The fast Brownian motility of DAG in B cell immunological synapse.

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The fast Brownian motility of DAG in B cell immunological synapse. The fast Brownian motility of DAG in B cell immunological synapse. (A and B) Representative TIRFM images of DT40-WT B cells expressing PKCθ-C1–GFP (A) or GFP-DGKζ (B) on PLBs containing anti-IgM surrogate antigens (left). FI profiles of BCR and DAG or GFP-DGKζ (middle) along the white line in the left images. Correlated pixel FI plot of BCR and DAG or GFP-DGKζ (right) in the boxed area in the left images. Scale bars, 2 μm. (C to H) Single-molecule tracking of DAG (PKCθ-C1–mEos3.2) or PIP2 (mEos3.2–PLC-δ–PH) in DT40-WT B cells on PLBs tethering anti-IgM surrogate antigens. (C) Trajectories were pseudo-colored according to the value of the diffusion coefficients (n is given above the figures). (D) Mean square displacement (MSD) plots. Bar represents mean ± SEM. (E) Diffusion coefficients with the median indicated by black bars. (F) Cumulative probability of diffusion coefficients of DAG and PIP2. (G) The probability distribution of diffusion coefficients and fitting to two populations showing slow (red line) and fast (green line) diffusion. The mean diffusion coefficients of fast and slow population are given above the plots. (H) The percentage of the slow population in DAG or PIP2. (I to K) (I) DAG or PIP2 trajectories are randomly colored and are plotted on the background of BCR microclusters. The right panels are the magnified regions from the boxed areas (left) with time series. Scale bars, 2 μm. (J) The percentage of trajectories crossing the BCR microcluster borders. (K) The percentage of the trajectories showing the direction from inside to outside the BCR microclusters. n = 6 cells. Bar represents mean ± SEM. **P < 0.01, ***P < 0.001 in two-tailed t tests. Data are representative of three independent experiments. Chenguang Xu et al. Sci. Immunol. 2017;2:eaan0787 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works