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

Abstract

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on "outside-in" signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function-associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling-induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

Figure 1.. Crk proteins promote LFA-1 adhesion strengthening and actin reorganization

A and B) CD4⁺ T cells were allowed to adhere for 20 mins 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. Signal intensity is represented by a heat map. Scale bar, 10μm. Data pooled from 3 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 1ug/mL condition. D) CD4⁺ T cells were allowed to adhere to surfaces for 20 mins 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, 10μm. Data were pooled from 2 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 mean +/− SD from 3 experiments. F) Percentage of T cells that rolled or adhered with 100s⁻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 upon the LFA-1/ICAM-1 interaction. Data are mean +/− SD from 3 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 experiment of 3. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; by one-way ANOVA for comparison of multiple groups, or by a t-test for comparison of two groups.

Figure 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 mins, washed, and time-lapse images were taken of migrating cells using DIC optics. Scale bar, 10μm. (see movies S1 and S2). Darts 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 1μm total stack at the coverslip interface, with Lifeact-GFP intensity represented by a heat map. Scale bar, 10μm. (see Supplemental Movies 3 and 4). C-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 mean +/− SD from 3 experiments. D) Average speed and E) Directionality of migrating T cells, calculated as net displacement divided by track length. Data were pooled from 4 experiments. F) Tracks from a single experiment of individual migrating T cells centered at the same starting point. *p<0.05; **p<0.01; ***p<0.001 by a t-test.

Figure 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 mins, washed, and the percentage of migrating cells was calculated after 5 min pre-treatment with DMSO or with increasing concentrations of (A) Y-27632 or (B) S-nitro-blebbistatin. Data are mean +/− SD from 3 experiments. C) Representative images of migrating CD4⁺ T cells treated with DMSO, 10μM Y-27632, or 10μM S-nitro-blebbistatin. Cells were fixed and stained with phalloidin. Scale bar, 10μm. 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 mean +/− SD from 3 experiments. *p<0.05; **p<0.01; ***p<0.001 by one-way ANOVA for comparison of multiple groups, or by a t-test for comparison of two groups.

Figure 4.. LFA-1 dependent Cdc42 activation is blunted in DKO T cells

A-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, 10μm. 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 GST-PAK-PBD to pulldown GTP-bound GTPases. Representative western blot (C) and quantification of Cdc42-GTP (D, left) and Rac1-GTP (D, right). Data are mean +/− SD from 4 experiments. Densitometry was normalized to that of the WT untreated condition. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; by one-way ANOVA.

Figure 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 panel) and pERK (right panel). Values were normalized to the WT Mn²⁺ condition. Data represent mean +/− SD from 3 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 inhibitors IC87114 (PI3Kδ) or CZC24832 (PI3Kγ). Cells were fixed, stained with fluorescent phalloidin, and total phalloidin staining per cell was quantified. Data were pooled from 3 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 vs 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, 10μm. Data were pooled from 3 experiments. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; by one-way ANOVA for comparison of multiple groups, or by a t-test for comparison of two groups.

Figure 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 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 3–5 experiments.

Figure 7.. LFA-1 dependent mechanosensing requires Crk proteins

A-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, 10μm. B) Quantification of total phalloidin staining per cell. Values were normalized to WT, 50kPa condition. Data is pooled from 2 experiments. C) CD4⁺ T cells were treated with Mn²⁺, followed by exposure to soluble or surface bound ICAM-1 for 20 mins, 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. E) Quantification of pCasL abundance in cells plated on hydrogels of different stiffnesses. Data are mean +/− SD from 3 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 mean +/− SD from 3 experiments. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; by one-way ANOVA.

Figure 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 (SFK) 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 (GEF) 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). Phospho-CasL then serves as a scaffold for additional signaling molecules that direct cellular responses to the perceived mechanical cues.