Figure 1
Figure 1. LFA-1 stimulation induces actin cloud formation. (A) Difference in actin rearrangement upon stimulation with TCR and LFA-1. Jurkat cells were plated on a glass-based dish coated with anti-CD3, anti–LFA-1, or control Ab for 30 minutes (“Assay for actin rearrangement”). Cells were fixed and stained with phalloidin–Alexa 568. The spreading area and the actin cloud area were 294 ± 125 μm2 and 64 ± 21 μm2, respectively. The average diameter of the actin cloud was 10 to 11 μm (n = 20). (B) Quantitative analysis of actin cloud formation versus peripheral actin rearrangement upon stimulation with anti–LFA-1 or anti-CD3, respectively. The bottom histograms show the fluorescence intensity of the section that is shown by red arrows in the corresponding top panels. (C) Intracellular localization of actin cloud. Three-dimensional localization of actin cloud was analyzed by confocal microscopy. (D) Actin cloud is specifically induced by LFA-1 stimulation. Jurkat cells were stimulated in plates coated with Ab against LFA-1, CD45, or MHC class I for 30 minutes. (E) Actin cloud formation upon stimulation with ICAM-1. Jurkat cells were plated on coverslips coated with human ICAM-1/Ig or control Ig in the presence of SDF-1 (100 ng/mL) or Mn2+ (1 mM) for 30 minutes. (F) Actin cloud formation in normal T cells. Human PBLs that were stimulated with anti-CD3 in the presence of IL-2 for 4 days and rested for 3 days, or mouse naive splenic T cells were stimulated with immobilized anti–LFA-1 for 30 minutes. Each of the CD4+ and CD8+ human T cells were further analyzed by staining with CD4–Alexa 488 or CD8-APC together with phalloidin–Alexa 568. Approximately 10% of the cells formed actin clouds. Among them, approximately 25% were by CD4+ T cells and 75% were by CD8+ T cells (by counting 50 cells). (G) Accumulation of tyrosine-phosphorylated proteins within the actin cloud. Jurkat cells were stimulated with anti–LFA-1 or anti-CD3 for 10 minutes, fixed, and stained with phalloidin–Alexa 488 and anti-pY–biotin Ab, followed by streptavidin-Cy3. More than 100 cells were analyzed, and one T cell is shown as a representative. White arrows indicate actin cloud.

LFA-1 stimulation induces actin cloud formation. (A) Difference in actin rearrangement upon stimulation with TCR and LFA-1. Jurkat cells were plated on a glass-based dish coated with anti-CD3, anti–LFA-1, or control Ab for 30 minutes (“Assay for actin rearrangement”). Cells were fixed and stained with phalloidin–Alexa 568. The spreading area and the actin cloud area were 294 ± 125 μm2 and 64 ± 21 μm2, respectively. The average diameter of the actin cloud was 10 to 11 μm (n = 20). (B) Quantitative analysis of actin cloud formation versus peripheral actin rearrangement upon stimulation with anti–LFA-1 or anti-CD3, respectively. The bottom histograms show the fluorescence intensity of the section that is shown by red arrows in the corresponding top panels. (C) Intracellular localization of actin cloud. Three-dimensional localization of actin cloud was analyzed by confocal microscopy. (D) Actin cloud is specifically induced by LFA-1 stimulation. Jurkat cells were stimulated in plates coated with Ab against LFA-1, CD45, or MHC class I for 30 minutes. (E) Actin cloud formation upon stimulation with ICAM-1. Jurkat cells were plated on coverslips coated with human ICAM-1/Ig or control Ig in the presence of SDF-1 (100 ng/mL) or Mn2+ (1 mM) for 30 minutes. (F) Actin cloud formation in normal T cells. Human PBLs that were stimulated with anti-CD3 in the presence of IL-2 for 4 days and rested for 3 days, or mouse naive splenic T cells were stimulated with immobilized anti–LFA-1 for 30 minutes. Each of the CD4+ and CD8+ human T cells were further analyzed by staining with CD4–Alexa 488 or CD8-APC together with phalloidin–Alexa 568. Approximately 10% of the cells formed actin clouds. Among them, approximately 25% were by CD4+ T cells and 75% were by CD8+ T cells (by counting 50 cells). (G) Accumulation of tyrosine-phosphorylated proteins within the actin cloud. Jurkat cells were stimulated with anti–LFA-1 or anti-CD3 for 10 minutes, fixed, and stained with phalloidin–Alexa 488 and anti-pY–biotin Ab, followed by streptavidin-Cy3. More than 100 cells were analyzed, and one T cell is shown as a representative. White arrows indicate actin cloud.

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