Fig 2: Arp2/3 inhibition impairs actin dynamics in CTLs.(A) Confocal projections of OT-I CTLs treated with 90 µM of CK689 or CK666 mixed with OVA-loaded EL4 for 25 minutes; cells were fixed and labeled with antibodies against CD8 (green), actin (red), and ?-tubulin (white) to mark the centrosome (white arrows). A representative 3D reconstruction of en face interaction (white box) of the actin phenotype at the interface between the OT-I CTLs and its target. Scale bars: 5 µm. (B) Quantitation derived from images as exemplified in A and showing the percentages of conjugates displaying the different actin reorganization phenotypes at the synapse (left panel, see Supplemental Figure 1) or the centrosome distance relative to the synapse (right panel: CK689 CTLs = 190, conjugates = 91, CK666 CTLs = 179, conjugates = 80, mean of 3 independent experiments, error bars indicate SEM). (C) Actin dynamics and centrosome position (white arrows) in OT-I CTLs expressing EGFP-Lifeact (green) and PACT-mRFP (red) during interaction with EL4 blue at various time point (min:s) from the first contact. Images are confocal projections from Supplemental Videos 1 and 2. Scale bars: 5 µm. (D) OT-I CTL motility while migrating on ICAM-1 following treatment with 90 µM CK666 or CK689. The speeds of cell migration were analyzed with time-lapse images (see Methods). The graph includes plots of 28 cells for CK689 and 18 cells for CK666 from 3 videos each. ***P < 0.0001, unpaired t test.

Fig 3: Two-photon optogenetic manipulation of calcium signals using eOS1 actuator.a B3Z T cell clones expressing either the OS1 or eOS1 calcium actuators were stained with Indo-1 and deposited on ICAM-1-coated surface. Cells were visualized using a two-photon laser tuned at 720 nm. PA: 940 nm; laser power 20%; galvano scanner at 10 µs/pixel. Scale bar: 30 μm. Representative of two independent experiments. b Nuclear translocation of NFAT following two-photon photoactivation of eOS1. A B3Z T cell clone expressing the eOS1 actuator and a NFAT-mCherry reporter was visualized using a two-photon laser tuned at 1040 nm. PA: 940 nm; laser power 20%; galvano scanner at 100 µs/pixel. Scale bar: 10 μm. Representative of two independent experiments. Quantification (bottom) of the translocation score, reflecting the increased intensity of mCherry fluorescence as NFAT accumulated in the nucleus. Results are shown as mean ±SEM (n = 78 cells). c, d Spatiotemporal patterning of photoactivation. An eOS1-expressing B3Z clone was stained with Indo-1 and deposited on ICAM-1-coated surface. c Images corresponding to three repeated photoactivations of a region of interest are shown (left) together with the calcium responses in the photoactivated area (right). PA: 940 nm; laser power 20%; galvano scanner at 10 µs/pixel. Scale bar: 30 μm. Representative of five movies in two independent experiments. d Left, photoactivation of two individual cells was performed at 940 nm with the laser power set at 10% and using the Tornado scanning mode (100 µs/pixel). Scale bar: 30μm. Right, quantification of calcium responses in photoactivated (#1 and #2) and non photoactivated cells (#3 and #4). Rare cells with high intracellular calcium before photoactivation corresponded to very transient events or to dead cells. e B3Z T cells expressing mScarlet-eOS1 were stained with Indo-1 and visualized using a two-photon laser tuned at 720 and 1040 nm (for Indo-1 and mScarlet imaging, respectively). PA: 940 nm; laser power 15%; galvano scanner at 20 µs/pixel. Note that mScarlet fluorescence redistributes close to the plasma membrane upon STIM-1 aggregation (arrow). Scale bar: 30 μm. Representative of two independent experiments. f, g B3Z T cells expressing the eOS1 actuator and the Twitch2B calcium indicator were visualized using a two-photon laser tuned at 830 nm. PA: 940 nm; laser power 15 or 20%; galvano scanner at 10 µs/pixel. Time-lapse images f and mean calcium signals g are shown before or after photoactivations. Scale bar: 30 μm. Representative of two independent experiments. Source data are provided as a Source Data File.

Fig 4: Optogenetic manipulation of calcium signals in migrating T cells in vitro.a eOS1-expressing B3Z T cell clones were deposited on Poly-L-Lysine (PLL) and ICAM-1-coated dishes and visualized by videomicroscopy and photoactivated with a 100 ms pulse of blue light. a Left, representative images showing the morphology of B3Z cells during ameboid migration (before PA), arrest and rounding up (after PA), and subsequent spreading and adhesion. Scale bar: 10 μm. Times are in min:sec. Middle, quantification of B3Z cells roundness and spreading (cell area) before and after PA. Each dot represents one cell. Bars represent mean values. Groups were compared using a two-tailed Mann–Whitney test (***p < 0.001). Representative of three independent experiments. Right, mean velocity of B3Z cells graphs as the function of time. Representative of six independent experiments. b, c Calcium influx is required for B3Z T cell arrest and adhesion. b B3Z cells were stained with Indo-1, resuspended in medium containing the indicated concentration of EGTA to totally (1 mM) or partially (0.5 mM) chelate extracellular Ca2+ and analyzed by flow cytometry. Thapsigargin was added at the indicated time point during the acquisition. Representative of two independent experiments. c Velocity and cell area of eOS1-expressing B3Z cells migrating on PLL + ICAM-1-coated surface and in the presence of the indicated concentration of EGTA are quantified before and after photoactivation. Representative of four independent experiments. Bars represent mean values. Groups were compared using a two-tailed Mann–Whitney test (**p < 0.01, ***p < 0.001). d eOS1-expressing B3Z cells were visualized migrating on PLL alone or PLL + ICAM-1-coated surfaces and subjected to photoactivation. Representative images (left) and cell area quantification (right) showing B3Z cell spreading after photoactivation occurs only in the presence of ICAM-1. Scale bar: 20 μm. Times are in min:sec. Representative of four independent experiments. Bars represent mean values. Groups were compared using a two-tailed Mann–Whitney test (ns p = 0.4715, *p = 0.01, ***p < 0.001). e Repeated photoactivations prolong B3Z T cell arrest (left) and limit cell spreading (right). B3Z cells were subjected to a single or multiple photoactivation (100 ms every 5 min). Representative of three independent experiments. Bars represent mean values. Groups were compared using a two-tailed Mann–Whitney test (ns p = 0.9257 and 0.4821, ***p < 0.001). f CD8+ T cells were activated and transduced to express eOS1, then deposited on PLL + ICAM-1-coated dishes before being subjected to a single or repeated (every 5 min) photoactivations. Representative of two independent experiments. Source data are provided as a Source Data File.

Fig 5: Expression of mAb24 and KIM127 binding epitopes (β2 integrin E+ and H+ conformations) in arresting mouse neutrophils(A) qDF imaging of the first 150 nm above the coverslip of mouse neutrophils rolling on P-selectin/ICAM-1 substrate. CXCL-1 (10 ng/mL) was perfused at about −20 s in the presence of 1 μg/mL mAb24-Alexa Fluor 488 (mAb24-AF488) and 1 μg/mL KIM127-Dylight 550 (KIM127-DL550). Merge shows mAb24 (green) and KIM127 (red). Scale bar, 5 μm.(B) Total cluster area, number of clusters, and average cluster area of mAb24-AF488 and KIM127-DL550 antibody-labeled clusters (red, E+; green, H+; yellow, E+H+) are depicted as a function of time before and after arrest at 0 s.(C–E) Total cluster area (C), number of clusters (D), and average cluster area (E) of mAb24-AF488 and KIM127-DL550 antibody-labeled clusters (red, E+; green, H+; yellow, E+H+) before (−10 s) and at the time of arrest at 0 s. Mean ± SEM of 7 cells from three independent experiments are shown.**p < 0.01; ***p < 0.001; and ****p < 0.0001.