Endocytosis inhibition during H2O2-induced apoptosis in yeast

R E S EA RCH AR T I C L E

Endocytosis inhibition during H2O2-induced apoptosis in yeast

Clara Pereira, Claudia Bessa & Lucılia Saraiva

REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal

Correspondence: Clara Pereira, Laboratory

of Microbiology, Department of Biological

Sciences, Faculty of Pharmacy, University of

Porto, Rua Jorge Viterbo Ferreira, 228, 4050-

313 Porto, Portugal. Tel.: +351 220 428 584;

fax: +351 226 093 390; e-mail:

[email protected]

Received 5 January 2012; revised 18 June

2012; accepted 25 June 2012.

DOI: 10.1111/j.1567-1364.2012.00825.x

Editor: Jens Nielsen

Keywords

yeast; endocytosis; H2O2; apoptosis.

Abstract

Yeast revealed to be a versatile organism for studying endocytosis. Here, inhibi-

tion of endocytosis by H2O2 and its correlation with apoptotic cell death were

ascertained in Saccharomyces cerevisiae. We found that H2O2 causes alterations

in vacuolar morphology and a concentration-dependent inhibition of endocy-

tosis. We also found that H2O2-induced endocytosis inhibition is a reversible

process that occurs in the early phase of the apoptotic cascade, preceding chro-

matin condensation and DNA fragmentation. Additionally, mutants affecting

early steps of the endocytic pathway display sensitivity to H2O2. As endocytosis

inhibition was also observed with acetic acid, it may be a broader cellular dys-

function of oxidative stress-induced toxicity in yeast.

Introduction

Endocytosis is a general mechanism by which eukaryotic

cells internalize extracellular molecules through the

formation of vesicles from the plasma membrane. The

opposite process, expel of specific molecules and delivery

of lipids and proteins to the plasma membrane by the

fusion of internal membranes, is called exocytosis. It is

the control of these two processes that regulates the inter-

action between the cell and its environment. Besides its

role in cell physiology, vesicular trafficking has also been

connected to the regulation of signaling pathways and

complex programs like cell cycle, mitosis, and apoptosis.

Consequently, it has been implicated in human diseases

(Scita & Fiore, 2010).

Reflecting its importance for the eukaryotic cell, many

features of the vesicular trafficking pathways are evolu-

tionarily conserved. Many studies on endocytosis have

been performed in Saccharomyces cerevisiae, which has

proven to be an extremely versatile model. For instance,

several components of the endocytic process have been

identified through genetic studies in yeast. It is consen-

sual that the ability to describe this process in detail in

yeast can lead to considerable understanding of endocyto-

sis in higher eukaryotes (Novick et al., 1980; Shaw et al.,

2001; Burston et al., 2009).

In mammals, the vesicular trafficking system appears to

be affected by oxidative stress caused by reactive oxygen

species (ROS) such as hydrogen peroxide (H2O2). Like all

aerobically growing organisms, yeast also suffers exposure

to moderate oxidative stress and has developed multiple

mechanisms for preventing and counteracting its effects,

including adaptation to increased resistance and wide-

spread changes in gene expression (Collinson & Dawes,

1992). However, for high levels of oxidative stress, cells

undergo a form of apoptotic cell death, exhibiting fea-

tures like chromatin condensation and DNA fragmenta-

tion (Madeo et al., 1999). In this study, the impact of

H2O2 on endocytosis and its relation with apoptotic cell

death were addressed in yeast.

Materials and methods

Yeast strains, plasmids and growth conditions

Saccharomyces cerevisiae strains and plasmids used in this

study are listed in Table 1. W303 strain was transformed

with the YX232-mtGFP plasmid by the lithium acetate

method. Yeast cultures were grown in synthetic complete

(SC) medium with 0.67% (w/v) yeast nitrogen base w/o

amino acids (Difco), 2% (w/v) glucose (Sigma-Aldrich)

and 0.2% (w/v) dropout (mix; Sigma-Aldrich), and the

FEMS Yeast Res && (2012) 1–6 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

YEA

ST R

ESEA

RC

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required amino acids. Transformed yeasts were grown in

selective SC medium lacking tryptophan. For the assays

with the endocytic mutants, cells were grown in rich

[YEPD; 1% (w/v) yeast extract, 2% (w/v) peptone, 2%

(w/v) glucose] medium. Cells were grown to exponential

phase with continuous shacking (160 r.p.m.) at 30 °C.

Viability and calcofluor white sensitivity assays

For H2O2 viability assays, exponential cultures were trea-

ted with 1–10 mM H2O2 for up to 5 h or with 80 and

140 mM acetic acid for 1 h with shaking at 30 °C. Viabil-ity was assessed by colony-forming unit (CFU) counts

after 2 days incubation at 30 °C on Sabouraud Dextrose

Agar (Difco) plates and expressed as percentage of time

zero. Sensitivity to calcofluor white was assessed basically

as described by Ram et al. (1994). Briefly, cells were

grown overnight in YEPD, and serial dilutions plated on

Sabouraud Dextrose Agar plates containing 0 or

50 lg mL�1 calcofluor white (Fluka; Sigma-Aldrich).

Plates were incubated in the dark at 30 °C and photo-

graphed after 2 days.

Staining with FM4-64

Endocytosis was assessed using the lipophilic dye FM4-64

(N-(3-thiethylammoniumpropyl)-4-(p-diethyl-aminophe-

nylhexatrienyl) pyridinium dibromide; Molecular Probes),

basically as described (Vida & Emr, 1995). Briefly, cells

were incubated with 3 lg mL�1 FM4-64 for 1 h at 30 °C(time required for FM4-64 to reach the vacuole in

untreated wild-type (wt) cells), and thereafter observed

under a microscope. The percentage of cells with endocy-

tic inhibition (lacking vacuolar staining) was estimated by

counting at least 200 cells per sample.

Cell death assays

ROS production was assessed using dihydroethidium

(DE; Sigma-Aldrich). W303 cells were incubated with

10 lg mL�1 DE for 30 min at 30 °C, washed once, and

visualized under a microscope. Nuclear staining was

performed in fixed cells with 2 lg mL�1 4,6-diamido-2-

phenyl-indole (DAPI; Sigma-Aldrich) as described (Silva

et al., 2005). DNA fragmentation was assessed by TUNEL

(In Situ Cell Death Detection Kit, Fluorescein; Roche

Applied Science) as described (Silva et al., 2005). Mito-

chondria were visualized using a mitochondria-localized

green fluorescent protein (mtGFP) encoded by YX232-

mtGFP. For the determination of apoptotic phenotypes,

at least 200 cells per sample were evaluated.

Fluorescence microscopy

Samples were observed under an Eclipse E400 fluores-

cence microscope (Nikon) under appropriate filter

setting. Images were captured by Digital Sight camera

(Nikon DS-5Mc) with software for image acquisition

(Nikon ACT-2U).

Statistical analysis

Statistical analysis was performed by Paired t-test with

the software GraphPad Prism. Statistical significance was

accepted at P < 0.05.

Results

Oxidative stress disturbs endocytosis in a

concentration-dependent manner

The effect of two inducers of oxidative stress and apopto-

sis, H2O2 and acetic acid (Madeo et al., 1999; Pereira

et al., 2010), on fluid-phase endocytosis was investigated

in the W303 strain using the fluorescent membrane probe

FM4-64. As reported by Vida & Emr (1995), we observed

that the dye initially stains the plasma membrane, fol-

lowed by the cytoplasmic endocytic intermediates and

finally, after 1 h incubation, the vacuolar membrane

(Fig. 1a, 0 mM). However, for 1 h incubation with the

dye, cells exposed to low concentrations (1–3 mM) of

H2O2 exhibited defects in the movement through endocy-

tic intermediates to the vacuole, visible by the presence of

dot-like staining as described in (Vida & Emr, 1995;

Fig. 1a, 3 mM). Additionally, for higher concentrations of

H2O2, cells only displayed plasma membrane staining,

indicating a defect in the early movement of the dye from

the plasma membrane to endocytic intermediates (Fig. 1a,

5 and 10 mM). When the percentage of cells with

Table 1. Yeast strains and plasmids used in this study

S. cerevisiae

W303 Mata ura3-1 leu2–3,

112 his3–11,

15 trp1-1 ade2-1 can1–100

Lab collection

BY4741 Mat a; his3D 1; leu2D 0;

met15D 0; ura3D 0

EUROSCARF collection

ede1D By4741; YBL047c::kanMX4 EUROSCARF collection

sla1D By4741; YBL007c::kanMX4 EUROSCARF collection

end3D By4741; YNL084c::kanMX4 EUROSCARF collection

ent1D By4741; YDL161w::kanMX4 EUROSCARF collection

rvs161D By4741; YCR009c::kanMX4 EUROSCARF collection

bzz1D By4741; YHR114w::kanMX4 EUROSCARF collection

vps21D By4741; YOR089c::kanMX4 EUROSCARF collection

vps41D By4741; YDR080w::kanMX4 EUROSCARF collection

ypt7D By4741; YML001w::kanMX4 EUROSCARF collection

Plasmid

YX232-GFP mtGFP under control of TP1

promoter

Westermann &

Neupert (2000)

ª 2012 Federation of European Microbiological Societies FEMS Yeast Res && (2012) 1–6Published by Blackwell Publishing Ltd. All rights reserved

2 C. Pereira et al.

endocytic inhibition (lacking vacuolar staining) was quan-

tified, a close correlation with the loss of viability

(assessed by CFU counts) was observed (Fig. 1b).

Together, the results obtained showed that H2O2 causes a

concentration-dependent inhibition of yeast endocytosis

(Fig. 1a and b). The same results were obtained for the

BY4741 strain (data not shown).

Additionally, the vacuolar morphology was also

affected, with a swollen and less fragmented vacuole than

that observed in untreated cells (Fig. 1a, 3 mM). A simi-

lar effect on yeast endocytosis was also observed for acetic

acid. Indeed, when cells were treated with 80 and

140 mM acetic acid for 1 h, an inhibition of endocytosis,

more severe for the highest concentration tested, was also

detected (Fig. 1c).

Interestingly, for longer incubation times (4 h), a res-

toration of the vacuolar staining in cells exposed to

low concentrations of H2O2 was observed (Fig. 2).

However, this was not observed for higher concentra-

tions of H2O2 (5 mM). In this case, endocytosis

remained impaired except if the oxidant is removed

and cells allowed to recover for 3 h in fresh media.

The recovery of endocytosis occurred before the recov-

ery in clonogenicity, which required an additional 2 h

(data not shown).

Endocytosis is an early event in the H2O2-

induced apoptotic program

To correlate endocytosis impairment with the apoptotic

program, ROS production, mitochondrial network

fragmentation, chromatin condensation and DNA

fragmentation were monitored upon exposure to 5 mM

H2O2. For time zero, the percentage of cells displaying

these apoptotic features was close to zero (Fig. 3). How-

ever, with only 30 min treatment, the majority of cells

exhibited ROS production and an almost simultaneous

mitochondrial network fragmentation (Fig. 3a and b). At

this time point, about half of the cells (57%) displayed a

blockage in endocytosis as evidenced by the absence of

A B

C

Fig. 1. H2O2 and acetic acid lead to yeast endocytosis inhibition in a concentration-dependent manner. W303 cells were treated with the

indicated concentration of H2O2 and acetic acid for 1 h and compared to control yeast (untreated cells; 0 mM). (a) Representative

photomicrographs of H2O2-treated cells stained with FM4-64 (left side) and respective bright-field images (right side). (b) Quantification of

viability and endocytosis inhibition (lacking vacuolar staining) for H2O2-treated cells. Values are mean ± SE (n = 3). (c) Representative

photomicrographs of acetic acid-treated cells stained with FM4-64. Bar, 10 lm.

Fig. 2. Inhibition of endocytosis is a reversible process. W303 cells

were treated with 3 mM H2O2 for 1 and 4 h. Representative

photomicrographs of FM4-64 stained cells (left side) and respective

bright-field images (right side). Bar, 5 lm.


Endocytosis impairment during H2O2-induced apoptosis 3

vacuolar staining (Fig. 3b). After 1 h treatment, although

the majority of the cells exhibited endocytic inhibition,

the number of cells with chromation condensation and

DNA fragmentation was still low. Indeed, a significant

increase in the percentage of cells exhibiting these two

apoptotic markers was only achieved with longer incuba-

tion times (5 h; Fig. 3b).

Endocytic mutants display different responses

to H2O2-induced apoptosis

To assess the impact of vesicular trafficking on the cellu-

lar response to H2O2, several null yeast mutants with

decreased endocytosis were used. From the proteins

recruited at early phases of endocytosis, Ede1p (early

immobile phase), Ent1p, End3p, Sla1p (Mid/late immo-

bile phase) and Rvs161p, Bzz1p (Actin/mobile phase)

(Boettner et al., 2011) were studied. From the proteins

recruited at late steps of apoptosis, Vps21p required for

vesicle transport, Vps41p and Ypt7 involved in transport

from late endosomes to the vacuole (Lachmann et al.,

2011), were studied (Fig. 4a).

For 1-h treatment with 3 mM H2O2, end3Δ and

rvs161Δ showed a significant sensitivity, around 28% and

34%, respectively (Fig. 4b). The remaining deletion

mutants exhibited a response to H2O2 not significantly

different from the wt. There was no correlation between

H2O2 sensitivity and mutants with obvious defects in the

endomembrane system (namely ypt7Δ, vps41Δ and

vps21Δ; Fig. 5a). Because several mutants with decreased

endocytosis (namely rvs161Δ, bzz1Δ, ent1Δ and ede1Δ;

Fig. 5a) do not exhibit accumulation of endocytic inter-

mediates, H2O2-induced endocytosis inhibition in wt cells

may be more severe.

Because many endocytosis mutants exhibit alteration in

the cell wall, which could affect the response to H2O2,

sensitivity assays using calcofluor white were performed

to discard this hypothesis. Mutants with defects in the cell

wall are generally more sensitive to this anionic dye that

interferes with the construction and stress response of the

cell wall (Ram et al., 1994). The results obtained showed

a strong sensitivity to calcofluor for sla1Δ and end3Δstrains, moderate for ede1Δ and bzz1Δ strains, and nor-

mal for the remaining mutants (Fig. 5b). As such, a cor-

relation between cell wall defects and H2O2 sensitivity

was not observed.

Discussion

Internalization of FM4-64 is a fast and straightforward

way to monitor endocytosis in yeast. The uptake of

FM4-64 is time, temperature, and adenosine-5′-triphos-

(a) (b)

(c)

Fig. 3. Endocytosis is an early event in H2O2-induced yeast apoptosis. W303 cells were incubated with 5 mM H2O2 for up to 5 h.

(a) Photomicrographs illustrative of apoptotic markers were obtained with untreated (control) and H2O2-treated cells for 5 h. Bar,

10 lm. (b) Quantification of endocytosis inhibition and apoptotic markers is expressed as percentage of total cells. (c) Quantification

of cell viability. In (b) and (c), values are mean ± SE (n = 3).


4 C. Pereira et al.

phate (ATP)-dependent and can be divided into two

stages: movement from the plasma membrane to endocy-

tic intermediates (stage 1) and from endocytic intermedi-

ates to the vacuole (stage 2; Ram et al., 1994). Herein, it

is shown for the first time that H2O2 prevents both

stages, stage 2 for low concentrations and stage 1 for

higher concentrations. Recovery of endocytic trafficking

upon H2O2 removal indicates that endocytosis is not per-

manently damaged but instead inhibited/delayed. Inhibi-

tion of endocytosis can be correlated with loss of

clonogenicity, and it occured after ROS production but

before chromatin condensation or DNA fragmentation.

Chromatin condensation and specially DNA fragmenta-

tion are typically late apoptotic events, which occur after

(b)(a)

Fig. 4. H2O2 sensitivity for endocytosis mutants. (a) Schematic illustration of the role of the proteins under study in the endocytic pathway. Ede1

is an early factor with a role in the initiation of endocytic sites. It is followed by the assembly of the clathrin coat promoted by Ent1, followed by

actin recruitment. This is promoted by a complex containing End3 and Sla1 (among others). Sla1 is a negative regulator preventing premature

actin assembly. Its inhibition may be lifted by the competitive binding of Bzz1. Bzz1 along with actin generated tension and proteins like Rvs161

promotes invagination and vesicle scission. After vesicle release, the endocytic coat is disassembled and the endocytic vesicle becomes associated

with actin cables and moves into the cell. The endocytic vesicle then fuses with early endosomes, a process promoted by Vps21. Early endosomes

matures into late endosomes, Ypt7 is recruited and finally along with Vps41 participates in the fusion with the vacuole (adapted from Boettner

et al., 2011; Lachmann et al., 2011). (b) Quantification of viability in wt (BY4741) and endocytosis mutant strains (deleted in indicated proteins)

treated with H2O2 for 1 h. Values are mean ± SE (n = 4).

(a) (b)

Fig. 5. Depiction of the endocytic mutants for fluid-phase endocytosis and sensitivity to calcofluor white. (a) Representative photomicrographs of

wt (BY4741) and endocytosis mutant strains (deleted in indicated proteins) stained with FM4-64 (left side) and respective bright-field images

(right side). Bar, 10 lm. (b) Sensitivity to calcofluor white. Wt and endocytosis mutant strains (deleted in indicated proteins) were spotted on

Sabouraud plates containing 0 (data not shown) or 50 lg mL�1 of calcofluor white and monitored after 2 days of growth. For sla1Δ and end3Δ

cell concentrations were adjusted to compensate the growth defects.


Endocytosis impairment during H2O2-induced apoptosis 5

the majority of cells have already lost clonogenicity

(Pereira et al., 2007). Additionally, endocytosis inhibition

occurred almost simultaneously with mitochondrial net-

work fragmentation that is an early dysfunction. Because

both of these processes are highly dependent on the actin

cytoskeleton (Pereira et al., 2010; Scita & Fiore, 2010),

and it is known that H2O2 causes actin depolymerization

(Vilella et al., 2005), a process with a role in apoptosis

progression in yeast and other organisms (Leadsham

et al., 2010). This suggests that actin damage may be

involved in the observed endocytic inhibition. This

hypothesis is supported by the fact that the endocytic

mutants with a strong sensitivity to H2O2, rvs161Δ and

end3Δ lead to strong defects in actin organization

(Benedetti et al., 1994; Munn et al., 1995). Although

Slap1 is also involved in actin organization, it plays a dis-

tinct role from Rvs161p and End3. In fact, unlike

Rvs161p and End3, Sla1p is an inhibitor of the actin

polymerization process (Holtzman et al., 1993). Because

endocytic inhibition was also observed in response to ace-

tic acid and was further reported for ethanol and heat

shock (Meaden et al., 1999), it may be a cellular dysfunc-

tion common to several stressors in yeast.

As H2O2-dependent changes in endocytic trafficking in

yeast can be compared with those reported for mamma-

lian cells, the knowledge provided in this study may also

contribute to the understanding of the toxicity mecha-

nisms of oxidative stress in human diseases.

Acknowledgements

We thank B. Westermann for providing the plasmid

YX232-mtGFP. This work was supported by FCT through

REQUIMTE (grant no. PEst-C/EQB/LA0006/2011) and

C. Pereira (SFRH/BPD/44209/2008) fellowship.

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Endocytosis inhibition during H2O2-induced apoptosis in yeast

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