Toplak Tim, Palmieri Benoit, Juanes-García Alba, Vicente-Manzanares Miguel, Grant Martin, Wiseman Paul W
Department of Physics, McGill University, Montréal, Québec, Canada.
Universidad Autonoma de Madrid School of Medicine/IIS-Princesa Diego de Leon, Madrid, Spain.
PLoS One. 2017 Oct 19;12(10):e0186058. doi: 10.1371/journal.pone.0186058. eCollection 2017.
We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales.
我们引入并使用多尺度小波成像（WIMS）来改进荧光相关光谱技术，以测量在多个长度尺度上发生的物理过程和特征。在本研究中，细胞图像的小波变换用于表征细胞和亚细胞水平（即粘着斑）的分子动力学。我们通过将WIMS应用于表达荧光标记粘着蛋白的迁移性骨肉瘤细胞的图像时间序列，展示了该技术的实用性，这使我们能够表征从光学分辨率尺度到粘着斑和整个细胞大小尺度的细胞不同组分。使用WIMS，我们测量了粘着斑数量、方向以及细胞边界在回缩和突出时的速度。我们还确定了单个粘着斑在组装、拆卸或伸长过程中的内部动力学。因此，正如之前所表明的，WIMS揭示了粘着斑数量与细胞突出区域面积密切相关，建立了突出大小与粘着动力学之间的相关性。我们还应用该技术来表征表达肌球蛋白IIB突变形式（1935D）的中国仓鼠卵巢细胞中粘着斑、肌动蛋白和肌球蛋白的行为，该突变形式显示出细丝稳定性降低并损害细胞前后极性。我们发现突变型和野生型形式在每个粘着斑极点处都有单独的肌动蛋白和肌球蛋白群体。然而，我们发现与表达野生型肌球蛋白IIB的细胞中这些群体保持静止相反，在突变情况下这些群体迅速向内相互移动。这两个系统获得的结果证明了WIMS有潜力揭示属于不同尺度的选定参数之间的新相关性。
