Project acronym AutoClean
Project Cell-free reconstitution of autophagy to dissect molecular mechanisms
Researcher (PI) Claudine Simone Kraft
Host Institution (HI) UNIVERSITAETSKLINIKUM FREIBURG
Call Details Consolidator Grant (CoG), LS1, ERC-2017-COG
Summary Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.
Summary
Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.
Max ERC Funding
1 955 666 €
Duration
Start date: 2018-06-01, End date: 2023-05-31
Project acronym BACTERIAL SYRINGES
Project Protein Translocation Through Bacterial Syringes
Researcher (PI) Stefan Raunser
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS1, ERC-2013-CoG
Summary "The main objective of this application is to study the molecular basis of cellular infection by bacterial ABC-type toxins (Tc). Tc complexes are important virulence factors of a range of bacteria, including Photorhabdus luminescens and Yersinia pseudotuberculosis that infect insects and humans. Belonging to the class of pore-forming toxins, tripartite Tc complexes perforate the host membrane by forming channels that translocate toxic enzymes into the host.
In our previous cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we now intend to unravel the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We will use a hybrid approach, including biochemical reconstitution, structural analysis by cryo-EM and X-ray crystallography, fluorescence-based assays and site-directed mutagenesis to provide a comprehensive description of the molecular mechanism of infection at an unprecedented level of molecular detail.
Our results will be paradigmatic for understanding the mechanism of action of ABC-type toxins and will shed new light on the interactions of bacterial pathogens with their hosts."
Summary
"The main objective of this application is to study the molecular basis of cellular infection by bacterial ABC-type toxins (Tc). Tc complexes are important virulence factors of a range of bacteria, including Photorhabdus luminescens and Yersinia pseudotuberculosis that infect insects and humans. Belonging to the class of pore-forming toxins, tripartite Tc complexes perforate the host membrane by forming channels that translocate toxic enzymes into the host.
In our previous cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we now intend to unravel the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We will use a hybrid approach, including biochemical reconstitution, structural analysis by cryo-EM and X-ray crystallography, fluorescence-based assays and site-directed mutagenesis to provide a comprehensive description of the molecular mechanism of infection at an unprecedented level of molecular detail.
Our results will be paradigmatic for understanding the mechanism of action of ABC-type toxins and will shed new light on the interactions of bacterial pathogens with their hosts."
Max ERC Funding
1 999 992 €
Duration
Start date: 2014-07-01, End date: 2019-06-30
Project acronym BENDER
Project BiogENesis and Degradation of Endoplasmic Reticulum proteins
Researcher (PI) Friedrich Förster
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Consolidator Grant (CoG), LS1, ERC-2016-COG
Summary The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Summary
The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Max ERC Funding
2 496 611 €
Duration
Start date: 2017-04-01, End date: 2022-03-31
Project acronym BRAIN-MATCH
Project Matching CNS Lineage Maps with Molecular Brain Tumor Portraits for Translational Exploitation
Researcher (PI) Stefan PFISTER
Host Institution (HI) DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG
Call Details Consolidator Grant (CoG), LS2, ERC-2018-COG
Summary Brain tumors represent an extremely heterogeneous group of more than 100 different molecularly distinct diseases, many of which are still almost uniformly lethal despite five decades of clinical trials. In contrast to hematologic malignancies and carcinomas, the cell-of-origin for the vast majority of these entities is unknown. This knowledge gap currently precludes a comprehensive understanding of tumor biology and also limits translational exploitation (e.g., utilizing lineage targets for novel therapies and circulating brain tumor cells for liquid biopsies).
The BRAIN-MATCH project represents an ambitious program to address this challenge and unmet medical need by taking an approach that (i) extensively utilizes existing molecular profiles of more than 30,000 brain tumor samples covering more than 100 different entities, publicly available single-cell sequencing data of normal brain regions, and bulk normal tissue data at different times of development across different species; (ii) generates unprecedented maps of normal human CNS development by using state-of-the art novel technologies; (iii) matches these molecular portraits of normal cell types with tumor datasets in order to identify specific cell-of-origin populations for individual tumor entities; and (iv) validates the most promising cell-of-origin populations and tumor-specific lineage and/or surface markers in vivo.
The expected outputs of BRAIN-MATCH are four-fold: (i) delivery of an unprecedented atlas of human normal CNS development, which will also be of great relevance for diverse fields other than cancer; (ii) functional validation of at least three lineage targets; (iii) isolation and molecular characterization of circulating brain tumor cells from patients´ blood for at least five tumor entities; and (iv) generation of at least three novel mouse models of brain tumor entities for which currently no faithful models exist.
Summary
Brain tumors represent an extremely heterogeneous group of more than 100 different molecularly distinct diseases, many of which are still almost uniformly lethal despite five decades of clinical trials. In contrast to hematologic malignancies and carcinomas, the cell-of-origin for the vast majority of these entities is unknown. This knowledge gap currently precludes a comprehensive understanding of tumor biology and also limits translational exploitation (e.g., utilizing lineage targets for novel therapies and circulating brain tumor cells for liquid biopsies).
The BRAIN-MATCH project represents an ambitious program to address this challenge and unmet medical need by taking an approach that (i) extensively utilizes existing molecular profiles of more than 30,000 brain tumor samples covering more than 100 different entities, publicly available single-cell sequencing data of normal brain regions, and bulk normal tissue data at different times of development across different species; (ii) generates unprecedented maps of normal human CNS development by using state-of-the art novel technologies; (iii) matches these molecular portraits of normal cell types with tumor datasets in order to identify specific cell-of-origin populations for individual tumor entities; and (iv) validates the most promising cell-of-origin populations and tumor-specific lineage and/or surface markers in vivo.
The expected outputs of BRAIN-MATCH are four-fold: (i) delivery of an unprecedented atlas of human normal CNS development, which will also be of great relevance for diverse fields other than cancer; (ii) functional validation of at least three lineage targets; (iii) isolation and molecular characterization of circulating brain tumor cells from patients´ blood for at least five tumor entities; and (iv) generation of at least three novel mouse models of brain tumor entities for which currently no faithful models exist.
Max ERC Funding
1 999 875 €
Duration
Start date: 2019-05-01, End date: 2024-04-30
Project acronym CancerHetero
Project Dissection of tumor heterogeneity in vivo
Researcher (PI) Haikun Liu
Host Institution (HI) DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG
Call Details Consolidator Grant (CoG), LS2, ERC-2014-CoG
Summary It is now widely accepted that tumors are composed of heterogeneous population of cells, which contribute
to many aspects of treatment resistance observed in clinic. Despite the acknowledgment of the tumor cell
heterogeneity, little evidence was shown about complexity and dynamics of this heterogeneity in vivo,
mainly because of lacking flexible genetic tools which allow sophisticated analysis in primary tumors. We
recently developed a very efficient mouse somatic brain tumor model which have a full penetrance of high
grade glioma development. Combination of this model with several transgenic mouse lines allow us to
isolate and track different population of cells in primary tumors, most importantly, we also confirmed that
this can be done on single cell level. Here I propose to use this set of valuable genetic tools to dissect the
cellular heterogeneity in mouse gliomas. First we will perform several single cell lineage tracing experiment
to demonstrate the contribution of brain tumor stem cell, tumor progenitors as well as the relatively
differentiated cells, which will provide a complete data sets of clonal dynamics of different tumor cell types.
Second we will further perform this tracing experiment with the presence of conventional chemotherapy.
Third we will perform single cell RNA sequencing experiment to capture the molecular signature, which
determines the cellular heterogeneity, discovered by single cell tracing. This result will be further validated
by analysis of this molecular signatures in human primary tumors. We will also use our established in vivo
target validation approach to manipulate the candidate molecular regulators to establish the functional
correlation between molecular signature and phenotypic heterogeneity. This project will greatly improve our
understanding of tumor heterogeneity, and possibly provide novel approaches and strategies of targeting
human glioblastomas.
Summary
It is now widely accepted that tumors are composed of heterogeneous population of cells, which contribute
to many aspects of treatment resistance observed in clinic. Despite the acknowledgment of the tumor cell
heterogeneity, little evidence was shown about complexity and dynamics of this heterogeneity in vivo,
mainly because of lacking flexible genetic tools which allow sophisticated analysis in primary tumors. We
recently developed a very efficient mouse somatic brain tumor model which have a full penetrance of high
grade glioma development. Combination of this model with several transgenic mouse lines allow us to
isolate and track different population of cells in primary tumors, most importantly, we also confirmed that
this can be done on single cell level. Here I propose to use this set of valuable genetic tools to dissect the
cellular heterogeneity in mouse gliomas. First we will perform several single cell lineage tracing experiment
to demonstrate the contribution of brain tumor stem cell, tumor progenitors as well as the relatively
differentiated cells, which will provide a complete data sets of clonal dynamics of different tumor cell types.
Second we will further perform this tracing experiment with the presence of conventional chemotherapy.
Third we will perform single cell RNA sequencing experiment to capture the molecular signature, which
determines the cellular heterogeneity, discovered by single cell tracing. This result will be further validated
by analysis of this molecular signatures in human primary tumors. We will also use our established in vivo
target validation approach to manipulate the candidate molecular regulators to establish the functional
correlation between molecular signature and phenotypic heterogeneity. This project will greatly improve our
understanding of tumor heterogeneity, and possibly provide novel approaches and strategies of targeting
human glioblastomas.
Max ERC Funding
2 000 000 €
Duration
Start date: 2015-06-01, End date: 2020-05-31
Project acronym cenRNA
Project The role of RNA in centromere biology and genome integrity
Researcher (PI) Sylvia Erhardt
Host Institution (HI) RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG
Call Details Consolidator Grant (CoG), LS1, ERC-2015-CoG
Summary One of the most astonishing processes in the life of a cell is the division into two daughter cells. Such a highly organized process would presumably be regulated tightly by the underlying centromeric DNA sequence; however, the sites of chromosome attachment to the microtubule spindle are regulated by epigenetic mechanisms. The best-characterized epigenetic mark for centromeres is the histone H3-variant CENP-A, which replaces H3 in some of the nucleosomes within centromeric chromatin. Centromeres are embedded in pericentromeric heterochromatin and it has become apparent in recent years that heterochromatin is transcribed into non-coding RNAs. We have recently shown that a long non-coding RNA from pericentromeric heterochromatin of the X chromosome (SATIII) in Drosophila melanogaster localizes in trans to centromeres of all other chromosomes and is an essential component for correct loading and maintenance of CENP-A and, therefore, genome stability. Additional RNAs in Drosophila and RNAs from other species have been linked to centromeric chromatin, but their function is not understood. We propose that a complex, RNA-based epigenetic mechanism regulates centromere establishment and function.
This proposal is designed to the precise function of SATIII RNA by identifying the associated protein complexes as well as structural and post-transcriptional features of SATIII. We will evaluate the mechanisms by which SATIII functions as a heritable mark of centromeres through generations, during the developing germ line, and species separation. In parallel, we will systematically identify and characterize centromere-associated RNAs (cenRNAs) in Drosophila and human cells. We will elucidate their function in centromere biology and chromosome segregation, essentially as we have done and propose to do for SATIII. These experiments are designed to provide a detailed understanding of the essential, RNA-based epigenetic regulation of centromeres.
Summary
One of the most astonishing processes in the life of a cell is the division into two daughter cells. Such a highly organized process would presumably be regulated tightly by the underlying centromeric DNA sequence; however, the sites of chromosome attachment to the microtubule spindle are regulated by epigenetic mechanisms. The best-characterized epigenetic mark for centromeres is the histone H3-variant CENP-A, which replaces H3 in some of the nucleosomes within centromeric chromatin. Centromeres are embedded in pericentromeric heterochromatin and it has become apparent in recent years that heterochromatin is transcribed into non-coding RNAs. We have recently shown that a long non-coding RNA from pericentromeric heterochromatin of the X chromosome (SATIII) in Drosophila melanogaster localizes in trans to centromeres of all other chromosomes and is an essential component for correct loading and maintenance of CENP-A and, therefore, genome stability. Additional RNAs in Drosophila and RNAs from other species have been linked to centromeric chromatin, but their function is not understood. We propose that a complex, RNA-based epigenetic mechanism regulates centromere establishment and function.
This proposal is designed to the precise function of SATIII RNA by identifying the associated protein complexes as well as structural and post-transcriptional features of SATIII. We will evaluate the mechanisms by which SATIII functions as a heritable mark of centromeres through generations, during the developing germ line, and species separation. In parallel, we will systematically identify and characterize centromere-associated RNAs (cenRNAs) in Drosophila and human cells. We will elucidate their function in centromere biology and chromosome segregation, essentially as we have done and propose to do for SATIII. These experiments are designed to provide a detailed understanding of the essential, RNA-based epigenetic regulation of centromeres.
Max ERC Funding
1 896 250 €
Duration
Start date: 2016-07-01, End date: 2021-06-30
Project acronym CHROMATINREPAIRCODE
Project CHROMATIN-REPAIR-CODE: Hacking the chromatin code for DNA repair
Researcher (PI) Haico Van Attikum
Host Institution (HI) ACADEMISCH ZIEKENHUIS LEIDEN
Call Details Consolidator Grant (CoG), LS2, ERC-2013-CoG
Summary "Our cells receive tens of thousands of different DNA lesions per day. Failure to repair these lesions will lead to cell death, mutations and genome instability, which contribute to human diseases such as neurodegenerative disorders and cancer. Efficient recognition and repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged, through histone and non-histone proteins, into a condensed structure called chromatin. The DNA repair machinery has to circumvent this barrier to gain access to the damaged DNA and repair the lesions. Our recent work suggests that chromatin-modifying enzymes (CME) help to overcome this barrier at sites of DNA damage. However, the identity of these CME, their mode of action and interconnections with DNA repair pathways remain largely enigmatic. The aim of this project is to systematically identify and characterize the CME that operate during DNA repair processes in both yeast and human cells. To reach this goal we will use a cross-disciplinary approach that combines novel and cutting-edge genomics approaches with bioinformatics, genetics, biochemistry and high-resolution microscopy. Epigenetics-IDentifier (Epi-ID) will be used as a tool to unveil novel CME, whereas RNAi-interference and genetic interaction mapping studies will pinpoint the CME that may potentially regulate repair of DNA damage. A series of functional assays will eventually characterize their role in distinct DNA repair pathways, focusing on those that counteract DNA strand breaks and replication stress. Together these studies will provide insight into how CME assist cells to repair DNA damage in chromatin and inform on the relevance of CME to maintain genome stability and counteract human diseases."
Summary
"Our cells receive tens of thousands of different DNA lesions per day. Failure to repair these lesions will lead to cell death, mutations and genome instability, which contribute to human diseases such as neurodegenerative disorders and cancer. Efficient recognition and repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged, through histone and non-histone proteins, into a condensed structure called chromatin. The DNA repair machinery has to circumvent this barrier to gain access to the damaged DNA and repair the lesions. Our recent work suggests that chromatin-modifying enzymes (CME) help to overcome this barrier at sites of DNA damage. However, the identity of these CME, their mode of action and interconnections with DNA repair pathways remain largely enigmatic. The aim of this project is to systematically identify and characterize the CME that operate during DNA repair processes in both yeast and human cells. To reach this goal we will use a cross-disciplinary approach that combines novel and cutting-edge genomics approaches with bioinformatics, genetics, biochemistry and high-resolution microscopy. Epigenetics-IDentifier (Epi-ID) will be used as a tool to unveil novel CME, whereas RNAi-interference and genetic interaction mapping studies will pinpoint the CME that may potentially regulate repair of DNA damage. A series of functional assays will eventually characterize their role in distinct DNA repair pathways, focusing on those that counteract DNA strand breaks and replication stress. Together these studies will provide insight into how CME assist cells to repair DNA damage in chromatin and inform on the relevance of CME to maintain genome stability and counteract human diseases."
Max ERC Funding
1 999 575 €
Duration
Start date: 2014-03-01, End date: 2019-02-28
Project acronym CiliaTubulinCode
Project Self-organization of the cilium: the role of the tubulin code
Researcher (PI) Gaia PIGINO
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS1, ERC-2018-COG
Summary This project aims at understanding of the role of the tubulin code for self-organization of complex microtubule based structures. Cilia turn out to be the ideal structures for the proposed research.
A cilium is a sophisticated cellular machine that self-organizes from many protein complexes. It plays motility, sensory, and signaling roles in most eukaryotic cells, and its malfunction causes pathologies. The assembly of the cilium requires intraflagellar transport (IFT), a specialized bidirectional motility process that is mediated by adaptor proteins and direction specific molecular motors. Work from my lab shows that anterograde and retrograde IFT make exclusive use of the B-tubules and A-tubules, respectively. This insight answered a long standing question and shows that functional differentiation of tubules exists and is important for IFT.
Tubulin post-translational modifications (PTMs) contribute to a tubulin code, making microtubules suitable for specific functions. Mutation of tubulin-PTM enzymes can have dramatic effects on cilia function and assembly. However, we do not understand of the role of tubulin-PTMs in cilia. Therefore, I propose to address the hypotheses that the tubulin code contributes to regulating bidirectional IFT motility, and more generally, that the tubulin code is a key player in structuring complex cellular assembly processes in space and time.
This proposal aims at (i) understanding if tubulin-PTMs are necessary and/or sufficient to regulate the bidirectionality of IFT (ii) examining how the tubulin code regulates the assembly of cilia and (iii) generating a high-resolution atlas of tubulin-PTMs and their respective enzymes.
We will combine advanced techniques encompassing state-of-the-art cryo-electron tomography, biochemical imaging, fluorescent microscopy, and in vitro assays to achieve molecular and structural understanding of the role of the tubulin code in the self-organization of cilia and of microtubule based cellular structures.
Summary
This project aims at understanding of the role of the tubulin code for self-organization of complex microtubule based structures. Cilia turn out to be the ideal structures for the proposed research.
A cilium is a sophisticated cellular machine that self-organizes from many protein complexes. It plays motility, sensory, and signaling roles in most eukaryotic cells, and its malfunction causes pathologies. The assembly of the cilium requires intraflagellar transport (IFT), a specialized bidirectional motility process that is mediated by adaptor proteins and direction specific molecular motors. Work from my lab shows that anterograde and retrograde IFT make exclusive use of the B-tubules and A-tubules, respectively. This insight answered a long standing question and shows that functional differentiation of tubules exists and is important for IFT.
Tubulin post-translational modifications (PTMs) contribute to a tubulin code, making microtubules suitable for specific functions. Mutation of tubulin-PTM enzymes can have dramatic effects on cilia function and assembly. However, we do not understand of the role of tubulin-PTMs in cilia. Therefore, I propose to address the hypotheses that the tubulin code contributes to regulating bidirectional IFT motility, and more generally, that the tubulin code is a key player in structuring complex cellular assembly processes in space and time.
This proposal aims at (i) understanding if tubulin-PTMs are necessary and/or sufficient to regulate the bidirectionality of IFT (ii) examining how the tubulin code regulates the assembly of cilia and (iii) generating a high-resolution atlas of tubulin-PTMs and their respective enzymes.
We will combine advanced techniques encompassing state-of-the-art cryo-electron tomography, biochemical imaging, fluorescent microscopy, and in vitro assays to achieve molecular and structural understanding of the role of the tubulin code in the self-organization of cilia and of microtubule based cellular structures.
Max ERC Funding
1 986 406 €
Duration
Start date: 2019-03-01, End date: 2024-02-29
Project acronym CohesinLooping
Project Cohesin-mediated chromosomal looping: From linear paths to 3D effects
Researcher (PI) Benjamin Rowland
Host Institution (HI) STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Call Details Consolidator Grant (CoG), LS2, ERC-2017-COG
Summary The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.
Summary
The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.
Max ERC Funding
1 998 375 €
Duration
Start date: 2018-04-01, End date: 2023-03-31
Project acronym ComplexAssembly
Project The birth of protein complexes
Researcher (PI) Martin BECK
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Consolidator Grant (CoG), LS2, ERC-2016-COG
Summary Protein complexes are central to many cellular functions but our knowledge of how cells assemble protein complexes remains very sparse. Biophysical and structural data of assembly intermediates are extremely rare. Particularly in higher eukaryotes, it has become clear that complex assembly by random collision of subunits cannot cope with the spatial and temporal complexity of the intricate architecture of many cellular machines. Here I propose to combine systems biology approaches with in situ structural biology methods to visualize protein complex assembly. I want to investigate experimentally in which order the interfaces of protein complexes are formed and to which extent structures of assembly intermediates resemble those observed in fully assembled complexes. I want develop methods to systematically screen for additional factors involved in assembly pathways. I furthermore want to test the hypothesis that mechanisms must exist in eukaryotes that coordinate local mRNA translation with the ordered formation of protein complex interfaces. I believe that in order to understand assembly pathways, these processes, that so far are often studied autonomously, need to be considered jointly and in a protein complex centric manner. The research proposed here will bridge across these different scientific disciplines. In the long term, a better mechanistic understanding of protein complex assembly and the structural characterization of critical intermediates will be of high relevance for scenarios under which a cell’s protein quality control system has to cope with stress, such as aging and neurodegenerative diseases. It might also facilitate the more efficient industrial production of therapeutically relevant proteins.
Summary
Protein complexes are central to many cellular functions but our knowledge of how cells assemble protein complexes remains very sparse. Biophysical and structural data of assembly intermediates are extremely rare. Particularly in higher eukaryotes, it has become clear that complex assembly by random collision of subunits cannot cope with the spatial and temporal complexity of the intricate architecture of many cellular machines. Here I propose to combine systems biology approaches with in situ structural biology methods to visualize protein complex assembly. I want to investigate experimentally in which order the interfaces of protein complexes are formed and to which extent structures of assembly intermediates resemble those observed in fully assembled complexes. I want develop methods to systematically screen for additional factors involved in assembly pathways. I furthermore want to test the hypothesis that mechanisms must exist in eukaryotes that coordinate local mRNA translation with the ordered formation of protein complex interfaces. I believe that in order to understand assembly pathways, these processes, that so far are often studied autonomously, need to be considered jointly and in a protein complex centric manner. The research proposed here will bridge across these different scientific disciplines. In the long term, a better mechanistic understanding of protein complex assembly and the structural characterization of critical intermediates will be of high relevance for scenarios under which a cell’s protein quality control system has to cope with stress, such as aging and neurodegenerative diseases. It might also facilitate the more efficient industrial production of therapeutically relevant proteins.
Max ERC Funding
1 957 717 €
Duration
Start date: 2018-02-01, End date: 2023-01-31
