Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging
Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging
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DOI：
10.1073/pnas.0710336105
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发表时间：
2007-12-18
影响因子：
11.1
通讯作者：
Li, Rong
中科院分区：
文献类型：
--
作者：
Slaughter, Brian D.;Schwartz, Joel W.;Li, Rong
荧光相关光谱（FCS），荧光互相关光谱（FCCS）和光子计数直方图（PCH）是波动的方法，最近出现的潜在有用的工具，用于获得参数的分子动力学，相互作用，和寡聚体在体内。在这里，我们报告成功实施FCS，FCCS和PCH在活酵母细胞中使用荧光蛋白标记的蛋白质表达从他们的天然染色体位点，检查胞质动力学和有丝分裂原活化蛋白激酶（MAPK）级联反应，广泛发生的信号基序，在响应交配信息素的组件之间的相互作用。FCS分析详细描述了MAPK蛋白的扩散特性和移动的浓度。使用EGFP和mCherry标记的蛋白质对的FCCS分析观察到Ste 7（MAPK激酶）与MAPK Fus 3的相互作用。或Kss 1，以及支架蛋白，Ste 5，与细胞溶质中的Ste 7和Ste 11（MAPK激酶激酶），提供它们结合平衡的体内常数。在胞质溶胶中Ste 5与Fus 3的相互作用低于检测限，表明弱相互作用（如果存在的话），K-d > 400-500 nM。使用PCH，我们表明，细胞溶质的Ste 5主要是单体。如PCH所证实的，使用二聚化标签的Ste 5的人工二聚化刺激了Ste 5和Fus 3之间的相互作用。天然Ste 5被发现优先结合Fus 3在信息素处理的细胞中的皮质，通过荧光共振能量转移（FRET）检测。这些结果提供了体内MAPK复合物的定量空间图，并直接支持膜缔合和调节Ste 5支架是MAPK活化的关键步骤的模型。
Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3. or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K-d > 400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.