Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM).
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 10nm 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.
Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK.
Elizabeth M H
EM
Duke
Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
Minoo
M
Razi
Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
Anne
A
Weston
Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, 12489 Berlin, Germany.
Peter
P
Guttmann
Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, 12489 Berlin, Germany.
Stephan
S
Werner
Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, 12489 Berlin, Germany.
Katja
K
Henzler
Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Institute for Soft Matter and Functional Materials, 12489 Berlin, Germany.
Gerd
G
Schneider
Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
Sharon A
SA
Tooze
Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK. Electronic address: lucy.collinson@cancer.org.uk.
Lucy M
LM
Collinson
D000818:Animals; D002460:Cell Line; D011992:Endosomes / Q000648:ultrastructure*; D005453:Fluorescence; D006046:Gold / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D008322:Mammals / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*; D010588:Phagosomes / Q000648:ultrastructure*; D014056:Tomography, X-Ray / Q000379:methods*
D006046:Gold
Autophagosomes; Cryo-CLXM; Cryo-fluorescence; Cryo-soft X-ray tomography; Endosomes; Omegasomes