T Cell Signal Regulation by the Actin Cytoskeleton
T Cell Signal Regulation by the Actin Cytoskeleton
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DOI：
10.1074/jbc.m109.097311
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发表时间：
2010-05-07
影响因子：
4.8
通讯作者：
Rodgers, William
中科院分区：
文献类型：
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作者：
Chichili, Gurunadh R.;Westmuckett, Andrew D.;Rodgers, William
T细胞形成维持和调节细胞刺激信号的免疫突触（IS）。在IS中富集的是Src家族激酶Lck。相反，激活Src家族激酶的膜磷酸酶CD 45被排除在外，这是避免T细胞受体磷酸信号淬灭所必需的。数据表明，这种安排发生的胆固醇依赖筏在IS的富集。然而，筏在构建IS中的作用仍然不清楚。为了解决这个问题，我们使用荧光共振能量转移（FRET）来询问IS的纳米级结构。FRET探针由膜锚定的荧光蛋白组成，具有对筏的不同亲和力。筏和nonraft探针表现出集群的IS。然而，筏供体-受体对的共聚类比筏-非筏对的共聚类大10倍。我们测量了干扰筏在IS上的CD 45定位和LCK调节的效果，通过用菲律宾肽处理刺激的T细胞。菲律平特异性地破坏了IS中筏FRET对的共聚簇，并允许CD 45靶向IS和Lck的调节酪氨酸的去磷酸化。簇筏探针也是敏感的latrunctulin B，破坏肌动蛋白丝。引人注目的是，丰富的皮质细胞骨架，使用jasplakinaline维持筏探针共聚类，CD 45排斥，和LCK调节后，在IS的菲律宾。这些数据表明，肌动蛋白细胞骨架维持膜筏环境中的IS，促进LCK的调节，排除CD 45。
T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.