Research ArticleImmunology

Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1

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Science Signaling  11 Dec 2018:
Vol. 11, Issue 560, eaat3178
DOI: 10.1126/scisignal.aat3178
  • Fig. 1 Crk proteins promote LFA-1 adhesion strengthening and actin reorganization.

    (A and B) CD4+ T cells were allowed to adhere for 20 min to ICAM-1–coated coverslips in the presence or absence of (A) Mn2+ or (B) PMA, fixed, and stained with phalloidin. (Right) Quantification of phalloidin staining of individual cells normalized to the WT untreated condition. Signal intensity is represented by a heat map. Scale bars, 10 μm. Data were pooled from three experiments. (C) CD4+ T cells were allowed to adhere to coverslips coated with increasing concentrations of ICAM-1, and phalloidin staining of individual cells was quantified and normalized to the 1 μg/ml condition. (D) CD4+ T cells were allowed to adhere to surfaces for 20 min that were coated with ICAM-1, anti-CD3, or both. Cells were fixed and stained with phalloidin. (Right) Quantification of phalloidin staining of individual cells normalized to the WT ICAM-1 condition. Scale bar, 10 μm. Data were pooled from two experiments. (E) CD4+ T cell adhesion to ICAM-1 was measured using a standard plate-based adhesion assay. T cells were treated with PMA (to bypass inside-out signaling) or Mn2+ (to exogenously induce integrin conformational change). Data are means ± SD from three experiments. (F) Percentage of T cells that rolled or adhered with 100 s−1 shear rate on surfaces coated with ICAM-1, P-selectin, and SDF-1. Treatment with anti–LFA-1 antibody blocked all adhesion, showing that adhesion is dependent on the LFA-1/ICAM-1 interaction. Data are means ± SD from three experiments. (G) Percentage of adhered cells in (F) that subsequently spread. (H) CD4+ T cells were allowed to adhere in the absence of shear, and then the shear rate was increased every 2 min. For each shear rate, the percentage of remaining cells adhered was calculated. Data represent one of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way analysis of variance (ANOVA) for comparison of multiple groups or by a t test for comparison of two groups. ns, not significant.

  • Fig. 2 Crk proteins are required for leading edge formation and normal migration.

    (A) CD4+ T cells were allowed to adhere to ICAM-1–coated coverslips for 20 min and washed, and time-lapse images were taken of migrating cells using differential interference contrast (DIC) optics (see movies S1 and S2). Scale bar, 10 μm. Arrowheads highlight multiple cell protrusions. (B) CD4+ T cells expressing Lifeact-GFP were treated as in (A), and time-lapse images were taken using confocal microscopy. Images are a projection of a 1-μm total stack at the coverslip interface, with Lifeact-GFP intensity represented by a heat map (see movies S3 and S4). Scale bar, 10 μm. (C to F) CD4+ T cells were treated as in (A) and tracked over a 10-min period using DIC optics. (C) Percentage of adhered T cells that were migratory. Data are means ± SD from three experiments. Average speed (D) and directionality (E) of migrating T cells calculated as net displacement divided by track length. Data were pooled from four experiments. (F) Tracks from a single experiment of individual migrating T cells centered at the same starting point. ***P < 0.001 by a t test.

  • Fig. 3 Migration of DKO T cells is more dependent on myosin activity.

    (A and B) CD4+ T cells were allowed to adhere to ICAM-1–coated coverslips for 20 min and washed, and the percentage of migrating cells was calculated after 5-min pretreatment with dimethyl sulfoxide (DMSO) or with increasing concentrations of (A) Y-27632 or (B) (S)-nitro-blebbistatin. Data are means ± SD from three experiments. (C) Representative images of migrating CD4+ T cells treated with DMSO, 10 μM Y-27632, or 10 μM (S)-nitro-blebbistatin. Cells were fixed and stained with phalloidin. Scale bar, 10 μm. (D) Length/width ratios of individual cells migrating under the different treatments. Cells that were not migratory were predominantly round and, thus, excluded from the analysis. Data are means ± SD from three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA for comparison of multiple groups or by a t test for comparison of two groups.

  • Fig. 4 LFA-1–dependent Cdc42 activation is blunted in DKO T cells.

    (A and B) CD4+ T cells were allowed to migrate on ICAM-1–coated surfaces, fixed, and probed with fluorescent phalloidin to label F-actin and with an antibody that recognizes (A) open-WASp or (B) total WAVE2. Scale bar, 10 μm. (C and D) CD4+ T cells were treated with Mn2+ to activate LFA-1, followed by exposure to soluble or surface-bound ICAM-1 for 20 min. Lysates were mixed with glutathione S-transferase (GST)–PAK–PBD to pull down GTP-bound GTPases. Representative Western blot (C) and quantification of Cdc42-GTP (D, left) and Rac1-GTP (D, right). Data are means ± SD from four experiments. Densitometry was normalized to that of the WT untreated condition. **P < 0.01 and ***P < 0.001 by one-way ANOVA. AU, arbitrary units; WCL, whole-cell lysate; IB, immunoblotting.

  • Fig. 5 Crk proteins promote integrin-mediated PI3K signaling.

    (A) CD4+ T cells were treated as in Fig. 4C, and lysates were immunoblotted with indicated antibodies. (B) Quantification of pAKT (left) and pERK (right). Values were normalized to the WT Mn2+ condition. Data represent means ± SD from three experiments. (C) CD4+ T cells were treated with Mn2+ and allowed to adhere to ICAM-1–coated surfaces in the presence or absence of the pan-PI3K inhibitor LY-294002 or the specific inhibitor IC87114 (PI3Kδ) or CZC24832 (PI3Kγ). Cells were fixed and stained with fluorescent phalloidin, and total phalloidin staining per cell was quantified. Data were pooled from three experiments. (D) CD4+ T cells expressing the PIP3 biosensor GRP1-PH-GFP were imaged while migrating on ICAM-1. GRP1-PH-GFP intensity is represented as a heat map. (Right) Ratio of average GRP1-PH-GFP intensity at the leading edge versus the trailing edge on a cell-by-cell basis. Note that signal intensity for each cell was adjusted individually to best reveal front-rear asymmetry. Scale bar, 10 μm. Data were pooled from three experiments. ***P < 0.001 by one-way ANOVA for comparison of multiple groups or by a t test for comparison of two groups.

  • Fig. 6 Crk proteins are necessary for phosphorylation of c-Cbl and its interaction with p85.

    (A) CD4+ T cells were treated with Mn2+ and then allowed to adhere to ICAM-1–coated plates for 20 min. Lysates were immunoprecipitated (IP) with anti-p85 and immunoblotted for pTyr. (B) Cells were treated as in (A), except lysates were immunoprecipitated with anti-CrkL and immunoblotted for pTyr. (C) CD4+ T cells were treated with Mn2+ followed by exposure to soluble or surface-bound ICAM-1 for 20 min. Lysates were immunoprecipitated with anti-pTyr and immunoblotted for c-Cbl and Cbl-b. (D) Cells were treated as in (C), except lysates were immunoblotted for Pyk2. (E) CD4+ T cells were treated with Mn2+ and allowed to adhere to ICAM-1–coated plates in the presence of the indicated drugs for 20 min. Lysates were immunoprecipitated with anti-pTyr and immunoblotted for c-Cbl. Immunoblots are representative of three to five experiments.

  • Fig. 7 LFA-1–dependent mechanosensing requires Crk proteins.

    (A and B) CD4+ T cells were treated with Mn2+ and allowed to adhere to ICAM-1–coated hydrogels of increasing stiffness. Cells were fixed, stained with fluorescent phalloidin, and imaged. Phalloidin intensity is represented as a heat map. Scale bar, 10 μm. (B) Quantification of total phalloidin staining per cell. Values were normalized to WT, 50-kPa condition. Data were pooled from two experiments. (C) CD4+ T cells were treated with Mn2+, followed by exposure to soluble or surface-bound ICAM-1 for 20 min, lysed, and immunoprecipitated with anti-pTyr, followed by immunoblot for CasL. (D) CD4+ T cells were treated with Mn2+ and allowed to adhere to ICAM-1–coated hydrogels of increasing stiffness, lysed, immunoprecipitated with anti-pTyr, and immunoblotted for CasL. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Quantification of pCasL abundance in cells plated on hydrogels of different stiffnesses. Data are means ± SD from three experiments. (F) CD4+ T cells were treated with Mn2+ and allowed to adhere to ICAM-1–coated surfaces for 10 min, followed by treatment with the indicated drugs for an additional 10 min. Cells were lysed, immunoprecipitated with anti-pTyr, and immunoblotted for CasL and c-Cbl. (Right) Quantification of pCasL abundance. Data are means ± SD from three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA. LatB, latrunculin B.

  • Fig. 8 Proposed model for Crk-dependent integrin signaling.

    Our data suggest a model in which engagement of LFA-1 by immobilized ligands activates Src family kinases (SFKs) and induces the binding of preformed Crk/CasL complexes to c-Cbl. Formation of this complex promotes SFK-dependent phosphorylation of c-Cbl (yellow dots), which, in turn, creates a binding site for the p85 subunit of PI3K. This activates PI3K catalytic function, resulting in localized production of PIP3. Recruitment of guanine exchange factors (GEFs) to PIP3-rich membrane regions, and possibly also to the Crk/c-Cbl complex itself, then induces Rho GTPase activation, resulting in actin polymerization and leading edge formation. Actin polymerization also provides the force necessary to open the CasL substrate domain and enable its phosphorylation (most likely also by SFKs). pCasL then serves as a scaffold for additional signaling molecules that direct cellular responses to the perceived mechanical cues.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/560/eaat3178/DC1

    Fig. S1. Mass spectrometry coverage of c-Cbl and Cbl-b.

    Fig. S2. ICAM-1 coating is consistent between hydrogels of different stiffness.

    Fig. S3. Interaction of CrkL with CasL and c-Cbl.

    Movie S1. WT CD4+ T cell migrating on ICAM-1.

    Movie S2. DKO CD4+ T cell migrating on ICAM-1.

    Movie S3. Lifeact-GFP–expressing WT CD4+ T cell migrating on ICAM-1.

    Movie S4. Lifeact-GFP–expressing DKO CD4+ T cell migrating on ICAM-1.

  • The PDF file includes:

    • Fig. S1. Mass spectrometry coverage of c-Cbl and Cbl-b.
    • Fig. S2. ICAM-1 coating is consistent between hydrogels of different stiffness.
    • Fig. S3. Interaction of CrkL with CasL and c-Cbl.
    • Legends for movies S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). WT CD4+ T cell migrating on ICAM-1.
    • Movie S2 (.avi format). DKO CD4+ T cell migrating on ICAM-1.
    • Movie S3 (.avi format). Lifeact-GFP–expressing WT CD4+ T cell migrating on ICAM-1.
    • Movie S4 (.avi format). Lifeact-GFP–expressing DKO CD4+ T cell migrating on ICAM-1.

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