# 2008 The Authors

Journal compilation # 2008 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2008.00753.xTraffic 2008; 9: 1060–1072Blackwell Munksgaard

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A Cryosectioning Procedure for the UltrastructuralAnalysis and the Immunogold Labelling of YeastSaccharomyces cerevisiae

Janice Griffith, Muriel Mari, Ann De Maziere

and Fulvio Reggiori*

Department of Cell Biology, University Medical CentreUtrecht, 3584 CX Utrecht, the Netherlands*Corresponding author: Fulvio Reggiori,f.reggiori@umcutrecht.nl

Yeast Saccharomyces cerevisiae has been a crucial model

system for the study of a multitude of cellular processes

because of its amenability to genetics, molecular biology

and biochemical procedures. By contrast, the morphologi-

cal analysis of this organism by immunoelectron micros-

copy (IEM) has remained in a primordial phase preventing

researchers to routinely incorporate this technique into

their investigations. Here, in addition to simple but detailed

protocols to perform conventional electron microscopy

(EM) on plastic embedded sections, we present a new

IEM procedure adapted from the Tokuyasu method to

prepare cryosections from mildly fixed cells. This novel

approach allows an excellent cell preservation and the

negatively stained membranes create superb contrast that

leads to a unique resolution of the yeast morphology. This,

plus the optimal preservation of the epitopes, permits

combined localization studies with a fine resolution of

protein complexes, vesicular carriers and organelles at an

ultrastructural level. Importantly, we also show that this

cryo-immunogold protocol can be combined with high-

pressure freezing and therefore cryofixation can be

employed if difficulties are encountered to immobilize

a particular structure with chemical fixation. This new IEM

techniquewill be a valuable tool for the large community of

scientists using yeast as a model system, in particular for

those studying membrane transport and dynamics.

Key words: electron microscopy, high-pressure freezing,

immunogold labelling, rehydration method, Tokuyasu

cryosectioning, yeast

Received 22 February 2008, revised and accepted for

publication 17 April 2008, uncorrected manuscript pub-

lished online 21 April 2008, published online 20 May 2008

The budding yeast Saccharomyces cerevisiae has been

extensively used as a model organism to study a multitude

of cellular pathways because of its intrinsic advantages as

an experimental system (1). This unicellular eukaryote has

excellent classical and molecular genetics, a fast growth

rate and it is experimentally tractable, which gives complete

control over its chemical and physical environment. Being

a micro-organism, yeast shares with bacteria the simplicity

and rapidity of growth and the suitability for biochemical and

genetic methods that allows the application of the full range

of molecular technology (2–5). Being a eukaryote, it shares

with its multicellular cousins many fundamental housekeep-

ing processes and typical subcellular compartmentalization.

Therefore, lessons from this organism are very often trans-

posable to higher eukaryotes and identification of orthologue

proteins isparticularlywell facilitated.More recently, yeasthas

been the primary eukaryote of choice for the development of

genome-wide methodologies to study cellular functions. The

complete sequence of the yeast genome appeared in 1996

and was the first among eukaryotes (6). That, plus the very

efficient molecular genetic techniques, which permit any of

the6000genes tobe replacedwithamutantallele, completely

deleted or genomically modified, has boosted S. cerevisiae

genomic studies (2,5). By genomically tagging all the open

reading frames for example, it has been possible to deter-

mine the global protein expression and localization (7,8).

Despite the variety of possible experimental approaches

and all the acquired knowledge, the ultrastructural analysis

of S. cerevisiae by immunoelectron microscopy (IEM) has

remained difficult. IEM has been proven to be a powerful

investigational tool in higher eukaryotes because this is the

only technique that has the capacity to simultaneously

show the precise localization of a protein and the ultra-

structure of the organelle and environment wherein it is

embedded (9,10). Fluorescence light microscopy approaches

lack this resolution (9,10). For example, fluorescence-based

analyses have shown that numerous proteins localize to

early endosomes but only IEM has been able to reveal that

several of them are organized in subdomains that reflect

specialized retrograde transport events (11,12). Another

example has been the use of IEM to demonstrate that coat

protein II (COPII) coats give rise to COPII-coated transport

vesicles in vivo and do not just form endoplasmic reticulum

(ER) subdomains that collect proteins for transport via non-

coated carriers (13).

The major difficulty encountered in the development of

a satisfactory procedure for IEM in yeast has been the high

protein concentrations in the cytoplasm of this organism.

Most contrast methods for EM exploit the fact that the

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protein concentration in the membranes or in specific

macromolecular complexes is higher than that of the

cytoplasm. This difference enhances the contrast that

leads to the morphological resolution of the various sub-

cellular structures and compartments. Because in yeast

this difference is limited, the obtained contrast is low and

membranes are only marginally outlined. We have circum-

vented this problem by adapting the Tokuyasu cryosec-

tioning method (14) to yeast. The main reason to opt for

this approach is that the negative staining method em-

ployed in this technique leads to an outstanding definition

of membranes, which permits to also visualize small

membranous structures such as vesicles. Because cryo-

sectioning is the electron microscopic (EM) approach that

best preserves epitopes (10, 14), the obtained prepara-

tions can be efficiently immunogold labelled. Here, we

illustrate the power of this approach by providing a new

detailed protocol for the analysis of yeast.

Results

Cryosectioning permits a unique resolution of

yeast ultrastructure

The overall ultrastructure of S. cerevisiae is preserved when

fixed and subsequently embedded in resins such as Spurr’s

and Epon (15,16). As shown in Figure 1, not only the more

prominent subcellular compartments, for instance the

nucleus, the vacuole and the ER but also the plasma

membrane and the cell wall, are clearly identifiable. Mito-

chondria are also recognizable even if their morphology is

not well preserved. However, small organelles typical of

yeast such as the different Golgi compartments or the

various classes of endosomes, but also vesicular carriers,

cannot be easily seen because of the small difference in

contrast between membranes and cytosol. Nevertheless,

these conventional EM approaches can be very useful

to monitor apparent morphological changes such as, for

example, the proliferation of the ER, Golgi apparatus

expansions, endosome enlargements or autophagosome

formation (15–19). Another general limitation of these two

conventional EM protocols is that only a limited number of

proteins can be labelled with immunological reactions

because fixation conditions, dehydration procedures, resin

properties and the resin polymerization temperature have

cumulative denaturing effects on epitopes. To circumvent

these problems and obtain high-resolution yeast prepara-

tions that can be immunolabelled, we turned to the

Tokuyasu cryosectioning method (14).

The general procedure to prepare samples for the Tokuyasu

cryosectioning can be divided in four main steps: (i)

fixation, (ii) embedding into gelatine, (iii) cryoprotection by

sucrose infiltration and (iv) freezing in liquid nitrogen (14).

Figure 1: Spurr’s and Epon embed-

ded yeast cells. Wild-type cells

(SEY6210) grown to logarithmic (log)

phase were embedded in either

Spurr’s (panels A, B and C) or Epon

resin (panels D, E and F) as described in

Material and methods. Panels (C) and

(D) are image insets of panels (A) and

(F), respectively. M, mitochondria; N

nucleus; V, vacuole. Black bar, 500 nm.

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Immunoelectron Microscopy of Yeast

Cryosectioning of these samples then permits one to obtain

ultrathin preparations that are either directly imaged after

colouration or immunolabelled to localize specific proteins

(10). Our strategy to develop a protocol suitable for the

analysis of yeast has been to start from the one that we

routinely use for mammalian cells and optimize it (10,14).

Our first experiment was to explore if the employed

fixatives could have a role in preserving the yeast mor-

phology. We tried three different mixtures commonly

employed for IEM preparations: 4% paraformaldehyde

(PFA), 2% PFA/0.2% acrolein and 2% PFA/0.2% glutaral-

dehyde (GA), all in 0.1 M phosphate buffer (pH 7.4). In 4%

PFA-fixed cells, the cytoplasm was granular and the

membranes were not well preserved making the identifi-

cation of organelles more difficult (Figure 2A,B). In addi-

tion, numerous cells were damaged in the proximity of the

plasma membrane (Figure 2A,B, not shown). Since 4%

PFA is known to be a mild fixative, this result is probably

caused by the loss of some cellular material. An indirect

confirmation of this conclusion was from the fact that the

vacuole lumens were lost when cells were incubated with

4% PFA. The interior of the vacuole is mostly composed of

water making its fixation more difficult. The 2% PFA/0.2%

acrolein mixture gave a better result (Figure 2C,D). Cells

were better preserved and membranes were visible even

if their resolution was not satisfactory. Without doubt, the

best preparations were obtained with 2% PFA/0.2% GA:

The cytoplasm was homogenously fixed generating a clear

contrast with the white profiles of well-preserved mem-

branes (Figure 2E,F). This positive outcome is probably

because of the major fixation strength of GA, which was

also indicated by the fact that vacuole lumens were often

preserved. Based on this test result, we opted to use the

3-h 2% PFA/0.2% GA fixation further in developing our

protocol.

A common feature in all the samples prepared for this

comparative analysis was the detachment of the cell wall

and/or its flipping over the section (Figure 2). This was

more apparent in cells mildly fixed with 4% PFA than in

those treated with 2% PFA/0.2% GA, where this structure

Figure 2: Effects of different fixatives on yeast cryosections. The wild-type strain (SEY6210) grown to log phase was fixed for 2 h with

either 4% PFA (panels A and B), 2% PFA/0.2% acrolein (panels C and D) or 2% PFA/0.2% GA (panels E and F) all in 0.1 M phosphate buffer

before being prepared for cryosectioning. Panels (B), (D) and (E) are image insets of panels (A), (C) and (E), respectively. PM, plasma

membrane; M, mitochondria; N, nucleus; V, vacuole; CW, cell wall. Black bar, 500 nm; white bar 200 nm.

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Griffith et al.

is strongly cross-linked to the cell body and therefore

remains more effectively in its original place. The yeast

cell wall is a rigid extracellular layered meshwork com-

posed of glucans, chitin and mannoproteins (20), and the

observed phenomenon could be caused by the structural

tension release during the cutting of the sections. Sucrose

infiltration is one of the most critical steps of the Tokuyasu

method because the cryoprotectant properties of this

sugar allow the freezing of biological material without ice

crystal formation that damage morphology. Thus, another

critical concern about the cell wall is that it hampers

sucrose infiltration (21). To bypass the problem of bad

penetration, two different approaches have been used in

the past to embed yeast cells with viscous EM chemicals.

In the first approach, the cell wall is removed by enzymatic

activity before fixing the resulting spheroplasts (22). In the

second, cells are first fixed and then treated with sodium

metaperiodate. This oxidative agent alters the glucan

matrix composing the cell wall and permits the release of

cell wall proteins, effectively making this structure more

permeable to viscous compounds (21). Importantly, these

two methods could also provide a solution to the problem

of the cell wall detachment and flipping over encountered

during the cutting. We decided to choose the second

approach because the metaperiodate treatment is per-

formed after cell fixation. This has the practical advantage

that experiments can be terminated with the fixation and

this prevents physiological and morphological alterations

because of other prefixation treatments.

Accordingly, our next experiment was to prepare cells for

cryosectioning with or without a periodate treatment after

a 2-h fixation in 2% PFA/0.2% GA in 0.1 M phosphate

buffer. The periodate treatment was performed with either

periodic acid (H5IO5) or sodium metaperiodate (NaIO4). As

already shown, without chemical treatment, the cell wall

was either detaching from the cell body and thus damaging

the cellular surface or flipping over the preparation and

consequently covering both the plasma membrane and the

adjacent ER (Figure 3A,B). In contrast, incubation in the

presence of 1% periodic acid for 1 h allowed intact preser-

vation of the cell wall and consequently also the vast

majority of the cells (Figure 3C,D). Crucially, the ultrastruc-

ture of these cells was better preserved probably because

of a more complete infiltration of sucrose leaving the cellular

structures more protected during the freezing (Figure 3C,D).

Identical results were obtained with sodium periodate (not

shown). Based on this result, we decided to include the

postfixation periodic acid treatment in our protocol.

We also verified if the buffer used to carry out the fixation

and the periodic acid treatment affected yeast morphol-

ogy. Accordingly, we compared the results with the 0.1 M

phosphate buffer with those obtained using a modified

0.1 M PHEM buffer (20 mM PIPES, 50 mM HEPES, pH 6.9,

20 mM EGTA, 4 mM MgCl2). Although we did not observe

striking differences (Figure 3C–F), the impression is that

the PHEM buffer gave a finer resolution. Importantly, in

our follow-up experiments, we found that, in general,

PHEM buffer better preserves the immunoreactivity of

the yeast preparations (not shown). In addition, it allows

the use of uranyl acetate-containing pickup mixtures to

enhance contrast of the sections (see below), whereas

phosphate buffer leads to the formation of precipitates that

cover the sections. Consequently, we chose to incorporate

the PHEM buffer in our standard protocol.

In summary, our tests revealed that the most optimal

procedure to prepare yeast cells for cryosectioning is to fix

them for 3 h in 2% PFA/0.2% GA in 0.1 M PHEM buffer,

then to treat them with 1% periodic acid in the same buffer

for 1 h after which they are embedded in gelatine and

subjected to sucrose infiltration (see Material andMethods

for the detailed protocol). As shown in Figure 4, this

approach permits one to preserve and identify without

difficulty all major yeast organelles: the nucleus and

its pores, the vacuole, mitochondria, the peripheral ER

and the plasma membrane. Importantly, a multitude of

vesicles, sometimes clustered together, can be observed

in the cytoplasm, which very likely not only represent

vesicular carriers but also organelles such as endosomes

and Golgi compartments (Figure 4).

The contrast obtained with the new protocol is highly

satisfactory, but to even further facilitate the identification

of small membranous structures, we decided to explore if

it was possible to enhance them. It has been shown that

addition of uranyl acetate to the solution used for the

pickup of cryosections further enhances the contrast (23).

Therefore, we compared the results obtained with the

standard pickup solution (1% methyl cellulose, 1.15 M

sucrose in 0.1 M PHEM buffer) with those achieved using

either low (0.6% uranyl acetate, 1% methyl cellulose,

1.15 M sucrose in 0.1 M PHEM buffer) or high (3.3% uranyl

acetate, 1% methyl cellulose in 0.1 M PHEM buffer)

concentrations of uranyl acetate. As illustrated in Figure 5,

augmentation of the uranyl strength in the pickup solution

leads to a darker staining of the cytoplasmic and luminal

contents creating better contrasts with the white mem-

branes as illustrated by showing examples of mitochondria

and clusters of vesicles (Figure 5). In the case of 3.3%

uranyl acetate, an enlargement of the lipid bilayer profiles

accompanied the staining (Figure 5C). Thus, the addition of

uranyl acetate in the pickup mixture is a good solution to

facilitate morphological analyses. The presence of this

chemical in the cryosections, however, often increases

unspecific immunoreactions (not shown). Consequently,

the inclusion of uranyl acetate in the pickup solution when

performing IEM needs to be weighed against the possible

loss of specific immunostaining.

The new protocol permits the specific immunogold

labelling of yeast cryosections

Once the experimental conditions leading to an optimal

resolution of the yeast ultrastructure were determined, we

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Immunoelectron Microscopy of Yeast

investigated if these preserved the reactivity of the epitopes

and the specificity of the immunoreactions. Therefore,

sections prepared from wild-type cells using the devel-

oped protocol were labelled using various antibodies. As

shown in Figure 6, ER/nucleus (panels A and B), vacuole

(panels C and D), plasma membrane (panels E and F),

mitochondria (panels G and H) and lipid droplets (panels I

and L) were specifically recognized using antibodies

against the established protein markers Kar2, Prc1/CPY,

Pma1, Por1 and Erg6, respectively. The quantification of

the immunogold labelling of these antibodies is resumed in

Table 1. These images undoubtedly demonstrate that our

protocol can be used for the IEM analysis of yeast and

combine an unique ultrastructural resolution with efficient

immunolabelling. The morphology of the yeast Golgi

apparatus has largely remained elusive (24). We chose to

analyse the ultrastructure of this compartment using our

procedure to test its resolution power. Because of the lack

of antibodies against protein markers of this organelle, we

genomically tagged VRG4, a gene encoding for an anti-

porter mostly localized to the cis-Golgi compartments (24),

with the DNA sequence of the triple hemagglutinin (3xHA)

epitope. The strain expressing the Vrg4-3�HA fusion was

processed following our protocol and the obtained ultrathin

sections were incubated with anti-HA antibodies. As

shown in Figure 7, our analysis reveals that the yeast Golgi

consists of an autonomous cisterna that often has a circular

conformation.

We then decided to explore if the resolution of our protocol

allows the visualization of protein complexes as well. After

synthesis, the precursor protease Ape1 forms a large cyto-

plasmic oligomer that is transported into the vacuole lumen

through the cytoplasm to vacuole targeting (Cvt) pathway

(25). To detect the cytoplasmic oligomer, cryosections

obtained from wild-type cells were labelled with a polyclonal

anti-Ape1 antiserum. As shown in Figure 6M,N, the anti-

body specifically recognized (Table 1) an evident dark

Figure 3: Effects of the periodic acid treatment and the employed buffers on yeast cryosections. A, B). Periodic acid treatment

stabilizes the yeast cell wall. The wild-type strain (SEY6210) grown to log phase, fixed with 2% PFA/0.2% GA in 0.1 M phosphate buffer for

2 h before being treated (panels C and D) or not (panels A and B) with periodic acid and prepared for cryosectioning. E, F) The use of PHEM

buffer improves the resolution of the preparations. Wild-type cells (SEY6210) grown as above were fixed with 2% PFA/0.2% GA in 0.1 M

PHEM buffer before being treated with periodic acid and processed for cryosectioning. Panels (B), (D) and (E) are image insets of panels

(A), (C) and (E), respectively. PM, plasma membrane; M, mitochondria; N nucleus; V, vacuole; CW, cell wall. Black bar, 500 nm; white bar

200 nm.

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Griffith et al.

cytoplasmic structure. The circular form and the large size,

100–150 nm, confirm that this structure is the Ape1 oligo-

mer (25). From this result, we concluded that our procedure

also allows the detection of large protein complexes.

The rehydration method can be combined with the

yeast cryosectioning procedure

Chemical fixation and cryofixation are the two principal

approaches commonly used to immobilize biological sam-

ples. High-pressure freezing (HPF) is generally acknowl-

edged as being the method of choice for cryofixation

(26,27). The main advantage of HPF is that samples are

structurally stabilized within a few milliseconds, whereas

chemicals have a lag time in completely penetrating into

cells and tissues. This time lag allows cellular processes to

proceed for a short while and that occasionally might result

in structural and distributional artefacts. The disadvantage

of HPF is that subsequent to freezing, to prepare samples

Figure 4: Details of the yeast mor-

phology in cryosection prepara-

tions. Wild-type cells (SEY6210)

grown to log phase were fixed with

2% PFA/0.2% GA in 0.1 M PHEM

buffer, treated with periodic acid and

cryosectioned. Multiple examples of

the ultrastructural resolution of struc-

tures and organelles obtained with the

newly developed procedure. PM,

plasma membrane; M, mitochondria;

N nucleus; N.pore, nuclear pore; V,

vacuole; CW, cell wall; ER, endoplas-

mic reticulum; MVB, multivesicular

bodies; ?, newly visualized structure.

Black bar, 200 nm.

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Immunoelectron Microscopy of Yeast

for EM analyses, fixation, dehydration and resin embed-

ding are needed. Compared with the non-embedded

sections obtained by the Tokuyasu method, visualization

of yeast membranes in resin sections is relatively poor and

the efficiency of the immunolabelling is generally much

lower or destroyed. Therefore, a procedure has recently

been developed, the rehydration method, which combines

the advantages of HPF and those of the Tokuyasu technique

(28).

Apart from occasionally observing altered shape of the

vacuole, our protocol appears to preserve the morphology

of all the organelles in growing yeast. To confirm this

notion and also to have an alternative cryosectioning

procedure that could allow future studies of structures

potentially affected by chemical fixation or yeast grown in

experimental conditions making it more resilient to chem-

ical immobilization, we decided to apply the rehydration

method to yeast. Growing wild-type cells were first sub-

jected to HPF and then rehydrated as described in Material

and Methods (28). The resolution and the overall yeast

morphology of cryosections obtained with this procedure

were indistinguishable from the chemically fixed samples

(Figure 8A,B). In most of the cells, however, we observed

the detachment of the cell wall and/or its flipping over

the sections. In addition, the cytoplasmic material was

not homogeneously preserved indicating a non-perfect

sucrose infiltration or cryo-artefacts because of damages

during freezing. All that is very likely because of the fact

that cells were not treated with periodic acid during the

process of rehydration.

In parallel, those preparations were also incubated with

anti-Kar2 antibodies. The specific labelling of the ER proved

that the efficiency of the immunological reactions on

cryosections prepared using the standard protocol or the

rehydration procedure is almost identical (Figure 8C),

although more unspecific background labelling was pres-

ent in the rehydrated samples. As already observed

(Figure 5; unpublished observations), this phenomenon is

probably caused by the presence of uranyl acetate in the

yeast preparations.

Taking all these observations together, we concluded that

the rehydration method can be used for the analysis of

yeast. In turn, the examination of HPF samples confirmed

that the new cryosectioning protocol, which includes

chemical fixation and periodic acid treatment, is altering

neither the S. cerevisiae ultrastructure nor the immuno-

genicity of the epitopes.

Discussion

Morphological analysis of yeast cells by EM

Because of its structure and composition, yeastS. cerevisiae

has proven to be a challenge for electron microscopists.

Nevertheless, some EM procedures to analyse this uni-

cellular organism at an ultrastructural level have been

developed and allow the study of certain physiological

aspects of this eukaryote (15–19). Permanganate fixation

followed by embedding in the Spurr’s resin is one of them

and the preparations are of good quality because of the

excellent membrane contrasts (Figure 1A–C). This ap-

proach is relatively rapid and the results are much better

than those obtained with chemical fixation (Figure 1D–F).

Fine specimens are also generated if cells are first immo-

bilized with HPF and then embedded in Epon resin

(25,29,30). The new protocol presented here leads to

a superb resolution of the yeast morphology as well

(Figures 3E,F, 4 and 6). Membranes are very well pre-

served and their white profiles prominently emerge from

Figure 5: Addition of uranyl acetate in the mixture for the pickup of cryosections improves contrast. Wild-type cells (SEY6210)

grown to log phase were fixed with 2% PFA/0.2% GA in 0.1 M PHEM buffer, treated with periodic acid and cryosectioned. Cryosections

were then picked up with 0%, 0.6% or 3.3% uranyl acetate in 1% methyl cellulose/1.15 M sucrose in PHEM buffer. Mitochondria (row 1)

and clusters of vesicles (row 2) are shown in each panel to highlight the difference in colouration and contrast between the three pickup

solutions. Bar, 100 nm.

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Griffith et al.

Figure 6: Legend on next page.

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Immunoelectron Microscopy of Yeast

the dark cytoplasmic background typical of yeast. This has

the major advantage of revealing small membranous

structures such as vesicle carriers that are more difficult

to detect with other methods (Figures 4 and 5). This is

a crucial asset of our procedure because the yeast endo-

somes and Golgi compartments have vesicular conforma-

tions. Consequently, our protocol opens the possibility to

study them at an ultrastructural level. Importantly, the

contrast in our preparations can be further enhanced by

increasing the percentage of uranyl acetate in the pickup

solution, thus facilitating the visualization of small mem-

branous structures (Figure 5). The white profile of the

membranes seen in the sections prepared following the

Tokuyasu method is because of the loss of lipids. This

procedure is consequently not the optimal solution if there

is a particular interest in studying the morphology of lipid

bilayers; resins generate better results in this particular

case. The necessity of having experienced personnel and

sophisticated infrastructure is the only limiting factor that

could impede the incorporation of the newly developed

protocol in every laboratory.

Cryofixation is undoubtedly the best approach to immobi-

lize yeast for EM processing because after freezing, cells

can be warmed and normal growth can be resumed (30).

This means that the ultrastructure of the yeast cell has

been immobilized in its native state. Therefore, the images

generated using HPF may be as close as we can observe

the native cell structure at an EM level and they can

consequently serve as the ‘gold standard’ by which all

other specimen preparation methods for yeast can be

judged (30). Our new procedure employs chemical fixation

and consequently criticisms could arise about the preser-

vation of the yeast ultrastructure. However, the fact that

the overall morphology of yeast cells prepared with our

protocol is largely indistinguishable from that obtained

after HPF and rehydration (Figure 8A,B), proves the reli-

ability of our method. Because of this ascertainment, plus

the facts that the HPF/rehydration procedure is more time

consuming and includes the risks of ice crystal damage,

we have opted to routinely use chemical fixation.

Immunogold labelling of yeast EM sections

In the past, most of the immunogold labelling reactions

have been carried out on yeast EM sections prepared by

embedding cells in resins such as Lowicryl, LR Gold or LR

White after either chemical fixation or HPF (21,25, 29,31–33).

These resins do not optimally preserve the yeast ultra-

structure and/or as in the case of Epon and Spurr’s,

membrane contrasts are not always optimal making the

analysis of small vesicular structures difficult. As just

discussed, the Tokuyasu cryosectioning method allows

a better resolution. Crucially, this is the EM approach that

best preserves epitopes (10,14) and therefore our pro-

cedure has an additional evident advantage over the use of

resins, especially when analysing the distribution of non-

abundant proteins. Importantly, the oxidation of glycosyl

residues by the periodic acid treatment has the additional

advantage to reduce the background labelling on the cell

wall and on the vacuole (not shown). However, it should be

kept in mind that if the used antibodies are raised against

the cell wall glucan or recognize a glycan on a protein, they

will very likely not work on preparations obtained following

our procedure. A different adaptation of the Tokuyasu

method has previously been applied to yeast revealing its

potentialities in both resolution and immunogold labelling

(34–37). Our optimization has generated a better protocol

that permits an efficient and specific labelling of very well

preserved organelles and also protein complexes (Fig-

ure 6). Obviously, double labelling can also be performed

(not shown). Thus, we can now provide researchers with

a technology that basically allows investigating every

membrane trafficking and/or organelle biogenesis event

at an ultrastructural level. During the preparation of this

manuscript, a study that included an analysis of the yeast

endocytic transport route by IEM using a procedure almost

identical to ours has been published (38). This confirms the

unique investigative possibilities offered by our protocol.

It is important to note that the developed procedure

possesses a certain flexibility that can allow researchers

Figure 6: Immunogold labelling of yeast cryosections. Wild-type cells (SEY6210) grown to log phase were cryosectioned following the

optimized protocol. Preparations where first incubated with anti-Kar2 (panels A and B), anti-Prc1 (panels C and D), anti-Pma1 (panels E and

F), anti-Por1 (panels G and H), anti-Erg6 (panels I and L) and anti-Ape1 (panels M and N) antibodies, and then with protein A-10 nm gold

particle conjugates before collecting the images. PM, plasma membrane; M, mitochondria; CW, cell wall; N nucleus; V, vacuole; ER,

endoplasmic reticulum; LD, lipid droplets. Bar, 200 nm.

Table 1: Quantification of the immunogold labellingsa

Protein marker Organelle Organelle

labelling

Background

labelling

Ratio

Labelling density (gold particles/mm2)

Prc1 Vacuole 13.25 1.14 12

Erg6 Lipid droplets 55.33 0.91 61

Ape1 Ape1 oligomer 326.61 1.62 202

Linear density (gold particles/mm)

Kar2 Endoplasmic

reticulum

3.15 0.47 7

Pma1 Plasma

membrane

10.88 0.21 52

Por1 Mitochondria 1.54 0.29 6

aThe labelling and the linear densities were determined as

described in Material and methods. The lumen and the limiting

membrane of the nucleus were used to establish the background

labelling. In the case of Kar2, which also localizes to the nuclear

envelop, the plasma membrane was used to ascertain the

background. The ratio between the organelle and background

labellings is calculated by dividing the corresponding values.

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Griffith et al.

to circumvent eventual practical obstacles. For example,

if a determined structure cannot be properly immobilized

in its native form with chemicals, our protocol offers the

alternative of using cryosectioning combined with HPF

(Figure 8A,B). The immunolabelling efficiency of the ob-

tained sections is comparable with that of chemically fixed

preparations (Figure 8C). This approach, however, will

need further optimization to avoid the unspecific back-

ground labelling and the problems connected with the cell

wall. These latter could probably be solved by treating the

rehydrated cells with periodic acid before proceeding with

the Tokuyasu method adapted to yeast. Not all antibodies

work on GA-fixed material and this problem can be over-

come by fixing yeast cells in 4% PFA in PHEM buffer. The

quality of the cryosections prepared in this way is still

satisfactory even if lower than that obtained with the 2%

PFA/0.2% GA treatment (not shown). In general, as

a common routine, an antibody used for the first time

has to be checked at least on 2% PFA/0.2% GA- and 4%

PFA-fixed preparations in different dilutions. This test

allows determining which conditions preserve the epitope

immunogenicity the most and do not lead to unspecific

labelling. When the distribution of the immunolocalized

protein is unknown, it is particularly difficult to conclude if

the observed labelling is genuine. Consequently, the

eventual possibility of analysing a knockout strain in parallel

is extremely useful. However, if antibodies do not react on

any chemically fixed preparation or are not available, yeast

genetics offers an exceptional opportunity. Protein tags

can be genomically inserted at either the 50 or 30 end of

yeast genes using simple polymerase chain reaction

(PCR)-based integration methods (2,5,39). That permits

the creation of fusion proteins under the control of native

promoters that can be detected using commercially avail-

able antibodies (Figure 7). Our laboratory has selected

both monoclonal and polyclonal antibodies recognizing

green fluorescent protein, myc, HA, FLAG and V5 tags

that work on 2% PFA/0.2% GA-fixed material and do not

cross-react with epitopes present on endogenous yeast

proteins. We routinely use them for single and double

Figure 7: Ultrastructure and im-

munogold labelling of the yeast

early Golgi compartments. The

FRY366 strain grown to log phase

was cryosectioned following the opti-

mized protocol. Preparations where

first incubated with anti-HA anti-

bodies and successively with protein

A-10 nm gold particle conjugates

before being photographed. Bar,

200 nm.

Figure 8: HPF and rehydration of yeast cells. Wild-type cells (SEY6210) grown to log phase were fixed by HPF and rehydrated as

described in Material and Methods. Cryosections were directly imaged (panels A and B) or first incubated with anti-Kar2 antibodies and

then protein A-10 nm gold (panel C). PM, plasma membrane; M, mitochondria; CW, cell wall; N nucleus; ER, endoplasmic reticulum; MVB,

multivesicular bodies. Bar, 200 nm.

Traffic 2008; 9: 1060–1072 1069

Immunoelectron Microscopy of Yeast

immunolabellings of cryosections obtained from engi-

neered strains.

Conclusions

The fact that our protocol permits the obtaining of high-

quality cryosections from organisms surrounded by a cell

wall makes it suitable for the analysis of other yeast as

well. Therefore, it could be used for investigations in other

model systems such as Schizosaccharomyces pombe and

Pichia pastoris mostly exploited for the analysis of the cell

cycle regulation and peroxisome biogenesis/degradation,

respectively (40,41). In addition, our procedure can be

a precious tool to study pathological yeast such as Candida

albicans or Cryptococcus neoformans (42,43).

During the past several years, the use of electron tomo-

graphy has increased because it allows one to perform

three-dimensional analyses of a certain subcellular envir-

onment. This technology has also been successfully

applied to yeast (44). Importantly, it has recently been

shown that thicker, 300- to 400-nm immunogold-labelled

cryosections can be resolved by electron tomography

(12,13,45). Consequently, our method can be integrated

into three-dimensional tomography analyses. The com-

bination of immunolabelling and electron tomography

uniquely allows the recognition of specific structures and

the reconstruction of their membranous surrounding.

In summary, here we describe a new protocol that exploits

the high resolution and the efficient immunogold labelling

of the Tokuyasu cryosectioning for the analysis of yeast

S. cerevisiae. That, plus the possibility of combining this

procedure with other EM methods such as HPF and

tomography, will provide researchers with a new precious

tool for investigations in this broadly used model organism.

Material and Methods

Strains, plasmids and mediaThe S. cerevisiae wild-type strain used in this study was SEY6210 (MATaura3-52 leu2-3,112 his3-D200 trp1-D901ys2-801 suc2-D9 mel GAL) (46).

PCR-based integration of the sequence coding for 3�HA epitope at the 30

end of VRG4 was used to generate the FRY366 strain expressing the Vrg4-

3�HA fusion under the control of the native promoter (47). The template for

integration was pFA6a-3HA-His3MX6 (47). PCR verification and western

blot were used to confirm the integration at the correct locus.

Cells were grown in a rich medium (YPD; 1% yeast extract, 2% peptone,

2% glucose) to log phase and collected by centrifugation before being

processed for EM.

Permanganate fixation of yeast and embedding

with Spurr’s resin (16)Fifteen optical density (OD)600 unit equivalents of cells were resuspended

by pipetting in 1 mL of freshly prepared ice-cold 1.5% KMnO4 (Sigma) and

transferred into a 1.5 mL microfuge tube. After topping up the tube with

0.5 mL of the same solution to exclude air, samples were mixed on

a rotatory wheel for 30 min at 48C. This operation was repeated once more

before washing the pellets five times with 1 mL of distilled water. Cells

were then dehydrated in increasing amounts of acetone (10, 30, 50, 70, 90,

95 and three times 100%) by incubation on a rotatory wheel for at least

20 min at room temperature at each step. After centrifugation, pellets were

resuspended in 33% Spurr’s resin in acetone and mixed on the same

device for 1 h at room temperature. This operation was repeated twice

overnight and successively during all the day in 100% Spurr’s resin. The

Spurr’s resin mixture was prepared by mixing 10 g of 4-vinylcyclohexene

dioxide (or ERL4206), 4 g of epichlorohydrin-polyglycol epoxy (DER) resin

736, 26 g of (2-nonen-1-yl)succinic anhydride (NSA) and 0.4 g of N,N-

diethylethanolamine (all from Sigma). Incubating the preparations overnight

at 708C polymerized the Spurr’s resin. About 65–80 nm sections were then

cut using an Ultracut E ultramicrotome (Leica Microsystems) and trans-

ferred on Formvar carbon-coated copper grids. Sections where then stained

first with 6% uranyl acetate for 30 min at room temperature and then with

a lead-citrate solution (80 mM lead nitrate, 120 mM sodium citrate, pH 12)

for 2 min before being viewed.

Fixation of yeast and embedding with Epon resinTen OD600 unit equivalents of cells were resuspended in 2% GA in 0.1 M

cacodylate buffer (pH 7.4) for at least 2 h. Cells were then centrifuged,

washed three times with the 0.1 M cacodylate buffer before being post-

fixed in 1% OsO4, 1.5% ferrocyanide at 48C for 60 min. The pellet was then

washed five times with distilled water and left in the last wash for 30 min

before being centrifuged and resuspended in warm 2% low melting point

agarose (Roche) and immediately spun down. After solidification of the agar

on ice, the tip containing the cells was cut into small 1 mm3 blocks. These

blocks were then dehydrated by immerging them into increasing amounts

of ethanol (50, 70, 80, 90, 96 and three times 100%) by incubation on

a rotatory wheel for at least 15 min at room temperature at each step.

These amalgamations were followed by others: 1,2-propylene oxide

(Merck):Epon resin (3:1) for 30 min, 1,2-propylene oxide:Epon resin (1:1)

for 30 min, 1,2-propylene oxide-Epon (3:1) for 60 min and Epon resin

overnight. The Epon solution was prepared by mixing 12 g of glycid ether

100, 8 g of 2-dodecenylsuccinic acid anhydride, 5 g of methyl nadic

anhydride and 560 mL of benzyldimethylamine(all from Serva). The Epon

resin was then replaced the following day with freshly made one and the

incubation continued for 4 h. After centrifugation at 3 000 � g for 10 min,

the Epon resin was polymerized by heating the sample at 638C for 3 days.

About 65- to 80-nm sections were then cut using an Ultracut E ultramicro-

tome (Leica Microsystems) and transferred on Formvar carbon-coated

copper grids. Sections where then stained as described for those embed-

ded in Spurr’s resin.

Adaptation of the Tokuyasu method for yeast (14)Growing cultures at approximately 1 OD600 were rapidly mixed with an

equal volume of double-strength fixative [4% (w/v) PFA, 0.4% (v/v) GA in

0.1 M PHEM buffer (20 mM PIPES, 50 mM HEPES, pH 6.9, 20 mM EGTA,

4 mM MgCl2)] and incubated for 15–20 min at room temperature on a roller.

The fixative was then replaced by fresh standard strength fixative and

fixation proceeded for 3 h at room temperature. Cells were then resus-

pended in 1 mL of 0.1 M PHEM buffer and transferred in a 1.5-mL

microfuge tube where they were washed three times with the same

buffer. Pellets were resuspended in 1 mL of freshly prepared 1% periodic

acid (J.T. Baker, Phillipsburg, NJ) in 0.1 M PHEM buffer and incubated at

room temperature for 1 h on a roller. Cells were washed again three times

with 1 mL of 0.1 M PHEM buffer before adding 12% gelatine dissolved in

0.1 M PHEM buffer at 378C. This resuspension was then kept at 378C for

10 min to properly infiltrate the clumps of yeast cells.

After solidification at 48C, blocks of about 1 mm3 were trimmed under

a dissection microscope at 48C. These gelatine-embedded blocks were

immersed overnight in 2.3 M sucrose in rotating vials at 48C. They were

then mounted on ultramicrotome specimen holders and frozen by plunging

1070 Traffic 2008; 9: 1060–1072

Griffith et al.

into liquid nitrogen. After trimming to a suitable block shape, 45- to 60-nm

ultrathin sections were cut at �1208C on dry diamond knives (Diatome AG)

using either an UC6 or an UCT ultramicrotome (Leica). Flat ribbons of

sections were shifted from the knife-edge with an eyelash and picked up in

a wire loop filled with a drop of 1% (w/v) methyl cellulose, 1.15 M sucrose in

PBS buffer. Sections were thawed on the pickup droplet and transferred,

sections downwards, to Formvar carbon-coated copper grids.

Adaptation of the rehydration method (28) for yeastGrowing cells were centrifuged to concentrate them 10 times and loaded

into the flat specimen carrier (0.2 mm deep and 1.2 mm in diameter) of the

EM-PACT-1 HPF apparatus (Leica Microsystems) (27). Cryoimmobilization

at a pressure of 2000 bar (2 � 108 Pa) was performed according to the

manufacturer’s manual within 1 min after.

The cryofixed samples were transferred to the substitution medium (0.1%

uranyl acetate, 0.5% GA, 4% water in acetone) in 1.5-mL microtubes

placed at �908C in a cryosubstitution apparatus (AFS; Leica Microsystems).

The samples were dehydrated and fixed in this solution according to the

following protocol: at �908C for 25 h, in 15 h the temperature was raised to

�608C (28C/h); at �608C for 8 h, in 15 h the temperature was raised to

�308C (28C/h) and at �308C for 1 h. Uranyl acetate was then washed off by

rinsing the samples four times for 30 min at �308C with 0.5% GA, 4%

water in acetone. The tubes were finally removed from the substitution

apparatus and placed for 1 h on ice in the substitution medium minus

uranyl acetate.

After the dehydration and fixation during cryosubstitution, the samples

were rehydrated on ice in eight steps of 10 min each: 95% acetone/0.5%

GA, 90% acetone/0.5% GA, 80% acetone/0.5%, GA, 70% acetone/0.5%

GA, 50% acetone/0.1 M PHEM buffer/0.5% GA, 30 acetone/0.1 M PHEM

buffer/0.5% GA and 0.1 M PHEM buffer/0.5% GA. Samples were

then processed as described for the Tokuyasu method adapted to yeast

(see above).

ImmunolabellingSections on copper or nickel grids from either the Tokuyasu or the

rehydration procedure can be stored for several years in the cold still

covered by the pickup droplet and protected from humidity (48). For the

immunolabelling, grids were placed on a plate of 2% gelatine in 0.1 M

phosphate buffer (pH 7.4) and then warmed to 378C for 30 min to let the

pickup solution diffuse away together with the gelatine. Next, the grids

were passed over a series of droplets of washing, blocking, antibody and

protein A-gold solutions for routine labelling procedures (49). After a final

wash in distilled water, the sections were left for 10 min on 2% uranyl

oxalate (pH 7) (50) and transferred, via a few seconds on a puddle of distilled

water, to a mixture of 1.8% methyl cellulose (pH 4) and 0.6% uranyl

acetate. After 10 min, the grids were looped out, the excess viscous

solution was drained away and the sections were allowed to dry (51).

ImmunoreagentsImmunological reactions were performed using mouse anti-porin (Mol-

ecular Probes), anti-Pma1 (EnCor Biotechtologies) and anti-HA (a kind gift of

Guojun Gu, Washington University) antibodies, and rabbit anti-Prc1 (a kind

gift of Andreas Conzelmann, University of Fribourg), anti-Kar2 (a kind gift of

Ineke Braakman, Utrecht University), anti-Erg6 (a kind gift of Guenther

Daum, Graz University of Technology) and anti-Ape1 (a kind gift of Ignacio

Sandoval and Marıa Mazon, Universidad Autonoma de Madrid) antisera.

Protein A-gold conjugates were prepared according to the method

described by Slot and Geuze (52).

Image acquisitionSections were viewed in a JEOL 1010 or a JEOL 1200 electron micro-

scope (JEOL) and images were recorded on Kodak 4489 sheet films

(Kodak).

Statistical analysis of the immunogold labellingsThe specificity of antibodies recognizing proteins in the interior of a large

structure (Prc1, Erg6 and Ape1) was determined by calculating the labelling

density using the Point-Hit method, whereas that of proteins with trans-

membrane segments or contained in a narrow organelle as the ER (Kar2,

Pma1, Por1) by establishing the linear density (53).

Acknowledgments

The authors are grateful to Judith Klumperman for comments, and thank

Andreas Conzelmann, Ineke Braakman, Guenther Daum, Ignacio Sandoval

and Marıa Mazon for the antibodies. Authors also thank Jan Willem Slot,

Marcel Borgers, Patrick Marichal for support, and Marc van Peski and

Rene Scriwanek for assistance with the preparation of the Figures. F. R.

is supported by the Netherlands Organization for Health Research and

Development (ZonMW-VIDI-917.76.329) and by the Utrecht University

(High Potential grant). A. D. M. was sponsored by Janssen Pharmaceutica

(Beerse, Belgium).

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