Research ArticleIMMUNOLOGY

The chaperonin CCT controls T cell receptor–driven 3D configuration of centrioles

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Science Advances  02 Dec 2020:
Vol. 6, no. 49, eabb7242
DOI: 10.1126/sciadv.abb7242
  • Fig. 1 Pericentriolar Systems Biology regulation upon polarized T cell receptor and CD28 activation.

    (A) Workflow for IS formation, centrosome-enriched fraction isolation and in vitro polymerization assay, Western blot, and Erk1/2 activation. (B) Fluorescence from rhodamine–α-tubulin and γ-tubulin. Graph, rhodamine–α-tubulin in MTs (Ctrl, n = 66; IS, n = 76; Mann-Whitney). (C) iTRAQ (six replicates, 30 donors). Left graphs: Coordinated protein alterations in functional categories at centrosomes during IS. Zqa’ values, standardized log2 ratio averages of proteins (stimulated versus control). Right: Global distribution of the eight CCT subunits. Zq < 0, CCTs versus other proteins. BASC, super complex of BRCA1-associated proteins. (D) Systems Biology analysis of the six biological replicates showing coordinated increases/decreases. The SBT algorithm was applied. (E) Cytoscape map from the SBT analysis. (F) Western blot, centrosome-enriched fractions processed in (B) to (E). Graph, densitometric analysis. (G) CCT5 subunit localization in conjugates with APCs (*Raji cells). Scale bars, 10 μm. Graph, CCT5 at the IS (means ± SEM, Ctrl n = 23, IS n = 24; Mann-Whitney). (H and I) MT dynamics in activated Jurkat cells by TIRFm. (H) Fluorescence images, start point, and maximal projection of the time lapses (Δt); BF, bright field. Scale bar, 5 μm. (I) Imaris-generated track maps. Scale bars, 5 μm. Graphs, statistics for tracks (median ± interquartile range; n = 15; Mann-Whitney). A.U., arbitrary units; n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 CCT regulates tubulin dynamics at the IS.

    (A to C) Native immunoelectrophoresis of αβ-tubulin complexes in resting and activated siCtrl or siCCT Jurkat T cells detected with anti–α-tubulin (A) and PTMs for Δ1-α-tubulin (B) and Δ2-α-tubulin (C). (D and E) Native immunoelectrophoresis against total α-tubulin (E) and β-tubulin (D) antibodies, pE-tubulin (D), and acetylated K40 α-tubulin (E). n = 3. (F) Analysis of MT dynamics in activated Jurkat T cells cotransfected with MAP7-GFP and siCtrl or siCCT by TIRFm; n = 3. Scale bars, 5 μm. (G) Cells transfected with EB3-RFP were treated or not with cycloheximide (CHX) for 40 min and allowed to spread over a stimulating surface before TIRFm analysis. Imaris track analysis is shown (15 s; n = 16, siCtrl and CHX; Mann-Whitney). (H) Cells stably expressing low amounts of EB3-GFP were treated or not with bortezomib (BTZ; 520 nM) for 2 hours and allowed to spread over a stimulating surface before TIRFm analysis. Imaris track analysis is shown (30 s; Ctrl n = 15, BTZ n = 14; Mann-Whitney). (I and J) Native immunoelectrophoresis of αβ-tubulin complexes in resting and activated T cells treated with CHX (I) or BTZ (J) detected with anti–α-tubulin. n = 3, at least. Numbers correspond to normalized (Ctrl) densitometry analysis (Image Gauge software). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 CCT regulates the 3D T cell organization at synaptic contacts.

    3D reconstructions of cryo-SXT images showing resting (Ctrl) and IS-forming (IS) human primary CD4 T cells, treated with control siRNA (siCtrl) or siRNAs for CCT (siCCT). Blue, nucleus; transparent grid, plasma membrane; red, mitochondria; green, centrioles; white, vesicles; olive, lipid droplets (colors may change due to superposition of the nucleus). Asterisks indicate same corner for the two views. Insets: Slices of the reconstructed cryo-SXT imaging. N, nucleus; m, mitochondria. Scale bars, 500 nm. The size of the squares indicating the carbon surface is 5 μm.

  • Fig. 4 CCT regulates the 3D centriolar organization at synaptic contacts.

    (A) Gallery of CD4 T cells 3D reconstructed after cryo-SXT. Mitochondria are shown in red, centrioles in green, vesicles in white, and lipid droplets in yellow. The right images correspond to a section of the area of AIC, including the centrioles (thin arrows: longitudinal section and wide arrows, cross section). Scale bar, 500 nm. (B) Volume of the AIC. (C) Centrosome internal angle (a). (D) Angle (A) between the vector joining the center of mass of each centriole and the IS plane. (E) Angle (α) between the centriolar cross-vector and the IS plane. Graphs, median and interquartile range. Blue dots, mean (n = 12, control siCtrl; n = 12, control siCCT; n = 9 IS siCtrl; n = 8, IS siCCT). (F) Resonant scanner confocal videomicroscopy of whole cells showing growing MT tips toward the IS. (G) Model for centriolar and centrosomal disposition respect to the IS plane in resting (Ctrl) and TCR-stimulated T cells (IS).

  • Fig. 5 CCT regulation of tubulin dynamics is required for TCR signaling and IS formation.

    (A) TCR downstream signaling of siCtrl- and siCCT-treated Jurkat T cells during activation: pY83-CD3ζ, pY132-LAT and pY783-PLCγ1. Total proteins, loading controls. Graphs, means ± SEM; n = 5. Two-way ANOVA. (B) NFATc2 and pS468-NFκB in primary human CD4 T cells. Graphs, means ± SEM. n = 3 and 4. One-way ANOVA. (C) CD3ε distribution at the IS in Jurkat T cells during synapse; a maximal projection of CD3ε is shown, merged with bright-field (BF) image; 3D reconstruction was performed with Leica LASX. Scale bar, 5 μm. Graph, CD3ε ratio at the IS by “synapse measures” algorithm. Mean and SD. One-way ANOVA [n = 63 (siCtrl Ctrl), 47 (siCCT Ctrl), 127 (siCtrl IS), and 156 (siCCT IS) from three independent experiments]. (D) CD3ε surface expression in human primary CD4 and Jurkat T cells (n = 4 and 3, respectively). (E) CD3ζ-bearing vesicle dynamics in activated T cells by TIRFm as in Fig. 1 (H and I). Scale bars, 10 μm. Imaris track maps and statistical analysis are shown (30 s; median plus interquartile range; Mann-Whitney test; n = 30). Scale bars, 5 μm. **P < 0.01; *P < 0.05.

  • Fig. 6 CCT regulation of tubulin dynamics controls mitochondrial and metabolic tuning during IS formation.

    (A) Mitochondrial dynamics at the IS. Fluorescence (TIRFm) and bright-field (BF) images. Blue boxes, magnification of the insets (scale bars, 5 μm; n = 4). (B) STED 3D reconstruction of siCtrl and siCCT cells forming IS (45 min). Scale, distance to IS. Scale bar, 5 μm; n = 3. (C) Mitochondrial mass (n = 8). (D) Normalized ratio of mitochondrial membrane potential (ΔΨ) to total mitochondrial mass during IS. (E) ATP production during IS (n = 4; Mann-Whitney). (F) Mitochondrial respiration during IS (n = 4). (G) Mean fluorescence intensity (MFI) from IS-forming cells by TIRFm (Mitotracker Orange; frame lapse, 5 s; n = 3). (H) Mitochondrial localization at the IS (dotted lines, SEE-pulsed APCs). Scale bar, 10 μm. (I) mTORC1 signaling in human CD4 T cells. Graphs, normalized fold-induction ratios. Means ± SEM; one-way ANOVA (n = 4). (J) LAMP1+ vesicles in siCtrl and siCCT human CD4 T cells. Fluorescence maximal projection and BF merged image showing control or stimulating beads. Scale bar, 5 μm. Graph, LAMP1+ distance to the centrosome [n = 36 (siCtrl-Ctrl), 37 (siCCT-Ctrl), 40 (siCtrl-IS), and 43 (siCCT-IS); three donors; mean ± SD]; one-way ANOVA. (K) IL-2 production from siCtrl and siCCT cells (n = 4; one-way ANOVA; **P < 0.01; ****P < 0.0001).

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