Fig 1: S1P induces strong and sustained RhoA activation and brief, low-level Rac1 activation.(A-B) Analysis of RhoA and Rac1 activation by S1P and CCL19. Naïve CD4+ T cells were stimulated with S1P or CCL19 for the indicated times, then rapidly lysed and analyzed for GTP-bound RhoA and Rac1. Top panels show representative experiments; bottom panels show averages of multiple biological replicates (n=4 for RhoA; n=3 for Rac1). Fold activation was determined relative to the unstimulated controls. (C) Analysis of the Rac effector PAK1/2. T cells were stimulated with S1P or CCL19 for the indicated times, lysed with TritonX-100 lysis buffer, and analyzed by immunoblotting for phospho-PAK1/2, using GAPDH as a loading control. Top, representative Western Blot. Bottom, quantification from independent experiments (n=3). Fold increases were determined relative to the unstimulated controls. (D-E) Analysis of Rac1 activation in living cells. Purified human CD4+ T cells were transduced with Pak-PBD-mCherry, seeded onto fibronectin-coated glasses, and imaged before and after addition of 125nM S1P or 50mM CCL19 for up to 9min. Dark grayscale indicates Rac1-GTP levels. D, representative time-lapse images. E, Quantification of Rac1 activity at the membrane. Rac1-based protrusions were recorded, and their fluorescence intensity was compared to that of the same membrane area at steady state (dotted line). Plotted is the average Rac1 activity for S1P (blue) and CCL19 (red) over time. n = at least 20 separate cells for each condition, obtained from two separate experiments. For all panels, data represent means +/− StDev from multiple experiments. Statistical analysis was performed using an unpaired Student’s T test, relative to the unstimulated control. *, p<0.05; **, p<0.01; ***, p<0.005.
Fig 2: Long-range mutual activation establishes robust Rho and Rac polarity during primary T cell migration.a, Confocal image of a migrating human primary T cell expressing the polarity biosensors for Rac (PAK-PBD) and Rho (AnillinRBD). Activating either Rho or Rac leads to a robust establishment of polarity and that Rho and Rac both mutually reinforce each other at a distance. b, Primary human T cells expressing the Rac biosensor (Pak-PBD) to assay cell polarization following acute stimulation with the chemoattractant CCL19. c, Time-lapse confocal images of an unpolarized human primary T cell before and during CCL19 stimulation. Rac activation was monitored via the Rac biosensor Pak-PBD. d, Chemoattractants such as CCL19 are known to activate both Rho and Rac. e, Average time trace of Rac activation (measured by Pak-PBD level) at the cell front following addition of 25 nM CCL19 showing that CCL19 induces long-lasting polarization (mean ± 95% CI; n = 30, N = 3). f, To test whether long-lasting Rac partitioning depends exclusively on local Gαi-mediated activation or also requires long-range Rho/myosin signalling, cells were treated with 1 μg ml−1 Gαi inhibitor PTX, cells treated with 20 μM Y27 and a combination of PTX and 20 μM Y27. g, Average time trace of Rac activation (measured by Pak-PBD level) at the cell front following addition of 25 nM CCL19 in cells treated with either PTX, Y27 or a combination of both. PTX and Y27 treated cells polarized only transiently, while cells treated with both failed to polarize altogether when compared with control cells (mean ± 95% CI; n = 20 for PTX, n = 19 for Y27, n = 15 for PTX + Y27, N = 3). h, We prevent the long-range Rac to Rho communication using the mTORC2 inhibitor KU-0063794. i, Average time trace of Rac activation (measured by Pak-PBD level) at the cell front following addition of 25 nM CCL19 in cells treated with 10 μM KU-0063794 showing that cells with impaired mTORC2 signalling only polarize transiently in response to CCL19 (mean ± 95% CI; n = 20, N = 2). j, Ex vivo assay for human primary T cell chemotaxis. Cells are premixed with bovine dermal collagen and placed into linear channels. After collagen sets, the medium is added to one side of the channel (TCM) and medium with human CCL19 (100 ng total) and 10 μg ml−1 Dextran10k-AF647 (Dex647) is added to the other. Imaging takes place right on the edge of the well containing CCL19 so that T cell responses can be recorded as the CCL19 (as read out by Dex647) diffuses into the channel. k, Representative control experiment is shown at different time points with Dex647 fluorescence (top row) and corresponding tracks of T cells (bottom row); tracks display 3 min preceding time point listed. l–o, Average Y only component of velocity of all tracks at each time point recorded. Positive is considered towards the well containing CCL19. Control Volunteer N = 3, PTX Volunteer N = 3, Y27 Volunteer N = 2, KU Volunteer N = 2. For all panels, n, individual cell replicate, N, biological replicates.
Fig 3: Long-range mutual activation establishes robust Rho and Rac polarity in primary T cells.(A) (B) Time-lapse confocal images of an unpolarized human primary T cell before and during CCL19 stimulation. Rho activity was monitored via the Rho biosensor AnillinRBD. (C) Average time trace of Rho activity at the cell back following addition of 25 nM of CCL19 comparing control cells and cells treated with 10 μM of the mTORC2 inhibitor KU. (mean ± 95%CI; CTRL n = 30, KU n = 20, N = 3). (D) Primary human T cells expressing the Rac biosensor (Pak-PBD) to assay cell polarization following acute stimulation with the chemoattractant CCL19. CCL19 is known to activate both Rho and Rac. (E) Time-lapse confocal images of an unpolarized human primary T cell before and during CCL19 stimulation. Rac activation was monitored via the Rac biosensor Pak-PBD. (F) Average time trace of Rac activation at the cell front following addition of CCL19. (mean ± 95%CI; n = 30, N = 3) (G) (H) Cells are treated with GαI inhibitor to prevent direct Rac activation by CCL19. (I) Average time trace of Rac activation at the cell front of cells treated with 1 ug ml of GαI inhibitor following addition of CCL19. (mean ± 95%CI; n = 20, N = 3). (J)(K) Cells are treated with a combination of Y27 and PTX to prevent direct Rho and Rac activation by CCL19. (L) Average time trace of Rac activation at the cell front of cells treated with 1 ug ml of GαI inhibitor PTX together with 20 μM of Y27 following addition of CCL19 (mean ± 95%CI; n = 17, N = 3). (M)(N) Cells are treated with a combination of PTX and the PI3K inhibitor Duvelisib to disrupt the long-range Rho-mediated Rac activation described in this study. (O) Average time trace of Rac activation at the cell front of cells treated with 1 ug/ml of GαI inhibitor PTX together with 1µM PI3K inhibitor Duvelisib. (mean ± 95%CI; n = 13, N = 2). (P)(Q) Cells are treated with ROCK inhibitor Y27 to prevent Rho activation. (R) Average time trace of Rac activation at the cell front of cells treated with 20 μM of Y27 following addition of CCL19 (mean ± 95%CI; n = 19, N = 3). Data for (C), (F), (I) and (O) are similar as in Fig. 8 but separated into different panels for extra clarity. For all panels, n = individual cell replicate, N = biological replicates.
Fig 4: Long range mutual activation is necessary for chemotaxis in human primary T cells.Ex-vivo assay for human primary T cell chemotaxis. Cells are premixed with Bovine Dermal Collagen and placed into linear channels. After collagen sets, media is added to one side of the channel (TCM) and media with human CCL19 (100 ng total) and 10 μg ml−1 of Dextran10k-AF647 (Dex647) is added to the other. Imaging takes place right on the edge of the well containing CCL19 so that T cell responses can be recorded as the CCL19 (as read out by Dex647) diffuses into the channel. (A) Representative control, (B) Y27, (C) PTX, (D) KU experiment at 4 min, 90 min, 180 min with Dex647 fluorescence and corresponding tracks of T cells; tracks display 3 min preceding time point listed. (E) Average linear velocity of all tracks at each time point recorded. Control Volunteer N = 3, PTX Volunteer N = 3, Y27 Volunteer N = 2, KU Volunteer N = 2 (mean ± 95%CI).
Supplier Page from R&D Systems, a Bio-Techne Brand for CCL19/MIP-3 beta Protein
Available conjugates: Sizes Available: 25 ug