title: Membrane microdomains and cytoskeleton organization shape and regulate the IL-7 receptor signalosome in human CD4 T-cells.
abstract: Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France; Université Pierre et Marie Curie, Cellule Pasteur-UPMC, 25 Rue du Dr. Roux, 75015 Paris, France.
authors: Blanche
authors: B
authors: Tamarit
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors: Florence
authors: F
authors: Bugault
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France; Université Pierre et Marie Curie, Cellule Pasteur-UPMC, 25 Rue du Dr. Roux, 75015 Paris, France.
authors: Anne-Hélène
authors: AH
authors: Pillet
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors: Vincent
authors: V
authors: Lavergne
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS UMR3525, 25 Rue du Dr. Roux, 75015 Paris, France.
authors: Pascal
authors: P
authors: Bochet
authors: Leica Microsystems AG, Max Schmidheiny Strasse 201, CH-9435 Heerbrugg, Switzerland.
authors: Nathalie
authors: N
authors: Garin
authors: Leica Microsystems CMS GmbH, Am Friedrichplatz, D-68165 Mannheim, Germany.
authors: Ulf
authors: U
authors: Schwarz
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors: Jacques
authors: J
authors: Thèze
authors: Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Electronic address: rose@pasteur.fr.
authors: Thierry
authors: T
authors: Rose
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000276:immunology; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D053613:Janus Kinase 1 / Q000378:metabolism; D053616:Janus Kinase 3 / Q000378:metabolism; D007700:Kinetics; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D020395:Receptors, Interleukin-7 / Q000378:metabolism* / Q000502:physiology; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D020395:Receptors, Interleukin-7; D050799:STAT5 Transcription Factor; C415350:interleukin-7 receptor, alpha chain; D002772:Cholera Toxin; C507904:JAK1 protein, human; C507933:JAK3 protein, human; D053613:Janus Kinase 1; D053616:Janus Kinase 3