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:10.1101/2023.07.10.548468

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster

bioRxiv [Preprint]. 2023 Jul 11:2023.07.10.548468. doi: 10.1101/2023.07.10.548468.

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.

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 been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3^rd 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.

Publication types

Preprint

Abstract

Publication types

Grants and funding