Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
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DOI：
10.1073/pnas.1607674113
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发表时间：
2016-10-18
影响因子：
11.1
通讯作者：
Tani, Tomomi
中科院分区：
文献类型：
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作者：
Mehta, Shalin B.;McQuilken, Molly;Tani, Tomomi
在活细胞中，定向和构象等秩序调控驱动着大多数分子组合的功能，但仍然难以通过空间和时间精确测量。建立了瞬时荧光偏振显微镜，可同时对活细胞中荧光团的位置和方向进行成像，具有单分子灵敏度，时间分辨率为100 ms。我们开发了图像采集和分析方法来跟踪与高阶分子组合相互作用的单个粒子。我们追踪了分子的位置和方向的波动，从一个荧光团的集合水平下降到单个荧光团。我们使用荧光标记的DNA和f -肌动蛋白在体外测试了我们的系统，其中偏振荧光的集合取向是已知的。然后，我们在迁移的人角质形成细胞的前沿跟踪了稀疏标记的f -肌动蛋白网络的方向，揭示了相对于f -肌动蛋白网络局部逆行流动的肌动蛋白丝的各向异性分布。此外，我们还分析了丝状真菌生长菌丝中septin- gfp分子的位置和取向。我们的数据表明，septin-GFP分子在接近结合位点350 nm的范围内发生位置波动，在接近束中心方向30度的范围内发生角度波动。通过报告分子在形成动态高阶结构时的位置和方向，我们的方法可以深入了解活细胞中纳米级分子如何形成微米级有序组装。
Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within similar to 350 nm of the binding site and angular fluctuations within similar to 30 degrees of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.