Tracking movements of lipids and Thy1 molecules in the plasmalemma of living fibroblasts by fluorescence video microscopy with nanometer scale precision.
Tracking movements of lipids and Thy1 molecules in the plasmalemma of living fibroblasts by fluorescence video microscopy with nanometer scale precision.
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通过纳米级精度的荧光视频显微镜追踪活成纤维细胞质膜中脂质和 Thy1 分子的运动。
DOI：
10.1007/bf00236836
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Angelides,KJ
中科院分区：
文献类型：
--
作者：
Hicks,BW;Angelides,KJ
通过数字荧光显微照片中荧光乳胶微球（FS）质心的单粒子跟踪，监测了与N-生物素磷脂酰乙醇胺或糖基磷脂酰肌醇连接蛋白Thy 1结合的100 nm荧光乳胶微球（FS）在原代大鼠成纤维细胞质膜中的侧向扩散。硅增强靶相机被发现是上级优于慢扫描冷却CCD和增强行间转移CCD相机监测横向扩散的快速移动FS与纳米级的精度。为了估计最大跟踪精度，获得了包括固定到盖玻片的FS的120个图像的4秒序列。质心距原点的平均距离为7.5 ± 0.4 nm，没有质心距原点超过16 nm。然后使用SIT照相机来跟踪附着于成纤维细胞表面上的脂质和Thy 1分子的FS。脂质结合FS的横向扩散是不受约束的，系综平均扩散系数为0.80 × 10− 9 cm 2/sec。Thy 1结合的FS存在于两个流动人口，这两个表现出受限制的流动性。占总数61%的快速移动粒子群的系综扩散系数为6.1 × 10− 10 cm 2/sec，并且似乎仅限于平均长度约为700 nm的区域。缓慢移动的种群约占总数的39%，扩散系数为5.7 × 10− 12 cm 2/sec。这些结果表明，nanovid可以扩展到荧光显微镜的领域，并支持以前的研究表明，虽然至少一些脂质的横向流动性不限于小域的成纤维细胞质膜中的横向扩散的障碍，外周膜蛋白，这是唯一的脂质锚结合可以防止自由扩散。
The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to eitherN-biotinylphosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to slow scan cooled CCD and intensified interline transfer CCD cameras for monitoring lateral diffusion of rapidly moving FS with nanometer level precision. To estimate the maximum tracking precision, a 4 sec-sequence comprising 120 images of FS fixed to a cover glass was obtained. The mean distance of the centroids from the origin was 7.5 ± 0.4 nm, and no centroids were beyond 16 nm from the origin. The SIT camera was then used to track FS attached to lipids and Thy1 molecules on the surface of fibroblasts. The lateral diffusion of lipid-bound FS was unconstrained, and the ensemble averaged diffusion coefficient was 0.80 × 10−9cm2/sec. Thy1-bound FS existed in two mobility populations, both of which demonstrated constrained mobility. The rapidly moving population, comprising 61% of the total, had an ensemble diffusion coefficient of 6.1 × 10−10cm2/sec, and appeared to be restricted to domains with a mean length of about 700 nm. The slowly moving population, comprising about 39% of the total, had a diffusion coefficient of 5.7 × 10−12cm2/sec. These results demonstrate that nanovid can be extended to the realm of fluorescence microscopy and support previous studies indicating that while the lateral mobilities of at least some lipids are not constrained to small domains by barriers to lateral diffusion in the fibroblast plasmalemma, a peripheral membrane protein which is bound only by a lipid anchor can be prevented from diffusing freely.