Transport and degradation of marine particulate organic carbon (POC) has been widely studied in oceanography, yet most research has focused at the bulk scale. While it is known that microbes are key players in controlling the fate of POC, we lack a mechanistic understanding of the biochemical and physical interactions by which microbes drive POC degradation . In this project I will explore these microscale processes by focusing on the microbial dynamics around particles. To achieve this, I will develop a 'milli-fluidic' experimental system to permit the visualization of single POC aggregates in a closed-loop chamber. I will observe aggregate decomposition under flow as a function of time and treatment, and I will quantitatively characterize microscale bacterial attachment, bacterial growth, biofilm formation and enzyme function, as well as the coupling among these processes. These unique experimental data will inform a new mathematical model of POC degradation kinetics to predict the conditions and depths at which POC is remineralized in the ocean. By unraveling the dynamic microscale interplay among biology, biochemistry, and physics in the degradation of marine particles, this work will bring about a more fundamental, mechanism-based understanding of particle flux and the carbon cycle in the ocean.

Asthma is a clinically heterogeneous disorder caused by genetic and environmental influences that remain poorly understood. Promoter variations that induce over-expression of the Orosomucoid-like 3 gene (ORMDL3) in humans are the strongest genetic associations with severe childhood asthma. Orm-family proteins are a component of an essential enzyme complex, the SPOTS complex, for Serine Palmitoyl-transferase, Orm, Tsc3, and Sac1 complex, which catalyzes the rate-limiting step for de novo sphingolipid synthesis. Orm-family proteins act as inhibitors of Serine Palmitoyl-Transferase (SPT) and play an integral role in sphingolipid homeostasis. Elevated expression of ORMDL3 inhibits the synthesis of sphingolipids that appear to be protective against asthma and other inflammatory diseases. These observations raise the exciting possibility that sphingolipid synthesis may be a point of intervention that is orthogonal to existing therapies. However, the molecular architectures of the SPOTS complex and the Orm proteins, as well as the details of their functional roles in sphingolipid metabolism, are entirely unknown. I propose to determine the structure of the SPOTS complex in different functional conformations using cryo-electron microscopy (cryo-EM) and to characterize the mechanisms governing sphingolipid homeostasis with biochemical methods. These results will improve our understanding of sphingolipid homeostasis and the pathogenesis of asthma and other inflammatory diseases.

Pre-implantation development in mammals involves complex processes at the level of genomic organization, DNA methylation and transcriptional activity, leading to embryonic genome activation. How different epigenetic markers interact in single mouse blastomeres at early stages of development to influence the transcription of genes, and to what extent these processes vary from cell to cell and between embryos, is not fully understood. Although, in recent years a number of methods that enable single-cell omics measurement have been described, their integration to measure more than one entity in a single cell has remained challenging. Here, I propose a novel strategy for the integrated measurement of different epigenetic markers in combination with the measurement of nascent transcripts in single cells. The strategy relies on the use of a combination of specific restriction enzymes, for the measurement of 5-methylcytosine (MspJI), 5-hydroxymethylcytosine (AbaSI) and accessible chromatin (MseI and/or NlaIII), as well as 2’-fluoro GTP analogues in combination with specific RNases for the measurement of nascent transcripts. The application of these technologies to pre-implantation mouse embryos will shed light into the relationships of different epigenetic markers and their influence on transcription at the single cell level, and how these processes result in cell differentiation during early development. I further propose to use new deep-learning libraries, such as TensorFlow or Caffe, to quantitatively model and gain understanding of these dynamic processes in single blastomere cells.

The poor regenerative capacity of central nervous system (CNS) neurons leads to devastating and irreversible outcomes after injury or disease. The CNS is an immune privileged site, which maintains specialized crosstalk with the immune system. While the immune response to acute injury in the CNS is very similar to the wound healing process in other parts of the body, this response is oftentimes insufficient to achieve significant repair and restoration of neuronal function. One of the key challenges in analyzing this response is the diversity of the types of cells that participate in the process, which change with time and some of which may be unknown. Cutting-edge advances in massively parallel single-cell genomics, many pioneered by the Regev lab, will now enable me to analyze the entire injured tissue, at high, single-cell resolution, that takes on its complexity, heterogeneity, and temporal variation. Using this approach and comparing low- and high-regeneration states could help identify junctions where the immune response is insufficient to support regeneration, new cell types involved in the process, and targets for therapeutic intervention. Emerging cellular and molecular candidates will be perturbed experimentally in vitro and in vivo, using CRISPR/Cas9 technology and cell augmentation and depletion experiments, to validate their significance for neuronal regeneration. Human samples will be incorporated to establish validity in the clinical setting. The findings could be relevant for different types of CNS trauma, inflicted though mechanical or biochemical injury, autoimmune, neurodegenerative or vascular disease, at the brain, eye and spinal cord.

For individual genes, the number of transcripts per cell typically increases with cell size, yet how this is achieved is unknown. Recent image-based analysis suggests that both cell volume and surface area contribute to this regulation, with distinct classes of genes showing tighter correlations with either one of these two features. Here, I aim to go beyond these correlations – dissecting the mechanism by which cell geometry shapes the transcriptome. First, using quantitative RNA-sequencing and RNA metabolic labelling in cells with a range of defined shapes and sizes, I will define genome-wide classes of transcripts that scale with cell volume and surface area, and analyze the extent to which regulation of transcription and RNA stability contribute to this scaling. High-resolution mapping of changes in chromatin accessibility and modification state in these constrained cells will also be used to reveal the basis of transcriptional control and to suggest DNA-binding factors involved. Second, high-throughput image-based quantification of specific transcripts in single cells will be combined with genome-wide genetic screening. For each single-gene knockdown, this will generate a multivariate dataset containing the absolute abundance of a panel of selected transcripts in addition to hundreds of single-cell and population-context features. I will specifically identify and pursue genetic perturbations that disrupt the scaling of transcript abundance with either cell volume or area. Following these unbiased approaches with hypothesis-driven experiments and mathematical modeling will provide insight into how single cells precisely adapt genome expression to cell size and shape.

Sleep takes approximately one third of our lives yet its functions remain elusive. Extensive efforts have been made and hypotheses raised to link sleep with synaptic plasticity and memory consolidation. The sleep/wake pattern and sleep architecture evolve as development proceeds, and so does cortical connectivity, as featured by spinogenesis in early postnatal life and spine pruning during later adolescence. Several subcortical neuromodulators, including hypocretin, norepinephrine and dopamine, play critical roles in promoting arousal and thus control the sleep/wake cycle. We hypothesize that these arousal circuits help to shape the synaptic connections in cortex during development and the disruption of coherent sleep/wake cycle at certain developmental stages may cause defects in brain functions in adulthood. Mechanistically, synapses “tagged” by the arousal inputs during the previous wake phase may be selectively strengthened while other “untagged” synapses weakened in the following sleep. We will combine a series of state-of-art techniques including optogenetic and/or chemogenetic manipulations, viral tracing, two-photon live imaging as well as fiber photometry to interrogate these hypotheses, which will broaden and deepen our understanding towards sleep and brain plasticity in a developmental point of view and provide potential clinical insights on sleep and arousal disorders.

The proposed project will investigate the origin of animal cell contractility through both comparative and experimental evolution approaches in the choanoflagellate Salpingoeca rosetta. The project will address three scales of biological organization: (1) Gene expression. The differential transcriptomic profile of contractile and non-contractile S. rosetta cell types will be determined by single-cell RNAseq. In parallel, I will investigate the function in S. rosetta of the three key myogenic transcription factors of animals (Mef2, SRF and Myocardin) by ChIP-seq and CRISPR/Cas9-based genome editing, and I will determine their interaction partners by pulldown/mass spectrometry. (2) Cellular phenotype. Choanoflagellates show an intense contractile activity during settlement. I will test whether this contractility is controlled by membrane depolarization and calcium influx by live imaging of choanoflagellates expressing calcium/voltage sensors, pharmacological assays, and microelectrode voltage measurements. (3) Multicellular phenotype. S. rosetta develops into spherical colonies of up to 50 cells. In an experimental evolution approach, I will assess whether simple (actomyosin-mediated) departures from this spherical shape can be brought about by consistently applying, over hundreds of generations, selection pressures thought to have favored the early evolution of animal morphogenesis: selection for larger colonies or for benthic feeding from bacterial biofilms.

Due to its high prevalence among birds and high human fatality rate, avian influenza represents a serious and continuing pandemic threat, in particular via mutations that facilitate human infection, resulting in pathogenic strains. Mutations associated with human infection are concentrated in the PB2 subunit of influenza polymerase. This subunit is imported into the nucleus, where viral replication and transcription occurs, via a selective interaction with importin-alpha. Once in the nucleus, interaction of PB2 with protein ANP32A has been proposed to play an essential species-specific regulatory role.

The behavior of PB2 in solution reveals a high level of conformational flexibility that is essential to function. In addition intrinsically disordered domains of both ANP32A and importin-alpha are thought to play important roles in the interaction with PB2. This uncommonly high level of disorder presents particular challenges for structural studies, requiring development of state-of-the-art NMR-based technology.

My aim is to investigate the molecular basis of avian influenza adaptation to nuclear import and replication in human host. I will characterize structurally and dynamically key interactions between human-adapted avian influenza polymerase and human host proteins to contribute to a deeper understanding of viral replication, providing the basic information to facilitate design of innovative drugs.

The intestinal epithelium has the complex task of transporting water, ions and nutrients, while providing an efficient barrier to microbes and other potentially harmful luminal constituents. This delicate balance is notably altered in chronic inflammatory conditions of the gut, such as Crohn’s disease.
Luminal nutrients such as glucose have long been recognized as regulators of epithelial permeability. Recently, a role of the enteric nervous system in the control of barrier function has been proposed. Indeed, alterations to either enteric neurons or enteric glial cells leads to dysregulation of epithelial permeability. The structural basis for an interaction between the epithelium, glial cells and nerves has recently been provided, but neither the detailed nature nor the integration of this communication have been examined.
My project aims to investigate how nutrient-sensitive epithelial cells called enteroendocrine cells communicate with enteric glia and nerves in the mucosa to regulate epithelial permeability.
My approach will be to evaluate the signal integration, from the luminal stimulus to the epithelial response. We will use a combination of live cell imaging techniques and fluorescent reporters to visualize the propagation of the signals in living tissues. We will selectively block or release enteroendocrine cell mediators, inhibit enteric glial cells and/or block or stimulate enteric nerves to dissect the various components of this crosstalk.
My project will lead to a comprehensive understanding of signal integration in the intestine between the epithelium and the enteric nervous system, extending the concept of neural control of intestinal barrier function.

Extracellular interactome networks consisting of secreted and cell surface proteins coordinate complex biological processes, including embryonic development, homeostatic regulation and cancer progression. Identification of extracellular protein-protein interactions (PPIs) is central to biomedical science and therapeutics. For example, multiple cell surface and secreted proteins are targets for FDA-approved drugs. However, a description of extracellular PPIs for many of these proteins remains either unknown or incompletely known. We are particularly interested in Wnt signaling that controls the central nervous system (CNS) vascular development and blood-brain-barrier (BBB) formation and maintenance. The CNS vascular dysfunction including BBB breakdown is associated with brain trauma, stroke, multiple sclerosis and neurodegenerative disorders. So far information of extracellular PPIs of Wnt signaling for the development and regulation of CNS vasculature is limited. Here, we aim to utilize and improve an innovative technology for systematic and unbiased methods to identify novel extracellular PPIs related to Wnt signaling for the CNS vascularization and the BBB integrity. We expect to validate novel candidate PPIs using biochemical and biophysical assays. Additional experiments will assess these PPIs and their significance for CNS vascularization using cell culture systems and mouse genetic models. The information obtained from these studies will expand our knowledge of CNS vascular biology and bring a new approach to the study of extracellular interactomes.

The Hsp90 molecular chaperone helps fold and activate 10% of the proteome and 60% of the human kinome. Together with the kinase specific co-chaperone Cdc37 it facilitates the folding and regulation of its client-kinases. This is accompanied by transitions between open and closed Hsp90 conformations triggered by ATP hydrolysis. Recently the closed Hsp90ß:Cdc37:Cdk4 complex was solved by the Agard lab using cryoEM. It reveals that Hsp90ß inactivates Cdk4, through clamping an unfolded ß-sheet and thereby separating the two kinase lobes. I aim to elucidate the biochemical and structural basis for kinase capture and refolding by the chaperone machinery. This might either occur directly by kinase binding to a very different “open” Hsp90ß:Cdc37 complex or involve unfolding by Hsp70 and subsequent transfer to Hsp90ß via a “transfer complex” of Hsp70, Hop, Hsp90ß, Cdc37 and the kinase. I will reconstitute the chaperone system in-vitro to identify key steps and necessary protein components. The kinase folding state will be monitored using ligand/inhibitor binding as well as FRET between the lobes. Using this system, I will also study the role of several co-chaperones including FKBP51, Aha1 and PP5. To allow structural characterization of the important dynamic intermediates by cryoEM, I will trap these states using a variety of strategies and determine their structures to atomic resolution by state-of-the-art cryoEM. In vivo relevance will be assessed by disrupting the underlying interactions in cancer cell lines. This will offer the future perspective to pharmacologically target the identified structural states relevant in many human cancers.

Cardiac hypertrophy from different causes represents a major culprit for premature sudden cardiac death in humans. The infrequently feeding Burmese python exhibits a dramatic change in metabolism after a large meal that requires organs to grow, including physiologic cardiac hypertrophy much as is seen in athletes after exercise. Most interestingly, such growth is reversible and cyclically occurs at every meal. This could be clinically important since regression is not seen in most pathological hypertrophy. With this project, we aim to define the mechanisms that allow the python heart to revert hypertrophy, in the attempt to provide novel insights for regression of cardiac growth in mammals. We hypothesize the presence of one or more factors promoting regression in the python plasma after the peak of digestion. We will test this hypothesis by treating hypertrophied neonatal rat myocytes with python plasma and evaluate cellular dimension as a marker for regression of growth. The sponsor’s laboratory has established this protocol. Once we have defined the trigger responsible for regression of hypertrophy, we will study the mechanisms employed by the python to reduce cardiac mass. We envision the involvement of complex signalling cascades leading to apoptosis and autophagy that are needed to reduce cardiac mass in a controlled manner. Moreover, we will extend our research on established mammalian pathological models of cardiac hypertrophy. We will attempt to activate the same signalling pathways in mammalian models in order to reduce cardiac mass. The results of this project are expected to unveil novel pathways that may lead to therapeutics for cardiovascular diseases.

Aminoacyl-tRNA synthetases (aaRS) are the major translators of the genetic code by assigning the correct amino acids to the corresponding tRNAs and are thus of indisputable importance for protein biosynthesis. Additionally, several aaRS moonlight as inducer of cell signaling pathways. Recently, aaRS splice variants have been identified, which display unique cytokine-like properties. These splice variants are often truncated in their N- or C-terminus, thereby creating new termini. Terminal amine isotopic labeling of substrates (TAILS) was developed to map protease cleavage patterns. In the project suggested here, I will utilize TAILS to enrich and identify splice variants of AARS, especially of Arginyl-tRNA synthetase (RARS). Surprisingly, RARS signaling has not been studied yet, despite experimental evidence that RARS is present in the nucleus, signaling elicited by a RARS-splice variant, and the regulatory role of arginine in the immune system and cancer. Establishment of a timeline for RARS splicing events upon stimulation will decipher the underlying biological function and elucidate possible signaling functions of RARS. Investigation of RARS-induced signaling, interaction partners, and cellular dynamics will corroborate these results. As RARS splice variants lack export sequences, paracrine signaling could be mediated by exosomes. Dynamic regulation of RARS variants upon stimulation may lead to the identification of upstream regulators initiating splicing. Together, I suggest a new method for precise and quantitative detection of splice variants on protein level with a direct biological application for an enzyme class of very fundamental importance.

Touch is one of the primary sensory percepts. Although most well-studied in vertebrates, it is critical to most organism including insects which rely on touch to navigate through the world around them. Moths use a long proboscis, a specialized mouthpart to actively acquire tactile information from flowers during feeding. They unfurl their proboscis and probe the flower surface in a manner reminiscent of how rats use their whiskers to sense their environment, an extensively studied active sensing system. However unlike whiskers that have mechanosensors only the base, the proboscis has sensors along its entire length. Moreover, the mechanical properties of the proboscis (diameter and stiffness) can also be actively controlled by the moth during feeding. Thus we see a novel sensory system that can be tuned on the fly.
Moths also acquire rapid mechanosensory information about the relative position of flower from the proboscis to stabilize flight and feed even in windy conditions. They combine this tactile information with the proprioceptive information from the neck and proboscis to form a representation of flower shape and position. Despite the central role of touch in the plant-pollinator interaction, little is known about the proboscis neuromechanics. Using a combination of behavioral, biomechanical and electrophysiological and data analytic studies on the nocturnal hawkmoth, Manduca sexta, I aim to understand how proboscis mechanics govern the interaction with flowers, how the proboscis is actuated to acquire relevant information, and in turn, how tactile information is encoded by sensors to enable this essential ecosystem service.

Small regulatory RNAs carry out diverse functions in both bacterial and eukaryotic cell physiology. Despite a known role for sRNAs in ribosome biogenesis in eukaryotes, the potential role for sRNAs in bacterial ribosome biogenesis and/or (pre-) ribosome degradation and storage under stress conditions is unknown. Based on their functional properties and versatilities I hypothesize that small RNAs are involved in bacterial ribosome biogenesis. Using a combination of bioinformatic, cell biological and biochemical techniques in vitro and in vivo, I aim to identify small RNAs with physiologically relevant functions in ribosome assembly either as assembly co-factors or as regulators of ribosome biogenesis in response to environmental changes. Moreover, I will investigate at which conditions the identified sRNAs regulate ribosome biogenesis, which interaction partners are involved, which mechanisms are employed and which consequences result from sRNA function. To determine the influence of sRNAs on folding and processing of rRNA, I will use an in-vitro co-transcriptional assembly system.
In parallel, I aim to identify artificial small RNAs that could be used as effectors of ribosome biogenesis or as probes to monitor co-transcriptional folding of ribosomal RNA and thus the kinetics of ribosome assembly in mechanistic detail. The work will provide physiological and mechanistic insight into the function and regulation by sRNAs in ribosome biogenesis as well as into co-transcriptional rRNA folding.

Viral entry into its host cell may results in either successful or abortive infection. Abortive infection of permissive cells, which are able to support viral replication, is rarely discussed but is quite abundant. What makes one cell resist to infection while its neighbor succumb to it is the subject of this research proposal. This is important as it could lead to the discovery of novel cellular anti-viral modalities and deepen our understanding of host-virus interactions.
My goal is to characterize the anti-viral response to viral infections in single cells, its variability among genetically identical cells and how this relates to infection outcome (successful or abortive).
To do so, I will learn and implement new technologies developed by the Tay lab. The success of the project relies on using a combination of live-cell imaging with in-lab fabricated microfluidics devices that allow the retrieval of single cells with known fates for further analyses, such as single-cell RNA-sequencing. By using viruses that express fluorescent markers I will be able to monitor infection in living cells in real-time, extract and molecularly profile cells in specific time points, for example in the process of aborting viral infection (when fluorescent proteins level stop raising or begin to drop). The results from this screening approach will be validated using standard cell-biology and virology techniques. I also plan to use this technology to investigate the response of the known anti-viral pathways.
I expect to reveal new molecular participants in the anti-viral response as well as to explore the variability in the response among single cells and how it affects infection outcome.

The segmented body pattern of vertebrates is established in the early development through the periodic formation of somites, the vertebrae precursors, from the presomitic mesoderm (PSM). The rhythmic formation of somites is under control of a molecular oscillator, the segmentation clock, which controls the cyclic transcription of a group of genes within the PSM.
Previous microarray analyses and candidate gene approaches identify many cyclic genes in the PSM, mostly genes associated to the Wnt, Notch and FGF-signaling pathways. Yet, a comprehensive, dynamic and quantitative analysis of the transcriptional dynamics and regulation of cyclic genes is still lacking, mostly due to the challenge to obtain samples with unequivocal time resolution. By using a microfluidic system that allows controlling the rhythm and, thus, synchronizing samples in relation to an external reference time, we are now able to obtain nearly unlimited PSM cells accurately staged. With a combination of high-throughput sequencing methods, we plan to identify unknown cyclic genes and regulatory elements (enhancers/promoter) with a role in somitogenesis. We aim to establish a temporal relationship between genes and their regulatory regions, and identify molecular mechanisms that control the rhythmic formation of somites. For example we may discover that cyclic expression relies on the periodic interplay of multiple enhancers. Or, that it is caused by cyclic epigenomic modification within regulatory elements. Additionally, by identifying sequence motifs within these elements we will be able to connect signaling pathways to oscillatory gene expression.

Although the basis of nucleotide binding and oligomerization domain (Nod) receptors signaling has been discussed for several years, the molecular details that underlie this process are still being uncovered. Moreover its presence on brain neurons hasn’t been described yet. The role of Nod2 receptor goes beyond simply sensing the presence of a potential pathogen and activating the immune system. Its activation has been linked to stem cells cytoprotection, analgesic effects, protection from cerebral ischemia-reperfusion damage and altered sleeping patterns. Moreover, mutations on this gene have been associated with bipolar disorder and schizophrenia in humans. The cytosolic Nod2 receptor can be activated by muramyl dipeptides a product of the degradation of peptidoglycan, a polymer present in almost all bacteria cell wall and increasing evidence demonstrate that endogenous ligands like damage-associated molecular patters, can also activate Nod receptors. Particularly, not much is known about the impact of Nod2 activation on behavior or the mechanisms and the specific effects on brain physiology. Understanding the role of Nod2 on brain neurons can lead to better understanding of many neurological disorders. Preliminary data from Dr. Lledo lab have shown, the presence of Nod2 receptors in neurons from the striatal and thalamic areas of the brain. They also observed that Nod2 knock-outs show less anxiety, more social aggressiveness and lack of discrimination of olfactory cues in social interactions and that Nod2 signaling can selectively disrupt paradoxical sleep. Therefore the purpose of this proposal is to assess the impact of Nod2 signaling on brain activity and behavior.

While cancer has been shown to rely on the accumulation of multiple mutations, conditions such as injury have been also shown to trigger cancer. Consistently, a recurrent mutation in Squamous Cell Carcinoma (SCC) in the Ras oncogene is not sufficient to trigger SCC unless either injury or a second mutation occurs. Yet, it remains elusive how wound-triggered behaviors lead to SCC. This proposal aims to identify wound-induced epithelial and niche cell behaviors that are required for cancer initiation. My post-doctoral lab has recently established an imaging approach to study how epithelial stem cell behaviors are coordinated to close a wound in live mice. I will build on this understanding and combine the study of wound repair in the context of a mouse model expressing activated-Ras mutation in epithelial stem cells. Specifically, I will determine what changes in cell behaviors, such as proliferation and migration, occur in the Ras model and I will use drug-based and genetic manipulation models to determine the functional impact of examined mutant behaviors towards SCC initiation. Furthermore, I will interrogate and manipulate the immune response, a central component of both the wound repair process and SCC, in the epithelial Ras mutant model to dissect the immune cell role during SCC insurgence. Our innovative live imaging approaches to track my populations of interest in combination with manipulative approaches will allow me to identify the relevant cells and behaviors towards cancer initiation. All together my project will provide new insights into how the physiological process of wound repair can be hijacked into a process of malignant transformation.

My goal is to understand how the cadherin-catenin complex, a force-responsive protein assembly that links neighboring cells, transduces extracellular forces into intracellular signaling events. Thus far, absolute force magnitude has been considered the main control parameter in mechanotransduction; but because mechanical cues in tissues and organs are subject to tight temporal control, it is likely that force timescales complement force magnitude to modulate junctional remodeling and downstream signal transduction. For example, during Drosophila embryogenesis regular contraction forces mediate the formation of the ventral furrow and gastrulation. In this project, I will characterize, for the first time to my knowledge, the response of intercellular adhesions to not only force amplitude, but also its temporal variation. Specifically, I will combine single-molecule force spectroscopy and single-molecule fluorescence microscopy techniques to directly quantify the kinetics of individual cadherin-catenins complexes in cells upon temporally controlled mechanical stimuli. Using this approach, I will determine how the cadherin-catenin complex integrates temporal cues into the mechanotransduction process. As a working model, I propose that cadherin clustering responds both the force magnitude and timescale, and that clustering is the key event that drives downstream signal transduction. My long-term goal as an independent investigator is to use the approaches outlined in this proposal to understand how macromolecular complexes produce, detect and respond to forces to accomplish biological processes such as mitosis, cell migration and mechanotransduction.