Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events
Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events
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DOI：
10.1016/j.bbamem.2015.03.001
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发表时间：
2015-06-01
影响因子：
3.4
通讯作者：
Safinya, Cyrus R.
中科院分区：
文献类型：
--
作者：
Majzoub, Ramsey N.;Chan, Chia-Ling;Safinya, Cyrus R.
已知内体截留是使用阳离子脂质体-NA纳米颗粒（NP）成功细胞质递送核酸（NA）的主要瓶颈。由于野生型内体的亚分辨率尺寸和短寿命，已经证明难以定量测量早期内体（EE）内NP的分布。在这项研究中，我们使用Rab 5-GFP，在质膜和早期内体之间循环的GTP酶大家族的成员，荧光标记早期内体。使用表达Rab 5-GFP的细胞的荧光显微镜和定量图像分析，我们发现在早期时间点（t < 1小时），只有一部分（约35%）的RGD标记的NP（其靶向细胞表面整合素）与野生型EE共定位，与NP的膜电荷密度无关。相比之下，GTP水解缺陷突变体Rab 5-Q79 L延长了EE的大小和寿命，产生巨大的早期内体（GEE），使我们能够解析和定位GEE腔内发现的单个NP。值得注意的是，发现几乎所有的细胞内NP都被捕获在GEE内，这意味着在早期时间点很少或没有逃逸。观察到的NP和野生型Rab 5的小程度共定位与Rab 5-GDP向质膜的再循环一致，并且不指示NP从EE逃逸。来自晚期内体/溶酶体。我们的研究还表明Rab 5-Q79 L可以用于稳健的成像测定，其允许直接可视化NP与早期内体的腔膜的相互作用。(C)2015 Elsevier B. V.版权所有。
Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanopartides (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t < 1 h), only a fraction (approximate to 35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP's membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs.Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes. (C) 2015 Elsevier B.V. All rights reserved.