Em pact2

We followed the procedure described previously by Hall et al [28 (link)]. Yeast cells were collected from exponential growth cultures as follows: 10⁶ cells/ml of WT, GOA31 and GOA32 were inoculated respectively into 10-ml of pre-warmed YPD medium and grown for 6 h (WT, GOA32) or 8 h for GOA31 at 37°C. Cells were immediately frozen in liquid nitrogen under high pressure using a Leica EM PACT2 (Leica Microsystems) and transferred by Leica EM AFS2. Prior to TEM analysis, samples were sequentially warmed to −30°C in acetone/OsO₄ for 8h, then to 20°C for 3 h in acetone, and embedded in increasing amounts of Spurr (epoxy) resin for 24 h. Ultra-thin sections (100 nm) were stained with uranyl acetate and lead citrate and imaged with a Philips CM10 transmission electron microscope (FEI Life Sciences, Hillsboro, Oregon). To better evaluate the length of the cell wall and outer wall fibrils, 10 cells of each strain were randomly selected and 10 measurements from different sections of each cell were obtained using the GIMP version 2.8. Data are presented as the mean ± standard deviation of 100 measurements. Data were analyzed by One-Way ANOVA followed by Bonferroni’s test with a 99% confidence level and t-test using GraphPad Prism version 5.01.

High-pressure freezing (HPS)/freeze substitution (FS) was employed to ensure preservation of cellular structures, AgNP composition and ion distribution. Samples were placed in specimen carriers with 200 μm deep cavities, mounted in a 20% (wt/vol) solution of bovine serum albumin (Sigma) in αMEM, and frozen using a Leica EM PACT 2 (Leica Microsystems) high-pressure freezer. FS was performed in a Leica electron microscope AFS2 freeze substitution device using an anhydrous solution of acetone containing a 3% (vol/vol) glutaldehyde, 1% (wt/vol) osmium tetraoxide, and 0.5% (wt/vol) uranyl acetate at −90 °C for 8 h. Samples were then gradually warmed to 0 °C at 5 °C/h, washed twice in acetone, brought to room temperature, and infiltrated with resin, as described in the TEM methodology section.

High-pressure freezing (HPF) and freeze substitution sample preparation were performed at the Microscopy and Histology Facility at the University of Aberdeen, as described previously (60 (link)). Briefly, cells were washed 3 times in water, and then HPF was performed in a Leica EM PACT 2 (Leica Microsystems, Milton Keynes, UK). Freeze substitution was performed in a Leica AFS 2 in acetone-1% OsO₄. Samples were transferred to epoxy resin, and ultrathin sections were cut, stained with uranyl acetate and lead citrate, and imaged with an FEI Tecnai T12 Spirit TEM with Gatan camera. Cell walls were measured via FIJI with 5 measurements averaged per cell (n > 20 cells per condition).

The SN152 and hgt1Δ/Δ strains were grown in YP 0.1% glucose at 30°C. One drop of each overnight culture (40 μl) was processed for high pressure freezing cryofixation by Leica EM PACT 2 (Leica Microsystems, Milton Keynes, United Kingdom), according to the guidelines of the Microscopy facility of the University of Aberdeen (Lopes-Bezerra et al., 2018 (link)).