Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster

Joseph Y Kim; Jie E Yang; Josephine W Mitchell; Lauren A English; Sihui Z Yang; Tanner Tenpas; Erik W Dent; Jill Wildonger; Elizabeth R Wright

doi:10.1093/micmic/ozad125

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster

Microsc Microanal. 2023 Dec 21;29(6):2127-2148. doi: 10.1093/micmic/ozad125.

Authors

Joseph Y Kim^{1

2}, Jie E Yang^{1

3

4}, Josephine W Mitchell⁵, Lauren A English⁶, Sihui Z Yang⁷, Tanner Tenpas⁸, Erik W Dent⁸, Jill Wildonger⁹, Elizabeth R Wright^{1

3

4

10}

Affiliations

¹ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
² Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
³ Cryo-Electron Microscopy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁴ Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁵ Department of Chemistry and Biochemistry, Kalamazoo College, Kalamazoo, MI 49006, USA.
⁶ Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁷ Department of Biology, Stanford University, Stanford, CA 94305, USA.
⁸ Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁹ Departments of Pediatrics and Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
¹⁰ Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53715, USA.

PMID: 37966978
DOI: 10.1093/micmic/ozad125

Abstract

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Keywords: Drosophila melanogaster; chemical fixation; cryo-electron microscopy (cryo-EM); cryo-electron tomography (cryo-ET); fluorescence light microscopy; membrane blebs; micropatterning; primary neurons; varicosity.

MeSH terms

Animals
Cryoelectron Microscopy / methods
Drosophila melanogaster*
Electron Microscope Tomography / methods
Neurons* / ultrastructure

Abstract

MeSH terms

Grants and funding