Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: A fluorescent speckle microscopy study
Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: A fluorescent speckle microscopy study
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DOI：
10.1016/s0006-3495(03)74564-0
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发表时间：
2003-08-01
影响因子：
3.4
通讯作者：
Danuser, G
中科院分区：
文献类型：
--
作者：
Vallotton, P;Ponti, A;Danuser, G
荧光散斑显微镜（FSM）正在成为在体内分析聚合物组件，如细胞骨架的动力学的首选技术。通过这种方法产生的大量数据需要计算方法来恢复感兴趣的量;即，聚合和解聚活动以及细胞骨架随时间推移所经历的运动。实现这一目标的尝试已受到阻碍的有限的信号-噪声比的典型FSM数据，由不断出现和消失的斑点，由于聚合物周转，并通过存在的流动奇异性特征的许多细胞骨架聚合物组件。为了解决这些问题，我们提出了一种基于粒子的方法来跟踪荧光散斑的时间推移FSM图像序列，基于运筹学和图论的思想。我们的软件提供了连续帧之间的数千个散斑的位移，考虑到散斑可能出现和消失。在这篇文章中，我们利用这些信息来恢复散斑流场。首先，该软件进行了测试合成数据，以验证我们的方法。然后，我们将其应用到映射丝状肌动蛋白逆行流动在迁移蝾螈肺上皮细胞的前缘。我们的研究结果证实了先前发表的记波分析和手动跟踪此类FSM数据的结果，并说明了自动跟踪生成完整和定量流量测量的能力。第三，我们分析微管极向通量有丝分裂中期纺锤体组装在非洲爪蟾卵提取物，在这个系统中的微管组件的动态带来新的见解。
Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.