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. 2012 Mar 1;23(5):834–852. doi: 10.1091/mbc.E11-08-0731

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© 2012 Yi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Localization of endogenous F-actin structures and corresponding LP/LM markers at the Jurkat IS. (A) Phalloidin staining of endogenous F-actin at the IS in representative Jurkat T-cells stimulated on a supported planar bilayer (A1, A3) or on a glass coverslip coated with immobilized anti-CD3ε antibody (A5). (A2, A4, A6) Magnified images of the boxed regions (white) in A1, A3, and A5, respectively. The central zone, middle ring, and outer ring are indicated by brackets above A2, A4, and A6. (B) Overlap in localization of ICAM-1 with the middle ring of F-actin at the IS in a representative cell stimulated on a planar bilayer. Note that this image is a single frame from a movie of a cell expressing the F-actin reporter GFP–F-tractin-P (see the text) and not an image of a fixed, phalloidin-stained cell, as the distribution of ICAM-1 is disrupted by fixation. (B1) ICAM-1, (B3) GFP–F-tractin-P in the same cell, and (B5) merged image between B1 (green) and B3 (red). (B2, B4, B6) Magnified images of the boxed regions (white) in B1, B3, and B5, respectively. The outer and middle rings are indicated by brackets above B2, B4, and B6. (C) Overlap in localization of TCR MCs with the actin-depleted central zone at the IS in a representative cell stimulated on a planar bilayer. (C1) Anti-CD3ε antibody-labeled TCR MCs, (C2) phalloidin staining in the same cell, and (C3) merged image between C1 (green) and C2 (red). (D) Overlap in localization of the Arp2/3 complex with the outer ring of F-actin at the IS in a representative cell stimulated on a planar bilayer. (D1) Anti-p34 (Arp2/3 subunit) antibody staining, (D3) phalloidin staining in the same cell, and (D5) merged image between D1 (green) and D3 (red). (D2, D4, D6) Magnified images of the boxed regions (white) in D1, D3, and D5, respectively. The LP and LM regions are indicated by brackets above D2, D4, and D6. (E) Overlap in localization of myosin IIA with the middle ring of F-actin at the IS in a representative cell stimulated on planar bilayer. (E1) Anti–myosin IIA antibody staining, (E3) phalloidin staining in the same cell, and (E5) merged image between E1 (green) and E3 (red). (E2, E4, E6) Magnified images of the boxed regions (white) in E1, E3, and E5, respectively. The LP and LM regions are indicated by brackets above E2, E4, and E6. (F) Phalloidin staining at the plane of contact with the bilayer (F1) and 1 μm above the plane of contact with the bilayer (F2) in a representative cell stimulated on a planar bilayer. (F3) Graph showing the relative intensities of phalloidin fluorescence across the IS for the green line in F1 (and the corresponding green intensity trace in F3) and the red line in F2 (and the corresponding red intensity trace in F3). The positions of the LP/dSMAC, LM/pSMAC and cSMAC regions are indicated by brackets above F3. (G) Three-dimensional reconstructed views of phalloidin staining in a representative Jurkat cell stimulated on a planar bilayer. (G1) En face view of phalloidin fluorescence at the plane of contact with the bilayer. (G2) Cross-sectional view of phalloidin fluorescence in the LP/dSMAC region (corresponding to the red line in G1). (G3) Cross-sectional view of phalloidin fluorescence in the LM/pSMAC region (corresponding to the green line in G1). (H) Cartoon of the LP/dSMAC, LM/pSMAC, and cSMAC regions at the IS shown from an en face view (H1) and a side view (H2). Scale bars, 5 μm.