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: 2020-11-30
Project acronym CHROMABOLISM
Project Chromatin-localized central metabolism regulating gene expression and cell identity
Researcher (PI) Stefan KUBICEK
Host Institution (HI) CEMM - FORSCHUNGSZENTRUM FUER MOLEKULARE MEDIZIN GMBH
Call Details Consolidator Grant (CoG), LS3, ERC-2017-COG
Summary Epigenetics research has revealed that in the cell’s nucleus all kinds of biomolecules–DNA, RNAs, proteins, protein posttranslational modifications–are highly compartmentalized to occupy distinct chromatin territories and genomic loci, thereby contributing to gene regulation and cell identity. In contrast, small molecules and cellular metabolites are generally considered to passively enter the nucleus from the cytoplasm and to lack distinct subnuclear localization. The CHROMABOLISM proposal challenges this assumption based on preliminary data generated in my laboratory. I hypothesize that chromatin-bound enzymes of central metabolism and subnuclear metabolite gradients contribute to gene regulation and cellular identity.
To address this hypothesis, we will first systematically profile chromatin-bound metabolic enzymes, chart nuclear metabolomes across representative leukemia cell lines, and develop tools to measure local metabolite concentrations at distinct genomic loci. In a second step, we will then develop and apply technology to perturb these nuclear metabolite patterns by forcing the export of metabolic enzymes for the nucleus, aberrantly recruiting these enzymes to selected genomic loci, and perturbing metabolite patterns by addition and depletion of metabolites. In all these conditions we will measure the impact of nuclear metabolism on chromatin structure and gene expression. Based on the data obtained, we will model for the effects of cellular metabolites on cancer cell identity and proliferation. In line with the recent discovery of oncometabolites and the clinical use of antimetabolites, we expect to predict chromatin-bound metabolic enzymes that can be exploited as druggable targets in oncology. In a final aim we will validate these targets in leukemia and develop chemical probes against them.
Successful completion of this project has the potential to transform our understanding of nuclear metabolism in control of gene expression and cellular identity.
Summary
Epigenetics research has revealed that in the cell’s nucleus all kinds of biomolecules–DNA, RNAs, proteins, protein posttranslational modifications–are highly compartmentalized to occupy distinct chromatin territories and genomic loci, thereby contributing to gene regulation and cell identity. In contrast, small molecules and cellular metabolites are generally considered to passively enter the nucleus from the cytoplasm and to lack distinct subnuclear localization. The CHROMABOLISM proposal challenges this assumption based on preliminary data generated in my laboratory. I hypothesize that chromatin-bound enzymes of central metabolism and subnuclear metabolite gradients contribute to gene regulation and cellular identity.
To address this hypothesis, we will first systematically profile chromatin-bound metabolic enzymes, chart nuclear metabolomes across representative leukemia cell lines, and develop tools to measure local metabolite concentrations at distinct genomic loci. In a second step, we will then develop and apply technology to perturb these nuclear metabolite patterns by forcing the export of metabolic enzymes for the nucleus, aberrantly recruiting these enzymes to selected genomic loci, and perturbing metabolite patterns by addition and depletion of metabolites. In all these conditions we will measure the impact of nuclear metabolism on chromatin structure and gene expression. Based on the data obtained, we will model for the effects of cellular metabolites on cancer cell identity and proliferation. In line with the recent discovery of oncometabolites and the clinical use of antimetabolites, we expect to predict chromatin-bound metabolic enzymes that can be exploited as druggable targets in oncology. In a final aim we will validate these targets in leukemia and develop chemical probes against them.
Successful completion of this project has the potential to transform our understanding of nuclear metabolism in control of gene expression and cellular identity.
Max ERC Funding
1 980 916 €
Duration
Start date: 2018-05-01, End date: 2023-04-30
Project acronym ChromHeritance
Project Chromosome inheritance from mammalian oocytes to embryos
Researcher (PI) Kikue Tachibana-Konwalski
Host Institution (HI) INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Call Details Starting Grant (StG), LS3, ERC-2013-StG
Summary One of the most dramatic transitions in biology is the oocyte-to-zygote transition. This refers to the maturation of the female germ cell or oocyte, which undergoes two rounds of meiotic chromosome segregation and, following fertilization, is converted to a mitotically dividing embryo. We aim to establish an innovative research program that addresses fundamental questions about the molecular processes controlling the mammalian oocyte-to-zygote transition to ensure faithful inheritance of genomes from one generation to the next. We are taking an interdisciplinary approach combining germ cell and chromosome biology with cell cycle and epigenetic studies to understand how maternal factors regulate chromosome segregation in oocytes and chromatin organization in the zygote. A molecular understanding of key players regulating these processes is a requisite step for investigating how their deterioration contributes to maternal age-related aneuploidy and infertility. Aneuploidy is the leading cause of mental retardation and spontaneous miscarriage. The current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years. A better understanding of mammalian meiosis is therefore relevant to human reproductive health.
A special feature of the female germ line is that meiotic DNA replication occurs in the embryo but oocytes remain arrested until the first meiotic division is triggered months (mouse) or decades (human) later. The longevity of oocytes poses a challenge for the cohesin complex that must hold together sister chromatids from DNA synthesis until chromosome segregation. We specifically aim to: 1) elucidate how sister chromatid cohesion is maintained in mammalian oocytes, 2) identify mechanisms regulating cohesion in young and aged oocytes, and 3) investigate how the inheritance of genetic and resetting of epigenetic information is coordinated with cell cycle progression at the oocyte-to-zygote transition.
Summary
One of the most dramatic transitions in biology is the oocyte-to-zygote transition. This refers to the maturation of the female germ cell or oocyte, which undergoes two rounds of meiotic chromosome segregation and, following fertilization, is converted to a mitotically dividing embryo. We aim to establish an innovative research program that addresses fundamental questions about the molecular processes controlling the mammalian oocyte-to-zygote transition to ensure faithful inheritance of genomes from one generation to the next. We are taking an interdisciplinary approach combining germ cell and chromosome biology with cell cycle and epigenetic studies to understand how maternal factors regulate chromosome segregation in oocytes and chromatin organization in the zygote. A molecular understanding of key players regulating these processes is a requisite step for investigating how their deterioration contributes to maternal age-related aneuploidy and infertility. Aneuploidy is the leading cause of mental retardation and spontaneous miscarriage. The current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years. A better understanding of mammalian meiosis is therefore relevant to human reproductive health.
A special feature of the female germ line is that meiotic DNA replication occurs in the embryo but oocytes remain arrested until the first meiotic division is triggered months (mouse) or decades (human) later. The longevity of oocytes poses a challenge for the cohesin complex that must hold together sister chromatids from DNA synthesis until chromosome segregation. We specifically aim to: 1) elucidate how sister chromatid cohesion is maintained in mammalian oocytes, 2) identify mechanisms regulating cohesion in young and aged oocytes, and 3) investigate how the inheritance of genetic and resetting of epigenetic information is coordinated with cell cycle progression at the oocyte-to-zygote transition.
Max ERC Funding
1 499 738 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
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 DROSOPIRNAS
Project The piRNA pathway in the Drosophila germline a small RNA based genome immune system
Researcher (PI) Julius Brennecke
Host Institution (HI) INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Call Details Starting Grant (StG), LS3, ERC-2010-StG_20091118
Summary The discovery of RNA interference (RNAi) has revolutionized biology. As a technology it opened up new experimental and therapeutic avenues. As a biological phenomenon it changed our view on a diverse array of cellular processes. Among those are the control of gene expression, the suppression of viral replication, the formation of heterochromatin and the protection of the genome against selfish genetic elements such as transposons.
I propose to study the molecular mechanism and the biological impact of a recently discovered RNAi pathway, the Piwi interacting RNA pathway (piRNA pathway).
The piRNA pathway is an evolutionarily conserved small RNA pathway acting in the animal germline. It is the key genome surveillance system that suppresses the activity of transposons. Recent work has provided a conceptual framework for this pathway: According to this, the genome stores transposon sequences in heterochromatic loci called piRNA clusters. These provide the RNA substrates for the biogenesis of 23-29 nt long piRNAs. An amplification cycle steers piRNA production predominantly to those cluster regions that are complementary to transposons being active at a given time. Finally, piRNAs guide a protein complex centered on Piwi-proteins to complementary transposon RNAs in the cell, leading to their silencing.
In contrast to other RNAi pathways, the mechanistic framework of the piRNA pathway is largely unknown. Moreover, the spectrum of biological processes impacted by it is only poorly understood. piRNAs are for example not only derived from transposon sequences but also from various other genomic repeats that are enriched at telomeres and in heterochromatin.
We will systematically dissect the piRNA pathway regarding its molecular architecture as well as its biological functions in Drosophila. Our studies will be a combination of fly genetics, proteomics and genomics approaches. Throughout we aim at linking our results back to the underlying biology of germline development.
Summary
The discovery of RNA interference (RNAi) has revolutionized biology. As a technology it opened up new experimental and therapeutic avenues. As a biological phenomenon it changed our view on a diverse array of cellular processes. Among those are the control of gene expression, the suppression of viral replication, the formation of heterochromatin and the protection of the genome against selfish genetic elements such as transposons.
I propose to study the molecular mechanism and the biological impact of a recently discovered RNAi pathway, the Piwi interacting RNA pathway (piRNA pathway).
The piRNA pathway is an evolutionarily conserved small RNA pathway acting in the animal germline. It is the key genome surveillance system that suppresses the activity of transposons. Recent work has provided a conceptual framework for this pathway: According to this, the genome stores transposon sequences in heterochromatic loci called piRNA clusters. These provide the RNA substrates for the biogenesis of 23-29 nt long piRNAs. An amplification cycle steers piRNA production predominantly to those cluster regions that are complementary to transposons being active at a given time. Finally, piRNAs guide a protein complex centered on Piwi-proteins to complementary transposon RNAs in the cell, leading to their silencing.
In contrast to other RNAi pathways, the mechanistic framework of the piRNA pathway is largely unknown. Moreover, the spectrum of biological processes impacted by it is only poorly understood. piRNAs are for example not only derived from transposon sequences but also from various other genomic repeats that are enriched at telomeres and in heterochromatin.
We will systematically dissect the piRNA pathway regarding its molecular architecture as well as its biological functions in Drosophila. Our studies will be a combination of fly genetics, proteomics and genomics approaches. Throughout we aim at linking our results back to the underlying biology of germline development.
Max ERC Funding
1 500 000 €
Duration
Start date: 2010-09-01, End date: 2015-08-31
Project acronym ETAP
Project Tracing Evolution of Auxin Transport and Polarity in Plants
Researcher (PI) Jiri Friml
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA
Call Details Advanced Grant (AdG), LS3, ERC-2016-ADG
Summary Multicellularity in plants evolved independently from other eukaryotes and presents a unique, alternative way how to deal with challenges of life. A major plant developmental module is the directional transport for the plant hormone auxin. The crucial components are PIN auxin transporters, whose polar, subcellular localization determines directionality of auxin flow through tissues. PIN-dependent auxin transport 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. Despite decades of intensive research, the classical approaches in the established models are approaching their limits and many crucial questions remain unsolved, in particular related to PIN structure, regulatory motifs and evolutionary origin
I propose to start a new direction in my research using an evolutionary perspective. This promises to overcome current limitations and provides not only (i) interesting insights into PIN evolution and diversification, but also (ii) a unique opportunity to study how evolutionary conserved cellular mechanisms of e.g. endocytic trafficking evolved specific plug-ins to make them subject to plant-specific regulations. The characterization of (iii) prokaryotic PIN origin will provide a so urgently needed (iv) entry into PIN structural studies. To achieve these goals, we will also establish novel (v) genetic and cell biological models in the ancestral lineage of the land plants that will be of a great use for any plant evolutionary studies.
The intellectual and methodological challenges of such interdisciplinary strategy combining several lower and higher plant models are obvious, but our preliminary results at several fronts promise its feasibility and success to gain deeper understanding of exciting questions on evolution and mechanisms behind the coordination and specification of developmental programs.
Summary
Multicellularity in plants evolved independently from other eukaryotes and presents a unique, alternative way how to deal with challenges of life. A major plant developmental module is the directional transport for the plant hormone auxin. The crucial components are PIN auxin transporters, whose polar, subcellular localization determines directionality of auxin flow through tissues. PIN-dependent auxin transport 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. Despite decades of intensive research, the classical approaches in the established models are approaching their limits and many crucial questions remain unsolved, in particular related to PIN structure, regulatory motifs and evolutionary origin
I propose to start a new direction in my research using an evolutionary perspective. This promises to overcome current limitations and provides not only (i) interesting insights into PIN evolution and diversification, but also (ii) a unique opportunity to study how evolutionary conserved cellular mechanisms of e.g. endocytic trafficking evolved specific plug-ins to make them subject to plant-specific regulations. The characterization of (iii) prokaryotic PIN origin will provide a so urgently needed (iv) entry into PIN structural studies. To achieve these goals, we will also establish novel (v) genetic and cell biological models in the ancestral lineage of the land plants that will be of a great use for any plant evolutionary studies.
The intellectual and methodological challenges of such interdisciplinary strategy combining several lower and higher plant models are obvious, but our preliminary results at several fronts promise its feasibility and success to gain deeper understanding of exciting questions on evolution and mechanisms behind the coordination and specification of developmental programs.
Max ERC Funding
2 410 292 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
Project acronym GRADIENTSENSING
Project Cellular navigation along spatial gradients
Researcher (PI) Michael Karl Sixt
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA
Call Details Consolidator Grant (CoG), LS3, ERC-2016-COG
Summary Gradients of extracellular signalling molecules are a central concept in biology: for example gradients of guidance-cues such as chemokines position migrating cells in development, malignancy and immunity. Because immune cells are permanently motile, their function most critically depends on spatiotemporal orchestration by a large family of chemokines. To specify direction, concentration differences of the chemokine need to be interpreted by the migrating cell. Most mechanistic knowledge about eukaryotic gradient sensing is inferred from the amoeba Dictyostelium discoideum migrating towards soluble gradients of cyclicAMP. The biology of chemokines is much more diverse, e.g. gradients can take different shapes and, importantly, they do not only emerge in the soluble but also in the immobilized phase. In this proposal we suggest to address the principles of leukocyte chemotaxis using convergent system wide, cell biological and intravital approaches. Employing a newly developed, genetically tractable primary leukocyte system, we will test the contribution of spatial and temporal signalling paradigms of gradient sensing. Quantitative microscopy will be used to image cellular responses to engineered immobilized and soluble chemokine gradients of defined shape as well as to optogenetically triggered signals. In a complementary approach we will screen for proteins responding to chemokine signalling and perform the first genome wide genome editing-based loss of function screen for directionally persistent chemotaxis and haptotaxis. Findings will be validated in vivo to guarantee physiological relevance. In a support project we will precision-engineer the genome of primary leukocytes suitable for assaying migration. A unique combination of cellular, genetic, engineering and quantitative microscopy tools will allow this new and holistic approach to a question which is not only fundamental for immunology but also for understanding development and cancer biology.
Summary
Gradients of extracellular signalling molecules are a central concept in biology: for example gradients of guidance-cues such as chemokines position migrating cells in development, malignancy and immunity. Because immune cells are permanently motile, their function most critically depends on spatiotemporal orchestration by a large family of chemokines. To specify direction, concentration differences of the chemokine need to be interpreted by the migrating cell. Most mechanistic knowledge about eukaryotic gradient sensing is inferred from the amoeba Dictyostelium discoideum migrating towards soluble gradients of cyclicAMP. The biology of chemokines is much more diverse, e.g. gradients can take different shapes and, importantly, they do not only emerge in the soluble but also in the immobilized phase. In this proposal we suggest to address the principles of leukocyte chemotaxis using convergent system wide, cell biological and intravital approaches. Employing a newly developed, genetically tractable primary leukocyte system, we will test the contribution of spatial and temporal signalling paradigms of gradient sensing. Quantitative microscopy will be used to image cellular responses to engineered immobilized and soluble chemokine gradients of defined shape as well as to optogenetically triggered signals. In a complementary approach we will screen for proteins responding to chemokine signalling and perform the first genome wide genome editing-based loss of function screen for directionally persistent chemotaxis and haptotaxis. Findings will be validated in vivo to guarantee physiological relevance. In a support project we will precision-engineer the genome of primary leukocytes suitable for assaying migration. A unique combination of cellular, genetic, engineering and quantitative microscopy tools will allow this new and holistic approach to a question which is not only fundamental for immunology but also for understanding development and cancer biology.
Max ERC Funding
1 984 922 €
Duration
Start date: 2017-04-01, End date: 2022-03-31
Project acronym GROWTHPATTERN
Project Coordination Of Patterning And Growth In The Spinal Cord
Researcher (PI) Anna Kicheva
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA
Call Details Starting Grant (StG), LS3, ERC-2015-STG
Summary Individuals of the same species vary widely in size, but their organs have reproducible proportions and patterns of cell types. How cell fate specification and tissue growth are coordinated during embryonic development to achieve this reproducibility is a fundamental question in biology. Yet, surprisingly little is known about the underlying mechanisms. A major challenge has been to obtain the quantitative data required to assess the dynamics and variability in growth, pattern and signalling by morphogens – molecules that regulate both cell fate specification and tissue growth. I recently established experimental and theoretical approaches that allowed me to reconstruct with unprecedented resolution the three-dimensional growth and pattern of mouse and chick spinal cord. My data revealed a previously unanticipated role of tissue growth dynamics in controlling pattern reproducibility. This quantitative framework provides an exciting opportunity to elucidate the biophysical and molecular mechanisms of growth and pattern coordination. I will use this unique position to understand: 1) how signalling by multiple morphogens is integrated to control pattern, 2) how morphogens control cell cycle kinetics, 3) how morphogen source and target tissue are coupled to achieve pattern reproducibility. To address these issues, I will build on my experience with quantitative analyses to design novel assays where signalling, cell cycle dynamics and transcriptomes can be precisely measured and manipulated with single cell resolution. I will exploit state-of-the-art genome editing techniques to uncouple the critical feedback links and gain a novel perspective on pattern reproducibility and morphogen function. The project will advance our fundamental understanding of tissue morphogenesis and provide novel insights relevant to understanding information processing by signal transduction cascades, morphogen gradient activity, tissue engineering, and cancer biology.
Summary
Individuals of the same species vary widely in size, but their organs have reproducible proportions and patterns of cell types. How cell fate specification and tissue growth are coordinated during embryonic development to achieve this reproducibility is a fundamental question in biology. Yet, surprisingly little is known about the underlying mechanisms. A major challenge has been to obtain the quantitative data required to assess the dynamics and variability in growth, pattern and signalling by morphogens – molecules that regulate both cell fate specification and tissue growth. I recently established experimental and theoretical approaches that allowed me to reconstruct with unprecedented resolution the three-dimensional growth and pattern of mouse and chick spinal cord. My data revealed a previously unanticipated role of tissue growth dynamics in controlling pattern reproducibility. This quantitative framework provides an exciting opportunity to elucidate the biophysical and molecular mechanisms of growth and pattern coordination. I will use this unique position to understand: 1) how signalling by multiple morphogens is integrated to control pattern, 2) how morphogens control cell cycle kinetics, 3) how morphogen source and target tissue are coupled to achieve pattern reproducibility. To address these issues, I will build on my experience with quantitative analyses to design novel assays where signalling, cell cycle dynamics and transcriptomes can be precisely measured and manipulated with single cell resolution. I will exploit state-of-the-art genome editing techniques to uncouple the critical feedback links and gain a novel perspective on pattern reproducibility and morphogen function. The project will advance our fundamental understanding of tissue morphogenesis and provide novel insights relevant to understanding information processing by signal transduction cascades, morphogen gradient activity, tissue engineering, and cancer biology.
Max ERC Funding
1 499 119 €
Duration
Start date: 2016-07-01, End date: 2021-06-30
Project acronym LeukocyteForces
Project Cytoskeletal force generation and force transduction of migrating leukocytes
Researcher (PI) Michael Sixt
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA
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
