Project acronym BFTERRA
Project Biogenesis and Functions of Telomeric Repeat-containing RNA
Researcher (PI) Claus Maria Azzalin
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Summary
Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Max ERC Funding
1 602 600 €
Duration
Start date: 2009-10-01, End date: 2014-09-30
Project acronym BIOMEMOS
Project Higher order structure and function of biomembranes
Researcher (PI) Poul Nissen
Host Institution (HI) AARHUS UNIVERSITET
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies
Summary
The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies
Max ERC Funding
2 444 180 €
Duration
Start date: 2010-04-01, End date: 2015-03-31
Project acronym DEFACT
Project DNA repair factories how cells do biochemistry
Researcher (PI) Michael Lisby
Host Institution (HI) KOBENHAVNS UNIVERSITET
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary The integrity of a cell's genome is constantly challenged by DNA lesions such as base modifications and DNA strand breaks. A single double-strand break is lethal if unrepaired and may lead to loss-of-heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main cause of cancer and other genetic diseases. Homologous recombination is an error-free pathway for repairing DNA lesions such as single- and double-strand breaks, and for the restart of collapsed replication forks. This pathway is catalyzed by giga-Dalton protein complexes consisting of dozens of different proteins. These DNA repair factories are able to catalyze complex, multi-step biochemical processes, which have so far failed reconstitution in vitro. The aim of this project is to establish an understanding of how cells catalyze complex biochemical processes such as homologous recombination in vivo. To reach this goal, we will seek to define the complete set of RNA and protein components of DNA repair factories using a combination of genetic, cell biological and biochemical approaches in the yeast Saccharomyces cerevisiae. Further, we will characterize the molecular architecture of DNA repair factories using fluorescence resonance energy transfer (FRET) and by applying systematic hybrid loss-of-heterozygosity (LOH) to physical interactions among DNA repair proteins. Key findings will be extended to metazoans using the chicken DT40 model system. My aim is to determine the fundamental molecular principles that govern protein factories in living cells. As such, our results are likely to be directly relevant to other protein factories such as DNA replication factories, PML bodies, nuclear pore complexes and transcription clusters.
Summary
The integrity of a cell's genome is constantly challenged by DNA lesions such as base modifications and DNA strand breaks. A single double-strand break is lethal if unrepaired and may lead to loss-of-heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main cause of cancer and other genetic diseases. Homologous recombination is an error-free pathway for repairing DNA lesions such as single- and double-strand breaks, and for the restart of collapsed replication forks. This pathway is catalyzed by giga-Dalton protein complexes consisting of dozens of different proteins. These DNA repair factories are able to catalyze complex, multi-step biochemical processes, which have so far failed reconstitution in vitro. The aim of this project is to establish an understanding of how cells catalyze complex biochemical processes such as homologous recombination in vivo. To reach this goal, we will seek to define the complete set of RNA and protein components of DNA repair factories using a combination of genetic, cell biological and biochemical approaches in the yeast Saccharomyces cerevisiae. Further, we will characterize the molecular architecture of DNA repair factories using fluorescence resonance energy transfer (FRET) and by applying systematic hybrid loss-of-heterozygosity (LOH) to physical interactions among DNA repair proteins. Key findings will be extended to metazoans using the chicken DT40 model system. My aim is to determine the fundamental molecular principles that govern protein factories in living cells. As such, our results are likely to be directly relevant to other protein factories such as DNA replication factories, PML bodies, nuclear pore complexes and transcription clusters.
Max ERC Funding
1 700 030 €
Duration
Start date: 2009-12-01, End date: 2014-11-30
Project acronym DNAREPAIR
Project Defects in DNA strand break repair and links to inheritable disease
Researcher (PI) Stephen West
Host Institution (HI) THE FRANCIS CRICK INSTITUTE LIMITED
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary Our genetic material is continually subjected to damage, either from endogenous sources such as reactive oxygen species produced as by-products of oxidative metabolism, from the breakdown of replication forks during cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. To cope with DNA damage, cells employ elaborate and effective repair processes that specifically recognise a wide variety of lesions in DNA. These repair systems are essential for the maintenance of genome integrity. Unfortunately, some individuals are genetically predisposed to crippling diseases or cancers that are the direct result of mutations in genes involved in the DNA damage response. For several years our work has been at the forefront of basic biological research in the area of DNA repair, and in particular we have made significant contributions to the understanding of inheritable diseases such as breast cancer, Fanconi anemia, and the neurodegenerative disease Ataxia with Oculomotor Apraxia-1 (AOA-1). The focus of this ERC proposal is: (i) to define the phenotypic interplay between three inheritable cancer predisposition syndromes, Fanconi anemia, Bloom s syndrome and breast cancers caused by mutation of BRCA2, (ii) to determine the biological role of the newly discovered GEN1 Holliday junction resolvase in homologous recombination and repair, and (iii) to understand the actions of Aprataxin and Senataxin in relation to the inheritable neurodegenerative diseases AOA-1 and AOA-2, respectively. Our studies will provide an improved understanding of basic mechanisms of DNA repair and thereby underpin future therapeutic developments that will help individuals afflicted with these diseases.
Summary
Our genetic material is continually subjected to damage, either from endogenous sources such as reactive oxygen species produced as by-products of oxidative metabolism, from the breakdown of replication forks during cell growth, or by agents in the environment such as ionising radiation or carcinogenic chemicals. To cope with DNA damage, cells employ elaborate and effective repair processes that specifically recognise a wide variety of lesions in DNA. These repair systems are essential for the maintenance of genome integrity. Unfortunately, some individuals are genetically predisposed to crippling diseases or cancers that are the direct result of mutations in genes involved in the DNA damage response. For several years our work has been at the forefront of basic biological research in the area of DNA repair, and in particular we have made significant contributions to the understanding of inheritable diseases such as breast cancer, Fanconi anemia, and the neurodegenerative disease Ataxia with Oculomotor Apraxia-1 (AOA-1). The focus of this ERC proposal is: (i) to define the phenotypic interplay between three inheritable cancer predisposition syndromes, Fanconi anemia, Bloom s syndrome and breast cancers caused by mutation of BRCA2, (ii) to determine the biological role of the newly discovered GEN1 Holliday junction resolvase in homologous recombination and repair, and (iii) to understand the actions of Aprataxin and Senataxin in relation to the inheritable neurodegenerative diseases AOA-1 and AOA-2, respectively. Our studies will provide an improved understanding of basic mechanisms of DNA repair and thereby underpin future therapeutic developments that will help individuals afflicted with these diseases.
Max ERC Funding
2 449 091 €
Duration
Start date: 2010-06-01, End date: 2015-05-31
Project acronym ECCENTRIC
Project Epigenetic challenges in centromere inheritance during the cell cycle
Researcher (PI) Geneviève Almouzni - Pettinotti
Host Institution (HI) INSTITUT CURIE
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary Studies concerning the mechanism of DNA replication have advanced our understanding of genetic transmission through multiple cell cycles. Recent work has shed light on possible means to ensure the stable transmission of information beyond just DNA and the concept of epigenetic inheritance has emerged. Considering chromatin-based information, key candidates have arisen as epigenetic marks including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Thus, understanding the dynamics and stability of these marks following disruptive events during replication and repair and throughout the cell cycle becomes of critical importance for the maintenance of any given chromatin state. To approach these issues, we propose to study the maintenance of heterochromatin at centromeres, key chromosomal regions for the proper chromosome segregation. Our current goal is to access to the sophisticated mechanisms that have evolved in order to facilitate inheritance of epigenetic marks not only at the replication fork, but also at other stages of the cell cycle, during repair and development. Beyond inheritance of DNA methylation, understanding how inheritance of histone variants and their modifications can be controlled either coupled or not coupled to DNA replication will be a major focus of this project. Our studies will build on the expertise and tools developed over the years in a strategy that integrates molecular, cellular, and biochemical approaches. This will be combined with the use of new technologies to monitor cell cycle (Fucci), protein dynamics (SNAP-Tagging) together with single molecule analysis involving DNA and chromatin combing. We wish to define a possible framework for an understanding of both the stability and reversibility of epigenetic marks and their dynamics at centromeres. These lessons may teach us general principles of inheritance of epigenetic states.
Summary
Studies concerning the mechanism of DNA replication have advanced our understanding of genetic transmission through multiple cell cycles. Recent work has shed light on possible means to ensure the stable transmission of information beyond just DNA and the concept of epigenetic inheritance has emerged. Considering chromatin-based information, key candidates have arisen as epigenetic marks including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Thus, understanding the dynamics and stability of these marks following disruptive events during replication and repair and throughout the cell cycle becomes of critical importance for the maintenance of any given chromatin state. To approach these issues, we propose to study the maintenance of heterochromatin at centromeres, key chromosomal regions for the proper chromosome segregation. Our current goal is to access to the sophisticated mechanisms that have evolved in order to facilitate inheritance of epigenetic marks not only at the replication fork, but also at other stages of the cell cycle, during repair and development. Beyond inheritance of DNA methylation, understanding how inheritance of histone variants and their modifications can be controlled either coupled or not coupled to DNA replication will be a major focus of this project. Our studies will build on the expertise and tools developed over the years in a strategy that integrates molecular, cellular, and biochemical approaches. This will be combined with the use of new technologies to monitor cell cycle (Fucci), protein dynamics (SNAP-Tagging) together with single molecule analysis involving DNA and chromatin combing. We wish to define a possible framework for an understanding of both the stability and reversibility of epigenetic marks and their dynamics at centromeres. These lessons may teach us general principles of inheritance of epigenetic states.
Max ERC Funding
2 490 483 €
Duration
Start date: 2010-06-01, End date: 2015-12-31
Project acronym EUKARYOTIC RIBOSOME
Project Structural studies of the eukaryotic ribosome by X-ray crystallography
Researcher (PI) Nenad Ban
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.
Summary
The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.
Max ERC Funding
2 446 725 €
Duration
Start date: 2010-07-01, End date: 2015-06-30
Project acronym EUKDNAREP
Project The Initiation of Eukaryotic DNA Replication: Mechanism, Regulation and Role in Genome Stability
Researcher (PI) John Diffley
Host Institution (HI) THE FRANCIS CRICK INSTITUTE LIMITED
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary In each cell cycle, eukaryotic cells must faithfully replicate large genomes in a relatively short time. This is accomplished by initiating DNA replication from many replication origins distributed along chromosomes. Ensuring that each origin is efficiently activated once and only once per cell cycle is crucial for maintaining the integrity of the genome. Recent evidence indicates that defects in the regulation of origin firing may be important contributors to genome instability in cancer. Strict once per cell cycle DNA replication is achieved by a two-step mechanism. DNA replication origins are first licensed by loading an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). This can only occur during G1 phase. Initiation then occurs during S phase, triggered by cyclin dependent kinases (CDKs) and Dbf4-dependent kinase (DDK), which promote recruitment of proteins required for helicase activation and replisome assembly. Research proposed herein will lead to a deeper understanding of the mechanism and regulation of DNA replication. We have reconstituted the licensing reaction with purified proteins which will be used to characterise the mechanism of licensing and the mechanism by which licensing is regulated in the cell cycle. We will also use this system to reconstitute events leading to the initiation of DNA replication. We will use genetic and biochemical approaches to characterise the mechanisms by which perturbed licensing causes gross chromosome rearrangements. We will also explore mechanisms involved in regulating the temporal programme of origin firing and how origin firing is regulated in response to DNA damage. Work in budding yeast and mammalian cells will be pursued in parallel to exploit the specific advantages of each system.
Summary
In each cell cycle, eukaryotic cells must faithfully replicate large genomes in a relatively short time. This is accomplished by initiating DNA replication from many replication origins distributed along chromosomes. Ensuring that each origin is efficiently activated once and only once per cell cycle is crucial for maintaining the integrity of the genome. Recent evidence indicates that defects in the regulation of origin firing may be important contributors to genome instability in cancer. Strict once per cell cycle DNA replication is achieved by a two-step mechanism. DNA replication origins are first licensed by loading an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). This can only occur during G1 phase. Initiation then occurs during S phase, triggered by cyclin dependent kinases (CDKs) and Dbf4-dependent kinase (DDK), which promote recruitment of proteins required for helicase activation and replisome assembly. Research proposed herein will lead to a deeper understanding of the mechanism and regulation of DNA replication. We have reconstituted the licensing reaction with purified proteins which will be used to characterise the mechanism of licensing and the mechanism by which licensing is regulated in the cell cycle. We will also use this system to reconstitute events leading to the initiation of DNA replication. We will use genetic and biochemical approaches to characterise the mechanisms by which perturbed licensing causes gross chromosome rearrangements. We will also explore mechanisms involved in regulating the temporal programme of origin firing and how origin firing is regulated in response to DNA damage. Work in budding yeast and mammalian cells will be pursued in parallel to exploit the specific advantages of each system.
Max ERC Funding
2 449 999 €
Duration
Start date: 2010-05-01, End date: 2015-04-30
Project acronym FORCEFULACTIN
Project Coordinated regulation of actin dynamics in cell motility and morphogenesis : from molecules to reconstituted biomimetic assays
Researcher (PI) Marie-France Carlier Épouse Pantaloni
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary An impressive variety of motile and morphogenetic processes are driven by site-directed polarized asssembly of actin filaments. In the past ten years, breathtaking advances coming from cell biology, cell biophysics, and biochemistry have brought insight into the molecular bases for production of force and movement by site-directed actin polymerization. Yet, we do not know, with the detail sufficient to understand how force is produced, by which molecular mechanisms the filaments are nucleated or created by branching. We do not know by which elementary steps insertional polymerization of barbed ends of filaments against the membrane is performed by different protein machineries, nor how these machineries work in a coordinated fashion. Here we propose a multiscale and interdisciplinary approach of the mechanisms used by the major actin nucleators to organize the motile response of actin. The elementary reactions involved in the processive walk of formin at the growing barbed ends of filaments and the role of ATP hydrolysis in force production will be analyzed by a combination of biochemical solution studies and physical methods using functionalized GUVs and optical tweezers. The multifunctionality of WH2 domains involved in actin sequestration, filament nucleation severing and processive elongation will be similarly examined in an interdisciplinary perspective from structural biology at atomic resolution to physics at the mesoscopic scale. Biochemical and structural methods and single molecule measurements (TIRFM) will shed light into the elementary steps and structural mechanism of filament branching. Biomimetic assays with functionalized GUVs associated with biophysical methods like FRAP or fluorescence correlation spectroscopy will elucidate how different filament initiating machineries segregate in the membrane as a consequence of their interactions with growing filaments and function in a coordinated fashion during actin-based motility.
Summary
An impressive variety of motile and morphogenetic processes are driven by site-directed polarized asssembly of actin filaments. In the past ten years, breathtaking advances coming from cell biology, cell biophysics, and biochemistry have brought insight into the molecular bases for production of force and movement by site-directed actin polymerization. Yet, we do not know, with the detail sufficient to understand how force is produced, by which molecular mechanisms the filaments are nucleated or created by branching. We do not know by which elementary steps insertional polymerization of barbed ends of filaments against the membrane is performed by different protein machineries, nor how these machineries work in a coordinated fashion. Here we propose a multiscale and interdisciplinary approach of the mechanisms used by the major actin nucleators to organize the motile response of actin. The elementary reactions involved in the processive walk of formin at the growing barbed ends of filaments and the role of ATP hydrolysis in force production will be analyzed by a combination of biochemical solution studies and physical methods using functionalized GUVs and optical tweezers. The multifunctionality of WH2 domains involved in actin sequestration, filament nucleation severing and processive elongation will be similarly examined in an interdisciplinary perspective from structural biology at atomic resolution to physics at the mesoscopic scale. Biochemical and structural methods and single molecule measurements (TIRFM) will shed light into the elementary steps and structural mechanism of filament branching. Biomimetic assays with functionalized GUVs associated with biophysical methods like FRAP or fluorescence correlation spectroscopy will elucidate how different filament initiating machineries segregate in the membrane as a consequence of their interactions with growing filaments and function in a coordinated fashion during actin-based motility.
Max ERC Funding
2 434 195 €
Duration
Start date: 2010-05-01, End date: 2015-10-31
Project acronym INCEL
Project Revealing the molecular architecture of integrin mediated cell adhesion
Researcher (PI) Ohad Medalia
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary Cell adhesions play an important role in the organization, growth, maturation, and function of living cells. Interaction of cells with the extracellular matrix (ECM) plays an essential role in a variety of disease states , inflammation, and repair of damaged tissues. At the cellular level, many of the biological responses to external stimuli originate at adhesion loci, such as focal adhesions (FA), which link cells to the ECM . Cell adhesion is mediated by receptor proteins such as cadherins and integrins. The precise molecular composition, dynamics and signalling activity of these adhesion assemblies determine the specificity of adhesion-induced signals and their effects on the cell. However, characterization of the molecular architecture of FAs is highly challenging, and it thus remains unclear how these molecules function together, how they are recruited to the adhesion site, how they are turned over, and how they function in vivo. In this project, I aim to conduct an interdisciplinary study that will provide a quantum step forward in the understanding of the functional organization of FAs. We will analyze, for the first time, the three-dimensional structure of FAs in wild-type cells and in cells deficient in the specific proteins involved in the cell-adhesion machinery. We will study the effect of specific geometries on the functional architecture of focal adhesions in 3D. A combination of state-of-the-art technologies, such cryo-electron tomography of intact cells, gold cluster chemistry for in situ labeling, and modulation of the underlying matrix using micro- and nano-patterned adhesive surfaces, together with correlative light, atomic force and electron microscopy, will provide a hybrid approach for dissecting out the complex process of cell adhesion.In summary, this project addresses the properties of FAs across a wide range of complexities and dimensions, from macroscopic cellular phenomena to the physical nature of these molecular assemblies
Summary
Cell adhesions play an important role in the organization, growth, maturation, and function of living cells. Interaction of cells with the extracellular matrix (ECM) plays an essential role in a variety of disease states , inflammation, and repair of damaged tissues. At the cellular level, many of the biological responses to external stimuli originate at adhesion loci, such as focal adhesions (FA), which link cells to the ECM . Cell adhesion is mediated by receptor proteins such as cadherins and integrins. The precise molecular composition, dynamics and signalling activity of these adhesion assemblies determine the specificity of adhesion-induced signals and their effects on the cell. However, characterization of the molecular architecture of FAs is highly challenging, and it thus remains unclear how these molecules function together, how they are recruited to the adhesion site, how they are turned over, and how they function in vivo. In this project, I aim to conduct an interdisciplinary study that will provide a quantum step forward in the understanding of the functional organization of FAs. We will analyze, for the first time, the three-dimensional structure of FAs in wild-type cells and in cells deficient in the specific proteins involved in the cell-adhesion machinery. We will study the effect of specific geometries on the functional architecture of focal adhesions in 3D. A combination of state-of-the-art technologies, such cryo-electron tomography of intact cells, gold cluster chemistry for in situ labeling, and modulation of the underlying matrix using micro- and nano-patterned adhesive surfaces, together with correlative light, atomic force and electron microscopy, will provide a hybrid approach for dissecting out the complex process of cell adhesion.In summary, this project addresses the properties of FAs across a wide range of complexities and dimensions, from macroscopic cellular phenomena to the physical nature of these molecular assemblies
Max ERC Funding
1 294 000 €
Duration
Start date: 2009-11-01, End date: 2015-10-31
Project acronym JTOMO
Project Study of the molecular organization of cell junctions by cryo-electron tomography
Researcher (PI) Achilleas Frangakis
Host Institution (HI) JOHANN WOLFGANG GOETHE-UNIVERSITATFRANKFURT AM MAIN
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary Cells sense, affect and respond to their environment through the fundamental function of adhesion. Several types of adhesion sites, which are mediated via dynamically maintained multi-protein structures, anchor extracellular-matrix proteins to the cytoskeleton. Despite considerable efforts, the long-standing questions of how adhesion sites are formed, structured and regulated remain unanswered. In this research plan we will investigate desmosomes and adherens junctions by cryo-electron tomography of cells and tissue. The principal objectives are: (a) to visualize the molecular architecture and reveal the structural differences of the adhesion sites under various conditions and influences, i.e. mutations, wounds, etc. (b) to reveal their molecular association to the cytoskeleton (intermediate and actin filaments respectively), and to chart the network of interactions underlying cellular adhesion, and (c) to develop novel pattern recognition and classification techniques in order to structurally characterize the adhesion sites in toto by cryo-electron tomography of vitreous sections. We will use pattern recognition techniques and locally averaged cryo-electron sub-tomograms to quantify the macromolecular complexes in terms of stoichiometry and protein interactions in situ at high resolution (~3 nm). In particular, we aim to reveal how a pool of constituent proteins is organized in the two adhesion sites. Significant amounts of information coming from immunogold electron microscopy, fragments from X-ray structures, force measurements with atomic force microscopy, and structural bioinformatics will be integrated into our cryo-electron tomograms. This research will pioneer structural comparisons of protein networks at nanometer resolution in situ and in toto. The experimental and theoretical methods that will be developed would be indispensable for studying any spatially constrained protein network whose state depends on local properties.
Summary
Cells sense, affect and respond to their environment through the fundamental function of adhesion. Several types of adhesion sites, which are mediated via dynamically maintained multi-protein structures, anchor extracellular-matrix proteins to the cytoskeleton. Despite considerable efforts, the long-standing questions of how adhesion sites are formed, structured and regulated remain unanswered. In this research plan we will investigate desmosomes and adherens junctions by cryo-electron tomography of cells and tissue. The principal objectives are: (a) to visualize the molecular architecture and reveal the structural differences of the adhesion sites under various conditions and influences, i.e. mutations, wounds, etc. (b) to reveal their molecular association to the cytoskeleton (intermediate and actin filaments respectively), and to chart the network of interactions underlying cellular adhesion, and (c) to develop novel pattern recognition and classification techniques in order to structurally characterize the adhesion sites in toto by cryo-electron tomography of vitreous sections. We will use pattern recognition techniques and locally averaged cryo-electron sub-tomograms to quantify the macromolecular complexes in terms of stoichiometry and protein interactions in situ at high resolution (~3 nm). In particular, we aim to reveal how a pool of constituent proteins is organized in the two adhesion sites. Significant amounts of information coming from immunogold electron microscopy, fragments from X-ray structures, force measurements with atomic force microscopy, and structural bioinformatics will be integrated into our cryo-electron tomograms. This research will pioneer structural comparisons of protein networks at nanometer resolution in situ and in toto. The experimental and theoretical methods that will be developed would be indispensable for studying any spatially constrained protein network whose state depends on local properties.
Max ERC Funding
1 724 400 €
Duration
Start date: 2010-01-01, End date: 2015-12-31
