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ANALYSIS OF CELLULAR RESPONSES TO HEAVY METAL-INDUCED STRESS IN Saccharomyces
cerevisiae
Pei-Ju Chin
Molecular Genetics and Biochemistry ProgramDepartment of Biology
Georgia State UniversityAtlanta, GA 30303
Research Interest in Houghton Lab
Model Organism: Saccharomyces cerevisiae (baker’s yeast)
Cellular response with heavy metal treatments Chromium (Cr), Cadmium (Cd) and Copper (Cu)
Oxidative Stress
Metal Exposure
Apoptosis Autophagy
Cell Fate Decision
Heavy Metals- Beneficial but Harmful
Widely used in industries
Electroplating
Anti-corrosion
Rechargeable batteries
Hybrid/Electric car
Painting
Yellow color given
Photo diode (CdS)
Photo drum
Light sensor in smart phone
Improper disposal harms the public health
Itai-Itai (Pain-Pain) Disease-Pandemic, Accidental Cadmium Exposure
1945
Kakioma mine with zinc ore
Waste was flushed to Jinzu river
Weaken bone and joint
Kidney failure
Courtesy of Kanazawa Medical University, Japan and University of California, Santa Cruz, USA
Yeast Is Chosen as the Model System for the Simplicity and Homology with Higher Eukaryotes
Advantages
Easy to handle and manipulate
Annotated genome
Available mutant library
Available GFP-tag library
Homology
Homology with higher eukaryotes
Apoptosis and autophagy pathway is well-investigated Chin , PJ and Houghton, JE. Unpublished data
Why is Yeast Chosen as the Model System?
Braun RJ (2012) Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Frontiers in oncology 2:182.
Environmental Stress Triggers Apoptotic Responses by Accumulating ROS Inside Cells
Madeo F. et. al. 2004. Cur. Opin. Microbiol. 7:655-660
Metallicion
Metal-induced ROS Generation- Direct and Indirect
Metals
Redox-active Redox-inactive
Directly generate
ROS
•Indirectly generate ROS
•Metal replacement from metalloenzymes
•Deplete antioxidant defenses
Cu, Cr, Fe Cd, Pb, Hg
Fenton ReactionCu2+ + H2O2 →Cu3+ + OH·+ OH−
Hanas, JS. And Gunn CG. Nucleic Acids Res. 1996, 24, 924-930
Avery. Adv Appl Microbiol. 2001;49:111-42
Metals and other oxidant stressors such as H2O2 generate ROS
(reactive oxygen species – superoxide, peroxide, hydroxyl radicals)
Highly Reactive ROS Oxidizes Cellular Components
Metal-induced Response in S. cerevisiae-Acute and lethal
• 30 μ M Cd(NO3)2• 8mM Cu(NO3)2
Seedingculture
100mLYEPD
O/N
incubation
2OD600
1 hour
DIC
PI
Merge0.1
1
10
BM
H1
BM
H2
CD
C25
DN
M1
ER
F2
FIS
1
HX
T17
LTE
1
PH
B1
PH
B2
RT
G2
SH
R5
Fo
ld c
ha
ng
es
Cd-30 minsCd-60 mins
0.1
1
10
AS
F1
BIR
1C
DC
48
FU
N34
HX
T17
IFM
1K
AP
120M
CA
1O
AF
1R
SM
23R
TT
107
SN
L1
ST
M1
SV
F1
SX
M1
UT
H1
WW
M1
YO
P1
Fo
ld c
ha
ng
es
Cd-30 mins
Cd-60 mins
24~48 hours
Cd 1+3Cu 1+3
Post-incubate
3 hours
Wash
Yeast Genome Pathway Analysis, 2006
The Presence of Reduced-glutathione (GSH) Indicates Cell Suffering from Oxidative Stress
0.1
1
10
100
BU
D9
FB
A1
FB
P1
GN
D1
GN
D2
PF
K1
PF
K2
PG
I1P
GM
1P
GM
2P
RS
1P
RS
2P
RS
3P
RS
4P
RS
5R
BK
1R
KI1
RP
E1
SO
L1
SO
L3
SO
L4
SP
C1
TA
L1
TK
L1
TK
L2
TK
L2
VH
S1
ZW
F1
Fo
ld c
han
ges
Cd-30 mins Cd-60 mins
0
5
10
15
20
25
30
35
40
0 5 15 30 60 90
Rel
ativ
e co
nce
ntr
atio
n (
uM
/A)
Exposure time (min)
GSH (Reduced)GSSG (Oxidized)
Antioxidant Defense Mechanism upon Cadmium Exposure
ZWF1 and GND1 are induced
GSH/GSSG increases during Cd exposure
1
10
GND1 ZWF1
Fo
ld C
ha
ng
es
Cd-30 mins
Cd-60 mins
Post-Exposure Cell Fate Decision
Survival Route Antioxidant Defense Mechanism (eg. GSH) Autophagy
Elimination of harmful or damaged molecules
Suicide Route Damage is far beyond recoverable Altruistic, restrain the dissemination of damage to
entire population Programmed Cell Death/Type-I Cell Death Autophagy-associated cell death/Type-II Cell Death
Apoptosis-A Suicide Program with Tight Regulation
Programmed Cell Death Under control Irreversible
A mechanism to remove abnormal or unhealthy cells Embryogenesis Infection Damaged cells which cannot be
repaired
Morphological characteristics Cell shrinkage, appearance of
apoptotic bodies Chromatin condensation,
hyperpolarization of mitochondria, increase of membrane permeability, accumulation of reactive oxygen species (ROS)
Caspase-dependent Apoptosis
Courtesy of Philip Yau
Zoli et al. Breast Cancer Res. 7:R681
Classification of Cysteine-dependent Asparate-directed Proteases (Caspases)
MacKenzie, S.H. and Clark A.C. Death by Caspase Dimerzation. Protein Dimerization and Oligomerization in Biology. ISBN: 978-1-4614-3228-9. Landes Bioscience
Yeast Caspase 1 (Yca1p), a Metacaspase with Caspase 3-like activity
Wong, AH et. al. 2012. J Biol Chem 287: 29251-29259
Like its executioner orthologues, Yca1 is inactivated until the pro-domain is cleaved
Cell-Death
Pro
Cell-Death
Pro
ApoptosisCascade
Heavy Metals Such as Cadmium Results in the Accumulation of ROS and the Activation of Yca1p, an Only-known Caspase in Yeast
30 μM Cd 0+4 hrs 30 μM Cd 1+3 hrs
DHR
SR_FLICA
Nargund A. et al. 2008. Apoptosis 13: 811-821
Yca1p appears to have DEVD like activity.
Zvad-fmk Proves a Second Caspase-like Activity
IETD and DEVD activity
Nargund, A. Unpublished data
Pan-CaspaseInhibitor
Cysteine proteases in yeast
1. RIM13 – calpain like protease
2. ATG4 – involved in autophagy3. YCA1 – known function - yeast caspase4. SNO4
5. HSP33
6. HSP32
7. ESP1 – known function-acts as separase8. OTU2
Candidates for Second Caspase
Atg4p, second caspase-like protease invloved in Cd –induced apoptosis
IETD and DEVD activity
ATG4 shows very little IETD like activity in Cd treated YCA1Δ
Defensive Mechanisms before Triggering PCD
Antioxidant Defense Mechanism Superoxide dismutase (SOD), peroxidase, reduced glutathione
(GSH)…
Lysosomal/Ubiquitinylation Proteasome System (UPS) Clean up misfolded, damaged or harmful proteins
Stress granule (SG) formation Stalled translational pre-initiation complex (eIF2α, eIF4G,
PABP…)
Quick response after cell has recovered
Autophagy Degrade damaged organelles (eg. Mitochondria)
Autophagy (mitophagy) can lead to PCD as well (autophagic cell death/Type-II cell death)
Autophagy (Self-Eating) –Cellular Homeostasis Mechanism
Klionsky DJ et al. 2007. Autophagy 3:5 413-416
Are autophagic initiators required for apoptotic response in S. cerevisiae?
Aim I.
0.1
1
10
100A
TG
1
AT
G2
AT
G3
AT
G4
AT
G5
AT
G6
AT
G7
AT
G8
AT
G9
AT
G10
AT
G11
AT
G12
AT
G13
AT
G14
AT
G15
AT
G16
AT
G17
AT
G18
AT
G19
AT
G2
0
AT
G2
1
AT
G2
2
AT
G2
3
AT
G2
4
AT
G2
6
AT
G2
7
AT
G2
9
AT
G31
AT
G32
AT
G33
Fo
ld C
ha
ng
es n
orm
alize
d b
y u
ntre
ate
d
Cd 30 mins
Cd 60 mins
Autophagic Genes Are Induced upon Cadmium Exposure in S. cerevisiae
1
10Fo
ld C
ha
ng
es N
orm
alize
d b
y u
ntre
ate
dCd 30 mins
Cd 60 mins
J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86
(Atg6)
Atg4p, Atg6p and Atg8p are Involved in the
Maturation of Autophagosome Biosynthesis
J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86
Are the Autophagy Initiators required for Yca1p cleavage?
Chin, P.J. and Houghton, J.E. Unpublished data
Are the Autophagy Initiators required for Apoptotic Response in Yeast upon Metal Exposure?
Chin, P.J. and Houghton, J.E. Unpublished data
Population of propidium iodine (PI) positive
Summary of Aim I
Atg4p contributes to Caspase 8-like activity (Nargund A. Unpublished data)
Autophagic initiators: Atg4p, Atg6p and Atg8p are required for Yca1p-dependent apoptosis upon metal exposure
Does autophagosome apparatus facilitate Yca1p activation?
Aim II.
Yca1p Is Insoluble in vivo
Natural property per se (unlikely) E. coli-expressed His-tagged Yca1p is soluble
Be sequestered in vacuoles
[NP-40]
Yca1p is Trans-localized to Acidic Vacuoles upon Cadmium Treatment
DIC Yca1-GFP
MDC(Acidic vacuoles including
autophagosome )
Crude Isolation of Autophagosome
To investigate the presence of Yca1p in autophagosome
Yca1-GFP strain instead of wild type was used Anti-Yca1 WB requires specialized
lysis condition (detergent)
Anti-GFP was used for WB
Copper, instead of cadmium was used Yca1-GFP strain reacts poorly with
cadmium treatment
Ficoll15%
Ficoll8%
Ficoll4%
Ficoll0%
Autophagosome
Cell debrisInsoluble Fraction
Yca1p-GFP was Absent from Debris Sedimentation after Cu Treatment
25%
50%
100%
Fic
oll
0-4
%
Fic
oll
4-8
%
Sed
imen
tati
on
Yca
1-G
FP
ex
pre
ssio
n le
vel
Cu 0+4
Cu 1+3
(GAPDH-GFP)
Summary of Aim II.
Yca1p-GFP was present in autophagosome in both Cu-untreated or treated cells Autophagosome vacuoles sequester
the activity of Yca1p
Autophagosome facilitates the process of Yca1p
Yca1p-GFP was absent from the cell debris fractionation after Cu treatment Solubility of Yca1-GFP is increased
after Cu treatment
Accessible for initiators/self-cleavage process
Autophagosome
Yca1p
Cu/Cd
Soluble
insoluble
Is autophagy a life-saver or kiler in yeast undergoing heavy metal-induced oxidative stress ?
Aim III.
Intercommunication of autophagy and apoptosis in higher eukaryotic cells-Autophagic proteins as cytoprotector
Autophagy induces cytoprotection in neuron cells by removing aggregated huntingtin (Hara T. et. al. Nature 441:885-889)
Atg6 helps to resolve DNA-damaged foci (Mathew R. et. al. Genes Dev:21 1367-1381)
C. elegans lives longer in caloric restriction condition while autophagy plays an essential part (Jia K. et. al. Autophagy 3:597-599 )
Life span is prolonged in yeast and delay of chronological aging by rapamycin (Alvers et al. Autophagy 5:847-849)
ROS reduces the activity of Atg4 to de-lipidize Atg8-PE,therefore facilitates autophagy (Scherz-Shouval R. et. al. EMBO J 26:1749-1760)
Intercommunication of autophagy and apoptosis in higher eukaryotic cells-Autophagic proteins facilitate apoptosis
Atg12 cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to Bcl-2 and causes apoptosis (Rubinstein AD et. al. Mol Cell 44:698-709)
Atg4D cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to mitochondria, release cytochrome C and causes apoptosis (Virginie et. al. Autophagy 5: 1057-1059)
Atg6 cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to mitochondria, release cytochrome C and causes apoptosis while defects its role to conjugate Atg5-Atg12(Luo S et. al. Cell Death Differ 17:268-277)
Autophagy selectively eliminates catalase and causes the accumulation of ROS (Yu L et. Al. PNAS 103:452-4957)
Distinguish pathways exist in S. cerevisiae
No Bcl-2.
No potent caspase-3 cleavage site of autophagic proteins
No BH3 domain in autophagic proteins
Autophagy, A Life-saver or Killer?
Debates do exist
Cell type-dependent Cells from different tissue or genotype
Autophagy inducer/inhibitor -dependent Rapamycin, 3-MA, bafilomycin A1
Stress type-dependent Hypoxia, starvation, ROS, misfold protein accumulation
Time/Occasion-dependent When to introduce autophagy and apoptosis
What if the role of autophagy is decided by the stage it is introduced?
Rapamycin Induces Autophagic Response by Inhibiting the Hyper-phosphorylation of Atg13p Caused by TORC1
Rapamycin-induced Autophagy is Demonstrated by the Increased Atg8-PE to Atg8 Ratio in Dose-independent Manner
Kiel Jan A. K. W. 2010. Phil. Trans R. Soc. 365: 819-830
[rapa]
Treatment Schema
Independent Events Autophagy only
Apoptosis only
Two-Stage Events Autophagy->Apoptosis
vice versa
Overlapping Events Treatment A-D
The Duplicitous Role of Autophagy in S. cerevisiae
Rescue Introduced prior to Cd
High autophagic activity
Kill Introduced after Cd
Basal autophagic Activity
One activity impedes another
Autophagy Facilitates Cd-induced Apoptotic Response-Type-II Cell Death
All PI has MDC Type-II CD
Not all MDC has PI Survival?
Summary of Aim III.
Duplicitous role of autophagy Autophagy prior to Cd-induced apoptosis: Protective
Autophagy follows Cd-induced apoptosis: Destructive
The trend of apoptosis and autophagy activity is mutually exclusive
Is the appearance of granularity after rapamycininduction a useful marker for monitoring autophagicflow?
Aim IV.
Rapamycin
Untreated Cd Untreated Cd
Side Scatter Detector (SSC)
5.6% 1.3%
Heterogeneity Serves as a Potential Marker for Monitoring Rapamycin–induced Autophagy in S. cerevisiae
S. cerevisiae BY4741 wt
Rapamycin Wash Cd Treatment
The Core Principle: Detection of Cellular Size (Volume) and Complexity (Granularity)
Courtesy of RIC Facility, Brigham Young University, MRC Clinical Sciences Centre, and iGEM 2010 website Sysmex.com
TEM Atg8(LC3) tagging orAcid vacuole staining
Transform/Turnoverof Atg8p by WB
Direct evidence of autophagophore formation
Most descriptive
Time-saving Easy to perform Dynamic (Toxicity of dyes)
Direct evidence of autophagic flux
Golden standard
Time consumingSophisticated facilities/well-trained personnel requiredAdequate matrix to avoid statistic biasNon-dynamic
Non-specific result of acid organelle labelingArtificial effects of GFPAdequate matrix to avoid statistic bias
Time consumingLabor intensiveNon-dynamic
Takeshige, A., et. Al. J. Cell. Biol. 119:301-311. Mizushima, N. Int . J. Biochem. Cell. Biol. 36:2491-2502. Mizushima, N., Yoshimori, T. Autophagy 3:542-545.
The Evaluation of Methodologies Measuring Autophagic Activities
[rapamycin] XY-Plot HistogramFSC HistogramSSC
0.0
1.0
2.0
4.0
8.0
FACS Profile Cell volume
Complexity
WT yeast cell responses to rapamycin
Cell Volume
Co
mp
lexity
The Augmentation of Q2 Population was Found in Wild Type upon Rapamycin Treatment
wtWild type atg4Δ
atg6Δ atg8Δ
The Character of Q2 Augmentation was Absent in Autophagic Mutants upon Rapamycin Treatment
Q2 Population Represents Autophagic Population
Wt was treated by 4 μg rapamycin for 2 hours
Q3 and Q2 population was sorted by FACS Aria
Sample was lysed and analyzed by WB
Cell Volume
Co
mp
lexity
Courtesy of NIA/IRP Flow Cytometry Unit
The Dynamic Monitoring of Autophagic Activities by Cytometry-based Method
Wild type, 4 μg/mL rapamycin treatment
The cells were sampled every 15 minutes
Cell Volume
Co
mp
lexity
Evaluation of the Performance between Immunoblotand Cytometry-based Methods in Routine Practice
Crude Protein Extraction/Quantification (1 hour)
SDS-PAGE (2 hours)
Atg8-probed Immunoblot (8 hours)
Sample Collection
Data Acquisition
Apoptotic Staining (optional)(1 minutes~2 hours)
Flow Cytometry
Summary
The fluctuation of Q2 population dynamically reflects the autophagic potential S. cerevisiae tends to increase its cell size and complexity upon
rapamycin treatment, which phenomenon was not observed in autophagic mutant
Q2 has higher Atg8-PE to Atg8 ratio than Q3
Pros of the cytometry-based technique Staining is not required Quick, easy and dynamic Cells are viable for downstream analysis
Cons of the cytometry-based technique Other cellular activities than autophagy which increase Q2 may
interfere the result. The specific staining may be required for excluding other activities Well-established control is required
General Discussions and Conclusions
The entire autophagy pathway are necessary for Yca1p processing in S. cerevisiae
Autophagic initiators are required for Yca1 activation
Solubility of Yca1p determines its accessibility
For the role of autophagic responses in apoptosis, the stage may be critical in S. cerevisiae
Early: autophagy helps cell to survive
Late (autophagic cell death, Type II Cell Death): autophagy further facilitates apoptosis
Granularity is a useful marker for monitoring autophagic flow
Thorough experimental design needs to be practiced
Clearance of damaged organelles
Cell recovered and survived
Anti-apoptotic Autophagy
Autophagy Initialization(Atg4p, Atg6p, Atg8p)
Apoptosis(Type I Cell Death)
Apoptosis Autophagy ApoptosisAutophagy
High autophagy flux(Atg8-PE/Atg8)
Yca1 Pro
Yca1 Pro
Yca1 Pro
Insoluble
soluble
Autophagosome
Acknowledgement Dissertation Committee
Dr. John E. Houghton Dr. Susanna F. Greer Dr. Irene T. Weber
Lab Alumni Dr. Anupama Shanmuganathan Dr. Amrita Nargund Rupa Koduru Abhishikta Madireddy Chelsea Hagan Yi Peng
Core Facility Debby Walthall Sonya R. Young Dr. Hyuk-Kyu Seoh Ping Jiang Gemeia Cameron
Biology Department Dr. Phang C. Tai Dr. Zehava Eichenbaum LaTesha M. Warren
Friends@Metro Atlanta Area
Funding Support NIH (GM579450) Georgia Research Alliance Molecular Basis of Disease, Georgia State University
Family Ping-Hwei Chin Chiu-Lan Huang Su-Ying Chin Hsuan Liu