LFA-1 is present in cytosolic clusters similar to those containing RhoB and tubulin in migrating T lymphocytes, and reducing RhoB abundance impairs the.

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LFA-1 is present in cytosolic clusters similar to those containing RhoB and tubulin in migrating T lymphocytes, and reducing RhoB abundance impairs the localization of LFA-1. LFA-1 is present in cytosolic clusters similar to those containing RhoB and tubulin in migrating T lymphocytes, and reducing RhoB abundance impairs the localization of LFA-1. (A and B) Representative confocal images of T lymphocytes attached to immobilized ICAM-1 stained with antibodies against LFA-1 (green), RhoB (red), and actin (blue) or tubulin (blue). Magnified areas (1, top right; 2, bottom right) are indicated with dashed rectangles in large image. White arrows indicate fluorescence overlap between LFA-1 and RhoB or between LFA-1 and actin in (A) and between LFA-1, RhoB, and tubulin in (B). Images are deconvoluted and Gaussian-fitted. Scale bars, 5 μm (low magnification) and 2 μm (high magnification). Images are representative of five experiments with 15 to 20 cells analyzed per experiment. See also fig. S2 for triple-color overlaps and quantification. (C) High-magnification image from a representative STED image of a T lymphocyte attached to ICAM-1 and stained with antibodies against RhoB (red) and tubulin (green). White arrows indicate fluorescence overlap; n = 3 experiments. Scale bar, 1 μm. (D) Representative confocal images of T lymphocytes transfected with scrambled control or RhoB-specific siRNA attached to immobilized ICAM-1 and stained with antibody against LFA-1. Marked cell area (3 × 3 μm) taken for analysis: uropod (white squares) and lamella (yellow squares). Top images are grayscale and bottom images are rainbow scale of graded fluorescence intensity. z1, low z-position; z2, middle z-position; and z3, high z-position; n = 3 experiments [15 to 20 cells per small interfering RNA (siRNA) per experiment]. Scale bar, 5 μm. (E and F) Mean fluorescence intensity (MFI) of LFA-1 from cells in experiments similar to those in (D) in the uropod (E) and in the lamellipodium (F). Pooled data of designated cell area (3 × 3 μm) from two independent experiments (n = 40 cells) are shown. Normalized to cells with control siRNA. Means ± SEM; n = 2 experiments. (G) Representative confocal image of HSB-2 cells expressing green fluorescent protein (GFP)–tagged wild-type (WT)–RhoB (green) and stained for internalized (Int.) LFA-1 (red). Magnified area (bottom) is indicated with dashed rectangles in the top left image; white arrows indicate fluorescence overlap. Scale bars, 5 μm (low magnification) and 2 μm (high magnification); n = 3 experiments (with 10 cells per experiment). *P < 0.05, ****P < 0.001, unpaired (two-tailed) t test. Malin Samuelsson et al., Sci. Signal. 2017;10:eaai8629 ©2017 by American Association for the Advancement of Science