High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells
High-Speed Imaging of Rab Family Small GTPases Reveals Rare Events in Nanoparticle Trafficking in Living Cells
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DOI：
10.1021/nn204448x
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发表时间：
2012-02-01
期刊：
影响因子：
17.1
通讯作者：
Dawson, Kenneth A.
中科院分区：
文献类型：
--
作者：
Sandin, Peter;Fitzpatrick, Laurence W.;Dawson, Kenneth A.
尽管纳米材料在诊断和治疗中的应用越来越多，但研究纳米颗粒与活细胞中亚细胞结构相互作用的方法仍然相对不发达。在这里，我们描述了一个强大的和定量的方法，允许精确跟踪所有的细胞相关的纳米粒子，因为它们通过在活细胞内吞隔室。使用快速的3D活细胞共聚焦荧光显微镜，结合标记各种内吞膜的小GTP酶的瞬时过表达，我们的研究揭示了纳米颗粒通过早期内体到晚期内体和溶酶体的运输动力学。我们发现，内化后，40 nm的聚苯乙烯纳米粒子首先通过早期的核内体中间装饰Rab 5，但这些纳米粒子迅速转移到晚期核内体和最终溶酶体标记Rab 9和Rab 7，分别。直径为100 nm的较大纳米颗粒也到达酸性Rab 9-和Rab 7-阳性隔室，尽管与较小的40 nm纳米颗粒相比速率较慢。我们的工作还揭示了相对较少的纳米颗粒能够进入内吞再循环途径，这是通过缺乏与Rab 11的显着共定位来判断的。最后，我们证明，这种定量方法是足够敏感的，能够检测到罕见的事件在纳米粒子的运输，特别是存在的纳米粒子在Rab 1A标记的结构，从而揭示了广泛的细胞内相互作用的纳米粒子和细胞内环境。
Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments In a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as Judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events In nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment.