Project acronym APPL
Project Anionic PhosPhoLipids in plant receptor kinase signaling
Researcher (PI) Yvon Jaillais
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS3, ERC-2013-StG
Summary "In plants, receptor kinases form the largest family of plasma membrane (PM) receptors and they are involved in virtually all aspects of the plant life, including development, immunity and reproduction. In animals, key molecules that orchestrate the recruitment of signaling proteins to membranes are anionic phospholipids (e.g. phosphatidylinositol phosphate or PIPs). Besides, recent reports in animal and yeast cells suggest the existence of PM nanodomains that are independent of cholesterol and lipid phase and rely on anionic phospholipids as well as electrostatic protein/lipid interactions. Strikingly, we know very little on the role of anionic phospholipids in plant signaling. However, our preliminary data suggest that BKI1, an inhibitory protein of the steroid receptor kinase BRI1, interacts with various PIPs in vitro and is likely targeted to the PM by electrostatic interactions with these anionic lipids. These results open the possibility that BRI1, but also other receptor kinases, might be regulated by anionic phospholipids in plants. Here, we propose to analyze the function of anionic phospholipids in BRI1 signaling, using the root epidermis as a model system. First, we will ask what are the lipids that control membrane surface charge in this tissue and recruit BR-signaling component to the PM. Second, we will probe the presence of PIP-enriched nanodomains at the plant PM using super-resolution microscopy techniques and investigate the roles of these domains in BRI1 signaling. Finally, we will analyze the function of the BKI1-related plant-specific family of anionic phospholipid effectors in plant development. In summary, using a transversal approach ranging from in vitro studies to in vivo validation and whole organism physiology, this work will unravel the interplay between anionic phospholipids and receptor signaling in plants."
Summary
"In plants, receptor kinases form the largest family of plasma membrane (PM) receptors and they are involved in virtually all aspects of the plant life, including development, immunity and reproduction. In animals, key molecules that orchestrate the recruitment of signaling proteins to membranes are anionic phospholipids (e.g. phosphatidylinositol phosphate or PIPs). Besides, recent reports in animal and yeast cells suggest the existence of PM nanodomains that are independent of cholesterol and lipid phase and rely on anionic phospholipids as well as electrostatic protein/lipid interactions. Strikingly, we know very little on the role of anionic phospholipids in plant signaling. However, our preliminary data suggest that BKI1, an inhibitory protein of the steroid receptor kinase BRI1, interacts with various PIPs in vitro and is likely targeted to the PM by electrostatic interactions with these anionic lipids. These results open the possibility that BRI1, but also other receptor kinases, might be regulated by anionic phospholipids in plants. Here, we propose to analyze the function of anionic phospholipids in BRI1 signaling, using the root epidermis as a model system. First, we will ask what are the lipids that control membrane surface charge in this tissue and recruit BR-signaling component to the PM. Second, we will probe the presence of PIP-enriched nanodomains at the plant PM using super-resolution microscopy techniques and investigate the roles of these domains in BRI1 signaling. Finally, we will analyze the function of the BKI1-related plant-specific family of anionic phospholipid effectors in plant development. In summary, using a transversal approach ranging from in vitro studies to in vivo validation and whole organism physiology, this work will unravel the interplay between anionic phospholipids and receptor signaling in plants."
Max ERC Funding
1 797 840 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
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
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 BACEMO
Project Bacterial Cell Morphogenesis
Researcher (PI) Rut Carballido Lopez
Host Institution (HI) INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT
Call Details Starting Grant (StG), LS3, ERC-2012-StG_20111109
Summary In bacteria, the though external cell wall and the intracellular actin-like (MreB) cytoskeleton are major determinants of cell shape. The biosynthetic pathways and chemical composition of the cell wall, a three dimensional polymer network that is one of the most prominent targets for antibiotics, are well understood. However, despite decades of study, little is known about the complex cell wall ultrastructure and the molecular mechanisms that control cell wall morphogenesis in time and space. In rod-shaped bacteria, MreB homologues assemble into dynamic structures thought to control shape by serving as organizers for the movement and assembly of macromolecular machineries that effect sidewall elongation. However, the mechanistic details used by the MreB cytoskeleton to fulfil this role remain to be elucidated. Furthermore, development of high-resolution microscopy techniques has led to new breakthroughs this year, published by our lab and others, which are shaking the model developed over the last decade and re-questioning the MreB “actin cytoskeleton” designation.
The aim of this project is to combine powerful genetic, biochemical, genomic and systems biology approaches available in the model bacterium Bacillus subtilis with modern high-resolution light microscopic techniques to study the dynamics and mechanistic details of the MreB cytoskeleton and of CW assembly. Parameters measured by the different approaches will be combined to quantitatively describe the features of bacterial cell morphogenesis.
Summary
In bacteria, the though external cell wall and the intracellular actin-like (MreB) cytoskeleton are major determinants of cell shape. The biosynthetic pathways and chemical composition of the cell wall, a three dimensional polymer network that is one of the most prominent targets for antibiotics, are well understood. However, despite decades of study, little is known about the complex cell wall ultrastructure and the molecular mechanisms that control cell wall morphogenesis in time and space. In rod-shaped bacteria, MreB homologues assemble into dynamic structures thought to control shape by serving as organizers for the movement and assembly of macromolecular machineries that effect sidewall elongation. However, the mechanistic details used by the MreB cytoskeleton to fulfil this role remain to be elucidated. Furthermore, development of high-resolution microscopy techniques has led to new breakthroughs this year, published by our lab and others, which are shaking the model developed over the last decade and re-questioning the MreB “actin cytoskeleton” designation.
The aim of this project is to combine powerful genetic, biochemical, genomic and systems biology approaches available in the model bacterium Bacillus subtilis with modern high-resolution light microscopic techniques to study the dynamics and mechanistic details of the MreB cytoskeleton and of CW assembly. Parameters measured by the different approaches will be combined to quantitatively describe the features of bacterial cell morphogenesis.
Max ERC Funding
1 650 050 €
Duration
Start date: 2013-02-01, End date: 2019-01-31
Project acronym BacForce
Project Quantifying minute forces: How mechanoregulation determines the behavior of pathogenic bacteria
Researcher (PI) Benedikt SABASS
Host Institution (HI) LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Call Details Starting Grant (StG), LS3, ERC-2019-STG
Summary Bacteria can generate mechanical forces that are important for the colonization of surfaces, formation of biofilms, and infection of host cells. This proposal addresses the fundamental question of how bacteria can control their force generation to robustly respond to chemo-mechanical cues on complex surfaces. Currently, a knowledge gap exists between the molecular regulation pathways on the one hand and the mechanical behavior on the other hand. One major impediment for understanding of how behavior is connected to control is, to date, the impossibility of studying bacterial force directly in unconstrained situations. Based on an initial study, I propose employing new methods for the unperturbed, high-resolution measurement of bacterial traction forces on wide spatiotemporal scales. Thus, the force-generation linking behavior to control can be investigated directly.
The objectives are to (A) gain access to nanoscopic mechanical phenomena through the development of cutting-edge super-resolution traction force microscopy, (B) employ the methods to characterize how Pseudomonas aeruginosa controls pilus-generated forces while responding to chemical cues, and (C) establish how surface rigidity affects force generation by P. aeruginosa during biofilm formation. In an interdisciplinary approach, I will combine traction measurements with genetic perturbations, molecule labeling, and computer simulations to produce functional models of the mechanocontrol strategies.
Altogether, I will establish a novel technique, opening up the possibility of studying nanoscopic force generation in many types of cells. Through these advances, I will characterize a set of mechanoregulation strategies in P. aeruginosa that are paradigmatic for diverse Gram-negative pathogens employing the same type of pili. Broadly, I expect that the studied bacterial control strategies have a generic, minimal nature and can appear as basic motives throughout development, homeostasis, and disease.
Summary
Bacteria can generate mechanical forces that are important for the colonization of surfaces, formation of biofilms, and infection of host cells. This proposal addresses the fundamental question of how bacteria can control their force generation to robustly respond to chemo-mechanical cues on complex surfaces. Currently, a knowledge gap exists between the molecular regulation pathways on the one hand and the mechanical behavior on the other hand. One major impediment for understanding of how behavior is connected to control is, to date, the impossibility of studying bacterial force directly in unconstrained situations. Based on an initial study, I propose employing new methods for the unperturbed, high-resolution measurement of bacterial traction forces on wide spatiotemporal scales. Thus, the force-generation linking behavior to control can be investigated directly.
The objectives are to (A) gain access to nanoscopic mechanical phenomena through the development of cutting-edge super-resolution traction force microscopy, (B) employ the methods to characterize how Pseudomonas aeruginosa controls pilus-generated forces while responding to chemical cues, and (C) establish how surface rigidity affects force generation by P. aeruginosa during biofilm formation. In an interdisciplinary approach, I will combine traction measurements with genetic perturbations, molecule labeling, and computer simulations to produce functional models of the mechanocontrol strategies.
Altogether, I will establish a novel technique, opening up the possibility of studying nanoscopic force generation in many types of cells. Through these advances, I will characterize a set of mechanoregulation strategies in P. aeruginosa that are paradigmatic for diverse Gram-negative pathogens employing the same type of pili. Broadly, I expect that the studied bacterial control strategies have a generic, minimal nature and can appear as basic motives throughout development, homeostasis, and disease.
Max ERC Funding
1 498 864 €
Duration
Start date: 2020-08-01, End date: 2025-07-31
Project acronym BACTERIAL SPORES
Project Investigating the Nature of Bacterial Spores
Researcher (PI) Sigal Ben-Yehuda
Host Institution (HI) THE HEBREW UNIVERSITY OF JERUSALEM
Call Details Starting Grant (StG), LS3, ERC-2007-StG
Summary When triggered by nutrient limitation, the Gram-positive bacterium Bacillus subtilis and its relatives enter a pathway of cellular differentiation culminating in the formation of a dormant cell type called a spore, the most resilient cell type known. Bacterial spores can survive for long periods of time and are able to endure extremes of heat, radiation and chemical assault. Remarkably, dormant spores can rapidly convert back to actively growing cells by a process called germination. Consequently, spore forming bacteria, including dangerous pathogens, (such as C. botulinum and B. anthracis) are highly resistant to antibacterial treatments and difficult to eradicate. Despite significant advances in our understanding of the process of spore formation, little is known about the nature of the mature spore. It is unrevealed how dormancy is maintained within the spore and how it is ceased, as the organization and the dynamics of the spore macromolecules remain obscure. The unusual biochemical and biophysical characteristics of the dormant spore make it a challenging biological system to investigate using conventional methods, and thus set the need to develop innovative approaches to study spore biology. We propose to explore the nature of spores by using B. subtilis as a primary experimental system. We intend to: (1) define the architecture of the spore chromosome, (2) track the complexity and fate of mRNA and protein molecules during sporulation, dormancy and germination, (3) revisit the basic notion of the spore dormancy (is it metabolically inert?), (4) compare the characteristics of bacilli spores from diverse ecophysiological groups, (5) investigate the features of spores belonging to distant bacterial genera, (6) generate an integrative database that categorizes the molecular features of spores. Our study will provide original insights and introduce novel concepts to the field of spore biology and may help devise innovative ways to combat spore forming pathogens.
Summary
When triggered by nutrient limitation, the Gram-positive bacterium Bacillus subtilis and its relatives enter a pathway of cellular differentiation culminating in the formation of a dormant cell type called a spore, the most resilient cell type known. Bacterial spores can survive for long periods of time and are able to endure extremes of heat, radiation and chemical assault. Remarkably, dormant spores can rapidly convert back to actively growing cells by a process called germination. Consequently, spore forming bacteria, including dangerous pathogens, (such as C. botulinum and B. anthracis) are highly resistant to antibacterial treatments and difficult to eradicate. Despite significant advances in our understanding of the process of spore formation, little is known about the nature of the mature spore. It is unrevealed how dormancy is maintained within the spore and how it is ceased, as the organization and the dynamics of the spore macromolecules remain obscure. The unusual biochemical and biophysical characteristics of the dormant spore make it a challenging biological system to investigate using conventional methods, and thus set the need to develop innovative approaches to study spore biology. We propose to explore the nature of spores by using B. subtilis as a primary experimental system. We intend to: (1) define the architecture of the spore chromosome, (2) track the complexity and fate of mRNA and protein molecules during sporulation, dormancy and germination, (3) revisit the basic notion of the spore dormancy (is it metabolically inert?), (4) compare the characteristics of bacilli spores from diverse ecophysiological groups, (5) investigate the features of spores belonging to distant bacterial genera, (6) generate an integrative database that categorizes the molecular features of spores. Our study will provide original insights and introduce novel concepts to the field of spore biology and may help devise innovative ways to combat spore forming pathogens.
Max ERC Funding
1 630 000 €
Duration
Start date: 2008-10-01, End date: 2013-09-30
Project acronym BACTIN
Project Shaping the bacterial cell wall: the actin-like cytoskeleton, from single molecules to morphogenesis and antimicrobials
Researcher (PI) Rut CARBALLIDO LOPEZ
Host Institution (HI) INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT
Call Details Consolidator Grant (CoG), LS3, ERC-2017-COG
Summary One of the ultimate goals in cell biology is to understand how cells determine their shape. In bacteria, the cell wall and the actin-like (MreB) cytoskeleton are major determinants of cell shape. As a hallmark of microbial life, the external cell wall is the most conspicuous macromolecule expanding in concert with cell growth and one of the most prominent targets for antibiotics. Despite decades of study, the mechanism of cell wall morphogenesis remains poorly understood. In rod-shaped bacteria, actin-like MreB proteins assemble into disconnected membrane-associated structures (patches) that move processively around the cell periphery and are thought to control shape by spatiotemporally organizing macromolecular machineries that effect sidewall elongation. However, the ultrastructure of MreB assemblies and the mechanistic details underlying their morphogenetic function remain to be elucidated.
The aim of this project is to combine ground-breaking light microscopy and spectroscopy techniques with cutting-edge genetic, biochemical and systems biology approaches available in the model rod-shaped bacterium Bacillus subtilis to elucidate how MreB and cell wall biosynthetic enzymes collectively act to build a cell. Within this context, new features of MreB assemblies will be determined in vivo and in vitro, and a “toolbox” of approaches to determine the modes of action of antibiotics targeting cell wall processes will be developed. Parameters measured by the different approaches will be used to refine a mathematical model aiming to quantitatively describe the features of bacterial cell wall growth. The long-term goals of BActin are to understand general principles of bacterial cell morphogenesis and to provide mechanistic templates and new reporters for the screening of novel antibiotics.
Summary
One of the ultimate goals in cell biology is to understand how cells determine their shape. In bacteria, the cell wall and the actin-like (MreB) cytoskeleton are major determinants of cell shape. As a hallmark of microbial life, the external cell wall is the most conspicuous macromolecule expanding in concert with cell growth and one of the most prominent targets for antibiotics. Despite decades of study, the mechanism of cell wall morphogenesis remains poorly understood. In rod-shaped bacteria, actin-like MreB proteins assemble into disconnected membrane-associated structures (patches) that move processively around the cell periphery and are thought to control shape by spatiotemporally organizing macromolecular machineries that effect sidewall elongation. However, the ultrastructure of MreB assemblies and the mechanistic details underlying their morphogenetic function remain to be elucidated.
The aim of this project is to combine ground-breaking light microscopy and spectroscopy techniques with cutting-edge genetic, biochemical and systems biology approaches available in the model rod-shaped bacterium Bacillus subtilis to elucidate how MreB and cell wall biosynthetic enzymes collectively act to build a cell. Within this context, new features of MreB assemblies will be determined in vivo and in vitro, and a “toolbox” of approaches to determine the modes of action of antibiotics targeting cell wall processes will be developed. Parameters measured by the different approaches will be used to refine a mathematical model aiming to quantitatively describe the features of bacterial cell wall growth. The long-term goals of BActin are to understand general principles of bacterial cell morphogenesis and to provide mechanistic templates and new reporters for the screening of novel antibiotics.
Max ERC Funding
1 902 195 €
Duration
Start date: 2019-02-01, End date: 2024-01-31
