GoKawiilUnless otherwise noted, chemicals and buffer components were purchased from Sigma-Aldrich.
Cell culture
PtK2 cells (ATCC, CCL-56) were cultured in MEM (Gibco) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 1× non-essential amino acids (Gibco), and 1× antibiotic–antimycotic (Gibco) at 37 °C in 5% CO 2 . All cell lines were obtained from ATCC, which validates lines by STR profiling. We did not further validate them after purchase. Cells were monitored for mycoplasma contamination with the ATCC universal mycoplasma detection kit (a PCR-based assay) and tested negative. No commonly misidentified cell lines were used in this study.
Cell line generation
For generation of a doxycycline-inducible F-tractin–mScarlet and zyxin–LDO–mNeongreen PtK2 cell line, HEK293T (ATCC, CRL-3216) cells were co-transfected with lentiviral plasmids and packing plasmids (PMD2.G and PsPAX2) using Lipofectamine 2000 (Thermo Fisher Scientific). Six hours post-transfection, the culture medium was refreshed. Forty-eight hours post-transfection, the culture medium containing lentivirus was collected and filtered. Lentivirus infection with polybrene (5 μg ml−1) followed immediately after collection. Three types of lentivirus were added to PtK2 cells: F-tractin–mScarlet, zyxin–LDO–mNeongreen, and TetOn. Twenty-four hours post-infection, cells that stably integrated the lentiviral plasmids were selected with puromycin and blasticidin for one week.
Cellular cryo-ET grid preparation
C-flat 1.2/1.3 holey carbon Au 200-mesh grids (Electron Microscopy Sciences) were sputter coated with 22 nm of carbon and baked in a dry oven at 60 °C overnight. Grids were plasma cleaned and coated with 10 μg ml−1 fibronectin (Sigma, FC010) in DPBS for 1–3 h at 37 °C in a tissue culture incubator. Fibronectin-coated grids were washed with DPBS and stored in fresh PtK2 culture medium in a 35 mm tissue culture dish for immediate use.
Doxycycline-inducible F-tractin–mScarlet and zyxin–LDO–mNeongreen PtK2 cells were grown to 90% confluency before passaging. Cells were thoroughly trypsinized and filtered through a 0.45-μm filter to remove clumps. Thirty thousand cells were added to each 35 mm tissue culture dish containing the fibronectin-coated grids. Twenty-four hours later, the medium was replaced with fresh PtK2 medium containing 100 ng ml−1 of doxycycline to induce expression of F-tractin–mScarlet and zyxin–LDO–mNeongreen. The next day, grids were prepared for plunge freezing. Grids for the majority of tomograms (presented in Fig. 1b and Extended Data Fig. 1c,d) were treated with 1 µg ml−1 Rho Activator II (Cytoskeleton, CN03) for 3.5 h, while the grid for the tomogram presented in Extended Data Fig. 1b (tomogram 11 in Extended Data Fig. 1d) was untreated. The grids were then washed with DPBS and loaded onto a Leica EM-GP plunge freezer operating at 25 °C. Three microlitres of DPBS was added to the front of the grid, followed by blotting from the back using a Whatman no. 5 filter paper for 7 s, then flash frozen in liquid ethane.
Cryo-fluorescence imaging
Grids were mounted on a Leica Cryo-CLEM microscope. Epifluorescence microscopy was performed using a 50× 0.9 NA air objective at −180 °C. z-Stacks of images were captured at a depth of 16 bit. Image acquisition was performed using LAS X software (Leica). Images were post-processed with THUNDER (Leica) to denoise, then maximum intensity projected.
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