Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL^+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL_1/2, 1∶20,000), and anti-detyrosinated tubulin (L₄, 1∶20,000).</p

Analysis of C-termini of recombinant EB1 forms overexpressed in wild type and TTL-deficient fibroblasts.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) and of fractions of immunoprecipitation experiments carried out on cells transfected with cDNA encoding different EB1 forms. EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. (A) transfection of fibroblasts (NIH3T3) with plasmids encoding tyrosinated EB1 fused with GFP at the N-terminus (GFP-EB1), followed by immunoprecipitation using anti-GFP antibody and analysis using anti-Tyr EB1 (1∶15000) and anti-deTyr EB1 (1∶200). No detyrosinated GFP-EB1 could be detected. (B) Transfection of fibroblasts with cDNA encoding GFP-EB1 ending with the C-terminus of α-tubulin GEEEGEEY (GFP-EB1-CterTub), followed by immunoprecipitation with anti-GFP antibody and analysis using anti-Tyr Tub (1∶20,000) and anti-deTyr Tub (1∶20,000). NIH3T3 were used as TTL^+/+ cells and MEFs isolated from TTL null mice were used as TTL^−/−. A very low quantity of detyrosinated protein ending with α-tubulin residues was detected (upper band in IP fractions of lower panel).</p

Analysis of developed anti-EB1 antibodies compared to the known anti-tubulin antibodies.

<p>(A) Western-blot analysis of the indicated proteins (15 ng) separated on 10% SDS-PAGE using a commercial anti-EB1 antibody (anti-total EB1, raised against amino-acids 107–268 of mouse EB1), the presently developed antibodies (anti-Tyr EB1 and anti-deTyr EB1), and tubulin antibodies. Detyrosinated EB1 was obtained from recombinant EB1 using carboxypeptidase A. Tyrosinated and detyrosinated tubulin were obtained from purified brain tubulin, using respectively TTL and carboxypeptidase A. Both anti-Tyr EB1 and anti-deTyr EB1 are highly specific. (B) Double immunostaining with anti-total EB1 antibody and anti-Tyr EB1 on fibroblasts after transfection of plasmids allowing expression of either tyrosinated or detyrosinated EB1 with EGFP at the N-terminus. The transfected cells were detected by EGFP signal. Anti-Tyr EB1 is highly specific of tyrosinated form of EB1. (C) Immunostaining of endogenous EB1 in astrocytes with anti-Tyr EB1 and anti-total EB1.</p

7 DIV neurons spread over DW4 patterns.

<p>Dendritic marker MAP2 (green), microtubules (tubulin in red) and nuclei (Hoechst staining, blue). Scale bar = 40 µm. Once formed, the axon developed freely over hundreds of microns along either straight or curved paths following the global organization of DW4 patterns arranged in a network.</p

Effect of soma constraints on centrosome position.

<p>(A) Microtubule labeling (green), highlighting the different organizations of microtubules in DC, BmS, and DS patterns. Nuclei (blue) and centrioles (red) stained with antibodies against γ-tubulin. Red arrows point to the centrioles. (B) Superimposition of density maps for centrioles and corresponding patterns (<i>n</i> = 154, 168, and 160 from stage 2 neurons for the DC, DS, and BmS patterns, respectively). (C) Centrosome distribution in stage 2 (1 DIV) and 3 (3 DIV) neurons grown on BmS patterns. Upper panel: Scheme of BmS pattern indicating the regions of interest Z0-Z3; with the scatter plot of centriole distribution superimposed (red dots, stage 3). Percentages of centrioles in each region of interest with inset showing the density map of the upper scatter plots superimposed on dashed lines delimiting the patterns. (<i>n</i> = 160, stage 2 neurons; <i>n</i> = 184, stage 3 neurons). (D) Centriole positioning (red dots) and axonal localization in neurons grown over the BmS pattern. (<i>n</i> = 31, 12, and 20 neurons for the L1, L2, and L3 directions, respectively).</p

Influence of neurite curvature on axonal polarization.

<p>(A) DW4 set of patterns of increasing curvature along directions L2–L4; Scale bars, 20 µm. (B) Partial and complete unhookings observed on fixed cells (microtubules: green, F-actin: red). White arrows point to partial unhooking, characterized by a displaced neuritic shaft still attached to the substrate by a large lamellipodium. The yellow arrow indicates a complete unhooking characterized by a high density of MTs crossing the pattern arch and remaining entities strictly following the curved adhesive line. Scale bar, 10 µm. (C) Time-lapse experiment (indicated in minutes, beginning 30 hours after plating) of a neurite developing on a DW4 pattern showing partial unhooking (white arrow). The black arrowhead points to the neurite tip and the green dashed line marks the position of the adhesive pattern. Scale bars, 20 µm. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033623#pone.0033623.s007" target="_blank">Movie S1</a> for the original time-lapse sequence. (D) Physical modeling of a curved neurite (in red) as an elastic wire under tension Τ. Curvature is reflected by the angle γ (see text), and Fγ = 2 T sin γ (black arrow) is the force experienced by the elastic wire. (E) Percentages of neurons displaying unhookings when grown over DW0 (0%), DW4-1 (4.7%), DW4-2 (7.0%), DW4-3 (10.6%), and DW4 (22.7%) patterns. (<i>n</i> = 117, 129, 128, 132, and 132, respectively). (F) Preferential axonal specification along the straight direction L1 were plotted from stage 3 neurons plated over DW0 (24.4%), DW4-1 (32.6%), DW4-1 (34.7%), DW4-3 (45.5%), and DW4 (52.3%) patterns. (<i>n</i> = 115, 285, 225, 330, and 216, respectively). ***, significantly different from random, <i>p</i><0.001.</p

Combined action of drugs and micropatterns on axonal polarization.

<p>(A–B) Immunolabeling of stage 3 neurons on DW4 patterns grown in the presence of 0.5 µM cytochalasin D (A) or 3 nM taxol (B): axon (tau staining, red), microtubules (tubulin staining, green) and nuclei (Hoechst staining, blue). Both drugs induced multiple axon (MA) formation as revealed by a tau positive staining of several neurites (white arrows). Scale bar, 20 µm. (C) Percentages of multiple axon (MA) neurons grown over DW0 or DW4 micropatterns, in sham conditions or in the presence of cytochalasin D (0.5 µM) or taxol (3 nM); (sham <i>n</i> = 117; CD, <i>n</i> = 112; Tx, <i>n</i> = 150 for DW0 and sham <i>n</i> = 109; CD, <i>n</i> = 153; Tx, <i>n</i> = 319 for DW4). ***, significantly different from DW0, <i>p</i><0.001. (D) Axonal preference along L1 for neurons grown on DW4 micropatterns, in the presence of DMSO, 45 nM nocodazole (Nz), 0.5 µM cytochalasin D (CD) or 3 nM taxol (Tx) (<i>n</i> = 107, 146, 104, and 237 neurons with a unique axon, respectively). Blue dotted lines represent the predicted preference along L1 in the presence of CD or Tx as determined with the probabilistic model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033623#pone.0033623.s008" target="_blank">Text S1</a>). *, significantly different from the expected distribution, <i>p</i><0.05.</p

Effect of soma constraints on axonal polarization.

<p>(A) Design of patterns DC, BmS, and DS; L1–L3 directions are indicated. (B) Immunolabelings of stage 3 neurons on DC, BmS and DS patterns: axon (tau staining, red), microtubules (tubulin staining, green) and nuclei (Hoechst staining, blue). The shape of the cells reflects the global organization of DC/DS patterns in a hexagonal network. Scale bar, 20 µm. (C) Results of axonal polarization, <i>i.e.</i> percentages of stage 3 neurons with their axon along L1–L3 directions (<i>n</i> = 194, 176 and 267 for the DC, BmS and DS patterns, respectively). *, significantly different from random (blue dotted line, 33.3% in each direction), <i>p</i><0.05.</p

Regeneration of the olfactory epithelium.

<p>Mean epithelial OMP positive area before and after regeneration at the three levels 4, 5, 6 in WT and STOP null mice at two different ages. The x-axis refers to the three levels studied, from rostral (level 4) to caudal (level 6), where turbinates are most developed and olfactory epithelium most abundant. There is no difference in the ability of olfactory epithelium to regenerate at the three levels studied between WT and STOP null mice in 3 month-old (A) and 10 month-old (B) animals. All values are represented as mean +/− SEM, *p<0.05. The photomicrographs in A and B illustrate OMP immunostaining in the olfactory epithelium of animals after regeneration. Scale bar: 500 µm.</p

Ultrastructure of olfactory bulb glomeruli.

<p>Electron microscopy micrographs of olfactory bulb glomeruli in WT (A) and STOP null (B–F) mice at 3 to 6 months of age. In STOP null mice, olfactory axon endings are filled with autophagic-like structures (B, arrow), tubulovesicular profiles (C, arrowhead), or both (D). When few autophagic structures were present (arrows) (E, F), olfactory axons endings could be identified by the presence of synaptic vesicles and postsynaptic densities (arrowheads) (E, F). De: dentrite; OA: olfactory axon. Scale bar: 0,5 µm (A, C, E, F); 1 µm (B, D).</p