Project acronym 3DCellPhase-
Project In situ Structural Analysis of Molecular Crowding and Phase Separation
Researcher (PI) Julia MAHAMID
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm.
Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
Summary
This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm.
Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
Max ERC Funding
1 228 125 €
Duration
Start date: 2018-02-01, End date: 2023-01-31
Project acronym ATG9_SOLVES_IT
Project In vitro high resolution reconstitution of autophagosome nucleation and expansion catalyzed byATG9
Researcher (PI) Sharon TOOZE
Host Institution (HI) THE FRANCIS CRICK INSTITUTE LIMITED
Call Details Advanced Grant (AdG), LS1, ERC-2017-ADG
Summary Autophagy is a conserved, lysosomal-mediated pathway required for cell homeostasis and survival. It is controlled by the master regulators of energy (AMPK) and growth (TORC1) and mediated by the ATG (autophagy) proteins. Deregulation of autophagy is implicated in cancer, immunity, infection, aging and neurodegeneration. Autophagosomes form and expand using membranes from the secretory and endocytic pathways but how this occurs is not understood. ATG9, the only transmembrane ATG protein traffics through the cell in vesicles, and is essential for rapid initiation and expansion of the membranes which form the autophagosome. Crucially, how ATG9 functions is unknown. I will determine how ATG9 initiates the formation and expansion of the autophagosome by amino acid starvation through a molecular dissection of proteins resident in ATG9 vesicles which modulate the composition and property of the initiating membrane. I will employ high resolution light and electron microscopy to characterize the nucleation of the autophagosome, proximity-specific biotinylation and quantitative Mass Spectrometry to uncover the proteome required for the function of the ATG9, and optogenetic tools to acutely regulate signaling lipids. Lastly, with our tools and knowledge I will develop an in vitro reconstitution system to define at a molecular level how ATG9 vesicle proteins, membranes that interact with ATG9 vesicles, and other accessory ATG components nucleate and form an autophagosome. In vitro reconstitution of autophagosomes will be assayed biochemically, and by correlative light and cryo-EM and cryo-EM tomography, while functional reconstitution of autophagy will be tested by selective cargo recruitment. The development of a reconstituted system and identification proteins and lipids which are key components for autophagosome formation will provide a means to identify a new generation of targets for translational work leading to manipulation of autophagy for disease related therapies.
Summary
Autophagy is a conserved, lysosomal-mediated pathway required for cell homeostasis and survival. It is controlled by the master regulators of energy (AMPK) and growth (TORC1) and mediated by the ATG (autophagy) proteins. Deregulation of autophagy is implicated in cancer, immunity, infection, aging and neurodegeneration. Autophagosomes form and expand using membranes from the secretory and endocytic pathways but how this occurs is not understood. ATG9, the only transmembrane ATG protein traffics through the cell in vesicles, and is essential for rapid initiation and expansion of the membranes which form the autophagosome. Crucially, how ATG9 functions is unknown. I will determine how ATG9 initiates the formation and expansion of the autophagosome by amino acid starvation through a molecular dissection of proteins resident in ATG9 vesicles which modulate the composition and property of the initiating membrane. I will employ high resolution light and electron microscopy to characterize the nucleation of the autophagosome, proximity-specific biotinylation and quantitative Mass Spectrometry to uncover the proteome required for the function of the ATG9, and optogenetic tools to acutely regulate signaling lipids. Lastly, with our tools and knowledge I will develop an in vitro reconstitution system to define at a molecular level how ATG9 vesicle proteins, membranes that interact with ATG9 vesicles, and other accessory ATG components nucleate and form an autophagosome. In vitro reconstitution of autophagosomes will be assayed biochemically, and by correlative light and cryo-EM and cryo-EM tomography, while functional reconstitution of autophagy will be tested by selective cargo recruitment. The development of a reconstituted system and identification proteins and lipids which are key components for autophagosome formation will provide a means to identify a new generation of targets for translational work leading to manipulation of autophagy for disease related therapies.
Max ERC Funding
2 121 055 €
Duration
Start date: 2018-07-01, End date: 2023-06-30
Project acronym AutoClean
Project Cell-free reconstitution of autophagy to dissect molecular mechanisms
Researcher (PI) Claudine Simone Kraft
Host Institution (HI) UNIVERSITAETSKLINIKUM FREIBURG
Call Details Consolidator Grant (CoG), LS1, ERC-2017-COG
Summary Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.
Summary
Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.
Max ERC Funding
1 955 666 €
Duration
Start date: 2018-06-01, End date: 2023-05-31
Project acronym BEFINE
Project mechanical BEhavior of Fluid-INduced Earthquakes
Researcher (PI) Marie, Estelle, Solange VIOLAY
Host Institution (HI) ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Call Details Starting Grant (StG), PE10, ERC-2017-STG
Summary Fluids play an important role in fault zone and in earthquakes generation. Fluid pressure reduces the normal effective stress, lowering the frictional strength of the fault, potentially triggering earthquake ruptures. Fluid injection induced earthquakes (FIE) are direct evidence of the effect of fluid pressure on the fault strength. In addition, natural earthquake sequences are often associated with high fluid pressures at seismogenic depths. Although simple in theory, the mechanisms that govern the nucleation, propagation and recurrence of FIEs are poorly constrained, and our ability to assess the seismic hazard that is associated with natural and induced events remains limited. This project aims to enhance our knowledge of FIE mechanisms over entire seismic cycles through multidisciplinary approaches, including the following:
- Set-up and installation of a new and unique rock friction apparatus that is dedicated to the study of FIEs.
- Low strain rate friction experiments (coupled with electrical conductivity measurements) to investigate the influence of fluids on fault creep and earthquake recurrence.
- Intermediate strain rate friction experiments to investigate the effect of fluids on fault stability during earthquake nucleation.
- High strain rate friction experiments to investigate the effect of fluids on fault weakening during earthquake propagation.
- Post-mortem experimental fault analyses with state-of-art microstructural techniques.
- The theoretical friction law will be calibrated with friction experiments and faulted rock microstructural observations.
These steps will produce fundamental discoveries regarding natural earthquakes and tectonic processes and help scientists understand and eventually manage the occurrence of induced seismicity, an increasingly hot topic in geo-engineering. The sustainable exploitation of geo-resources is a key research and technology challenge at the European scale, with a substantial economical and societal impact.
Summary
Fluids play an important role in fault zone and in earthquakes generation. Fluid pressure reduces the normal effective stress, lowering the frictional strength of the fault, potentially triggering earthquake ruptures. Fluid injection induced earthquakes (FIE) are direct evidence of the effect of fluid pressure on the fault strength. In addition, natural earthquake sequences are often associated with high fluid pressures at seismogenic depths. Although simple in theory, the mechanisms that govern the nucleation, propagation and recurrence of FIEs are poorly constrained, and our ability to assess the seismic hazard that is associated with natural and induced events remains limited. This project aims to enhance our knowledge of FIE mechanisms over entire seismic cycles through multidisciplinary approaches, including the following:
- Set-up and installation of a new and unique rock friction apparatus that is dedicated to the study of FIEs.
- Low strain rate friction experiments (coupled with electrical conductivity measurements) to investigate the influence of fluids on fault creep and earthquake recurrence.
- Intermediate strain rate friction experiments to investigate the effect of fluids on fault stability during earthquake nucleation.
- High strain rate friction experiments to investigate the effect of fluids on fault weakening during earthquake propagation.
- Post-mortem experimental fault analyses with state-of-art microstructural techniques.
- The theoretical friction law will be calibrated with friction experiments and faulted rock microstructural observations.
These steps will produce fundamental discoveries regarding natural earthquakes and tectonic processes and help scientists understand and eventually manage the occurrence of induced seismicity, an increasingly hot topic in geo-engineering. The sustainable exploitation of geo-resources is a key research and technology challenge at the European scale, with a substantial economical and societal impact.
Max ERC Funding
1 982 925 €
Duration
Start date: 2018-03-01, End date: 2023-02-28
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 BURSTREG
Project Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of transcriptional bursting and its effects on cellular fitness
Researcher (PI) Tineke LENSTRA
Host Institution (HI) STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression influences cell fate decisions and disease progression. Interestingly, gene expression variability is not the same for every gene: noise can vary by several orders of magnitude across transcriptomes. The reason for this transcript-specific behavior is that genes are not transcribed in a continuous fashion, but can show transcriptional bursting, with periods of gene activity followed by periods of inactivity. The noisiness of a gene can be tuned by changing the duration and the rate of switching between periods of activity and inactivity. Even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. Here, I will use cutting-edge single-molecule RNA imaging techniques to directly observe and measure transcriptional bursting in living yeast cells. First, bursting properties will be quantified at different endogenous and mutated genes to evaluate the contribution of cis-regulatory promoter elements on bursting. Second, the role of trans-regulatory complexes will be characterized by dynamic depletion or gene-specific targeting of transcription regulatory proteins and observing changes in RNA synthesis in real-time. Third, I will develop a new technology to visualize the binding dynamics of single transcription factor molecules at the transcription site, so that the stability of upstream regulatory factors and the RNA output can directly be compared in the same cell. Finally, I will examine the phenotypic effect of different bursting patterns on organismal fitness. Overall, these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.
Summary
Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression influences cell fate decisions and disease progression. Interestingly, gene expression variability is not the same for every gene: noise can vary by several orders of magnitude across transcriptomes. The reason for this transcript-specific behavior is that genes are not transcribed in a continuous fashion, but can show transcriptional bursting, with periods of gene activity followed by periods of inactivity. The noisiness of a gene can be tuned by changing the duration and the rate of switching between periods of activity and inactivity. Even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. Here, I will use cutting-edge single-molecule RNA imaging techniques to directly observe and measure transcriptional bursting in living yeast cells. First, bursting properties will be quantified at different endogenous and mutated genes to evaluate the contribution of cis-regulatory promoter elements on bursting. Second, the role of trans-regulatory complexes will be characterized by dynamic depletion or gene-specific targeting of transcription regulatory proteins and observing changes in RNA synthesis in real-time. Third, I will develop a new technology to visualize the binding dynamics of single transcription factor molecules at the transcription site, so that the stability of upstream regulatory factors and the RNA output can directly be compared in the same cell. Finally, I will examine the phenotypic effect of different bursting patterns on organismal fitness. Overall, these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.
Max ERC Funding
1 950 775 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
Project acronym C-CLEAR
Project Complement: to clear or not to clear
Researcher (PI) Piet Gros
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Advanced Grant (AdG), LS1, ERC-2017-ADG
Summary Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear.
My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry.
Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes.
Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.
Summary
Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear.
My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry.
Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes.
Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.
Max ERC Funding
2 332 500 €
Duration
Start date: 2018-07-01, End date: 2023-06-30
Project acronym CHROMDOM
Project Chromosomal domain formation, compartmentalization and architecture
Researcher (PI) Johannes STIGLER
Host Institution (HI) LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary The three-dimensional organization of chromosomes is necessary for hereditary fidelity and gene regulation. Recent studies have found that eukaryotic interphase chromosomes are spatially organized in compartments, chiefly topologically associated domains (TADs), in a hierarchical order of nested chromatin loops, coining the term “chromosome folding”. TADs are clusters of genes and regulatory elements that are confined to their genomic compartment by spatially constricting their accessible range of action. The folded structure of chromosomes through long-range loops enables mutual interactions of distant genomic loci that otherwise would not be in contact.
While crosslinking-based chromosome conformation capture (3C) techniques have revealed the underlying structure of interphase chromosomes, the molecular mechanism of how chromosome-organizing proteins, such as the insulator CTCF or the structural maintenance of chromosomes (SMC) complex cohesin build the chromosomal scaffold and contribute to genomic organization, is not understood.
Due to the complexity of the processes involved, biochemical information on how chromosomal proteins contribute to the establishment of TADs is scarce. I have previously demonstrated that single molecule techniques can be used to study the interactions of single cohesin complexes with DNA, chromatin and DNA-bound proteins and to resolve processes that are inaccessible in bulk biochemical experiments. In this project, I will use and expand the high-throughput single molecule technique of DNA curtains to study the molecular details of how chromosomal scaffolding proteins and genetic insulators form the basis for the three-dimensional folding of chromosomes. My experiments will build a novel experimental platform to study the dynamics of chromosomal configuration and maintenance in a reconstituted single molecule assay and will reveal the molecular details that drive the organization of chromosomes into hierarchically organized structures.
Summary
The three-dimensional organization of chromosomes is necessary for hereditary fidelity and gene regulation. Recent studies have found that eukaryotic interphase chromosomes are spatially organized in compartments, chiefly topologically associated domains (TADs), in a hierarchical order of nested chromatin loops, coining the term “chromosome folding”. TADs are clusters of genes and regulatory elements that are confined to their genomic compartment by spatially constricting their accessible range of action. The folded structure of chromosomes through long-range loops enables mutual interactions of distant genomic loci that otherwise would not be in contact.
While crosslinking-based chromosome conformation capture (3C) techniques have revealed the underlying structure of interphase chromosomes, the molecular mechanism of how chromosome-organizing proteins, such as the insulator CTCF or the structural maintenance of chromosomes (SMC) complex cohesin build the chromosomal scaffold and contribute to genomic organization, is not understood.
Due to the complexity of the processes involved, biochemical information on how chromosomal proteins contribute to the establishment of TADs is scarce. I have previously demonstrated that single molecule techniques can be used to study the interactions of single cohesin complexes with DNA, chromatin and DNA-bound proteins and to resolve processes that are inaccessible in bulk biochemical experiments. In this project, I will use and expand the high-throughput single molecule technique of DNA curtains to study the molecular details of how chromosomal scaffolding proteins and genetic insulators form the basis for the three-dimensional folding of chromosomes. My experiments will build a novel experimental platform to study the dynamics of chromosomal configuration and maintenance in a reconstituted single molecule assay and will reveal the molecular details that drive the organization of chromosomes into hierarchically organized structures.
Max ERC Funding
1 499 350 €
Duration
Start date: 2018-07-01, End date: 2023-06-30
Project acronym CIRCOMMUNICATION
Project Deciphering molecular pathways of circadian clock communication
Researcher (PI) gad ASHER
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Consolidator Grant (CoG), LS1, ERC-2017-COG
Summary The overarching objective of this interdisciplinary project is to elucidate mechanisms through which billions of individual clocks in the body communicate with each other and tick in harmony. The mammalian circadian timing system consists of a master clock in the brain and subsidiary oscillators in almost every cell of the body. Since these clocks anticipate environmental changes and function together to orchestrate daily physiology and behavior their temporal synchronization is critical.
Our recent finding that oxygen serves as a resetting cue for circadian clocks points towards the unprecedented involvement of blood gases as time signals. We will apply cutting edge continuous physiological measurements in freely moving animals, alongside biochemical/molecular biology approaches and advanced cell culture setup to determine the molecular role of oxygen, carbon dioxide and pH in circadian clock communication and function.
The intricate nature of the mammalian circadian system demands the presence of communication mechanisms between clocks throughout the body at multiple levels. While previous studies primarily addressed the role of the master clock in resetting peripheral clocks, our knowledge regarding the communication among clocks between and within peripheral organs is rudimentary. We will reconstruct the mammalian circadian system from the bottom up, sequentially restoring clocks in peripheral tissues of a non-rhythmic animal to (i) obtain a system-view of the peripheral circadian communication network; and (ii) study novel tissue-derived circadian communication mechanisms.
This integrative proposal addresses fundamental aspects of circadian biology. It is expected to unravel the circadian communication network and shed light on how billions of clocks in the body function in unison. Its impact extends beyond circadian rhythms and bears great potential for research on communication between cells/tissues in various fields of biology.
Summary
The overarching objective of this interdisciplinary project is to elucidate mechanisms through which billions of individual clocks in the body communicate with each other and tick in harmony. The mammalian circadian timing system consists of a master clock in the brain and subsidiary oscillators in almost every cell of the body. Since these clocks anticipate environmental changes and function together to orchestrate daily physiology and behavior their temporal synchronization is critical.
Our recent finding that oxygen serves as a resetting cue for circadian clocks points towards the unprecedented involvement of blood gases as time signals. We will apply cutting edge continuous physiological measurements in freely moving animals, alongside biochemical/molecular biology approaches and advanced cell culture setup to determine the molecular role of oxygen, carbon dioxide and pH in circadian clock communication and function.
The intricate nature of the mammalian circadian system demands the presence of communication mechanisms between clocks throughout the body at multiple levels. While previous studies primarily addressed the role of the master clock in resetting peripheral clocks, our knowledge regarding the communication among clocks between and within peripheral organs is rudimentary. We will reconstruct the mammalian circadian system from the bottom up, sequentially restoring clocks in peripheral tissues of a non-rhythmic animal to (i) obtain a system-view of the peripheral circadian communication network; and (ii) study novel tissue-derived circadian communication mechanisms.
This integrative proposal addresses fundamental aspects of circadian biology. It is expected to unravel the circadian communication network and shed light on how billions of clocks in the body function in unison. Its impact extends beyond circadian rhythms and bears great potential for research on communication between cells/tissues in various fields of biology.
Max ERC Funding
1 999 945 €
Duration
Start date: 2018-03-01, End date: 2023-02-28
Project acronym COLD
Project Climate Sensitivity of Glacial Landscape Dynamics
Researcher (PI) Dirk SCHERLER
Host Institution (HI) HELMHOLTZ ZENTRUM POTSDAM DEUTSCHESGEOFORSCHUNGSZENTRUM GFZ
Call Details Starting Grant (StG), PE10, ERC-2017-STG
Summary How do erosion rates in glacial landscapes vary with climate change and how do such changes affect the dynamics of mountain glaciers? Providing quantitative constraints towards this question is the main objective of COLD. These constraints are so important because mountain glaciers are sensitive to climate change and their deposits provide a unique history of Earths terrestrial climate that allows reconstructing leads and lags in the climate system.
The climate sensitivity of mountain glaciers is influenced by debris on their surface that impedes ice melting. Theoretical models of frost-related bedrock fracturing predict that rates of debris production are temperature-sensitive and that its supply to mountain glaciers increases during warming periods. Thus a previously unrecognized negative feedback emerges that lowers ice melt rates and potentially buffers part of the ice retreat due to warming. However, the temperature-sensitivity of debris production in glacial landscapes is poorly understood. Specifically, we lack robust erosion rate estimates for these landscapes, which are key for testing models of frost-related bedrock fracturing.
Here, I propose an innovative combination of new tools that capitalize on recent developments in cosmogenic nuclide geochemistry, landscape evolution modelling, and planetary-scale remote sensing analysis. I will use these tools to quantify headwall erosion rates in mountainous glacial landscapes and to gauge the sensitivity of mountain glaciers to variations in debris supply. Expected results will provide a basis for assessing the impacts of global warming, for improved predictions of valley glacier evolution, and for palaeoclimate interpretations of glacial landforms. COLD will focus on glacial landscapes, but the inverse modelling approach I will develop is applicable to any landscape on Earth and has the potential to fundamentally transform how we use cosmogenic nuclides to constrain Earth surface dynamics.
Summary
How do erosion rates in glacial landscapes vary with climate change and how do such changes affect the dynamics of mountain glaciers? Providing quantitative constraints towards this question is the main objective of COLD. These constraints are so important because mountain glaciers are sensitive to climate change and their deposits provide a unique history of Earths terrestrial climate that allows reconstructing leads and lags in the climate system.
The climate sensitivity of mountain glaciers is influenced by debris on their surface that impedes ice melting. Theoretical models of frost-related bedrock fracturing predict that rates of debris production are temperature-sensitive and that its supply to mountain glaciers increases during warming periods. Thus a previously unrecognized negative feedback emerges that lowers ice melt rates and potentially buffers part of the ice retreat due to warming. However, the temperature-sensitivity of debris production in glacial landscapes is poorly understood. Specifically, we lack robust erosion rate estimates for these landscapes, which are key for testing models of frost-related bedrock fracturing.
Here, I propose an innovative combination of new tools that capitalize on recent developments in cosmogenic nuclide geochemistry, landscape evolution modelling, and planetary-scale remote sensing analysis. I will use these tools to quantify headwall erosion rates in mountainous glacial landscapes and to gauge the sensitivity of mountain glaciers to variations in debris supply. Expected results will provide a basis for assessing the impacts of global warming, for improved predictions of valley glacier evolution, and for palaeoclimate interpretations of glacial landforms. COLD will focus on glacial landscapes, but the inverse modelling approach I will develop is applicable to any landscape on Earth and has the potential to fundamentally transform how we use cosmogenic nuclides to constrain Earth surface dynamics.
Max ERC Funding
1 499 308 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
