MOESM1 of Source location and mechanism analysis of an earthquake triggered by the 2016 Kumamoto, southwestern Japan, earthquake

Additional file 1. This file includes additional figures (Figures S1–3) and a table (Table S1)

MOESM1 of Evaluation of accuracy of synthetic waveforms for subduction-zone earthquakes by using a land–ocean unified 3D structure model

Additional file 1: Table S1. FDM parameters. Table S2. Estimated FAMT source parameters. Table S3. Estimated best double couples based on FAMT solutions. Figure S1. Residuals versus origin-time correction. Figure S2. Horizontal source locations estimated by the FAMT and other analyses. Figure S3. Comparisons of the observed and synthetic velocity waveforms for EV2003. Figure S4. Comparisons of the observed and synthetic velocity waveforms for EV2007. Figure S5. Comparisons of the first body wave part of the observed and synthetic velocity waveforms for EV2003. Figure S6. Comparisons of the first body wave part of the observed and synthetic velocity waveforms for EV2007

GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

<div><p>The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis. </p> </div

Dynamics of TC10-containing vesicles.

<p>(<i><b>A</b></i>) N1E-115 cells expressing mCherry-TC10 were serum-starved for 6 h and then imaged every 20 sec. Representative mCherry images superimposed with corresponding DIC images at the indicated time points (in min:sec) are shown. A bar, 10 μm. (<i><b>B</b></i>) The time-lapse TIRF images (3 × 3 μm) showing two examples of fusion events in the cell bodies of PC12 cells expressing mTFP-TC10. Images were obtained at 200 msec intervals. Time point zero was set to the first frame showing the highest intensity of the vesicles. The bottom row in each example shows a plot of fluorescence intensity scanned across the center of a fusing vesicle. This TIRF imaging cannot be executed in growth cones because of their structural fragility. </p

Effect of TC10 depletion on NGF-induced neurite outgrowth in PC12 cells.

<p>PC12 cells were transfected with pCAGGS-Flag-resi-TC10-WT or its mutants in combination with a TC10-targeted shRNA vector. After selection with puromycin, the selected cells were cultured with 50 ng/ml NGF for 2 d and fixed for microscopy. At least 100 cells were assessed in each experiment, and the experiments were repeated three times. (<i><b>A</b></i>) Representative DIC images of the control cells (top-left), TC10-depleted cells (top-right), resi-TC10-WT expressing TC10-depleted cells (bottom-left), and resi-TC10-Q75L expressing TC10-depleted cells (bottom-right) are shown. A bar, 15 μm. (<i><b>B</b></i>) Cells with neurites longer than their cell body lengths were scored as neurite-bearing cells. The results are expressed as the mean plus SE of the percentage of neurite-bearing cells. The symbols indicate the results of a one-way ANOVA followed by Dunnett’s post-hoc test; **p < 0.01.</p

Hypothetical mechanism underlying the neuritogenesis-promoting functions of TC10.

<p>The bottom panel exhibits an enlarged view of the boxed region in the top panel, which shows the migration and subsequent fusion of vesicles for membrane expansion in an extending growth cone. In this model, most TC10 on vesicles is GTP-bound and associated with effectors including Exo70. Vesicles containing GTP-TC10 migrate to the plasma membrane. At their destination, vesicle tethering occurs through a full assembly of the exocyst complex containing Exo70 pre-bound to GTP-TC10. Next, GTP hydrolysis of TC10 on the tethered vesicles induces release of Exo70 and exocyst disassembly, which contributes to vesicle fusion.</p

Distribution of TC10 activity at the plasma membrane of extending neurites.

<p>(<i><b>A</b></i>) PC12 cells expressing Raichu-TC10/K-RasCT were treated with 50 ng/ml NGF for 24 h and then imaged every 2 min for 1 h. Representative images of the ratio of FRET/CFP at the indicated time points (in min) are shown in an intensity-modulated display (IMD) mode with the corresponding DIC images. In the IMD mode, eight colors from red to blue are used to represent the FRET/CFP ratio, with the intensity of each color indicating the mean intensity of FRET and CFP. The upper and lower limits of the ratio images are shown on the right. A bar, 5 μm. (<i><b>B</b></i>) Hippocampal neurons expressing Raichu-TC10/K-RasCT were imaged every 3 min for 30 min. Representative ratio images of FRET/CFP at the indicated time points (in min) are shown as described for (<i><b>A</b></i>). A bar, 10 μm.</p

Colocalization of TC10 with Rab11 on vesicles.

<p>N1E-115 cells expressing mCherry-Rab11 and mTFP-TC10 were serum-starved for 6 h and imaged every 10 sec. (<i><b>A</b></i>) A representative image of the subcellular distribution of TC10 and Rab11 in a growth cone. A merged image (bottom-right) shows remarkable colocalization between TC10 and Rab11 on vesicles. A bar, 5 μm. (<i><b>B</b></i>) A scatter plot depicts the percentage of Rab11-containing vesicles that also have TC10. A blue bar indicates the median value (n = 35). (<i><b>C</b></i>) mTFP (TC10), mCherry (Rab11), and merged images corresponding to the boxed region in (<i><b>A</b></i>) at the indicated time points are shown. Blue arrowheads mark the co-migration of TC10 and Rab11 on vesicles. (<i><b>D</b></i>) Representative images of vesicular localization of TC10 and Rab11 in a neurite shaft at the indicated time points. The movement of one vesicle containing both TC10 and Rab11 is traced with a dotted line. A bar, 5 μm.</p

Spatiotemporal changes in TC10 activity at the plasma membrane following NGF or dbcAMP stimulation.

<p>(<i><b>A and B</b></i>) PC12 cells expressing Raichu-TC10/K-RasCT were starved for 30 min and then stimulated with 50 ng/ml NGF. Images were obtained every 2 min for 20 min after NGF addition. (<i><b>A</b></i>) Representative images of the ratio of FRET/CFP at the indicated time points (in min) after NGF stimulation are shown as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079689#pone-0079689-g001" target="_blank">Figure 1A</a>. A bar, 10 μm. (<i><b>B</b></i>) The mean FRET/CFP ratios averaged over the whole cell were determined by measuring the relative increase compared with the reference value, which was averaged over 10 min before NGF stimulation. Error bars show the SE (n = 55). The blue symbol indicates the result of a Student’s <i>t</i> test analysis (**p < 0.01). (<i><b>C</b></i>) PC12 cells were cotransfected with pRaichu-TC10/K-RasCT and pERedNLS-Rac1-S17N, stimulated with NGF, and then imaged every 2 min. The mean FRET/CFP ratios averaged over the whole cell are expressed in the same manner as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079689#pone-0079689-g002" target="_blank">Figure 2B</a>. Error bars show the SE (n = 58). The blue symbol indicates a significant difference between the value at 10 min after NGF addition in the control cells (<i><b>B</b></i>) and the one in the Rac1-S17N-expressing cells in a Student’s <i>t</i> test analysis (**p < 0.01). (<i><b>D</b></i>) PC12 cells expressing Raichu-TC10/K-RasCT were treated with 10 μM apocynin for 30 min, stimulated with NGF, and then imaged every 2 min. The mean FRET/CFP ratios averaged over the whole cell are expressed in the same manner as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079689#pone-0079689-g002" target="_blank">Figure 2B</a>. Error bars show the SE. The number of experiments for the control condition was 14 and for the apocynin pretreatments was 24. The blue symbol indicates the result of a Student’s t test analysis (**p < 0.01). (<i><b>E and F</b></i>) PC12 cells expressing Raichu-TC10/K-RasCT were starved for 30 min and then treated with 1 mM dbcAMP. Images were obtained every 2 min for 20 min after dbcAMP addition. (<i><b>E</b></i>) Representative images of the ratio of FRET/CFP at the indicated time points (in min) after dbcAMP treatment are shown as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079689#pone-0079689-g001" target="_blank">Figure 1A</a>. A bar, 10 μm. (<i><b>F</b></i>) The mean FRET/CFP ratios averaged over the whole cell are expressed in the same manner as in to legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079689#pone-0079689-g002" target="_blank">Figure 2B</a>. Error bars show the SE (n = 30). The blue symbol indicates the result of a Student’s <i>t</i> test analysis (**p < 0.01).</p

Exo70 distribution in growth cones and effects of TC10 depletion on Exo70-containing vesicles.

<p>(<i><b>A</b></i>) N1E-115 cells expressing EGFP-Exo70 were serum-starved for 6 h, fixed, and confocal images were obtained. Representative DIC and EGFP images of a growth cone and a neurite shaft are shown in a maximum intensity projection mode. A bar, 10 μm. (<i><b>B and C</b></i>) PC12 cells were transfected with control or TC10-targeted shRNA vector. After selection with puromycin for 2 d, the cells were immunostained with anti-Exo70 antibody and confocal images were obtained. (<i><b>B</b></i>) A representative Exo70 image of a control or TC10-depleted cell with black and white reversed. A bar, 10 μm. (<i><b>C</b></i>) A scatter plot depicts the number of vesicles in individual cells in control or TC10 knockdown cells. Bars indicate the median values (n = 20). The blue symbol indicates the result of a Student’s <i>t</i> test analysis (**p < 0.01). (<i><b>D</b></i>) N1E-115 cells expressing EGFP-Exo70 and mCherry-TC10 were serum-starved for 6 h and imaged. Representative images of the subcellular distribution of Exo70 and TC10 in growth cones are shown. Merged images (bottom-right) show remarkable colocalization of Exo70 with TC10 on vesicles. A bar, 10 μm.</p