Expansion Microscopy on Saccharomyces cerevisiae Artemis G. Korovesi1*, Leonor Morgado1*, Marco Fumasoni1, Ricardo Henriques1,2, Hannah S. Heil1§, Mario Del Rosario1§ 1Instituto Gulbenkian de Ciência, Oeiras, Portugal 2MRC-Laboratory for Molecular Cell Biology. University College London, London, United Kingdom §To whom correspondence should be addressed: hsheil@igc.gulbenkian.pt; mrosario@igc.gulbenkian.pt *These authors contributed equally. Abstract The unicellular eukaryote Saccharomyces cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts is a crucial step to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae. 5/4/2022 - Open Access ----!@#$NewPage!@#$---- Figure 1. Yeast expansion microscopy overview a) Scheme of expansion microscopy protocol workflow. b) Maximum intensity projection of DIC/DAPI composite image of yeast, acquired with a Nikon HCS using a 100x/1.45 oil objective. c) Maximum intensity projection of fluorescence image of post-expanded yeast, acquired with a Zeiss LSM 980 using a 40x/1.1 water objective and airyscan detection. Signals of DAPI and DIC/NHS-Ester are represented in magenta and grey, respectively. Scale bars: 5 µm. Samples were assessed by manual segmentation and measurement of the area and circularity between expanded (n=13) and non-expanded (n=27) specimens. The average expansion factor across all experiments was 3.52 ± 0.42 (mean ± standard deviation) while the circularity was retained within the level 20% of the population’s standard deviation. Description The unicellular eukaryote S. cerevisiae represents an invaluable resource for the study of basic eukaryotic cellular and molecular processes. The combination of a high genetic amenability, numerous genetic tools, and vast genomic resources makes it one of the most versatile model organisms, employed in a wide range of basic research disciplines. However, its small size compared to other eukaryotic organisms has limited its use for the study of sub-cellular structures. The diameter of unbudded yeast cells ranges approximately between 4 μm in haploids and 6 μm in diploids (Milo and Phillips 2015), complicating conventional diffraction-limited light microscopy approaches. The last decade witnessed the rise of super-resolution (SR) techniques that enable sub-diffraction resolution fluorescence imaging of cellular structures. Expansion microscopy (ExM), for instance, allows for increasing imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel and is then expanded isotropically in water (Wassie et al. 2019). By doing so, fluorescently-labelled structures that previously would be too small or too close together to be distinguished can be resolved in 3 dimensions. The increased z-resolution can be exploited by z-sectioning, using confocal or light-sheet microscopy. In addition, ExM can also be used in combination with other super-resolution methods, such as airyscan confocal microscopy and Structured Illumination Microscopy (SIM), further enhancing resolution. ExM holds great potential to study the cellular structures of yeast, especially when paired with pan-labelling techniques to visualise total protein content inside cells. ExM protocols present a robust tool to study cellular organelles, obtaining super- resolution results with the advantage of employing inexpensive and straightforward equipment compared to other techniques that require more complex and expensive setups, such as optical SR approaches and electron microscopy. ExM can be applied in various organisms and cellular models, offering outstanding outcomes with expansion factors ranging from 4x to 20x (Faulkner et al. 2020, Truckenbrodt et al. 2018). However, certain cellular organelles are resistant to these processes, such as the cell wall of fungi and Gram-positive bacteria. These organisms present a cell wall composed of peptidoglycans or glycoproteins that are highly resistant to denaturation and conventional digestion processes. The digestion of the cell wall and the maintenance of the structure of the resulting protoplasts are crucial steps to achieve isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available (Götz et al. 2020). In particular, only one study describes an ExM protocol applied to S. cerevisiae, showing different yeast structures in high resolution (Chen et al. 2021). Here we describe an adapted and extended ExM protocol for S. cerevisiae (Figure 1) with a final expansion factor of ~3.5x. The major difference regarding to the protocol of Chen et al., is the staining strategy. While the previously published protocol is based on immunolabeling to visualise and super-resolve specific target proteins, here, samples are stained with DAPI and the pan-labelling reagent NHS-Ester BODIPY FL. This allows to provide an overview of the ultrastructural context of the whole cells. However, both protocols complement each other and could be combined, in principle. Here, we have compiled detailed step-by-step instructions for ExM to visualise the ultrastructure of S. cerevisiae, supported by supplementary video documentation (Supplementary Video 1). Briefly, the first day begins with the preparation and fixation of yeast cells, which includes the digestion of the cell wall with zymolyase. The generated protoplasts are mounted on the coverslip, and linking takes place overnight, during which linker molecules will attach to the sample’s proteins, acting as anchors for the next step. On the second day, the first step is gelation, where the sample is embedded in a monomer solution that polymerases and forms a matrix to which the anchor molecules will be connected. This is followed by the denaturation step, where the sample structures are disintegrated. The samples are then left expanding overnight in water, swelling the polymerised gel, which will push the anchored molecules apart in an isotropic manner. Finally, on the third day, staining with DAPI and NHS-Ester takes place. Samples are left again expanding overnight, and they can be imaged from the next day onwards. Assessing the expansion factor and specimen cellular integrity following sample preparation is a critical step that requires consideration. During sample expansion, the denaturation/digestion steps facilitate isotropic expansion of the sample but different organelle structures might possess specific requirements that need to be identified during the experimental planning 5/4/2022 - Open Access ----!@#$NewPage!@#$---- phase of the experiment. The simplest solution to assess specimen integrity and expansion factor is to acquire microscopy data of the same field of view and specimen before and after expansion. This process will allow the user to detect immediate changes in the sample but carries the complication of being difficult to execute since finding the same sample, particularly in yeast, can be difficult and time-consuming. Other options, such as the one included in this protocol, image multiple expanded and non-expanded cells and compare their features (area and circularity) as means to assess expansion (average circularity 0.892 ± 0.063 on expanded samples (mean ± standard deviation, n=13) and 0.906 ± 0.035 on non-expanded ones (n=27)). Individual organelle expansion should be obtained via specific labelling of the structure and determining before and after expansion sizes and compare it to the expansion factor of the whole cell. Methods Preparation of stock solutions: Sorbitol buffer: ● 1.2 M sorbitol solution in 0.1 M KH2PO4. Linking solution: ● 0.1 mg/mL acryloyl X-SE in PBS. Store in aliquots at -20 °C. Monomer solution: Stocks: ● 38% sodium acrylate (w/w, diluted with ddH2O): 25 g in 65.79 mL, store at -20 °C. ● 40% acrylamide stock: 20 g in 50 mL ddH2O, store at -20°C. ● 2% N,N’-Methylenebisacrylamide : 0.2 g in 10 mL ddH2O, store at -20 °C. Reagent Final concentration PBS 1x Sodium acrylate* 19 g/100 mL Acrylamide 10 g/100 mL N,N’-Methylenebisacrylamide 0.1 g/100 mL Store in 493 µL aliquots at -20 °C. *Sodium acrylate is provided with variable quality levels. One should test it before using it: the reagent should be fully dissolved and not show impurities in the solution, in the form of precipitates or discolouring. Denaturation buffer: Reagent Final concentration ddH2O - SDS 200 mM NaCl 200 mM Tris* 50 mM 5/4/2022 - Open Access ----!@#$NewPage!@#$---- Store in falcon tubes at -20 °C. *Adjust pH to 9. YPD Media: 1. Dissolve 5 g of yeast extract and 10 g of peptone in 375 mL of ddH2O by manually stirring the flask and autoclave the solution. 2. Add 100 mL of ddH2O, 20 mL of autoclaved 50% Glucose (Dextrose) and 5 mL 1% Adenine + 1% Tryptophan. Coverslip cleaning Notes: i) This procedure should be performed under a fume hood, and one should use safety gloves when handling chloroform. ii) The chloroform and NaOH solutions can be reused. iii) The preparation of NaOH is an exothermic reaction, thus the NaOH stock solution should be prepared on an ice bath. iv) Other coverslip cleaning protocols are available, such as a chloroform- free protocol (Pereira et al. 2015). 1. Arrange single coverslips in a coverslip rack. 2. Place a glass container in an ultrasonic bath and fill the bath with water. 3. Place the rack in the glass container. 4. Fill the glass container with chloroform and sonicate for 1 hour. 5. Remove the rack with a tweezers/forceps and place it in an empty glass container. The coverslips will dry in a few minutes as the chloroform evaporates quickly. 6. Pour the remaining chloroform back into the bottle through a funnel. 7. Reinstall the rack in the glass container in the ultrasonic bath. 8. Fill the glass container with a 5 M NaOH solution and sonicate for 1 hour. 9. Pass the coverslips into a beaker with ddH2O and transfer the NaOH solution back into the bottle. 10. Wash the coverslips three times in a volume of 500 mL fresh ddH2O. 11. Pre-dry the coverslips with a nitrogen or filtered air gun and then place them in a drying cabinet until they are completely dried. 12. Store in 100 % ethanol in a glass Petri dish. Coverslip poly-L-lysine coating 1. Dry the previously cleaned coverslips. 2. Place the coverslips in a clean plastic plate, for example, in a 6-well plate. 3. Add 300 µL of 0.1 mg/mL poly-L-lysine on each coverslip and incubate at room temperature (RT) for 10 minutes. 4. Add water to each well until the coverslip is covered and incubate at RT for 5 minutes. 5. Dry the coverslips. 6. Store coverslips on a rack at 4 °C until needed for cell/gel mounting. Day 1 Yeast cells cultivation and fixation The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used in this protocol. 1. Culture yeast cells in the conditions required to assess the relevant biological question. (In our case, we incubate yeast cells in fresh YPD media supplemented with adenine and tryptophan at 30 °C and 220 rpm of agitation until the culture growth reaches the exponential phase). 2. Count yeast cells with a hemocytometer and calculate the volume required to collect 6x107 cells. 3. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube. 5/4/2022 - Open Access ----!@#$NewPage!@#$---- 4. Discard the supernatant and resuspend in 3 mL of PBS 1X. 5. Centrifuge cells at 4,000 rpm for 2 minutes in a Falcon tube. 6. Resuspend the cells in 3 mL of 4% paraformaldehyde (PFA), and fix them at 4 °C for 30 minutes in a multi-rotator. 7. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes. 8. Discard the supernatant and resuspend in 3 mL of PBS 1X. 9. Centrifuge the fixed yeast cells at 4,000 rpm for 2 minutes. 10. Discard the supernatant and resuspend in 6 mL of sorbitol buffer. Cell wall digestion 1. Add 0.3 μL of zymolyase (5 U/μL) to 200 μL of fixed cells solution (2x106 cells) for a final concentration of 0.0075 U/ µL and incubate the reaction at 30 °C for 3 minutes*. 2. Place the samples on ice to block the enzymatic reaction. * The length of the reaction provided is optimised for a population of exponentially growing cells. Samples collected in other conditions may require an adjusted treatment to optimise the cell wall digestion. Mounting and fixating the cells on the coverslip 1. Place the poly-L-lysine coated coverslips on a 6-well plate. 2. Add 200 µL of treated cells to each coverslip and incubate them at RT for 10 minutes to allow the sample to sediment. 3. Under a fume hood, add enough of the 4% PFA/1% Glutaraldehyde (GA) fixative to cover the sample and incubate at RT for 10 minutes. 4. Wash 3x with PBS. 5. Inspect the sample under a microscope to confirm that the cells are attached (i.e., cells are immobile and not floating) and if the majority of cells present no cell wall. 6. If needed to calculate the cell expansion factor, image the cells at this time point to measure dimensions such as area or diameter of cells before expansion. Linking 1. Remove PBS, add 1 mL of 0.1 mg/mL acryloyl X-SE solution in PBS to each well and incubate for 12 hours (overnight) at RT. Day 2 Gelation Note: Proceed quickly through steps 3 to 5, as after the gelation solution is prepared, it will polymerise in minutes. For this reason, it is recommended to proceed with these steps with a maximum of four samples each time. Reagents and slides are maintained on ice to prevent premature polymerisation. 1. Cool the reagents (monomer solution, TEMED, and APS) and the gelation chamber slides (see supplementary figure 1) on ice for at least 10 minutes. 2. Remove the excess solution from the coverslip with a lint-free wipe and place it next to the slide on which it will be mounted (Note: the coverslip side with the sample should be facing up). 3. Prepare the gelation solution: Add first the TEMED to the monomer solution, then APS and vortex. (Note: it is important to follow this order, as when APS is added the polymerisation reaction starts). Volume Reagents Stock concentration Final concentration 5/4/2022 - Open Access ----!@#$NewPage!@#$---- 493 µL Monomer solution - - 5 µL APS 50% 0.5% 2.5 µL TEMED 99% 0.5% 500 µL Final Volume - - 4. Add 120 µL of gelation solution in each gelation chamber, place the coverslip on top, with the side with the sample facing the solution, and gently press it. Carefully remove excess with a lint-free wipe. 5. Keep the slides for 5 more minutes on ice. 6. Incubate for 1 hour at 37 °C in a humidified chamber. Denaturation 1. Preheat a heating block to 95 °C. 2. Thaw the denaturation buffer in a warm water bath and proceed only when the solution is clear. 3. Carefully remove the coverslip from the slide and transfer the polymerised gel to a new 6-well plate (Note: sometimes the coverslip comes attached to the gel, and it can be transferred along). 4. Measure the width and length of the polymerised gel before expansion. 5. Add 2 mL of denaturation buffer to each well and incubate for 15 minutes at RT. 6. Prepare an 1.5 mL Eppendorf tube for each sample and add 1 mL of fresh denaturation buffer in each of them. 7. Transfer the gel into the Eppendorf tube with a metal spatula and incubate at 95 °C for 90 minutes. Expansion 1. Carefully pour the gel from the Eppendorf tube to a big clean plastic plate (e.g., Petri dish with a diameter of at least 60 mm) and remove the excess of denaturation buffer solution. 2. Add ddH2O to Petri dish until the gel is completely submerged. 3. Change ddH2O 2x after 30 minutes. 4. Expand in ddH2O overnight at RT. Day 3 NHS and DAPI staining 1. Remove ddH2O. 2. Measure the width and length of the gel after expansion. Divide these values by the ones measured before expansion to calculate the macro-expansion factor, which should be approximately 4x. 3. Cut gel in small pieces using the custom cutter shape (see supplementary figure 2) and a razor blade. 4. Select the pieces to be stained, cut the correct corner for reference of the gel orientation (see supplementary figure 1), and transfer them to a new 6-well plate with a metal spatula. 5. Wash 2x for 15 minutes with PBS. (Note: the gels will shrink by 50%.) 6. Prepare 10 µg/mL NHS Ester and 300 µM DAPI in PBS (Note: confirm that the NHS and DAPI solution is completely homogenised). 7. Add 2 mL to each well, wrap it in aluminium foil and incubate for 90 minutes at RT on a rocking platform. 8. Wash 3x for 15 minutes with PBS+Tween-20 0.1%. 5/4/2022 - Open Access ----!@#$NewPage!@#$---- 9. Add ddH2O to each well until the gel is completely submerged. 10. Change ddH2O 2x after 30 minutes. 11. Expand in ddH2O overnight at RT. Imaging day Immobilisation of the gel 1. Place a poly-L-Lysine coated coverslip in an Attofluor™ Cell Chamber (or another comparable mounting device). 2. Carefully dry a piece of gel on a spatula using lint-free wipes and transfer it to the coverslip, with the sample facing the coverslip (use cut corner as reference of gel orientation). 3. Incubate at RT for 7 minutes. Confirm if the gel is immobilised on the coverslip. 4. Add ddH2O until the gel is completely submerged. Reagents Resources Supplier Article number Acrylamide Sigma A9099 Acryloyl-X SE Invitrogen A20770 Adenine Sigma A9126 APS - Ammonium Persulfate Roth 9592.3 DAPI Invitrogen D1306 DMAA - N,N’-Methylenebisacrylamide Sigma M7279-25G GA - Glutaraldehyde Sigma G5882-10X1ML Glucose (Dextrose) Sigma G7021 KH2PO4 - Potassium dihydrogen phosphate Sangon Biotech A100781 NaCl Roth HN00.2 NHS-Ester Thermo Fisher Scientific D2184 Peptone Thermo Fisher Scientific 211820 PFA – Paraformaldehyde Thermo Fisher Scientific 43368 Poly-L-Lysine Sigma P8920 SDS – Sodium Dodecyl Sulfate Sigma L3771 Sodium acrylate Sigma 408220 5/4/2022 - Open Access ----!@#$NewPage!@#$---- Sorbitol solution Sangon Biotech A100691 TEMED – N,N,N′,N′-Tetramethylethylenediamine Sigma T9281 Tris Carl Roth 5429.1 Tryptophan Sigma T0254 Tween-20 Roth 9127.1 Yeast extract Thermo Fisher Scientific 288620 Zymolyase enzyme Zymo research E1004 Attofluor™ Cell Chamber Thermo Fisher Scientific A7816 Coverslip (24 mm round #1.5) Marienfeld 117640 Coverslip rack Diversified Biotech WSDR-1000 Razor blade Carl Roth CK08.1 Extended Data Description: Supplementary figures of custom tools and troubleshooting table.. Resource Type: Text. File: ExM_yeast_Supp.docx. DOI: 10.22002/D1.20159 Description: Supplementary Video 1. The video contains footage and instructions showing how to proceed throughout the protocol, focusing on the most critical steps. . Resource Type: Audiovisual. File: YeastExM_SuppVideo.mp4. DOI: 10.22002/D1.20160 References Chen L, Yao L, Zhang L, Fei Yn, Mi L, Ma J. 2021. Applications of Super Resolution Expansion Microscopy in Yeast. Frontiers in Physics. 9:650353. DOI: 10.3389/fphy.2021.650353 Faulkner EL, Thomas SG, Neely RK. 2020. An introduction to the methodology of expansion microscopy. Int J Biochem Cell Biol 124: 105764. PubMed ID: 32407880 Götz R, Panzer S, Trinks N, Eilts J, Wagener J, Turrà D, Di Pietro A, Sauer M, Terpitz U. 2020. Expansion Microscopy for Cell Biology Analysis in Fungi. Front Microbiol 11: 574. PubMed ID: 32318047 Milo, R., & Phillips, R. 2015. Cell Biology by the numbers (1st ed.). Garland Science. DOI: 10.1201/9780429258770 Pereira PM, Almada P, Henriques R. 2015. High-content 3D multicolor super-resolution localization microscopy. Methods Cell Biol 125: 95-117. PubMed ID: 25640426 Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO. 2018. X10 expansion microscopy enables 25-nm resolution on conventional microscopes. EMBO Rep 19: . PubMed ID: 29987134 Wassie AT, Zhao Y, Boyden ES. 2019. Expansion microscopy: principles and uses in biological research. Nat Methods 16: 33- 41. PubMed ID: 30573813 Funding: This work was supported by the Gulbenkian Foundation (AGK, LM, RH, MF, HSH, MDR) and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. [101001332] to RH), the European Molecular Biology Organization (EMBO) (Installation Grant EMBO-2020-IG-4734 to RH and the postdoctoral fellowship ALTF 499-2021 to HSH) and the Wellcome Trust 5/4/2022 - Open Access ----!@#$NewPage!@#$---- (203276/Z/16/Z to RH). MF is supported by The European Horizon 2020 Framework Programme, H2020-MSCA-IF-2020 - 101030203/ GENMAINEVO. LM is kindly supported by a joint-collaboration between Abbelight and the Instituto Gulbenkian de Ciência. AGK would like to acknowledge support from the Integrative Biology and Biomedicine (IBB) PhD programme from Instituto Gulbenkian de Ciência. Author Contributions: Artemis G. Korovesi: investigation, visualization, writing - original draft. Leonor Morgado: investigation, visualization, writing - original draft. Marco Fumasoni: conceptualization, writing - review editing. Ricardo Henriques: conceptualization, writing - review editing. Hannah S. Heil: supervision, data curation, writing - review editing, conceptualization. Mario Del Rosario: supervision, data curation, writing - review editing, conceptualization. Reviewed By: Ulrich Terpitz History: Received March 21, 2022 Revision Received April 28, 2022 Accepted May 3, 2022 Published May 4, 2022 Copyright: © 2022 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Korovesi, AG; Morgado, L; Fumasoni, M; Henriques, R; Heil, HS; Del Rosario, M (2022). Expansion Microscopy on Saccharomyces cerevisiae. microPublication Biology. 10.17912/micropub.biology.000566 5/4/2022 - Open Access