Project acronym 3DNANOMECH
Project Three-dimensional molecular resolution mapping of soft matter-liquid interfaces
Researcher (PI) Ricardo Garcia
Host Institution (HI) AGENCIA ESTATAL CONSEJO SUPERIOR DEINVESTIGACIONES CIENTIFICAS
Call Details Advanced Grant (AdG), PE4, ERC-2013-ADG
Summary Optical, electron and probe microscopes are enabling tools for discoveries and knowledge generation in nanoscale sicence and technology. High resolution –nanoscale or molecular-, noninvasive and label-free imaging of three-dimensional soft matter-liquid interfaces has not been achieved by any microscopy method.
Force microscopy (AFM) is considered the second most relevant advance in materials science since 1960. Despite its impressive range of applications, the technique has some key limitations. Force microscopy has not three dimensional depth. What lies above or in the subsurface is not readily characterized.
3DNanoMech proposes to design, build and operate a high speed force-based method for the three-dimensional characterization soft matter-liquid interfaces (3D AFM). The microscope will combine a detection method based on force perturbations, adaptive algorithms, high speed piezo actuators and quantitative-oriented multifrequency approaches. The development of the microscope cannot be separated from its applications: imaging the error-free DNA repair and to understand the relationship existing between the nanomechanical properties and the malignancy of cancer cells. Those problems encompass the different spatial –molecular-nano-mesoscopic- and time –milli to seconds- scales of the instrument.
In short, 3DNanoMech aims to image, map and measure with picoNewton, millisecond and angstrom resolution soft matter surfaces and interfaces in liquid. The long-term vision of 3DNanoMech is to replace models or computer animations of bimolecular-liquid interfaces by real time, molecular resolution maps of properties and processes.
Summary
Optical, electron and probe microscopes are enabling tools for discoveries and knowledge generation in nanoscale sicence and technology. High resolution –nanoscale or molecular-, noninvasive and label-free imaging of three-dimensional soft matter-liquid interfaces has not been achieved by any microscopy method.
Force microscopy (AFM) is considered the second most relevant advance in materials science since 1960. Despite its impressive range of applications, the technique has some key limitations. Force microscopy has not three dimensional depth. What lies above or in the subsurface is not readily characterized.
3DNanoMech proposes to design, build and operate a high speed force-based method for the three-dimensional characterization soft matter-liquid interfaces (3D AFM). The microscope will combine a detection method based on force perturbations, adaptive algorithms, high speed piezo actuators and quantitative-oriented multifrequency approaches. The development of the microscope cannot be separated from its applications: imaging the error-free DNA repair and to understand the relationship existing between the nanomechanical properties and the malignancy of cancer cells. Those problems encompass the different spatial –molecular-nano-mesoscopic- and time –milli to seconds- scales of the instrument.
In short, 3DNanoMech aims to image, map and measure with picoNewton, millisecond and angstrom resolution soft matter surfaces and interfaces in liquid. The long-term vision of 3DNanoMech is to replace models or computer animations of bimolecular-liquid interfaces by real time, molecular resolution maps of properties and processes.
Max ERC Funding
2 499 928 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
Project acronym 3MC
Project 3D Model Catalysts to explore new routes to sustainable fuels
Researcher (PI) Petra Elisabeth De jongh
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Consolidator Grant (CoG), PE4, ERC-2014-CoG
Summary Currently fuels, plastics, and drugs are predominantly manufactured from oil. A transition towards renewable resources critically depends on new catalysts, for instance to convert small molecules (such as solar or biomass derived hydrogen, carbon monoxide, water and carbon dioxide) into more complex ones (such as oxygenates, containing oxygen atoms in their structure). Catalyst development now often depends on trial and error rather than rational design, as the heterogeneity of these composite systems hampers detailed understanding of the role of each of the components.
I propose 3D model catalysts as a novel enabling tool to overcome this problem. Their well-defined nature allows unprecedented precision in the variation of structural parameters (morphology, spatial distribution) of the individual components, while at the same time they mimic real catalysts closely enough to allow testing under industrially relevant conditions. Using this approach I will address fundamental questions, such as:
* What are the mechanisms (structural, electronic, chemical) by which non-metal promoters influence the functionality of copper-based catalysts?
* Which nanoalloys can be formed, how does their composition influence the surface active sites and catalytic functionality under reaction conditions?
* Which size and interface effects occur, and how can we use them to tune the actitivity and selectivity towards desired products?
Our 3D model catalysts will be assembled from ordered mesoporous silica and carbon support materials and Cu-based promoted and bimetallic nanoparticles. The combination with high resolution characterization and testing under realistic conditions allows detailed insight into the role of the different components; critical for the rational design of novel catalysts for a future more sustainable production of chemicals and fuels from renewable resources.
Summary
Currently fuels, plastics, and drugs are predominantly manufactured from oil. A transition towards renewable resources critically depends on new catalysts, for instance to convert small molecules (such as solar or biomass derived hydrogen, carbon monoxide, water and carbon dioxide) into more complex ones (such as oxygenates, containing oxygen atoms in their structure). Catalyst development now often depends on trial and error rather than rational design, as the heterogeneity of these composite systems hampers detailed understanding of the role of each of the components.
I propose 3D model catalysts as a novel enabling tool to overcome this problem. Their well-defined nature allows unprecedented precision in the variation of structural parameters (morphology, spatial distribution) of the individual components, while at the same time they mimic real catalysts closely enough to allow testing under industrially relevant conditions. Using this approach I will address fundamental questions, such as:
* What are the mechanisms (structural, electronic, chemical) by which non-metal promoters influence the functionality of copper-based catalysts?
* Which nanoalloys can be formed, how does their composition influence the surface active sites and catalytic functionality under reaction conditions?
* Which size and interface effects occur, and how can we use them to tune the actitivity and selectivity towards desired products?
Our 3D model catalysts will be assembled from ordered mesoporous silica and carbon support materials and Cu-based promoted and bimetallic nanoparticles. The combination with high resolution characterization and testing under realistic conditions allows detailed insight into the role of the different components; critical for the rational design of novel catalysts for a future more sustainable production of chemicals and fuels from renewable resources.
Max ERC Funding
1 999 625 €
Duration
Start date: 2015-09-01, End date: 2020-08-31
Project acronym ABCTRANSPORT
Project Minimalist multipurpose ATP-binding cassette transporters
Researcher (PI) Dirk Jan Slotboom
Host Institution (HI) RIJKSUNIVERSITEIT GRONINGEN
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Summary
Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym ABCvolume
Project The ABC of Cell Volume Regulation
Researcher (PI) Berend Poolman
Host Institution (HI) RIJKSUNIVERSITEIT GRONINGEN
Call Details Advanced Grant (AdG), LS1, ERC-2014-ADG
Summary Cell volume regulation is crucial for any living cell because changes in volume determine the metabolic activity through e.g. changes in ionic strength, pH, macromolecular crowding and membrane tension. These physical chemical parameters influence interaction rates and affinities of biomolecules, folding rates, and fold stabilities in vivo. Understanding of the underlying volume regulatory mechanisms has immediate application in biotechnology and health, yet these factors are generally ignored in systems analyses of cellular functions.
My team has uncovered a number of mechanisms and insights of cell volume regulation. The next step forward is to elucidate how the components of a cell volume regulatory circuit work together and control the physicochemical conditions of the cell.
I propose construction of a synthetic cell in which an osmoregulatory transporter and mechanosensitive channel form a minimal volume regulatory network. My group has developed the technology to reconstitute membrane proteins into lipid vesicles (synthetic cells). One of the challenges is to incorporate into the vesicles an efficient pathway for ATP production and maintain energy homeostasis while the load on the system varies. We aim to control the transmembrane flux of osmolytes, which requires elucidation of the molecular mechanism of gating of the osmoregulatory transporter. We will focus on the glycine betaine ABC importer, which is one of the most complex transporters known to date with ten distinct protein domains, transiently interacting with each other.
The proposed synthetic metabolic circuit constitutes a fascinating out-of-equilibrium system, allowing us to understand cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life. Analysis of this circuit will address many outstanding questions and eventually allow us to design more sophisticated vesicular systems with applications, for example as compartmentalized reaction networks.
Summary
Cell volume regulation is crucial for any living cell because changes in volume determine the metabolic activity through e.g. changes in ionic strength, pH, macromolecular crowding and membrane tension. These physical chemical parameters influence interaction rates and affinities of biomolecules, folding rates, and fold stabilities in vivo. Understanding of the underlying volume regulatory mechanisms has immediate application in biotechnology and health, yet these factors are generally ignored in systems analyses of cellular functions.
My team has uncovered a number of mechanisms and insights of cell volume regulation. The next step forward is to elucidate how the components of a cell volume regulatory circuit work together and control the physicochemical conditions of the cell.
I propose construction of a synthetic cell in which an osmoregulatory transporter and mechanosensitive channel form a minimal volume regulatory network. My group has developed the technology to reconstitute membrane proteins into lipid vesicles (synthetic cells). One of the challenges is to incorporate into the vesicles an efficient pathway for ATP production and maintain energy homeostasis while the load on the system varies. We aim to control the transmembrane flux of osmolytes, which requires elucidation of the molecular mechanism of gating of the osmoregulatory transporter. We will focus on the glycine betaine ABC importer, which is one of the most complex transporters known to date with ten distinct protein domains, transiently interacting with each other.
The proposed synthetic metabolic circuit constitutes a fascinating out-of-equilibrium system, allowing us to understand cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life. Analysis of this circuit will address many outstanding questions and eventually allow us to design more sophisticated vesicular systems with applications, for example as compartmentalized reaction networks.
Max ERC Funding
2 247 231 €
Duration
Start date: 2015-07-01, End date: 2020-06-30
Project acronym ASAP
Project Thylakoid membrane in action: acclimation strategies in algae and plants
Researcher (PI) Roberta Croce
Host Institution (HI) STICHTING VU
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Life on earth is sustained by the process that converts sunlight energy into chemical energy: photosynthesis. This process is operating near the boundary between life and death: if the absorbed energy exceeds the capacity of the metabolic reactions, it can result in photo-oxidation events that can cause the death of the organism. Over-excitation is happening quite often: oxygenic organisms are exposed to (drastic) changes in environmental conditions (light intensity, light quality and temperature), which influence the physical (light-harvesting) and chemical (enzymatic reactions) parts of the photosynthetic process to a different extent, leading to severe imbalances. However, daily experience tells us that plants are able to deal with most of these situations, surviving and happily growing. How do they manage? The photosynthetic membrane is highly flexible and it is able to change its supramolecular organization and composition and even the function of some of its components on a time scale as fast as a few seconds, thereby regulating the light-harvesting capacity. However, the structural/functional changes in the membrane are far from being fully characterized and the molecular mechanisms of their regulation are far from being understood. This is due to the fact that all these mechanisms require the simultaneous presence of various factors and thus the system should be analyzed at a high level of complexity; however, to obtain molecular details of a very complex system as the thylakoid membrane in action has not been possible so far. Over the last years we have developed and optimized a range of methods that now allow us to take up this challenge. This involves a high level of integration of biological and physical approaches, ranging from plant transformation and in vivo knock out of individual pigments to ultrafast-spectroscopy in a mix that is rather unique for my laboratory and will allow us to unravel the photoprotective mechanisms in algae and plants.
Summary
Life on earth is sustained by the process that converts sunlight energy into chemical energy: photosynthesis. This process is operating near the boundary between life and death: if the absorbed energy exceeds the capacity of the metabolic reactions, it can result in photo-oxidation events that can cause the death of the organism. Over-excitation is happening quite often: oxygenic organisms are exposed to (drastic) changes in environmental conditions (light intensity, light quality and temperature), which influence the physical (light-harvesting) and chemical (enzymatic reactions) parts of the photosynthetic process to a different extent, leading to severe imbalances. However, daily experience tells us that plants are able to deal with most of these situations, surviving and happily growing. How do they manage? The photosynthetic membrane is highly flexible and it is able to change its supramolecular organization and composition and even the function of some of its components on a time scale as fast as a few seconds, thereby regulating the light-harvesting capacity. However, the structural/functional changes in the membrane are far from being fully characterized and the molecular mechanisms of their regulation are far from being understood. This is due to the fact that all these mechanisms require the simultaneous presence of various factors and thus the system should be analyzed at a high level of complexity; however, to obtain molecular details of a very complex system as the thylakoid membrane in action has not been possible so far. Over the last years we have developed and optimized a range of methods that now allow us to take up this challenge. This involves a high level of integration of biological and physical approaches, ranging from plant transformation and in vivo knock out of individual pigments to ultrafast-spectroscopy in a mix that is rather unique for my laboratory and will allow us to unravel the photoprotective mechanisms in algae and plants.
Max ERC Funding
1 696 961 €
Duration
Start date: 2011-12-01, End date: 2017-11-30
Project acronym BENDER
Project BiogENesis and Degradation of Endoplasmic Reticulum proteins
Researcher (PI) Friedrich Förster
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Consolidator Grant (CoG), LS1, ERC-2016-COG
Summary The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Summary
The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Max ERC Funding
2 496 611 €
Duration
Start date: 2017-04-01, End date: 2022-03-31
Project acronym BioCircuit
Project Programmable BioMolecular Circuits: Emulating Regulatory Functions in Living Cells Using a Bottom-Up Approach
Researcher (PI) Tom Antonius Franciscus De greef
Host Institution (HI) TECHNISCHE UNIVERSITEIT EINDHOVEN
Call Details Starting Grant (StG), PE4, ERC-2015-STG
Summary Programmable biomolecular circuits have received increasing attention in recent years as the scope of chemistry expands from the synthesis of individual molecules to the construction of chemical networks that can perform sophisticated functions such as logic operations and feedback control. Rationally engineered biomolecular circuits that robustly execute higher-order spatiotemporal behaviours typically associated with intracellular regulatory functions present a unique and uncharted platform to systematically explore the molecular logic and physical design principles of the cell. The experience gained by in-vitro construction of artificial cells displaying advanced system-level functions deepens our understanding of regulatory networks in living cells and allows theoretical assumptions and models to be refined in a controlled setting. This proposal combines elements from systems chemistry, in-vitro synthetic biology and micro-engineering and explores generic strategies to investigate the molecular logic of biology’s regulatory circuits by applying a physical chemistry-driven bottom-up approach. Progress in this field requires 1) proof-of-principle systems where in-vitro biomolecular circuits are designed to emulate characteristic system-level functions of regulatory circuits in living cells and 2) novel experimental tools to operate biochemical networks under out-of-equilibrium conditions. Here, a comprehensive research program is proposed that addresses these challenges by engineering three biochemical model systems that display elementary signal transduction and information processing capabilities. In addition, an open microfluidic droplet reactor is developed that will allow, for the first time, high-throughput analysis of biomolecular circuits encapsulated in water-in-oil droplets. An integral part of the research program is to combine the computational design of in-vitro circuits with novel biochemistry and innovative micro-engineering tools.
Summary
Programmable biomolecular circuits have received increasing attention in recent years as the scope of chemistry expands from the synthesis of individual molecules to the construction of chemical networks that can perform sophisticated functions such as logic operations and feedback control. Rationally engineered biomolecular circuits that robustly execute higher-order spatiotemporal behaviours typically associated with intracellular regulatory functions present a unique and uncharted platform to systematically explore the molecular logic and physical design principles of the cell. The experience gained by in-vitro construction of artificial cells displaying advanced system-level functions deepens our understanding of regulatory networks in living cells and allows theoretical assumptions and models to be refined in a controlled setting. This proposal combines elements from systems chemistry, in-vitro synthetic biology and micro-engineering and explores generic strategies to investigate the molecular logic of biology’s regulatory circuits by applying a physical chemistry-driven bottom-up approach. Progress in this field requires 1) proof-of-principle systems where in-vitro biomolecular circuits are designed to emulate characteristic system-level functions of regulatory circuits in living cells and 2) novel experimental tools to operate biochemical networks under out-of-equilibrium conditions. Here, a comprehensive research program is proposed that addresses these challenges by engineering three biochemical model systems that display elementary signal transduction and information processing capabilities. In addition, an open microfluidic droplet reactor is developed that will allow, for the first time, high-throughput analysis of biomolecular circuits encapsulated in water-in-oil droplets. An integral part of the research program is to combine the computational design of in-vitro circuits with novel biochemistry and innovative micro-engineering tools.
Max ERC Funding
1 887 180 €
Duration
Start date: 2016-08-01, End date: 2021-07-31
Project acronym BIOGRAPHENE
Project Sequencing biological molecules with graphene
Researcher (PI) Gregory Schneider
Host Institution (HI) UNIVERSITEIT LEIDEN
Call Details Starting Grant (StG), PE4, ERC-2013-StG
Summary Graphene – a one atom thin material – has the potential to act as a sensor, primarily the surface and the edges of graphene. This proposal aims at exploring new biosensing routes by exploiting the unique surface and edge chemistry of graphene.
Summary
Graphene – a one atom thin material – has the potential to act as a sensor, primarily the surface and the edges of graphene. This proposal aims at exploring new biosensing routes by exploiting the unique surface and edge chemistry of graphene.
Max ERC Funding
1 499 996 €
Duration
Start date: 2014-05-01, End date: 2019-04-30
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