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.
Institut Pasteur, Département Infection et Epidémiologie, Département d'Immunologie, Unité d'Immunogénétique Cellulaire, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
J Biol Chem. 2013 Mar 22;288(12):8691-8701. doi: 10.1074/jbc.M113.449918. Epub 2013 Jan 17.
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.
白细胞介素 (IL)-7 是中央和外周水平 CD4 T 淋巴细胞（辅助细胞）的主要稳态调节剂。在被 IL-7 激活后，几种信号通路，主要是 JAK/STAT、PI3K/Akt 和 MAPK，诱导参与 T 细胞分化、激活和增殖的基因表达。我们分析了 IL-7 对 CD4 T 细胞激活的早期事件。我们已经表明，IL-7 在最初的几分钟内诱导富含胆固醇的膜微区的形成，这些膜微区分隔其激活的受体并启动其与细胞骨架的锚定，支持信号复合物，即信号体，在 IL-7 受体细胞质结构域上的形成。在这里，我们通过受激发射耗尽显微镜描述了膜微区和细胞骨架瞬态组织在 IL-7 调节的 JAK/STAT 信号通路中的关键作用。我们使用适当的抑制剂沿着 IL-7 激活动力学对磷酸化 STAT5 和细胞骨架成分进行成像。我们表明，脂质筏抑制剂延迟并减少 IL-7 诱导的 JAK1 和 JAK3 磷酸化。药物诱导的细胞骨架解聚抑制磷酸化 STAT5 的形成、运输和易位到细胞核，从而控制参与 T 细胞激活和增殖的基因的转录。我们将这些定量分析的结果拟合在一起，并提出以下机制。嵌入在膜微区中的激活的 IL-7 受体诱导肌动蛋白微丝网格的形成，锚定微管，微管从富含筏的受体径向生长到核膜。由信号体磷酸化的 STAT5 被动力蛋白加载并沿着微管滑行穿过细胞质，在 IL-7 刺激后 2 分钟到达细胞核。径向微管在 15 分钟后消失，而横向微管，独立于磷酸化 STAT5 的运输，开始从微管组织中心萌芽。
