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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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.

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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'

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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

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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

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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

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[gene j][Agg

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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