role of protein aggregation in cellular degeneration a systems biology approach ariel b. lindner,...
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Role of protein aggregation in cellular degenerationa systems biology approach
Ariel B. Lindner, Ph.D.INSERM Junior Researcher
Candidate for CR1 position, INSERM CSS2
INSERM U571: Laboratory of Evolutive & Medical Molecular Genetics
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old poles
new poles
Lindner et al (1993) J. Org. Chem. Lindner et al (2008) Proc. Acad. Sci. U.S.A.
M. Sc. & Ph.D. ('93-'95;'96-'01)
Weizmann Institute of Science, Israel
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Lessons from Catalytic Antibodies: Enzymology, Immunology, Biotechnology
2.8Å2.9Å
TyrH100D AsnL34
inhibitor
kcat/kuncat: 2.6x105
Ag+Ab' (Ab-Ag)'.
(Ab-Ag)Ag+Ab
Antibody catalysts as structural & functional models of esterases
Lindner et al (2004) in Catalytic antibodies
Lindner et al (2002) J. Mol. Biol.
Antibody multi-specificity mediated by conformational diversity
Lindner et al (1999) J. Mol. Biol.
Tawfik, Lindner et al (1997) Eur. J. Biochem.
Highly efficient enzyme mimicks
Kim et al (1997) Mol. Immunol.
Biotechnological applications: - Protein micro-array - Drugs & explosives biosensor
Lindner et al. US & European patents licensed to Biosensor Ltd. (Sweden), Quantomix Ltd. (Israel)
Levit-Binun*, Lindner* et al (2002) J. Biophys.
MRC, Cambridge, UK Scripps Institute USA
Postdoc ('02-'05), EMBO & Marie Curie fellow Zharadka et al (2005) Nature
Deciphering DNA repair mechanism of Deinococcus radiodurans
‘extended synthesis-dependent strand annealing’ (ESDSA) mechanism:
Deinococcal robustness could inspire approaches in anti-ageing research and regenerative medicine; promising tool for genome shuffling.
C 0 1 4 4+UV
INSERM U571, Evolutionary Biotechnology group (Head: Prof. M. Radman, PhD student: D. Slade)
Strand invasion serves as 'primers' for ssDNA extension synthesis
Synthesis of complementary strands
Cross-overs for chromosome maturation
+BrdU
Anti-BrdU Ab
Time (hrs)
Babic, Lindner et al (2008) Science
INSERM U571, Evolutionary Biotechnology group (A. Babic PhD student)
Harnessing SeqA-yfp ability to label hemi-methylated dsDNA
Direct visualisation of horizontal gene transfer
Key observations:
Transfer at distance
High efficiency of integration (96%) mediated by RecA,BC
Variable DNA degradation patterns (all RecBCD dependent).
High frequency of inter-chromosomal exchanges.
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Opens door to future in vivo studies of DNA repair and chromosomal instabilities in single cells
Postdoc ('02-'05), EMBO & Marie Curie fellow
10'
0'
donor
recipient
Lindner et al (2008) patent application
INSERM Jr. Researcher ('06-'09') Lindner et al (2008) review in preparation
INSERM U571, Evolutionary systems biology group; Head - Dr. F. Taddei
Systems approach to phenotypic variability
population individuals
Phenotype Genotype Environment
Possible sources / consequences:
Stochasticity / Noise
Post-replication errors / Aging
Epigenetic switches / Adaptability
phenotypic variability: the distribution of a given trait under constant genetic and environmental conditions
SexStressPersistenceAggregationInfectionAging…
bacterial
Robert et al (2008) in preparation
Bacteria: E. coli (K12)
Chromosomal Fluorescent Reporters
Automated time lapse microscopy
Image analysis =>
Lineage reconstruction, growth
& fluorescence quantification
Experimental setup:
INSERM Jr. Researcher ('06-'09') Lindner et al (2008) in preparation
Persistence to antibiotics
Results:Unidirectional phenotypic switch from persisting to sensitive cells governed by differential permeability Accumulation of protein aggregates in sensitive cellsClonal death of sensitive lineages
YFP
ibp
Persistence: cells of a genetically homogeneous microbial population surviving antibiotic treatment yet,
when re-grown they remain as sensitive to the antibiotic.
INSERM Jr. Researcher ('06-'09') Stewart et al (2005) PLoS BiolLindner et al. (2008) PNAS
INSERM U571, Evolutionary systems biology group; Dr. Eric Stewart, post-doc
Escherichia coli as model organism for aging research
Aging:
Reduced fitness as function of time
Reduced metabolism Decreased offspring production Increased chance of death
Age: consecutive divisions as old pole cell
Consecutive old/new pole divisions
Nor
mal
ized
gro
wth
rat
e
new
old
INSERM Jr. Researcher ('06-'09') Lindner et al. (2008) PNAS
INSERM U571, Evolutionary systems biology group
Understanding bacterial aging:
Hidden molecular asymmetry
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.
Experimental system:
Correlate aggregates presence with growth-rate in lineage context
Asymmetric segregation of protein aggregates is associated with cellular aging
Mother cell1
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Gen
erat
ion
s New pole cellOld pole cell
0.90 0.95 1.00 1.05 1.10Relative growth rate
Cycles of aging and rejuvenation
IbpA-YFP translational fusion
INSERM Jr. Researcher ('06-'09') Lindner et al (2008) PNAS
INSERM U571, Evolutionary systems biology group
Asymmetric segregation of protein aggregates is associated with cellular aging
INSERM Jr. Researcher ('06-'09') Lindner et al (2008) PNAS
INSERM U571, Evolutionary systems biology group
Asymmetric segregation of protein aggregates is associated with cellular aging
Results:
Stochastic aggregate appearance leads to deterministic accumulation in old poles
Aggregate asymmetric segregation follows growth rate pattern decrease
Aggregate accumulation rate accedes cellular growth rate
Asymmetric inheritance of damaged proteins accounts for 40% of observed aging
New pole cells
Old pole cells
New pole cells
Old pole cells
Old pole cells
New pole cells
Proposed project
Role of protein aggregation in cellular degeneration: a systems biology approach
Objectives:
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.
The ubiquitous folding & disaggregation network
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flow
flow
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Proposed project Xu, L. et al. (2007) Nano Lett.
Automated colony screening
Old cell retention 'home' (unlimited generations)
Microfluidics: Design & Implementation
Old cell retention 'home' (unlimited generations)
Biological system:
Reference bacterial strain reporting aggregate state
Single knock-out E. coli library
Tuneable, fluorescent protein coupled, chromosomal expression of folding/ disaggregation network genes
Over-expression E. coli library
[genei]
[gene j][Agg
.]
Role of protein aggregation in cellular degeneration: ‘Lab on Chip’
'lab on chip'Y. Chen & D. Baigl
ENS /CNRSCNRS grant ANR grant
Biochemistry of aggregationS. Dukan
CNRS, Marseille
A systems approach to individual differences in longevity
Axa Chair (decision 07/2008)
T. Kirkwood, L. Partridge, J. Vaupel, F. TaddeiNewcastle U., UC London, Max Planck, Rostock, Paris Descatres U. & INSERM
INRIA/INSERM grant
H. Berry, & H. de Jong"Action d’Envergure" : aging
INRIA
Statistical lineage modelsPY Bourguignon, V. Schachter
Genoscope, CEA
Image analysisL. Moisan
Paris Descartes U.
Modeling asymmetry & agingG. Paul
ETH
High resolution microscopyH. Dong
Chinese academy of sciences
FBS, Paris Centre
Synthetic Biology1st prize foundational research MIT
Paris iGEM team; French, European network
Project collaborations & finances
Post-doc fellows: Swiss federal fellow, Chinese academy of sciences; PhD fellow:Human Frontiers