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. Author manuscript; available in PMC: 2022 Sep 15.
Published in final edited form as: J Cell Sci. 2021 Aug 23;134(16):jcs258462. doi: 10.1242/jcs.258462

Figure 2. BBS1 is required for centrosome and endosomal TCR translocation to the IS.

Figure 2

(A,B) Immunofluorescence analysis of γ—tubulin (A) or PCNT (B) in conjugates of control (ctr) and BBS1KD (J KD) Jurkat cells (A), or control (ctr) and BBS1KO primary T cells (T KO) (B), with SEE/SAg (SEA+SEB+SEE)-loaded Raji cells (APCs). Conjugates were co-stained for nesprin-2 (B). Left, Representative images (medial optical sections) of conjugates formed in the presence of SEE/SAg (see Fig.S4A,B for SEE/SAg-independent conjugates). Right, Quantification (%) of antigen-specific conjugates harboring γ—tubulin (A) or PCNT (B) staining at the IS (≥25 cells/sample, n=3, unpaired two-tailed Student’s t-test). (C,D) Measurement of the distance (μm) of the centrosome (γ—tubulin or PCNT) from the T cell-APC contact site in 15-min conjugates of control (ctr) and BBS1KD (J KD) Jurkat cells (C), or control (ctr) and BBS1KO primary T cells (T KO) (D), with Raji cells in the absence or presence of SEE/SAg (≥10 cells/sample, n=3, Kruskal-Wallis test). (E) Measurement of the distance (μm) of the centrosome (PCNT) from the nucleus (nesprin-2) in 15-min conjugates of control (ctr) and BBS1KO (T KO) primary T cells with Raji cells in the absence or presence of SAg (≥10 cells/sample, n=3, Kruskal-Wallis test). Centrosomal area was magnified to highlight the parameters used for quantification (detailed in Fig.S4C). (F,G) Immunofluorescence analysis of CD3ζ in conjugates of control (ctr) and BBS1KD (J KD) Jurkat (F), or control (ctr) and BBS1KO primary T cells (T KO) (G), with SEE/SAg-loaded Raji cells (APCs). Representative images (medial optical sections) of the conjugates formed in the presence of SEE/SAg are shown (see Fig.S4A,D for SEE/SAg-independent conjugates). (H,I) Left, Quantification (%) of antigen-specific conjugates harboring CD3ζ staining at the IS (≥25 cells/sample, n=3, unpaired two-tailed Student’s t-test). Middle, Relative CD3ζ fluorescence intensity at the IS in 15 min-conjugates of control (ctr) and BBS1KD (J KD) Jurkat cells (H), or control (ctr) and BBS1KO primary T cells (T KO) (I), with SEE/SAg-loaded Raji cells (≥10 cells/sample, n=3, Kruskal-Wallis test). Right, Measurement of the distance (μm) of the endosomal TCR-CD3 pool (eCD3ζ) from the T cell-APC contact site in 15-min conjugates of control (ctr) and BBS1KD (J KD) Jurkat cells (H), or control (ctr) and BBS1KO primary T cells (T KO) (I), with Raji cells in the absence or presence of SEE/SAg (≥10 cells/sample, n=3, Kruskal-Wallis test). Size bar, 5 μm. The data are expressed as mean±SD. ****P≤0.0001; ***P≤0.001; **P≤0.01; n.s., not significant.