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Awardees

2022 -
Cross Disciplinary Fellowships - CDF

Understanding and controlling the sub-motors of bacterial rotary nanomachines

RIEU Martin (.)

. - University of Oxford - Oxford - United Kingdom

BERRY Richard (Host supervisor)
The bacterial flagellar motor (BFM) consists of hundreds of proteins that assemble into a transmembrane rotary nanomachine. It propels the flagella that drive bacterial swimming. New structural studies suggest that the motor is itself powered by smaller rotary motors. The latter, called stator complexes, are now thought to be autonomous rotary machines, powered by transmembrane ion flux, that drive the rest of the BFM. They share the structural motif of a pentamer surrounding a dimer (5:2), but it is not yet known whether these complexes really are rotary machines, and if so how rotation drives their functions. Here, we will deliver the stators into Droplets on Hydrogel Bilayers, which will allow single-molecule fluorescence imaging and membrane energization. Coupled to FRET and precise sodiometry based on organic sensors, this will allow us to determine the relation between the ion flux, the conformation and the rotation of the units. Finally, we will decipher their interaction with the whole BFM by fast measurements of their discrete dynamics: attaching gold nanorods to the BFM and following rotation in vivo by polarization microscopy, with angular resolution of a few degrees at sub-microsecond timescales. This project will be the first to characterize the molecular mechanisms behind the activation and the dynamics of the sub-motors of the BFM, using advanced techniques from bilayer biochemistry and single-molecule fluorescence, and will bring a new understanding of large rotary machines. It will also open avenues for synthetic biology, making important steps towards the synthesis of artificial cells endowed with the ability to self-propel.
2022 -
Long-Term Fellowships - LTF

Revealing the control of epithelial mechanics during wound healing using in vivo force manipulation

ROGALLA Svana (.)

. - Instituto Biofisika, Basque Centre for Biophysics (UPV/EHU, CSIC) - Leioa - SPAIN

SOLON Jerome (Host supervisor)
Wound healing is a dynamic process in which a living organism replaces lost or damaged tissue based on molecular, hormonal and cellular responses. Understanding the mechanics behind the cellular responses during wound healing processes is a key question in biology and biomedicine, however, the contribution of tissue mechanics (setting tissue deformation upon forces) to wound healing in live animals remains poorly understood. In this project, I will use live Drosophila melanogaster embryos as a model system to reveal how changes in epithelial mechanics are controlled during wound repair. Combining Drosophila genetics with laser dissection, and a novel procedure allowing the application of controlled forces on a single magnetic particle embedded within the tissue in vivo, I will measure the changes in epithelial mechanics within the wounded tissue and link these to specific wound healing regulatory signals. The project will comprise three consecutive stages: 1) I will measure changes in mechanical forces during the different phases of wound healing and correlate them with activation of biochemical signals, such as Hippo and JnK pathways. 2) Based on these measurements and in collaboration with theoretical physicists, I will create a biophysical model of wound healing processes. 3) I will investigate the effect of ectopic forces on the kinetics of wound healing and on cell response, particularly focusing on potential modulation in gene activity by mechanical perturbation. Altogether, these experiments will allow to reveal the control of wound healing in vivo encompassing mechanical and biochemical signals and the resulting changes in epithelial mechanics and cellular forces.
2022 -
Long-Term Fellowships - LTF

Deciphering the nature of genomic conflict using locus-specific chromatin perturbation and capture

RUDNIZKY Sergei (.)

. - Johns Hopkins University School of Medicine - Baltimore - United States

HA Taekjip (Host supervisor)
Chromatin modulates DNA accessibility and hence serves as a fundamental layer in the complex regulation of gene expression and DNA integrity. However, the role of chromatin organization in resolving a genomic conflict between two seemingly competing processes – transcription and DNA damage response (DDR) is not clear. This stems from the dynamic nature of chromatin that is transcribed and repaired in an asynchronous and position-dependent manner. Moreover, locus-specific purification of mammalian chromatin, usually found with only two copies per cell, presents a major challenge regarding the necessary assay specificity and sensitivity. To overcome these limitations, I will develop a single-molecule (SM) pulldown approach based on multi-step enrichment with CRISPR Cas9 and Cas12a, followed by Oligopaint to capture and dissect any chromatin locus of interest. Using light-activated vfCRISPR, I will introduce lesions at transcriptionally active or silent regions to induce DDR with high spatiotemporal control. Captured chromatin will be characterized by SM and super-resolution imaging regarding its composition and organization and will be used as a native substrate to access the dynamics of transcription and DNA repair factors in real-time. Coordination between transcription, DDR, and local chromatin will be examined by guiding vfCRISPR to the promoter and gene body of the heat-shock induced Hsp70 model gene. The developed method can be expanded to mouse models and can be broadly applied to genomic regions of high relevance to health and disease, providing an exciting opportunity for understanding the fundamentals of transcription and DDR in vivo at unprecedented resolution.
2022 -
Long-Term Fellowships - LTF

Are endothelial cells regulated differently during limb regeneration than during development?

SAVAGE Aaron (.)

. - President & Fellows of Harvard College - Cambridge - United States

WHITED Jessica (Host supervisor)
Biological question My question is: how is vascular regrowth regulated during limb regeneration? I aim to understand— at a precise molecular, genetic, and cellular level— how vascular regeneration is regulated in axolotls; whether by angiogenesis or novel mechanisms remains unclear. Scientific content This multi-disciplinary project spans developmental biology, genomics, and regenerative medicine, combining cutting-edge transcriptomic, imaging, and molecular biology practices analyze vascular regeneration in axolotl limbs. Specific aims: 1) Generate the first axolotl vasculature map, using CRISPR knockin-generated transgenics, contrasting native vascular limb patterning to post-regeneration limb patterning; 2) Analyze regenerating vascular cells transcriptionally to elucidate key genes/pathways, using single cell RNA sequencing; 3) Perform in vitro and in vivo assays (morpholino/CRISPRi knockdown; mutagenesis, etc.) to characterize how interplay between key genes/pathways regulates how blood vessels and other tissues, such as fibroblasts interact, informing limb regeneration. Novel transgenics will express both vascular-specific fluorescent proteins and the TVA receptor, enabling visualization of expression and spatiotemporal delivery of molecular modulators, for precise control of gene expression. Significance To understand limb regrowth, it is essential to understand complex interactions between different cell types during regeneration. This project will pioneer characterization of regenerating vessels, informing both wider understanding of limb regeneration and how vessels can be reactivated, generally, to expand in response to injury and disease.
2022 -
Long-Term Fellowships - LTF

The ecological role of bacterial specialized metabolites in bacteria-microalgae interactions

SCHLEYER Guy (.)

. - Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI) - Jena - GERMANY

HERTWECK Christian (Host supervisor)
Phytoplankton are marine photosynthetic microorganisms that thrive in the photic zones of the oceans. They form the basis of marine food-webs and greatly influence global biogeochemical cycles and the climate. Many studies highlighted the importance of microbial interactions in shaping community composition, especially bacteria-phytoplankton interactions. Over the past decade, chemical communication was found to be a key aspect in mediating these interactions, such as mutualism and pathogenicity. Although bacteria are prolific producers of specialized metabolites, also known as natural products, the involvement of such metabolites in bacteria-phytoplankton interactions has been largely overlooked. Moreover, these metabolites have been studied mostly for pharmaceutical purposes rather than for their role in nature. This research is aimed at studying bacterial specialized metabolites and their involvement in mediating bacteria-phytoplankton interactions. To achieve this, I will study ecologically-relevant bacteria-phytoplankton systems, both mutualistic and pathogenic, mediated by chemical communication. The systems will be developed in collaboration with the Pohnert group, which has been studying numerous systems of bacteria-algae interactions. I will identify bacterial specialized metabolites that mediate these interactions using analytical chemistry tools and will further characterize their biosynthetic pathways and their cellular targets using diverse biochemical techniques. These findings will shed light on the role of specialized metabolites in governing bacteria-microalgae interactions and allow us to better understand their ecological role in nature.
2022 -
Long-Term Fellowships - LTF

Using computational models to link dynamics of brain plasticity with behavioral changes over time

SCHURR Roey (.)

. - President & Fellows of Harvard College - Cambridge - United States

GERSHMAN Samuel (Host supervisor)
The ultimate goal of neuroscience is to provide a mechanistic explanation of the cognitive processes that drive human behavior. I propose to study the link between changes in one’s brain connectivity and structure and one’s behavioral changes, by integrating computational models, behavior and neuroimaging. Specifically, I will study the dynamical changes of behavioral and neuroimaging measurements to uncover the computational infrastructure of core cognitive processes. Computational model of behavioral yield subject-specific parameters. These parameters provide a mechanistic explanation to the cognitive process and to inter-subject variability. However, no large-scale longitudinal study has approached the source of observed changes in the estimated parameters over time. Is it measurement noise or true variability in the cognitive process? I will collect longitudinal data of behavior and neuroimaging from subjects who will perform seven behavioral tasks with established computational models. By studying the covariance of computational parameters over time, I will identify latent factors that may represent core cognitive process (e.g. working memory). I will explore the hypothesis that these latent factors reflect shared neural circuitry across tasks. In particular, I will test whether the dynamical changes in the computational phenotype are associated with functional and structural changes in specific brain networks. Finding such a link between model-based parameters and neuroimaging measurements will be a major step towards a mechanistic understanding of the computational and anatomical infrastructures that underlie core cognitive processes in the human brain.
2022 -
Long-Term Fellowships - LTF

Unravelling the mechanisms of bis(monoacylglycero)phosphate synthesis and function in endolysosomes

SINGH Shubham (.)

. - Sloan Kettering Institute for Cancer Research - New York - United States

FARESE Robert (Host supervisor)
Bis(monoglycero)phosphates (BMP) are a structurally unique orphan lipids exclusively localized at endolysosomal systems in the cells. Studies suggests that BMP might play a crucial role in the endolysosomal system by regulating lysosomal hydrolase activity, cholesterol transport and budding of lysosomal intralumenal vesicles. Interestingly, BMP levels are deregulated in various neuropathological conditions characterized by defective endolysosomal system like Niemann-Pick Disease Type C, Gaucher’s disease, Fabry disease, and dementia, etc. Though BMP were discovered five decades back and studies suggest their indispensable role in the endolysosomal physiology, nothing is known about their exact function, metabolism and how their deregulated levels associate with the neurodegenerative diseases. In this proposal, I aim to identify the enzymes that synthesize and degrade BMP in mammalian cells, and exhaustively characterize their role in the endolysosomal systems. I will use chemical genetic and siRNA strategies in conjunction with lipidomics to identify the enzymes that regulate BMP levels. Using in vitro reconstitution, I will determine the function of candidate enzymes and regulators in BMP metabolism. I will use genetic manipulations targeting enzymes to selectively modulate BMP levels in the cells/rodents, and examine their role in the endolysosomal physiology especially in the neurons. Overall, the results of the proposed studies will not only help in mapping BMP metabolism in cells but will also aid in understanding of the role of BMP in the endolysosomal physiology.
2022 -
Cross Disciplinary Fellowships - CDF

Tumour homing immune cells for cavitation therapy

SMITH Cameron (.)

. - California Institute of Technology - Pasadena - United States

SHAPIRO Mikhail (Host supervisor)
One of the great difficulties in the treatment of cancer is the inaccessibility of the tumour core. Due to poor vascularity, hypoxia, and high interstitial pressure, many conventional cancer therapeutics are unable to treat this significant portion of the target tumour, leading to less successful therapies. In this project we will address these limitations by utilising recent developments in synthetic biology and ultrasound. Our approach will take advantage of the Shapiro laboratory’s work on genetically encoded air-filled proteins called gas vesicles (GVs), which we will express in engineered tumour-infiltrating macrophages and T-cells in response to tumour-specific biomarkers such as VEGF, Her2, or claudin-6. The resulting GVs will act as seeds for highly selective and localised therapy when combined with focused ultrasound. We hypothesise that high energy inertial cavitation – the formation and violent collapse of bubbles – will be seeded by GVs to damage surrounding tumour cells. This would both allow for selective killing of tumour cells as well as the potential release of antigens which would make the tumour more susceptible to attack by the immune system, while additionally having the potential to allow the immune system to recognise and attack metastases. Successful completion of this project will produce a new class of local and targeted anti-tumour therapy and create a scientific and technological foundation for future developments and therapies utilising the combination of engineered cells and ultrasound.
2022 -
Long-Term Fellowships - LTF

Enhancing receptor signaling by liquid condensate formation

SONG Daesun (.)

. - The Board of Trustees of the Leland Stanford Junior University - Redwood City - United States

LIN Michael (Host supervisor)
Transmembrane receptors activate intracellular signaling pathways mediating growth, survival, or differentiation. Liquid condensate formation by T cell receptors enhances downstream signaling, but whether condensation can generally enhance receptor function is unknown. I propose to study the ability of condensation to generally enhance receptor function using the epidermal growth factor receptor (EGFR) as a model, with the following aims: 1. Design inducible EGFR condensates. Fusions of cryptochrome 2 (CRY2) and the Fused-in-Sarcoma intrinsically disordered region (IDR) form droplets in response to blue light, while cryptochrome variants CRY2olig and GFP-CRY2high without IDR create visible accumulations of unknown liquidity. I will fuse EGFR-GFP to CRY2-IDR, CRY2olig, or CRY2high and assess liquidity of light-induced accumulations by time-lapse morphology and FRAP. 2. Compare signaling –/+ EGFR condensation. I will activate the above proteins –/+ light –/+ EGF, comparing to EGFR-GFP, while measuring amplitude and kinetics of EGFR/Akt/ERK phosphorylation, Shc-RFP recruitment, and receptor internalization. This will determine if condensation enhances signaling, and if so at what step and in what manner. 3. Determine if any observed signaling enhancement also augments function by assessing proliferation of keratinocytes expressing EGFR-EGFP or the above fusions –/+ light –/+ EGF. This project will combine my existing expertise with liquid condensates from my Ph.D. training with the expertise of the Lin Lab at Stanford in CRY2 biochemistry and EGFR signaling to obtain insights into the role of condensation in organizing and amplifying biological signals.
2022 -
Long-Term Fellowships - LTF

Functional cell atlas of neural crest cell contribution to newt development and regeneration

SUZUKI Miyuki (.)

. - California Institute of Technology - Pasadena - United States

BRONNER Marianne (Host supervisor)
During development, neural crest stem cells arise within the forming central nervous system but then migrate into and throughout the periphery where they differentiate into diverse cell types. In addition to their importance in development, neural crest cells have recently been implicated in organ regeneration (e.g. heart regeneration in adult zebrafish) raising the intriguing possibility that they may be required in multiple regenerating structures. Salamanders have the remarkable regenerative ability and represent an excellent model for exploring the role of neural crest cells in both organogenesis and organ regeneration. To identify neural crest-derived cells that potentially re-express a neural crest gene regulatory program during organ regeneration, I have established Sox10::EGFP and Cre/loxP transgenic newts, Pleurodeles waltl, that genetically label and enable visualization of Sox10-expressing cells in vivo. With this in hand, I will explore two pressing questions: 1. Do neural crest-derived cells contribute to adult limb and heart regeneration in the newt? 2. If so, what are the underlying molecular mechanisms? To address these questions, in Aim 1, I will generate an atlas of cell types formed by neural crest-derived cells using Cre/loxP technique; in Aim 2, I will evaluate the involvement of neural crest-derived cells in organogenesis and organ regeneration using in vivo cell ablation technique. Aim 3 will generate a single-cell RNA-seq transcriptome profile during the regenerative process. This comprehensive atlas of cellular fates and functions of neural crest in newts promises to deepen understanding of development and regeneration in vertebrates.
2022 -
Long-Term Fellowships - LTF

Investigating the regulomic basis of major evolutionary transitions

TAYLOR Benjamin (.)

. - Purdue University - West Lafayette - United States

HARPUR Brock (Host supervisor)
The evolution of life on Earth has been extensively shaped by major transitions in biological complexity. The evolution of insect eusociality is one such transition and has occurred multiple times independently in recent evolutionary history. Unfortunately, the mechanistic basis of this transition is poorly understood. One major hypothesis proposes that eusociality is associated with substantial increases in gene regulatory complexity, but no study to date has provided a robust empirical test of this possibility. I propose to provide such a test. Using a novel meta-analytic machine learning approach, I will integrate new and existing multi-omic datasets from eusocial, subsocial, and solitary hymenopteran species to identify phylogenetically-robust signatures of the transition to eusociality. I predict that eusocial species will exhibit substantial expansions in regulatory complexity compared to related solitary taxa, in the form of increases in the proportion of the genome that is subject to alternative splicing or epigenetic regulation. Additionally, I will combine population genomic data and lab-based transgenic experiments to validate the functional role of regulatory loci, thereby corroborating the causal role of the regulome in generating complex social phenotypes. This project will integrate my experience in machine learning and gene expression analysis with expertise in alternative splicing and functional genomics provided by Dr Harpur’s group. Doing so will allow us to greatly advance our understanding of how social life evolves and diversifies by answering a fundamental biological question: how can biological complexity evolve without increases in gene content?
2022 -
Long-Term Fellowships - LTF

Templated polypeptide synthesis inside a nanopore cavity

TOPARLAK Omer duhan (.)

. - University of Oxford - Oxford - United Kingdom

BAYLEY Hagan (Host supervisor)
Genetically-coded protein synthesis is the universal hallmark of cellular life, where the ribosome is the central figure, linking nucleic acids to protein catalysts. Yet, our understanding of templated polypeptide synthesis is restricted to the extant ribosome and its engineered derivatives, with its limited chemistry. In order to facilitate the design of artificial ribosomes by simplifying the complex nature of the task, we propose to study peptide bond formation and the DNA-templated polymerization of amino acids within a nanotube cavity. Recent developments in nanopore technologies have unlocked the potential of single molecule chemistry with angstrom-level precision. To this end, we will utilize phi29 DNA packaging motor nanopores, which will allow the base pairing between template DNA and aminoacylated polynucleotides as activated substrates. Inspired by non-ribosomal peptide synthesis and prebiotic reactions on the early Earth, we will explore thiol-thioester, thiol-disulfide and S-to-N acyl transfer reactions. We will make use of thioester-activated (e.g. acetyl-CoA) and aminoacylated trinucleotides as transfer RNA mimics to drive polymerization. Modified or unnatural amino acid side chains on the nanopore wall can enable otherwise uphill condensation reactions. The newly synthesized polymers will not be restricted to the inclusion of natural amino acid side chains or conventional polypeptide backbones. The synthesis will take place continuously, at a useful rate and without the need for an external fuel. The ultimate chemistry we propose will enable (bio)chemists to produce efficient protein synthesis devices that will serve as a foundation for synthetic cells.
2022 -
Cross Disciplinary Fellowships - CDF

A multi-scale all-optical platform for the investigation of membrane potential dynamics

TORTAROLO Giorgio (.)

. - Swiss Federal Institute of Technology Lausanne (École Polytechnique Fédérale de Lausanne, (EPFL)) - Lausanne - SWITZERLAND

MANLEY Suliana (Host supervisor)
Membrane potential is a ubiquitous cellular feature, underlying important functions including the release of neurotransmitters upon arrival of an action potential, and calcium signaling related to cell proliferation control. Voltage imaging is becoming the standard to investigate membrane potentials, offering significant advantages over electrophysiology: it is less invasive, scalable, and suited to examine intracellular organelles. Although recent imaging implementations capture fast action potentials, they rely on averaging multiple events over a reduced field of view and are limited in temporal resolution. Hence, they can detect the single action potential but miss its propagation dynamics and, significantly, its modulation of synaptic transmission. Furthermore, intracellular propagation of membrane potentials remains unstudied. To overcome these limitations, I propose an all-optical optogenetic approach that leverages “smart” controlling architectures and state-of-the-art detectors to realize a paradigm change: from voltage imaging to on-demand voltage tracking. Upon further developing hardware in the host laboratory, I will investigate the propagation of organellar and plasma membrane potential signals, spontaneous or evoked, in non-excitable cells and neurons. I anticipate that this approach will uncover unprecedented insights into the intra- and inter-cellular signaling mechanisms and their modulation during activity. Indeed, these signatures are hypothesized to be altered by multiple sclerosis and other neurodegenerative diseases, and have so far proved elusive to measure.
2022 -
Long-Term Fellowships - LTF

Chemical and optogenetic approaches to identify and quantify the membrane sources of autophagosomes

UEMATSU Masaaki (.)

. - Cornell University - Ithaca - United States

BASKIN Jeremy (Host supervisor)
Autophagy is one of the main systems for the degradation of intracellular components. In this dynamic process, an ad hoc membranous structure called an autophagosome is transiently created to engulf cytoplasmic cargo to be degraded. Previous studies have identified many proteins essential for autophagy. However, the membrane sources of autophagosomes are still under debate, in large part due to the lack of direct methods to measure inter-organelle lipid transport with high spatiotemporal resolution. I propose to develop a method to locally label lipids on donor organelle membranes and detect them on the destination organelle (autophagosome) after lipid transfer. I will use a unique system, developed by my host laboratory and termed optoPLD, a light-controlled enzymatic tool that can produce labeled lipids from phosphatidylcholine (PC) on specific organelle membranes. For the labeling, I will engineer optoPLD to produce deuterated PCs on the donor membrane. For detection, I will generate a second optoPLD with an orthogonal optogenetic tool that can be activated with different wavelength of light to release the deuterated portion of PCs on the destination membrane via hydrolysis. I will measure the deuterated fraction of the released moiety to quantify the amount of transferred lipid. Combining this method with the acute knockdown of autophagy-related proteins to accumulate autophagosomes will determine the fractional contribution of organelles to the membrane sources of autophagosomes. Beyond autophagy, I envision that this method will be applicable to investigate any inter-organelle lipid transport, providing a much-needed tool to understand lipid homeostasis.
2022 -
Long-Term Fellowships - LTF

Haemogenic gastruloids: a novel approach to generate and study blood stem cells in vitro

VAN DEN BRINK Susanne carina (.)

. - Institut Hospital del Mar d'Investigacions Mèdiques (Hospital del Mar Medical Research Institute, IMIM) - Barcelona - SPAIN

BIGAS Anna (Host supervisor)
Bone marrow haematopoietic stem cell (HSC) transplants are life-saving treatments in a plethora of haematological and non-haematological malignancies. The availability of HSC transplants is limited, and there has been little success in producing or expanding HSCs in vitro. Rare instances of in vitro production of HSCs are of low efficiency and require ectopic expression of multiple genes, limiting their clinical applicability. During embryonic development, HSCs are formed in a specific niche which is key to HSC production. The study of the cellular and molecular contributions of the niche to mammalian HSC production remains restricted to in vivo mouse embryos, which are onerous to manipulate and not amenable to high-throughput screenings. Here, I propose to reconstruct the HSC generating niche in vitro using gastruloids; stem cell aggregates that self-organize into embryo-like structures with spatial and temporal precision. By modifying the original mouse gastruloids protocol, we have been able to recapitulate the emergence of haematopoietic clusters in the wall of vascular structures, with coordinated successive production of haemogenic endothelial and HSC-like cells. By systematically comparing haemogenic gastruloids with embryos I will build on these observations to: 1) obtain a tractable model system that can be used to study the embryonic HSC-forming niche in vitro 2) use this model to study how niche signals regulate HSC formation 3) achieve robust in vitro production of transgene-free, engraftable HSCs. Altogether, I will generate a model that will a) increase our understanding of the HSC niche and b) allow scalable in vitro production of tissue compatible HSCs.
2022 -
Cross Disciplinary Fellowships - CDF

Targeted protein degradation and electrophysiology to study the function of the proteasome

WHITTAKER Joanna (.)

. - University of Groningen - Groningen - NETHERLANDS

MAGLIA Giovanni (Host supervisor)
TYCH Katarzyna (Host supervisor)
The proteasome, a large protein complex capable of proteolysis, is the primary means by which misfolded or unnecessary proteins are degraded in eukaryotes and archaea. Due to its size and complexity, the mechanisms underlying its function, including the way in which it processes substrates with different primary and secondary structure content, are poorly understood. Accordingly, experimental methods must be used that enable the spatio-temporal measurement of these functions. Biological nanopores are created when a transmembrane protein forms a nanometre-scale hole in a membrane. Building on their effective use in a broad range of applications, planned developments using nanopores include their integration into biosensors for the detection of analytes from blood. Recently, the group of Prof. Maglia developed a proteasome nanopore, paving the way to protein sequencing using nanopores. This significant development also provides an excellent platform for this proposed fundamental study into the function of the proteasome. In this work, PROTACs (proteolysis-targeting chimeras) will be used to target proteins with different primary and secondary structure content for polyubiquitination by the E3 ubiquitin ligase. Electrochemical measurements will be used to observe the impact of physico-chemical properties and structure on unfolding and degradation by the proteasome in real time. PROTACs enable selected proteins to be targeted for degradation from mixtures or biological samples. This project is expected to have a significant impact on our understanding of the function of the proteasome, as well as in the areas of peptide sequencing and biosensors for tailored medicine.
2022 -
Long-Term Fellowships - LTF

Defining mechanisms of metabolic-epigenetic crosstalk that drive cancer initiation

XIAO Yi (.)

. - The University of Texas Southwestern Medical Center (UT Southwestern) - Dallas - United States

MCBRAYER Samuel (Host supervisor)
Recent discoveries of recurrent mutations in metabolic genes across human cancers have revealed a central role of altered metabolism in cancer initiation. Pathogenic metabolic gene mutations trigger accumulation of a class of biomolecules called “oncometabolites”, which impair the activities of enzymes that catalyze the demethylation of histones and DNA. Despite these advances, the specific chromosomal regions and genes that represent the functional effectors linking oncometabolites with cancer initiation are poorly understood. This knowledge gap persists in part due to the difficulty of using primary tumor specimens to retrospectively identify mechanisms of malignant transformation. To circumvent this challenge, the host lab has created the first genetically engineered mouse (GEM) model of lower grade glioma driven by the isocitrate dehydrogenase 1 (IDH1) R132H mutant oncoprotein, which produces the oncometabolite R-2-hydroxyglutarate (R-2HG). I propose to use this model together with isogenic, IDH1 wild-type companion GEM models to elucidate the dynamics of changes in chromatin structure, gene expression, and cell populations induced by R-2HG in vivo. I will conduct ATAC-seq, bulk RNA-seq, and single-cell RNA-seq analyses of engineered neural cells isolated throughout the process of metabolism-dependent malignant transformation. My studies will provide a conceptual framework for understanding the deterministic and stochastic functions of oncometabolite signaling to chromatin. My studies will also identify specific oncogenic mechanisms that operate at the interface of metabolome and epigenome to drive malignancy, which could nominate new therapeutic targets in glioma.
2022 -
Cross Disciplinary Fellowships - CDF

Revealing the fundamental regulators of cell mechanical properties by single cell microfluidics

XU Catherine (.)

. - Max Planck Institute for the Science of Light (Max-Planck-Institut für die Physik des Lichts) - Erlangen - GERMANY

GUCK Jochen (Host supervisor)
Changes in mechanical properties of cells are key in a range of processes, including cell migration and development, and are frequently altered in disease states such as cancers. Yet, despite their key role, the gene regulatory networks underlying these processes are currently largely unresolved. Thus, the central aim of my proposed project is to gain a detailed understanding of how cellular mechanical properties are controlled, by developing microfluidic technology to simultaneously measure the mechanical phenotype and transcriptome of single cells in high throughput. The advent of single cell sequencing methods has been transformational for our understanding of biology, and multimodal approaches such as those combining genome and transcriptome measurements of the same cell, are likely to be even more so. The physical dimension, however, remains largely unexplored, and its exploitation offers the prospect of revealing how the biochemical composition of cells relates to their physical properties. I will thus apply my PhD experience to develop a microfluidic platform that combines physical and biochemical cell analysis, using real-time deformability cytometry and droplet-based single cell RNA sequencing. By matching the transcriptomic profile of each cell with its brightfield image, which yields their mechanical and morphological features, I will identify genes involved in the regulation of mechanical properties and their generality across cell types. In addition to elucidating fundamental regulators of cell mechanics, this technology will allow the investigation of their interplay with gene expression during both physiological and pathological cell state changes.
2022 -
Long-Term Fellowships - LTF

Molecular and material design principles for meiotic microtubule-organizing centers

YAGUCHI Kan (.)

. - The University of Texas Southwestern Medical Center (UT Southwestern) - Dallas - United States

WOODRUFF Jeffrey (Host supervisor)
Formation of viable oocytes requires meiotic microtubule-organizing centers (MMOCs) lacking centrosomes. Multiple MMOCs assemble in oocyte cytoplasm and coalesce at two opposite spindle poles. While much is known about centrosomes, how MMOCs assemble and resist microtubule-mediated stresses is largely unexplored. I hypothesize that MMOC scaffolds begin as liquid droplets formed through demixing of proteins from the cytoplasm (phase separation model). In this case, these scaffold droplets could then fuse with each other and resist forces through regulated material hardening. Alternatively, MMOCs might assemble via diffusion-limited accretion (network percolation model) that require no hardening to resist forces. To test these hypotheses, this proposal aims to quantify the material properties of MMOCs and define the compositions for functional MMOC assembly. First, I will measure the liquidity and strength of MMOCs over time in living C. elegans oocytes. This can be accomplished using an optical technique pioneered in my host lab to induce nano-scale shear force (FLUCS). Second, I will use in vitro reconstitution system combined with FLUCS to determine the minimal protein motifs sufficient for MMOC assembly and material property. The host lab’s unique FlexiBAC expression system allows me to purify truncated candidate proteins known to localize at MMOCs. Finally, I will assess the fidelity of meiotic chromosome segregation when the material properties or components of MMOCs are altered in living oocytes. I expect these results to identify molecular and material designs for MMOC function. They will also represent a key first step toward creating a fully synthetic oocyte.
2022 -
Long-Term Fellowships - LTF

Physiological functions and molecular mechanisms of neuronal ER-phagy

YPERMAN Klaas (.)

. - Leibniz-Forschungsinstitut für Molekulare Pharmakologie - Berlin - GERMANY

HAUCKE Volker (Host supervisor)
Neurons are highly polarized cells with a complex architecture that underlies their function in information processing in the brain. The fact that neurons are long-lived post-mitotic cells poses special challenges for the pathways that preserve the integrity of their functional proteome. How neuronal proteostasis is maintained over the lifetime of an organism is a major unanswered question in neuroscience. The proposed project combines and extends my prior expertise in molecular cell biology in plants with recent findings from the host lab to dissect the physiological functions and molecular mechanisms of the turnover of the endoplasmic reticulum (ER), the largest neuronal organelle, via autophagy. Work in the host lab has shown that loss of the essential autophagy protein ATG5 causes the selective accumulation of tubular ER in axons and a concomitant increase in excitatory neurotransmission as a consequence of elevated calcium release from ER stores. Major questions regarding the physiological triggers for neuronal ER-phagy and the machinery involved remain unsolved. In the proposed project, I will use an unbiased proteomic approach utilizing novel proximity labeling strategies to identify the factors that control neuronal ER-phagy in the central nervous system. Moreover, I will develop live correlative light and electron microscopy approaches in genome-engineered mouse hippocampal and stem cell-derived human neurons to unravel the physiological stimuli that trigger axonal ER-phagy. The results obtained will provide fundamental new insights into the role of ER-phagy in maintaining brain function and aid the development of therapies to combat neurodegeneration.