Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Summary

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Abstract

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Introduction

The endosomal system is a major membrane sorting apparatus that plays multiple crucial cellular roles including trafficking of lysosomal enzyme sorting receptors and the recycling of plasma membrane (PM) receptors^1,2. Endosomes are divided in three different compartments, i.e. the early endosomes (EE), the late endosomes (LE) and the recycling endosomes. This classification is based on the time it takes for endocytosed material to reach them, on specific marker proteins and on their morphology. Membranes, i.e. protein and lipid bilayers, internalized from the PM can either be delivered to lysosomes via endosomes for degradation or be recycled back. Membranes are also transported to endosomes from the Golgi and similarly, either continue to lysosomes or be retrieved back to the Golgi. Furthermore, proteins can be sorted into luminal vesicles budding inward from the endosomal limiting membrane, a process that leads to the formation of a subcategory of LE, the multivesicular bodies.

The yeast endosomal system is relatively less complex than the one of high eukaryotic cells. Yeast endosomes are divided into EE and LE. In contrast to mammalian cells they do not contain recycling endosomes but also tissue-specific lysosome-related organelles. Consequently they have a less complex network of endosomal trafficking routes^3,4. Therefore yeast has represented and still represents an advantageous experimental system to study some of the principles underlying membrane traffic in the endosomal system. This advantage is emphasized by the fact that numerous genes involved in the endosomal pathways have been initially isolated with genetic screens in yeast⁵. While the yeast endosomal system in wild type and mutant cells has been extensively studied using biochemical and fluorescence microscopy approaches, its investigation at the ultrastructural level has only been minimal. Morphological analyses are particularly relevant in yeast because most of the endosomal organelles are detected as punctuate structures by fluorescence microscopy, which make difficult their unequivocal identification⁶. Unfortunately only a limited number of antisera recognizing yeast endosomal protein markers is working in immuno-electron-microscopy (IEM) preparations^7-10. For some proteins, this problem has been circumvented by the endogenous tagging of the gene of interest and the use of an antibody recognizing the tag to detect it^7,11,12. Often, however, proteins are undetectable by IEM because of their low expression levels. Their overexpression is not a solution because this approach can induce mis-localizations and/or alterations in the organelle morphology/functions. Thus the labelling of the endocytic compartments with a probe detectable by electron microscopy EM is an effective option. This is an optimal solution especially if the probe is entering the endocytic route in a time-dependent manner, which allows to know when it will mark a specific organelle⁶.

The uptake of positively charged nanogold by yeast spheroplasts (i.e. yeast where the cell wall has been enzymatically removed) has successfully been used to identify the yeast endosomal compartments¹⁰. These particles strongly bind to the negatively charged lipids composing the biological membranes. Thus the positively charged nanogold associates with the PM, penetrates the cell by endocytosis and passes through the EE and LE before reaching the vacuole. These small gold particles, however, do not have an appropriate size to be seen by EM. To render them visible, their size can be enlarged by chemical reactions that lead to the deposition of silver or gold around the gold probe^13-15. We have developed and successfully applied an IEM approach based on the Tokuyasu method to perform subcellular localization studies^8,16. This method allows performing immunogold labelling on yeast preparations with an excellent resolution of the morphology^8,17-24. We have also established a procedure combining this IEM protocol with the nanogold labelling of the yeast endosomal system compartments⁶. Using this approach we have morphologically characterized different subclasses of endosomes and ultrastructurally examined mutants with an endosomal trafficking defect^6,25. Moreover, we have demonstrated that this nanogold labelling can be combined with immunogold labelings providing the possibility to explore the distribution of a protein of interest on the different endosome subpopulations. Here we present how the labelling of the yeast endosomal system with positively charged nanogold is practically performed.

Protocol

1. Spheroplast Preparation

1. Incubate the yeast overnight at 30 °C in 10 ml of the appropriate medium determined by the design of the experiment.
2. The day after, measure the optical density of the culture at 600 nm (OD₆₀₀) using a photometer, Dilute cells in the same culture medium to an OD₆₀₀ of 0.2-0.4 and grow them to an exponential growth phase unless the design of the experiment requires a different condition. Cells are in the exponential phase when the culture has an OD₆₀₀ of 1-2.
3. Collect 10 OD₆₀₀ equivalents of cells by centrifugation at 3,500 x g for 5 min in a 50 ml tube. After centrifugation, discard the supernatant.
4. Resuspend the cell pellet in 5 ml of 100 mM PIPES (pH 9.6), 10 mM dithiothreitol and incubated at 30 °C for 10 min.
5. Collect again the cells by centrifugation at 3,500 x g for 5 min. Discard the supernatant.
6. Resuspend the cells in 5 ml of medium (determined by the design of the experiment) containing 1 M sorbitol and 5 mg of lytic enzyme, and incubate the mixture at 30 °C with gentle shaking for 30 min.
7. Centrifuge the cell suspension at 300 x g for 5 min to collect the pellet fraction, which corresponds to the spheroplasts. Discard the supernatant.
8. Resuspend the spheroplasts in 960 µl of ice cold media (medium determined by the design of the experiment) containing 1 M sorbitol and transfer the mixture into an ice cold 2 ml microcentrifuge tube.

2. Nanogold Uptake

1. Using a pipette, gently mix the spheroplast with 4 nmol of positively charged nanogold particles resuspended in 40 µl of water. The final volume of the mixture has to be of 1 ml.
2. Place the obtained cell suspension on ice for 15 min.
3. Transfer the suspension at room temperature and incubate it for the required time to allow nanogold internalization by endocytosis.
   NOTE: A 5 min uptake will principally lead to the labelling of endocytic vesicles and early endosomal compartments, while a 15 min uptake will permit to mark the entire endosomal system (early and late endosomes). Longer incubation times (more than 30 min) will also permit to label the vacuole.
   NOTE: Pulse-chase labelling experiments cannot be performed with this method. Therefore, when labelling for the analysis of LE, nanogold will also be found at the PM and inEE.

3. Fixation and Sectioning

1. Stop the nanogold uptake by adding 1 ml of double strength fixative [4% paraformaldehyde (PFA), 0.4% glutaraldehyde (GA) in 0.1 M PHEM buffer (20 mM PIPES, 50 mM HEPES, pH 6.9, 20 mM EGTA, 4 mM MgCl₂)] containing 1 M sorbitol to the cell suspension. Kept the tube at room temperature.
2. Gently invert manually the micocentrifuge tubes several times during 30 min (this will permit to keep the spheroplasts in suspension) and then centrifuge it twice at 1,700 x g for 25 sec.
3. Replace the fixative by discarding the supernatant and by adding 1 ml of fresh standard strength fixative (2% PFA, 0.2% GA in 0.1 M PHEM buffer) containing 1 M sorbitol and incubate for 2 hr at room temperature on a slowly rotating wheel.
4. Process the cells for cryo-sectioning as described previously⁶. NOTE: For the nanogold uptake and silver enhancement procedures, the periodic acid treatment described in the indicated protocol has not to be performed. The periodic acid treatment is to better permeabilize the cell wall²⁶ but this structure has been eliminated during the generation of spheroplasts.
5. Cut 50 nm thin cryosections at -120 °C with dry diamond knife using an UCT ultramicrotome as previously described⁸ and place them on Formvar carbon coated 50 meshes nickel grids.

4. Silver Enhancement for Nanogold Particle Visualization

NOTE: The procedure to prepare the reaction mixtures is basically the one indicated by the manufacturer. Practical adaptations using this protocol, makes the silver enhancement procedure effective and reliable on cryosections.

1. Remove the silver enhancement kit from the freezer and thaw it in a 37 °C incubator or water bath. When thawed, place the kit in a 24 °C incubator placed into a dark room until use (see 4.7).
2. Place the heating plate in the dark room and warm it to the final temperature of 24 °C.
3. Cover the top of heating plate with the surface protector, shiny side up, and secure it with a tape.
4. Place the Parafilm on the Benchkote and mark the edges with a black marker to be able to see the Parafilm edges in the dark.
5. Tape a thermometer on the top of the Benchkote to monitor the temperature of the heating plate. Gradually adjust the temperature if not 24 °C.
6. Place the 50 ml tube with double distilled water and the silver enhancement kit inside.
7. Fill the small Petri dishes with double distilled water, pre-warmed at 37 °C. Place the grids in the water, specimen side down, for 30 min.
8. Repeat this 4.7 step one more time.
9. Transfer the grids in the storage box and bring them to the dark room.
10. Rinse the grids again by passing them (with the specimen side down) on several drops of double distilled water at 24 °C placed on the Parafilm that has been fixed on the heating plate.
11. Make sure that 9 additional double distilled water drops are ready on the Parafilm, for the rinsing after the silver enhancement reaction.
12. Turn the light off and make sure that all lights are off. Turn the red light on.
13. Take out the A and B solutions of the silver enhancement kit from the 24 °C incubator.
14. Put first 6 drops of the A solution and then 6 drops of the B solution in a 1.5 ml microcentrifuge tube, and mix well with a glass Pasteur pipette avoiding to make bubbles. The solution is quite thick and it has to be manually mixed with a pipette before finally vortexing it briefly.
15. Put the A and B solutions back in the 24 °C incubator and take out the C solution.
16. Add 6 drops of the C solution to the A/B mix. Then mix again first with a Pasteur pipette and then by vortexing, avoid making bubbles.
17. Use immediately the final mixture by placing drops (~20 µl/drop) on the Parafilm.
18. With a clean, anti-magnetic tweezers, place the grids on the mixture (e.g., silver enhancing reaction) for 6 to15 min depending of the enhancement (i.e. size of the gold particles) wished to be obtained.
19. Remove the grids from the mixture and pass them on the top of the double distilled water drops at 24 °C for washes. First, use in rapidly succession 6 drops and then 3 drops with 7 min incubation time per drop, before turning the lights on.
20. Proceed to the next step, either immuno-gold labeling or membrane staining²⁷ in order to visualize the result for an EM investigation.

Results

According to the presented protocol, the morphology of the yeast endosome system can be access by transmission EM. Figure 1 shows different types of endosomal compartments that have been reached and therefore labeled with nanogold. The silver-enhanced nanogold can be clearly seen as electron dense particles. The optimal settings established for the silver enhancement reaction allows having gold particles in a homogenous size range between 5 and 15 nm, which does not interfere with the ultrastructure of t...

Discussion

Immuno-electron-microscopy is a technique that allows combining localization of proteins with the ultrastructural resolution of the carriers and organelles where these proteins reside. This is particularly crucial when studying the yeast endosomal system because its compartments appear as punctuate structures in fluorescence microscopy. It is therefore difficult to distinguish them. For this reason the use of a probe detectable by EM and entering the endocytic route in a time dependent manner is crucial to specifically m...

Materials

Name	Company	Catalog Number	Comments
PIPES (piperazin-1,4-bis (2-ethansulfensaure)	Merck, Darmstadt, Germany	1,102,200,250
HEPES	Merck, Darmstadt, Germany	391,340
EGTA	Sigma, St louis, MO	E4378
MgCl2.6H2O	Merck, Darmstadt, Germany	1,058,330,250
DL-Dithiothreitol	Sigma, St louis, MO	D0632
Sorbitol	Sigma, St louis, MO	S1876
Positively charged Nanogold	Nanoprobes, Yaphank, NY	2022	store at -20 ̊C
HQ-silver™ enhancement kit	Nanoprobes, Yaphank, NY	2012	store at -20 ̊C
Paraformaldehyde (PFA)	Sigma, St louis, MO	441244
Glutaraldehyde 8% EM grade	Polyscience, Inc. , Warrington, PA	216	store at 4 ̊C
Lytic enzyme	MP Biomedicals, Santa Ana, CA	153526	store at 4 ̊C
Parafilm M	Sigma, St louis, MO	P7793
50 meshes nickel grids	Agar Scientific, Stansted, United Kingdom	G209N
Cryo-immuno diamond knife 35 º	Diatome AG, Biel, Switzerland	N.A.
UCT ultramicrotome	Leica, Solms, Germany	N.A.
Formvar 15/95 resine	Electron Microscopy Sciences, Hatfield, PA	15800
50 meshes nickel grids	Agar Scientific, Stansted, United Kingdom	AGG2050N
Parafilm	Sigma, St louis, MO	P7793
Grids storage box	Leica, Solms, Germany	162-50
Falcon 100mmx15mm petri dishe	Corning, Corning, NY	351029
Pasteur capillary pipettes 150 mm	Van Bruggen Glas, Rotterdam, The Netherlands	10216234
1.5 ml microcentrifuge	Sarstedt, Nümbrecht, Germany	72690001
50 ml tube	Bio-one, Alphen aan den Rijn, The Netherlands	210296
Benchkote surface protector	Whatman, Maidstone, United Kingdom	1159201