1. ANALYSIS OF CELLULAR RESPONSES TO HEAVY
METAL-INDUCED STRESS IN Saccharomyces
cerevisiae
Pei-JuChin
Molecular Genetics and Biochemistry Program
Department of Biology
Georgia State University
Atlanta,GA 30303

2. 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

3. 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

4. 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 MedicalUniversity, Japan and University of California, Santa Cruz, USA

5. 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 Unpublished data

6. Why is Yeast Chosen as the Model System?
Braun RJ (2012) Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding ofneurodegeneration.
Frontiers in oncology 2:182.

7. Environmental Stress Triggers Apoptotic
Responses by Accumulating ROS Inside Cells
Madeo F. et. al. 2004. Cur. Opin. Microbiol. 7:655-660
Metallic
ion

8. 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 Reaction
Cu2+ + H2O2 → Cu3+ + OH·+ OH−
Hanas, JS. And Gunn CG. Nucleic Acids Res. 1996, 24, 924-930

9. 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

10. Metal-induced Response in S. cerevisiae-
Acute and lethal
• 30 μ M Cd(NO3)2
• 8mM Cu(NO3)2
Seeding
culture
100mL
YEPD
O/N
incubation
2OD600
1 hour
DIC
PI
Merge0.1
1
10
BMH1
BMH2
CDC25
DNM1
ERF2
FIS1
HXT17
LTE1
PHB1
PHB2
RTG2
SHR5
Foldchanges
Cd-30 mins
Cd-60 mins
0.1
1
10
ASF1
BIR1
CDC48
FUN34
HXT17
IFM1
KAP120
MCA1
OAF1
RSM23
RTT107
SNL1
STM1
SVF1
SXM1
UTH1
WWM1
YOP1
Foldchanges
Cd-30 mins
Cd-60 mins
24~48 hours
Cd 1+3
Cu 1+3
Post-incubate
3 hours
Wash

11. Yeast Genome PathwayAnalysis, 2006
The Presence of Reduced-glutathione (GSH) Indicates
Cell Suffering from Oxidative Stress

12. 0.1
1
10
100
BUD9
FBA1
FBP1
GND1
GND2
PFK1
PFK2
PGI1
PGM1
PGM2
PRS1
PRS2
PRS3
PRS4
PRS5
RBK1
RKI1
RPE1
SOL1
SOL3
SOL4
SPC1
TAL1
TKL1
TKL2
TKL2
VHS1
ZWF1
Foldchanges
Cd-30 mins Cd-60 mins
0
5
10
15
20
25
30
35
40
0 5 15 30 60 90
Relativeconcentration(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
FoldChanges
Cd-30 mins
Cd-60 mins

13. 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

14. 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 PhilipYau
Zoli et al. Breast Cancer Res. 7:R681

15. 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

16. 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
Apoptosis
Cascade

17. 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

18. Yca1p appears to have DEVD like activity.
Zvad-fmk Proves a Second Caspase-like Activity
IETD and DEVD activity
Nargund,A. Unpublished data
Pan-Caspase
Inhibitor

19. Cysteine proteases in yeast
1. RIM13 – calpain like protease
2. ATG4 – involved in autophagy
3. YCA1 – known function - yeast caspase
4. SNO4
5. HSP33
6. HSP32
7. ESP1 – known function-acts as separase
8. OTU2
Candidates for Second Caspase

20. Atg4p, second caspase-like protease invloved in Cd –induced
apoptosis
IETD and DEVD activity
ATG4 shows very little IETD like activity in Cd treatedYCA1Δ

21. 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)

22. Autophagy (Self-Eating) –
Cellular Homeostasis Mechanism
Klionsky DJ et al. 2007. Autophagy 3:5 413-416

23. Are autophagic initiators required for apoptotic
response in S. cerevisiae?
Aim I.

24. 0.1
1
10
100 ATG1
ATG2
ATG3
ATG4
ATG5
ATG6
ATG7
ATG8
ATG9
ATG10
ATG11
ATG12
ATG13
ATG14
ATG15
ATG16
ATG17
ATG18
ATG19
ATG20
ATG21
ATG22
ATG23
ATG24
ATG26
ATG27
ATG29
ATG31
ATG32
ATG33
FoldChangesnormalizedbyuntreated
Cd 30 mins
Cd 60 mins
Autophagic Genes Are Induced upon Cadmium
Exposure in S. cerevisiae
1
10
FoldChangesNormalizedbyuntreated
Cd 30 mins
Cd 60 mins
J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86
(Atg6)
Atg4p,Atg6pandAtg8pare Involvedin the
MaturationofAutophagosome Biosynthesis
J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86

25. Are the Autophagy Initiators required for Yca1p cleavage?
Chin, P.J. and Houghton, J.E. Unpublished data

26. 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

27. Summary of Aim I
 Atg4p contributes to Caspase 8-like activity
(Nargund A. Unpublished data)
 Autophagic initiators: Atg4p,Atg6p and
Atg8p are required forYca1p-dependent
apoptosis upon metal exposure

28. Does autophagosome apparatus facilitateYca1p
activation?
Aim II.

29. Yca1p Is Insoluble in vivo
 Natural property per se (unlikely)
 E. coli-expressed His-taggedYca1p is soluble
 Be sequestered in vacuoles
[NP-40]

30. Yca1p is Trans-localized to Acidic Vacuoles upon
Cadmium Treatment
DIC Yca1-GFP
MDC
(Acidic vacuoles including
autophagosome )

31. 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
Ficoll
15%
Ficoll
8%
Ficoll
4%
Ficoll
0%
Autophagosome
Cell debris
Insoluble Fraction

32. Yca1p-GFP was Absent from Debris Sedimentation after Cu
Treatment
25%
50%
100%
Ficoll0-4%
Ficoll4-8%
Sedimentation
Yca1-GFPexpressionlevel
Cu 0+4
Cu 1+3
(GAPDH-GFP)

33. Summary of Aim II.
 Yca1p-GFP was present in
autophagosome in both Cu-
untreated or treated cells
 Autophagosome vacuoles sequester
the activity ofYca1p
 Autophagosome facilitates the
process ofYca1p
 Yca1p-GFP was absent from the
cell debris fractionation after Cu
treatment
 Solubility ofYca1-GFP is increased
after Cu treatment
 Accessible for initiators/self-cleavage
process
Autophagosome
Yca1p
Cu/Cd
Soluble
insoluble

34. Is autophagy a life-saver or kiler in yeast undergoing
heavy metal-induced oxidative stress ?
Aim III.

35. Intercommunication of autophagy and apoptosis in
higher eukaryotic cells-
Autophagic proteins as cytoprotector
 Autophagy induces cytoprotection in neuron cells by removing
aggregated huntingtin (HaraT. 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-lipidizeAtg8-PE,therefore
facilitates autophagy (Scherz-Shouval R. et. al. EMBO J 26:1749-
1760)

36. 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 (RubinsteinAD et.
al. MolCell 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 conjugateAtg5-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

37. 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, bafilomycinA1
 Stress type-dependent
 Hypoxia, starvation, ROS, misfold protein accumulation
 Time/Occasion-dependent
 When to introduce autophagy and apoptosis

38. What if the role of autophagy is decided by the
stage it is introduced?

39. Rapamycin Induces Autophagic Response by Inhibiting
the Hyper-phosphorylation of Atg13p Caused by TORC1

40. 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]

41. Treatment Schema
 Independent Events
 Autophagy only
 Apoptosis only
 Two-Stage Events
 Autophagy->Apoptosis
 vice versa
 Overlapping Events
 Treatment A-D

42. 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

43. Autophagy Facilitates Cd-induced Apoptotic Response-
Type-II Cell Death
 All PI has MDC
 Type-II CD
 Not all MDC has
PI
 Survival?

44. 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

45. Is the appearance of granularity after rapamycin
induction a useful marker for monitoring autophagic
flow?
Aim IV.

46. 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 CdTreatment

47. The Core Principle:
Detection of Cellular Size (Volume) and Complexity (Granularity)
Courtesy of RIC Facility, BrighamYoung University, MRC
Clinical Sciences Centre, and iGEM 2010 website
Sysmex.com

48. TEM Atg8(LC3) tagging or
Acid vacuole staining
Transform/Turnover
of 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 consuming
Sophisticated
facilities/well-trained
personnel required
Adequate matrix to avoid
statistic bias
Non-dynamic
Non-specific result of acid
organelle labeling
Artificial effects of GFP
Adequate matrix to avoid
statistic bias
Time consuming
Labor intensive
Non-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

49. [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
CellVolume
Complexity
The Augmentation of Q2 Population was Found in Wild Type upon
Rapamycin Treatment

50. wtWild type atg4Δ
atg6Δ atg8Δ
The Character of Q2 Augmentation was Absent in
Autophagic Mutants upon Rapamycin Treatment

51. 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 byWB
CellVolume
Complexity
Courtesy of NIA/IRP Flow Cytometry Unit

52. The Dynamic Monitoring of Autophagic Activities
by Cytometry-based Method
 Wild type, 4 μg/mL rapamycin treatment
 The cells were sampled every 15 minutes
CellVolume
Complexity

53. Evaluation of the Performance between Immunoblot
and Cytometry-based Methods in Routine Practice

54. 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

55. 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

56. General Discussions and Conclusions
 The entire autophagy pathway are necessary forYca1p
processing in S. cerevisiae
 Autophagic initiators are required forYca1 activation
 Solubility ofYca1p 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

57. 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

58. 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