测量 3D 细胞突起中的肌动蛋白流。
Measuring actin flow in 3D cell protrusions.
机构信息
Department of Developmental and Cell Biology, Laboratory for Fluorescence Dynamics, University of California, Irvine, California.
出版信息
Biophys J. 2013 Oct 15;105(8):1746-55. doi: 10.1016/j.bpj.2013.07.057.
Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast.
肌动蛋白动力学对于确定细胞形状、张力和迁移非常重要。荧光斑点显微镜和时空图像相关光谱学等方法已被用于在二维空间中捕获细胞内高分辨率的肌动蛋白周转率动态。然而，由于分辨率较低和对比度较差，这些方法不能直接应用于 3D。在这里，我们提出通过快速光束振荡的纳米级精确成像和波动光谱技术相结合，以高时空分辨率捕获 3D 中的肌动蛋白流动。为了测量在 I 型胶原基质中培养的表达肌动蛋白-eGFP 的细胞中沿细胞突起的肌动蛋白流动，激光围绕突起旋转，并以垂直于突起的四叶草形状调制其轨迹。轨道也沿着突起轴的两个紧密间隔的位置交替。对来自这两个位置的荧光波动应用双体交叉相关函数，以捕获肌动蛋白的流动。对非运动细胞突起尖端的测量结果表明，在两个轨道位置处没有双体相关，表明 F-肌动蛋白束没有流动。然而，在一些突起中，双体相关方法揭示了在突起表面附近 F-肌动蛋白束的定向流动，流速在 1μm/min 的范围内，与使用荧光斑点显微镜在二维空间中的结果相当。此外，我们发现肌动蛋白流动速率与突起尖端的距离有关。在这些肌动蛋白运动过程中，我们还通过同时用反射探测来检测胶原纤维，观察到胶原的变形。快速光束振荡方法的纳米级精确成像与双体交叉相关函数方法的结合，以四叶草形轨迹实现，为在对比度较差的条件下定量捕获细胞迁移过程中的动态流动和蛋白质组织提供了一种方法。
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