Myosin-actin Interaction in Dictyostelium Cells Revealed by GFP-based Strain Sensor and Validated Linear Spectral Unmixing
Myosin-actin Interaction in Dictyostelium Cells Revealed by GFP-based Strain Sensor and Validated Linear Spectral Unmixing
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基于 GFP 的应变传感器和经过验证的线性光谱分离揭示了盘基网柄菌细胞中肌球蛋白-肌动蛋白的相互作用
DOI：
10.1002/cyto.a.20900
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
and T. Q. P. Uyeda
中科院分区：
文献类型：
--
作者：
Iwai;S.;and T. Q. P. Uyeda
肌球蛋白是一种以肌动蛋白为基础的运动蛋白，参与广泛的细胞运动过程。虽然肌球蛋白-肌动蛋白相互作用的体外性质已被广泛研究，但体内相互作用仍然知之甚少。最近，我们开发了一种基于GFP的应变传感器，称为PriSSM（基于PRIM的应变传感器模块），通过使用邻近成像（PRIM）技术，检测两个GFP分子直接接触的光谱变化。利用PriSSM-肌球蛋白II融合蛋白，可以在网柄藻细胞中检测到肌球蛋白II与F-肌动蛋白之间的相互作用。在PriSSM的光谱测量中，为了将表达传感器蛋白的细胞的测量光谱分解为传感器和背景自体荧光的贡献，我们应用了线性光谱解混方法，该方法基于每个波长处的误差是独立的假设。然而，细胞自发荧光通常包括系统误差，因此解混过程可能导致有偏估计。在这里，为了验证我们的光谱解混程序，我们估计通过解混光谱获得的荧光比值中可能的最大误差，包括这种系统误差。这种估计提供了一个通用的标准，以验证通过线性解混光谱，包括串行相关的误差项所获得的结果。使用所提出的标准和PriSSM-肌球蛋白II融合蛋白，我们研究了在几种不同条件下网柄藻细胞中肌球蛋白II和F-肌动蛋白之间的相互作用。光谱结果以及表达蛋白质的细胞的显微镜观察结果表明，通过尾区形成肌球蛋白丝对F‐actin的结合只有轻微的影响，但对肌球蛋白II的皮质定位有显著影响。© 2010国际细胞计数促进学会
Myosin is an actin‐based motor protein that is involved in a wide range of cellular motile processes. Although in vitro properties of the myosin‐actin interaction have been extensively studied, the interaction in vivo remains poorly understood. Recently, we developed a GFP‐based strain sensor termed PriSSM (PRIM‐based strain sensor module), by using the proximity imaging (PRIM) technique, which detects spectral changes of two GFP molecules that are in direct contact. Using PriSSM‐myosin II fusion proteins, the interaction between myosin II and F‐actin can be detected inDictyosteliumcells. In the spectroscopic measurements of PriSSM, to decompose the measured spectra of the cells expressing the sensor proteins into the contributions from the sensor and the background autofluorescence, we applied the linear spectral unmixing approach, which was based on the assumption that the errors at each wavelength were independent. Cellular autofluorescence, however, often includes systematic errors, so that the unmixing procedures might lead to biased estimates. Here, to validate our spectral unmixing procedures, we estimate the possible maximum errors in the fluorescence ratio values that are obtained by unmixing spectra including such systematic errors. This estimation provided a general criterion to validate the results obtained by linear unmixing of spectra including serially correlated error terms. Using the proposed criterion and PriSSM‐myosin II fusion proteins, we examined the interaction between myosin II and F‐actin inDictyosteliumcells under several different conditions. The spectroscopic results, together with the microscopic observations of the cells expressing the proteins, suggest that the formation of myosin filaments through the tail region has only a slight effect on binding to F‐actin but has significant effects on the cortical localization of myosin II. © 2010 International Society for Advancement of Cytometry