Patterned two-photon photoactivation illuminates spatial reorganization in live cells.
Patterned two-photon photoactivation illuminates spatial reorganization in live cells.
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DOI：
10.1021/jp108295s
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发表时间：
2011-04-28
影响因子：
2.9
通讯作者：
Groves, Jay T.
中科院分区：
文献类型：
--
作者：
Smith, Adam W.;Smoligovets, Alexander A.;Groves, Jay T.
可光激活的荧光蛋白提供了以高空间和时间分辨率光学标记和跟踪处于明亮状态的分子的位置的可能性。自从绿色荧光蛋白的可光激活变体(PaGFP)的开发和PaGFP的双光子激活被证明以来，已经出现了几个图案化光激活的报道。然而，到目前为止，还没有开发出量化光激活种群的空间重组的方法。在这里，我们报告了使用奇异值分解(SVD)来跟踪光活化后荧光团随时间的分布。该方法被用来描述原代小鼠T细胞中活细胞肌动蛋白的细胞骨架行为，其中一个动态细胞骨架负责响应抗原肽识别而对膜蛋白进行重组。该方法还用于观察永生化猴肾(Cos-7)细胞，细胞骨架较稳定。将PaGFP与Utrin的F-肌动蛋白结合域(UtrCH)融合，分别转染两种细胞。通过将图像转换为径向分布分布的时间序列来分析具有圆形图案的一对电流计扫描镜的样品中的光活化图案。前两个SVD分量状态很好地描述了剖面的时间演化过程。对于T细胞，我们发现肌动蛋白细丝具有高度的流动性。在1−2 S时间尺度上观察到从光激活区向内的转运，这与逆行循环相一致。对于Cos-7细胞，我们发现肌动蛋白相对稳定，并没有像静止的成纤维细胞预期的那样经历显著的向心流。图案化光激活和奇异值分解分析的结合提供了一种独特的方法来测量活细胞内的空间再分布动态。
Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value decomposition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1−2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.
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影响因子：
3.4
作者：
DeMond, Andrew L.;Mossman, Kaspar D.;Groves, Jay T.
通讯作者：
Groves, Jay T.
影响因子：
3
作者：
Nguyen, Quoc-Thang;Tsai, Philbert S.;Kleinfeld, David
通讯作者：
Kleinfeld, David
影响因子：
48
作者：
Riedl, Julia;Crevenna, Alvaro H.;Wedlich-Soldner, Roland
通讯作者：
Wedlich-Soldner, Roland
DOI：
10.1073/pnas.0902621106
发表时间：
2009-08-04
影响因子：
11.1
作者：
Hartman, Nina C.;Nye, Jeffrey A.;Groves, Jay T.
通讯作者：
Groves, Jay T.
影响因子：
3.5
作者：
Post, JN;Lidke, KA;Arndt-Jovin, DJ
通讯作者：
Arndt-Jovin, DJ