Journal: The Journal of Cell Biology

Article Title: Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism

doi: 10.1083/jcb.201409036

Figure Lengend Snippet: GFP-tagged and endogenous α-tubulin have similar properties. (A) Bright-field (a) and TIRF (b and c) images of a live cell expressing GFP–α-tubulin. The two focal planes show cilia (b) and cell body microtubules (c). Bar, 1 µm. (B) Western blot analysis of wild type (WT) and the GFP–α-tubulin expressing strain GFP-Tub1. Whole cells (WC), cell bodies (CB), isolated cilia (FLA), axonemes (AX), and MM fractions were loaded and probed with antibodies to GFP and α-tubulin. The bands corresponding to α-tubulin, GFP–α-tubulin, and the uncleaved ble-GFP–α-tubulin are marked. (C) Western blots probed with anti–α-tubulin and anti-GFP showing different dilutions of the cell body (CB) sample in comparison to the undiluted cilia sample (FLA). The amounts of endogenous and tagged tubulin in cilia correspond to ∼20 and ∼10% of the respective cell body tubulin. (D) Western blot showing a dilution series of axonemes (AX) and undiluted MM of strain GFP-Tub1 to determine the distribution of tagged and endogenous tubulin inside cilia. The blot was probed with antibodies to α-tubulin and the matrix protein IFT139. (E) Western blots of wild-type and GFP-Tub1 axonemes probed with antibodies to α-tubulin, acetylated α-tubulin (6-11B-1), polyglutamylated tubulin (GT335), GFP, and, as a loading control, IC2, an outer arm dynein intermediate chain. (F and G) Schematic presentations of the distribution of GFP-tagged tubulin and endogenous tubulin in whole cells (F) and cilia (G).

Article Snippet: The following primary antibodies were used: mouse anti-acetylated tubulin (clone 6-11B-1; 1:10,000; Sigma-Aldrich), mouse anti–α-tubulin (clone B-5-1-2; 1:10,000; Sigma-Aldrich), mouse anti-IC2 (1:50; ), mouse anti-IFT81 (1:200; ), mouse anti-IFT139 (1:50; ), and mouse anti-IFT172 (1:50; ), mouse GT335 (anti-glutamylated tubulin; 1:2,000; AdipoGen), rabbit anti–β-tubulin (1:2,000; ), rabbit anti-FAP12 (1:1,000; provided by D. Cole, University of Idaho, Moscow, ID), rabbit anti-GFP (1:500; Invitrogen), and rabbit anti-NAB1 (1:5,000; Agrisera).

Techniques: Expressing, Western Blot, Isolation, Comparison, Control

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: MT detyrosination near KTs increases with the establishment of correct, more stable, MT attachments. (A) Confocal analysis of U2OS cells by immunofluorescence for detyrosinated and tyrosinated α-tubulin (detyr-tub and tyr-tub, respectively), and KTs with ACAs. Maximum intensity 3D projection of representative cells for each attachment configuration (zoomed images highlight monotelic/syntelic attachments—monastrol, amphitelic attachments—DMSO and hyper-stable amphitelic attachments—taxol). (B) Single sections from the respective confocal series shown in A with dashed lines indicating line-scanned representative KT fibers (k-fibers). Scale bars, 5 µm. Line-scan plots of the selected KT fibers indicated in B. (C and D) Interquartile representation of the mean cumulative fluorescence intensity ratio between detyrosinated/tyrosinated α-tubulin within 1 µm distance from the KT centroid in monotelic/syntelic attachments (monastrol: n = 391 k-fibers, ∼20 k-fibers/cell, five cells/experiment, four independent experiments) and hyper-stable amphitelic attachments (taxol: n = 199 k-fibers, ∼20 k-fibers/cell, five cells/experiment, two independent experiments), respectively, compared with untreated amphitelic (DMSO: n = 396 k-fibers, ∼20 k-fibers/cell, five cells/experiment, four independent experiments) attachments. (E and F) Interquartile representation of the fluorescence intensity ratio between detyrosinated/tyrosinated α-tubulin as a function of distance from the KT centroid from the same dataset used in C and D. Means are represented by “+.” ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, nonsignificant; unpaired two-tailed t test. AU, arbitrary units. KT, kinetochore.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Immunofluorescence, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: TTL depletion increases α-tubulin detyrosination on astral MTs and in the vicinity of KTs without affecting inter-KT distance. (A) Protein lysates from parental U2OS cells collected 72 h after RNAi transfection in control and after siTTL were immunoblotted for tyrosinated tubulin, detyrosinated tubulin, and TTL. GAPDH was used as loading control. (B) Quantification of the fluorescence intensity ratio between detyrosinated/tyrosinated α-tubulin along astral MTs in control and siTTL metaphase cells ( n = 150 astral MTs/condition; 10 astral MTs/cell, 5 cells/experiment, pool of three independent experiments. ****, P < 0.0001, unpaired two-tailed t test). (C) Quantification of the fluorescence intensity of detyrosinated α-tubulin relative to ACA at the KTs in control siTTL U2OS cells ( n [control] = 2,224 KTs, 19 metaphase cells, pool of two independent experiments; n [siTTL] = 2,507 KTs, 19 metaphase cells, pool of two independent experiments. ****, P < 0.0001, unpaired two-tailed t test). The mean fluorescence intensity of tubulin detyrosination at KTs normalized by the mean of control cells is represented. Error bars represent SD. (D ) Quantification of the inter-KT distance in control, siTTL, and nocodazole (1 µM)–treated U2OS parental cells ( n [control] = 762 KT pairs, n [siTTL] = 795 KT pairs, and n [nocodazole] = 695 KT pairs, from 6–11 cells/condition, pool of three independent experiments. ****, P < 0.0001, unpaired two-tailed t test).

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Transfection, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Cellular adaptation to the chronic loss of TTL. (A) Protein lysates of control (sgCtrl) and TTL KO (sgTTL) U2OS cells were immunoblotted for cas9, polyglutamylated tubulin (polyglut-α/β-tub), α-tubulin (α-tub), TTL, tyrosinated α-tubulin (Tyr-tub), and detyrosinated α-tubulin (Detyr-tub). GAPDH was used as loading control. (B) Confocal/CH-STED analysis of control (sgControl) and TTL KO (sgTTL) U2OS cells by immunofluorescence for Detyr-tub (confocal) and Tyr-tub (CH-STED). Red-hot lookup table was used to map extremes in fluorescence intensity for Detyr-tub. Scale bars, 5 µm. (C) Protein lysates of control (sgCtrl) and TTL KO (sgTTL) U2OS H2B-GFP/mCherry-tub cells were immunoblotted for cas9, mCherry, Tyr-tub, Detyr-tub, and TTL. α-tubulin was used as loading control. (D) Quantification of the percentage of anaphase cells with lagging chromosomes in control (sgCtrl) and TTL KO (sgTTL) U2OS H2B-GFP/mCherry-tub cells by live-cell imaging between 72 and 168 h after lentiviral transduction ( n [sgCtrl] = 266 cells, n [sgTTL] = 241 cells, pool of two independent experiments, with two and nine replicates, respectively, *, P < 0.05, logistic regression). (E) Quantification of the percentage of anaphase cells with lagging chromosome between 72 and 192 h after lentiviral transduction in control (sgCtrl) and TTL KO (sgTTL) U2OS cells by immunofluorescence in fixed cells ( n [sgCtrl] 72 h = 324 cells, n [sgCtrl] 96 h = 311 cells, n [sgCtrl] 120 h = 280 cells, n [sgCtrl] 144 h = 264 cells, n [sgCtrl] 168 h = 320 cells, n [sgCtrl] 192 h = 356 cells; n [sgTTL] 72 h = 301 cells, n [sgTTL] 96 h = 377 cells, n [sgTTL] 120 h = 462 cells, n [sgTTL] 144 h = 256 cells, n [sgTTL] 168 h = 367 cells, n [sgTTL] 192 h = 386 cells, pool of three independent experiments. ****, P < 0.0001; **, P < 0.01; *, P < 0.05; ns, nonsignificant; logistic regression). (F) Protein lysates of control (sgControl) and TTL KO (sgTTL) U2OS cells were collected at different time points after lentiviral transduction and immunoblotted for cas9, Detyr-tub, TTL, Tyr-tub, and α-tubulin. GAPDH was used as loading control.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Immunofluorescence, Fluorescence, Live Cell Imaging, Transduction

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Constitutively high α-tubulin detyrosination in the vicinity of KTs impairs faithful chromosome segregation. (A) Confocal/CH-STED analysis of U2OS cells by immunofluorescence for detyrosinated tubulin (confocal), tyrosinated tubulin (CH-STED), and KTs/ACA (CH-STED). Dashed lines and arrowheads indicate representative line-scanned k-fibers and astral MTs, respectively. Scale bars, 5 µm. (B) Graphic representation of the line scans indicated in A. (C) Quantification of the fluorescence intensity ratio between detyrosinated/tyrosinated α-tubulin along k-fibers ( n = 150 k-fibers; ∼10 k-fibers/cell; five cells/experiment, three independent experiments for each condition). “0” corresponds to the KT centroid. The mean values are represented by a thicker line and the SD associated to each point is represented by a shaded band. (D) Protein lysates of U2OS cells stably expressing H2B-GFP/mCherry-α-tubulin 72 h after RNAi transfection were immunoblotted for detyr-tub, tyr-tub, TTL, and β-tubulin. GAPDH was used as loading control. (E) Representative images from spinning-disk confocal time-series illustrating different stages of mitosis in control and TTL RNAi U2OS cells stably expressing H2B-GFP (cyan)/mCherry-α-tubulin (red). Pixels were saturated to allow the visualization of lagging chromosomes (arrowhead). Scale bars, 5 µm. Time is hours:minutes. (F) Quantification of the percentage of anaphase cells with lagging chromosomes in control (11.7%) and TTL-deficient (siTTL; 44.3%) U2OS H2B-GFP/mCherry-α-tubulin cells ( n > 60 cells, pool of three independent experiments. ****, P < 0.0001, logistic regression). (G) Confocal/CH-STED analysis of a representative anaphase U2OS cell after siTTL by immunofluorescence for tyrosinated tubulin (CH-STED) and KTs/ACA (CH-STED). DNA was counterstained with DAPI (confocal). Inserts highlight KT negative (KT-) and positive (KT+) lagging chromosomes. Scale bars, 5 µm. A 4× magnified maximum projection (five z-stacks) of the insert KT+ highlights the bi-orientation and stretching of a merotelic KT. Scale bars, 1 µm. (H) Quantification of the relative percentage of KT- and KT+ lagging chromosomes in control and TTL RNAi cells ( n > 1,000 anaphase cells, pool of two independent experiments, three replicates per experiment). (I) Quantification of the percentage of anaphase cells with lagging chromosomes in control and TTL-depleted (siTTL) cells in an asynchronous (untreated) cell population (control: n = 566 anaphase cells; siTTL: n = 386 anaphase cells, pool of three independent experiments. ****, P < 0.0001, logistic regression) and upon monastrol washout (control: n = 3,977 anaphase cells; siTTL: n = 3,130 anaphase cells, pool of 11 independent experiments. ****, P < 0.0001, logistic regression). AU, arbitrary units.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Immunofluorescence, Fluorescence, Stable Transfection, Expressing, Transfection

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: The catalytic activity of TTL is critical for α-tubulin retyrosination and prevents chromosome missegregation. (A) Immunoblot analysis of tyrosinated and detyrosinated α-tubulin, TTL, GFP, and TTL-YFP levels after GFP, TTL-YFP, TTL-YFP R , and TTL[CD]-YFP R exogenous expression. GAPDH was used as loading control. The expression of the TTL-YFP constructs was determined using an anti-GFP antibody. Representative immunoblot of three independent experiments. (B) Confocal analysis of U2OS cells expressing TTL-YFP R and TTL[CD]-YFP R . Detyrosinated and tyrosinated α-tubulin and TTL-YFP R /TTL[CD]-YFP R expression was analyzed by indirect immunofluorescence and by direct detection of the YFP signal, respectively. Maximum intensity 3D projection of representative cells for each condition. Scale bars, 10 µm. (C) Quantification of the percentage of anaphase cells with lagging chromosomes in control cells transiently expressing low-mild levels of GFP (control: 10.5% and siTTL: 28.4% [ n = 171 and n = 155, respectively]), TTL-YFP R (control: 12.1% and siTTL: 13.6% [ n = 141 and n = 103, respectively]), TTL[CD]-YFP R (control: 15.3% and siTTL; 30% [ n = 85 and n = 110, respectively]). A pool of three independent experiments for each condition was used. ****, P < 0.0001, unpaired one-tailed t test.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Activity Assay, Western Blot, Expressing, Construct, Immunofluorescence, One-tailed Test

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Modulation of α-tubulin detyrosination levels by manipulation of vasohibins- SVBP impacts chromosome segregation fidelity. (A, C, and D) Immunoblot analysis of detyrosinated α-tubulin and total α-tubulin levels after VASH1-SVBP overexpression, RNAi mediated knockdown of TTL or VASH1 and VASH2 (with/without SVBP), respectively. GAPDH was used as loading control. The expression of VASH1-GFP and SVBP-myc was determined using anti-GFP and anti-myc antibodies, respectively. (B and E) Representative scanning confocal microscopic images of metaphase spindles immunostained with antibodies against detyrosinated and tyrosinated α-tubulin for each condition. DNA was counterstained with DAPI. Scale bars, 10 µm. (F) Representative images from spinning-disk confocal time-series displaying different stages of mitosis in U2OS cells stably expressing H2B-GFP (cyan)/mCherry-α-tubulin (red) transfected with the indicated plasmids and siRNAs. White arrowhead points to a segregation error during anaphase. Scale bars, 10 µm. Time in hours:minutes. (G) Quantification of the percentage of anaphase cells with segregation defects (lagging chromosomes and DNA bridges not discriminated) from spinning-disk confocal imaging of U2OS cells stably expressing H2B-GFP/mCherry-α-tubulin transfected with the indicated plasmids and siRNAs. Error bars indicate SD of the mean. N (number of cells, number of independent experiments): control(GFP)(38, 4); VASH1-GFP+SVBP-myc (38, 4); siTTL (28, 2); siControl (121, 5); siVASH1/2 (91, 4); and siVASH1/2+siSVBP (45, 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Western Blot, Over Expression, Expressing, Stable Transfection, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: The timing of centrosome separation at NEB is indistinguishable between control and TTL-depleted cells. (A) Representative immunoblot to confirm TTL depletion efficiency by RNAi. (B) Live U2OS cells stably expressing H2B-GFP/mRFP-α-tubulin seeded on a horizontal, 10-µm-width line micropattern, at the moment of NEB, in control and after siTTL. Scale bars, 10 µm. (C) Polar plot showing centrosome positioning relative to the long nuclear axis at NEB for control and siTTL cells ( n [control] = 22 cells, n [siTTL] = 19 cells, pool of three independent experiments, nonsignificant differences between conditions, nonparametric Kolmogorov–Smirnov test). Nuclear shape is represented by a blue ellipse, and centrosomes are represented by red circles.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Western Blot, Stable Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: MT detyrosination does not interfere with global kMT dynamics . (A) Representative images of DIC and time-lapse fluorescent images of metaphase spindles in control, TTL-deficient (siTTL) and MCAK-deficient (siMCAK) U2OS cells stably expressing photoactivatable-GFP/mCherry-tubulin before photoactivation (Pre-PA) and at the indicated time points (min) after photoactivation of GFP-tubulin fluorescence. Scale bars, 5 µm. (B) Normalized fluorescence intensity after photoactivation in all conditions for metaphase cells. Data points represent mean ± SD. (C) Relative distribution of kMTs and non-kMTs obtained for each experimental condition after photoactivation (D) Calculated kMT t 1/2 under different conditions. Bars indicate mean values/cell, and error bars represent SD. Control-prometaphase: n = 31, pool of five independent experiments; control-metaphase: n = 32 cells, pool of six independent experiments; siTTL-prometaphase: n = 28, pool of six independent experiments; siTTL-metaphase: n = 17 cells, pool of three independent experiments; siMCAK-prometaphase: n = 15, pool of three independent experiments; siMCAK-metaphase: n = 30 cells, pool of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; unpaired two-tailed t test.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Distinct distribution of detyrosinated α-tubulin after TTL or MCAK depletion. (A) Representative scanning confocal microscopic images of metaphase spindles of U2OS cells immunostained with antibodies against detyrosinated and tyrosinated α-tubulin for each condition. DNA was counterstained with DAPI. Scale bars, 5 µm. (B) Protein lysates from parental U2OS cells 72 h after RNAi were immunoblotted for MCAK, detyrosinated and tyrosinated. α-tubulin, β-tubulin, and TTL. β-Tubulin were used as loading control. (C) Quantification of the percentage of spindles with enriched detyrosinated α-tubulin at the poles in all conditions (control: n = 697 anaphase cells, siTTL: n = 678 anaphase cells, siMCAK: n = 608 anaphase cells, pool of three independent experiments for each condition; *, P < 0.05, logistic regression). (D) Quantification of the fold change of anaphase cells with lagging chromosomes after monastrol washout for each condition relative to controls (control: n = 877 anaphase cells, siTTL: n = 1,142 anaphase cells, siMCAK: n = 743 anaphase cells, pool of three independent experiments for each condition. ****, P < 0.0001, logistic regression).

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques:

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: MCAK depolymerizing activity at centromeres is required and sufficient to rescue error correction in response to a constitutive increase of MT detyrosination. (A) Diagram of the MCAK constructs overexpressed in this study. *** indicates point mutations that inactivate the motor domain. (B) Protein lysates of U2OS cells 24–36 h after DNA transfection with the different MCAK constructs after TTL depletion were immunoblotted for GFP, endogenous (endo) and tagged ectopic MCAK, Tyr-tub, Detyr-tub, and β-tubulin. GAPDH was used as loading control. (C) 3D-deconvolution analysis of U2OS cells overexpressing different EGFP-MCAK constructs and immunostained for tyrosinated α-tubulin (red-hot look-up table). Scale bars, 5 µm. (D) Quantification of the fold change of anaphase cells with lagging chromosomes relative to controls in rescue experiments using MCAKFL overexpression ( n > 1,500 anaphase cells, pool of five independent experiments. ****, P < 0.0001, logistic regression) and MCAK pharmacological enhancement with UMK57 ( n > 1,000 anaphase cells, pool of four independent experiments. ****, P < 0.0001, logistic regression). (E) Quantification of the fold change of anaphase cells with lagging chromosomes relative to controls in rescue experiments using different EGFP-MCAK constructs. Significant rescue was observed upon overexpression of MCAKFL ( n > 1,500 anaphase cells, pool of five independent experiments. ****, P < 0.0001, logistic regression) and GCPBM ( n > 900 anaphase cells, *, P = 0.011, pool of four independent experiments, logistic regression). (F) Quantification of the metaphase spindle length 24-48 h after transfection with different EGFP-MCAK constructs (GFP-control: n = 47 cells, MCAKFL: n = 27 cells, MCAKhypir: n = 47 cells, ΔNMCAK: n = 33 cells, GCPBM: n = 46 cells, GBPBMhypir: n = 38 cells, pool of three independent experiments for each condition, except for GCPBMhypir [pool of two independent experiments]. **, P < 0.01, unpaired two-tailed t test). (G) Quantification of the astral MT length (~10 astral MTs/cell; GFP-control: n = 471 astral MTs, MCAKFL: n = 264 astral MTs, MCAKhypir: n = 470 astral MTs, ΔNMCAK: n = 326 astral MTs, GCPBM: n = 456 astral MTs, GBPBMhypir: n = 378 astral MTs, pool of three independent experiments for each condition, except for GCPBMhypir [pool of two independent experiments]. ****, P < 0.0001, unpaired two-tailed t test). MT, microtubule.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Activity Assay, Construct, Transfection, Over Expression, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Overexpression of the kinesin-13 Kif2b rescues chromosome missegregation due to increased α-tubulin detyrosination. (A) 3D-deconvolution microscopy analysis of U2OS cells overexpressing GFP, EGFP-Kif2a, and EGFP-Kif2b and immunostained for tyrosinated α-tubulin. Scale bars, 5 µm. (B) Quantification of the fold change of anaphase cells with lagging chromosomes relative to controls (GFP) in rescue experiments using Kif2a and Kif2b overexpression. n (GFP) = 509, n (GFP+siTTL) = 530, n (Kif2a) = 296, n (Kif2a+siTTL) = 221, n (Kif2b) = 404, and n (Kif2b+siTTL) = 295, pool of two independent experiments. ns, nonsignificant; **, P < 0.01; ****, P < 0.0001; logistic regression.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Over Expression, Microscopy

Journal: The Journal of Cell Biology

Article Title: α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

doi: 10.1083/jcb.201910064

Figure Lengend Snippet: Centromeric MCAK does not significantly alter global kMT dynamics. (A) Representative images of DIC and time-lapse fluorescent images of metaphase spindles of U2OS cells stably expressing mEos-α-tubulin. Photoconversion was performed after overexpression of GFP (control) and GCPBM. Images before photoconvertion (Pre-PC) and at the indicated times points (min) after photoconversion are displayed. Scale bars, 5 µm. (B) Normalized fluorescence intensity after photoconversion of mEos-α-tubulin in U2OS cells in metaphase (GFP-control: n = 9 cells from five independent experiments and GCPBM: n = 6 cells from three independent experiments). Data points represent mean ± SD. (C) Calculated kMT t 1/2 in control and GCPBM-overexpressing cells in prometaphase (GFP-control: n = 21 cells, and GCPBM: n = 25 cells, pool of five independent experiments, P = 0.0847, unpaired two-tailed t test) and metaphase (GFP-control: n = 9 cells from five independent experiments and GCPBM: n = 6 cells from three independent experiments, P = 0.4351, unpaired two-tailed t test) using the fluorescence decay after photoconversion. Only kMT t 1/2 between prometaphase and metaphase in control cells varied significantly. ***, P < 0.001. Bars indicate mean values/cell, and error bars represent SD. (D) Proposed model for the discrimination of mitotic errors by MT detyrosination (see Discussion for description). O.E., overexpression.

Article Snippet: TTL and tubulins were probed using the following antibodies: rabbit polyclonal anti-TTL (1:2,000, ProteinTech), rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:2,000, Bio-Rad), rabbit polyclonal anti-detyrosinated α-tubulin , mouse monoclonal anti-polyglutamilated tubulin (1:1,000, Adipogen), mouse monoclonal anti-α-tubulin B-5-1-2 clone (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-β-tubulin clone T5201 (1:2,000, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Over Expression, Fluorescence, Two Tailed Test

Journal:

Article Title: Infiltrating CTLs in Human Glioblastoma Establish Immunological Synapses with Tumorigenic Cells

doi: 10.2353/ajpath.2009.081034

Figure Lengend Snippet: MTOC is polarized toward tumor cells in CD8+ T cells. Two synapses between a CD8+ T cell and a tumor cell are shown in detail. Each synapse was stained for nuclei (DAPI) (blue), CD8+ T cells (green), vimentin (red), and MTOC (γ-Tub) (magenta). The panels illustrate three synapses between a T cell and a tumor cell. γ-Tub/DAPI and the overlapping of all four channels (MERGE) are also shown for each case. In A, a tumor cell with an aberrant nucleus is contacted by a CD8+ T cell. A magnification of the MERGE is shown in Aa and the CD8+ T cell is shown at higher magnification in images 1–4 [vimentin, (1) CD8 (2), γ-tubulin (3), and γ-tubulin+DAPI(4)]. The CD8+ T cell(2) is establishing a synapsing contact with the tumor cell (1) and the MTOC (γ-Tub)(3) is oriented toward the tumor cell. The MTOC is located in the indentation that the T cell nucleus forms and is oriented toward the intercellular contact (4). Image Ab illustrates a schematic drawing of the intercellular contact. B illustrates another tumor cell contacted by a CD8+ T cell, illustrated in the same way as in A. Magnification of MERGE is shown in Ba. Polarization of the MTOC (γ-Tub) (magenta) toward a tumor cell can be observed in T cell. The MTOC of the tumor cell can also be seen in the same optical plane. Magnification of the T cell is illustrated in images 1–4. The CD8+ T cell (2) in contact with the tumor cell (1) displays its MTOC (3) polarized toward the tumor cell and is also located at the notch formed by the T cell nucleus. (4) A schematic representation of the synapse is illustrated in Bb. Scale bar = 15 μm.

Article Snippet: Immunocytochemical Procedures The 60-μm tumor sections were cut serially through the entire sample, and immunofluorescence or diaminobenzidine detection was performed as described previously, 4 , 18 , 19 using the following primary antibodies: human CD3 (1:100, Rabbit, Dako, Glostrup, Denmark), phosphorylated ZAP-70 (1:50, rabbit, Cell Signaling, Danvers, MA), vimentin (1:400, Mouse IgM, Sigma, St Louis, MO), α-tubulin (1:100, Mouse IgG1, Sigma, St Louis, MO), γ-tubulin (1:500, Rabbit, Sigma, St Louis, MO), granzyme-B (GzmB, 1:50, Rabbit, Abcam, Cambridge, UK), human CD8 (1:100, Mouse IgG2b, Serotec, Kidlington, UK), human CD4 (1:20, Mouse IgG1, Novocastra, Newcastle, UK), human CD56 (1:100, Mouse IgG1, Novocastra), human CD11b (1:100, Rat, Abcam, Cambridge, UK), and cleaved caspase-3 (cCASP3, 1:250, Rabbit, Promega, Madison, WI).

Techniques: Staining

Journal:

Article Title: Infiltrating CTLs in Human Glioblastoma Establish Immunological Synapses with Tumorigenic Cells

doi: 10.2353/ajpath.2009.081034

Figure Lengend Snippet: T cells polarize the α-tubulin cytoskeleton and form CD3 central microclusters. Confocal analysis of three synapses between T cells and tumor cells in human GBM is shown in detail. A: Confocal pictures of the immunohistochemistry of CD3 (green), α-tubulin (α-Tub) (red), vimentin (Vimt) (magenta), and DAPI (blue) as a counterstain. In the left column, the interaction between the T cell (CD3) and tumor cell (Vimt) is depicted schematically over the grayscale of α-tubulin staining (grayscale). The confocal images show three cases of synapses where CD3 and α-tubulin present an area of higher density at the center of the intercellular contact (white arrows). B: Measurements of the relative fluorescence of CD3 and α-tubulin at the membrane of the three T cells analyzed in (A). In all three cases, CD3 and α-tubulin have a similar pattern of fluorescence, with a higher signal at the center of the intercellular contact (white arrows). C: A schematic representation of the interaction between the T cell and the tumor cell. D: A three-dimensional study of the three synapses analyzed in A and B. The channels for CD3, vimentin, CD3/vimentin/DAPI (MERGE), and CD3/vimentin (CD3/Vimt) are shown. In the CD3/Vimt column of images, the plane of the interface (broken yellow line) and the angle of vision of the three-dimensional rendering (broken yellow triangle) is depicted. Each stack of images was rendered three dimensionally and clipped at the interface. The white arrow indicates the area of maximum relative fluorescence of CD3. In the column of images 3D/CD3/Vimt, the three-dimensional reconstruction of the three synapses is shown. Two perpendicular planes are illustrated crossing the interface (broken yellow arrow) and the area of maximum fluorescence of CD3 (white arrow). The plane orientation (PO) is depicted for each synapse. The plane of the intercellular contact (interface) is illustrated and the white arrow indicates the cluster of maximum CD3 fluorescence at the center of the interface.

Article Snippet: Immunocytochemical Procedures The 60-μm tumor sections were cut serially through the entire sample, and immunofluorescence or diaminobenzidine detection was performed as described previously, 4 , 18 , 19 using the following primary antibodies: human CD3 (1:100, Rabbit, Dako, Glostrup, Denmark), phosphorylated ZAP-70 (1:50, rabbit, Cell Signaling, Danvers, MA), vimentin (1:400, Mouse IgM, Sigma, St Louis, MO), α-tubulin (1:100, Mouse IgG1, Sigma, St Louis, MO), γ-tubulin (1:500, Rabbit, Sigma, St Louis, MO), granzyme-B (GzmB, 1:50, Rabbit, Abcam, Cambridge, UK), human CD8 (1:100, Mouse IgG2b, Serotec, Kidlington, UK), human CD4 (1:20, Mouse IgG1, Novocastra, Newcastle, UK), human CD56 (1:100, Mouse IgG1, Novocastra), human CD11b (1:100, Rat, Abcam, Cambridge, UK), and cleaved caspase-3 (cCASP3, 1:250, Rabbit, Promega, Madison, WI).

Techniques: Immunohistochemistry, Staining, Fluorescence

Journal:

Article Title: Infiltrating CTLs in Human Glioblastoma Establish Immunological Synapses with Tumorigenic Cells

doi: 10.2353/ajpath.2009.081034

Figure Lengend Snippet: Cells in contact with T cells show fragmentation of the α-tubulin cytoskeleton in tumor cells and expression of cCASP3. A: A confocal analysis of a human GBM region stained to visualize CD3, α-tubulin, and vimentin, with DAPI as a nuclear counterstain. The overlapping of all four channels is shown in MERGE. (B) shows the region but only superposing the channels for DAPI, CD3, and α-tubulin. Two areas of the region are illustrated in detail (1 and 2). Area 1 has no T cells and area 2 has T cells. Images 1′ and 2′ show a higher magnification of areas 1 and 2. The confocal resolution of the images is sufficient to distinguish the different appearance of the α-tubulin cytoskeleton of tumor cells whether or not they are synapsing with T cells. Graphs show the relative fluorescence measurements of α-tubulin cytoskeleton along the cell membrane (broken yellow arrow) of a tumor cell not in contact with T cells (a [cropped from 1]) and a tumor cell in contact with a T cell (b [cropped from 2]). The measurements of the relative fluorescence of α-tubulin in the cell not in contact with T cells (a) shows uniform fluorescence levels, while the tumor cell in contact with a T cell (b) displays broken fluorescence levels. C: The quantification of the percentage of tumor cells that present a cleaved appearance of α-tubulin, whether they are in contact or not with T cells. Pie charts show that tumor cells in contact with T cells present a cleaved α-tubulin cytoskeleton more frequently than the tumor cells not in contact with T cells (63% vs 24%). A total of 167 cells were analyzed for this quantification. D illustrates two CD8+ T cells in contact with a cCASP3+ cell. The top row shows a CD8+ T cell in contact with a cCASP3+ cell displaying a fragmented nucleus. The bottom row illustrates a T cell in contact with a cCASP3+ cell showing a condensed and reduced nucleus. Scale bar = 15 mm.

Article Snippet: Immunocytochemical Procedures The 60-μm tumor sections were cut serially through the entire sample, and immunofluorescence or diaminobenzidine detection was performed as described previously, 4 , 18 , 19 using the following primary antibodies: human CD3 (1:100, Rabbit, Dako, Glostrup, Denmark), phosphorylated ZAP-70 (1:50, rabbit, Cell Signaling, Danvers, MA), vimentin (1:400, Mouse IgM, Sigma, St Louis, MO), α-tubulin (1:100, Mouse IgG1, Sigma, St Louis, MO), γ-tubulin (1:500, Rabbit, Sigma, St Louis, MO), granzyme-B (GzmB, 1:50, Rabbit, Abcam, Cambridge, UK), human CD8 (1:100, Mouse IgG2b, Serotec, Kidlington, UK), human CD4 (1:20, Mouse IgG1, Novocastra, Newcastle, UK), human CD56 (1:100, Mouse IgG1, Novocastra), human CD11b (1:100, Rat, Abcam, Cambridge, UK), and cleaved caspase-3 (cCASP3, 1:250, Rabbit, Promega, Madison, WI).

Techniques: Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: Motor innervation directs the correct development of the mouse sympathetic nervous system

doi: 10.1038/s41467-024-51290-0

Figure Lengend Snippet: a Current model of sympathetic ganglia development from early stream of free neural crest migration. Free-NCCs (yellow) become primed toward autonomic fate (yellow-blue hybrid cells) as they approach the dorsal aorta region, where they differentiate into sympathetic neurons (blue) and coalesce to form the sympathetic ganglia chain. b Transverse sections of the same E10.5 Hb9-GFP embryo (representative of 3 embryos) from sacral to thoracic levels reveal the developmental progression of motor axons ( Hb9-GFP labeling) exiting the ventral neural tube (outlined) and intersecting the stream of free NCCs (SOX10 + ). NCCs are recruited on motor nerves as SCPs (arrowheads) leaving a “gap” with freely migrating cells. Scatterplots of measurement of ( c ) the angle created by intersecting the line bisecting the NCC migratory stream, with the dorsoventral axis bisecting the neural tube ( d ) gap between nerve-associated SCP and the nearest free NCC, ( e ) mediolateral thickness of the NCC/SCP streams (distance between most medial and lateral SOX10 + cells just ventrolateral to the neural tube), perpendicular to ( c ). The red, blue, and green colors in ( c – e ) represent measurements from individual E10.5 embryos (n = 3). Linear regression assessed correlation coefficients and p-values. f E10.5 transverse optical sections (representative of 3 embryos) show ITGA4 + /SOX10 + SCP associated with axons (2H3, Neurofilament) at thoracic level, and free migrating ITGA4 - /SOX10 + NCCs at sacral level. Single channels are magnified from the boxed regions in the merge image. The nerve bundle is outlined. g , h Transverse sections of Hb9-GFP embryos. At E10.5 ( g ), SCPs (SOX10 + , gray) migrate along GFP + /TUJ1 + motor axons that form the ventral root before sensory axons (GFP - /TUJ1 + ) have begun to extend from the DRG (representative of 3 embryos). At E11.5 ( h ), SCPs are associated with both motor and sensory fascicles in the main nerve bundle (blue arrowheads). Along the white ramus connecting to sympathetic ganglia (PHOX2B + , blue), SCPs are largely associated with motor axons (red arrows), which form the bulk of the nerve (white arrowheads). The boxed regions are magnified in the right panels. Representative of 6 embryos. i Transverse sections representative of n = 5 E12.5 Hb9-GFP embryos with TRKA staining of sensory projections (red) co-extending with motor axons (GFP + , green). Individual channels are shown in the right panels. The boxed area, magnified in the bottom panel, shows the white ramus (arrowhead) formed predominantly by preganglionic motor axons, with minimal contribution from viscerosensory fibers. PHOX2B (blue) marks sympathetic ganglia. j Schematic showing equal distribution of SCPs (yellow) on motor (green) and sensory (red) fibers in axial and intercostal nerves, while SCPs migrating toward sympathetic chain ganglia (blue) are almost exclusively recruited on motor axons of the white ramus. DA dorsal aorta, DRG dorsal root ganglia, MN motor neurons, NCCs neural crest cells, NT neural tube, SCPs Schwann cell precursors, SG sympathetic chain ganglia, WR white ramus communicans. Scale bars: b : 100 μm; f : 50 μm; g – i : 100 μm.

Article Snippet: Rabbit polyclonal anti-TH (1:800, Pel-Freez Biologicals, #P40101-150, RRID:AB_2617184), sheep polyclonal anti-TH (1:2000, Novus Biologicals, #NB300-110), chicken polyclonal anti-TH (1:500, Abcam, #ab76442, RRID:AB_1524535), rabbit polyclonal anti-Hb9 (1:8000, gift from Samuel Pfaff’s laboratory ), mouse monoclonal anti-bIII tubulin/TUJ1 (1:500, Promega, #G712A), mouse monoclonal anti-bIII tubulin/TUJ1 (1:1000, Abcam #ab7751, clone TU-20, RRID:AB_306045), rabbit polyclonal anti-bIII tubulin/TUJ1 (1:1000, Synaptic Systems, cat#302302, RRID:AB_10637424), chicken polyclonal anti-GFP (1:500, Aves Labs Inc., #GFP-1020, RRID:AB_10000240), chicken polyclonal anti-GFP (1:1000, Abcam, ab13970, polyclonal, RRID:AB_300798), rabbit polyclonal anti-GFP (1:5000, Thermo/LifeTech, #A6455, lot#2126798, RRID:AB_221570), goat polyclonal anti-PHOX2B (R&D, 1:1000, #AF4940), mouse monoclonal anti-Neurofilament/NF200 (1:200, Developmental Hybridoma Studies Bank, clone 2H3), goat polyclonal anti-SOX10 (1:500, Santa-Cruz, #sc-17342), rabbit monoclonal anti-SOX10 (1:2000, Abcam, # ab155279, clone EPR4007, RRID:AB_2650603), goat anti-human SOX10 (1:800, R&D Systems, #AF2864, RRID:AB_442208), rabbit polyclonal anti-PRPH (1:500, Chemicon, #AB1530, RRID:AB_90725), rabbit monoclonal anti-KI67 (1:500, Thermo Scientific, #RM-9106, clone SP6, RRID:AB_2341197), rat monoclonal anti-PECAM (1:300, BD Pharmingen, #553370, RRID:AB_394816), goat polyclonal anti-PECAM (1:300, R&D systems #AF3628, RRID:AB_2161028), rabbit monoclonal anti-ITGA4/CD49d (1:500, Invitrogen, #MA5-27947, clone RM268, RRID:AB_2744984), rabbit monoclonal anti-Cleaved Caspase 3/Asp175 (1:500, Cell signaling #96645, clone 5A1E), rabbit polyclonal anti-TrkA (1:500, # 06-574 Sigma-Aldrich, RRID:AB_310180).

Techniques: Migration, Labeling, Staining

Journal: Nature Communications

Article Title: Motor innervation directs the correct development of the mouse sympathetic nervous system

doi: 10.1038/s41467-024-51290-0

Figure Lengend Snippet: a Transverse sections showing complete loss of motor neuron cell bodies and axons (asterisks) in E11.5 Olig2-Cre; DTA embryos (right, n = 3) compared to control littermates (left, n = 2). Motor neurons are labeled with the cell-specific transgenic reporter MN (218-2) -GFP . In mutants, SCPs (SOX10 + , red) migrate exclusively along sensory nerves (GFP - /TUJ1 + ). Individual labeling from the boxed regions are shown separately in the right panels. b Transverse sections at E12.5 showing mispatterning of viscerosensory projections (TRKA + /TUJ1 + ; arrowheads) in Olig2-Cre; DTA (bottom, n = 3) compared to controls (top, n = 3). PHOX2B (blue) identifies sympathetic ganglia. The boxed regions are magnified in middle and right panels. TRKA (gray) is shown separately in the right panels. c Transverse view of SCPs (SOX10, red) migrating on motor nerves (arrowheads) in E12.5 controls (left). SCP delivery to sympathetic ganglia (PHOX2B, TH) along the white ramus is interrupted (asterisks) in Olig2-Cre; DTA (right). SOX10 (gray) is shown separately in the right panels. d Area of glial cells (SOX10 + pixels) in sympathetic ganglia from E12.5 controls (black dots) and Olig2-Cre; DTA (blue dots). Mean (normalized to control) ± SEM, Unpaired two-sided t test (**) p = 0.0017; controls n = 4, mutants n = 4. e Sympathetic chain ganglia area (PHOX2B+ px) measured in transverse sections from E12.5 controls and Olig2-Cre; DTA . Mean (normalized to control) ± SEM, Unpaired two-sided t test (*) p = 0.0226; controls n = 7, mutants n = 7. f Time course of autonomic priming identified by PHOX2B + cells associated with peripheral nerves (TUJ1 + ) outside sympathetic ganglia (arrowheads) in E10.5, E11.5, and E12.5 Olig2-Cre; DTA (bottom) and control (top) embryos. PHOX2B is shown separately in gray. Images are representative of at least 3 embryos per genotype, per stage. g Ectopic autonomic priming (PHOX2B + cells) along sensory nerves (TRKA + /TUJ1 + ) away from the ganglia chain in E12.5 Olig2-Cre; DTA (bottom), but not in controls (top). The merged channels in the boxed regions are shown separately in the right panels. Average number of nerve-associated primed SCPs (PHOX2B + cells outside the ganglia) ( h ) and distance from ganglia ( i ) in controls and Olig2-Cre; DTA . Mean ± SEM, Unpaired two-sided t test (*) p = 0.0136; (**) p = 0.0027; controls n = 3, mutants n = 3. j Schematics of autonomic priming (yellow-blue hybrid cells) on motor axons of the white ramus in the vicinity of the dorsal aorta in controls (left) compared to uncontrolled aberrant priming along sensory nerves far away from the sympathetic chain in motor nerve-ablated mutants (right). DA dorsal aorta, DRG dorsal root ganglia, MN motor neurons, NT neural tube, SCPs Schwann cell precursors, SG sympathetic chain ganglia, WR white ramus communicans. Scale bars: 100 µm.

Article Snippet: Rabbit polyclonal anti-TH (1:800, Pel-Freez Biologicals, #P40101-150, RRID:AB_2617184), sheep polyclonal anti-TH (1:2000, Novus Biologicals, #NB300-110), chicken polyclonal anti-TH (1:500, Abcam, #ab76442, RRID:AB_1524535), rabbit polyclonal anti-Hb9 (1:8000, gift from Samuel Pfaff’s laboratory ), mouse monoclonal anti-bIII tubulin/TUJ1 (1:500, Promega, #G712A), mouse monoclonal anti-bIII tubulin/TUJ1 (1:1000, Abcam #ab7751, clone TU-20, RRID:AB_306045), rabbit polyclonal anti-bIII tubulin/TUJ1 (1:1000, Synaptic Systems, cat#302302, RRID:AB_10637424), chicken polyclonal anti-GFP (1:500, Aves Labs Inc., #GFP-1020, RRID:AB_10000240), chicken polyclonal anti-GFP (1:1000, Abcam, ab13970, polyclonal, RRID:AB_300798), rabbit polyclonal anti-GFP (1:5000, Thermo/LifeTech, #A6455, lot#2126798, RRID:AB_221570), goat polyclonal anti-PHOX2B (R&D, 1:1000, #AF4940), mouse monoclonal anti-Neurofilament/NF200 (1:200, Developmental Hybridoma Studies Bank, clone 2H3), goat polyclonal anti-SOX10 (1:500, Santa-Cruz, #sc-17342), rabbit monoclonal anti-SOX10 (1:2000, Abcam, # ab155279, clone EPR4007, RRID:AB_2650603), goat anti-human SOX10 (1:800, R&D Systems, #AF2864, RRID:AB_442208), rabbit polyclonal anti-PRPH (1:500, Chemicon, #AB1530, RRID:AB_90725), rabbit monoclonal anti-KI67 (1:500, Thermo Scientific, #RM-9106, clone SP6, RRID:AB_2341197), rat monoclonal anti-PECAM (1:300, BD Pharmingen, #553370, RRID:AB_394816), goat polyclonal anti-PECAM (1:300, R&D systems #AF3628, RRID:AB_2161028), rabbit monoclonal anti-ITGA4/CD49d (1:500, Invitrogen, #MA5-27947, clone RM268, RRID:AB_2744984), rabbit monoclonal anti-Cleaved Caspase 3/Asp175 (1:500, Cell signaling #96645, clone 5A1E), rabbit polyclonal anti-TrkA (1:500, # 06-574 Sigma-Aldrich, RRID:AB_310180).

Techniques: Control, Labeling, Transgenic Assay

Journal: Nature Communications

Article Title: Motor innervation directs the correct development of the mouse sympathetic nervous system

doi: 10.1038/s41467-024-51290-0

Figure Lengend Snippet: Transverse sections at E11.5 ( a ) and E13.5 ( b ) showing misplaced sympathoblasts differentiating into sympathetic neurons (TH + , yellow) along sensory nerves (TUJ1 + , cyan) and forming ectopic mini-ganglia in Olig2-Cre; DTA (arrowheads) but not in controls. SOX10 labels SCPs (magenta). TH staining is shown separately in the bottom panels (gray). Arrows point to fragmented sympathetic chain ganglia. Representative images of 6 embryos per genotype at E11.5 and 4 embryos per genotype at E13.5. c Dorsal view of a whole mount immunostaining for TH, Neurofilament (2H3), and vascular marker CD31 of E12.5 control embryo. The region encompassing the sympathetic chain visualized in (d) is outlined. d Dorsal view of a whole mount immunostaining for TH in control (left) and Olig2-Cre; DTA (right) E11.5 (top), E12.5 (middle), and E13.5 (bottom) embryos. Arrowheads point to sensory fiber-associated TH + sympathetic neurons distant from the chain ganglia. Premature and aberrant extension of sympathetic fibers is visible in mutants. Representative images of at least n = 5 embryos per genotype at each stage. e Quantification of misplaced sensory nerve-attached sympathetic neurons per DRG between brachial and lumbar levels, averaged per embryo. Mean ± SEM, Unpaired two-sided t test (****) p < 0.0001; controls n = 3, mutants n = 3. f Quantification of misplaced TH + cells outside the chain ganglia at E12.5, per embryo. Mean ± SEM, Unpaired two-sided t test (**) p = 0.0066; controls n = 3, mutants n = 4. g Distribution of the distances between misplaced TH + cells and the borders of the chain ganglia. Each color of dot represents a different embryo (n = 3 controls, n = 4 mutants). Sagittal view of thoracic ( h ) and lumbar ( i ) regions from whole mount TH immunostaining of E12.5 (top) and E13.5 (bottom) Olig2-Cre; DTA embryos (right) and control littermates (left). Yellow arrowheads point to fragmented sympathetic chain in mutants; red arrowheads indicate abnormal sympathetic axon growth. Representative images of at least n = 5 embryos per genotype at each stage. j Volume measurements of sympathetic chain ganglia, cervical ganglia, and adrenal/paraganglia. Datapoints represent the average volume of ganglia from individual embryos (n = 3 control, n = 4 mutant). Mean ± SEM, one-way ANOVA using Šidák correction for multiple comparisons (**) p = 0.0032; (*) p = 0.0251; ns: p = 0.9815. k Oblique view of whole mount immunostaining for TH, Neurofilament (2H3), and vascular marker CD31 of E12.5 control embryo. The outlined forelimb region is shown in ( l ). l Forelimbs of E11.5 (top) and E12.5 (bottom) controls (left) and Olig2-Cre; DTA (right) littermates. Red arrowheads point to ectopic TH + neurons extending axons aberrantly into the limb. m Length of TH + sympathetic axons innervating the forelimb in control littermates vs Olig2-Cre; DTA embryos (at E11, controls n = 4, mutants n = 3; at E12.5, controls n = 3, mutants n = 4). Mean ± SEM, one-way ANOVA using Šidák correction for multiple comparisons (***) p = 0.0003; (*) p = 0.0118. n Schematic showing both ectopic and normally positioned sympathetic neurons (blue) projecting prematurely and along inappropriate paths in association with sensory fibers in motor-ablated mutants. The sympathetic chain is fragmented. DRG dorsal root ganglia, MN motor neurons, NT neural tube, OZ organ of Zuckerkandl, SCPs Schwann cell precursors, SG sympathetic chain ganglia. Scale bars: a , b : 100 µm, c : 500 µm; d : 200 µm (top and middle), 300 µm (bottom); h , i : 100 µm (top), 200 µm (bottom); k : 500 µm; l : 200 µm (top), 300 µm (bottom).

Article Snippet: Rabbit polyclonal anti-TH (1:800, Pel-Freez Biologicals, #P40101-150, RRID:AB_2617184), sheep polyclonal anti-TH (1:2000, Novus Biologicals, #NB300-110), chicken polyclonal anti-TH (1:500, Abcam, #ab76442, RRID:AB_1524535), rabbit polyclonal anti-Hb9 (1:8000, gift from Samuel Pfaff’s laboratory ), mouse monoclonal anti-bIII tubulin/TUJ1 (1:500, Promega, #G712A), mouse monoclonal anti-bIII tubulin/TUJ1 (1:1000, Abcam #ab7751, clone TU-20, RRID:AB_306045), rabbit polyclonal anti-bIII tubulin/TUJ1 (1:1000, Synaptic Systems, cat#302302, RRID:AB_10637424), chicken polyclonal anti-GFP (1:500, Aves Labs Inc., #GFP-1020, RRID:AB_10000240), chicken polyclonal anti-GFP (1:1000, Abcam, ab13970, polyclonal, RRID:AB_300798), rabbit polyclonal anti-GFP (1:5000, Thermo/LifeTech, #A6455, lot#2126798, RRID:AB_221570), goat polyclonal anti-PHOX2B (R&D, 1:1000, #AF4940), mouse monoclonal anti-Neurofilament/NF200 (1:200, Developmental Hybridoma Studies Bank, clone 2H3), goat polyclonal anti-SOX10 (1:500, Santa-Cruz, #sc-17342), rabbit monoclonal anti-SOX10 (1:2000, Abcam, # ab155279, clone EPR4007, RRID:AB_2650603), goat anti-human SOX10 (1:800, R&D Systems, #AF2864, RRID:AB_442208), rabbit polyclonal anti-PRPH (1:500, Chemicon, #AB1530, RRID:AB_90725), rabbit monoclonal anti-KI67 (1:500, Thermo Scientific, #RM-9106, clone SP6, RRID:AB_2341197), rat monoclonal anti-PECAM (1:300, BD Pharmingen, #553370, RRID:AB_394816), goat polyclonal anti-PECAM (1:300, R&D systems #AF3628, RRID:AB_2161028), rabbit monoclonal anti-ITGA4/CD49d (1:500, Invitrogen, #MA5-27947, clone RM268, RRID:AB_2744984), rabbit monoclonal anti-Cleaved Caspase 3/Asp175 (1:500, Cell signaling #96645, clone 5A1E), rabbit polyclonal anti-TrkA (1:500, # 06-574 Sigma-Aldrich, RRID:AB_310180).

Techniques: Staining, Immunostaining, Marker, Control, Mutagenesis

Journal: Nature Communications

Article Title: Motor innervation directs the correct development of the mouse sympathetic nervous system

doi: 10.1038/s41467-024-51290-0

Figure Lengend Snippet: a Transverse embryo sections at E15.5 showing fusion of sympathetic (TH + , magenta) and dorsal root ganglia (TRKA + , cyan) in Olig2-Cre; DTA (right), but not in control littermates (left). TRKA and TH are shown separately in middle and bottom panels, respectively. b Distance between sympathetic and dorsal root ganglia in Olig2-Cre; DTA and controls. Mean ± SEM, Unpaired two-sided t test (****) p < 0.0001; controls n = 9, mutants n = 7. c Immunostaining for TH (magenta), SOX10 (yellow), and 2H3 (cyan) in sagittal sections of Olig2-Cre; DTA embryos and control littermates. The dorsal root ganglion is outlined with a dotted line. Boxed regions are magnified in the insets showing interspersed sensory neurons in sympathetic ganglia (white arrowheads). Yellow arrowheads point to fusion of DRG and sympathetic ganglia in mutants. d Fraction of ganglia pairs showing aberrant organization such as misplaced sympathoblasts around the sensory ganglia, or misplaced sensory neurons in the sympathetic ganglia. Mean ± SEM, Unpaired two-sided t test (**) p = 0.0046; controls n = 3, mutants n = 3. e Average cross-sectional area of DRGs per embryo. Mean ± SEM, Unpaired two-sided t test (*) p = 0.0196; controls n = 4, mutants n = 5. f Transverse view of fragmented DRG (TRKA + , cyan) (arrowheads) in E18.5 Olig2-Cre; DTA (bottom) but not controls (top). TH (magenta) and TUJ1 (yellow) mark sympathetic projections. Representative images of n = 4 controls and n = 5 mutants. g Schematic showing intermixing of sympathetic (blue) and sensory (red) ganglia in the absence of motor nerves (green). NT neural tube, SG sympathetic ganglia, DRG dorsal root ganglia, MN motor neurons. Scale bars: a : 100 µm; c : 200 µm (insets 50 µm); f : 100 µm.

Article Snippet: Rabbit polyclonal anti-TH (1:800, Pel-Freez Biologicals, #P40101-150, RRID:AB_2617184), sheep polyclonal anti-TH (1:2000, Novus Biologicals, #NB300-110), chicken polyclonal anti-TH (1:500, Abcam, #ab76442, RRID:AB_1524535), rabbit polyclonal anti-Hb9 (1:8000, gift from Samuel Pfaff’s laboratory ), mouse monoclonal anti-bIII tubulin/TUJ1 (1:500, Promega, #G712A), mouse monoclonal anti-bIII tubulin/TUJ1 (1:1000, Abcam #ab7751, clone TU-20, RRID:AB_306045), rabbit polyclonal anti-bIII tubulin/TUJ1 (1:1000, Synaptic Systems, cat#302302, RRID:AB_10637424), chicken polyclonal anti-GFP (1:500, Aves Labs Inc., #GFP-1020, RRID:AB_10000240), chicken polyclonal anti-GFP (1:1000, Abcam, ab13970, polyclonal, RRID:AB_300798), rabbit polyclonal anti-GFP (1:5000, Thermo/LifeTech, #A6455, lot#2126798, RRID:AB_221570), goat polyclonal anti-PHOX2B (R&D, 1:1000, #AF4940), mouse monoclonal anti-Neurofilament/NF200 (1:200, Developmental Hybridoma Studies Bank, clone 2H3), goat polyclonal anti-SOX10 (1:500, Santa-Cruz, #sc-17342), rabbit monoclonal anti-SOX10 (1:2000, Abcam, # ab155279, clone EPR4007, RRID:AB_2650603), goat anti-human SOX10 (1:800, R&D Systems, #AF2864, RRID:AB_442208), rabbit polyclonal anti-PRPH (1:500, Chemicon, #AB1530, RRID:AB_90725), rabbit monoclonal anti-KI67 (1:500, Thermo Scientific, #RM-9106, clone SP6, RRID:AB_2341197), rat monoclonal anti-PECAM (1:300, BD Pharmingen, #553370, RRID:AB_394816), goat polyclonal anti-PECAM (1:300, R&D systems #AF3628, RRID:AB_2161028), rabbit monoclonal anti-ITGA4/CD49d (1:500, Invitrogen, #MA5-27947, clone RM268, RRID:AB_2744984), rabbit monoclonal anti-Cleaved Caspase 3/Asp175 (1:500, Cell signaling #96645, clone 5A1E), rabbit polyclonal anti-TrkA (1:500, # 06-574 Sigma-Aldrich, RRID:AB_310180).

Techniques: Control, Immunostaining