Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: HPK1 overexpression affects SLP76 microclusters. (A) J14-SLP76-YFP cells were transfected with HPK1-mCherry. The day after transfection, cells were activated for 3 min on anti-CD3–coated coverslips, fixed, and analyzed by confocal microscopy. A single optical section at the level of the contact surface is shown. The plot shows the number of microclusters in cells expressing or not expressing the HPK1-mCherry construct. (B) J14-SLP76-YFP cells were transfected with HPK1-KD-mCherry and analyzed as in A. (C) J14-SLP76-YFP cells were transfected with HPK1-Y379F-mCherry and analyzed as in A. For all panels, images are representative of more than three independent experiments. Horizontal lines and error bars show means and SEM. Bars, 5 µm.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Over Expression, Transfection, Confocal Microscopy, Expressing, Construct

Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: HPK1 reduces the persistence of SLP76 microclusters. (A) J14-SLP76-YFP cells transfected with HPK1-mCherry were activated on anti-CD3–coated coverslips and imaged with a spinning-disk confocal microscope at one image/20 s. Frames from Video 1 at different time points are shown. One untransfected control (CTRL) cell (closed arrowhead) and one cell expressing HPK1-mCherry (open arrowhead) are visible in each frame. (B). Lifetime of single SLP76-YFP microclusters was determined by manual tracking using time-lapse images of J14-SLP76-YFP cells, acquired as described in A. The plot shows SLP76 microcluster lifetime in untransfected cells and in cells expressing HPK1-mCherry or HPK1-KD-mCherry. (C, top) Z projection of the HPK1-mCherry–expressing cell shown in A, imaged after 80 s of activation. The boxed area highlights a microcluster containing both SLP76-YFP (green) and HPK1-mCherry (red). (bottom) Magnification of the boxed area shown in the top image at different time points. Time 0 corresponds to the first detection of this SLP76-YFP microcluster (i.e., 40 s in the time lapse displayed in A). Similar results were obtained in three independent experiments. (D) J14-SLP76-YFP cells transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were stimulated on anti-CD3 coverslips and analyzed as described in A. Knockdown efficiency was assessed by flow cytometry ( Fig. S1 ). (E) SLP76 microcluster lifetime assessed in cells treated as in D. The plot shows pooled data of three experiments. Horizontal lines and error bars show means and SEM. Bars: (A, C [top], and D) 5 µm; (C, bottom) 1 µm.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Transfection, Microscopy, Control, Expressing, Activation Assay, Knockdown, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: HPK1 uncouples SLP76 from LAT microclusters. (A) J14-SLP76-YFP cells transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were activated for 3 min on anti-CD3–coated coverslips, fixed, stained with antiphospho-LAT (Y191) mAbs, and imaged by confocal microscopy. Results are representative of three independent experiments. (B and C) J14 cells expressing SLP76-YFP were transfected as in A and stimulated for the indicated time points. SLP76-YFP (B) and phospho-LAT (p-LAT; C) microclusters were imaged and automatically counted (see Materials and methods). Circles represent the number of microclusters/cell of pooled data from three independent experiments. (D) Human primary CD4 + T cells, transfected with control or HPK1-specific siRNAs, were activated as in A, fixed, and stained with antiphospho-SLP76 (Y128) and antiphospho-LAT (Y191) mAbs. Images were acquired as in A. HPK1 knockdown efficiency was assessed by flow cytometry ( Fig. S2 B ). (E and F) Human primary CD4 + T cells were transfected and stimulated as in D and fixed at the indicated time points. Phospho-SLP76 (p-SLP76) and phospho-LAT microclusters were counted as in B. (G) J14-SLP76-WT cells expressing FLAG-SLP76 and transfected with control or HPK1-specific siRNAs were stimulated by anti-CD3 antibodies for the indicated time points, lysed, and immunoprecipitated (IP) by the anti-FLAG mAb. Proteins were separated by electrophoresis in nonreducing conditions and analyzed by immunoblotting (IB) as indicated. Comparable results have been obtained in two independent experiments. See Fig. S3 A for analysis of HPK1 knockdown efficiency. (H) J14 cells transiently transfected with SLP76-FLAG and/or HPK1-MYC constructs were stimulated for the indicated time points, lysed, and immunoprecipitated by anti-FLAG antibodies. Proteins were analyzed by immunoblotting as in G. Mobility of molecular mass markers (in kilodaltons) is shown on the left. Horizontal lines and error bars show means and SEM. Bars, 5 µm.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Transfection, Control, Staining, Confocal Microscopy, Expressing, Knockdown, Flow Cytometry, Immunoprecipitation, Electrophoresis, Western Blot, Construct

Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: Persistence of SLP76 microclusters is partially dependent on 14-3-3 proteins binding to phosphorylated Ser376. (A) J14-SLP76-YFP cells were activated for 3 min on anti-CD3–coated coverslips, fixed, and stained with anti-SLP76 and anti–14-3-3ζ antibodies. SLP76-YFP microclusters (left; green in right image) and SLP76–14-3-3ζ complexes detected by in situ PLA (middle; red in right image) were visualized by confocal microscopy. A z-stack projection is shown. Bottom image represents an x-z projection of the cells shown in top images. Images are representative of three independent experiments. Bars, 5 µm. (B) J14 cells were transiently transfected with SLP76-YFP-WT or -S376A constructs. Cells were activated as in A, fixed, and imaged by confocal microscopy. SLP76-YFP microclusters were automatically counted (see Materials and methods) in cells expressing comparable levels of YFP labeling. Horizontal lines and error bars show means and SEM. (C) J14-SLP76-WT cells stably expressing FLAG-SLP76 were stimulated with anti-CD3 mAbs for the indicated time points, lysed, and immunoprecipitated (IP) with anti-FLAG mAbs. Samples were sequentially analyzed by GST (left) and GST–14-3-3ζ (middle) overlay assays followed by immunoblotting with anti-SLP76 and anti-GADS antibodies (right). Similar results were obtained in four independent experiments. Open arrowheads indicate heavy chains of precipitating antibodies. Mobility of molecular mass markers (in kilodaltons) is shown on the left. (D) Human CD4 + T cells were treated as in C, immunoprecipitated with anti-SLP76 antibodies, and subjected to overlay assays and immunoblotting as in C. This result is representative of two independent experiments. Mobility of molecular mass markers is shown on the right in kilodaltons.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Binding Assay, Staining, In Situ, Confocal Microscopy, Transfection, Construct, Expressing, Labeling, Stable Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: HPK1-dependent binding of 14-3-3ζ to GADS. (A) HPK1-HA constructs, wild type (WT) or kinase deficient (KD), were transfected in COS7 cells and immunoprecipitated (IP) with anti-HA mAbs. Recombinant MBP or MBP-GADS was added to either sample and incubated with [ 32 P]ATP. Supernatants were then spotted onto Whatman filters, and incorporated radioactivity was measured. Comparable results have been obtained in three independent experiments. Error bars indicate SDs of triplicates. (B) J14-SLP76-WT cells expressing FLAG-SLP76 and transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were stimulated by anti-CD3 mAbs for the indicated time points, lysed, and immunoprecipitated with anti-FLAG mAbs. Samples were analyzed by GST (top) and GST–14-3-3ζ (middle) overlay assay followed by Western blotting with anti-SLP76 and anti-GADS antibodies (bottom). Similar results have been obtained in three independent experiments. Open arrowheads indicate heavy chains of the precipitating antibodies. HPK1 knockdown efficiency was assessed by immunoblotting ( Fig. S1 C ). Mobility of molecular mass markers is shown on the right in kilodaltons. (C) Quantification of GST–14-3-3ζ binding to SLP76 (left) and GADS (right) in overlay assays shown in B. Bands in B (middle and bottom) were acquired and quantified as outlined in Materials and methods. Intensity of bound GST–14-3-3ζ bands was normalized by the relative amount of SLP76 (left) or GADS (right) in the same lane. (D) Jurkat cells transfected with control or HPK1-specific siRNAs were activated for 10 min on anti-CD3–coated coverslips, fixed, and stained with the antibody pairs indicated on the left and by DAPI for visualizing nuclei (blue). Protein complexes (red) were detected by in situ PLA. A z-stack projection is shown in each image. Bars, 5 µm. (E) Images obtained by in situ PLA experiments as shown in D were analyzed using the BlobFinder software to automatically count spots generated by protein–protein interactions. Histograms show means and SEM of the number of spots per cell from two independent experiments. The number of cells analyzed is indicated beside each bar.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Binding Assay, Construct, Transfection, Immunoprecipitation, Recombinant, Incubation, Radioactivity, Expressing, Control, Overlay Assay, Western Blot, Knockdown, Staining, In Situ, Software, Generated, Protein-Protein interactions

Journal: The Journal of Cell Biology

Article Title: Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

doi: 10.1083/jcb.201103105

Figure Lengend Snippet: Mutation of 14-3-3ζ binding sites of SLP76 and GADS leads to additive increase in SLP76 microclusters and NFAT activation. (A) J14 cells were transiently transfected with SLP76-YFP, -WT, or -S376A together with CFP-GADS constructs, either WT or a T254A mutant. Cells were stimulated for 5 min on anti-CD3–coated coverslips, fixed, and imaged by confocal microscopy. The number of SLP76 microclusters was automatically assessed in selected cells showing homogeneous levels of YFP and CFP fluorescence. A similar analysis was performed on phospho-LAT microclusters ( Fig. S5 A ). Horizontal lines and error bars show means and SEM. (B) J14 cells stably expressing FLAG-SLP76 (J14-WT) or FLAG-SLP76-S376A (J14-S376A) were transiently cotransfected with the indicated CFP-GADS construct and an NFAT-luciferase reporter plasmid. Cells were stimulated with the indicated concentrations of plate-bound anti-CD3 and 1 µg/ml of soluble anti-CD28 mAbs for 5 h. Control samples were stimulated with PMA and Ca 2+ ionophore to assess maximal NFAT-dependent transcription. Luciferase activity was measured in cell lysates. Data represent means ± SD of normalized luciferase activity from quadruplicate samples expressed as a percentage of maximal luciferase activity. Comparable expression levels of all constructs are shown in Fig. S5 B.

Article Snippet: In brief, cells were dropped onto glass coverslips (MatTek or LabTek) coated with anti-CD3 mAb (UCHT-1, MEM-92, or HIT-3a) or on superantigen-pulsed dendritic cells.

Techniques: Mutagenesis, Binding Assay, Activation Assay, Transfection, Construct, Confocal Microscopy, Fluorescence, Stable Transfection, Expressing, Luciferase, Plasmid Preparation, Control, Activity Assay

Journal: The Journal of cell biology

Article Title: Fusion of lysosomes to plasma membrane initiates radiation-induced apoptosis.

doi: 10.1083/jcb.201903176

Figure Lengend Snippet: Figure 2. Calcium-dependent lysosomal fusion with the plasma membrane mediates ionizing radiation–induced ASMase secretion and apoptosis. (A) 10 Gy–induced ASMase surface translocation measured using anti-ASMase antibody is inhibited by removal of extracellular calcium and supplementation with 2 mM EGTA. Controls with calcium are the same as those in Fig. 1 A. (B) Representative confocal fluorescence microscopy images of LAMP1 (yellow) surface staining using anti-LAMP1 antibody following 10 Gy (DAPI counterstain [magenta], left) and quantification (right). Scale bars: 10 µm. Data (mean ± 95% confidence interval) are derived from 12 cells per group. This experiment was performed four times with similar results. (C) Secreted β-hexosaminidase activity detected in cleared cell medium 2 min following 10 Gy with and without BAPTA-AM pretreatment. (D) 10 Gy–induced CRP formation 2 min after ionizing radiation as measured by anti-ceramide surface staining is inhibited by removal of extracellular calcium with EGTA supplementation. (E) Secreted ASMase activity detected in cleared cell medium using [14C]sphingomyelin substrate at 2 min following 10-Gy exposure is abrogated by BAPTA-AM pretreatment. (F) Apoptosis quantified by bisbenzimide nuclear staining following 10 Gy is diminished following BAPTA-AM pretreatment. Data (mean ± SD) are from five representative fields per group. In A and C–E, data represent mean ± SEM collated from three to four independent experiments; for A–F, *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test versus −Ca2+ (A), 0 min (B), 0 mM BAPTA-AM (C and E), 10 Gy (D), or +BAPTA-AM (F).

Article Snippet: Cells were washed into PBS + Ca2+ and allowed to attached to poly-L-lysine–coated chamber glass slides (LabTek #1.5 German borosilicate, 155382, Thermo Fisher Scientific).

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Fluorescence, Microscopy, Staining, Derivative Assay, Activity Assay, Two Tailed Test