Project acronym AutoRecon
Project Molecular mechanisms of autophagosome formation during selective autophagy
Researcher (PI) Sascha Martens
Host Institution (HI) UNIVERSITAT WIEN
Call Details Consolidator Grant (CoG), LS3, ERC-2014-CoG
Summary I propose to study how eukaryotic cells generate autophagosomes, organelles bounded by a double membrane. These are formed during autophagy and mediate the degradation of cytoplasmic substances within the lysosomal compartment. Autophagy thereby protects the organism from pathological conditions such as neurodegeneration, cancer and infections. Many core factors required for autophagosome formation have been identified but the order in which they act and their mode of action is still unclear. We will use a combination of biochemical and cell biological approaches to elucidate the choreography and mechanism of these core factors. In particular, we will focus on selective autophagy and determine how the autophagic machinery generates an autophagosome that selectively contains the cargo.
To this end we will focus on the cytoplasm-to-vacuole-targeting pathway in S. cerevisiae that mediates the constitutive delivery of the prApe1 enzyme into the vacuole. We will use cargo mimetics or prApe1 complexes in combination with purified autophagy proteins and vesicles to reconstitute the process and so determine which factors are both necessary and sufficient for autophagosome formation, as well as elucidating their mechanism of action.
In parallel we will study selective autophagosome formation in human cells. This will reveal common principles and special adaptations. In particular, we will use cell lysates from genome-edited cells in combination with purified autophagy proteins to reconstitute selective autophagosome formation around ubiquitin-positive cargo material. The insights and hypotheses obtained from these reconstituted systems will be validated using cell biological approaches.
Taken together, our experiments will allow us to delineate the major steps of autophagosome formation during selective autophagy. Our results will yield detailed insights into how cells form and shape organelles in a de novo manner, which is major question in cell- and developmental biology.
Summary
I propose to study how eukaryotic cells generate autophagosomes, organelles bounded by a double membrane. These are formed during autophagy and mediate the degradation of cytoplasmic substances within the lysosomal compartment. Autophagy thereby protects the organism from pathological conditions such as neurodegeneration, cancer and infections. Many core factors required for autophagosome formation have been identified but the order in which they act and their mode of action is still unclear. We will use a combination of biochemical and cell biological approaches to elucidate the choreography and mechanism of these core factors. In particular, we will focus on selective autophagy and determine how the autophagic machinery generates an autophagosome that selectively contains the cargo.
To this end we will focus on the cytoplasm-to-vacuole-targeting pathway in S. cerevisiae that mediates the constitutive delivery of the prApe1 enzyme into the vacuole. We will use cargo mimetics or prApe1 complexes in combination with purified autophagy proteins and vesicles to reconstitute the process and so determine which factors are both necessary and sufficient for autophagosome formation, as well as elucidating their mechanism of action.
In parallel we will study selective autophagosome formation in human cells. This will reveal common principles and special adaptations. In particular, we will use cell lysates from genome-edited cells in combination with purified autophagy proteins to reconstitute selective autophagosome formation around ubiquitin-positive cargo material. The insights and hypotheses obtained from these reconstituted systems will be validated using cell biological approaches.
Taken together, our experiments will allow us to delineate the major steps of autophagosome formation during selective autophagy. Our results will yield detailed insights into how cells form and shape organelles in a de novo manner, which is major question in cell- and developmental biology.
Max ERC Funding
1 999 640 €
Duration
Start date: 2016-03-01, End date: 2021-02-28
Project acronym AuxinER
Project Mechanisms of Auxin-dependent Signaling in the Endoplasmic Reticulum
Researcher (PI) Jürgen Kleine-Vehn
Host Institution (HI) UNIVERSITAET FUER BODENKULTUR WIEN
Call Details Starting Grant (StG), LS3, ERC-2014-STG
Summary The phytohormone auxin has profound importance for plant development. The extracellular AUXIN BINDING PROTEIN1 (ABP1) and the nuclear AUXIN F-BOX PROTEINs (TIR1/AFBs) auxin receptors perceive fast, non-genomic and slow, genomic auxin responses, respectively. Despite the fact that ABP1 mainly localizes to the endoplasmic reticulum (ER), until now it has been proposed to be active only in the extracellular matrix (reviewed in Sauer and Kleine-Vehn, 2011). Just recently, ABP1 function was also linked to genomic responses, modulating TIR1/AFB-dependent processes (Tromas et al., 2013). Intriguingly, the genomic effect of ABP1 appears to be at least partially independent of the endogenous auxin indole 3-acetic acid (IAA) (Paque et al., 2014).
In this proposal my main research objective is to unravel the importance of the ER for genomic auxin responses. The PIN-LIKES (PILS) putative carriers for auxinic compounds also localize to the ER and determine the cellular sensitivity to auxin. PILS5 gain-of-function reduces canonical auxin signaling (Barbez et al., 2012) and phenocopies abp1 knock down lines (Barbez et al., 2012, Paque et al., 2014). Accordingly, a PILS-dependent substrate could be a negative regulator of ABP1 function in the ER. Based on our unpublished data, an IAA metabolite could play a role in ABP1-dependent processes in the ER, possibly providing feedback on the canonical nuclear IAA-signaling.
I hypothesize that the genomic auxin response may be an integration of auxin- and auxin-metabolite-dependent nuclear and ER localized signaling, respectively. This proposed project aims to characterize a novel auxin-signaling paradigm in plants. We will employ state of the art interdisciplinary (biochemical, biophysical, computational modeling, molecular, and genetic) methods to assess the projected research. The identification of the proposed auxin conjugate-dependent signal could have far reaching plant developmental and biotechnological importance.
Summary
The phytohormone auxin has profound importance for plant development. The extracellular AUXIN BINDING PROTEIN1 (ABP1) and the nuclear AUXIN F-BOX PROTEINs (TIR1/AFBs) auxin receptors perceive fast, non-genomic and slow, genomic auxin responses, respectively. Despite the fact that ABP1 mainly localizes to the endoplasmic reticulum (ER), until now it has been proposed to be active only in the extracellular matrix (reviewed in Sauer and Kleine-Vehn, 2011). Just recently, ABP1 function was also linked to genomic responses, modulating TIR1/AFB-dependent processes (Tromas et al., 2013). Intriguingly, the genomic effect of ABP1 appears to be at least partially independent of the endogenous auxin indole 3-acetic acid (IAA) (Paque et al., 2014).
In this proposal my main research objective is to unravel the importance of the ER for genomic auxin responses. The PIN-LIKES (PILS) putative carriers for auxinic compounds also localize to the ER and determine the cellular sensitivity to auxin. PILS5 gain-of-function reduces canonical auxin signaling (Barbez et al., 2012) and phenocopies abp1 knock down lines (Barbez et al., 2012, Paque et al., 2014). Accordingly, a PILS-dependent substrate could be a negative regulator of ABP1 function in the ER. Based on our unpublished data, an IAA metabolite could play a role in ABP1-dependent processes in the ER, possibly providing feedback on the canonical nuclear IAA-signaling.
I hypothesize that the genomic auxin response may be an integration of auxin- and auxin-metabolite-dependent nuclear and ER localized signaling, respectively. This proposed project aims to characterize a novel auxin-signaling paradigm in plants. We will employ state of the art interdisciplinary (biochemical, biophysical, computational modeling, molecular, and genetic) methods to assess the projected research. The identification of the proposed auxin conjugate-dependent signal could have far reaching plant developmental and biotechnological importance.
Max ERC Funding
1 441 125 €
Duration
Start date: 2015-06-01, End date: 2020-05-31
Project acronym CELLREPROGRAMMING
Project Uncovering the Mechanisms of Epigenetic Reprogramming of Pluripotent and Somatic Cell States
Researcher (PI) Yaqub Hanna
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS3, ERC-2011-StG_20101109
Summary The generation of animals by nuclear transfer demonstrated that the epigenetic state of somatic cells could be reset to an embryonic state, capable of directing the development of a new organism. The nuclear cloning technology is of interest for transplantation medicine, but any application is hampered by the inefficiency and ethical problems. A breakthrough solving these issues has been the in vitro derivation of reprogrammed Induced Pluripotent Stem “iPS” cells by the ectopic expression of defined transcription factors in somatic cells. iPS cells recapitulate all defining features of embryo-derived pluripotent stem cells, including the ability to differentiate into all somatic cell types. Further, recent publications have demonstrated the ability to directly trans-differentiate somatic cell types by ectopic expression of lineage specification factors. Thus, it is becoming increasingly clear that an ultimate goal in the stem cell field is to enable scientists to have the power to safely manipulate somatic cells by “reprogramming” their behavior at will. However, to frame this challenge, we must understand the basic mechanisms underlying the generation of reprogrammed cells in parallel to designing strategies for their medical application and their use in human disease specific research. In this ERC Starting Grant proposal, I describe comprehensive lines of experimentation that I plan to conduct in my new lab scheduled to open in April 2011 at the Weizmann Institute of Science. We will utilize exacting transgenic mammalian models and high throughput sequencing and genomic screening tools for in depth characterization of the molecular “rules” of rewiring the epigenome of somatic and pluripotent cell states. The proposed research endeavors will not only contribute to the development of safer strategies for cell reprogramming, but will also help decipher how diverse gene expression programs lead to cellular specification during normal development.
Summary
The generation of animals by nuclear transfer demonstrated that the epigenetic state of somatic cells could be reset to an embryonic state, capable of directing the development of a new organism. The nuclear cloning technology is of interest for transplantation medicine, but any application is hampered by the inefficiency and ethical problems. A breakthrough solving these issues has been the in vitro derivation of reprogrammed Induced Pluripotent Stem “iPS” cells by the ectopic expression of defined transcription factors in somatic cells. iPS cells recapitulate all defining features of embryo-derived pluripotent stem cells, including the ability to differentiate into all somatic cell types. Further, recent publications have demonstrated the ability to directly trans-differentiate somatic cell types by ectopic expression of lineage specification factors. Thus, it is becoming increasingly clear that an ultimate goal in the stem cell field is to enable scientists to have the power to safely manipulate somatic cells by “reprogramming” their behavior at will. However, to frame this challenge, we must understand the basic mechanisms underlying the generation of reprogrammed cells in parallel to designing strategies for their medical application and their use in human disease specific research. In this ERC Starting Grant proposal, I describe comprehensive lines of experimentation that I plan to conduct in my new lab scheduled to open in April 2011 at the Weizmann Institute of Science. We will utilize exacting transgenic mammalian models and high throughput sequencing and genomic screening tools for in depth characterization of the molecular “rules” of rewiring the epigenome of somatic and pluripotent cell states. The proposed research endeavors will not only contribute to the development of safer strategies for cell reprogramming, but will also help decipher how diverse gene expression programs lead to cellular specification during normal development.
Max ERC Funding
1 960 000 €
Duration
Start date: 2011-11-01, End date: 2016-10-31
Project acronym DIVIMAGE
Project Bridging spatial and temporal resolution gaps in the study of cell division
Researcher (PI) Daniel Wolfram Gerlich
Host Institution (HI) INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Call Details Starting Grant (StG), LS3, ERC-2011-StG_20101109
Summary Cell division underlies the growth and development of all living organisms. Following partitioning of bulk cytoplasmic contents by cleavage furrow ingression, dividing animal cells split by a distinct process termed abscission. Whereas a number of factors required for abscission have been identified in previous studies, it is not known by which mechanism they mediate fission of the intercellular bridge between the nascent sister cells. Here, we will establish correlative workflows of time-lapse imaging, super resolution fluorescence microscopy, electron tomography, and electrophysiological assays to bridge spatial and temporal resolution gaps in the study of abscission. We will further develop computational tools for image-based RNAi screening. With this, we aim to:
1) elucidate how membrane and cytoskeletal dynamics coordinately split the intercellular bridge;
2) uncover the signaling pathways controlling abscission timing.
Failure in abscission can lead to aneuploidy and cancer. Elucidating its mechanism and temporal control is therefore of general biological and medical relevance. The computational and correlative imaging methods developed in this project will further provide the research community new possibilities for mechanistic studies in intact cells.
Summary
Cell division underlies the growth and development of all living organisms. Following partitioning of bulk cytoplasmic contents by cleavage furrow ingression, dividing animal cells split by a distinct process termed abscission. Whereas a number of factors required for abscission have been identified in previous studies, it is not known by which mechanism they mediate fission of the intercellular bridge between the nascent sister cells. Here, we will establish correlative workflows of time-lapse imaging, super resolution fluorescence microscopy, electron tomography, and electrophysiological assays to bridge spatial and temporal resolution gaps in the study of abscission. We will further develop computational tools for image-based RNAi screening. With this, we aim to:
1) elucidate how membrane and cytoskeletal dynamics coordinately split the intercellular bridge;
2) uncover the signaling pathways controlling abscission timing.
Failure in abscission can lead to aneuploidy and cancer. Elucidating its mechanism and temporal control is therefore of general biological and medical relevance. The computational and correlative imaging methods developed in this project will further provide the research community new possibilities for mechanistic studies in intact cells.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-03-01, End date: 2017-02-28
Project acronym LeukocyteForces
Project Cytoskeletal force generation and force transduction of migrating leukocytes
Researcher (PI) Michael Sixt
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGYAUSTRIA
Call Details Starting Grant (StG), LS3, ERC-2011-StG_20101109
Summary Cell migration is a universal feature of all metazoan life and crucially involved in most developmental, homeostatic and pathological processes. Most efforts to understand its molecular and mechanical aspects were focused on the “haptokinetic” paradigm. Here cells generate traction by coupling the protrusive and contractile forces of the actomyosin cytoskeleton via transmembrane receptors to the extracellular environment. Our recent work demonstrated that leukocytes, the class of animal cells that migrates with highest speed and efficiency, violate this paradigm. Once embedded in physiological three-dimensional matrices they instantaneously shift between adhesive and non-adhesive modes to transduce force. This proposal suggests a combined cell biological and biophysical approach to elucidate the molecular and mechanical principles underlying such plasticity. We will focus on the machinery most proximate to force generation and use genetics and pharmacology to characterize how nucleation, elongation, depolymerization and crosslinking of actin filaments act in leukocytes migrating through environments of varying geometry and adhesive properties (Postdoc 1). Mechanical manipulations in conjunction with high resolution monitoring of substrate deformations will reveal how cytoskeletal force is transduced to the extracellular environment (Postdoc 2). In a technical support project (Technician) we will develop a cell-system with optimized access to stable genetic manipulations. Technically, these questions will be addressed by employing advanced live cell fluorescence imaging in combination with artificial environments engineered using microfluidics and substrate micropatterning. Importantly, findings will ultimately be challenged in living tissues. This multidisciplinary approach will generate an integrated view of locomotion-plasticity that will not only impact basic cell biology and immunology but also developmental and cancer biology.
Summary
Cell migration is a universal feature of all metazoan life and crucially involved in most developmental, homeostatic and pathological processes. Most efforts to understand its molecular and mechanical aspects were focused on the “haptokinetic” paradigm. Here cells generate traction by coupling the protrusive and contractile forces of the actomyosin cytoskeleton via transmembrane receptors to the extracellular environment. Our recent work demonstrated that leukocytes, the class of animal cells that migrates with highest speed and efficiency, violate this paradigm. Once embedded in physiological three-dimensional matrices they instantaneously shift between adhesive and non-adhesive modes to transduce force. This proposal suggests a combined cell biological and biophysical approach to elucidate the molecular and mechanical principles underlying such plasticity. We will focus on the machinery most proximate to force generation and use genetics and pharmacology to characterize how nucleation, elongation, depolymerization and crosslinking of actin filaments act in leukocytes migrating through environments of varying geometry and adhesive properties (Postdoc 1). Mechanical manipulations in conjunction with high resolution monitoring of substrate deformations will reveal how cytoskeletal force is transduced to the extracellular environment (Postdoc 2). In a technical support project (Technician) we will develop a cell-system with optimized access to stable genetic manipulations. Technically, these questions will be addressed by employing advanced live cell fluorescence imaging in combination with artificial environments engineered using microfluidics and substrate micropatterning. Importantly, findings will ultimately be challenged in living tissues. This multidisciplinary approach will generate an integrated view of locomotion-plasticity that will not only impact basic cell biology and immunology but also developmental and cancer biology.
Max ERC Funding
1 458 125 €
Duration
Start date: 2012-04-01, End date: 2017-03-31
Project acronym PeroxiSystem
Project Systematic exploration of peroxisomal structure and function
Researcher (PI) Maya Benyamina Schuldiner
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Consolidator Grant (CoG), LS3, ERC-2014-CoG
Summary Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. This key organelle propagates cellular signals for differentiation, development and metabolism, and thus it is no surprise that a large number of diseases, including metabolic disorders, have been linked to peroxisomal dysfunction. Despite the importance of peroxisomes many fundamental questions remain open. For example, we do not know the entire proteome of peroxisomes, the extent of their metabolic functions, how peroxisomes change to meet cellular requirements or how they interact and communicate with other cellular organelles. In this proposal we suggest to employ our expertise and unique toolsets, successfully applied in the study of whole organelles, to shed new light on peroxisomes as a cellular unit – a PeroxiSystem. We propose to combine state-of-the art high content tools with mechanistic studies to uncover new peroxisomal proteins under a variety of growth conditions (Aim1), map the functions of unstudied peroxisomal proteins using both systematic and hypothesis driven approaches (Aim 2) and unravel how peroxisomes communicate with other organelles (Aim 3). To perform these studies we will build on expertise attained during an ERC StG: combining high throughput genetic manipulations of yeast libraries alongside high content screens. Importantly, we will try to bridge the knowledge gap in peroxisomal biology by creating new tools that can be applied to this unique organelle. Our findings should make an important step towards an unprecedented, thorough and multifaceted understanding of peroxisomes, their cellular geography and roles as well as their regulation when presented with various metabolic conditions. More broadly, the approaches presented here can be easily applied to study any organelle of choice, thus providing a conceptual framework in the study of cell biology.
Summary
Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. This key organelle propagates cellular signals for differentiation, development and metabolism, and thus it is no surprise that a large number of diseases, including metabolic disorders, have been linked to peroxisomal dysfunction. Despite the importance of peroxisomes many fundamental questions remain open. For example, we do not know the entire proteome of peroxisomes, the extent of their metabolic functions, how peroxisomes change to meet cellular requirements or how they interact and communicate with other cellular organelles. In this proposal we suggest to employ our expertise and unique toolsets, successfully applied in the study of whole organelles, to shed new light on peroxisomes as a cellular unit – a PeroxiSystem. We propose to combine state-of-the art high content tools with mechanistic studies to uncover new peroxisomal proteins under a variety of growth conditions (Aim1), map the functions of unstudied peroxisomal proteins using both systematic and hypothesis driven approaches (Aim 2) and unravel how peroxisomes communicate with other organelles (Aim 3). To perform these studies we will build on expertise attained during an ERC StG: combining high throughput genetic manipulations of yeast libraries alongside high content screens. Importantly, we will try to bridge the knowledge gap in peroxisomal biology by creating new tools that can be applied to this unique organelle. Our findings should make an important step towards an unprecedented, thorough and multifaceted understanding of peroxisomes, their cellular geography and roles as well as their regulation when presented with various metabolic conditions. More broadly, the approaches presented here can be easily applied to study any organelle of choice, thus providing a conceptual framework in the study of cell biology.
Max ERC Funding
2 000 000 €
Duration
Start date: 2015-09-01, End date: 2020-08-31
Project acronym PSDP
Project POLARITY AND SUBCELLULAR DYNAMICS IN PLANTS
Researcher (PI) Jirí Friml
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGYAUSTRIA
Call Details Starting Grant (StG), LS3, ERC-2011-StG_20101109
Summary Plant life strategy is marked by acquisition of highly flexible development that adapts plants’ phenotype to the environment. Various environmental signals are integrated into the endogenous signalling networks involving the versatile phytohormone auxin. The intercellular auxin transport mediates a large variety of adaptive plant growth responses. Subcellular polar distribution of PIN auxin transporters determines directionality of auxin flow and thus have potential to integrate internal and external signals via the redirection of auxin fluxes and translate them into modulation of development. Auxin transport thus represents a unique model for studying the functional link between basic cellular processes, such as vesicle trafficking and cell polarity, and their developmental outcome at the level of the multicellular organism.
We will employ approaches of cell biology, molecular genetics and chemical genomics in Arabidopsis thaliana to identify the cellular and molecular mechanisms regulating the directional throughput of auxin flow a integration of environmental signals into subcellular dynamics of PIN auxin transporters as well as endogenous feed-back regulations of this mechanism.
In our proposal, we will focus on four main research directions.
1. Novel regulators of cell polarity identified by chemical genomics
2. Cellular mechanisms of cell polarity maintenance
3. Integration of signals into subcellular dynamics of auxin transport
4. Mathematical modelling of regulatory circuits for adaptive development
The results will demonstrate the viability of genetics approaches for addressing cell biological questions in plants, open new horizons in plant cell biology and plant hormone fields and inspire researchers also in non-plant fields. The expected output has clear application potential for targeted modulation of plant development. The project will further strengthen our position of world-leading laboratory in plant hormone and plant cell biology fields.
Summary
Plant life strategy is marked by acquisition of highly flexible development that adapts plants’ phenotype to the environment. Various environmental signals are integrated into the endogenous signalling networks involving the versatile phytohormone auxin. The intercellular auxin transport mediates a large variety of adaptive plant growth responses. Subcellular polar distribution of PIN auxin transporters determines directionality of auxin flow and thus have potential to integrate internal and external signals via the redirection of auxin fluxes and translate them into modulation of development. Auxin transport thus represents a unique model for studying the functional link between basic cellular processes, such as vesicle trafficking and cell polarity, and their developmental outcome at the level of the multicellular organism.
We will employ approaches of cell biology, molecular genetics and chemical genomics in Arabidopsis thaliana to identify the cellular and molecular mechanisms regulating the directional throughput of auxin flow a integration of environmental signals into subcellular dynamics of PIN auxin transporters as well as endogenous feed-back regulations of this mechanism.
In our proposal, we will focus on four main research directions.
1. Novel regulators of cell polarity identified by chemical genomics
2. Cellular mechanisms of cell polarity maintenance
3. Integration of signals into subcellular dynamics of auxin transport
4. Mathematical modelling of regulatory circuits for adaptive development
The results will demonstrate the viability of genetics approaches for addressing cell biological questions in plants, open new horizons in plant cell biology and plant hormone fields and inspire researchers also in non-plant fields. The expected output has clear application potential for targeted modulation of plant development. The project will further strengthen our position of world-leading laboratory in plant hormone and plant cell biology fields.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-02-01, End date: 2017-01-31
Project acronym REGENERATEACROSS
Project A Cross Species Approach to Understand the Mechanism and Evolution of Limb Regeneration Capacity
Researcher (PI) Elly Margaret Tanaka
Host Institution (HI) FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Call Details Advanced Grant (AdG), LS3, ERC-2011-ADG_20110310
Summary Limb regeneration capacity varies among vertebrates, ranging from full regeneration in salamanders, to stage-restricted premetamorphic regeneration in frogs, to finger-tip regeneration in newborn mice and humans.
The molecular and cellular basis for these differences in regeneration ability is not known and it is still unclear if these different regenerative events are tied by some common mechanisms with progressive restriction due to extracellular signals or cell intrinsic changes.
Connective tissue fibroblasts or their progenitors play a key role in regenerating the patterned salamander limb. They harbor critical positional information and can reconstitute not only the connective tissue but also a complete, patterned skeleton. In contrast, such cells typically contribute to scar tissue during mammalian wound healing. Their role in mouse finger tip regeneration is unknown.
I seek to determine the differences in fibroblast biology that account for the differences in regeneration between salamander, frog and mouse. To define the differences in composition of the connective tissue population, I will perform parallel lineage tracing of different fibroblast populations and their progenitors during wound healing and regeneration in salamander, frog and mouse. To determine differences in cell intrinsic potential versus extracellular cues required for regeneration I will perform cross-species transplantation of lineage-labeled cells between salamander and frog coupled with expression profiling to identify molecular changes that occur in cells in a regenerative versus non-regenerative context. Finally I will use this expression profiling and our molecular knowledge of limb regeneration factors to test whether frog and mouse cells can acquire regenerative traits at the cellular level by the forced exposure to intracellular and extracellular regeneration cues or by the downregulation of putative inhibitory factors.
Summary
Limb regeneration capacity varies among vertebrates, ranging from full regeneration in salamanders, to stage-restricted premetamorphic regeneration in frogs, to finger-tip regeneration in newborn mice and humans.
The molecular and cellular basis for these differences in regeneration ability is not known and it is still unclear if these different regenerative events are tied by some common mechanisms with progressive restriction due to extracellular signals or cell intrinsic changes.
Connective tissue fibroblasts or their progenitors play a key role in regenerating the patterned salamander limb. They harbor critical positional information and can reconstitute not only the connective tissue but also a complete, patterned skeleton. In contrast, such cells typically contribute to scar tissue during mammalian wound healing. Their role in mouse finger tip regeneration is unknown.
I seek to determine the differences in fibroblast biology that account for the differences in regeneration between salamander, frog and mouse. To define the differences in composition of the connective tissue population, I will perform parallel lineage tracing of different fibroblast populations and their progenitors during wound healing and regeneration in salamander, frog and mouse. To determine differences in cell intrinsic potential versus extracellular cues required for regeneration I will perform cross-species transplantation of lineage-labeled cells between salamander and frog coupled with expression profiling to identify molecular changes that occur in cells in a regenerative versus non-regenerative context. Finally I will use this expression profiling and our molecular knowledge of limb regeneration factors to test whether frog and mouse cells can acquire regenerative traits at the cellular level by the forced exposure to intracellular and extracellular regeneration cues or by the downregulation of putative inhibitory factors.
Max ERC Funding
2 447 600 €
Duration
Start date: 2012-06-01, End date: 2017-12-31
Project acronym RevMito
Project Deciphering and reversing the consequences of mitochondrial DNA damage
Researcher (PI) Cory Dunn
Host Institution (HI) HELSINGIN YLIOPISTO
Call Details Starting Grant (StG), LS3, ERC-2014-STG
Summary Mitochondrial DNA (mtDNA) encodes several proteins playing key roles in bioenergetics. Pathological mutations of mtDNA can be inherited or may accumulate following treatment for viral infections or cancer. Furthermore, many organisms, including humans, accumulate significant mtDNA damage during their lifespan, and it is therefore possible that mtDNA mutations can promote the aging process.
There are no effective treatments for most diseases caused by mtDNA mutation. An understanding of the cellular consequences of mtDNA damage is clearly imperative. Toward this goal, we use the budding yeast Saccharomyces cerevisiae as a cellular model of mitochondrial dysfunction. Genetic manipulation and biochemical study of this organism is easily achieved, and many proteins and processes important for mitochondrial biogenesis were first uncovered and best characterized using this experimental system. Importantly, current evidence suggests that processes required for survival of cells lacking a mitochondrial genome are widely conserved between yeast and other organisms, making likely the application of our findings to human health.
We will study the repercussions of mtDNA damage by three different strategies. First, we will investigate the link between a conserved, nutrient-sensitive signalling pathway and the outcome of mtDNA loss, since much recent evidence points to modulation of such pathways as a potential approach to increase the fitness of cells with mtDNA damage. Second, we will explore the possibility that defects in cytosolic proteostasis are precipitated by mtDNA mutation. Third, we will apply the knowledge and concepts gained in S. cerevisiae to both candidate-based and unbiased searches for genes that determine the aftermath of severe mtDNA damage in human cells. Beyond the mechanistic knowledge of mitochondrial dysfunction that will emerge from this project, we expect to identify new avenues toward the treatment of mitochondrial disease.
Summary
Mitochondrial DNA (mtDNA) encodes several proteins playing key roles in bioenergetics. Pathological mutations of mtDNA can be inherited or may accumulate following treatment for viral infections or cancer. Furthermore, many organisms, including humans, accumulate significant mtDNA damage during their lifespan, and it is therefore possible that mtDNA mutations can promote the aging process.
There are no effective treatments for most diseases caused by mtDNA mutation. An understanding of the cellular consequences of mtDNA damage is clearly imperative. Toward this goal, we use the budding yeast Saccharomyces cerevisiae as a cellular model of mitochondrial dysfunction. Genetic manipulation and biochemical study of this organism is easily achieved, and many proteins and processes important for mitochondrial biogenesis were first uncovered and best characterized using this experimental system. Importantly, current evidence suggests that processes required for survival of cells lacking a mitochondrial genome are widely conserved between yeast and other organisms, making likely the application of our findings to human health.
We will study the repercussions of mtDNA damage by three different strategies. First, we will investigate the link between a conserved, nutrient-sensitive signalling pathway and the outcome of mtDNA loss, since much recent evidence points to modulation of such pathways as a potential approach to increase the fitness of cells with mtDNA damage. Second, we will explore the possibility that defects in cytosolic proteostasis are precipitated by mtDNA mutation. Third, we will apply the knowledge and concepts gained in S. cerevisiae to both candidate-based and unbiased searches for genes that determine the aftermath of severe mtDNA damage in human cells. Beyond the mechanistic knowledge of mitochondrial dysfunction that will emerge from this project, we expect to identify new avenues toward the treatment of mitochondrial disease.
Max ERC Funding
1 497 160 €
Duration
Start date: 2015-04-01, End date: 2020-03-31
Project acronym sRNA-EMB
Project Small RNA regulation of the body plan and epigenome in Arabidopsis embryos
Researcher (PI) Michael Nodine
Host Institution (HI) GREGOR MENDEL INSTITUT FUR MOLEKULARE PFLANZENBIOLOGIE GMBH
Call Details Starting Grant (StG), LS3, ERC-2014-STG
Summary Small RNAs are short non-coding RNAs that regulate gene expression in plants and animals. Although small RNAs are essential for proper differentiation and epigenome regulation, little is known regarding their embryonic functions, especially in plants. Arabidopsis thaliana is a leading system to study the regulatory roles of small RNAs because of the abundance of genetic, genomic and epigenomic resources. Moreover, Arabidopsis embryos undergo invariant division patterns and rapidly differentiate to generate the most basic plant cell-types arranged in correct positions. Early Arabidopsis embryos are therefore morphologically simple structures composed of diverse cell types making them ideal for determining the influence of small RNAs on fundamental cellular differentiation and reprogramming events. The objectives of the proposed research are designed to assess the regulatory roles of small RNAs in establishing both the basic body plan and epigenome in plant embryos. We will utilize modified next-generation sequencing technologies to identify small RNAs present in developing embryos. Because we will generate these RNA profiles from a mixture of cell-types, we will also use a fluorescent protein-based approach to quantify specific miRNA repressive activities in individual cell-types. To determine the functions of individual miRNA/target interactions during embryogenesis, we will identify miRNAs required for embryo development and use genome-wide approaches to study specific miRNA/target interactions in greater detail. Lastly, we will use a fusion of genetic and genomic methods to determine how small RNAs influence the nascent epigenome during early embryogenesis.
Summary
Small RNAs are short non-coding RNAs that regulate gene expression in plants and animals. Although small RNAs are essential for proper differentiation and epigenome regulation, little is known regarding their embryonic functions, especially in plants. Arabidopsis thaliana is a leading system to study the regulatory roles of small RNAs because of the abundance of genetic, genomic and epigenomic resources. Moreover, Arabidopsis embryos undergo invariant division patterns and rapidly differentiate to generate the most basic plant cell-types arranged in correct positions. Early Arabidopsis embryos are therefore morphologically simple structures composed of diverse cell types making them ideal for determining the influence of small RNAs on fundamental cellular differentiation and reprogramming events. The objectives of the proposed research are designed to assess the regulatory roles of small RNAs in establishing both the basic body plan and epigenome in plant embryos. We will utilize modified next-generation sequencing technologies to identify small RNAs present in developing embryos. Because we will generate these RNA profiles from a mixture of cell-types, we will also use a fluorescent protein-based approach to quantify specific miRNA repressive activities in individual cell-types. To determine the functions of individual miRNA/target interactions during embryogenesis, we will identify miRNAs required for embryo development and use genome-wide approaches to study specific miRNA/target interactions in greater detail. Lastly, we will use a fusion of genetic and genomic methods to determine how small RNAs influence the nascent epigenome during early embryogenesis.
Max ERC Funding
1 499 989 €
Duration
Start date: 2015-07-01, End date: 2020-06-30
