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Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons

Chen, Wen-Shin, Chen, Yi-Ju, Huang, Yung-An, Hsieh, Bing-Yuan, Chiu, Ho-Chieh, Kao, Pei-Ying, Chao, Chih-Yuan, Hwang, Eric·2017·Scientific Reports
DOI 10.1038/srep42297PMC PMC5304320
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Abstract

The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. First, neurite elongation is compromised in TPX2-depleted neurons. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons.

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Figures

Figure 1 TPX2 depletion affects neurite elongation. Dissociated hippocampal neurons were transfected with plasmid expressing non-targeting control shRNA (A) or Tpx2-targeting shRNA (B) at 2DIV and fixed at 5DIV. The insert shows a magnified image of the soma. Images were inverted and γ-corrected (γ = 0.05, in insert γ = 0.1) to enhance visualization. The scale bar presents 100 μm. Quantification of total neurite length per neuron (C), branch number per 200 μm neurite (D), and primary neurite number (E) of control and TPX2-depleted neurons. *p < 0.05 by two-tailed Student’s t-test. Error bars represent SEM from 3 independent experiments. More than 70 neurons were analyzed for each group from 3 independent experiments. (F) Schematic diagram of the neuron replating protocol. (G) Histogram of primary neurite number distribution of replated neurons. Neuron number of each group was normalized to total neuron number counted (non-targeting = 99, Tpx2-targeting = 92).
Figure 1 TPX2 depletion affects neurite elongation. Dissociated hippocampal neurons were transfected with plasmid expressing non-targeting control shRNA (A) or Tpx2-targeting shRNA (B) at 2DIV and fixed at 5DIV. The insert shows a magnified image of the soma. Images were inverted and γ-corrected (γ = 0.05, in insert γ = 0.1) to enhance visualization. The scale bar presents 100 μm. Quantification of total neurite length per neuron (C), branch number per 200 μm neurite (D), and primary neurite number (E) of control and TPX2-depleted neurons. *p < 0.05 by two-tailed Student’s t-test. Error bars represent SEM from 3 independent experiments. More than 70 neurons were analyzed for each group from 3 independent experiments. (F) Schematic diagram of the neuron replating protocol. (G) Histogram of primary neurite number distribution of replated neurons. Neuron number of each group was normalized to total neuron number counted (non-targeting = 99, Tpx2-targeting = 92).CC BY · PubMed Central
Figure 2 TPX2 localization in dissociated hippocampal neurons. (A) 5DIV hippocampal neurons were fixed and stained with anti-TPX2 (left), β-III-tubulin antibody (middle), and DAPI. (B) 1~4DIV hippocampal neurons in dissociated culture stained with anti-TPX2 (top row) and anti-γ-tubulin (center row) antibodies. All scale bars represent 10 μm.
Figure 2 TPX2 localization in dissociated hippocampal neurons. (A) 5DIV hippocampal neurons were fixed and stained with anti-TPX2 (left), β-III-tubulin antibody (middle), and DAPI. (B) 1~4DIV hippocampal neurons in dissociated culture stained with anti-TPX2 (top row) and anti-γ-tubulin (center row) antibodies. All scale bars represent 10 μm.CC BY · PubMed Central
Figure 3 TPX2 localizes to the microtubules along the neurite shaft. Representative images of PLA for TPX2 and β-III-tubulin in 1DIV (A) or 8DIV (B) dissociated hippocampal neurons. Dylight 488-conjugated secondary antibody was applied after PLA to stain β-III-tubulin antibody and to detect neurons (shown in green). DAPI stain was used to detect the nucleus (shown in blue). (C) PLA puncta were produced along the neurite shaft only when anti-TPX2 and anti-β-III-tubulin antibodies were both present. Alexa 405-conjugated secondary antibody was applied after PLA to stain β-III-tubulin antibody and to facilitate neurite visualization (shown in blue). All scale bars represent 10 μm.
Figure 3 TPX2 localizes to the microtubules along the neurite shaft. Representative images of PLA for TPX2 and β-III-tubulin in 1DIV (A) or 8DIV (B) dissociated hippocampal neurons. Dylight 488-conjugated secondary antibody was applied after PLA to stain β-III-tubulin antibody and to detect neurons (shown in green). DAPI stain was used to detect the nucleus (shown in blue). (C) PLA puncta were produced along the neurite shaft only when anti-TPX2 and anti-β-III-tubulin antibodies were both present. Alexa 405-conjugated secondary antibody was applied after PLA to stain β-III-tubulin antibody and to facilitate neurite visualization (shown in blue). All scale bars represent 10 μm.CC BY · PubMed Central
Figure 4 TPX2 depletion reduces microtubule emanating frequency at the tip and base of the neurite. Cortical neurons were cotransfected with plasmids expressing EB3-mCherry and non-targeting or Tpx2-targeting shRNA before plating, incubated for 4 days before subjected to live cell imaging. (A) The image of a 4DIV cortical neuron expressing EB3-mCherry. The color windows indicated the region from which kymographs were generated. The scale bar represents 10 μm. (B) Kymographs of EB3-mCherry in indicated regions of the neurite. (C) Quantification of EB3-mCherry dynamics in control and TPX2-depleted neurons. *p < 0.05, two-tailed Student’s t-test. Error bars represent SEM from 3 independent repeats. At least 10 neurons were analyzed per condition per repeat.
Figure 4 TPX2 depletion reduces microtubule emanating frequency at the tip and base of the neurite. Cortical neurons were cotransfected with plasmids expressing EB3-mCherry and non-targeting or Tpx2-targeting shRNA before plating, incubated for 4 days before subjected to live cell imaging. (A) The image of a 4DIV cortical neuron expressing EB3-mCherry. The color windows indicated the region from which kymographs were generated. The scale bar represents 10 μm. (B) Kymographs of EB3-mCherry in indicated regions of the neurite. (C) Quantification of EB3-mCherry dynamics in control and TPX2-depleted neurons. *p < 0.05, two-tailed Student’s t-test. Error bars represent SEM from 3 independent repeats. At least 10 neurons were analyzed per condition per repeat.CC BY · PubMed Central
Figure 5 RanGTP is enriched in the tip of the neurite and the soma. (A,B) Images of 3DIV hippocampal neurons immunofluorescence stained with antibody against Ran (A) and RanGTP (B), respectively. The inset in middle panel A shows the same image around the soma region with reduced image contrast. All images have the same scale and the scale bar represents 10 μm. (C) Ran (green) or RanGTP (red) intensity linescan along a 10 μm stretch from the neurite tip in 3DIV hippocampal neurons. RanGTP accumulated significantly more compared to Ran in the neurite tip. Mean ± SEM from 3 independent experiments, ***p < 0.001, two-way ANOVA followed by Bonferroni post-test. (D) To obtain the intensity change at the neurite tip, Ran or RanGTP intensity average within 1 μm from the neurite tip was divided by the intensity along the entire neurite. A ratio above 1.0 indicates an increase, while a ratio below 1.0 indicates a decrease of Ran at the neurite tip. The intensity ratios were classified into three groups. The group with ratio equal to or greater than 1.5 is shown in red, the group with ratio in between 1.5 and 1.0 is shown in orange, and the group with ratio in between 1.0 and 0 is shown in yellow. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. A total of 131 and 120 neurites were analyzed for Ran and RanGTP from three independent repeats, respectively.
Figure 5 RanGTP is enriched in the tip of the neurite and the soma. (A,B) Images of 3DIV hippocampal neurons immunofluorescence stained with antibody against Ran (A) and RanGTP (B), respectively. The inset in middle panel A shows the same image around the soma region with reduced image contrast. All images have the same scale and the scale bar represents 10 μm. (C) Ran (green) or RanGTP (red) intensity linescan along a 10 μm stretch from the neurite tip in 3DIV hippocampal neurons. RanGTP accumulated significantly more compared to Ran in the neurite tip. Mean ± SEM from 3 independent experiments, ***p < 0.001, two-way ANOVA followed by Bonferroni post-test. (D) To obtain the intensity change at the neurite tip, Ran or RanGTP intensity average within 1 μm from the neurite tip was divided by the intensity along the entire neurite. A ratio above 1.0 indicates an increase, while a ratio below 1.0 indicates a decrease of Ran at the neurite tip. The intensity ratios were classified into three groups. The group with ratio equal to or greater than 1.5 is shown in red, the group with ratio in between 1.5 and 1.0 is shown in orange, and the group with ratio in between 1.0 and 0 is shown in yellow. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. A total of 131 and 120 neurites were analyzed for Ran and RanGTP from three independent repeats, respectively.CC BY · PubMed Central
Figure 6 Overexpressing importin-α reduces microtubule emanating frequency and compromises neuronal morphogenesis. Cortical neurons were cotransfected with plasmids overexpressing importin-α (KPNA1, 4, 6) and EB3-mCherry before plating, incubated for 2 days before subjected to live cell imaging. (A) Kymographs of EB3-mCherry at the tip of neurites. (B–D) Quantification of EB3-mCherry dynamics in EGFP (control) and importin-α-overexpressing neurons. At least 10 neurons were analyzed per condition per repeat. (E) Representative images of dissociated hippocampal neurons transfected with plasmid overexpressing KPNA1, KPNA4, or KPNA6 at 2DIV and fixed at 4DIV. Images were inverted to enhance visualization. The scale bar presents 50 μm. Quantification of total neurite length (F), branch number per 200 μm neurite (G), and primary neurite number (H) of EGFP- and importin-α-overexpressing neurons. More than 50 neurons were analyzed for each group from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Dunnett’s comparison to the control group. Error bars represent SEM from 3 independent experiments.
Figure 6 Overexpressing importin-α reduces microtubule emanating frequency and compromises neuronal morphogenesis. Cortical neurons were cotransfected with plasmids overexpressing importin-α (KPNA1, 4, 6) and EB3-mCherry before plating, incubated for 2 days before subjected to live cell imaging. (A) Kymographs of EB3-mCherry at the tip of neurites. (B–D) Quantification of EB3-mCherry dynamics in EGFP (control) and importin-α-overexpressing neurons. At least 10 neurons were analyzed per condition per repeat. (E) Representative images of dissociated hippocampal neurons transfected with plasmid overexpressing KPNA1, KPNA4, or KPNA6 at 2DIV and fixed at 4DIV. Images were inverted to enhance visualization. The scale bar presents 50 μm. Quantification of total neurite length (F), branch number per 200 μm neurite (G), and primary neurite number (H) of EGFP- and importin-α-overexpressing neurons. More than 50 neurons were analyzed for each group from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Dunnett’s comparison to the control group. Error bars represent SEM from 3 independent experiments.CC BY · PubMed Central
Figure 7 Importazole reduces microtubule emanating frequency at the tip and the base of the neurite. Cortical neurons were transfected with plasmid expressing EB3-mCherry before plating, incubated for 4 days before subjected to live cell imaging in the presence of 15 μM importazole (IPZ). (A) Kymographs of EB3-mCherry in indicated regions of the neurite. (B) Quantification of EB3-mCherry dynamics in control (DMSO) or 15 μM IPZ (IPZ) treated neurons. EB3 dynamic properties before IPZ application (pre) and those after IPZ application (post) are compared. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from 3 independent repeats. 6 neurons were analyzed per condition per repeat.
Figure 7 Importazole reduces microtubule emanating frequency at the tip and the base of the neurite. Cortical neurons were transfected with plasmid expressing EB3-mCherry before plating, incubated for 4 days before subjected to live cell imaging in the presence of 15 μM importazole (IPZ). (A) Kymographs of EB3-mCherry in indicated regions of the neurite. (B) Quantification of EB3-mCherry dynamics in control (DMSO) or 15 μM IPZ (IPZ) treated neurons. EB3 dynamic properties before IPZ application (pre) and those after IPZ application (post) are compared. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from 3 independent repeats. 6 neurons were analyzed per condition per repeat.CC BY · PubMed Central
Figure 8 Schematic model for the Ran-activated TPX2 in microtubule nucleation. MT-bound TPX2 molecules are activated by the RanGTP gradient, which is concentrated at the tip of the neurite and in the soma.
Figure 8 Schematic model for the Ran-activated TPX2 in microtubule nucleation. MT-bound TPX2 molecules are activated by the RanGTP gradient, which is concentrated at the tip of the neurite and in the soma.CC BY · PubMed Central

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Authors
Chen, W.-S., Chen, Y.-J., Huang, Y.-A., Hsieh, B.-Y., Chiu, H.-C., Kao, P.-Y., Chao, C.-Y., & Hwang, E.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-02-13
Volume
7
Issue
1
ISSN
2045-2322
DOI
10.1038/srep42297
PMC
PMC5304320
Cited by
41
References
55
Language
en
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