Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM).
Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM).
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DOI：
10.1016/j.ultramic.2013.10.006
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发表时间：
2014-08
期刊：
影响因子：
2.2
通讯作者：
Collinson, Lucy M.
中科院分区：
文献类型：
--
作者：
Duke, Elizabeth M. H.;Razi, Minoo;Weston, Anne;Guttmann, Peter;Werner, Stephan;Henzler, Katja;Schneider, Gerd;Tooze, Sharon A.;Collinson, Lucy M.
低温软x射线断层扫描（cryo-SXT）是一种强大的成像技术，可以从完整的、未染色的哺乳动物细胞中提取尽可能接近活体状态的超微结构信息。亚细胞细胞器包括细胞核、高尔基体和线粒体，由于与透射电子显微照片的相似性，仅通过形态学就可以识别。在这项研究中，我们使用冷冻- sxt成像核内体和自噬体，细胞器在电子显微镜样品制备过程中特别容易受到化学固定伪影的影响。我们使用了两种方法来识别这些隔间。对于早期和可循环的内体，我们使用了抗转铁蛋白受体抗体偶联到10 nm金颗粒上。对于外部应用标记无法访问的自噬体，我们开发了相关的冷冻荧光和冷冻sxt工作流程（cryo-CLXM）来定位GFP-LC3和RFP-Atg9。我们在家庭实验室中使用了一个独立的冷冻荧光分期来定位克隆的荧光团，然后在同步加速器上进行低温软x射线断层扫描来分析细胞的超微结构。我们绘制了内吞和自噬结构的三维超微结构，并发现了从内质网的“热点”产生的大粒体簇。因此，免疫金标记和cryo-CLXM可用于分析使用其他成像方式无法获得的细胞过程。我们使用冷冻软x射线断层成像完整的，未染色的哺乳动物细胞。内体用转铁蛋白受体的金标记物进行鉴定。一种新的相关冷冻荧光和冷冻sxt的工作流程被用于定位早期自噬体。内体，内质网和形成自噬体之间的相互作用在3D中绘制。在内质网的“热点”处形成多个大粒体。
Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities. We image whole, unstained mammalian cells using cryo-soft X-ray tomography. Endosomes are identified using a gold marker for the transferrin receptor. A new workflow for correlative cryo-fluorescence and cryo-SXT is used to locate early autophagosomes. Interactions between endosomes, endoplasmic reticulum and forming autophagosomes are mapped in 3D. Multiple omegasomes are shown to form at ‘hotspots’ on the endoplasmic reticulum.
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影响因子：
2.9
作者：
Gu, Weiwei;Etkin, Laurence D.;Larabell, Carolyn A.
通讯作者：
Larabell, Carolyn A.
影响因子：
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