L6 cell PREM membrane images supporting "The structure and spontaneous curvature of clathrin lattices at the plasma membrane"

dataset

posted on 2021-03-22, 20:33 authored by Kem SochackiKem Sochacki, Bridgette Heine, Gideon Haber, John R. Jimah, Marco A. Alfonzo-Méndez, Aleah D. Roberts, Bijeta Prasai, Agila Somasundaram, Jenny E. Hinshaw, Justin W. Taraska

This dataset is part of a collection of platinum replica electron microscopy data (https://doi.org/10.25444/nhlbi.c.5334212) from the manuscript entitled:

"The structure and spontaneous curvature of clathrin lattices at the plasma membrane" by Sochacki et al. 2021 (https://doi.org/10.1016/j.devcel.2021.03.017).

Figure 1-2 of this manuscript compare the structure of clathrin in eight different cell types: PC12-GR5 (https://doi.org/10.25444/nhlbi.14195507; male rat adrenal gland, pheochromocytoma), 3T3-L1 (https://doi.org/10.25444/nhlbi.14195129; ATCC® CL-173™, male mouse embryo fibroblast), BS-C-1 (https://doi.org/10.25444/nhlbi.14195216; ATCC® CCL-26™, Cercopithecus aethiops, kidney epithelial, sex unknown), HeLa (https://doi.org/10.25444/nhlbi.14195480; ATCC® CCL-2™, female human cervix epithelial, adenocarcinoma), L6 (https://doi.org/10.25444/nhlbi.14195489; ATCC® CRL-1458™, male rat skeletal muscle, myoblast), MCF7 (https://doi.org/10.25444/nhlbi.14195504; ATCC® HTB-22™, female human mammary gland, epithelial, adenocarcinoma), RAW264.7 (https://doi.org/10.25444/nhlbi.14195537; ATCC® TIB-71™, male mouse, Abelson murine leukemia virus-induced tumor, monocyte/macrophage), and U-87 MG https://doi.org/10.25444/nhlbi.14195561; ATCC® HTB-14™, male human, brain, epithelial, likely glioblastoma).

All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm).

L6 (ATCC® CRL-1458™, male rat skeletal muscle, myoblast) cells were grown in DMEM media supplemented with 10% fetal bovine serum , Glutamax, and sodium pyruvate. They were grown on Poly-lysine coated coverslips for one day prior to unroofing. They were rinsed briefly with unroofing buffer (30mM HEPES, 70mM KCl, 5mM MgCl2, 3mM EGTA at pH 7.4) prior to unroofing. They were unroofed by brief sonication pulse in the presence of 0.5% paraformaldehyde or direct syringe flow in the presence of 2% paraformaldehyde. The coverslips were then moved immediately into unroofing buffer containing 2% glutaraldehyde.

Cells were kept in glutaraldehyde at 4 degs C until ready for staining and dehydration; less than two days. The cells were stained with 0.1% tannic acid for 20 minutes, rinsed, then stained with 0.1% uranyl acetate for 20 minutes and dehydrated slowly through a series of increasing ethanol concentration to 100% ethanol. The coverslips were then dried with critical point drying and rotary shadowed with platinum and carbon. The replicas were released from the coverslip with hydrofluoric acid and placed onto a TEM grid for imaging. TEM imaging of replicas was performed on a JEOL 1400 equipped with SerialEM freeware for montaging.