Project acronym AAA
Project Adaptive Actin Architectures
Researcher (PI) Laurent Blanchoin
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Advanced Grant (AdG), LS3, ERC-2016-ADG
Summary Although we have extensive knowledge of many important processes in cell biology, including information on many of the molecules involved and the physical interactions among them, we still do not understand most of the dynamical features that are the essence of living systems. This is particularly true for the actin cytoskeleton, a major component of the internal architecture of eukaryotic cells. In living cells, actin networks constantly assemble and disassemble filaments while maintaining an apparent stable structure, suggesting a perfect balance between the two processes. Such behaviors are called “dynamic steady states”. They confer upon actin networks a high degree of plasticity allowing them to adapt in response to external changes and enable cells to adjust to their environments. Despite their fundamental importance in the regulation of cell physiology, the basic mechanisms that control the coordinated dynamics of co-existing actin networks are poorly understood. In the AAA project, first, we will characterize the parameters that allow the coupling among co-existing actin networks at steady state. In vitro reconstituted systems will be used to control the actin nucleation patterns, the closed volume of the reaction chamber and the physical interaction of the networks. We hope to unravel the mechanism allowing the global coherence of a dynamic actin cytoskeleton. Second, we will use our unique capacity to perform dynamic micropatterning, to add or remove actin nucleation sites in real time, in order to investigate the ability of dynamic networks to adapt to changes and the role of coupled network dynamics in this emergent property. In this part, in vitro experiments will be complemented by the analysis of actin network remodeling in living cells. In the end, our project will provide a comprehensive understanding of how the adaptive response of the cytoskeleton derives from the complex interplay between its biochemical, structural and mechanical properties.
Summary
Although we have extensive knowledge of many important processes in cell biology, including information on many of the molecules involved and the physical interactions among them, we still do not understand most of the dynamical features that are the essence of living systems. This is particularly true for the actin cytoskeleton, a major component of the internal architecture of eukaryotic cells. In living cells, actin networks constantly assemble and disassemble filaments while maintaining an apparent stable structure, suggesting a perfect balance between the two processes. Such behaviors are called “dynamic steady states”. They confer upon actin networks a high degree of plasticity allowing them to adapt in response to external changes and enable cells to adjust to their environments. Despite their fundamental importance in the regulation of cell physiology, the basic mechanisms that control the coordinated dynamics of co-existing actin networks are poorly understood. In the AAA project, first, we will characterize the parameters that allow the coupling among co-existing actin networks at steady state. In vitro reconstituted systems will be used to control the actin nucleation patterns, the closed volume of the reaction chamber and the physical interaction of the networks. We hope to unravel the mechanism allowing the global coherence of a dynamic actin cytoskeleton. Second, we will use our unique capacity to perform dynamic micropatterning, to add or remove actin nucleation sites in real time, in order to investigate the ability of dynamic networks to adapt to changes and the role of coupled network dynamics in this emergent property. In this part, in vitro experiments will be complemented by the analysis of actin network remodeling in living cells. In the end, our project will provide a comprehensive understanding of how the adaptive response of the cytoskeleton derives from the complex interplay between its biochemical, structural and mechanical properties.
Max ERC Funding
2 349 898 €
Duration
Start date: 2017-09-01, End date: 2022-08-31
Project acronym ACTMECH
Project Emergent Active Mechanical Behaviour of the Actomyosin Cell Cortex
Researcher (PI) Stephan Wolfgang Grill
Host Institution (HI) TECHNISCHE UNIVERSITAET DRESDEN
Call Details Starting Grant (StG), LS3, ERC-2011-StG_20101109
Summary The cell cortex is a highly dynamic layer of crosslinked actin filaments and myosin molecular motors beneath the cell membrane. It plays a central role in large scale rearrangements that occur inside cells. Many molecular mechanisms contribute to cortex structure and dynamics. However, cell scale physical properties of the cortex are difficult to grasp. This is problematic because for large scale rearrangements inside a cell, such as coherent flow of the cell cortex, it is the cell scale emergent properties that are important for the realization of such events. I will investigate how the actomyosin cytoskeleton behaves at a coarse grained and cellular scale, and will study how this emergent active behaviour is influenced by molecular mechanisms. We will study the cell cortex in the one cell stage C. elegans embryo, which undergoes large scale cortical flow during polarization and cytokinesis. We will combine theory and experiment. We will characterize cortex structure and dynamics with biophysical techniques such as cortical laser ablation and quantitative photobleaching experiments. We will develop and employ novel theoretical approaches to describe the cell scale mechanical behaviour in terms of an active complex fluid. We will utilize genetic approaches to understand how these emergent mechanical properties are influenced by molecular activities. A central goal is to arrive at a coarse grained description of the cortex that can predict future dynamic behaviour from the past structure, which is conceptually similar to how weather forecasting is accomplished. To date, systematic approaches to link molecular scale physical mechanisms to those on cellular scales are missing. This work will open new opportunities for cell biological and cell biophysical research, by providing a methodological approach for bridging scales, for studying emergent and large-scale active mechanical behaviours and linking them to molecular mechanisms.
Summary
The cell cortex is a highly dynamic layer of crosslinked actin filaments and myosin molecular motors beneath the cell membrane. It plays a central role in large scale rearrangements that occur inside cells. Many molecular mechanisms contribute to cortex structure and dynamics. However, cell scale physical properties of the cortex are difficult to grasp. This is problematic because for large scale rearrangements inside a cell, such as coherent flow of the cell cortex, it is the cell scale emergent properties that are important for the realization of such events. I will investigate how the actomyosin cytoskeleton behaves at a coarse grained and cellular scale, and will study how this emergent active behaviour is influenced by molecular mechanisms. We will study the cell cortex in the one cell stage C. elegans embryo, which undergoes large scale cortical flow during polarization and cytokinesis. We will combine theory and experiment. We will characterize cortex structure and dynamics with biophysical techniques such as cortical laser ablation and quantitative photobleaching experiments. We will develop and employ novel theoretical approaches to describe the cell scale mechanical behaviour in terms of an active complex fluid. We will utilize genetic approaches to understand how these emergent mechanical properties are influenced by molecular activities. A central goal is to arrive at a coarse grained description of the cortex that can predict future dynamic behaviour from the past structure, which is conceptually similar to how weather forecasting is accomplished. To date, systematic approaches to link molecular scale physical mechanisms to those on cellular scales are missing. This work will open new opportunities for cell biological and cell biophysical research, by providing a methodological approach for bridging scales, for studying emergent and large-scale active mechanical behaviours and linking them to molecular mechanisms.
Max ERC Funding
1 500 000 €
Duration
Start date: 2011-12-01, End date: 2017-08-31
Project acronym ACTOMYO
Project Mechanisms of actomyosin-based contractility during cytokinesis
Researcher (PI) Ana Costa Xavier de Carvalho
Host Institution (HI) INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR-IBMC
Call Details Starting Grant (StG), LS3, ERC-2014-STG
Summary Cytokinesis completes cell division by partitioning the contents of the mother cell to the two daughter cells. This process is accomplished through the assembly and constriction of a contractile ring, a complex actomyosin network that remains poorly understood on the molecular level. Research in cytokinesis has overwhelmingly focused on signaling mechanisms that dictate when and where the contractile ring is assembled. By contrast, the research I propose here addresses fundamental questions about the structural and functional properties of the contractile ring itself. We will use the nematode C. elegans to exploit the power of quantitative live imaging assays in an experimentally tractable metazoan organism. The early C. elegans embryo is uniquely suited to the study of the contractile ring, as cells dividing perpendicularly to the imaging plane provide a full end-on view of the contractile ring throughout constriction. This greatly facilitates accurate measurements of constriction kinetics, ring width and thickness, and levels as well as dynamics of fluorescently-tagged contractile ring components. Combining image-based assays with powerful molecular replacement technology for structure-function studies, we will 1) determine the contribution of branched and non-branched actin filament populations to contractile ring formation; 2) explore its ultra-structural organization in collaboration with a world expert in electron microcopy; 3) investigate how the contractile ring network is dynamically remodeled during constriction with the help of a novel laser microsurgery assay that has uncovered a remarkably robust ring repair mechanism; and 4) use a targeted RNAi screen and phenotype profiling to identify new components of actomyosin contractile networks. The results from this interdisciplinary project will significantly enhance our mechanistic understanding of cytokinesis and other cellular processes that involve actomyosin-based contractility.
Summary
Cytokinesis completes cell division by partitioning the contents of the mother cell to the two daughter cells. This process is accomplished through the assembly and constriction of a contractile ring, a complex actomyosin network that remains poorly understood on the molecular level. Research in cytokinesis has overwhelmingly focused on signaling mechanisms that dictate when and where the contractile ring is assembled. By contrast, the research I propose here addresses fundamental questions about the structural and functional properties of the contractile ring itself. We will use the nematode C. elegans to exploit the power of quantitative live imaging assays in an experimentally tractable metazoan organism. The early C. elegans embryo is uniquely suited to the study of the contractile ring, as cells dividing perpendicularly to the imaging plane provide a full end-on view of the contractile ring throughout constriction. This greatly facilitates accurate measurements of constriction kinetics, ring width and thickness, and levels as well as dynamics of fluorescently-tagged contractile ring components. Combining image-based assays with powerful molecular replacement technology for structure-function studies, we will 1) determine the contribution of branched and non-branched actin filament populations to contractile ring formation; 2) explore its ultra-structural organization in collaboration with a world expert in electron microcopy; 3) investigate how the contractile ring network is dynamically remodeled during constriction with the help of a novel laser microsurgery assay that has uncovered a remarkably robust ring repair mechanism; and 4) use a targeted RNAi screen and phenotype profiling to identify new components of actomyosin contractile networks. The results from this interdisciplinary project will significantly enhance our mechanistic understanding of cytokinesis and other cellular processes that involve actomyosin-based contractility.
Max ERC Funding
1 499 989 €
Duration
Start date: 2015-07-01, End date: 2020-06-30
Project acronym ACTOMYOSIN RING
Project Understanding Cytokinetic Actomyosin Ring Assembly Through Genetic Code Expansion, Click Chemistry, DNA origami, and in vitro Reconstitution
Researcher (PI) Mohan Balasubramanian
Host Institution (HI) THE UNIVERSITY OF WARWICK
Call Details Advanced Grant (AdG), LS3, ERC-2014-ADG
Summary The mechanism of cell division is conserved in many eukaryotes, from yeast to man. A contractile ring of filamentous actin and myosin II motors generates the force to bisect a mother cell into two daughters. The actomyosin ring is among the most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate contractile properties remains one of the great challenges in cell biology. Efforts to generate a comprehensive understanding of the mechanism of actomyosin ring assembly have been hampered by the lack of structural information on the arrangement of actin, myosin II, and actin modulators in the ring in its native state. Fundamental questions such as how actin filaments are assembled and organized into a ring remain actively debated. This project will investigate key issues pertaining to cytokinesis in the fission yeast Schizosaccharomyces pombe, which divides employing an actomyosin based contractile ring, using the methods of genetics, biochemistry, cellular imaging, DNA origami, genetic code expansion, and click chemistry. Specifically, we will (1) attempt to visualize actin filament assembly in live cells expressing fluorescent actin generated through synthetic biological approaches, including genetic code expansion and click chemistry (2) decipher actin filament polarity in the actomyosin ring using total internal reflection fluorescence microscopy of labelled dimeric and multimeric myosins V and VI generated through DNA origami approaches (3) address when, where, and how actin filaments for cytokinesis are assembled and organized into a ring and (4) reconstitute actin filament and functional actomyosin ring assembly in permeabilized spheroplasts and in supported bilayers. Success in the project will provide major insight into the mechanism of actomyosin ring assembly and illuminate principles behind cytoskeletal self-organization.
Summary
The mechanism of cell division is conserved in many eukaryotes, from yeast to man. A contractile ring of filamentous actin and myosin II motors generates the force to bisect a mother cell into two daughters. The actomyosin ring is among the most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate contractile properties remains one of the great challenges in cell biology. Efforts to generate a comprehensive understanding of the mechanism of actomyosin ring assembly have been hampered by the lack of structural information on the arrangement of actin, myosin II, and actin modulators in the ring in its native state. Fundamental questions such as how actin filaments are assembled and organized into a ring remain actively debated. This project will investigate key issues pertaining to cytokinesis in the fission yeast Schizosaccharomyces pombe, which divides employing an actomyosin based contractile ring, using the methods of genetics, biochemistry, cellular imaging, DNA origami, genetic code expansion, and click chemistry. Specifically, we will (1) attempt to visualize actin filament assembly in live cells expressing fluorescent actin generated through synthetic biological approaches, including genetic code expansion and click chemistry (2) decipher actin filament polarity in the actomyosin ring using total internal reflection fluorescence microscopy of labelled dimeric and multimeric myosins V and VI generated through DNA origami approaches (3) address when, where, and how actin filaments for cytokinesis are assembled and organized into a ring and (4) reconstitute actin filament and functional actomyosin ring assembly in permeabilized spheroplasts and in supported bilayers. Success in the project will provide major insight into the mechanism of actomyosin ring assembly and illuminate principles behind cytoskeletal self-organization.
Max ERC Funding
2 863 705 €
Duration
Start date: 2015-11-01, End date: 2020-10-31
Project acronym Age Asymmetry
Project Age-Selective Segregation of Organelles
Researcher (PI) Pekka Aleksi Katajisto
Host Institution (HI) HELSINGIN YLIOPISTO
Call Details Starting Grant (StG), LS3, ERC-2015-STG
Summary Our tissues are constantly renewed by stem cells. Over time, stem cells accumulate cellular damage that will compromise renewal and results in aging. As stem cells can divide asymmetrically, segregation of harmful factors to the differentiating daughter cell could be one possible mechanism for slowing damage accumulation in the stem cell. However, current evidence for such mechanisms comes mainly from analogous findings in yeast, and studies have concentrated only on few types of cellular damage.
I hypothesize that the chronological age of a subcellular component is a proxy for all the damage it has sustained. In order to secure regeneration, mammalian stem cells may therefore specifically sort old cellular material asymmetrically. To study this, I have developed a novel strategy and tools to address the age-selective segregation of any protein in stem cell division. Using this approach, I have already discovered that stem-like cells of the human mammary epithelium indeed apportion chronologically old mitochondria asymmetrically in cell division, and enrich old mitochondria to the differentiating daughter cell. We will investigate the mechanisms underlying this novel phenomenon, and its relevance for mammalian aging.
We will first identify how old and young mitochondria differ, and how stem cells recognize them to facilitate the asymmetric segregation. Next, we will analyze the extent of asymmetric age-selective segregation by targeting several other subcellular compartments in a stem cell division. Finally, we will determine whether the discovered age-selective segregation is a general property of stem cell in vivo, and it's functional relevance for maintenance of stem cells and tissue regeneration. Our discoveries may open new possibilities to target aging associated functional decline by induction of asymmetric age-selective organelle segregation.
Summary
Our tissues are constantly renewed by stem cells. Over time, stem cells accumulate cellular damage that will compromise renewal and results in aging. As stem cells can divide asymmetrically, segregation of harmful factors to the differentiating daughter cell could be one possible mechanism for slowing damage accumulation in the stem cell. However, current evidence for such mechanisms comes mainly from analogous findings in yeast, and studies have concentrated only on few types of cellular damage.
I hypothesize that the chronological age of a subcellular component is a proxy for all the damage it has sustained. In order to secure regeneration, mammalian stem cells may therefore specifically sort old cellular material asymmetrically. To study this, I have developed a novel strategy and tools to address the age-selective segregation of any protein in stem cell division. Using this approach, I have already discovered that stem-like cells of the human mammary epithelium indeed apportion chronologically old mitochondria asymmetrically in cell division, and enrich old mitochondria to the differentiating daughter cell. We will investigate the mechanisms underlying this novel phenomenon, and its relevance for mammalian aging.
We will first identify how old and young mitochondria differ, and how stem cells recognize them to facilitate the asymmetric segregation. Next, we will analyze the extent of asymmetric age-selective segregation by targeting several other subcellular compartments in a stem cell division. Finally, we will determine whether the discovered age-selective segregation is a general property of stem cell in vivo, and it's functional relevance for maintenance of stem cells and tissue regeneration. Our discoveries may open new possibilities to target aging associated functional decline by induction of asymmetric age-selective organelle segregation.
Max ERC Funding
1 500 000 €
Duration
Start date: 2016-05-01, End date: 2021-04-30
Project acronym APOQUANT
Project The quantitative Bcl-2 interactome in apoptosis: decoding how cancer cells escape death
Researcher (PI) Ana Jesús García Sáez
Host Institution (HI) EBERHARD KARLS UNIVERSITAET TUEBINGEN
Call Details Starting Grant (StG), LS3, ERC-2012-StG_20111109
Summary The proteins of the Bcl-2 family function as key regulators of apoptosis by controlling the permeabilization of the mitochondrial outer membrane. They form an intricate, fine-tuned interaction network which is altered in cancer cells to avoid cell death. Currently, we do not understand how signaling within this network, which combines events in cytosol and membranes, is orchestrated to decide the cell fate. The main goal of this proposal is to unravel how apoptosis signaling is integrated by the Bcl-2 network by determining the quantitative Bcl-2 interactome and building with it a mathematical model that identifies which interactions determine the overall outcome. To this aim, we have established a reconstituted system for the quantification of the interactions between Bcl-2 proteins not only in solution but also in membranes at the single molecule level by fluorescence correlation spectroscopy (FCS).
(1) This project aims to quantify the relative affinities between an reconstituted Bcl-2 network by FCS.
(2) This will be combined with quantitative studies in living cells, which include the signaling pathway in its entirety. To this aim, we will develop new FCS methods for mitochondria.
(3) The structural and dynamic aspects of the Bcl-2 network will be studied by super resolution and live cell microscopy.
(4) The acquired knowledge will be used to build a mathematical model that uncovers how the multiple interactions within the Bcl-2 network are integrated and identifies critical steps in apoptosis regulation.
These studies are expected to broaden the general knowledge about the design principles of cellular signaling as well as how cancer cells alter the Bcl-2 network to escape cell death. This systems analysis will allow us to predict which perturbations in the Bcl-2 network of cancer cells can switch signaling towards cell death. Ultimately it could be translated into clinical applications for anticancer therapy.
Summary
The proteins of the Bcl-2 family function as key regulators of apoptosis by controlling the permeabilization of the mitochondrial outer membrane. They form an intricate, fine-tuned interaction network which is altered in cancer cells to avoid cell death. Currently, we do not understand how signaling within this network, which combines events in cytosol and membranes, is orchestrated to decide the cell fate. The main goal of this proposal is to unravel how apoptosis signaling is integrated by the Bcl-2 network by determining the quantitative Bcl-2 interactome and building with it a mathematical model that identifies which interactions determine the overall outcome. To this aim, we have established a reconstituted system for the quantification of the interactions between Bcl-2 proteins not only in solution but also in membranes at the single molecule level by fluorescence correlation spectroscopy (FCS).
(1) This project aims to quantify the relative affinities between an reconstituted Bcl-2 network by FCS.
(2) This will be combined with quantitative studies in living cells, which include the signaling pathway in its entirety. To this aim, we will develop new FCS methods for mitochondria.
(3) The structural and dynamic aspects of the Bcl-2 network will be studied by super resolution and live cell microscopy.
(4) The acquired knowledge will be used to build a mathematical model that uncovers how the multiple interactions within the Bcl-2 network are integrated and identifies critical steps in apoptosis regulation.
These studies are expected to broaden the general knowledge about the design principles of cellular signaling as well as how cancer cells alter the Bcl-2 network to escape cell death. This systems analysis will allow us to predict which perturbations in the Bcl-2 network of cancer cells can switch signaling towards cell death. Ultimately it could be translated into clinical applications for anticancer therapy.
Max ERC Funding
1 462 900 €
Duration
Start date: 2013-04-01, End date: 2019-03-31
Project acronym APOSITE
Project Apoptotic foci: composition, structure and dynamics
Researcher (PI) Ana GARCIA SAEZ
Host Institution (HI) EBERHARD KARLS UNIVERSITAET TUEBINGEN
Call Details Consolidator Grant (CoG), LS3, ERC-2018-COG
Summary Apoptotic cell death is essential for development, immune function or tissue homeostasis, and it is often deregulated in disease. Mitochondrial outer membrane permeabilization (MOMP) is central for apoptosis execution and plays a key role in its inflammatory outcome. Knowing the architecture of the macromolecular machineries mediating MOMP is crucial for understanding their function and for the clinical use of apoptosis.
Our recent work reveals that Bax and Bak dimers form distinct line, arc and ring assemblies at specific apoptotic foci to mediate MOMP. However, the molecular structure and mechanisms controlling the spatiotemporal formation and range of action of the apoptotic foci are missing. To address this fundamental gap in our knowledge, we aim to unravel the composition, dynamics and structure of apoptotic foci and to understand how they are integrated to orchestrate function. We will reach this goal by building on our expertise in cell death and cutting-edge imaging and by developing a new analytical pipeline to:
1) Identify the composition of apoptotic foci using in situ proximity-dependent labeling and extraction of near-native Bax/Bak membrane complexes coupled to mass spectrometry.
2) Define their contribution to apoptosis and its immunogenicity and establish their assembly dynamics to correlate it with apoptosis progression by live cell imaging.
3) Determine the stoichiometry and structural organization of the apoptotic foci by combining single molecule fluorescence and advanced electron microscopies.
This multidisciplinary approach offers high chances to solve the long-standing question of how Bax and Bak mediate MOMP. APOSITE will provide textbook knowledge of the mitochondrial contribution to cell death and inflammation. The implementation of this new analytical framework will open novel research avenues in membrane and organelle biology. Ultimately, understanding of Bax and Bak structure/function will help develop apoptosis modulators for medicine.
Summary
Apoptotic cell death is essential for development, immune function or tissue homeostasis, and it is often deregulated in disease. Mitochondrial outer membrane permeabilization (MOMP) is central for apoptosis execution and plays a key role in its inflammatory outcome. Knowing the architecture of the macromolecular machineries mediating MOMP is crucial for understanding their function and for the clinical use of apoptosis.
Our recent work reveals that Bax and Bak dimers form distinct line, arc and ring assemblies at specific apoptotic foci to mediate MOMP. However, the molecular structure and mechanisms controlling the spatiotemporal formation and range of action of the apoptotic foci are missing. To address this fundamental gap in our knowledge, we aim to unravel the composition, dynamics and structure of apoptotic foci and to understand how they are integrated to orchestrate function. We will reach this goal by building on our expertise in cell death and cutting-edge imaging and by developing a new analytical pipeline to:
1) Identify the composition of apoptotic foci using in situ proximity-dependent labeling and extraction of near-native Bax/Bak membrane complexes coupled to mass spectrometry.
2) Define their contribution to apoptosis and its immunogenicity and establish their assembly dynamics to correlate it with apoptosis progression by live cell imaging.
3) Determine the stoichiometry and structural organization of the apoptotic foci by combining single molecule fluorescence and advanced electron microscopies.
This multidisciplinary approach offers high chances to solve the long-standing question of how Bax and Bak mediate MOMP. APOSITE will provide textbook knowledge of the mitochondrial contribution to cell death and inflammation. The implementation of this new analytical framework will open novel research avenues in membrane and organelle biology. Ultimately, understanding of Bax and Bak structure/function will help develop apoptosis modulators for medicine.
Max ERC Funding
2 000 000 €
Duration
Start date: 2019-04-01, End date: 2024-03-31
Project acronym ARFMEMBRANESENSORS
Project Membrane sensors in the Arf orbit
Researcher (PI) Bruno Antonny
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Advanced Grant (AdG), LS3, ERC-2010-AdG_20100317
Summary Cellular organelles are continuously remodelled by numerous cytosolic proteins that associate transiently with their lipid membrane. Some distort the bilayer, others change its composition, extract lipids or bridge membranes at distance. Previous works from my laboratory have underlined the importance of membrane sensors, i.e. elements within proteins that help to organize membrane-remodelling events by sensing the physical and chemical state of the underlying membrane. A membrane sensor is not necessarily of well-folded domain that interacts with a specific lipid polar head: some intrinsically unfolded motifs harboring deceptively simple sequences can display remarkable membrane adhesive properties. Among these are some amphipathic helices: the ALPS motif with a polar face made mostly by small uncharged polar residues, the Spo20 helix with several histidines in its polar face and, like a mirror image of the ALPS motif, the alpha-synuclein helix with very small hydrophobic residues. Using biochemistry and molecular dynamics, we will compare the membrane binding properties of these sequences (effect of curvature, charge, lipid unsaturation); using bioinformatics we will look for new motifs, using cell biology we will assess the adaptation of these motifs to the physical and chemical features of organelle membranes. Concurrently, we will use reconstitution approaches on artificial membranes to dissect how membrane sensors contribute to the organization of vesicle tethering by golgins and sterol transport by ORP proteins. We surmise that the combination of a molecular ¿switch¿, a small G protein of the Arf family, and of membrane sensors permit to organize these complex reactions in time and in space.
Summary
Cellular organelles are continuously remodelled by numerous cytosolic proteins that associate transiently with their lipid membrane. Some distort the bilayer, others change its composition, extract lipids or bridge membranes at distance. Previous works from my laboratory have underlined the importance of membrane sensors, i.e. elements within proteins that help to organize membrane-remodelling events by sensing the physical and chemical state of the underlying membrane. A membrane sensor is not necessarily of well-folded domain that interacts with a specific lipid polar head: some intrinsically unfolded motifs harboring deceptively simple sequences can display remarkable membrane adhesive properties. Among these are some amphipathic helices: the ALPS motif with a polar face made mostly by small uncharged polar residues, the Spo20 helix with several histidines in its polar face and, like a mirror image of the ALPS motif, the alpha-synuclein helix with very small hydrophobic residues. Using biochemistry and molecular dynamics, we will compare the membrane binding properties of these sequences (effect of curvature, charge, lipid unsaturation); using bioinformatics we will look for new motifs, using cell biology we will assess the adaptation of these motifs to the physical and chemical features of organelle membranes. Concurrently, we will use reconstitution approaches on artificial membranes to dissect how membrane sensors contribute to the organization of vesicle tethering by golgins and sterol transport by ORP proteins. We surmise that the combination of a molecular ¿switch¿, a small G protein of the Arf family, and of membrane sensors permit to organize these complex reactions in time and in space.
Max ERC Funding
1 997 321 €
Duration
Start date: 2011-05-01, End date: 2015-04-30
Project acronym ArtifiCell
Project Synthetic Cell Biology: Designing organelle transport mechanisms
Researcher (PI) James Edward Rothman
Host Institution (HI) UNIVERSITY COLLEGE LONDON
Call Details Advanced Grant (AdG), LS3, ERC-2014-ADG
Summary Imagine being able to design into living cells and organisms de novo vesicle transport mechanisms that do not naturally exist? At one level this is a wild-eyed notion of synthetic biology.
But we contend that this vision can be approached even today, focusing first on the process of exocytosis, a fundamental process that impacts almost every area of physiology. Enough has now been learned about the natural core machinery (as recognized by the award of the 2013 Nobel Prize in Physiology or Medicine to the PI and others) to take highly innovative physics/engineering- and DNA-based approaches to design synthetic versions of the secretory apparatus that could someday open new avenues in genetic medicine.
The central idea is to introduce DNA-based functional equivalents of the core protein machinery that naturally form (coats), target (tethers), and fuse (SNAREs) vesicles. We have already taken first steps by using DNA origami-based templates to produce synthetic phospholipid vesicles and complementary DNA-based tethers to specifically capture these DNA-templated vesicles on targeted bilayers. Others have linked DNA oligonucleotides to trigger vesicle fusion.
The next and much more challenging step is to introduce such processes into living cells. We hope to break this barrier, and in the process start a new field of research into “synthetic exocytosis”, by introducing Peptide-Nucleic Acids (PNAs) of tethers and SNAREs to re-direct naturally-produced secretory vesicles to artificially-programmed targets and provide artificially-programmed regulation. PNAs are chosen mainly because they lack the negatively charged phosphate backbones of DNA, and therefore are more readily delivered into the cell across the plasma membrane. Future steps, would include producing the transport vesicles synthetically within the cell by externally supplied origami-based PNA or similar cages, and - much more speculatively - ultimately using encoded DNA and RNAs to provide these functions.
Summary
Imagine being able to design into living cells and organisms de novo vesicle transport mechanisms that do not naturally exist? At one level this is a wild-eyed notion of synthetic biology.
But we contend that this vision can be approached even today, focusing first on the process of exocytosis, a fundamental process that impacts almost every area of physiology. Enough has now been learned about the natural core machinery (as recognized by the award of the 2013 Nobel Prize in Physiology or Medicine to the PI and others) to take highly innovative physics/engineering- and DNA-based approaches to design synthetic versions of the secretory apparatus that could someday open new avenues in genetic medicine.
The central idea is to introduce DNA-based functional equivalents of the core protein machinery that naturally form (coats), target (tethers), and fuse (SNAREs) vesicles. We have already taken first steps by using DNA origami-based templates to produce synthetic phospholipid vesicles and complementary DNA-based tethers to specifically capture these DNA-templated vesicles on targeted bilayers. Others have linked DNA oligonucleotides to trigger vesicle fusion.
The next and much more challenging step is to introduce such processes into living cells. We hope to break this barrier, and in the process start a new field of research into “synthetic exocytosis”, by introducing Peptide-Nucleic Acids (PNAs) of tethers and SNAREs to re-direct naturally-produced secretory vesicles to artificially-programmed targets and provide artificially-programmed regulation. PNAs are chosen mainly because they lack the negatively charged phosphate backbones of DNA, and therefore are more readily delivered into the cell across the plasma membrane. Future steps, would include producing the transport vesicles synthetically within the cell by externally supplied origami-based PNA or similar cages, and - much more speculatively - ultimately using encoded DNA and RNAs to provide these functions.
Max ERC Funding
3 000 000 €
Duration
Start date: 2015-09-01, End date: 2021-08-31
Project acronym ASYMMEM
Project Lipid asymmetry: a cellular battery?
Researcher (PI) André NADLER
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Starting Grant (StG), LS3, ERC-2017-STG
Summary It is a basic textbook notion that the plasma membranes of virtually all organisms display an asymmetric lipid distribution between inner and outer leaflets far removed from thermodynamic equilibrium. As a fundamental biological principle, lipid asymmetry has been linked to numerous cellular processes. However, a clear mechanistic justification for the continued existence of lipid asymmetry throughout evolution has yet to be established. We propose here that lipid asymmetry serves as a store of potential energy that is used to fuel energy-intense membrane remodelling and signalling events for instance during membrane fusion and fission. This implies that rapid, local changes of trans-membrane lipid distribution rather than a continuously maintained out-of-equilibrium situation are crucial for cellular function. Consequently, new methods for quantifying the kinetics of lipid trans-bilayer movement are required, as traditional approaches are mostly suited for analysing quasi-steady-state conditions. Addressing this need, we will develop and employ novel photochemical lipid probes and lipid biosensors to quantify localized trans-bilayer lipid movement. We will use these tools for identifying yet unknown protein components of the lipid asymmetry regulating machinery and analyse their function with regard to membrane dynamics and signalling in cell motility. Focussing on cell motility enables targeted chemical and genetic perturbations while monitoring lipid dynamics on timescales and in membrane structures that are well suited for light microscopy. Ultimately, we aim to reconstitute lipid asymmetry as a driving force for membrane remodelling in vitro. We expect that our work will break new ground in explaining one of the least understood features of the plasma membrane and pave the way for a new, dynamic membrane model. Since the plasma membrane serves as the major signalling hub, this will have impact in almost every area of the life sciences.
Summary
It is a basic textbook notion that the plasma membranes of virtually all organisms display an asymmetric lipid distribution between inner and outer leaflets far removed from thermodynamic equilibrium. As a fundamental biological principle, lipid asymmetry has been linked to numerous cellular processes. However, a clear mechanistic justification for the continued existence of lipid asymmetry throughout evolution has yet to be established. We propose here that lipid asymmetry serves as a store of potential energy that is used to fuel energy-intense membrane remodelling and signalling events for instance during membrane fusion and fission. This implies that rapid, local changes of trans-membrane lipid distribution rather than a continuously maintained out-of-equilibrium situation are crucial for cellular function. Consequently, new methods for quantifying the kinetics of lipid trans-bilayer movement are required, as traditional approaches are mostly suited for analysing quasi-steady-state conditions. Addressing this need, we will develop and employ novel photochemical lipid probes and lipid biosensors to quantify localized trans-bilayer lipid movement. We will use these tools for identifying yet unknown protein components of the lipid asymmetry regulating machinery and analyse their function with regard to membrane dynamics and signalling in cell motility. Focussing on cell motility enables targeted chemical and genetic perturbations while monitoring lipid dynamics on timescales and in membrane structures that are well suited for light microscopy. Ultimately, we aim to reconstitute lipid asymmetry as a driving force for membrane remodelling in vitro. We expect that our work will break new ground in explaining one of the least understood features of the plasma membrane and pave the way for a new, dynamic membrane model. Since the plasma membrane serves as the major signalling hub, this will have impact in almost every area of the life sciences.
Max ERC Funding
1 500 000 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
