Transcript Lindner et al (2008) PNAS

Role of protein aggregation in cellular degeneration
a systems biology approach
Ariel B. Lindner, Ph.D.
INSERM Junior Researcher
1mM
Lindner et al (1993) J. Org. Chem.
Aggregates
Candidate for CR1 position, INSERM CSS2
Lindner et al (2008) Proc. Acad. Sci. U.S.A.
INSERM U571: Laboratory of Evolutive & Medical Molecular Genetics
M. Sc. & Ph.D. ('93-'95;'96-'01)
Lessons from Catalytic Antibodies: Enzymology, Immunology, Biotechnology
Highly efficient enzyme mimicks
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Tawfik, Lindner et al (1997) Eur. J. Biochem.
Kim et al (1997) Mol. Immunol.
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Lindner et al (2002) J. Mol. Biol.
Lindner et al (2004) in Catalytic antibodies
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oxyanion hole
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Products
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Ester
Substrates
Antibody catalysts as structural &
functional models of esterases
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AsnL34
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Antibody multi-specificity mediated by
conformational diversity
substrate
recognition
Lindner et al (1999) J. Mol. Biol.
2.8Å
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Biotechnological applications:
- Protein micro-array
- Drugs & explosives biosensor
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inhibitor
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kcat/kuncat:
Ag+Ab
(Ab-Ag)
Ag+Ab'
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Lindner et al. US & European patents
licensed to Biosensor Ltd. (Sweden), Quantomix Ltd. (Israel)
Levit-Binun*, Lindner* et al (2002) J. Biophys.
Weizmann Institute of Science, Israel
MRC, Cambridge, UK
Scripps Institute USA
Zharadka et al (2005) Nature
Postdoc ('02-'05), EMBO & Marie Curie fellow
Deciphering DNA repair mechanism of Deinococcus radiodurans
‘extended synthesis-dependent strand
annealing’ (ESDSA) mechanism:
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Time (hrs)
0 1 4 4+UV
Strand invasion serves as 'primers' for
ssDNA extension synthesis
Synthesis of complementary strands
Cross-overs for chromosome maturation
Deinococcal robustness could inspire
approaches in anti-ageing research and
regenerative medicine; promising tool for
genome shuffling.
+BrdU
Anti-BrdU Ab
INSERM U571, Evolutionary Biotechnology group (Head: Prof. M. Radman, PhD student: D. Slade)
Postdoc ('02-'05), EMBO & Marie Curie fellow
Babic, Lindner et al (2008) Science
Lindner et al (2008) patent application
Direct visualisation of horizontal gene transfer
Harnessing SeqA-yfp ability to label
hemi-methylated dsDNA
0'
Key observations:
Transfer at distance
High efficiency of integration (96%)
mediated by RecA,BC
donor
recipient
Variable DNA degradation patterns
(all RecBCD dependent).
High frequency of
inter-chromosomal exchanges.
Opens door to future in vivo studies of
DNA repair and chromosomal instabilities
in single cells
INSERM U571, Evolutionary Biotechnology group (A. Babic PhD student)
10'
1mm
Lindner et al (2008) review in preparation
Robert et al (2008) in preparation
INSERM Jr. Researcher ('06-'09')
Systems approach to phenotypic variability
phenotypic variability: the distribution of
a given trait under constant genetic and
environmental conditions
Possible sources / consequences:
Stochasticity
/ Noise
Post-replication errors / Aging
Epigenetic switches / Adaptability
Phenotype
bacterial
Genotype  Environment
population individuals
Experimental setup:
Bacteria: E. coli (K12)
Chromosomal Fluorescent Reporters
Automated time lapse microscopy
Image analysis =>
Lineage reconstruction, growth
& fluorescence quantification
INSERM U571, Evolutionary systems biology group; Head - Dr. F. Taddei
Sex
Stress
Persistence
Aggregation
Infection
Aging…
Lindner et al (2008) in preparation
INSERM Jr. Researcher ('06-'09')
Persistence to antibiotics
ibp
Persistence: cells of a genetically
homogeneous microbial population
surviving antibiotic treatment yet,
when re-grown they remain as sensitive
to the antibiotic.
Results:
Unidirectional phenotypic switch from
persisting to sensitive cells governed
by differential permeability
Accumulation of protein aggregates in
sensitive cells
Clonal death of sensitive lineages
YFP
INSERM Jr. Researcher ('06-'09')
Stewart et al (2005) PLoS Biol
Lindner et al. (2008) PNAS
Escherichia coli as model organism for aging research
Aging:
Reduced metabolism
Decreased offspring production
Increased chance of death
Reduced fitness as function of time
Normalized growth rate
Age: consecutive divisions as old pole cell
new
old
Consecutive old/new pole divisions
INSERM U571, Evolutionary systems biology group; Dr. Eric Stewart, post-doc
Lindner et al. (2008) PNAS
INSERM Jr. Researcher ('06-'09')
Asymmetric segregation of protein aggregates is associated with cellular aging
Understanding bacterial aging:
Cycles of aging and rejuvenation
Hidden molecular asymmetry
0.90
1
Generations
Context:
Carbonylated proteins retained in
yeast mother cells
Age-related protein misfolding
diseases
putative stem cells exhibit lower
aggregation level as compared with
differentiated cells.
Relative growth rate
0.95
1.00
1.05
2
Mother cell
Old pole cell
New pole cell
3
4
5
IbpA-YFP translational fusion
Experimental system:
Correlate aggregates presence with
growth-rate in lineage context
INSERM U571, Evolutionary systems biology group
1.10
INSERM Jr. Researcher ('06-'09')
Lindner et al (2008) PNAS
Asymmetric segregation of protein aggregates is associated with cellular aging
INSERM U571, Evolutionary systems biology group
Lindner et al (2008) PNAS
INSERM Jr. Researcher ('06-'09')
Asymmetric segregation of protein aggregates is associated with cellular aging
Results:
Stochastic aggregate appearance
leads to deterministic accumulation in
old poles
New pole cells
Old pole cells
Aggregate asymmetric segregation
follows growth rate pattern decrease
Old pole cells
Aggregate accumulation rate accedes
cellular growth rate
Asymmetric inheritance of damaged
proteins accounts for 40% of
observed aging
INSERM U571, Evolutionary systems biology group
New pole cells
Old pole cells
New pole cells
Proposed project
Role of protein aggregation in cellular degeneration: a systems biology approach
Objectives:
The ubiquitous folding & disaggregation network
Quantitative assessment of
aggregates effect on cellular
degeneration through a
systematic in vivo study of the
related functional networks.
Explore how cellular factors,
genetic backgrounds &
environmental variables affect
cellular degeneration.
Adapted from Baneyx (2004) Nat. Biotech.
Proposed project
Xu, L. et al. (2007) Nano Lett.
Role of protein aggregation in cellular degeneration: ‘Lab on Chip’
Biological system:
Reference bacterial
aggregate state
Microfluidics: Design & Implementation
strain
Automated colony screening
reporting
Tuneable, fluorescent protein coupled,
chromosomal expression of folding/
disaggregation network genes
Old cell retention 'home' (unlimited generations)
Single knock-out E. coli library
Over-expression E. coli library
Old cell retention 'home' (unlimited generations)
1mm
flow
flow
50mm
Project collaborations & finances
'lab on chip'
Y. Chen & D. Baigl
ENS /CNRS
Image analysis
L. Moisan
Paris Descartes U.
CNRS grant
Modeling asymmetry & aging
G. Paul
ETH
ANR grant
Statistical lineage models
PY Bourguignon, V. Schachter
Genoscope, CEA
"Action d’Envergure" : aging
H. Berry, & H. de Jong
INRIA
High resolution microscopy
H. Dong
Chinese academy of sciences
INRIA/INSERM grant
Synthetic Biology
1st prize foundational research MIT
Paris iGEM team; French, European network
Biochemistry of aggregation
S. Dukan
CNRS, Marseille
FBS, Paris Centre
A systems approach to individual differences in longevity
T. Kirkwood, L. Partridge, J. Vaupel, F. Taddei
Newcastle U., UC London, Max Planck, Rostock, Paris Descatres U. & INSERM
Axa Chair (decision 07/2008)
Post-doc fellows: Swiss federal fellow, Chinese academy of sciences; PhD fellow:Human Frontiers