Protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination are important for controlling the cell cycle and preventing genome instability. Using the budding yeast Saccharomyces cerevisiae, I will characterize the significance of PTMs by developing an approach in which I can bring an enzymatic activity that removes those PTMs into close proximity to a substrate in trans.
As my first aim, I will generate a system with which to co-localize a phosphatase or deubiquitinase (DUB) activity and potential substrates. To accomplish this, I will create fusions between phosphatases/DUBs and an IgG constant region, and this fusion will be expressed in a strain where the potential substrate is fused to the TAP tag, which associates with the IgG constant region. I will validate this system using well characterized phosphorylated/ubiquitinated proteins.
As my second aim, I will transform plasmids that express these DUB/phosphatase fusions under an inducible promoter into a pre-existing library of yeast strains in which every gene is fused to the TAP tag. I will screen for slow growth or DNA damage sensitivity after induction of the DUB/phosphatase fusions.
As my third aim, I will determine the significance of the identified PTMs. In order to do this, modified sites for each candidate protein will be mapped and mutated.

The aim of this proposal is to better understand the role of the intestinal mucosal barrier in metabolic diseases as obesity and type-2 diabetes, which cause more than 3 million deaths per year. While the gut microbiota has been found to be a crucial factor for obesity and insulin resistance in mice and humans, the underlying disease-causing mechanisms are still unclear.
The human intestine is colonized by a complex microbial community. While its composition is specific for each individual, a core gut microbiota is shared between humans. However, the composition of gut microbiota is altered in individuals who are obese or diabetic.
The high density of the gut microbiota is a permanent challenge for the gut epithelium. As only one single, mucus-covered cell layer separates microbes from human tissue the mucosal barrier is essential in preventing microbial translocation. One epithelial defense mechanism is the secretion of antimicrobial peptides (AMPs). These endogenous peptide antibiotics have broad-spectrum antimicrobial activity and can modulate the composition of gut microbiota.
In this proposal I will focus on the intestinal barrier between host and microbiota. I will test if and how the shifted microbial composition in metabolic diseases is associated with alterations in mucosal AMP production and function. Furthermore, I will analyze mucosa-associated microbiota in metabolic disease by using mouse models and samples from human patients. Also, I will compare the peptide repertoire in the mucus of different mouse models and human diabetes patients to identify alterations in metabolic disease.
The planned investigations will help to better understand the relationship between the mucosal host barrier and metabolic diseases.

Microbes are present within and significantly affect the dynamics of all natural habitats, playing vital roles in a plethora of medical, ecological and industrial contexts. Consequently, a better understanding of the fundamental functions of these important organisms is crucial. Here, I focus on one of the most fundamental microbial functions – the uptake of nutrients – and ask how this is affected by a universal environmental parameter – the flow of the fluid that microbes live in. It is predicted that fluid flow relative to a microbe could enhance the nutrient uptake. Yet, this ubiquitous, fundamental process has, to date, eluded experimental investigation and our current knowledge rests entirely on theoretical predictions. The archetypal problem in this context – uptake of a single compound by a single microbial cell when the relative motion of the fluid and the cell is laminar and unidirectional – remains unsolved, despite its deceiving simplicity, and will represent the core goal of this project. I propose to develop the first experimental framework and perform seminal experiments to understand flow-enhanced uptake quantitatively, based on a combination of cutting edge approaches including microfluidics, optical tweezers, advanced microscopy, Raman spectroscopy, and particle tracking. This will provide a quantitative experimental approach to this unsolved problem, ground theoretical predictions in observational data, reveal any processes not accounted for in current theoretical frameworks, and help resolve existing inconsistencies in the theory. Further afield, the approach I will develop will trigger solutions for investigating the full range of realistic, more complex scenarios of flow, nutrients, and population structure.

The main cause of mortality in human cancers is the formation of metastases, in which tumor cells exhibit resistance to established anti-cancer therapies. My postdoctoral lab (PI: CS Mitsiades) has documented that this observation may be due, at least in part, to the fact that nonmalignant cells in metastatic sites can function as "accessories" to the tumor cells, conferring to them increased threshold to treatment-induced cell death. My research will characterize the molecular basis of this crosstalk between cells of colorectal cancer (CRC), one of the leading causes of cancer-related deaths), and hepatocytes, as key "accessory" cells in the liver, a major site of CRC metastases. I will also examine how the genetic features of CRC can influence the consequences of their interaction with hepatocytes; and, conversely, how the latter cells alter the sub-clonal architecture of the CRC cell population. My studies will involve 2- and 3-dimensional in vitro co-cultures of CRC cells and hepatocytes; and "orthometastatic" in vivo models to simulate the CRC liver metastases. In addition, I will apply a novel combined marking of individual cells with sequence-based "barcoding" and "Brainbow"-based multicolor labeling, to monitor how hepatocytes alter the clonal architecture of CRC cell populations, and sort out individual clones with differential responsiveness to hepatocytes. Using these models and my prior work on molecular pathways associated with CRC, I will determine which molecular pathways are essential for the hepatocyte-induced increased in the threshold of metastatic CRC for cell death. This study will provide new insights into the biology of metastatic cancers.

Cell differentiation is a fundamental biological process that underlies all multicellular organisms. It is clear that mammalian differentiation requires DNA methylation; However, we still don't know why certain sequences are methylated and others are not. In addition, recent studies propose that not just methylation status, but also methylation variability is relevant in differentiation, because erroneous methylation variability can lead to disease. The canonical role of DNA methylation in parental imprinting and X-inactiviation is to maintain already silenced DNA, not to establish silencing. DNA methylation may have a similar role in development, namely, to maintain already silenced DNA. What, then, is the driving cellular signal that determines epigenetic status? One possibility is transcription factors, which are known to drive differentiation. To study this interaction, I propose to query simultaneously DNA methylation status, transcription factor binding, and gene expression at many time points through a cellular differentiation process. This unprecedented time resolution and combination of experiments will enable me to identify causal relationships between factor binding and DNA methylation. Furthermore, I will measure not only mean DNA methylation, but also its variability. To aid in this task, I will develop a computational model to capture important sources of variation and apply this to the experimental data. Using the model of granulocyte differentiation, combining these experiments will yield new insights into causal interactions between TFs and either mean DNA methylation status or variability of DNA methylation.

Reprogramming of somatic cells to pluripotency using ectopic expression of defined factors is both a long and inefficient process, which consists of many unknown epigenetic changes. In contrast, somatic cell nuclear transfer (SCNT) is rapid and more efficient process, which results in pluripotent stem cells that are indistinguishable from their naturally occurring embryonic stem cells counterparts. In my postdoctoral work, I intend to comprehensively study the differences between the two processes, consequently aiming at identifying factors that may, upon ectopic expression, reprogram somatic cells to pluripotency in a more rapid and deterministic fashion. I therefore intend to conduct single cell analyses in untreated mouse oocytes and SCNT embryos to identify possible facilitators of SCNT-mediated cellular reprogramming. Next, to comprehensively compare between gene activation during iPS formation and SCNT, I intend to generate mouse embryonic fibroblasts (MEFs) carrying cassettes of predictive markers for both processes, thus enabling single-cell population tracing and cell-type specific gene-expression analyses. My ultimate goal will be to test whether the newly identified factors may enhance cellular reprogramming upon ectopic expression in somatic cells. I thus intend to generate secondary reprogrammable MEFs, harboring different combinations of factors, and assess their ability to reprogram somatic cells to pluripotency.

A fundamental question in biology is why different individuals show different behaviors. Behavioral variability among individuals may arise from genetic heterogeneity, the effects of different external environments, or experience. However, phenotypic variation, including in behavior, is widely observed even between genetically identical individuals that have been raised in the same environment. This variability suggests that epigenetic or other non-genetic differences between individuals may induce different behavioral states.
My overarching goal is to study the non-genetic differences underlying behavioral variability among individuals, and to investigate how different environments, in particular stressful conditions, affect this inter-individual variability.
C. elegans is an ideal model for studying these issues due to its relatively simple nervous system, the substantial knowledge about its behavior, and the available genetic and molecular tools, as well as high-throughput methods for quantitative, simultaneous monitoring of behavior in many individual worms.
As my first specific aim, I will quantitatively characterize inter-individual behavioral variability in isogenic C. elegans populations exposed to various environments. In my second aim, I will use these behavioral paradigms to decipher neuronal circuits involved in generating variability. As my third aim, I will identify potential molecular components underlying these neuronal functions, with an initial focus on epigenetic mechanisms.
Identifying the non-genetic sources of behavioral variability among individuals may have a major impact for advancing basic research on individuality, epigenetics, and behavior.

Viruses are completely reliant on the host cell machinery and have evolved many mechanisms to hijack it for their propagation. At the same time our cells have developed defense mechanisms to cope with infections. A key way by which viruses influence host physiology is through interaction with the cellular translation machinery. Not only viruses must co-opt this machinery to translate their own mRNAs, they also have to hinder host defenses that are aimed to inactivate the translation machinery. My research aims to elucidate the complex cellular changes that occur during these virus-cell encounters. I plan to concentrate on two major pathogens; a large DNA virus, Human cytomegalovirus (HCMV), and a small RNA virus, Influenza A. Although these viruses are very different, for both of these viruses the global effect of infection on the translation of host genes is still poorly defined. I therefore propose to tackle this important issue by employing a novel deep-sequencing technique termed ribosome profiling, which allows a comprehensive view of translation events. The robustness, scale and accuracy of this analysis will enable us to determine the identity and the relative levels of translation of each host protein during the course of infection and would therefore provide valuable information regarding translational control strategies used by these viruses to reprogram the cellular host translation machinery. I then plan to develop and apply more directed experiment to confirm translational changes and to elucidate their importance to viral replication. These experiments should provide a unique opportunity to understand the underlying changes that occur during infection.

Pr. A. Ting and coworkers have recently demonstrated the conceptual and technical feasability of spatially resolved proteomic mapping in live cells using an engineered Ascorbic PEroXidase (APEX). Such a methodology is meant to circumvent the limitations of classic MS spectrometry by extracting the desired proteomic information at the level of the living cell, with its structures preserved. This tagging methodology takes not more than 1 minute, paving the way potentially for spatially AND temporally resolved biological studies. In addition, certain biological compartment of high relevance like the mitochondrial intermembrane space (IMS) or mitochondria-ER junctions cannot be purified, consequently their proteomes are largely unknown. In this postdoctoral work we aim at establishing the proteomic maps of these two highly interesting cellular localizations which will certainly prove themselves illuminating in unraveling the biological processes in which they are involved (apoptosis, regulation of mitochondrial division, calcium and phospholipid exchange, protein import into mitochondria etc...). Another aim of this postdoctoral work is to develop ultra spatially resolved proteomic mapping methodologies to enable the mapping of macromolecular protein complexes found in very specific sub cellular localizations. Such endeavour requires developing bio-chemical approaches to restrict the tagging radius of the reactive species generated by APEX or to explore light based methodologies that serve the same purpose.

My goal is to elucidate the molecular mechanism of major histocompatibility class II (MHCII) trafficking to the immunological synapse (IS) between T-cells and dendritic cells (DCs). There is substantial evidence that in DCs peptide-bound MHCII is selectively trafficked from intracellular MHCII containing compartments (MIIC) to the IS where it interacts with the T-cell receptor for activation of T-cells. Nevertheless, the IS has been almost exclusively studied from the T-cell side and not from the DC and the molecular pathways that govern delivery of MHCII at the plasma membrane of DCs are still completely unknown. Specifically, the SNARE proteins, which are key trafficking proteins that catalyze intracellular membrane fusion, need to be identified. Based on my extensive experience in membrane trafficking in neuroendocrine cells, I propose to develop a new and innovative approach to identify the SNAREs responsible for MHCII trafficking to the IS. I will first position an IS between an antigen-specific T-cell and a DC parallel to the optical plane of a microscope with a micromanipulator, thereby enabling a high spatial resolution. I will then employ a combination of Förster resonance energy transfer (FRET)-based microscopy and functional assays to unequivocally identify the SNAREs that catalyze release of MIIC at the plasma membrane of DCs. Understanding how MHCII is selectively presented to the T-cell is essential for understanding the high sensitivity of T-cell activation by DCs. My study will also result in the development of new tools and insights that enable unraveling the release mechanisms of other surface exposed molecules and secreted cytokines from immune cells.

During multicellular development, cell differentiation is often controlled by morphogen gradients. Here, cells measure the local morphogen concentration to direct their cell fate. However, such gradients form by diffusion of a finite number of morphogen molecules and hence can exhibit strong stochastic fluctuations. How such ‘noisy’ morphogen gradients can induce cell fate patterns with the extreme precision observed during development is poorly understood. To address this question, we propose to study pattern formation by morphogen gradients in the simple nematode worm C. elegans, using a quantitative approach that relies on two innovative techniques: first, we will use a new microscopy technique, Single Plane Illumination Microscopy, to quantify with high precision the dynamics of both the morphogen gradient and the cellular response it generates. Then, using an information theory approach we will establish whether the cell’s response improves the information provided by the gradient and if so, what mechanisms are used. We will study a system where morphogen gradients provide the directional cue for asymmetric cell divisions, an important mechanism for generating spatial cell fate diversity. Here, cells measure the change in morphogen concentration across the cell surface to establish from what direction the gradient emanates. Such measurements are more challenging than measuring the local morphogen level alone and it is an open question how direction is sensed robustly from noisy morphogen gradients. Our research will likely elucidate general mechanisms underlying this robustness that are important for related processes such as cell polarity and eukaryotic chemotaxis.

Neural computations underlying critical brain functions, such as cognition and perception, rely on fundamental interactions between neural excitation and inhibition. However, neural function depends critically on behavioral state, such as sleep, wakefulness, arousal, and focused attention. GABAergic, inhibitory interneurons are subdivided into distinct classes with different synaptic targets, physiology, and molecular markers. Interneuron diversity may provide a mechanism for the functional flexibility of neuronal circuits, allowing dynamic sensory processing depending on computational goals (e.g. fine discrimination vs. detection of any input) and behavioral state. However, there is little data on the contribution of specific GABAergic interneurons in sensory cortex during distinct behavioral states, because until recently it was not possible to rigorously identify or manipulate these cells in the intact brain. We will perform extra- and intracellular recordings in primary visual cortex of awake mice expressing light-sensitive channels in specific interneuron classes, and will record physiological parameters to precisely quantify behavioral state with multivariate analysis. Using optogenetic tools, we will identify cells from genetically distinct classes and control their activity with millisecond precision. We will test how the temporal recruitment pattern of distinct interneuron classes differs across behavioral states (Aim 1) and controls synaptic integration in excitatory cells on a fine time-scale (Aim 2). Further, we aim to probe their causal roles in controlling dynamic transitions in cortical network activity (Aim 3).

Coordination of rhythmic behavior at the level of cell communities manifests the fundamental processing capability of eukaryotic organisms such as plants and animals. Biological rhythms often rely on genetic oscillators or clocks that operate in synchrony across time and space. One of the major challenges in modern biology is to quantitatively understand the dynamics of oscillatory behavior in plants and its role in the self-organization of multicellular patterning processes. I will address this grand challenge with the combination of computer model simulations, synthetic biology and the customized plant-tailored microfluidic platform. Initially, computer models of minimal genetic oscillatory circuits will be developed based on the integration of state-of-the-art molecular and structural information on hormonal signaling in plants. Predictions from these computer models will aid in the in vitro design of intracellular synthetic oscillator in the plant cell with the measurable hormone-responsive fluorescent output. Furthermore, the oscillator dynamics at the level of each individual plant cell as well as cell populations will be characterized with live cell imaging under variable environmental conditions in the microfluidics device. This pioneering framework for probing and quantifying coupled synthetic oscillators in plants will provide novel insights into the dynamics of hormone-mediated cellular rhythms and their role in shaping plants.

The microbiotic flora of the human gut contains a large number of Enterobacteriaceae including E. coli. While harmless in this environment, these bacteria may cause severe infections if they escape the gut. Recently, and across the globe, there have been an increasing number of outbreaks of carbapenem-resistant Enterobacteriaceae (CRE), which show resistance to the most potent of antibiotics, the carbapenems. Because resistance (and virulence) genes in bacterial cells are commonly carried on plasmids, it is vital that we understand how plasmids are segregated, partitioned and maintained over generations in bacterial cells. In this research, we propose to perform live cell imaging of the structures and inter-molecular dynamics involved in plasmid segregation using recently developed super-resolution PALM microscopy. We will focus on the type II (ParMRC) system for cells containing multiple pairs of R1 plasmids. We will investigate quantitatively the ParM spindles during plasmid segregation, observe the growth of filaments in bacteria, determine the number of filaments spawned from a single plasmid and count the number of ParR proteins on the same plasmid. In addition, we will track the dynamics of R1 plasmids within cells that contain more than a simple pair of plasmids. We will address whether the ParMRC system acts purely through pairwise interactions or if there are higher order effects. We will also probe the motion of plasmid bundles in bacterial cells. Our experiments will provides us a new (previously-inaccessible) level of quantitative information and a mechanistic understanding of the ParMRC segregation process.

The actin cortex is a contractile cytoskeletal network underneath the plasma membrane. Cortex contractility drives cell shape deformations during many cellular processes, including cell migration, cell rounding and cell division. When a cell divides, it forms an equatorial actomyosin ring that ingresses to separate the two prospective daughters, or poles, of the cell. The cortex of both poles is contractile, and a contractile imbalance between poles can result in cytoplasmic flows from one pole into the other, destabilising the position of the cleavage furrow and potentially leading to division failure. To ensure a stable cytokinetic cell shape, polar contractile forces must be mechanically balanced by opposing forces that resist cortex contractions. Here, we hypothesise that resistance of the plasma membrane to cortex contractions stabilises cell shape. To test this, we aim to: (1) characterise membrane dynamics during cortex contractions in dividing cells, combining cell biology and quantitative imaging; (2) measure mechanical membrane properties, using a combination of biophysical methods; (3) develop a theoretical model of the mechanics arising from membrane dynamics during cortex contractions, and verify this model using experimental perturbations. We will then test the model in other contexts where cortex contractions play a role, such as during cell rounding and cell migration. Because cell shape deformations in general are driven by cortex contractions, resistance of the membrane to cortex contractions could constitute a universal mechanism to stabilise cell shape in many different cell biological contexts.

53BP1 is a key protein implicated in immunoglobulin class-switching and DSB repair pathway choice. 53BP1 accumulation at DSB sites occurs rapidly after DSB formation, helping to promote non-homologous end joining (NHEJ) and antagonise DNA end resection. After DSB formation histones are specifically phosphorylated and ubiquitylated, which coincides with the recruitment of 53BP1. 53BP1 has been proposed to bind to modified chromatin directly, but the molecular details of this interaction are still unclear.

I plan to research how 53BP1 binds specifically to modified chromatin, investigating how histone post-translational marks are recognised directly in the context of a full nucleosome core particle (NCP). Recent work from my host laboratory has elucidated that 53BP1 reads a bivalent histone mark: H4 Lys20 di-methylation and H2A Lys15 ubiquitylation. I plan to crystallise the nucleosome-binding portion of 53BP1 in complex with an appropriately modified NCP, in order to discover the atomic details of this interaction. If successful this will be the first crystal structure of a protein bound to a post-translationally modified NCP. Furthermore, it will illuminate the remarkable selectivity of 53BP1 for the H2A Lys15 ubiquitin mark.

I plan to probe the functional consequence of 53BP1 binding to chromatin: is it just acting as a recruitment platform, or does 53BP1 also have a direct role in ordering chromatin? Using methods developed in the Durocher laboratory these questions can be answered directly through X-ray crystallography, biophysics and biochemistry.

One of the most fundamental functions of the brain is to learn and generate movements. Most neuronal activity is, in fact, related to motor output and the brain has a remarkable ability to acquire and improve motor skills. Despite the obvious relevance for shaping our own behavioral repertoire, the implementation of the learning process in the neuronal circuitry remains very much an open question. I suggest a tutor-student relationship of two important brain areas, with the motor cortex (MC) instructing the basal ganglia (BG), as a fundamental organizing mechanism in motor learning. Evidence from the host laboratory shows that while the execution of certain complex and precise motor sequences can be MC independent, their acquisition depends on both the BG and the MC. Together with preliminary data indicating that MC lesions lead to a loss of the suppression of disadvantageous actions, this suggests that MC tutors the BG during motor learning. To demonstrate this tutor-student relationship and to identify its means, I will employ state-of-the-art techniques. I will combine the host lab’s sophisticated motor skill learning paradigms with electrophysiological recordings and manipulations of neuronal activity. Key for this is a novel viral approach, developed during my PhD, for the specific targeting of cortico-striatal neurons, which provide MC’s input to the BG. Based on this approach, specific ablations of these projections, correlations of their activity with behavior and temporally precise optogenetic manipulations will identify MC as a tutor and characterize the nature of the tutoring signals. The proposed work will not only identify mechanisms underlying the interplay of defined neuronal circuits, but also reveal fundamental principles of motor skill learning and brain function.

The faithful transmission of epigenetic information is an essential process during development and cell differentiation. The mechanisms by which histone modifications and chromatin-associated complexes are re-deposited after DNA duplication, however, are not well understood. In the fission yeast Schizosaccharomyces pombe, repressive heterochromatic structures are assembled through the methylation of histone H3 at lysine 9 (H3K9) and the subsequent binding of HP1 proteins. At the centromeres, small non-coding RNAs guide the formation of heterochromatin and mediate silencing of centromeric transcripts. While the contributions of each of these components to the establishment of heterochromatin have been studied extensively, how these states are inherited remains an open question. Here we propose to analyse this process by using Chromatin in vivo Assay (CiA), a recently developed small molecule-based method to initiate heterochromatin formation in a reversible way. The key property of this approach is that the maintenance of silencing can be analysed in the absence of the initial trigger. This will allow us to perform a genetic screen to identify the components required for this process. Our goal is to identify determinants for epigenetic inheritance in this system and to use biochemistry to uncover the mechanism by which they function. We aim to be able to confront existing models on how epigenetic states are maintained.

Axon degeneration is a tightly controlled process of axon self-destruction that plays both a physiological role during development and pathophysiological role during neurodegeneration. Following a physical insult, such as a nerve transection or crush, the axon segment distal to the injury site undergoes rapid degeneration, a process that is called Wallerian degeneration. In addition to its role in axon degeneration distal to an injury site, Wallerian and Wallerian-like degeneration are also prominent features of numerous neurodegenerative diseases. However, the molecular mechanisms underlying Wallerian degeneration are poorly understood.
To identify molecules that are critical for Wallerian degeneration, I will perform a genome-wide RNAi screen. The physiological significance of the most promising candidates will be evaluated using cultured sensory neurons and genetically modified mice. I will further characterize functions of the candidates with biochemical and cell biological methods and define where the candidates reside in the Wallerian degeneration pathway. Previously, Prof. Tessier-Lavigne team found that the genetic deletion of SARM1 protects against Wallerian degeneration. However, the molecular function of SARM1 remains elusive. As such, SARM1 will also be analyzed using these methods. Finally, I will examine the function of SARM1 and the other Wallerian degeneration molecules in animal models of neuropathy. I expect that these studies will advance our understanding of the mechanisms regulating axon degeneration and will also provide insight into how to therapeutically target this pathway for the treatment of neurodegenerative diseases.

Recent microbiome studies open new opportunities to treat flora-related diseases. Here I propose to use evolutionary dynamics principles to reshape flora from a disease to a healthy state by altering expression patterns of the microbiome. Notably, this alteration of the flora can occur both at the level of species composition and at the level of genetic changes in each of these species. As a model system for microbiota reshaping I will use the lactose-intolerance (LI) condition in mice. I designed an unfavorable regime that would select against the bacteria that cause LI by fermenting lactose in the gut. This regime disfavors the bacteria that express lactose genes by putting a cost on activation of the bacterial lac-operon. By feeding mice a lactose analog that cannot be used to produce cellular energy, the cost of expressing lactose genes is expected to reduce lactose fermentation in the gut and thus to relieve LI symptoms. In parallel, I plan to apply similar evolutionary regimes on the bacterial lac-operon, yet in a more controlled system of lab-evolution experiments in E.coli cultures. These lab-evolution experiments will elucidate how promoter sequences evolve towards altered gene expression. Specifically, these experiments will follow, in real-time, the evolutionary process of de-novo promoter formation, from a random sequence to an active promoter and conversely from an active to an inactive promoter. To this end, cells with a randomized lac-promoter will be evolved in the presence of lactose, and in the opposite direction, cells with a native lac-promoter will be evolved in the presence of a lactose analog. Together, the proposed study will shed light on the evolution of transcription regulation, and on ways to reshape our flora by altering the microbiome transcriptome.