May 08, 2025
EM grid seeding for cryo-FIB milling
- kevin rose1
- 1UC Berkeley
Protocol Citation: kevin rose 2025. EM grid seeding for cryo-FIB milling. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpde65gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 23, 2025
Last Modified: May 08, 2025
Protocol Integer ID: 124861
Abstract
Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson’s disease by mutations in PINK1 and PRKN. Application of in situ cryo-electron tomography (cryo-ET) made it possible to visualize the consequences of mitochondrial depolarization at higher resolution than heretofore attainable. Parkin-expressing U2OS cells were treated with the depolarizing agents oligomycin and antimycin A (OA), subjected to cryo-FIB milling, and mitochondrial structure was characterized by in situ cryo-ET. Phagophores were visualized in association with mitochondrial fragments. Bridge-like lipid transporter (BLTP) densities potentially corresponding to ATG2A were seen connected to mitophagic phagophores. Mitochondria in OA-treated cells were fragmented and devoid of matrix calcium phosphate crystals. The intermembrane gap of cristae was narrowed and the intermembrane volume reduced, and some fragments were devoid of cristae. A subpopulation of ATP synthases re-localized from cristae to the inner boundary membrane (IBM) apposed to the outer membrane (OMM). The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined in situ by sub-tomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation is enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future in situ dissection of Parkin-dependent mitophagy.
EM grid preparation and cell seeding
EM grid preparation and cell seeding
Glow discharge carbon coated gold quantifoil R2-2 200 mesh EM grids at 15mA for 30 seconds
In a laminar flow hood, float EM grids carbon side down on 30 uL drops of 0.01% poly-L-lysine solution. Simultaneously, add 100 uL of 0.01% poly-L-lysine solution to each well of an 8-chamber plate with removable wells. Incubate both under UV for 30min at room temperature.
After incubation, split cells using 0.25% trypsin and resuspend to 100,000 cells/mL in complete media
Before seeding cells, remove poly-L-lysine solution from the 8 chamber plate and replace with 200uL complete media. Remove grids from atop the drops of poly-L-lysine and rinse them 3 times in complete media before placing into the bottoms of each of the wells of the 8 chamber plate.
Add 200 uL of cell solution to each well such that 20,000 cells per EM grid are seeded. Let cells recover overnight before treating and freezing.