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. 2022 Apr 11;11:e72805. doi: 10.7554/eLife.72805

Figure 1. ICAM-1 co-stimulation promotes the formation of actin arcs at the B-cell immune synapse.

(A–F) GFP-F-Tractin-expressing primary B cells on glass coated with anti-IgM alone (A, B, E1, E2) or with anti-IgM + ICAM-1 (C, D, F1, F2) and imaged using Airyscan (A, C) or TIRF-SIM (B, D, E1, E2, F1, F2). The white arrows in (A) and (B) indicate the thin outer rim of dendritic actin in the dSMAC. The blue bars in (A–D) indicate the pSMAC. (E2) and (F2) correspond to the boxed regions in (E1) and (F1), respectively. Of note, the cell shown in (E1/E2) is representative of ~70% of anti-IgM-stimulated cells, while the cell shown in (F1/F2) is representative of ~70% of anti-IgM + ICAM-1-stimulated cells. (G) Percent of cells with pSMAC actin arcs (N > 67 cells/condition from three experiments). (H, I) Percent of total synaptic F-actin (H) and percent of total IS footprint (I) contained within the dSMAC, pSMAC, and cSMAC portions of the synapse for primary B cells on anti-IgG/ICAM-1-coated glass (N = 44 cells/condition from six experiments). (J1, J2) GFP-F-Tractin-expressing A20 B cell on anti-IgG/ICAM-1-coated glass. (J2) corresponds to the boxed region in (J1). The magenta arrows in (A–D) and (J1) indicate actin arcs. Scale bars: 10 µm.

Figure 1.

Figure 1—figure supplement 1. Degree of alignment between the actin filaments in the pSMAC of B cells stimulated with anti-IgM alone versus anti-IgM and ICAM-1.

Figure 1—figure supplement 1.

(A1, A2) Shown are examples of how FibrilTool (Boudaoud et al., 2014) was used to measure pSMAC actin arc anisotropy by dividing the radially symmetric pSMAC into 10–12 trapezoid-shaped regions of interest (ROIs) of similar size so as to ‘linearize’ it (A1, stimulated with anti-IgM alone; A2, stimulated with anti-IgM and ICAM-1) (see also Murugesan et al., 2016). FibrilTool measures how well structures of interest (here actin filaments) within a given ROI (here the pSMAC) are arranged in parallel. Anisotropy values range from 0 when the orientation of the structures is completely random to 1.0 when all of the structures are perfectly aligned/parallel to each other. The red lines indicate the average orientation of actin in the ROI. Scale bar: 10 µm. All panels: TIRF-SIM. (A3) Frequency of measured anisotropy values. N = 267–358 ROIs from 22 to 23 cells from three experiments.