Project acronym BrokenGenome
Project Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.
Researcher (PI) Corentin CLAEYS BOUUAERT
Host Institution (HI) UNIVERSITE CATHOLIQUE DE LOUVAIN
Call Details Starting Grant (StG), LS1, ERC-2018-STG
Summary Sexual reproduction depends on the programmed induction of DNA double-strand breaks (DSBs) and their ensuing repair by homologous recombination. This complex process is essential for sexual reproduction because it ultimately allows the pairing and separation of homologous chromosomes during formation of haploid gametes. Although meiotic recombination has been investigated for decades, many of the underlying molecular processes remain unclear, largely due to the lack of biochemical studies. I have recently made important progress by, for the first time, successfully purifying proteins involved in two aspects of meiotic recombination: DSB formation and the final stage of formation of the crossovers that are a central raison-d’être of meiotic recombination. This has opened new avenues for future research that I intend to pursue in my own laboratory. Here, I propose a set of biochemical approaches, complemented by molecular genetics methods, to gain insights into four central problems: (i) How meiotic proteins collaborate to induce DSBs; (ii) How DSB proteins interact with components that form the axes of meiotic chromosomes; (iii) How proteins involved at later stages of recombination form crossovers; and (iv) How crossover proteins interact with components of synapsed chromosomes. For each problem, I will set up in vitro systems to probe the activities of the players involved, their interactions with DNA, and their assembly into macromolecular complexes. In addition, I propose to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis. My goal is to gain insights into the mechanisms that govern meiotic recombination. Importantly, these mechanisms are intimately linked not only to gamete formation, but also to the general recombination pathways that all cells use to maintain genome stability. In both contexts, our findings will be relevant to the development and avoidance of disease states.
Summary
Sexual reproduction depends on the programmed induction of DNA double-strand breaks (DSBs) and their ensuing repair by homologous recombination. This complex process is essential for sexual reproduction because it ultimately allows the pairing and separation of homologous chromosomes during formation of haploid gametes. Although meiotic recombination has been investigated for decades, many of the underlying molecular processes remain unclear, largely due to the lack of biochemical studies. I have recently made important progress by, for the first time, successfully purifying proteins involved in two aspects of meiotic recombination: DSB formation and the final stage of formation of the crossovers that are a central raison-d’être of meiotic recombination. This has opened new avenues for future research that I intend to pursue in my own laboratory. Here, I propose a set of biochemical approaches, complemented by molecular genetics methods, to gain insights into four central problems: (i) How meiotic proteins collaborate to induce DSBs; (ii) How DSB proteins interact with components that form the axes of meiotic chromosomes; (iii) How proteins involved at later stages of recombination form crossovers; and (iv) How crossover proteins interact with components of synapsed chromosomes. For each problem, I will set up in vitro systems to probe the activities of the players involved, their interactions with DNA, and their assembly into macromolecular complexes. In addition, I propose to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis. My goal is to gain insights into the mechanisms that govern meiotic recombination. Importantly, these mechanisms are intimately linked not only to gamete formation, but also to the general recombination pathways that all cells use to maintain genome stability. In both contexts, our findings will be relevant to the development and avoidance of disease states.
Max ERC Funding
1 499 075 €
Duration
Start date: 2019-07-01, End date: 2024-06-30
Project acronym CiliaTubulinCode
Project Self-organization of the cilium: the role of the tubulin code
Researcher (PI) Gaia PIGINO
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS1, ERC-2018-COG
Summary This project aims at understanding of the role of the tubulin code for self-organization of complex microtubule based structures. Cilia turn out to be the ideal structures for the proposed research.
A cilium is a sophisticated cellular machine that self-organizes from many protein complexes. It plays motility, sensory, and signaling roles in most eukaryotic cells, and its malfunction causes pathologies. The assembly of the cilium requires intraflagellar transport (IFT), a specialized bidirectional motility process that is mediated by adaptor proteins and direction specific molecular motors. Work from my lab shows that anterograde and retrograde IFT make exclusive use of the B-tubules and A-tubules, respectively. This insight answered a long standing question and shows that functional differentiation of tubules exists and is important for IFT.
Tubulin post-translational modifications (PTMs) contribute to a tubulin code, making microtubules suitable for specific functions. Mutation of tubulin-PTM enzymes can have dramatic effects on cilia function and assembly. However, we do not understand of the role of tubulin-PTMs in cilia. Therefore, I propose to address the hypotheses that the tubulin code contributes to regulating bidirectional IFT motility, and more generally, that the tubulin code is a key player in structuring complex cellular assembly processes in space and time.
This proposal aims at (i) understanding if tubulin-PTMs are necessary and/or sufficient to regulate the bidirectionality of IFT (ii) examining how the tubulin code regulates the assembly of cilia and (iii) generating a high-resolution atlas of tubulin-PTMs and their respective enzymes.
We will combine advanced techniques encompassing state-of-the-art cryo-electron tomography, biochemical imaging, fluorescent microscopy, and in vitro assays to achieve molecular and structural understanding of the role of the tubulin code in the self-organization of cilia and of microtubule based cellular structures.
Summary
This project aims at understanding of the role of the tubulin code for self-organization of complex microtubule based structures. Cilia turn out to be the ideal structures for the proposed research.
A cilium is a sophisticated cellular machine that self-organizes from many protein complexes. It plays motility, sensory, and signaling roles in most eukaryotic cells, and its malfunction causes pathologies. The assembly of the cilium requires intraflagellar transport (IFT), a specialized bidirectional motility process that is mediated by adaptor proteins and direction specific molecular motors. Work from my lab shows that anterograde and retrograde IFT make exclusive use of the B-tubules and A-tubules, respectively. This insight answered a long standing question and shows that functional differentiation of tubules exists and is important for IFT.
Tubulin post-translational modifications (PTMs) contribute to a tubulin code, making microtubules suitable for specific functions. Mutation of tubulin-PTM enzymes can have dramatic effects on cilia function and assembly. However, we do not understand of the role of tubulin-PTMs in cilia. Therefore, I propose to address the hypotheses that the tubulin code contributes to regulating bidirectional IFT motility, and more generally, that the tubulin code is a key player in structuring complex cellular assembly processes in space and time.
This proposal aims at (i) understanding if tubulin-PTMs are necessary and/or sufficient to regulate the bidirectionality of IFT (ii) examining how the tubulin code regulates the assembly of cilia and (iii) generating a high-resolution atlas of tubulin-PTMs and their respective enzymes.
We will combine advanced techniques encompassing state-of-the-art cryo-electron tomography, biochemical imaging, fluorescent microscopy, and in vitro assays to achieve molecular and structural understanding of the role of the tubulin code in the self-organization of cilia and of microtubule based cellular structures.
Max ERC Funding
1 986 406 €
Duration
Start date: 2019-03-01, End date: 2024-02-29
Project acronym DNAProteinCrosslinks
Project DNA-protein crosslinks: endogenous origins and cellular responses.
Researcher (PI) Julian STINGELE
Host Institution (HI) LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Call Details Starting Grant (StG), LS1, ERC-2018-STG
Summary This project aims to address the most pressing questions in the emerging field of research on DNA-protein crosslinks (DPCs) and their repair. Covalent DPCs are highly toxic DNA lesions that block virtually all chromatin processes. DPCs are induced by various exogenous and endogenous agents, but dedicated repair mechanisms were unknown. It was previously assumed that DPCs are repaired by canonical DNA repair pathways. This has changed with my recent discovery of a specific and conserved DPC repair mechanism. I established that proteases of the SPRTN family degrade the protein components of DPCs, which maintains genome stability and ensures tumour suppression. Strikingly, DPC repair by SPRTN is essential for cellular viability, which suggests that cells are constantly challenged with substantial amounts of endogenous DPCs.
I hypothesize there is an entire unexplored pathway regulating protease-based DPC repair and that DPCs are key drivers of endogenous genome instability. I will employ genetic screening approaches and develop novel functional assays to systematically define the components and working principles of this novel DNA repair pathway in mammalian cells. I will determine how DPCs are detected in a chromatin context, how different repair activities are coordinated and connected to cellular processes such as replication or transcription. Moreover, I will identify the currently elusive origins of endogenous DPCs, by investigating the essential role of the SPRTN protease.
My results will not only provide insights into an essential cellular quality-control mechanism but also unravel processes causing genomic instability in human cells. Importantly, many chemotherapeutics used in the clinic exert their cytotoxicity by inducing DPCs. My results will thus have imminent implications for human health and have the potential to reveal novel drug target candidates for combination anti-cancer therapy.
Summary
This project aims to address the most pressing questions in the emerging field of research on DNA-protein crosslinks (DPCs) and their repair. Covalent DPCs are highly toxic DNA lesions that block virtually all chromatin processes. DPCs are induced by various exogenous and endogenous agents, but dedicated repair mechanisms were unknown. It was previously assumed that DPCs are repaired by canonical DNA repair pathways. This has changed with my recent discovery of a specific and conserved DPC repair mechanism. I established that proteases of the SPRTN family degrade the protein components of DPCs, which maintains genome stability and ensures tumour suppression. Strikingly, DPC repair by SPRTN is essential for cellular viability, which suggests that cells are constantly challenged with substantial amounts of endogenous DPCs.
I hypothesize there is an entire unexplored pathway regulating protease-based DPC repair and that DPCs are key drivers of endogenous genome instability. I will employ genetic screening approaches and develop novel functional assays to systematically define the components and working principles of this novel DNA repair pathway in mammalian cells. I will determine how DPCs are detected in a chromatin context, how different repair activities are coordinated and connected to cellular processes such as replication or transcription. Moreover, I will identify the currently elusive origins of endogenous DPCs, by investigating the essential role of the SPRTN protease.
My results will not only provide insights into an essential cellular quality-control mechanism but also unravel processes causing genomic instability in human cells. Importantly, many chemotherapeutics used in the clinic exert their cytotoxicity by inducing DPCs. My results will thus have imminent implications for human health and have the potential to reveal novel drug target candidates for combination anti-cancer therapy.
Max ERC Funding
1 497 375 €
Duration
Start date: 2019-02-01, End date: 2024-01-31
Project acronym INO3D
Project Mechanism of ATP Dependent Chromatin Modelling and Editing by INO80 Remodellers
Researcher (PI) Karl-Peter HOPFNER
Host Institution (HI) LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Call Details Advanced Grant (AdG), LS1, ERC-2018-ADG
Summary Nucleosomes, ~147 base pairs of DNA wrapped around an histone protein octamer, package and protect nuclear DNA but also carry important biological information. The position and composition of nucleosomes along chromosomal DNA is a key element of defining the state and identity of a cell. Chromatin remodellers are ATP dependent molecular machines that position, move or edit nucleosomes in a genome wide manner. Collectively, they shape the nucleosome landscape and play central roles in the maintenance and differentiation of cells, but also in pathological transformations. INO80, a megadalton large remodeller consisting of 15 or more subunits, is involved in replication, gene expression and DNA repair. It models chromatin by positioning barrier nucleosomes around nucleosome free regions, editing nucleosomes and generating nucleosome arrays. However, structural mechanisms for INO80 and other remodelling machines are poorly understood due to their complexity. To provide a comprehensive mechanistic framework, to understand how INO80 senses nucleosome free regions to position barrier nucleosomes and how it generates arrays or senses DNA breaks, I propose a challenging but ground-breaking endeavour using a combination of cryo-EM and functional approaches. We address structures of fungal and human INO80 complexes at promoter regions, on di-nucleosomes and at DNA ends and develop quantitative positioning assays to reveal common and distinct features of shaping chromatin in different species. We also explore cryo-EM as tool towards revealing distinct steps the chemo-mechanical remodelling reactions. The proposed research will help derive fundamental molecular principles underlying the modelling of the nucleosome landscape.
Summary
Nucleosomes, ~147 base pairs of DNA wrapped around an histone protein octamer, package and protect nuclear DNA but also carry important biological information. The position and composition of nucleosomes along chromosomal DNA is a key element of defining the state and identity of a cell. Chromatin remodellers are ATP dependent molecular machines that position, move or edit nucleosomes in a genome wide manner. Collectively, they shape the nucleosome landscape and play central roles in the maintenance and differentiation of cells, but also in pathological transformations. INO80, a megadalton large remodeller consisting of 15 or more subunits, is involved in replication, gene expression and DNA repair. It models chromatin by positioning barrier nucleosomes around nucleosome free regions, editing nucleosomes and generating nucleosome arrays. However, structural mechanisms for INO80 and other remodelling machines are poorly understood due to their complexity. To provide a comprehensive mechanistic framework, to understand how INO80 senses nucleosome free regions to position barrier nucleosomes and how it generates arrays or senses DNA breaks, I propose a challenging but ground-breaking endeavour using a combination of cryo-EM and functional approaches. We address structures of fungal and human INO80 complexes at promoter regions, on di-nucleosomes and at DNA ends and develop quantitative positioning assays to reveal common and distinct features of shaping chromatin in different species. We also explore cryo-EM as tool towards revealing distinct steps the chemo-mechanical remodelling reactions. The proposed research will help derive fundamental molecular principles underlying the modelling of the nucleosome landscape.
Max ERC Funding
2 201 875 €
Duration
Start date: 2019-10-01, End date: 2024-09-30
Project acronym MAMMALIANDEVELOPMENT
Project A systems-level understanding of the novel principle in early mammalian development
Researcher (PI) Takashi Hiiragi
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Early mammalian development is a unique process creating an extraembryonic structure. Despite its importance for understanding mammalian development and direct relevance to clinical practice, the mechanism underlying polarity establishment in the mammalian embryo has long been elusive. One of the major obstacles is the lack of description in molecular terms, since very few genes are known to specify the early lineages. Our recent studies provide a conceptual basis, suggesting that the mechanism is unique to mammals. The primary aim of this proposal is to elucidate the molecular program and the novel principle of early mammalian development at a systems level. To comprehensively identify molecules involved in early mouse development, we will conduct two complementary screens. One is a lentivirus-based promoter-trap screen: Venus-reporter is to be expressed under the endogenous control of the integrated genomic locus, which will be monitored using our live-embryo imaging system. Embryos showing a differential expression pattern will be selected for further analysis. As a complementary approach, single-blastomere-derived cRNAs are generated from embryos of various stages by the recently developed single-cell cRNA amplification method, followed by microarray analysis to statistically identify gene clusters differentially expressed in specific blastomeres. Function of the genes identified in two screens will be examined by RNAi and maternally conditional KO. Finally, the knowledge will be integrated into our computer simulation that successfully reconstitutes blastocyst morphogenesis. In the long term, the obtained tools (markers and Venus-trap lines) will provide a basis for functional siRNA screen. Genetic screen in early mouse embryos has never been achieved. Though we anticipate certain difficulties, we are confident that with the relevant expertise of collaborators and ourselves, these can be resolved and a substantial advance will be made in this important area.
Summary
Early mammalian development is a unique process creating an extraembryonic structure. Despite its importance for understanding mammalian development and direct relevance to clinical practice, the mechanism underlying polarity establishment in the mammalian embryo has long been elusive. One of the major obstacles is the lack of description in molecular terms, since very few genes are known to specify the early lineages. Our recent studies provide a conceptual basis, suggesting that the mechanism is unique to mammals. The primary aim of this proposal is to elucidate the molecular program and the novel principle of early mammalian development at a systems level. To comprehensively identify molecules involved in early mouse development, we will conduct two complementary screens. One is a lentivirus-based promoter-trap screen: Venus-reporter is to be expressed under the endogenous control of the integrated genomic locus, which will be monitored using our live-embryo imaging system. Embryos showing a differential expression pattern will be selected for further analysis. As a complementary approach, single-blastomere-derived cRNAs are generated from embryos of various stages by the recently developed single-cell cRNA amplification method, followed by microarray analysis to statistically identify gene clusters differentially expressed in specific blastomeres. Function of the genes identified in two screens will be examined by RNAi and maternally conditional KO. Finally, the knowledge will be integrated into our computer simulation that successfully reconstitutes blastocyst morphogenesis. In the long term, the obtained tools (markers and Venus-trap lines) will provide a basis for functional siRNA screen. Genetic screen in early mouse embryos has never been achieved. Though we anticipate certain difficulties, we are confident that with the relevant expertise of collaborators and ourselves, these can be resolved and a substantial advance will be made in this important area.
Max ERC Funding
1 150 000 €
Duration
Start date: 2008-07-01, End date: 2013-12-31
Project acronym MitoCRISTAE
Project Mitochondrial Cristae Biogenesis
Researcher (PI) Stefan Jakobs
Host Institution (HI) UNIVERSITAETSMEDIZIN GOETTINGEN - GEORG-AUGUST-UNIVERSITAET GOETTINGEN - STIFTUNG OEFFENTLICHEN RECHTS
Call Details Advanced Grant (AdG), LS1, ERC-2018-ADG
Summary Mitochondrial cristae biogenesis is an enigma ever since the first imaging of mitochondria, the ‘powerhouses’ of eukaryotic cells, by electron microscopy in the 1950s. The mitochondrial cristae, dynamic and structurally conserved invaginations of the mitochondrial inner membrane, are essential for respiratory ATP generation. Thereby, the form and function of the mitochondrial inner membrane are deeply intertwined. Indeed, irregular or disturbed cristae morphologies are believed to cause numerous human diseases, including neurodegeneration, cardiomyopathies, metabolic disorders and cancer.
Previous approaches to study cristae biogenesis have relied primarily on the use of 2D electron microscopy and biochemistry to analyse mutant cells defective in cristae formation. Based on striking pilot experiments, we propose to study cristae biogenesis by a radically different approach. We will induce synchronous cristae development in gene-edited cell lines initially defective in cristae formation. We will then follow de novo cristae biogenesis over time by combining a series of enabling approaches, including live cell and MINFLUX super-resolution microscopy, 3D (cryo) electron microscopy, label-free (SWATH) mass spectrometry, and single molecule counting. These technologies have just emerged in the last few years, and thus this proposal would not have been possible a few years ago. The primary aim of this proposal is to establish a deep, comprehensive and quantitative understanding of cristae biogenesis in human cells. Using theses insights, we will also investigate the effects of mutations in mitochondrial proteins associated with human diseases on cristae biogenesis.
Altogether, if successful, the outcome will represent a paradigm shift in our knowledge of how mitochondrial ultrastructure in healthy and diseased cells is generated and maintained. Our findings might spark innovative and novel strategies for the treatment of devastating human mitopathies.
Summary
Mitochondrial cristae biogenesis is an enigma ever since the first imaging of mitochondria, the ‘powerhouses’ of eukaryotic cells, by electron microscopy in the 1950s. The mitochondrial cristae, dynamic and structurally conserved invaginations of the mitochondrial inner membrane, are essential for respiratory ATP generation. Thereby, the form and function of the mitochondrial inner membrane are deeply intertwined. Indeed, irregular or disturbed cristae morphologies are believed to cause numerous human diseases, including neurodegeneration, cardiomyopathies, metabolic disorders and cancer.
Previous approaches to study cristae biogenesis have relied primarily on the use of 2D electron microscopy and biochemistry to analyse mutant cells defective in cristae formation. Based on striking pilot experiments, we propose to study cristae biogenesis by a radically different approach. We will induce synchronous cristae development in gene-edited cell lines initially defective in cristae formation. We will then follow de novo cristae biogenesis over time by combining a series of enabling approaches, including live cell and MINFLUX super-resolution microscopy, 3D (cryo) electron microscopy, label-free (SWATH) mass spectrometry, and single molecule counting. These technologies have just emerged in the last few years, and thus this proposal would not have been possible a few years ago. The primary aim of this proposal is to establish a deep, comprehensive and quantitative understanding of cristae biogenesis in human cells. Using theses insights, we will also investigate the effects of mutations in mitochondrial proteins associated with human diseases on cristae biogenesis.
Altogether, if successful, the outcome will represent a paradigm shift in our knowledge of how mitochondrial ultrastructure in healthy and diseased cells is generated and maintained. Our findings might spark innovative and novel strategies for the treatment of devastating human mitopathies.
Max ERC Funding
2 286 248 €
Duration
Start date: 2019-10-01, End date: 2024-09-30
Project acronym REGEXTRA
Project A Novel Level for the Regulation of Eukaryotic Gene Expression: Coupling Transcription to Translation
Researcher (PI) Katja Sträßer
Host Institution (HI) LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Eukaryotic gene expression is a highly regulated, fundamental cellular process encompassing distinct steps such as transcription, mRNA processing and nuclear export, translation and degradation of the mRNA. In this project a novel level in the regulation of gene expression will be analyzed. We propose that transcription and the correct processing as well as packaging of the newly synthesized mRNA into an mRNP control the translation of this mRNA in the cytoplasm thus coupling intranuclear events in mRNA biogenesis to translation. Recently, we showed that the transcription elongation factor Ctk1 functions as a positive factor in translation elongation by phosphorylating the ribosomal protein Rps2. According to our model, Ctk1 enhances the translation fidelity of mRNAs that have been correctly processed and assembled into mRNPs in the nucleus. In the project proposed here we will analyze how the function of Ctk1 couples transcription to translation and how Ctk1 functions in translation initiation, in support of which we have already obtained evidence. Importantly, we will identify additional players in coupling intranuclear mRNA biogenesis events to translation and unravel their molecular function. In addition, we will determine phosphorylated residues on ribosomal proteins and translation factors, identify their kinases and phosphatases, and analyze the biological significance of these phosphorylation events in translation. Moreover, the conservation in higher eukaryotes of the biological principles obtained with our model organism S. cerevisiae will be assessed. All these experiments are performed under the aspect that the cell uses phosphorylation of ribosomal proteins and translation factors to control the efficient and correct translation of a correctly processed mRNP providing a novel level of gene expression control in eukaryotes.
Summary
Eukaryotic gene expression is a highly regulated, fundamental cellular process encompassing distinct steps such as transcription, mRNA processing and nuclear export, translation and degradation of the mRNA. In this project a novel level in the regulation of gene expression will be analyzed. We propose that transcription and the correct processing as well as packaging of the newly synthesized mRNA into an mRNP control the translation of this mRNA in the cytoplasm thus coupling intranuclear events in mRNA biogenesis to translation. Recently, we showed that the transcription elongation factor Ctk1 functions as a positive factor in translation elongation by phosphorylating the ribosomal protein Rps2. According to our model, Ctk1 enhances the translation fidelity of mRNAs that have been correctly processed and assembled into mRNPs in the nucleus. In the project proposed here we will analyze how the function of Ctk1 couples transcription to translation and how Ctk1 functions in translation initiation, in support of which we have already obtained evidence. Importantly, we will identify additional players in coupling intranuclear mRNA biogenesis events to translation and unravel their molecular function. In addition, we will determine phosphorylated residues on ribosomal proteins and translation factors, identify their kinases and phosphatases, and analyze the biological significance of these phosphorylation events in translation. Moreover, the conservation in higher eukaryotes of the biological principles obtained with our model organism S. cerevisiae will be assessed. All these experiments are performed under the aspect that the cell uses phosphorylation of ribosomal proteins and translation factors to control the efficient and correct translation of a correctly processed mRNP providing a novel level of gene expression control in eukaryotes.
Max ERC Funding
899 713 €
Duration
Start date: 2008-09-01, End date: 2013-08-31
Project acronym REPLISOMEBYPASS
Project Challenges on the road to genome duplication: Single-molecule approaches to study replisome collisions
Researcher (PI) Karl DUDERSTADT
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Starting Grant (StG), LS1, ERC-2018-STG
Summary Faithful duplication and transmission of genetic and epigenetic information is the most vital cellular
function for the preservation and proliferation of life. In cells, this process is conducted by large
macromolecular complexes, known as replisomes, that coordinate the sequence of enzymatic events
during chromosome duplication. While recently developed single-molecule techniques promise
unprecedented access to the complex inner workings of these sophisticated machines, most studies
conducted have focused on individual factors, operating on non-physiological substrates, which has
provided an incomplete molecular picture.
My recent development of a multidimensional, single-molecule imaging approach that allows
for real-time visualisation of coordination during replication represents a significant breakthrough
in our ability to study macromolecular machines in vitro. Building on this success, here I describe
single-molecule imaging approaches to address one of the long-standing questions in chromosome
biology: How do replisomes maintain efficiency and coordination during collisions with obstacles
on the chromosome?
Our objective is to develop a complete molecular understanding of the consequences of
replisome collisions and the underlying mechanisms that allow for bypass or trigger replication fork
collapse. We will begin this long-term research effort by addressing several issues fundamental to
chromosome replication: How does replisome coordination and composition change during
encounters with topological barriers in chromosomes? What are the dynamic events that underlie
nucleosome disassembly by histone chaperones during replication? How does the eukaryotic
replisome collaborate with histone chaperones to ensure faithful inheritance of epigenetic
information encoded on histones?
These studies will provide a framework for understanding the dynamics of replisome collisions
and the molecular origin of chromosome damage underlying many diseases.
Summary
Faithful duplication and transmission of genetic and epigenetic information is the most vital cellular
function for the preservation and proliferation of life. In cells, this process is conducted by large
macromolecular complexes, known as replisomes, that coordinate the sequence of enzymatic events
during chromosome duplication. While recently developed single-molecule techniques promise
unprecedented access to the complex inner workings of these sophisticated machines, most studies
conducted have focused on individual factors, operating on non-physiological substrates, which has
provided an incomplete molecular picture.
My recent development of a multidimensional, single-molecule imaging approach that allows
for real-time visualisation of coordination during replication represents a significant breakthrough
in our ability to study macromolecular machines in vitro. Building on this success, here I describe
single-molecule imaging approaches to address one of the long-standing questions in chromosome
biology: How do replisomes maintain efficiency and coordination during collisions with obstacles
on the chromosome?
Our objective is to develop a complete molecular understanding of the consequences of
replisome collisions and the underlying mechanisms that allow for bypass or trigger replication fork
collapse. We will begin this long-term research effort by addressing several issues fundamental to
chromosome replication: How does replisome coordination and composition change during
encounters with topological barriers in chromosomes? What are the dynamic events that underlie
nucleosome disassembly by histone chaperones during replication? How does the eukaryotic
replisome collaborate with histone chaperones to ensure faithful inheritance of epigenetic
information encoded on histones?
These studies will provide a framework for understanding the dynamics of replisome collisions
and the molecular origin of chromosome damage underlying many diseases.
Max ERC Funding
1 500 000 €
Duration
Start date: 2019-01-01, End date: 2023-12-31
Project acronym RESEAL
Project Epithelial Resealing
Researcher (PI) Antonio Alfredo Coelho Jacinto
Host Institution (HI) FUNDACAO CALOUSTE GULBENKIAN
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Epithelia have the essential role of acting as a barrier that protects living organisms and its organs from the surrounding milieu. Therefore, it is crucial for epithelial tissues to have robust ways of maintaining its integrity despite the frequent damage caused by normal cell turnover, inflammation and injury. All epithelia have some capacity to repair themselves, however, the wound-healing process differs dramatically between the developmental stage and type of tissue involved. In this project we will focus on investigating the capacity that several simple epithelial tissues have to reseal small discontinuities very rapidly and efficiently. This repair mechanism that we call epithelial resealing is based on the contraction of an actomyosin purse string in the leading edge cells around the wound margin. Epithelial resealing seems to be a fundamental repair mechanism, acting in several types of simple embryonic and adult epithelia, in both vertebrates and invertebrates. The cell biology of epithelial resealing has started to be understood but there are still many open questions and the signalling cascades that regulate this process are largely unknown. We propose to investigate epithelial resealing using a combination of genetics and high resolution live imaging. The Drosophila embryonic epithelium will be our primary model system and we will start by characterizing in detail novel genes involved in resealing that have been identified in a pilot screen previously performed in the laboratory. We will also perform a new RNAi genetic screen based on a very large collections of transgenic lines to completely unravel the signalling network that controls epithelial resealing. In order to investigate how conserved in vertebrates are the epithelial resealing mechanisms, we will establish epithelial wounding assays in zebrafish simple epithelial tissues and we will study, in this vertebrate model system, the molecular mechanisms that we will uncover using Drosophila.
Summary
Epithelia have the essential role of acting as a barrier that protects living organisms and its organs from the surrounding milieu. Therefore, it is crucial for epithelial tissues to have robust ways of maintaining its integrity despite the frequent damage caused by normal cell turnover, inflammation and injury. All epithelia have some capacity to repair themselves, however, the wound-healing process differs dramatically between the developmental stage and type of tissue involved. In this project we will focus on investigating the capacity that several simple epithelial tissues have to reseal small discontinuities very rapidly and efficiently. This repair mechanism that we call epithelial resealing is based on the contraction of an actomyosin purse string in the leading edge cells around the wound margin. Epithelial resealing seems to be a fundamental repair mechanism, acting in several types of simple embryonic and adult epithelia, in both vertebrates and invertebrates. The cell biology of epithelial resealing has started to be understood but there are still many open questions and the signalling cascades that regulate this process are largely unknown. We propose to investigate epithelial resealing using a combination of genetics and high resolution live imaging. The Drosophila embryonic epithelium will be our primary model system and we will start by characterizing in detail novel genes involved in resealing that have been identified in a pilot screen previously performed in the laboratory. We will also perform a new RNAi genetic screen based on a very large collections of transgenic lines to completely unravel the signalling network that controls epithelial resealing. In order to investigate how conserved in vertebrates are the epithelial resealing mechanisms, we will establish epithelial wounding assays in zebrafish simple epithelial tissues and we will study, in this vertebrate model system, the molecular mechanisms that we will uncover using Drosophila.
Max ERC Funding
1 150 000 €
Duration
Start date: 2008-11-01, End date: 2014-10-31
Project acronym SEGCLOCKDYN
Project Collective and cell-autonomous dynamics of the genetic oscillators of the segmentation clock in zebrafish somitogenesis
Researcher (PI) Andrew Oates
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary The subdivision of the vertebrate embryo into repeating segments called somites, from which the muscles and bones will grow, is one of the oldest problems in developmental biology, recognized by Malpighi in the 1600s. This proposal focuses on the composition, function and dynamics of a population of genetic oscillators (termed the segmentation clock) that couples the spatial elongation of the embryo with the serial formation of somites. The research proposal outlines molecular, cellular, embryological and biophysical approaches to understanding these complex temporal and spatial phenomena in the zebrafish embryo. The work delves into three main areas: identification of oscillator components, control of oscillator frequency, and coordination of the oscillations in space and time. Novel methods and technologies to measure spatial growth rates, genetic oscillations, and signaling events in real-time in the growing embryo are proposed. Mathematical simulation and analytic physical theory to describe both morphological dynamics and microscopic biochemical mechanisms are to be developed in parallel with the biological experiments. The application of theory and experiment to the segmentation clock will give a unified framework in which to make and test physical theories of collective processes and their precision and robustness in developing systems. Coordination of the field of cells that gives rise to somites by synchronized genetic oscillations is a novel paradigm for patterning tissues, raising the possibility that genetic oscillations may be widely used in embryonic patterning, differentiation and morphogenesis. Analysis of the segmentation clock should provide the key concepts and methods to explore such systems.
Summary
The subdivision of the vertebrate embryo into repeating segments called somites, from which the muscles and bones will grow, is one of the oldest problems in developmental biology, recognized by Malpighi in the 1600s. This proposal focuses on the composition, function and dynamics of a population of genetic oscillators (termed the segmentation clock) that couples the spatial elongation of the embryo with the serial formation of somites. The research proposal outlines molecular, cellular, embryological and biophysical approaches to understanding these complex temporal and spatial phenomena in the zebrafish embryo. The work delves into three main areas: identification of oscillator components, control of oscillator frequency, and coordination of the oscillations in space and time. Novel methods and technologies to measure spatial growth rates, genetic oscillations, and signaling events in real-time in the growing embryo are proposed. Mathematical simulation and analytic physical theory to describe both morphological dynamics and microscopic biochemical mechanisms are to be developed in parallel with the biological experiments. The application of theory and experiment to the segmentation clock will give a unified framework in which to make and test physical theories of collective processes and their precision and robustness in developing systems. Coordination of the field of cells that gives rise to somites by synchronized genetic oscillations is a novel paradigm for patterning tissues, raising the possibility that genetic oscillations may be widely used in embryonic patterning, differentiation and morphogenesis. Analysis of the segmentation clock should provide the key concepts and methods to explore such systems.
Max ERC Funding
1 550 000 €
Duration
Start date: 2008-06-01, End date: 2013-05-31
