Journal: bioRxiv

Article Title: Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem

doi: 10.1101/504894

Figure Lengend Snippet: (A) Airyscan LSM Image MIP projections of 3T3 fibroblasts expressing both THD-EGFP (green) and mRby2-Paxillin (red). Bottom panel shows the merged image and the paxillin rich ROIs (red lines) with quantitative colocalization of both THD to paxillin in peripherally labelled paxillin rich focal adhesions. The images clearly show a diminishment of co-localized signal of THD to paxillin between the WT control and THD(R35E) mutant, which is further decreased in the THD(R118E) mutant. Quantitation results are displayed in (B) . The number of analyzed adhesions is indicated for each condition. (C) A model of talin1 F1 domain in the cytosol with a positively charged unstructured loop (green) and membrane-associated Rap1b-GTP by its C-terminal geranyl-geranyl (G-G) moiety (orange). Negatively charged phospholipids PI(4,5)P 2 are colored in red. On proximity of the plasma membrane, the low-affinity talin1 F1 Ras-associating (RA) domain probes for Rap1 and the F1 loop seeks negatively charged phospholipids. On contact with Rap1 and negatively charged phospholipids, the F1 RA domain interacts with Rap1 and the F1 loop helical state is favored resulting in cluster of positive charges on one side of the helix. View of THD (green) as seen from the membrane is displayed in the top right panel. Bottom panel shows view of THD as seen from the side. The views highlight the position of the THD F0, F1, F2 and F3 subdomains and the Rap1b (orange) bound to the F0 and F1 subdomains. Both Rap1b C-terminal geranyl-geranyl (G-G) moieties are pointing towards the membrane, as for the F1 loop, the F2 membrane orientation patch (MOP), the F3 association patch (FAP), and the position of the F3 β-integrin. The regions known to interact with the negatively charged phospholipids are shown in blue; the F1 fly-casting loop is shown as a helix, the F2 membrane orientation patch (MOP), and the F3 association patch (FAP). The β-integrin transmembrane and cytoplasmic tail is shown in red bound to the F3 subdomain. Two Rap1b (orange) molecules are shown with their C-terminal geranyl-geranyl (G-G) moiety inserted in the membrane. One Rap1b is bound to the F0 and one to the F1 subdomain. This model summarize the multiple known interactions of THD at the plasma membrane; the negatively charged PI(4,5)P 2 , Rap1 and integrin β-tails.

Article Snippet: Each image consisted of z-stacks of multiple frames that were first processed from a raw Airyscan image to a final integrated, corrected and deconvolved image that was then flattened as a maximum intensity projection using the ZEN software (Zeiss Inc.).

Techniques: Expressing, Control, Mutagenesis, Quantitation Assay, Membrane, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation

doi: 10.3389/fimmu.2023.1110482

Figure Lengend Snippet: ζ CAR engagement of antigen induced clustering without TCR integration. Anti−HER2 CAR T cells were labeled with either AF647−conjugated F(ab´) 2 goat anti−human IgG antibody that recognizes the CAR, anti−TCRα/β AF647-conjugated antibody, or AF647-conjugated anti−transferrin receptor (TfR) antibody. (A) Schematic representation of the used anti−HER2 CD28ζ CAR linked to GFP at the intracellular site (IC). (B) Cells were seeded onto surfaces coated with HER2 protein or anti−CD3 antibody OKT3 and contacts were formed for 20 minutes. Cells were fixed and TIRF microscopy was used to image the localization of CAR-GFP (green) and either CAR-AF647, TCR-AF647, or TfR-AF647 (red). Scale bar represents 5 µm. For each cell, the pixel-wise correlation of the brightness values recorded in the red and in the green channel were plotted along the x- and the y-axis, respectively. Pearson’s correlation coefficient (PCC) for each cell was calculated. Contact size (C) and Mean intensity (D) of anti−HER2 CAR-GFP plated on HER2 (blue) and OKT3 coated slides (grey) are displayed for n≥16 cells per group in a Whisker box plot. Statistical differences were determined by Wilcoxon-Mann-Whitney test with Python code (**p<0.01). (E) PCC for CAR-GFP and TCR-AF647 was calculated for anti−HER2 CAR T cells plated on HER2 (blue) and OKT3 (grey) coated slides and displayed for n≥24 cells per group in a Whisker box plot. Statistical differences were determined by Kruskal-Wallis test with Dunn’s post-hoc test performed with Python code (***p<0.001). (F) Labelled anti-HER2 CAR T cells were seeded on HER2 expressing N87 target cells plated on chambered coverslips. 3D fluorescence images of live anti-HER2 CAR T cells forming contacts with the tumor target were recorded in AiryScan Fast mode. Confocal images of each analyzed cell were recorded as controls. (G) Differential 3D distribution of CAR-GFP and either CAR-AF647, TCR-AF647, or TfR-AF647 in the synaptic contact region and the extrasynaptic membrane was normalized to total intensity (n CAR-GFP =11, n TCR-AF647 = 11, n CAR-AF647 = 10, n TfR-AF647 = 6; 3D images contained approximately 50-80 slices). (H) PCC for CAR-GFP and either CAR-AF647, TCR-AF647, or TfR-AF647 in the synaptic contact region and the extrasynaptic membrane was quantified separately for each slice and averaged for each individual cell and presented as the mean of multiple cells across 3 independent experiments. 2D AiryScan Fast images of unstimulated cells were used as control (n CAR_CAR =15, n TCR_CAR =7, n TfR_CAR =14). Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ns, not significant).

Article Snippet: Images were recorded with an LSM 880 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with an AiryScan/AiryScan Fast detection unit providing up to 120 nm lateral and 350 nm axial resolution ( ) and a high NA water immersion objective (C-Apochromat, 1.2 NA, 40x).

Techniques: Labeling, Microscopy, Whisker Assay, MANN-WHITNEY, Expressing, Fluorescence