Research ArticleLYMPHOCYTE MIGRATION

RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes

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Science Signaling  12 Dec 2017:
Vol. 10, Issue 509, eaai8629
DOI: 10.1126/scisignal.aai8629
  • Fig. 1 RhoB interacts with LFA-1, and a reduction in RhoB abundance impairs LFA-1–dependent migration in T lymphocytes.

    (A) Western blots (representative) from T lymphocytes transfected with scrambled control or RhoB-specific siRNA were incubated with antibodies against RhoB and actin (knockdown efficiency, 52.6 ± 8.9%; means ± SEM; n = 7 experiments). (B) T lymphocytes transfected with the indicated siRNAs were incubated on immobilized ICAM-1 before random migration was observed by time-lapse microscopy. Individual cells were tracked and plotted with a common origin. A representative pattern of a single cell from 25 cells in each of three independent experiments. (C) Quantification of mean cell migration speed from experiments similar to those shown in (B). Data are means ± SEM. (D) T lymphocytes were applied to an Ibidi μ-Slide containing a confluent human umbilical cord endothelial cell (HUVEC) layer with a shear stress of 1 dyne/cm2. The activity of interacting T lymphocytes was recorded at a rate of 60 frames/min during 3 min. T lymphocytes were manually scored according to their rate of interaction with HUVEC. Adherent: T lymphocytes adhered for more than 3 s. Transendothelial migration (TEM): T lymphocytes transmigrated beneath the HUVEC layer. Means ± SEM; n = 3 experiments. Statistical significance between siCTRL and siRhoB for both Adherent and TEM is indicated. (E) Guanosine triphosphate (GTP)–Rho isolated from T lymphocytes migrating on immobilized intercellular adhesion molecule–1 (ICAM-1) with or without CXCL12 was analyzed by Western blotting for RhoB. Mean horseradish peroxidase (HRP) intensity was normalized to unstimulated control (Unstim) of cells grown in suspension without CXCL12. Means ± SEM; n = 4 experiments. Statistical significance between Unstim and ICAM-1 and ICAM-1 + CXCL12 is indicated. See also fig. S1F for representative Western blotting analysis. (F) RhoB or rabbit immunoglobulin G (IgG) control immunoprecipitates (IP) from T lymphocytes grown in suspension were analyzed by Western blotting for the presence of RhoB and LFA-1. Representative blot; n = 2 experiments. The total cell lysate (TCL) and immunoprecipitated samples were from different parts of the same gel; intervening lanes have been spliced out. (G) GTP-Rho immunoprecipitates from T lymphocytes migrating on immobilized ICAM-1 (or unstimulated control cells grown in suspension without ICAM-1) were analyzed by Western blotting for the presence of LFA-1. Representative blot; n = 3 experiments. (H) Mean HRP intensity from experiments similar to those shown in (G), for Unstim and ICAM-1, respectively. Data are normalized to Unstim and shown as means ± SEM; n = 3 experiments. (I) Representative confocal images of a T lymphocyte attached to immobilized ICAM-1 and stained with antibodies against LFA-1 (green) or RhoB (red). Magnified areas (1, middle; 2, bottom) are indicated with dashed rectangles in the top left image. White arrows indicate fluorescence overlap. Images are deconvoluted and Gaussian-fitted. Scale bars, 5 μm (low magnification) and 2 μm (high magnification); n = 5 experiments (with 15 to 20 cells per experiment). (J) Mander’s colocalization coefficient (MCC) quantification of LFA-1 fluorescence colocalization with RhoB (left) and vice versa (right) in T lymphocytes. Mean overlap from three regions averaged per cell (total cell) and overlap from single regions grouped by location within the cell (uropod and lamellipodium). Pooled data from two independent experiments (n = 36 cells) are shown. Means ± SEM; n = 2 experiments. (K) High magnification of a representative STED image of a T lymphocyte attached to immobilized ICAM-1 and stained with antibodies against RhoB (red) or LFA-1 (green). White arrows indicate fluorescence overlap. Scale bar, 1 μm. *P <0.05, **P < 0.01, ***P < 0.001, unpaired (two-tailed) t test.

  • Fig. 2 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.

  • Fig. 3 LFA-1 locates within EEA-1–positive early endosomal structures and within Rab11-positive recycling endosomal structures in migrating T lymphocytes.

    (A) Representative confocal image of T lymphocytes attached to immobilized ICAM-1 and stained with antibodies against LFA-1 and EEA-1, Rab4, or Rab11. Magnified area (right) is indicated with dashed rectangles in the leftmost image. White arrows indicate fluorescence overlap. Scale bars, 5 μm (low magnification) and 2 μm (high magnification). Images are representative of three experiments, with 15 to 20 cells analyzed for each antibody per experiment. (B) T lymphocytes were stained with antibodies against RhoB and EEA-1 (top), Rab4 (middle), or Rab11 (bottom) as described in (A). (C) MCC quantification of fluorescence colocalization from experiments similar to those shown in (A). Mean overlap from three regions averaged per cell was pooled from two independent experiments (n = 25 to 36 cells). (D) Quantification of fluorescence colocalization in (B) as described in (C). Data from 25 to 36 cells are shown. In (C) and (D), data are presented as means ± SEM; n = 2 experiments. Statistical significance between EEA-1 versus Rab4 and Rab11 is indicated as asterisks above bars and between Rab4 and Rab11 is indicated by asterisks above black lines. **P < 0.01, ***P < 0.001, unpaired (two-tailed) t test.

  • Fig. 4 Dysfunctional RhoB impairs the LFA-1–mediated migration of HSB-2 cells.

    (A) HSB-2 cells expressing GFP, GFP-tagged WT-RhoB, GFP-tagged CA-RhoB, or GFP-tagged DN-RhoB were sorted (24 hours after transfection) based on GFP expression and incubated on immobilized ICAM-1 before random migration was observed by time-lapse microscopy. Individual cells were tracked and plotted with a common origin. A representative pattern tracked by a single cell from 45 cells from three experiments is shown. (B) Quantification of mean speed of the cells shown in (A). Means ± SEM; n = 3. Statistical significance between GFP-WT-RhoB and GFP-DN-RhoB (black line) is indicated. (C) Sorted HSB-2 cells expressing GFP, GFP-WT-RhoB, GFP-CA-RhoB, or GFP-DN-RhoB were stained with CellTracker Blue CMAC and incubated on immobilized ICAM-1. Excitation was measured, and nonadherent cells were removed by gentle washing before excitation was measured again. Adhesion index: Percentage of adherent cells = (average fluorescence intensity read in the washed wells)/(average fluorescence intensity read in the unwashed cells) × 100%. Means ± SEM; n = 4 experiments. Statistical significance between GFP-WT-RhoB and GFP-DN-RhoB (black line) is indicated. (D) Sorted HSB-2 cells expressing GFP, GFP-WT-RhoB, GFP-CA-RhoB, or GFP-DN-RhoB were analyzed by flow cytometry based on LFA-1 surface expression. Data are pooled and normalized to GFP. Means ± SEM; n = 3 experiments. Statistical significance between GFP-WT-RhoB and GFP-DN-RhoB (black line) is indicated. **P < 0.01, ***P < 0.001, unpaired (two-tailed) t test.

  • Fig. 5 Dysfunctional RhoB impairs LFA-1 internalization and recycling in HSB-2 cells.

    (A) Flow cytometric analysis of the cell surface expression of LFA-1 over time in sorted HSB-2 cells expressing GFP, GFP-tagged CA-RhoB, or GFP-tagged DN-RhoB. Pooled data normalized to 0 min (start point). Means ± SEM; n = 5 experiments. Statistical significance calculated by ordinary one-way analysis of variance (ANOVA) multiple comparisons test (*P < 0.05) between GFP and GFP-DN-RhoB is indicated. (B) LFA-1 surface expression from (A) was quantified at 120 min after staining. Data are normalized to GFP-expressing cells and are means ± SEM. Statistical significance between GFP and GFPDN-RhoB is indicated. (C and D) Sorted HSB-2 cells expressing GFP, GFP-tagged WT-RhoB, CA-RhoB, or DN-RhoB were attached to ICAM-1 and stained with antibodies against LFA-1 and Rab11 (C) or Rab4 (D). MCC quantification of confocal images of cells stained for LFA-1 and Rab11 (C) or Rab4 (D). Pooled data from three regions averaged per cell from 22 to 35 cells in two independent experiments are shown. Data are means ± SEM. Statistical significance of differences between means of GFP-WT-RhoB and GFP-DN-RhoB (black line) is indicated in (C). (B and C) *P < 0.05, ***P < 0.001, unpaired (two-tailed) t test.

  • Fig. 6 Reduced RhoB abundance impairs the localization of Rab11 in migrating T lymphocytes.

    (A) Representative confocal images of T lymphocytes transfected with scrambled control or RhoB-specific siRNA, attached to immobilized ICAM-1, and stained with antibodies against Rab11 (left), Rab5 (middle), or Rab7 (right). Dotted line represents cell shape. Left image is grayscale; right image is a rainbow scale of graded fluorescence intensity. Marked uropod cell area (3 × 3 μm) taken for analysis (yellow squares). Scale bar, 5 μm. Images are representative of two experiments with 15 cells per siRNA analyzed per experiment. (B) Mean fluorescence intensity of Rab11 (left), Rab5 (middle), and Rab7 (right) in the uropod. Pooled data of designated cell areas (3 × 3 μm) from 30 cells are shown. Normalized to cells with control siRNA. Means ± SEM; n = 2 experiments. (C) MCC quantification of LFA-1 fluorescence colocalization with Rab11 (left) and vice versa (right) in T lymphocytes transfected with the indicated siRNAs. Pooled data from three regions averaged per cell from 22 cells in two independent experiments. Means ± SEM; n = 2 experiments. **P = 0.01, ***P < 0.001, unpaired (two-tailed) t test.

  • Fig. 7 Reduced Rab11 abundance impairs LFA-1 recycling and consequently LFA-1–dependent migration in T lymphocytes.

    (A) Representative Western blots of T lymphocytes transfected with scrambled control or Rab11-specific siRNA were incubated with antibodies against total Rab11 and actin (mean knockdown efficiency, 88.7 ± 3.9%; means ± SEM; n = 3 experiments). (B) T lymphocytes transfected with the indicated siRNAs were incubated on immobilized ICAM-1 before random migration was observed by time-lapse microscopy. Individual cells were tracked and plotted with a common origin. A representative experiment of the pattern tracked by a single cell from 40 cells tracked is shown; n = 2 experiments. (C) Quantification of mean cell migration speed from the cells in experiments described in (B). Means ± SEM. (D) HSB-2 cells transfected with the indicated siRNAs and with biotinylated cell surface proteins were incubated on immobilized ICAM-1. The remaining biotin on surface-located cell membrane receptors was removed with reducing glutathione, and the internalized biotinylated LFA-1 was analyzed by Western blotting analysis of LFA-1 immunoprecipitates with streptavidin-HRP (first and second lanes). Reexposure of biotinylated LFA-1 to the surface was analyzed by reincubating T lymphocytes on immobilized ICAM-1 after the removal of biotinylated membrane receptors. Reexposed biotinylated membrane receptors were then removed with reducing glutathione, and the remaining intracellular biotinylated LFA-1 was subsequently analyzed by Western blotting analysis of LFA-1 immunoprecipitates with streptavidin-HRP (third and fourth lanes). (E) Mean band intensity from experiments similar to that represented in (D) (first and second lanes). Data are pooled and normalized to control. Means ± SEM; n = 3 experiments. (F) Mean band intensity from experiments similar to that represented in (D) (third and fourth lanes). Data are pooled and normalized to HRP intensity after internalization for siCTRL and siRhoB, as shown in (E). Means ± SEM; n = 3 experiments. (G) Representative Western blots from the analysis of isolated cytoplasmic (Cyt) or membrane (Mem) fractions from HSB-2 cells transfected with the indicated siRNAs. Blots were incubated with antibodies against total RhoB and LFA-1. (H) Quantification of the ratio between the mean band intensities of the cytoplasmic and membrane fractions from experiments similar to that shown in (G). Means ± SEM; n = 3 experiments. (I) Representative Western blots from isolated cytoplasmic or membrane fractions of HSB-2 cells transfected with scrambled control or RhoB siRNA and analyzed for total Rab11 and LFA-1. (J) Quantification of the mean band intensity ratio between the cytoplasmic fraction and the membrane fraction in (I). Left, Rab11; right, LFA-1. Pooled data from three independent experiments are shown. Means ± SEM. (H and J) *P < 0.05, **P < 0.01, ***P < 0.001, unpaired (two-tailed) t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/509/eaai8629/DC1

    Fig. S1. RhoB interacts with LFA-1, and a reduction in RhoB abundance impairs LFA-1–dependent migration in T lymphocytes.

    Fig. S2. LFA-1 is present in cytosolic clusters similar to those containing RhoB and tubulin in migrating T lymphocytes.

    Fig. S3. Both LFA-1 and RhoB interact with Rab11 in T lymphocytes.

    Fig. S4. Active RhoB directs LFA-1 between cytoplasmic and membrane compartments in HSB-2 cells.

    Fig. S5. Decreased abundance of functional RhoB impairs LFA-1 internalization and recycling.

    Fig. S6. RhoB activity regulates Rab11 localization, and both proteins control LFA-1 recycling in HSB-2 cells.

  • Supplementary Materials for:

    RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes

    Malin Samuelsson, Katarzyna Potrzebowska, Janne Lehtonen, Jason P. Beech, Ekatarina Skorova, Heli Uronen-Hansson, Lena Svensson*

    *Corresponding author. Email: lena_m.svensson{at}med.lu.se

    This PDF file includes:

    • Fig. S1. RhoB interacts with LFA-1, and a reduction in RhoB abundance impairs LFA-1–dependent migration in T lymphocytes.
    • Fig. S2. LFA-1 is present in cytosolic clusters similar to those containing RhoB and tubulin in migrating T lymphocytes.
    • Fig. S3. Both LFA-1 and RhoB interact with Rab11 in T lymphocytes.
    • Fig. S4. Active RhoB directs LFA-1 between cytoplasmic and membrane compartments in HSB-2 cells.
    • Fig. S5. Decreased abundance of functional RhoB impairs LFA-1 internalization and recycling.
    • Fig. S6. RhoB activity regulates Rab11 localization, and both proteins control LFA-1 recycling in HSB-2 cells.

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    Citation: M. Samuelsson, K. Potrzebowska, J. Lehtonen, J. P. Beech, E. Skorova, H. Uronen-Hansson, L. Svensson, RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes. Sci. Signal. 10, eaai8629 (2017).

    © 2017 American Association for the Advancement of Science

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