The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization - PubMed (original) (raw)
The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization
Audrey Gérard et al. J Cell Biol. 2007.
Abstract
Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.
Figures
Figure 1.
Constitutively active V12Rap1 induces T cell polarization. (A) Control, WTRap1-, V12Cdc42-, V12Rac1-, and V12Rap1-expressing BW5147 T lymphoma cells were fixed in suspension and stained for CD44, as described in Materials and methods. The histogram indicates percentage of polarized cells from three independent experiments. Error bars indicate the SD; P indicates the P value. (B) Polarized V12Rap1-expressing BW5147 T lymphoma cells were stained for the leading edge markers CXCR4, talin, and LFA-1, and for the uropod markers CD44 and ezrin. TI, transmission images. (C) V12Rap1 localization. Myc-V12Rap1–expressing BW5147 T lymphoma cells were stained for myc, CXCR4, and CD44. Bars: (A) 10 μm; (B and C) 5 μm.
Figure 2.
Intracellular localization and activation of Par polarity proteins in polarized and nonpolarized BW5147 T lymphoma cells. (A) Intracellular localization of PKCζ, Par3, and Cdc42. Control and polarized V12Rap1-expressing BW5147 T lymphoma cells were stained for Cdc42, PKCζ, Par3, CXCR4, and CD44. Bars, 5 μm. TI, transmission images. (B) V12Rap1 activates Cdc42. The activation of Cdc42 in control and V12Rap1-expressing BW5147 T lymphoma cells was determined by pulldown assays, as described in Materials and methods. (top) Activated GTP-bound Cdc42 and total Cdc42. (bottom) Immunoblot of V12Rap1 expression (exo, exogenous; endo, endogenous). (C) V12Rap1 induces PKCζ phosphorylation. Lysates of starved control and V12Rap1-expressing BW5147 T lymphoma cells were separated by SDS-PAGE and analyzed for the amount of total PKCζ and PKCζ phosphorylation on Thr410 residue. (top) Immunoblots of phosphorylated PKCζ and total PKCζ. (bottom) Immunoblot of Rap1 expression. (D) Lysates of starved control and V12Rap1-expressing BW5147 T lymphoma cells were fractionated in membranous (memb) and cytosolic (cyt) fractions and analyzed for the intracellular localization of PKCζ and Rap1 (top). (bottom) Fractionation control immunoblots Nf-κB (cytosolic fraction) and CD44 (membrane fraction). Sizes are indicated in kilodaltons.
Figure 3.
Activation of Cdc42 and PKCζ is necessary for V12Rap1-induced T cell polarity. (A) V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with dominant-negative N17Cdc42. Control and transduced cells were fixed in suspension and stained for CD44. Histogram indicates the percentage of polarized cells of three independent experiments. (B) Lysates of the cells described in A were separated by SDS-PAGE and analyzed for total PKCζ and PKCζ phosphorylation on Thr410 residue. (top) Phosphorylated PKCζ and total PKCζ. (bottom) Immunoblots of expressed N17Cdc42 and V12Rap1 (exo, exogenous; endo, endogenous). Sizes are indicated in kilodaltons. (C) Expression kinase-dead PKCζ and knockdown of Par3 inhibit V12Rap1-induced T cell polarization. V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with a kinase-dead mutant of PKCζ (PKCζKD-IRES-GFP), luciferase shRNA, or Par3shRNA (Par3sh-IRES-GFP). 72 h after infection, cells were fixed in suspension and stained for CD44. Polarization was determined in control and GFP-expressing cells. Histogram indicates the percentage of polarized cells from three independent experiments. (D) T lymphocytes derived from FVB mice were retrovirally transduced with a control vector encoding GFP alone or with V12Rap1-IRES-GFP. 48 h after infection, cells were treated with 2 μM PKCζ pseudosubstrate for 1 h and stained for CD44. Histogram represents the quantification of polarized cells from three independent experiments. Error bars indicate the SD; P indicates the P value. Bars: (A and C) 10 μm; (D) 5 μm.
Figure 4.
Rac activity is necessary for V12Rap1-induced polarity. (A) V12Rap1 and WT-PKCζ activate Rac. Pulldown Rac activity assays were performed with starved control, V12Rap1-, and WT-PKCζ-expressing BW5147 T lymphoma cells. (top) Activated GTP-bound Rac and total Rac. (bottom) Immunoblots of expressed WT-PKCζ and V12Rap1 (exo, exogenous; endo, endogenous). (B) Dominant-negative Cdc42 inhibits V12Rap1-induced Rac activation. V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with dominant-negative Cdc42 (N17Cdc42). 4 d after infection, cells were starved for 16 h and Rac activity was determined. (top) Activated GTP-bound Rac and total Rac. (bottom) Immunoblots of the Cdc42 and V12Rap1 proteins. (C) Kinase-dead PKCζ inhibits V12Rap1-induced Rac activation. V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with a kinase-dead PKCζ (PKCζ KD). 4 d after infection, cells were starved for 16 h and Rac activity was determined. (top) Activated GTP-bound Rac and total Rac. (bottom) Immunoblots of the expressed kinase-dead PKCζ and V12Rap1 proteins. Sizes are indicated in kilodaltons. (D) Dominant-negative N17Rac1 inhibits V12Rap1-induced T cell polarization. V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with N17Rac1 or an empty vector as a control, selected for 3 d, and subsequently analyzed by immunohistochemistry using CD44-specific antibody. Histogram represents the percentage of polarized cells from three independent experiments. Error bars indicate the SD; P indicates the P value.
Figure 5.
V12Rap1 interacts with Tiam1 and mediates the association of Tiam1 with the Par polarity complex. (A) V12Rap1, but not WTRap1, associates with endogenous Tiam1. GST pulldown in lysates of BW5147 T lymphoma cells with purified GST (control), GST-WTRap1, or GST-V12Rap1. Pulldowns were separated by SDS-PAGE, and proteins were identified by Western blotting using Tiam1- and GST-specific antibodies. Sizes are indicated in kilodaltons. (B) Tiam1 associates with Par3, activated PKCζ, and Rap1. Tiam1 was immunoprecipitated from starved control and V12Rap1-expressing BW5147 T lymphoma cells. Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting for Tiam1 and coimmunoprecipitated proteins (Par3, PKCζ, and Rap1). (right) Total lysates (exo, exogenous; endo, endogenous). Sizes are indicated in kilodaltons. (C) Intracellular localization of PKCζ and Tiam1. Control and polarized V12Rap1-expressing BW5147 T lymphoma cells were stained for CXCR4, V12Rap1, CD44, RhoA, PKCζ, and Tiam1. TI, transmission images. Bar, 5 μm.
Figure 6.
Tiam1-induced Rac activity is required for induction of T cell polarity. Dominant-negative Tiam1 (PHnCCex) inhibits V12Rap1-induced Rac activation (A) and cell polarization (B). (A) V12Rap1-expressing BW5147 T lymphoma cells were retrovirally transduced with PHnCCex fused to GFP. 4 d after infection, cells were starved for 16 h and Rac activity was determined. (top) Activated GTP-bound Rac and total Rac. (bottom) Immunoblots of expressed proteins (PhnCCex and Rap1; exo, exogenous; endo, endogenous). Sizes are indicated in kilodaltons. (B) Cells, as described in A, were fixed in suspension for 72 h after infection and stained for CD44. Histogram represents the percentage of polarized cells of three independent experiments. (C) Tiam1−/− T lymphocytes are impaired in V12Rap1-induced cell polarization. T lymphocytes derived from Tiam1+/+ and Tiam1−/− mice were retrovirally transduced with a control vector encoding GFP alone or with V12Rap1-IRES-GFP. 48 h after infection, cells were fixed in suspension and stained for CD44. Histogram represents the quantification of polarized cells of three independent experiments. Error bars indicate the SD; P represents the P value. Bars, 5 μm.
Figure 7.
Rap1 is a major activator of the Par complex upon chemokine stimulation. (A) Rap1 and the Par complex are activated by SDF1α. Lymphocytes from FVB mice were either nonstimulated or stimulated with 500 ng/ml SDF1α for the indicated time. Subsequently, Rap activity, Cdc42 activity, Rac activity, and PKCζ phosphorylation status were determined. (top) GTP-bound Rap and total Rap. (middle) GTP-bound Cdc42, total Cdc42, phosphorylated PKCζ, and total PKCζ. (bottom) GTP-bound Rac and total Rac. Sizes are indicated in kilodaltons. (B) Rap1 is localized at the leading edge upon chemokine-induced polarity. Myc-WTRap1A–expressing Jurkat cells were stimulated with 200 ng/ml SDF1α for 20 min, and then stained for CXCR4, ICAM-3, and myc. Bar, 5 μm. (C) Rap activity is necessary for chemokine-induced activation of Cdc42, PKCζ, and Rac. Myc-WTRap1A–expressing Jurkat cells were stimulated with 500 ng/ml SDF1α for 2 min. Subsequently, Cdc42 activity, Rac activity, and PKCζ phosphorylation status were determined. (top) GTP-bound Cdc42 and total Cdc42; (middle) phosphorylated PKCζ and total PKCζ; (bottom) GTP-bound Rac, total Rac, and Rap1-GAP. Sizes are indicated in kilodaltons. (D) Rap1-GAP–expressing Jurkat cells or control cells were stimulated for 20 min with SDF1α. Subsequently, the percentage of polarized cells was determined by ICAM3 staining and changes in morphology. Histogram represents the quantification of polarized cells from three independent experiments. Error bars indicate the SD; P represents the P value.
Figure 8.
Tiam1 is necessary for chemokine-induced T cell polarization and chemotaxis. (A) Rac activation is impaired in Tiam1−/− T lymphocytes upon stimulation with SDF1α. Lymphocytes from Tiam1+/+ and Tiam1−/− mice were either nonstimulated or stimulated with 500 ng/ml SDF1α for 1 min. Subsequently, Rac activity status was determined. (top) GTP-bound Rac and total Rac. (bottom) Tiam1. Sizes are indicated in kilodaltons. (B) Chemokine-induced polarization is strongly reduced in Tiam1−/− lymphocytes. T lymphocytes from Tiam1+/+ and Tiam1−/− mice were stimulated with 1 μg/ml SDF1α for 10 min. Cells were fixed in suspension and stained for CD44. Histogram represents the quantification of polarized cells of four independent experiments. P represents the P value. (C) Chemotaxis is reduced in Tiam1−/− lymphocytes. T lymphocytes from Tiam1+/+ and Tiam1−/− mice were used in Transwell chemotaxis assays. Chemotaxis was measured after 1 h using different concentrations of SDF1α. Results are presented as the percentage of the input cells and are derived from three independent experiments. Error bars indicate the SD. P values are the comparison of the chemotactic capacity of Tiam1+/+ and knockout cells toward the same concentration of SDF1α (*, P < 0.05; **, P < 0.01).
Figure 9.
Tiam1 cooperates with the Par complex in chemokine-induced T cell polarization and chemotaxis. (A) Tiam1 and the Par complex coimmunoprecipitate after SDF1α stimulation. T lymphocytes from FVB mice were either not stimulated or treated with SDF1α for 3 min. Tiam1 was immunoprecipitated from the cell lysates. Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting for Tiam1 and coimmunoprecipitated proteins (Par3 and PKCζ). (right) Total lysates. (B) Colocalization of Tiam1 and PKCζ in chemokine-stimulated T lymphocytes. Lymphocytes from Tiam1+/+ mice were stimulated for 20 min with 200 ng/ml SDF1α and stained for CD44, PKCζ, and Tiam1. Bar, 5 μm. (C) PKCζ activation is not impaired in Tiam1−/− T lymphocytes upon stimulation with SDF1α. Lymphocytes from Tiam1+/+ and Tiam1−/− mice were either nonstimulated or stimulated with 500 ng/ml SDF1α for 3 min. Subsequently, PKCζ phosphorylation status was determined. (top) Phosphorylated PKCζ and total PKCζ. (bottom) Tiam1. (D) PKCζ signaling is necessary for chemokine-induced Rac activation. Lymphocytes from FVB mice were either not treated or treated with 2 μM PKCζ pseudosubstrate for 1 h and stimulated with 500 ng/ml SDF1α for 3 min. Subsequently, Rac activity status was determined. (top) GTP-bound Rac. (bottom) Total Rac. PKCζ pseudosubstrate alters the polarization (E) and chemotactic capacity (F) of Tiam1+/+, but not Tiam1−/−, lymphocytes. (E) Lymphocytes of both genotypes were either nontreated or treated with 2 μM PKCζ pseudosubstrate for 1 h and stimulated with 1 μg/ml SDF1α for 10 min. Cells were fixed and stained for CD44. Histogram represents the quantification of polarized cells of four independent experiments. (F) Chemotaxis of Tiam1+/+ and Tiam1−/− lymphocytes either treated or untreated with 2 μM PKCζ pseudosubstrate was measured in response to 200 ng/ml SDF1α after 1 h. The results are presented as the percentage of the input cells and are based on four independent experiments. Sizes are indicated in kilodaltons. Error bars indicate the SD; P represents the P value.
Figure 10.
The Par polarity complex functions in Rap1- and chemokine-induced T cell polarization. Chemokines are able to activate Rap1 at a specific site on the cell, which is considered as the initiating event for the establishment of T cell polarity. Activated Rap1 recruits the Par polarity complex, which consists of Par3, Par6, and PKCζ. Rap1 activates Cdc42, which is able to activate the Par polarity complex, including PKCζ. The polarity complex subsequently activates Tiam1, and thereby Rac1. Tiam1 associates with active, but not inactive, Rap1 and with components of the Par complex, and may thereby function to connect the Par polarity to Rap1 at the site where T cell polarity is initiated. Furthermore, Tiam1 is required to activate Rac downstream of the Par complex, presumably to regulate actin remodeling required for T cell polarization. Arrows indicate activation steps.
References
- Bos, J.L. 2005. Linking Rap to cell adhesion. Curr. Opin. Cell Biol. 17:123–128. - PubMed
- Carman, C.V., and T.A. Springer. 2003. Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr. Opin. Cell Biol. 15:547–556. - PubMed
- Chen, X., and I.G. Macara. 2005. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1. Nat. Cell Biol. 7:262–269. - PubMed
- Chou, M.M., W. Hou, J. Johnson, L.K. Graham, M.H. Lee, C.S. Chen, A.C. Newton, B.S. Schaffhausen, and A. Toker. 1998. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1. Curr. Biol. 8:1069–1077. - PubMed
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