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Correlative single-molecule localization microscopy and electron tomography reveals endosome nanoscale domains.
Traffic ( IF 4.5 ) Pub Date : 2019-08-01 , DOI: 10.1111/tra.12671
Christian Franke 1 , Urska Repnik 1 , Sandra Segeletz 1 , Nicolas Brouilly 1, 2 , Yannis Kalaidzidis 1, 3 , Jean-Marc Verbavatz 1, 4 , Marino Zerial 1
Affiliation  

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

中文翻译:

相关的单分子定位显微镜和电子断层扫描揭示了内体纳米级域。

许多细胞器,包括内体,显示出分隔成不同的功能域,但是,不能通过衍射极限光学显微镜分辨。单分子定位显微镜(SMLM)提供纳米级分辨率,但是当缺少超微结构背景时,数据解释通常是不确定的。将SMLM与电子显微镜(EM)相结合的相关光电子显微镜(CLEM)使细胞器的功能性亚结构域与它们在纳米分辨率下的超微结构相关。但是,对EM样品制备的特定要求和对荧光单分子光开关的要求是对立的。在这里,我们开发了一种新颖的superCLEM工作流程,该工作流程结合了三色SMLM(dSTORM和 PALM)和电子断层扫描,使用半薄的Tokuyasu解冻了冰冻切片。我们应用superCLEM方法直接可视化HeLa细胞内体的纳米级间隔。内化的,荧光标记的转铁蛋白和EGF被解析为同一内体中形态不同的结构域。我们发现,小的GTPase Rab5在早期内体球状部分的纳米域中组织。在功能上不同的内体亚小室中同时可视化几种蛋白质,证明了superCLEM在纳米级分辨率下将细胞器超微结构与其分子组织联系起来的潜力。荧光标记的转铁蛋白和EGF被解析为同一内体中形态不同的结构域。我们发现,小的GTPase Rab5在早期内体球状部分的纳米域中组织。同时可视化功能独特的内体亚小室中的几种蛋白质,证明了superCLEM在纳米级分辨率下将细胞器超微结构与其分子组织联系起来的潜力。荧光标记的转铁蛋白和EGF被解析为同一内体中形态不同的结构域。我们发现,小的GTPase Rab5在早期内体球状部分的纳米域中组织。在功能上不同的内体亚小室中同时可视化几种蛋白质,证明了superCLEM在纳米级分辨率下将细胞器超微结构与其分子组织联系起来的潜力。
更新日期:2019-11-01
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