Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization.
Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization.
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DOI：
10.1093/jmcb/mjw027
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发表时间：
2016-12
影响因子：
5.5
通讯作者：
A. Sakamoto;T. Tsukamoto;Y. Furutani;Y. Sudo;K. Shimada;A. Tomita;H. Kiyoi;Takashi Kato;T. Funatsu
中科院分区：
文献类型：
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作者：
A. Sakamoto;T. Tsukamoto;Y. Furutani;Y. Sudo;K. Shimada;A. Tomita;H. Kiyoi;Takashi Kato;T. Funatsu
亲爱的编辑，MPL是一种细胞因子受体，调节髓系细胞，特别是巨核细胞和血小板的产生(Kaushansky，2005)。MPL的二聚化不仅对信号的启动很重要(Kaushansky，2005)，而且对最近治疗骨髓增生性肿瘤的耐药性也很重要(Koppikar等人，2012)。在目前的模型中，细胞因子受体以单体和二聚体的混合物形式存在，没有配体刺激，而配体结合诱导额外的受体二聚化(Kaushansky，2005)。然而，MPL二聚化的动力学仍然知之甚少，这归因于在高时空分辨率下分析细胞因子受体二聚化的方法的局限性。我们使用活细胞中MPL的单分子成像(Liu等人，2015)来解决这一方法学限制。对于MPL的单分子成像，MPL的N末端与小肽酰基载体蛋白(ACP)基因连锁(图1 A)。ACP-MPL在小鼠FDC-P2细胞系中稳定表达，该细胞系通常用于外源表达的细胞因子受体的功能分析(Koppikar等人，2012年)。与表达MPL的细胞相比，ACP-MPL表达的细胞对细胞因子血小板生成素(TPO)的反应在信号、增殖和存活方面得到了验证(补充图S1)。然后将ACP与荧光团以1：1的标记率进行酶标记(图1A)(George等人，2004年)。这种荧光标记对信号没有影响(补充图S1C)。通过全内反射荧光(TIRF)显微镜将荧光标记的ACP-MPL显示为离散的荧光点(图1B和C)，
Dear Editor, MPL is a cytokine receptor that regulates the production of myeloid cells, in particular megakaryocytes and platelets (Kaushansky, 2005). Dimerization of MPL is important not only for the initiation of signalling (Kaushansky, 2005) but also for the resistance to a recent therapy for myeloproliferative neoplasms (Koppikar et al., 2012). In the current model, cytokine receptors exist as a mixture of monomers and dimers without ligand stimulation, while ligand binding induces additional receptor dimerization (Kaushansky, 2005). However, the dynamics of MPL dimerization remains poorly understood, which is attributed to the limitation of methodologies for analysing the dimerization of a cytokine receptor at a high spatiotemporal resolution. We here employ singlemolecule imaging (Liu et al., 2015) of MPL in live cells to solve this methodological limitation.For single-molecule imaging of MPL, the N-terminus of MPL was genetically linked with small peptidic acyl carrier protein (ACP)(Figure 1 A). ACP–MPL was stably expressed in a mouse FDC-P2 cell line, which is conventionally used for functional analysis of exogenously expressed cytokine receptors (Koppikar et al., 2012). Responses of ACP–MPL-expressing cells to the cytokine thrombopoietin (TPO) were validated in terms of signalling, proliferation, and survival, compared to those of MPL-expressing cells (Supplementary Figure S1). ACP was then enzymatically conjugated with a fluorophore at a labelling ratio of 1: 1 (Figure 1 A)(George et al., 2004). Such fluorescence labelling had no influence on signalling (Supplementary Figure S1C). Fluorescently labelled ACP–MPL was visualized by total internal reflection fluorescence (TIRF) microscopy as discrete fluorescent spots (Figure 1 B and C),