Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles.

Autor:	Tillu VA; The University of Queensland, Institute for Molecular Bioscience, Brisbane, Queensland 4072, Australia., Redpath GMI; EMBL Australia Node in Single Molecule Science , School of Medical Sciences, University of New South Wales Sydney, New South Wales 2033, Australia., Rae J; The University of Queensland, Institute for Molecular Bioscience, Brisbane, Queensland 4072, Australia., Ruan J; University of New South Wales Sydney, Electron Microscope Unit , Mark Wainwright Analytical Centre, Sydney, New South Wales 2033, Australia., Yao Y; University of New South Wales Sydney, Electron Microscope Unit , Mark Wainwright Analytical Centre, Sydney, New South Wales 2033, Australia., Cagigas ML; University of New South Wales Sydney, School of Medical Sciences , Kensington, Sydney, New South Wales 2033, Australia., Whan R; University of New South Wales Sydney, Katharina Gaus Light Microscopy Facility , Mark Wainwright Analytical Centre, Sydney, New South Wales 2033, Australia., Hardeman EC; University of New South Wales Sydney, School of Medical Sciences , Kensington, Sydney, New South Wales 2033, Australia., Gunning PW; University of New South Wales Sydney, School of Medical Sciences , Kensington, Sydney, New South Wales 2033, Australia., Ananthanarayanan V; EMBL Australia Node in Single Molecule Science , School of Medical Sciences, University of New South Wales Sydney, New South Wales 2033, Australia., Parton RG; The University of Queensland, Institute for Molecular Bioscience, Brisbane, Queensland 4072, Australia.; The University of Queensland, Centre for Microscopy and Microanalysis, Brisbane, Queensland 4072, Australia., Ariotti N; The University of Queensland, Institute for Molecular Bioscience, Brisbane, Queensland 4072, Australia.; University of New South Wales Sydney, Electron Microscope Unit , Mark Wainwright Analytical Centre, Sydney, New South Wales 2033, Australia.; University of New South Wales Sydney, School of Medical Sciences , Kensington, Sydney, New South Wales 2033, Australia.
Jazyk:	angličtina
Zdroj:	Journal of cell science [J Cell Sci] 2024 Oct 15; Vol. 137 (20). Date of Electronic Publication: 2024 Oct 30.
DOI:	10.1242/jcs.262163
Abstrakt:	Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day. Competing Interests: Competing interests The authors declare no competing or financial interests. (© 2024. Published by The Company of Biologists Ltd.)
Databáze:	MEDLINE
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