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1.
Figure 7.

Figure 7. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

A model for the involvement of shootin1 in generation of an asymmetric signal for neuronal polarization. (A) A positive feedback loop between shootin1 accumulation in growth cone and shootin1-induced neurite elongation. (B) Competition among neurites for a limited amount of shootin1. (C) Shootin1 up-regulation triggers local positive feedback loops (red arrows) and negative regulations (blue arrows). Eventually, shootin1 will be asymmetrically accumulated in a single neurite and recruit PI 3-kinase activity there, thereby leading to neuronal polarization.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
2.
Figure 5.

Figure 5. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Inhibition of shootin1 transport prevents its asymmetric accumulation in hippocampal neurons. (A) Stage 2 hippocampal neurons treated with 50 μM blebbistatin for 1 h were double stained with anti-shootin1 antibody (red) and a volume marker CMFDA (green). Quantitative profiles show the relative fluorescence intensities of shootin1 and CMFDA and the relative concentration of shootin1 (shootin1 immunoreactivity/CMFDA staining) in neurites 1–4. The arrowheads and asterisks denote the minor processes and cell body, respectively. (B) Hippocampal neurons were treated with 50 μM blebbistatin at 14 h in vitro, further cultured for 36 h, and double stained with anti-shootin1 antibody (red) and CMFDA (green). Quantitative profiles show the relative fluorescence intensities of shootin1 and CMFDA and the relative concentration of shootin1 in neurites 1–6. The asterisks denote the cell body. Bars, 50 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
3.
Figure 2.

Figure 2. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Dynamic accumulation of EGFP-shootin1 in growth cones of hippocampal neurons. (A) A stage 2 hippocampal neuron expressing EGFP-shootin1 was observed under a time-lapse fluorescence microscope every 5 min. The full video is presented in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1). (B) The pictures represent a series of enlarged images of neurite 1 in A taken every 5 min. Asterisks indicate the front edge of the neurite. (C) Correlation between neurite elongation speed and EGFP-shootin1 levels in growth cones. Stage 2 hippocampal neurons expressing EGFP-shootin1 (green) and mRFP (red) was observed under a time-lapse fluorescence microscope every 5 min. Relative levels of EGFP-shootin1 and mRFP in growth cones were quantified using Multi Gauge (n = 315). Relative concentration of EGFP-shootin1 in growth cones was calculated by using mRFP as an internal standard (EGFP-shootin1/mRFP), and the neurite elongation speeds during the next 5 min were measured. (D–F) A hippocampal neuron expressing EGFP-shootin1 was observed by time-lapse fluorescence microscopy every 30 min. The full video is presented in Video 2. Arrows indicate EGFP-shootin1 accumulation in the growth cone of the nascent axon. (G) Elongation of neurites 1–5 shown in D. The circles denote growth cones with apparent shootin1 accumulation, and the diamonds indicate growth cones without the accumulation. The green shade denotes the period when the nascent axon (neurite 1) showed exclusive and continuous accumulation before rapid elongation. Bars, 20 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
4.
Figure 4.

Figure 4. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Shootin1 is anterogradely transported to the growth cones with wave-like structures and diffuses back to the soma. (A) Distal movements of EGFP-shootin1 within neurite shafts from the cell body to a growth cone. The arrows indicate boluses of EGFP-shootin1. (B) A stage 3 hippocampal neuron double stained with anti-shootin1 antibody and Rhodamine phalloidin. Shootin1- and F-actin–enriched wave is indicated by arrowheads. (C) Serial time-lapse images showing the effect of 50 μM blebbistatin on anterograde transport of EGFP-shootin1. Blebbistatin was applied to the medium for between 35 and 40 min. EGFP-shootin1 in the neurite shaft is indicated by arrowheads. (D) Disturbance of shootin1 transport by blebbistatin inhibits its accumulation in axonal growth cones. Stage 3 hippocampal neurons (cultured for 48 h) were incubated with 50 μM blebbistatin for 1 h and stained with anti-shootin1 antibody. (E) Shootin1 diffuses from the axonal growth cone to the cell body. Kaede-shootin1 expressed in stage 3 hippocampal neurons was converted from green to red using UV light. 1 h after the cessation of shootin1 transport by 50 μM blebbistatin, distributions of red Kaede-shootin1 and newly synthesized green Kaede-shootin1 were examined. Bars: (A and B) 20 μm; (C–E) 50 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
5.
Figure 3.

Figure 3. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Effects of shootin1 overexpression and RNAi on polarization of hippocampal neurons. (A) A neuron overexpressing EGFP-shootin1 was observed every 5 min under a time-lapse fluorescence microscope. The full video is presented in Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1). Arrows indicate EGFP-shootin1 accumulated at a high level in growth cones. Arrowheads indicate frequent transport of shootin1 from the cell body to the growth cones. (B) Stage 3 neurons overexpressing myc-shootin1 were immunostained by anti-myc antibody (red). Arrowheads indicate myc-shootin1 aberrantly accumulated in the growth cones of minor processes. (C) Hippocampal neurons overexpressing myc-shootin1 were cultured for 7 d and then double immunostained by anti-myc (green) and anti–tau-1 (red) antibodies. Arrowheads indicate axons labeled by anti–tau-1 antibody. (D) Quantification of the neurite lengths of the neurons (DIV7) with surplus axons. The lengths of the longest and secondary axons and dendrites were measured in neurons overexpressing myc-shootin1 (n = 26) or myc-GST (n = 27). Data present neurite length as means ± SE. (E) Quantification of the total neurite lengths of the neurons (DIV7) overexpressing myc-shootin1 or myc-GST. (F) Neurons transfected with the miRNA or a control miRNA vector were cultured for 50 h. They were then fixed and immunostained with anti-shootin1 antibody. The vectors for miRNA and control miRNA expressions are designed to coexpress EGFP. (G) Neurons prepared from E18 or E17 embryo and transfected with the miRNA or a control miRNA vector were cultured. Data present percentages of neurons bearing axons (stage 3 neurons) as means ± SE (**, P < 0.02; ***, P < 0.002; n = 3; a total of 752 neurons were examined). Black dots present percentages of nontransfected neurons bearing axons. (H and I) Quantification of the total neurite lengths of the neurons overexpressing myc-shootin1 or myc-GST (H) or expressing shootin1 miRNA or control miRNA (I). Data present neurite length as means ± SE (***, P < 0.0005; n = 3; a total of 761 neurons for H and 568 neurons for I were examined). Bars, 50 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
6.
Figure 6.

Figure 6. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Shootin1 regulates the localization of PI 3-kinase activity in hippocampal neurons. (A) Brain lysates from P5 rat brain were incubated with anti-shootin1 antibody, anti-p85 antibody, or control IgG. The immunoprecipitates were analyzed by immunoblotting with anti-shootin1 and anti-p85 antibodies as indicated. (B) Stage 3 hippocampal neurons were incubated with DMSO as control for 10 h and double stained with anti-shootin1 antibody and anti–P-Akt (Ser473) antibody. Arrows indicate an axonal growth cone. Note that PI 3-kinase activity was preferentially colocalized with shootin1 in the axonal growth cone (insets). (C) Neurons were cultured in the normal medium or in the presence of 20 μM LY294002 or DMSO. Data present percentages of neurons bearing axons (stage 3 neurons) as means ± SE (**, P < 0.005; ***, P < 0.002; n = 3; a total of 699 neurons were examined). (D) Stage 3 neurons transfected with the miRNA against shootin1 were double stained with anti-shootin1 antibody (blue) and anti–P-Akt antibody (red). The vector for the miRNA expressions is designed to coexpress EGFP (green). Arrows indicate a shootin1-immunonegative axonal growth cone without remarkable accumulation of P-Akt. (E) Shootin1 overexpressed in stage 3 neurons accumulated ectopically in minor processes together with P-Akt (arrowheads). (F) Stage 3 hippocampal neurons were incubated with 20 μM LY294002 for 10 h and double stained with anti-shootin1 antibody and anti–P-Akt antibody. Arrows indicate a P-Akt–immunonegative axonal growth cone with remarkable shootin1 accumulation. (G) Primary cultured hippocampal neurons were transfected by pCAGGS-myc-shootin1 or pCAGGS-myc-GST (control; left) or were transfected with the miRNA against shootin1 or a control miRNA (right) using Nucleofector. The efficiency of transfection was >80%. After 36 h in culture, cell lysates were collected and immunoblotted by anti–P-Akt antibody to monitor PI 3-kinase activity. (H) Hippocampal neurons overexpressing myc-shootin 1 were cultured for 7 d in the presence of 20 μM LY294002 or DMSO (n = 3; 198 neurons examined). Neurons transfected with pCAGGS–Myr–PI 3-kinase p110 plus the miRNA against shootin1 or pCAGGS–Myr–PI 3-kinase p110 plus a control-miRNA were also cultured for 7 d (n = 3; 225 neurons examined). Data present percentages of neurons with multiple axons as means ± SE. **, P < 0.001. Bars, 20 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.
7.
Figure 1.

Figure 1. From: Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.

Identification, structure, expression, and intracellular localization of shootin1. (A) Differential 2DE analysis of proteins in stage 2 (cultured for 14 h) and stage 3 (cultured for 62 h) hippocampal neurons. The arrows indicate the protein spot of shootin1 enriched in the stage 3 sample (stage 3/2 = 3.2; n = 12; P < 0.001). (B) Differential 2DE analysis of proteins in cell body/dendrite and axon samples. The arrows indicate the same protein spot shown in A, which is also enriched in the axon samples (axon/somatodendrite = 1.6; n = 7; P < 0.005). (C) Amino acid sequence of rat, human, and mouse shootin1. Sequences of the peptides identified by mass spectrometry analysis are underlined. (D) Schematic representation of rat shootin1, showing three coiled-coil domains (CC1–3) and a single proline-rich region. (E) Immunoblot analysis of purified recombinant shootin1 and shootin1 in cultured rat hippocampal neurons at different stages. Immunoblot data of synaptophysin are also shown. (F) Immunoblot analysis of shootin1 in adult rat tissues and P4 brain. (G) Immunoblot analysis of shootin1 in rat brains at various developmental stages. (H and I) Immunofluorescent localization of shootin1 in late stage 2 (H) and stage 3 (I) hippocampal neurons. Neurons were double stained with anti-shootin1 antibody (red) and a volume marker CMFDA (green). Quantitative profiles show the relative fluorescence intensities of shootin1 and CMFDA and relative concentration of shootin1 (shootin1 immunoreactivity/CMFDA staining). Arrows, arrowheads, and asterisks denote axonal growth cones, minor process growth cones, and cell bodies, respectively. Bars, 20 μm.

Michinori Toriyama, et al. J Cell Biol. 2006 October 9;175(1):147-157.

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