DOI: 10.25444/nhlbi.14195480
title: HeLa cell membranes at 0,2,5,10,20 minutes post-unroofing: PREM membrane images supporting "The structure and spontaneous curvature of clathrin lattices at the plasma membrane"

description: 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). <br> 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).<br><br>Figure 3 of this manuscript shows what happens to clathrin when HeLa cells are unroofed (the top of the cell and the cytoplasm is removed) and the remaining cell membrane is incubated in a minimal medium lacking energy or protein for up to 20 minutes (0,2,5,10,20 min time points). Flat clathrin curves into almost spherical buds over this time period.<br><br>All images are tiled montages acquired on an AMT XR111 ccd at 15000 x magnification (pixel size 1.2 nm). <br><br>The idea behind these experiments was to unroof cells and visualize the clathrin on their membranes either in their natural state (0 min, HeLa_membranes_1-4) or after they have been allowed to sit in a minimal media lacking any protein or energy source (HeLa_Xmin_postunroofing). From these experiments, we saw that flat clathrin can curve without added subunits or energy.<br><br>HeLa (ATCC® CCL-2™, female human cervix epithelial, adenocarcinoma, female) 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.For spontaneous curvature:HeLa (ATCC® CCL-2™, female human cervix epithelial, adenocarcinoma) 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 pulsebor direct syringe flow with no fixative present. The coverslips were then moved immediately into fresh unroofing buffer for the given amount of time. After either 2,5,10,20 min uncubation period, the cells were placed in 2% glutaraldehyde in unroofing buffer.<br>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.