Project acronym BioMatrix
Project Structural Biology of Exopolysaccharide Secretion in Bacterial Biofilms
Researcher (PI) Petya Violinova KRASTEVA
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary Bacterial biofilm formation is a paramount developmental process in both Gram-positive and Gram-negative species and in many pathogens has been associated with processes of horizontal gene transfer, antibiotic resistance development and pathogen persistence. Bacterial biofilms are collaborative sessile macrocolonies embedded in complex extracellular matrix that secures both mechanical resistance and a medium for intercellular exchange.
Biogenesis platforms for the secretion of biofilm matrix components - many of which controlled directly or indirectly by the intracellular second messenger c-di-GMP - are important determinants for biofilm formation and bacterial disease, and therefore present compelling targets for the development of novel therapeutics. During my Ph.D. and post-doctoral work I studied the structure and function of c-di-GMP-sensing protein factors controling extracellular matrix production by DNA-binding at the transcription initiation level or by inside-out signalling mechanisms at the cell envelope, as well as membrane exporters involved directly in downstream matrix component secretion.
Here, I propose to apply my expertise in microbiology, protein science and structural biology to study the structure and function of exopolysaccharide secretion systems in Gram-negative species. Using Pseudomonas aeruginosa, Vibrio spp. and Escherichia coli as model organisms, my team will aim to reveal the global architecture and individual building components of several expolysaccharide-producing protein megacomplexes. We will combine X-ray crystallography, biophysical and biochemical assays, electron microscopy and in cellulo functional studies to provide a comprehensive view of extracellular matrix production that spans the different resolution levels and presents molecular blueprints for the development of novel anti-infectives. Over the last year I have laid the foundation of these studies and have demonstrated the overall feasibility of the project.
Summary
Bacterial biofilm formation is a paramount developmental process in both Gram-positive and Gram-negative species and in many pathogens has been associated with processes of horizontal gene transfer, antibiotic resistance development and pathogen persistence. Bacterial biofilms are collaborative sessile macrocolonies embedded in complex extracellular matrix that secures both mechanical resistance and a medium for intercellular exchange.
Biogenesis platforms for the secretion of biofilm matrix components - many of which controlled directly or indirectly by the intracellular second messenger c-di-GMP - are important determinants for biofilm formation and bacterial disease, and therefore present compelling targets for the development of novel therapeutics. During my Ph.D. and post-doctoral work I studied the structure and function of c-di-GMP-sensing protein factors controling extracellular matrix production by DNA-binding at the transcription initiation level or by inside-out signalling mechanisms at the cell envelope, as well as membrane exporters involved directly in downstream matrix component secretion.
Here, I propose to apply my expertise in microbiology, protein science and structural biology to study the structure and function of exopolysaccharide secretion systems in Gram-negative species. Using Pseudomonas aeruginosa, Vibrio spp. and Escherichia coli as model organisms, my team will aim to reveal the global architecture and individual building components of several expolysaccharide-producing protein megacomplexes. We will combine X-ray crystallography, biophysical and biochemical assays, electron microscopy and in cellulo functional studies to provide a comprehensive view of extracellular matrix production that spans the different resolution levels and presents molecular blueprints for the development of novel anti-infectives. Over the last year I have laid the foundation of these studies and have demonstrated the overall feasibility of the project.
Max ERC Funding
1 499 901 €
Duration
Start date: 2018-08-01, End date: 2023-07-31
Project acronym CoSpaDD
Project Competition for Space in Development and Diseases
Researcher (PI) Romain LEVAYER
Host Institution (HI) INSTITUT PASTEUR
Call Details Starting Grant (StG), LS3, ERC-2017-STG
Summary Developing tissues have a remarkable plasticity illustrated by their capacity to regenerate and form normal organs despite strong perturbations. This requires the adjustment of single cell behaviour to their neighbours and to tissue scale parameters. The modulation of cell growth and proliferation was suggested to be driven by mechanical inputs, however the mechanisms adjusting cell death are not well known. Recently it was shown that epithelial cells could be eliminated by spontaneous live-cell delamination following an increase of cell density. Studying cell delamination in the midline region of the Drosophila pupal notum, we confirmed that local tissue crowding is necessary and sufficient to drive cell elimination and found that Caspase 3 activation precedes and is required for cell delamination. This suggested that a yet unknown pathway is responsible for crowding sensing and activation of caspase, which does not involve already known mechanical sensing pathways. Moreover, we showed that fast growing clones in the notum could induce neighbouring cell elimination through crowding-induced death. This suggested that crowding-induced death could promote tissue invasion by pretumoural cells.
Here we will combine genetics, quantitative live imaging, statistics, laser perturbations and modelling to study crowding-induced death in Drosophila in order to: 1) find single cell deformations responsible for caspase activation; 2) find new pathways responsible for density sensing and apoptosis induction; 3) test their contribution to adult tissue homeostasis, morphogenesis and cell elimination coordination; 4) study the role of crowding induced death during competition between different cell types and tissue invasion 5) Explore theoretically the conditions required for efficient space competition between two cell populations.
This project will provide essential information for the understanding of epithelial homeostasis, mechanotransduction and tissue invasion by tumoural cells
Summary
Developing tissues have a remarkable plasticity illustrated by their capacity to regenerate and form normal organs despite strong perturbations. This requires the adjustment of single cell behaviour to their neighbours and to tissue scale parameters. The modulation of cell growth and proliferation was suggested to be driven by mechanical inputs, however the mechanisms adjusting cell death are not well known. Recently it was shown that epithelial cells could be eliminated by spontaneous live-cell delamination following an increase of cell density. Studying cell delamination in the midline region of the Drosophila pupal notum, we confirmed that local tissue crowding is necessary and sufficient to drive cell elimination and found that Caspase 3 activation precedes and is required for cell delamination. This suggested that a yet unknown pathway is responsible for crowding sensing and activation of caspase, which does not involve already known mechanical sensing pathways. Moreover, we showed that fast growing clones in the notum could induce neighbouring cell elimination through crowding-induced death. This suggested that crowding-induced death could promote tissue invasion by pretumoural cells.
Here we will combine genetics, quantitative live imaging, statistics, laser perturbations and modelling to study crowding-induced death in Drosophila in order to: 1) find single cell deformations responsible for caspase activation; 2) find new pathways responsible for density sensing and apoptosis induction; 3) test their contribution to adult tissue homeostasis, morphogenesis and cell elimination coordination; 4) study the role of crowding induced death during competition between different cell types and tissue invasion 5) Explore theoretically the conditions required for efficient space competition between two cell populations.
This project will provide essential information for the understanding of epithelial homeostasis, mechanotransduction and tissue invasion by tumoural cells
Max ERC Funding
1 489 147 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
Project acronym DNATRAFFIC
Project DNA traffic during bacterial cell division
Researcher (PI) François-Xavier Andre Fernand Barre
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary The molecular mechanisms that serve to couple DNA replication, chromosome segregation and cell division are largely unknown in bacteria. This led a considerable interest to the study of Escherichia coli FtsK, an essential cell division protein that assembles into DNA-pumps to transfer chromosomal DNA between the two daughter cell compartments during septation. Indeed, our recent work suggests that FtsK might regulate the late stages of septation to ensure DNA is fully cleared from the septum before it is allowed to close. This would be the first example of a cell cycle checkpoint in bacteria.
FtsK-mediated DNA transfer is required in 15% of the cells at each generation in E. coli, in which it serves to promote the resolution of topological problems arising from the circularity of the chromosome by Xer recombination. However, the FtsK checkpoint could be a more general feature of the bacterial cell cycle since FtsK is highly conserved among eubacteria, including species that do not possess a Xer system. Indeed, preliminary results from the lab indicate that DNA transfer by FtsK is required independently of Xer recombination in Vibrio cholerae.
To confirm the existence and the generality of the FtsK checkpoint in bacteria, we will determine the different situations that lead to a requirement for FtsK-mediated DNA transfer by studying chromosome segregation and cell division in V. cholerae. In parallel, we will apply new fluorescent microscopy tools to follow the progression of cell division and chromosome segregation in single live bacterial cells. PALM will notably serve to probe the structure of the FtsK DNA-pumps at a high spatial resolution, FRET will be used to determine their timing of assembly and their interactions with the other cell division proteins, and TIRF will serve to follow in real time their activity with respect to the progression of chromosome dimer resolution, chromosome segregation, and septum closure.
Summary
The molecular mechanisms that serve to couple DNA replication, chromosome segregation and cell division are largely unknown in bacteria. This led a considerable interest to the study of Escherichia coli FtsK, an essential cell division protein that assembles into DNA-pumps to transfer chromosomal DNA between the two daughter cell compartments during septation. Indeed, our recent work suggests that FtsK might regulate the late stages of septation to ensure DNA is fully cleared from the septum before it is allowed to close. This would be the first example of a cell cycle checkpoint in bacteria.
FtsK-mediated DNA transfer is required in 15% of the cells at each generation in E. coli, in which it serves to promote the resolution of topological problems arising from the circularity of the chromosome by Xer recombination. However, the FtsK checkpoint could be a more general feature of the bacterial cell cycle since FtsK is highly conserved among eubacteria, including species that do not possess a Xer system. Indeed, preliminary results from the lab indicate that DNA transfer by FtsK is required independently of Xer recombination in Vibrio cholerae.
To confirm the existence and the generality of the FtsK checkpoint in bacteria, we will determine the different situations that lead to a requirement for FtsK-mediated DNA transfer by studying chromosome segregation and cell division in V. cholerae. In parallel, we will apply new fluorescent microscopy tools to follow the progression of cell division and chromosome segregation in single live bacterial cells. PALM will notably serve to probe the structure of the FtsK DNA-pumps at a high spatial resolution, FRET will be used to determine their timing of assembly and their interactions with the other cell division proteins, and TIRF will serve to follow in real time their activity with respect to the progression of chromosome dimer resolution, chromosome segregation, and septum closure.
Max ERC Funding
1 565 938 €
Duration
Start date: 2012-02-01, End date: 2017-01-31
Project acronym MECHABLASTO
Project Morphogenesis during pre-implantation development: molecular and mechanical regulation
Researcher (PI) Jean-Léon MAÎTRE
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS3, ERC-2017-STG
Summary During the first days of mammalian development, blastomeres organize themselves into the blastocyst, which implants the embryo into the maternal uterus. Failure to build the blastocyst will result in a miscarriage and yet the mechanisms underlying the construction of the blastocyst are mostly unknown. The blastocyst is sculpted by forces generated by its constituent cells. Without a tool to study the mechanics of the mammalian embryo, it is challenging to identify the molecules and cellular processes controlling morphogenetic forces. Using biophysical methods, I have recently measured the forces shaping the mouse blastocyst and identified cellular processes generating and controlling them. This approach enables the identification of the molecules controlling morphogenesis and constitutes the first step towards a complete theoretical modelling of blastocyst morphogenesis.
The aim of this project is to understand the molecular and mechanical aspects of blastocyst morphogenesis. By developing novel biophysical tools for the developing blastocyst, we will measure uncharacterized mechanical properties such as cytoplasmic and luminal pressure, adhesion strength and viscosity. The resulting mechanical map of the blastocyst will help understand the mechanisms of action of genes involved in its morphogenesis. To identify novel candidate genes involved in blastocyst morphogenesis, we will carry out a screen using live high-resolution confocal microscopy of mouse embryos injected with siRNA. Together, this will reveal the molecular, cellular and mechanical processes controlling blastocyst morphogenesis. I expect this to shed light on how blastomeres self-organize into the blastocyst and to reveal the physical laws underlying morphogenesis in general. Importantly, the knowledge and non-invasive biophysical techniques that we will develop will help developing Assisted Reproduction Technologies, which will be greatly beneficial to the fertility of the ageing European population.
Summary
During the first days of mammalian development, blastomeres organize themselves into the blastocyst, which implants the embryo into the maternal uterus. Failure to build the blastocyst will result in a miscarriage and yet the mechanisms underlying the construction of the blastocyst are mostly unknown. The blastocyst is sculpted by forces generated by its constituent cells. Without a tool to study the mechanics of the mammalian embryo, it is challenging to identify the molecules and cellular processes controlling morphogenetic forces. Using biophysical methods, I have recently measured the forces shaping the mouse blastocyst and identified cellular processes generating and controlling them. This approach enables the identification of the molecules controlling morphogenesis and constitutes the first step towards a complete theoretical modelling of blastocyst morphogenesis.
The aim of this project is to understand the molecular and mechanical aspects of blastocyst morphogenesis. By developing novel biophysical tools for the developing blastocyst, we will measure uncharacterized mechanical properties such as cytoplasmic and luminal pressure, adhesion strength and viscosity. The resulting mechanical map of the blastocyst will help understand the mechanisms of action of genes involved in its morphogenesis. To identify novel candidate genes involved in blastocyst morphogenesis, we will carry out a screen using live high-resolution confocal microscopy of mouse embryos injected with siRNA. Together, this will reveal the molecular, cellular and mechanical processes controlling blastocyst morphogenesis. I expect this to shed light on how blastomeres self-organize into the blastocyst and to reveal the physical laws underlying morphogenesis in general. Importantly, the knowledge and non-invasive biophysical techniques that we will develop will help developing Assisted Reproduction Technologies, which will be greatly beneficial to the fertility of the ageing European population.
Max ERC Funding
1 497 691 €
Duration
Start date: 2018-02-01, End date: 2023-01-31
Project acronym MOLSTRUCTTRANSFO
Project Molecular and Structural Biology of Bacterial Transformation
Researcher (PI) Rémi Fronzes
Host Institution (HI) INSTITUT PASTEUR
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary A common form of gene transfer is the vertical gene transfer between one organism and its offspring during sexual reproduction. However, some organisms, such as bacteria, are able to acquire genetic material independently of sexual reproduction by horizontal gene transfer (HGT). Three mechanisms mediate HGT in bacteria: conjugation, transduction and natural transformation. HGT and the selective pressure exerted by the widespread use antibiotics (in medicine, veterinary medicine, agriculture, animal feeding, etc) are responsible for the rapid spread of antibiotic resistance genes among pathogenic bacteria.
In this proposal, we focus on bacterial transformation systems, also named competence systems. Natural transformation is the acquisition of naked DNA from the extracellular milieu. It is the only programmed process for generalized genetic exchange found in bacteria. This highly efficient and regulated process promotes bacterial genome plasticity and adaptive response of bacteria to changes in their environment. It is essential for bacterial survival and/or virulence and greatly limits efficiency of treatments or vaccine against some pathogenic bacteria.
The architecture and functioning of the membrane protein complexes mediating DNA transfer through the cell envelope during bacterial transformation remain elusive. We want to decipher the molecular mechanism of this transfer. To attain this goal, we will carry out structural biology studies (X-ray crystallography and high resolution electron microscopy) as well as functional and structure-function in vivo studies. We have the ambition to make major contributions to the understanding of bacterial transformation. Ultimately, we hope that our results will also help to find compounds that could block natural transformation in bacterial pathogens.
Summary
A common form of gene transfer is the vertical gene transfer between one organism and its offspring during sexual reproduction. However, some organisms, such as bacteria, are able to acquire genetic material independently of sexual reproduction by horizontal gene transfer (HGT). Three mechanisms mediate HGT in bacteria: conjugation, transduction and natural transformation. HGT and the selective pressure exerted by the widespread use antibiotics (in medicine, veterinary medicine, agriculture, animal feeding, etc) are responsible for the rapid spread of antibiotic resistance genes among pathogenic bacteria.
In this proposal, we focus on bacterial transformation systems, also named competence systems. Natural transformation is the acquisition of naked DNA from the extracellular milieu. It is the only programmed process for generalized genetic exchange found in bacteria. This highly efficient and regulated process promotes bacterial genome plasticity and adaptive response of bacteria to changes in their environment. It is essential for bacterial survival and/or virulence and greatly limits efficiency of treatments or vaccine against some pathogenic bacteria.
The architecture and functioning of the membrane protein complexes mediating DNA transfer through the cell envelope during bacterial transformation remain elusive. We want to decipher the molecular mechanism of this transfer. To attain this goal, we will carry out structural biology studies (X-ray crystallography and high resolution electron microscopy) as well as functional and structure-function in vivo studies. We have the ambition to make major contributions to the understanding of bacterial transformation. Ultimately, we hope that our results will also help to find compounds that could block natural transformation in bacterial pathogens.
Max ERC Funding
1 405 149 €
Duration
Start date: 2012-01-01, End date: 2016-12-31
Project acronym NDOGS
Project Nuclear Dynamic, Organization and Genome Stability
Researcher (PI) Karine Marie Renée Dubrana
Host Institution (HI) COMMISSARIAT A L ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary The eukaryotic genome is packaged into large-scale chromatin structures occupying distinct domains in the cell nucleus. Nuclear compartmentalization has recently been proposed to play an important role in genome stability but the molecular steps regulated remain to be defined. Focusing on Double strand breaks (DSBs) in response to which cells activate checkpoint and DNA repair pathways, we propose to characterize the spatial and temporal behavior of damaged chromatin and determine how this affects the maintenance of genome integrity. Currently, most studies concerning DSBs signaling and repair have been realized on asynchronous cell populations, which makes it difficult to precisely define the kinetics of events that occur at the cellular level. We thus propose to follow the nuclear localization and dynamics of an inducible DSB concomitantly with the kinetics of checkpoint activation and DNA repair at a single cell level and along the cell cycle. This will be performed using budding yeast as a model system enabling the combination of genetics, molecular biology and advanced live microscopy. We recently demonstrated that DSBs relocated to the nuclear periphery where they contact nuclear pores. This change in localization possibly regulates the choice of the repair pathway through steps that are controlled by post-translational modifications. This proposal aims at dissecting the molecular pathways defining the position of DSBs in the nucleus by performing genetic and proteomic screens, testing the functional consequence of nuclear position for checkpoint activation and DNA repair by driving the DSB to specific nuclear landmarks and, defining the dynamics of DNA damages in different repair contexts. Our project will identify new players in the DNA repair and checkpoint pathways and further our understanding of how the compartmentalization of damaged chromatin into the nucleus regulates these processes to insure the transmission of a stable genome.
Summary
The eukaryotic genome is packaged into large-scale chromatin structures occupying distinct domains in the cell nucleus. Nuclear compartmentalization has recently been proposed to play an important role in genome stability but the molecular steps regulated remain to be defined. Focusing on Double strand breaks (DSBs) in response to which cells activate checkpoint and DNA repair pathways, we propose to characterize the spatial and temporal behavior of damaged chromatin and determine how this affects the maintenance of genome integrity. Currently, most studies concerning DSBs signaling and repair have been realized on asynchronous cell populations, which makes it difficult to precisely define the kinetics of events that occur at the cellular level. We thus propose to follow the nuclear localization and dynamics of an inducible DSB concomitantly with the kinetics of checkpoint activation and DNA repair at a single cell level and along the cell cycle. This will be performed using budding yeast as a model system enabling the combination of genetics, molecular biology and advanced live microscopy. We recently demonstrated that DSBs relocated to the nuclear periphery where they contact nuclear pores. This change in localization possibly regulates the choice of the repair pathway through steps that are controlled by post-translational modifications. This proposal aims at dissecting the molecular pathways defining the position of DSBs in the nucleus by performing genetic and proteomic screens, testing the functional consequence of nuclear position for checkpoint activation and DNA repair by driving the DSB to specific nuclear landmarks and, defining the dynamics of DNA damages in different repair contexts. Our project will identify new players in the DNA repair and checkpoint pathways and further our understanding of how the compartmentalization of damaged chromatin into the nucleus regulates these processes to insure the transmission of a stable genome.
Max ERC Funding
1 499 863 €
Duration
Start date: 2012-02-01, End date: 2018-01-31
Project acronym transtryp
Project Structural differences in mRNA translation machineries between eukaryotic pathogens and their mammalian hosts
Researcher (PI) Yaser HASHEM
Host Institution (HI) INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary mRNA translation consists on translating the genetic code to proteins by the ribosome that is universally conserved in all cells. However, its structure presents significant differences between bacteria and eukaryotes. Partly because of these differences, the bacterial ribosome can be targeted specifically by a number of antibiotics without affecting the eukaryotic host cells. However, the conservation of the ribosome among eukaryotes complicates the search for specific drugs against eukaryotic pathogens such as certain protozoa like plasmodium and kinetoplastids.
Our work along with other studies demonstrates the existence of significant structural differences between ribosomes of protozoa and mammals. Using Cryogenic electron microscopy, we endeavor to investigate such structural differences that are anticipated to affect some of the vital steps of mRNA translation, especially the initiation process, because of their position on the ribosome. 1. Thus we will focus on the structural differences in translation initiation between kinetoplastids and their mammalian hosts (i) by characterizing initiation complexes from several plasmodium and kinetoplastids species and compare them to their mammalian counterparts. (ii) We will also follow up on our previous works in solving the structures of various conventional, but also unconventional mammalian initiation complexes, in interaction with special mRNAs. 2. We will focus on the structure of protozoa-specific features characterized from elongating ribosomal complexes and (i) attempt to fish for regulators that they interact with from cell extracts. In addition, (ii) we will investigate the ribosomal structures from plasmodium at different stages of the parasite life cycle, as they vary according to the latter.
Our results will significantly advance our understanding of protein synthesis regulation in protozoa and will represent a promising step in the search for more efficient treatments against these eukaryotic pathogens.
Summary
mRNA translation consists on translating the genetic code to proteins by the ribosome that is universally conserved in all cells. However, its structure presents significant differences between bacteria and eukaryotes. Partly because of these differences, the bacterial ribosome can be targeted specifically by a number of antibiotics without affecting the eukaryotic host cells. However, the conservation of the ribosome among eukaryotes complicates the search for specific drugs against eukaryotic pathogens such as certain protozoa like plasmodium and kinetoplastids.
Our work along with other studies demonstrates the existence of significant structural differences between ribosomes of protozoa and mammals. Using Cryogenic electron microscopy, we endeavor to investigate such structural differences that are anticipated to affect some of the vital steps of mRNA translation, especially the initiation process, because of their position on the ribosome. 1. Thus we will focus on the structural differences in translation initiation between kinetoplastids and their mammalian hosts (i) by characterizing initiation complexes from several plasmodium and kinetoplastids species and compare them to their mammalian counterparts. (ii) We will also follow up on our previous works in solving the structures of various conventional, but also unconventional mammalian initiation complexes, in interaction with special mRNAs. 2. We will focus on the structure of protozoa-specific features characterized from elongating ribosomal complexes and (i) attempt to fish for regulators that they interact with from cell extracts. In addition, (ii) we will investigate the ribosomal structures from plasmodium at different stages of the parasite life cycle, as they vary according to the latter.
Our results will significantly advance our understanding of protein synthesis regulation in protozoa and will represent a promising step in the search for more efficient treatments against these eukaryotic pathogens.
Max ERC Funding
1 787 400 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
