Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae
Biotechnology for Biofuels
Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae
Sandra A Allen 0
William Clark 0
J Michael McCaffery 2
Zhen Cai 0
Alison Lanctot 0
Patricia J Slininger 1
Z Lewis Liu 1
Steven W Gorsich 0
0 Biology Department, Central Michigan University , Mt Pleasant, MI 48859 , USA
1 National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture , Peoria, IL 61604 , USA
2 Integrated Imaging Center, Department of Biology, Johns Hopkins University , Baltimore, MD 21218 , USA
Background: Biofuels offer a viable alternative to petroleum-based fuel. However, current methods are not sufficient and the technology required in order to use lignocellulosic biomass as a fermentation substrate faces several challenges. One challenge is the need for a robust fermentative microorganism that can tolerate the inhibitors present during lignocellulosic fermentation. These inhibitors include the furan aldehyde, furfural, which is released as a byproduct of pentose dehydration during the weak acid pretreatment of lignocellulose. In order to survive in the presence of furfural, yeast cells need not only to reduce furfural to the less toxic furan methanol, but also to protect themselves and repair any damage caused by the furfural. Since furfural tolerance in yeast requires a functional pentose phosphate pathway (PPP), and the PPP is associated with reactive oxygen species (ROS) tolerance, we decided to investigate whether or not furfural induces ROS and its related cellular damage in yeast. Results: We demonstrated that furfural induces the accumulation of ROS in Saccharomyces cerevisiae. In addition, furfural was shown to cause cellular damage that is consistent with ROS accumulation in cells which includes damage to mitochondria and vacuole membranes, the actin cytoskeleton and nuclear chromatin. The furfuralinduced damage is less severe when yeast are grown in a furfural concentration (25 mM) that allows for eventual growth after an extended lag compared to a concentration of furfural (50 mM) that prevents growth. Conclusion: These data suggest that when yeast cells encounter the inhibitor furfural, they not only need to reduce furfural into furan methanol but also to protect themselves from the cellular effects of furfural and repair any damage caused. The reduced cellular damage seen at 25 mM furfural compared to 50 mM furfural may be linked to the observation that at 25 mM furfural yeast were able to exit the furfural-induced lag phase and resume growth. Understanding the cellular effects of furfural will help direct future strain development to engineer strains capable of tolerating or remediating ROS and the effects of ROS.
The continued use of fossil fuels has raised
environmental, economical and political concerns and, as a result,
research into improving alternative and renewable
energy strategies is of great importance. Bioethanol is
one such alternative energy source. Most bioethanol
produced today takes advantage of ethanologenic
microorganisms fermenting agricultural products such as
cornstarch or sugar cane. Starch and sugar cane sources
are currently being used to produce competitively priced
ethanol in countries such as Brazil, Canada and the
USA. Unfortunately, these sources are not sufficient to
supply the world bioenergy needs due to the role they
play in human and livestock consumption [
]. Thus, the
goal of having a bioethanol fuel economy must include
in its vision the use of lignocellulosic-biomass waste
from agriculture, forests, industry and the municipalities.
Current technologies make the use of
lignocellulosicbiomass inefficient. However, programs using
agricultural and softwood biomass are currently producing
ethanol in Sweden, the USA and Canada, with the later
having established a committed plant for the production
of bioethanol from lignocellulose [
In order to release fermentable sugars from
lignocellulosic biomass, a weak acid pre-treatment step is often
employed. However, this process generates fermentation
inhibitors, which include aldehydes (furan aldehydes),
ketones, phenolics and organic acids [
]. Two furan
aldehydes are 2-furaldehyde (furfural) and
5-hydroxymethylfurfural (HMF), which are degradation products
of xylose and glucose, respectively. In order to protect
themselves yeast reduce these furan aldehydes to their
less toxic alcohol derivatives, furan methanol and furan
dimethanol, in NAD(P)H-dependent reactions. This
conversion occurs during the growth lag phase when
ethanol production and many enzymes are inhibited
]. Once these inhibitors are reduced, growth
resumes. In addition to detoxifying the furan aldehydes,
yeast cells must survive the toxic effects and repair any
damages caused by them. However, little is known
about the toxic effects of furan aldehydes on cells.
The NADPH producing pentose phosphate pathway
(PPP) plays an essential role in furfural tolerance [
When single PPP genes (ZWF1, GND1, TKL1 or RPE1)
are absent, yeast, that would normally allow growth
after a 24 hour lag, are unable to grow when
concentrations of furfural (25 mM) are present [
greatest growth defect is seen when the ZWF1 gene is
disrupted. ZWF1 encodes glucose-6-phosphate
dehydrogenase, which catalyzes the rate-limiting step of the
PPP and produces NADPH. This growth defect is
probably not due to an inability to reduce furfural, as
furfural can be reduced using NADH. However, the
PPP’s NADPH is also an important co-factor used to
protect cells against cellular stress caused by reactive
oxygen species (ROS).
ROS are generated in cells as metabolic byproducts,
the accumulation of which can be increased by
environmental conditions, genetic mutations and cell ageing
]. ROS include hydrogen peroxide (H2O2),
superoxide anion (O2 -), and the hydroxyl radical (OH ). ROS
are known to damage DNA, proteins, lipids and the
cytoskeleton and to induce programmed cell death
]. Cells can protect themselves from ROS by
activating certain genes, such as the PPP’s ZWF1, which
also is an essential gene for furfural tolerance. Yeast
lacking the rate limiting PPP gene, ZWF1, have an
increased sensitivity to ROS [
]. The role of the PPP in
ROS protection is likely due to the NADPH that it
]. The reducing power of NADPH is used by
many stress protection enzymes, such as those encoded
by OAR1, OYE2, TSA1 and GLR1, which encode the
enzymes mitochondrial 3-oxoacyl- [acyl-carrier-protein]
reductase, old yellow enzyme, thioredoxin peroxidase
and glutathione oxioreductase, respectively [
Since the PPP is necessary to protect cells against ROS
and furfural, we proposed that furfural’s role in cellular
toxicity involves ROS related damage. In this study, we
demonstrate that furfural causes an accumulation of
ROS and cellular damage to mitochondria, vacuoles,
actin and nuclear chromatin when healthy and
exponentially growing cells are transferred to furfural. The
damage is less severe in concentrations of furfural (25
mM) that normally allow growth after a 24 hour lag as
opposed to concentrations of furfural (50 mM) that
prevent growth completely (Additional file 1). This reduced
degree of cellular damage may be indicative of why
yeast can survive at lower concentrations of furfural.
Moreover, these data will be useful in the development
of more robust yeast strains.
Results and discussion
Accumulation of reactive oxygen species in cells
Yeast in exponential growth was transferred to media
containing no inhibitor, 25 or 50 mM furfural or 5 mM
hydrogen peroxide. Furfural addition immediately sent
the healthy cells into a growth lag phase even though
there were sufficient nutrients available (Additional file
1). Hydrogen peroxide present in the medium served as
a positive control of ROS. Cell cultures were allowed to
grow at 25°C and aliquots of cells were removed and
stained with the ROS detecting dye, 2’7’- DCF diacetate
(Figure 1). Cells staining positive for ROS were counted
in order to determine the percent of cells containing
accumulated ROS. For each sample at least 100 cells
were examined. At 0 h 4% of the cells had a positive
ROS signal. After 8 h of growth 10% of the cells had a
positive ROS signal with no furfural present. At the
same time point, 31% and 36% of cells stained positive
when 25 and 50 mM furfural were present, respectively.
This was consistent with the 32% of cells exposed to 5
mM hydrogen peroxide, which is a known inducer of
ROS (Figure 1). Interestingly, cells exposed to 50 mM
furfural had an aggregated staining pattern, which is
strikingly different from the even distribution of
fluorescence seen when cells are exposed to 25 mM furfural or
5 mM hydrogen peroxide. We speculate these
aggregates are either aggregated proteins or membranes
damaged by furfural. This extreme staining difference
may be a result of the stronger growth inhibition of
furfural at 50 mM [
] (Additional file 1).
Cellular damages determined by transmission electron
microscopy (TEM) analysis
ROS are known to damage DNA, proteins, lipids, and
the cytoskeleton [
]. We used TEM to test whether
furfural could induce similar internal cellular damage
(Figure 2). Fixed yeast cells, either exposed to 25 mM
furfural or no inhibitors, were processed for thin-section
TEM analysis. When no inhibitor was present,
mitochondrial structures were broadly distributed
around the cell periphery with a typical morphology and
contained internal cristae. In addition, vacuolar
structures were also typical, appearing as single dark
structures with smooth edges. In the presence of 25 mM
furfural, mitochondria appeared highly aggregated and
swollen with less structured cristae and were clustered
towards the cell interior. Though the vacuoles were
about the same size as the untreated cells, their edges
were not smooth, but rather lobular. Interestingly, the
furfural treated cells contained a lightly stained
background as opposed to the untreated cells that had a
clear background. We were unable to identify what this
was but we suspected that it was damaged cytoskeleton
or aggregated proteins; both would be consistent with
]. The nuclear membrane in furfural treated
and untreated cells appeared unaffected (Figure 2).
Interestingly, the yeast external cell wall did not appear
to be affected by either 25 or 50 mM furfural as
observed by scanning electron microscopy (data not
shown). This suggests that, prior to entering the cell,
furfural does not damage the cell wall.
Mitochondrial membrane damage
Yeast (SGY229) cells in exponential growth were either
not treated or treated with 25 mM or 50 mM furfural.
Cells containing a mitochondrial targeted green
fluorescent protein (pVT100U-mtGFP) allowed for
visualization of mitochondria, which usually appear as a tubular
network of membranes localized to the cells cortex [
At 0 h, cells contained the typical tubular shaped
mitochondria in 87% of observed cells (Figure 3, Table 1).
Mitochondria remained tubular in 80% of the untreated
cells at 6 h. However, in the presence of 25 or 50 mM
furfural mitochondria either fragmented evenly (41%
and 45%, respectively) or aggregated to one side of the
cell (9% and 45%, respectively). In the untreated cells
mitochondria remained tubular until 48 h when 79% of
cells contained evenly distributed fragments, which is
typical of mitochondria in cells going from exponential
to stationary growth phase. Cultures treated with 25
mM furfural at 24 h and 48 h contained mitochondria
that were predominately fragmented in 49% and 53% of
the cells. Yeast cultures treated with 50 mM furfural
continued to display predominantly aggregated
mitochondria that were not evenly distributed in 66% of
the cells at 24 h and 100% of the cells at 48 h.
Fragmented and aggregated mitochondria are phenotypes
associated with some yeast mutants like mgm1. These
mutants lose their mitochondrial DNA, making them
respiratory incompetent, and they exhibit poor growth
when dextrose is their carbon source and fail to grow
when glycerol is their carbon source [
]. In addition,
this mutant and its homologue mutants in Drosophilae,
Caenorhabditis elegans, and humans are more sensitive
to ROS [
Our observation that 25 mM furfural caused a less
severe phenotype compared to 50 mM furfural is
consistent with 25 mM furfural treated cells being able to
grow after a 24 h growth lag (Additional file 1). These
data are also consistent with our TEM images of
mitochondrial clusters (Figure 2).
Vacuole membrane damage
Yeast in exponential growth were either not treated or
treated with 25 or 50 mM furfural. At each time point
aliquots of cells were stained with the vacuole dye FM 4-64®
(Figure 4, Table 2). At 0 h, 96% of the cells contained
single and large normal vacuoles, which is the typical
appearance of the yeast vacuole [
]. When no inhibitor was
present vacuoles continued to predominately be a single
and large structure through 48 h. When 25 or 50 mM
furfural was added, vacuoles fragmented into two to four
medium sized vacuoles or greater than four smaller
vacuoles, respectively. As with mitochondria, the vacuoles
fragmented and aggregated more in the presence of 50
mM furfural. Our TEM images did not show fragmented
vacuoles. However, we suspect the lobular shape of the
vacuoles in the TEM images were showing small vacuoles
that were clustered together to produce the lobular affect
(Figure 2). The effect of furfural-induced vacuole
fragmentation is not clear, but it is known that drugs such as
nocodazole cause vacuoles to fragment, probably
Figure 3 Furfural causes mitochondrial membrane morphology
to go from tubules to aggregates. Exponentially growing yeast
cells expressing mitochondrial targeted green fluorescent protein
were untreated or treated with 25 mM or 50 mM furfural.
Representative images of yeast with no inhibitor (left column;
tubular), exposed to 25 mM furfural (middle column; evenly
distributed fragments) or 50 mM furfural (right column; aggregated)
are shown. Images of cells were taken 6 h after furfural treatment.
Prior to adding furfural, 87%, 12% and 1% of cells contained tubular,
fragmented and aggregated mitochondria, respectively. Data represent
averages of three experiments with standard error indicated. At least 100 cells
were examined at each time point.
* Tubular mitochondria are a network of evenly distributed tubules that are
often connected; fragmented mitochondria are small evenly distributed
spherical structures; aggregated mitochondria are small and clustered
spherical structures that are not evenly distributed.
associated with this drugs affect on microtubules [
Interestingly, vacuole fragmentation is also seen with some
mutants such as vac8, which also affects cellular
]. Whether or not furfural causes a block in
endocytosis or if blocking endocytosis helps protect yeast
is not known.
Nuclear chromatin disorganization
TEM analysis did not reveal any obvious nuclear
external damage (Figure 2). However, we were interested in
whether or not the nuclear chromatin inside the nucleus
was damaged. Yeast nuclei exposed to ROS are known
to become less compacted and appear larger and more
diffuse when stained with the DNA specific dye, DAPI
]. In order to test this in our furfural treated cells
we grew cells to exponential phase and treated them
with 25 or 50 mM furfural or with no inhibitor. At
various time points aliquots of cells were removed and
stained with DAPI. At 0 h, 7.5% of the cells contained
abnormal diffuse nuclear chromatin while the remaining
cells contained chromatin that appeared as normal
tightly compacted spheres. Upon adding 25 or 50 mM
furfural the nuclear chromatin became disorganized and
diffuse in 18.5% and 21.5% of the cells at 6 h,
respectively, and 11% and 23% of cells at 24 h, respectively
(Figure 5). Our DAPI observations are consistent with
cells undergoing ROS induced stress [
]. It is
interesting that by 24 h our data suggests that the nuclear
chromatin damage is recovering faster than the
mitochondrion and vacuole membrane damage (Figures 3
and 4). This difference in the time required to repair
each substrate (membrane versus chromatin) could be
linked to how fast the cell is capable of repairing
different cell components, the degree of damage to each cell
component or, possibly, the cell recognizes the
importance of repairing the DNA containing chromatin.
Together, our TEM and DAPI data suggest that furfural
does cause chromatin damage without causing obvious
nuclear structural damage.
Actin cytoskeleton damage
The actin cytoskeleton in a healthy growing yeast cell
will contain long thin actin cables in the mother cell
that extend into the daughter bud where they end as an
actin patch [
]. TEM analysis did not provide clear
evidence of any actin structure damage induced by furfural
(Figure 2). However, since actin damage is seen in ROS
induced apoptosis [
], we investigated the actin
structures in cells exposed to furfural. Yeast in exponential
growth were either not treated or treated with 25 or 50
mM furfural. At various time points aliquots of cells
were removed and their actin cytoskeleton stained with
Alexa Fluor® 568 phalloidin. At 0 h, 67% of the cells
contained actin structures consistent with normal
growth (actin cables in the mother cell and patches in
the daughter bud) and 33% lacked cables and only had
patches (Figure 6). In cell cultures without inhibitor
normal actin structures remained in most of the cells up
until 24 h. By 48 h many of these cells contained
abnormal actin, which is consistent with our previous
observations of cultures at stationary growth phase (data not
shown). Cultures containing 25 or 50 mM furfural
contained predominantly abnormal actin structures
(over 70% with only actin patches) through 48 h. The
25 mM treated cells contained less abnormal actin
compared to the 50 mM treated cells by 48 h. However, the
amount of abnormal actin from 6 h to 48 h in the 25
mM treated cells does not change significantly.
Mitochondria and vacuole membrane damage (Tables 1 and
2) appears to recover faster than actin damage. The
significance of this observation is unclear. Perhaps, since
actin cables are needed for cell budding, yeast cells do
not expend energy making actin cables until other
components are repaired and the cell is ready to bud.
Alternatively, actin patches are known to play a role in
endocytosis. Whether or not endocytosis is important in
furfural tolerance is not known. However, it is
noteworthy that the gene, BRE4, has been linked to both
endocytosis and furfural tolerance [
]. This link
needs to be further investigated.
Prior to this study it was not known if furfural causes
oxidative or internal cellular damage. The
lignocellulose-derived inhibitor, furfural, prevents yeast cells from
growing and producing ethanol until furfural is reduced
to furan methanol by NAD(P)H-dependent reactions.
However, what was happening to these yeast cells while
furfural was being reduced was not known. We show
that furfural does cause an accumulation of reactive
oxygen species (ROS) (Figure 1) and damages
mitochondrial and vacuole membranes (Figures 2, 3, 4), nuclear
chromatin (Figure 5) and the actin cytoskeleton (Figure
6). This is consistent with known targets of ROS
]. Surprisingly, preliminary results indicate that
furfural-induced ROS did not cause programmed cell
death (data not shown), which is often the final
consequence of ROS [
]. These programmed cell death
experiments are being further investigated. Moreover,
we suspect that the damage to the cell is only present
inside the cell, as no obvious damage to the external cell
wall was detected by scanning electron microscope
(SEM) analysis (data not shown). Alternatively, furfural
may damage the external side of the cell, but it remains
undetected by the current SEM assay.
Developing future furfural tolerant yeast strains will
probably involve one of two strategies. The first is to
engineer strains for the improved conversion of furfural
to furan methanol. A potential target gene for improved
conversion is YGL157W, which encodes an aldedyde
]. A similar strategy proved successful
when Petersson et al. (2006) overexpressed ADH6
(alcohol dehydrogenase) to improve HMF reduction [
The second strategy is to develop a strain that is able to
tolerate or remediate ROS and ROS-induced cellular
damage more effectively. Potential target genes to
engineer for an improved robustness include genes known
to function in stress tolerance such as OAR1, TSA1 and
]. In order to achieve maximal furfural
tolerance it is probable that both increased furfural
conversion and ROS tolerance will need to be considered in
future strain development strategies.
Yeast growth conditions and reagents
The budding yeast, S. cerevisiae, were grown using
standard laboratory conditions [
]. All standard
chemicals were purchased from Sigma-Aldrich (MO, USA).
For all experiments, strains derived from FY10 were
used . These include BY4741 (SGY110) (MATa
his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and SGY229 (MATa
his3Δ1 leu2Δ0 met15Δ0 ura3Δ0+ pVT100U-mtGFP).
BY4741 is a standard wild-type lab FY strain (S288C)
purchased from Open Biosystems (AL, USA). SGY229 is
BY4741 that contains plasmid pVT100U-mtGFP. The
plasmid pVT100U-mtGFP contains the URA3 gene as
an auxotrophic marker and encodes a mitochondrial
targeted GFP protein, which enables mitochondrial
]. Yeast were grown at 25°C in 3 ml of
medium with minimal shaking in 15 ml screw-capped
plastic centrifuge tubes (VWR 89039-664) in order to
provide a less aerobic environment that better mimics
industrial ethanol fermentation. For all experiments,
except the mitochondrial studies, yeast was grown in
liquid yeast extract-peptone-dextrose (YPD; 2% Bacto
peptone; 2% dextrose; 1% yeast extract; pH 5.5). In
mitochondria observation experiments yeast were grown
in either synthetic defined (SD)-complete or SD-URA
(synthetic medium composed of 0.67% yeast nitrogen
base, 2% dextrose, and supplemented with nucleic acids
and amino acids; pH 5.5) at 25°C. Yeast transformation
was performed using a standard lithium acetate
transformation protocol [
]. Exponentially growing yeast were
treated with 0 mM, 25 mM or 50 mM fresh furfural
(Sigma-Aldrich 185914) stored under nitrogen. For the
ROS experiments, 5 mM hydrogen peroxide
(SigmaAldrich H1009) was added to exponentially growing
yeast. This served as a positive control for ROS [
Aliquots of cells were removed for analysis at various
time points from 0 h to 48 h.
For SEM yeast were put through an ethanol dehydration
series. The samples were left in each step for 10 min at
concentrations of 30%, 50%, 70%, 95% and three
changes of 100% ethanol. Cells were collected onto
membrane filters with 0.22 micron holes, critical point
dried from CO2 with three different 10-min soaks and 2
min purges and transferred onto stubs with carbon tape.
Cells were then analysed using a JEOL 840A SEM. TEM
was performed as described in Rieder et al. using a
JOEL 12 EX TEM [
Fluorescence microscopy and cellular analysis
All fluorescence microscopy was performed using either
a Nikon 80i eclipse fluorescent light microscope or an
Olympus Fluoview 300 confocal microscope. Depending
upon the assay, one of the following three fluorescent
filters was used: FITC HYQ fluorescence filter (460-500
nm); TX RED HYQ fluorescent filter (532-587 nm); and
ultraviolet filter (325-375 nm).
ROS were measured by adding 10 μg of 2’
7’-dichlorofluorescein diacetate (DCF) (Sigma-35845) (using a 2.5
mg/ml stock in ethanol) to 107 cells and incubated at 30°C
for 2 h. Cells were washed with 1 ml of distilled water and
resuspended in 0.1 ml phosphate buffered saline (PBS) pH
]. Cells were observed using the FITC HYQ filter.
For each time point at least 100 cells were examined.
In order to view the mitochondrial membranes we
transformed pVT100U-mtGFP into BY4741 yeast.
Transformants with this plasmid express a
mitochondrial targeted GFP (pVT100U-mtGFP) [
] that allows
the direct visualization of mitochondria in living cells.
Mitochondria were visualized using a FITC HYQ
fluorescence filter (460-500 nm). Mitochondria were
classified in one of three categories: tubular, fragmented or
aggregated. Tubular mitochondria appear as a network
of evenly distributed tubules that are often connected.
Fragmented mitochondria appear as small spherical
structures that are evenly distributed throughout the
cell. Aggregated mitochondria appear as small spherical
structures that are clustered together and are not evenly
distributed in the cell. For each time point at least 100
cells were examined.
Vacuole morphology was visualized by taking 107 cells
and resuspending them in 250 μl YPD + 80 mM FM
464® in dimethyl sulphoxide (Invitrogen-Molecular Probes
T13320, CA, USA). FM 4-64 is a lipophilic vital stain
that becomes internalized and collects in vacuoles. Cells
were incubated at 30°C for 30-60 min, collected and
resuspended in 5 ml of YPD in a shake flask and
incubated at 30°C for 90-120 min. Cells were collected and
washed once with 5 ml of sterile deionized water. They
were then resuspended in 25 ml yeast nitrogen base
(0.67% ; pH 5.5). Vacuole stained cells were visualized
using the TX RED HYQ filter (532-587 nm) [
each time point at least 100 cells were examined.
Nuclear chromatin was visualized by taking 0.2 ODs
(A600; 1 cm) of cells and washing them one time with
deionized water. Cells were fixed by resuspending them
in 10 μl of deionized water and 190 μl of 100% ethanol.
One μl of a 2 mg/ml diaminophenylindole (DAPI;
Roche 10236276001, CA, USA) solution in deionized
water was added to the fixed cells and gently mixed.
Cells were then immediately collected and washed three
times with 200 μl deionized water. In the final wash
cells were resuspended in 50 μl of deionized water.
Nuclear chromatin was visualized using a UV-2E/C filter
(325-375 nm) [
]. Nuclear chromatin was classified as
either a tightly compacted sphere that covered a small
part of the cell or as a diffuse structure that covered a
large part of the cell. For each time point at least 100
cells were examined.
The actin cytoskeleton was visualized by taking 107
cells and resuspending them in 50 μl of 37%
formaldehyde and incubating for 15 min at 25°C. Cells were
collected and resuspended in a second fix solution (50 μl
of 37% formaldehyde and 500 ml of PBS (pH 7.0) and
incubated at room temperature for 1 h at 150
revolutions per minute. Cells were collected and washed three
times in 100 μl of PBS. In the final wash cells were
resuspended in 30 μl PBS and 8 μl of Alexa Fluor® 568
phalloidin dissolved in ethanol (Invitrogen-Molecular
Probes A12380), which is a high affinity probe specific
for F-actin. Cells were incubated at 4°C for 1 h in the
dark and then washed with 50 μl of PBS and
resuspended in a final volume of 50 μl PBS. The actin
cytoskeleton was visualized using the TX RED HYQ
fluorescent filter (532-587 nm) [
]. Actin cytoskeleton
structures were classified as either normal or abnormal.
Normal actin cytoskeleton contained tubules in the
mother cell that extended to he bud where they
terminated as circular stained patches. Abnormal actin
cytoskeleton lacked tubules and contained large actin patches
throughout the mother cell and bud. For each time
point at least 100 cells were examined.
Additional file 1: Figure S1 - Furfural causes exponentially growing
yeast to enter a growth lag phase. Exponentially growing yeast cells in
synthetic complete medium were either untreated (circle) or treated with
25 mM (square) or 50 mM furfural (triangle) and allowed to continue to
grow at 30°C. At the indicated time points aliquots of cells were
removed and cell density measured (A600).
Click here for file
DCF: dichlorofluorescein diacetate; GFP: green fluorescent protein; NADPH:
nicotinamide-adenine-dinucleotide phosphate (reduced form); PBS:
phosphate buffered saline; PPP: pentose phosphate pathway; ROS: reactive
oxygen species; SD: synthetic defines; SEM: scanning electron microscopy;
TEM: transmission electron microscopy; YNB: yeast nitrogen base; YPD: yeast
We are grateful to Sarah Lubitz and Megan Bolen for their assistance in SEM
experiments and Phil Oshel in the microscopy facility at Central Michigan
University for his assistance. This work was funded by Research Excellence
Funds and Faculty Research and Creative Endeavors grants from the Office
of Research and Sponsored Programs at Central Michigan University.
SAA performed the majority of the experiments including ROS analyses and
mitochondria, vacuole and chromatin damage studies. SAA also assisted
with analyses and manuscript writing. WC performed the actin staining and
analysis of those data. JMM performed all of the TEM and assisted in the
analysis of those data. ZC assisted with ROS and cell death experiments. AL
assisted with image gathering and analysis. PJS and ZLL contributed to data
analysis and manuscript revision. SWG directed the study and wrote the
manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
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