d) Histogram of run lengths of single dynein molecules moving along microtubules. The run length was fitted to a single exponential curve (N = 165). (e) TIRF microscopy of 605Q-dot-dynein(green) and 525Q-dot-LIS1 (red) are shown. Green and red arrowheads indicate dynein and LIS1, respectively. Scale bar, 2 µm. (f) Representative kymograph of 605Q-dot-dynein(green) and 525Q-dot-LIS1(red). The panels indicate each kymograph of dynein (upper), LIS1 (middle) and merged pattern (lower). Vertical scale bar, 2 µm; Horizontal scale bar, 1.0 sec. (g) A list of the duration of dynein-lis1 interaction. Within the limitations of recording time (150 s), we observed three types of events. i) Dynein was moving, followed by arrest of motion due to binding of LIS1 (GS: upper right). ii) Dynein-LIS1 complex sat on the microtubule, followed by a start of motion due to dissociation of LIS1 (SG: middle right). iii) Dynein was moving and stopped motion after binding of LIS1. Dynein starts to move again after dissociation of LIS1 (GSG: lower right). Schematic diagrams of three events are shown at the right. We obtained the rate parameter λ of the exponential distribution (duration of the LIS1-dynein complex) using our original computer simulation algorithm. The simulation algorithm was developed on VBA in Excel 2007 (Microsoft). (h) Images of 605Q-dot-dynein and 525Q-dot LIS1 by TIRF microcopy treated with vehicle control (left pane), Rab6a(T27N) (middle panel) and Rab6a(Q72L) (right panel) for ten minutes. Ratio: fraction of remaining LIS1-dynein complexes after ten minutes. N: total LIS1-dynein complexes. Scale bar, 2 µm. (i) Additional three examples of kymograph patterns of dynein (green), Rab6a(Q72L) (red) and LIS1 (blue) are shown. Vertical scale bar, 2 µm; Horizontal scale bar, 1.0 sec. TIRF; total internal reflection fluorescence.

5 Supplementary Figure S5 Supplementary Figure S5 Additional examples of Rab6a depleted DRG neurons. Immunocytochemistry data 48 h after transfection with each of the sirnas. Endogenous expression of Rab6a and DIC1 in the DRG neurons using the specific antibody for Rab6a and DIC1. Scale bar, 10 µm. sirna; small interfering RNA, DIC1; dynein intermediate chain 1, DRG neurons; dorsal root ganglion neurons.

6 Supplementary Methods DRG preparation DRGs with attached roots were dissected aseptically from postnatal mice (P2-P3) and collected in Hanks balanced salt solution (Sigma-Aldrich, MO, USA). The ganglia were carefully freed of roots and were treated with 0.5 mg/ml collagenase, 0.25% trypsin (Gibco) with 0.01% Deoxyribonuclease I (Sigma-Aldrich, MO, USA). Recombinant proteins purification Recombinant proteins for LIS1 and each of Rab6a s (wild-type, Q72L and T27N) were generated using baculovirus-insect cell expression system (Invitorgen) and pgex-4t expression vector (GE Healthcare Lifesciences, UK), respectively. Protein purification was performed using Glutathione Sepharose 4B (GE Healthcare Lifesciences, UK) according to the manufacturer's recommendation. To remove GST tag, we treated recombinant proteins with thrombin (GE Healthcare Lifesciences, UK), followed by absorption of thrombin by Benzamidine Sepharose 6B (GE Healthcare Lifesciences, UK). Immunoprecipitation Immunoprecipitation experiments were carried out with purified dynein, recombinant LIS1 and Rab6a mutants using the anti-dic antibody (clone: 74.1, Merk Millipore, MA, USA)(10 µg/ml) bound to protein G Sepharose (GE Healthcare) or anti-lis1 rabbit polyclonal antibody(20 µg/ml) bound to protein A Sepharose (GE Healthcare). The bound proteins were subjected to SDS-PAGE, followed by Western blotting with the anti-dic antibody (clone: 74.1)(x1000 dilution), anti-lis1 rabbit polyclonal

7 antibody (x 1000 dilution) or anti-rab6 rabbit polyclonal antibody (x1000 dilution) (Merk Millipore, MA, USA). Immunocytochemistry DRGs were fixed with 4%(w/v) ultra-pure electron microscopy-grade paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. Cells were then blocked with 5% (w/v) BSA and Block Ace Powder (DS Pharma Biomedical) in PBS and incubated with anti-rab6 antibod (x100 dilution)(sigma-aldrich, MO, USA) or anti-lis1 antibody (x 200 dilution), followed by incubation with Alexa546-conjugated anti-mouse IgG, Alexa488-conjugated anti-mouse IgG, Alexa488-conjugated anti-rabbit IgG (x 1000 dilution)(life Technologies, CA, USA). Each incubation was performed for 1 h at room temperature. Slides were mounted in ultra-pure electron microscopy-grade glycerin (Merck) containing 100 nm 4, 6-diamidino-2-phenylindole (DAPI)(0.2 µm). The images were observed and captured with a laser scanning confocal microscope (TCS-SP5, Leica, Germany). In vitro MT gliding assay Dynein, dynactins and tubulin were purified from fresh porcine brain as previously reported 9, with some modifications. Tubulin was labeled with TMR (FluoReporter TMR Protein Labeling Kit; Life Technologies, CA, USA). Dynein (12 µg) was applied to a flow chamber (Matsunami Glass, Osaka, Japan) and absorbed onto the glass slide for 5 min. TMR-labeled tubulins were polymerized in BRB80 buffer (40 mm PIPES; ph 7.2, 0.5 mm MgCl 2, 0.5 mm EGTA) with 1 mm ATP, 33 µm Taxol and an oxygen scavenger (4.5 mm glucose, 216 µg/ml glucose oxidase, 36 µg/ml catalase, 1% 2-mercaptoethanol) and introduced into the flow chamber. Each recombinant protein (LIS1; 1 µm x 20 µl, Rab6as; 1 µm x 20 µl) or purified dynactins (18 µg) were added

8 in sequence, and the gliding of TMR-labeled MTs were observed by a conventional inverted fluorescence microscope IX71 (Olympus, Tokyo, Japan) with an oil-immersion objective lens (UPlanSAPO, 100, NA=1.4, Olympus, Tokyo, Japan), captured with an EMCCD camera (ImagEM, Hamamatsu Photonics, Hamamatsu, Japan) and analyzed by AquaCosmos software (Hamamatsu Photonics, Hammamatsu, Japan). All experiments were performed at 37 C with a stage incubator (Tokai-Hit, Shizuoka, Japan).