TAGLN2 increases F-actin content and mediates long-lasting T cell adhesion and spreading. (A) J-GFP and J-TG2_GFP cells transfected with LifeAct (white) were loaded on anti-CD3/28 coverslips. After initial contact, time-lapse images were acquired, and single images representing 100-s time points are shown. Also, refer to Videos 5 and 6. Cell spreading areas were measured using FLUOVIEW program. EV, empty vector. (B) Cells were loaded on anti-CD3/28 coverslips, incubated for the indicated time points, and stained for F-actin, and the percentages of F-actin rings were scored and quantified. (left) Representative actin ring images were classified as ring (R), partial (P), and not cleared (N). (C) Cells were stimulated with soluble anti-CD3/28. At the indicated times, F-actin content was quantified using flow cytometry. The data are represented as relative fluorescence intensity compared with J-GFP cells at 0 min. (D) Cells were preincubated for 10 min with SEE-loaded Raji B cells (ICAM-1 [IC1]). The cells were then placed on PLL for the indicated time points. LifeAct redistribution was quantified as described in Materials and methods. (A–D) *, P < 0.05 versus J-GFP cells. (E–G) Jurkat T cells were transfected with either scrambled or siRNA targeting TAGLN2 (70 nM). (E) After 48 h, TAGLN2 expression was determined by Western blot. M, molecular mass. (F) Cells were loaded on anti-CD3/28 coverslips for indicated time points and then stained with TRITC-phalloidin. Cell spreading area and F-actin rings were analyzed as in B. (G) LifeAct redistribution at the IS was determined as in D. Graphical data shown are means of three experiments ± SD, with >50 cells. (F and G) *, P < 0.05 versus scrambled siRNA (si_SC). Bars, 10 µm.