Journal: bioRxiv

Article Title: Direct synthesis of EM-visible gold nanoparticles on genetically encoded tags for single-molecule visualization in cells

doi: 10.1101/520999

Figure Lengend Snippet: (a) The expression patterns of Ost4-GFP, Ost4-GFP-MTn, Nup124-GFP, Nup124-GFP-MTn, Sad1-GFP, and Sad1-GFP-MTn in S. pombe observed by live-cell fluorescence imaging (scale bar, 2 μm). The fluorescence images of GFP showed that the fusion proteins were correctly localized to expected subcellular locations: membranes of cortical ER and NE for Ost4, NPs for Nup124, the SPB for Sad1. (b) A cartoon shows the NE and cortical ER, NPs, and the SPB in a S. pombe cell. The regions of interest are labeled with numbers (I-VI) as references in other figures. (c) Procedures for the preparation of S. pombe cells for synthesizing AuNPs, three schemes labeled as 1 (“Cold MeOH”), 2 (“Oxidization + Chemical Fixation + Permeabilization”), 3 (“HPF/FSF + Oxidization + Chemical Fixation + Rehydration”) respectively, the Scheme 2 consists of four different sub-schemes (2a-2d), see the for details. (d) Synthesis of AuNPs in S. pombe cells treated by the “Cold MeOH” protocol with the optimized ANSM-based AuNPs synthesis protocol obtained with E. coli cells , the upper row shows the S. pombe cell suspension after AuNPs synthesis and the lower row shows the corresponding centrifuged cell pellets, apparently the colors of those MTn tags expressed cells are much darker than the corresponding control cells. (e) A typical EM image showing the unprecedented high labelling densities of AuNPs distributed on the cortical ER (II) and ER stacks (III) in a cell expressing Ost4-GFP-MTn prepared with the Scheme 1. (f) EM image showing better membranes preservation of an Ost4-GFP-MTn expressed cell treated with Scheme 2c than that in (e), the AuNPs could be clearly localized to outer membranes of the NE although the labeling densities of AuNPs reduced after the PFA fixation. (g) EM image showing the linear distributions of AuNPs along the membranes of cortical ER and ER stacks in a cell expressing Ost4-GFP-MTn treated with the Scheme 2a (with cold MeOH permeabilization), the structural preservation is better than that in (e) but with reduced labelling densities of AuNPs after the GA fixation. (h-i) EM images showing the AuNPs distributed along the membranes of NE (I) and ERs (II, IV) in cells expressing Ost4-GFP-MTn treated with Scheme 2b, lack of AuNPs in the nuclear pores (NPs), only a few background AuNPs scattered in cytosols, nuclei and mitochondria (M). (j) high densities of AuNPs formed on ER stacks in cells expressing Ost4-GFP-MTα treated with Scheme 2b (with 0.1% Triton X-100 2 min permeabilization). (k) A lot of AuNPs gathered in the SPB (among a dark density region), the specimen was prepared with Scheme 2a. (l) AuNPs synthesized in a cell expressing Nup124-GFP-MTn with Scheme 3a, grouped AuNPs formed on the NP (dark regions) with a regular interval along the NE. (m) Statistical analysis (one-way ANOVA followed by Brown-Forsythe test) of the AuNPs densities in ER (including NE), cytosol (Cyto), Nucleus (Nu) and mitochondrion (Mito) based on 20 EM images of 90 nm thick sections of S. pombe cells expressing Ost4-GFP-MTn (see Supplementary Chart 1 ).

Article Snippet: Cell pellet was then used to fill the 0.1 mm or 0.05 mm deep well of an aluminum carrier (Engineering Office of M. Wohlwend GmbH), quickly capped with a 3 mm × 0.16 mm sapphire disc, and loaded into a customized 3.05 mm × 0.66 mm HPF specimen holder for rapid freezing at ~2050 bar pressure for 340-360 milliseconds with a Wohlwend HPF Compact-01 high pressure freezing machine.

Techniques: Expressing, Fluorescence, Imaging, Labeling, Suspension, Control, Preserving, Synthesized