Maintenance, replication and expression of genetic information require intricate molecular mechanisms applied by a variety of molecular motors. Helicases are a large group of motors that catalyze the separation of nucleic acid strands, which is essential during replication, transcription, recombination and DNA repair. The physical basis of helicase activity is a fundamental yet poorly explored problem in biology, which we will address through a synergistic combination of complementary and unconventional rapid kinetic (temperature-jump/stopped-flow), force spectroscopic (single-molecule manipulation) and high-resolution AFM techniques. Based on their essential biological roles and wide-ranging mechanochemical activities, RecQ-family helicases (RFHs) are ideal models for studying helicase activity. RFHs are an important component in the maintenance of genomic integrity in organisms ranging from bacteria to humans: their mutations are characterized by severe chromosomal abnormalities. Five RFHs have been identified in humans: WRN, BLM, RECQ4, RECQ1 and RECQ5. Mutations in WRN, BLM and RECQ4 lead respectively to the heritable Werner’s, Bloom’s and Rothmund-Thomson syndromes, which are associated with chromosomal instability and cancer predisposition. In addition to unwinding duplex DNA, RFHs resolve multi-stranded DNA structures, e.g., displacement-loops and Holliday junctions, and interact with Topoisomerase III and single-stranded DNA binding protein to form an evolutionarily conserved complex with the ability to resolve double Holliday junctions (DHJs). The molecular details underlying these processes have yet to be elucidated and we lack a basic understanding of the physical basis of RFH activity, how this is activity is adapted to process complex DNA structures, and how these activities are modified and possibly regulated by specific binding partners. In Aim 1 we will determine the mechanochemical mechanism of DNA unwinding by human BLM and E. coli RecQ helicases. These studies will provide the basis for Aim 2 in which we will determine the changes in helicase activity required for processing of more complex structures. In Aim 3 we will determine how the interaction of RFHs with binding partners results in the complex activity of DHJ resolution. Completion of these aims will significantly advance our understanding of the mechanisms of RFHs and nucleic acid motors in general.

One fundamental question in biology is how the exact same genome gets partitioned into distinct gene expression profiles. This epigenetic mechanism is regulated by histone modifications and DNA methylation marks, which are established by enzymatic complexes. However, what attracts these complexes to distinct and specific sites and under different cellular contexts is not understood. It has long been suspected that non-coding RNA molecules provide site specificity to guide these complexes to their site of action.
Studies by our laboratory and others have discovered several examples of large non-coding RNA molecules that guide chromatin formation by physically associating with chromatin remodeling complexes (1-3). Thus, I hypothesize: (a) the physical association of long RNA molecules with epigenetic complexes may be a general mechanism for the establishment and maintenance of epigenetic states in development; (b) long RNAs play a key and global role that imparts specificity to chromatin modifying complexes.

In this proposal, I would like to address these issues by elucidating the mechanisms by which large non-coding RNAs are involved in regulating the epigenetic landscapes of the mammalian genome. I intend to focus on ES cells as model system with reliable readouts of differentiation to monitor how large non-coding RNAs influence cell fate decisions by undergoing different epigenetic conformations.

Vision plays as an essential role in controlling behaviors in a variety of animals. During this process, visual information is processed and transformed to specific motor outputs. Orienting behavior in zebrafish is an excellent model system in which to study such visuomotor transformation. The orienting behavior in zebrafish is known to require an intact optic tectum, a visual processing center, which receives visual input from the retina, and sends information to premotor network in the midbrain and hindbrain, called reticulospinal neurons. However, it remains to be elucidated how tectal neurons project to the premotor reticulospinal system to produce coordinated orienting behavior: how is each tectal neuron connected to reticulospinal neurons and how does it contribute to the production of orienting behavior?
To understand the neural bases of the visuomotor circuits controlling orienting behavior, I first plan to map neuronal connectivities of tectal neurons onto reticulospinal neurons using genetic labeling methods in zebrafish larvae, aiming at comprehensive analyses of their wiring patterns. Secondly, the morphological connectivities observed between tectal and reticulospinal neurons will be further tested for their physiological connectivities by calcium imaging. Finally, to examine the role of tectal neuron projections in visuomotor transformation, I will manipulate neural activities of genetically defined small subset of tectal neurons using optogenetic tools and simultaneously monitor their behaviors in living zebrafish. These approaches should give novel insights into neural mechanisms underlying sensorimotor coordination during animal behaviors.

Voltage-sensitive dye imaging in vivo provides unique insight into the correlated electrical activity of the neocortex offering millisecond temporal resolution and subcolumnar spatial resolution. Voltage-sensitive dye imaging has revealed propagating waves of activity and specific sequences of interactions between sensory and motor areas. Both spontaneous and sensory-evoked activity imaged with voltage-sensitive dye are profoundly regulated by different spontaneous behavioural states in mice. However, there have not yet been any reports of voltage-sensitive dye imaging in mice performing a learned task. Here, I therefore propose to image the cortical spatiotemporal dynamics of mice carrying out simple motor actions motivated by reward expectation predicted by a tactile whisker stimulus. During a training period, mice will learn to associate a whisker deflection with a reward that is delivered if the mouse licks or presses a lever within a few hundred milliseconds of the whisker stimulus. After mice have learned such a whisker detection paradigm, I will image the sensorimotor cortex and analyse trial-by-trial correlations of voltage-sensitive imaging data investigating events linked to the brain states, sensory processing and motor control. These measurements will be combined with electrophysiology to provide measures of spiking activity. In addition, I will carry out highly specific manipulations of cortical activity using optogenetic approaches through viral expression of channelrhodopsin 2 and halorhodopsin. My goal is to define the contributions of different interacting cortical areas during execution of a simple well-defined behavioural task.

In many different cell types, ranging from yeast to human epithelial cells, proper polarization is essential for cell function. Yeast needs to polarize for proliferation; epithelial cells that lose their polarization can become cancer cells. The polarization mechanisms differ in different cell types. Even closely related species use a variety of polarization machines. I propose to study how adaptable the polarization machine is. How many mutations are necessary to switch from one mechanism to another and how much do different mechanisms differ between species? I will address these questions in fungi, which are classical model systems to study cell polarization. I will destroy the polarization machinery in budding yeast and subsequently restore polarization by evolution experiments. By evolving different parallel lineages with different initial growth morphologies, my aim will be to evolve simultaneously different mechanisms. Next I will analyze the evolved cells to determine which new polarization mechanisms have evolved and compare these new mechanisms and the types of polarization they produce to fission yeast and Ashbya gossypii, which are related fungi. These evolution experiments will shed more light on the mechanism of polarization but also on how easy it is to switch from one polarization mechanism to another.

Our body is constantly exposed to microorganisms present in the environment and breaks in our skin integrity (wounds, burns or insect bites) can open doors for microbes and result in skin infection. Innate immune cells represent the front line of host immune defense and ensure that most infections are repelled without any symptoms of disease. Resident keratinocytes, macrophages and mast cells in the skin react to tissue damage and pathogen entry by secreting a cocktail of cytokines, chemokines, and soluble mediators. Among other things, these released factors initiate neutrophil recruitment from blood vessels and directed migration to the site of inflammation, an essential step in clearing infections. By employing advanced immunohistochemical methods, two-photon intravital microscopy, computational modeling, and genetic manipulation, I will investigate the crosstalk between innate immune cells and the sequence of in vivo events leading to neutrophil recruitment in the course of a sterile wound, as well as during bacterial and parasitic infections of the skin.

How a progenitor cell adopts a specific cell fate is a central question in developmental biology. The biochemical nature of transcription in addition to fluctuating surrounding signals implies the existence of developmental noise that must be filtered in order to ensure precision in cell specification. We would like to understand if «poising » genes for activation, within a progenitor/stem cell population, contributes to the buffering of noise. Recently, genome-wide studies have shown that many genes, despite being quiescent, are poised for activation with RNA polymerase II already present at the promoter for activation, this phenomenon is called Pol II stalling. Furthermore, the co-existence of both active and inactive chromatin modification marks (bivalent domains) in embryonic stem cells suggests that genes poised to choose either an active or repressed fate could be a general mechanism for cell fate decisions.
We would like to analyse the importance of these two mechanisms, stalled Pol II and bivalent chromatin marks, in cell specification during development. For this purpose, cell fate decisions within two developing organs will be examined, midgut and heart, using Drosophila melanogaster as a model.

The last decade has seen exponential growth in the pace of DNA sequencing and revolutions in microscopy, computing, and molecular visualization techniques. These advances have made it possible to dissect the inner workings of cells as they respond to internal or external cues. Despite the wealth of new data produced by these technologies, it remains remarkably difficult to predict how new mutations or new combinations of alleles will affect the physiology or morphology of an organism. Understanding how genetic variation is converted into observable differences between organisms is crucial for developing personalized medical treatments, bioengineering cellular machines or fuels, and predicting the evolutionary potentials of different alleles.

To attack this problem, we have assembled a team of new investigators with expertise in computational biology (Madan Babu), biochemistry and molecular population genetics and genomics (Christian Landry), and biophysics and evolutionary systems biology (Scott Rifkin). We will study how natural genetic variation in the budding yeast Saccharomyces cerevisiae affects two evolutionarily and ecologically important traits: the responses to mating pheromone and osmotic stress. In each case, the external signal triggers a cascade of gene activity which has been well-described. By combining detailed models of these signaling pathways with high-resolution measurements of how the external signals propagate through the networks, we will be able to predict the physiological and morphological consequences of genetic variation. This project applies cutting-edge technology to classic questions in evolutionary and cellular biology and will lay the groundwork for understanding the origin of phenotypic variation.

Cell signaling networks are the foundation of cell fate and behaviour and their aberrant activity is a key mechanism underlying the pathological behavior of cells during disease progression. However, signaling networks are remarkably complex, involving a myriad of dynamic interactions that flux in space and time. Thus, to understand how cell decisions arise requires a global view of cell signaling networks. We and others have demonstrated that to obtain systems-level insight into cell signaling a combination of evolutionary, experimental and computational exploration is needed. Here we aim to explore directly how signaling networks evolve. The Jun N-terminal kinase (JNK) is a subgroup of mitogen-activated protein (MAP) kinases activated by environmental stress, cell cycle and cytokines. JNK control several cellular processes including survival or death, proliferation, inflammation and stem cell differentiation. JNK is of high biomedical interest since mutations within it have been linked to different pathological conditions including cancer, diabetes and hepatitis. However, the regulatory network surrounding this kinase is highly context dependent and it is unclear how this result in so many biological roles for JNK. Thus we propose to study JNK signaling networks across evolutionary time to understand how signals are propagated through the network in response to perturbations such as kinase inhibitors or RNAi. By taking a systems level approach combining phosphoproteomics with computational and evolutionary biology we hope to determine how signalling systems operates and evolve at the network level.

Serotonin (5-HT) is an important neurochemical implicated in a variety of behavioral, cognitive and emotional processes and is a key pharmacological target in the treatment of many psychiatric and neurological diseases. The CNS 5-HT projection originates from the brainstem raphe nuclei. Because 5-HT release requires action potentials in RN neurons, critical insights into 5-HT function could be made by recording from the RN to establish when 5-HT cells fire. However, 5-HT cells are embedded in a heterogeneous network of cells utilizing a variety of neurotransmitters, therefore identifying the recorded cells in behaving animals is problematic. In addition, 5-HT cells are heterogeneous themselves; different subsets of 5-HT neurons having different anatomical and functional properties. To unlock the situation we propose an optogenetic approach to study the firing dynamics of identified 5-HT neurons in behaving animals. Specifically, I will focus my research on an identified subset of 5-HT neurons projecting to the medial prefrontal cortex (mPFC), since both brain areas are thought to be involved in the control of impulsivity. I will study the firing dynamics of identified mPFC projecting 5-HT neurons correlated to specific aspects of an impulse control task. Revealing not only 5-HT neurochemical identity, but being able to identify a sub-population of 5-HT cells based on anatomical projections could further our understanding of the involvement of the 5-HT system in higher brain functions. This fellowship would considerably advance my research career by allowing me to extend my knowledge of cellular physiology with behavioral and molecular approaches.

Object recognition is a difficult computational task solved by the nervous system. For a wide range of organisms object recognition occurs predominantly using the chemical senses. Behaviorally significant odor sources must be faithfully recognized despite being composed of an often complex chemical signature and being embedded in a complex and dynamic chemical background. The present proposal aims to understand the neural mechanisms that make such computations possible.

Increasing evidence suggests that object recognition can be understood as a process of probabilistic (Bayesian) inference. Although this idea is already being exploited in the visual system, it has yet had little impact on the field of olfaction. Our central hypothesis is that odor stimuli are represented using a probabilistic population code, and that odor representations are processed in Bayesian optimal way by the nervous system. This problem requires at least two computational processes: integration, which underlies recognition of multi-component odor sources, and marginalization, which is essential for invariance to irrelevant background odors.

Using this probabilistic framework, we will develop computational models for integration and marginalization in the olfactory system and test them using parallel experiments in rats and in Drosophila larvae. The rat will provide a model system that can be probed using psychophysics and multi-electrode recordings, allowing us to study the neural basis of marginalization in populations of olfactory cortex neurons. The Drosophila larva will provide a model system with a nervous circuit amenable to genetic manipulations: using chemotaxis as a behavioral paradigm, we will using molecular perturbations to decompose olfactory input into elementary parts mediated by single odorant receptors.

The probabilistic framework will be used to design and interpret experiments which will themselves inform refinement of the theoretical hypotheses. By comparing across species, we will test for the existence of conserved, and possibly optimal, solutions to ubiquitous problem of the odor recognition in naturalistic olfactory environments. This project is based on computational and systems neuroscience and includes strong contributions of the fields of mathematics and statistics (Pouget), biophysics and fly molecular biology (Louis), psychology and physiology (Mainen).

Innate immunity represents the first line of defence in animals and is essential for rapid protection and activation of the adaptive immunity. It is composed of a collection of biological processes that are highly conserved in metazoans. In the last 30 years, Drosophila has become a powerful model for dissecting the innate immune response at the organism level. Thus, in this study, I will use the fly as a model host to extend our understanding in innate defensive strategies used by all metazoans.

In flies, a systemic infection is the consequence of a cellular response mediated by haemocytes (macrophage-like cell) that engage in phagocytosis and a humoral response characterised by production of antimicrobial peptides (AMPs) in the fat body (equivalent to the mammalian liver). Often during infections, these two responses are coordinated and haemocytes have been suggested to play a role. However, the signals responsible for haemocyte-to-fat body communication remain unknown. To address this, I will restrict pathogen detection to haemocytes and analyse whether activation of these cells alone is sufficient to mount a systemic response. Afterwards, I will perform an in vivo RNAi screen to identify genes in haemocytes that alter AMP production and larvae survival upon infection. Finally, I will characterise the identified genes by various genetic and biochemical tools (e.g. generate null mutants).

This proposal, by combining studies at the single molecule level with a whole organism approach, can reveal novel modulators and signalling pathways, which may have significant implications for appreciating the biology of disease progression and development of new therapeutic agents.

Professional antigen presenting cells are comprised of dendritic cells (DC), macrophages and B cells. All three of these cell types have the ability to efficiently engage T cells, however, they differ in their ability to acquire antigen and process it into peptides for presentation to T cells. DC and macrophages efficiently acquire antigen through endocytosis, but only DC have endogenous pathways for cross-presentation of antigens to cytotoxic killer T cells. On the other hand, B cells are not as specialized at taking up material through endocytosis but can take up antigen through the B cell receptor and yet uptake of antigen alone is not sufficient for cross-priming cytotoxic killer T cells. New findings suggest that B cells are able to cross-present to T cells provided that secondary signals are supplied to activate cross-priming. Signaling pathways stemming from the activation of toll-like receptors (TLR) are one promising possibility to act as a necessary requirement. This project aims to develop a methodology to deliver antigen and TLR ligands specifically to B cells in vivo for the purposes of studying the ability of B cells to cross-prime cytotoxic killer T cells. Specifically, CD22, a B cell-specific marker, will be targeted using high affinity ligands that initiate endocytosis of CD22 and associated cargo. In such a manner, the requirement of TLR signaling pathways can be investigated systematically. This research will shed light on a fundamental aspect of the immune system while providing a tool that can be used to investigate many other aspects of B cell function in vivo.

Chemical genetics and de novo protein design are two complementary approaches in which chemical tools can help in shedding light on cell signaling pathways. The first approach utilizes small-molecules for targeting proteins, affecting their activity and is used to investigate their role in the cell. In contrast, in the second approach, the secondary and tertiary structures of synthetic oligomers elucidate the principles that govern folding, recognition and activity.
Our ultimate understanding of cell signaling pathways would be reflected in the ability to prepare artificial signaling systems that combine these methods and hence, can both mimic and modulate natural signal transtuction processes. Such protein mimetics will not only be able to bind proteins and affect their activity, but in doing so could simultaneously generate alternative signaling pathways.
As a first step toward achieving this goal, we will develop synthetic receptors that can mimic and thereby alter the signaling mechanism of receptor tyrosine kinases (RTKs). Similar to RTKs, they will possess a binding site and a catalytic domain, and the binding to growth factors will initiate receptors’ dimerization and catalysis. However, rather than stimulating RTK phosphorylation and down-stream cell signaling, binding to the synthetic receptors will trigger an alternative pathway resulting in the activation of nuclear receptors.
This will not only enhance our understanding of the parameters that govern biomolecular recognition and signaling, bur will open up a novel class of protein mimetics capable of inducing and responding to dynamic changes in the natural signal transduction processes, in real time

Protein motions play important roles in diverse biological processes, including folding, aggregation, catalysis and molecular recognition. In recent years, interest in protein motions has risen dramatically, driven by major advances in the experimental and computational characterizations of protein flexibility and dynamics. Thus, similar to how structural genomics has facilitated the field of structural bioinformatics, the large amounts of data on protein motions available means that large-scale bioinformatic analyses are now possible. We propose to collect and compare diverse experimental and predicted measures of protein flexibility and dynamics. This will allow us to assess the correlations between simple computational and/or experimental measures of protein flexibility and larger-scale protein dynamics, and to address important biological questions about the relationships between protein motions and function. In particular, we are interested in protein interactions: do unbound proteins sample conformations resembling their bound states, do flexible regions have greater tendencies to interact and can simple measures of protein flexibility be used to predict interaction sites and the conformations of bound proteins? In addition, we will investigate the role of flexibility in protein complex assembly, its relation to the topology of protein complexes and whether it can be used to improve predictions of protein aggregation. Thus, this work seeks to not only address fundamental questions of biological interest but may also lead to improved understandings of protein motions that could contribute to practical advances in fields such as protein engineering and drug design.

MicroRNAs (miRNAs) are an evolutionarily conserved group of small RNAs, regulating gene expression at the post-transcriptional level. Since their discovery, miRNAs has been shown to play essential roles in embryonic development and cell differentiation.
Embryonic stem (ES) cells are stable cell lines derived from the pre-implantation mouse embryo; they can differentiate, in vitro and in vivo, into all the cell types of the adult organism, an ability defined as Naive pluripotency.
Recently, Epistem cells (EpiSCs) have been derived from post-implantation embryos; these cells are in a more advance stage of development, called Primed pluripotency: they show commitment toward multiple cell lineages, but still retain a differentiation potential.
ES and EpiSCs are established in vitro models, extensively used for studying pluripotency and cell differentiation: the aim of my project is to understand how miRNAs regulate cell fate decisions in these different pluripotent states and the transition between them.
First, I’ll derive EpiSCs completely depleted of endogenous miRNAs, by inactivation an enzyme required for miRNA synthesis.
The characterization of these cells will highlight the global impact of miRNAs on cell commitment and pluripotency.
Then, to identify single miRNAs involved in these processes, I’ll compare the miRNA transcriptional profile of EpiSCs and ES cells.
MiRNAs differentially expressed will be functionally validated through knock-down studies.
Finally, I will investigate how miRNAs impinge on the core gene regulatory networks in ES cells and EpiSCs.
In sum, this project will extend our understanding of how ES cells control pluripotency and differentiation

The focus of the proposed research is to understand the genetics and cell biology that underlie neural tube morphogenesis in the mammalian embryo. Neural tube closure is one of the most complex morphological processes during embryogenesis in vertebrates, including humans. Failure in this process leads to Neural Tube Defects (NTDs), resulting in severe birth abnormalities such as anencephaly and spina bifida. However, the genetic risk factors contributing to the etiology of NTDs are still largely unknown.
The Niswander lab has developed an ex utero culture system to allow real time imaging of neural tube closure of mouse embryos. This has revealed extensive morphogenetic processes including cell shape changes, cell movements and formation of membrane protrusions and nano-tubes. My goal is to explore the cellular mechanisms, such as cytoskeletal rearrangement, adhesion and intracellular trafficking, regulating these dynamic processes in vivo. Toward this end, I will utilize this imaging technique combined with different genetic and cellular tools to follow the behavior of distinct intracellular components such as cytoskeleton-associated elements, various trafficking components and adhesion molecules during neural fold closure.
Chemical mutagenesis and forward genetic screening in the Niswander lab have identified a number of mouse mutants that display NTDs. I will focus on a mutation in the transcription factor grainy-head like 2 (grhl2) to determine its regulatory role at the cellular level during neural fold closure. Taken together, my goal is to provide specific genetic and cellular mechanisms that regulate neural fold closure in the mouse embryo.

Gram-negative bacterial pathogens use a type III secretion system to deliver effector proteins into eukaryotic cells. These effectors manipulate host cellular processes to promote infections that cause important diseases of both plants and humans.
The complexity of host-microbe interactions and, in particular, the low abundance of effectors, have prevented facile visualization of the spatial and temporal activities of these pathogen proteins during the natural infection process. Emerging imaging technologies promise to overcome these limitations, thereby providing new insights into the mechanisms involved in bacterial pathogenesis.
The use of well-characterized fluorophores (e.g., GFP) to tag effectors has largely failed, as they inhibit secretion and recently developed alternatives to these tags rely on substrates, toxic dyes or genetic manipulation of host cells, limiting their applicability in vivo.
I propose to develop the novel fluorescent tag iLOV as a tool to monitor the translocation of effectors into host cells. While its emission and excitation characteristics are identical to GFP, allowing the use of established imaging setups, iLOV is smaller and lacks a ß-barrel fold. In preliminary experiments I found an effector-iLOV fusion to be functional, based on its delivery into plant cells and activation of the host immune response.
The establishment of an autofluorescing tag that does not interfere with secretion will allow tracking of effector delivery and behavior in host cells during natural infections and enable the investigation of diverse a set of questions, including dynamics and order of effector secretion and their subcellular localization in host cells.

Homologous recombination is a vital cellular process that facilitates DNA repair and promotes evolutionary diversity. It entails the reciprocal exchange of single strands between homologous DNA duplexes, which can lead to the generation of a four-way branched intermediate generally known as Holliday junction (HJ). DNA molecules interconnected this way represent a covalent linkage between chromosomes, and therefore need to be separated to allow normal chromosome transmission during mitosis and meiosis. HJ resolution is mediated by structure-specific endonucleases that catalyse dual strand incisions across the junction or crossover point. Such enzymes have been isolated from bacteriophages, bacteria, archaea and yeast, while a mammalian HJ resolvase – although detectable as a biochemical activity – has remained elusive over decades of intensive search. Recently, this Holliday junction resolvase activity was identified as the product of the GEN1 gene. This has created the opportunity to clarify the mechanisms and the importance of HJ resolution in a cellular context. The primary focus of this proposal is to investigate the cellular roles of GEN1, with special emphasis on its predicted functions in facilitating recombinational DNA repair. The requirement for HJ resolution in the context of diverse processes such as DNA repair, DNA replication and meiotic recombination, implies that resolving enzymes must be coordinated with complex protein machines and their activity tightly controlled. We propose to investigate how GEN1 function is regulated and how its activity may be adapted in response to different cellular circumstances.

Blood-feeding mosquitoes, such as the yellow fever mosquito Aedes aegypti, use highly specialized and sensitive olfactory systems to locate their hosts. This sensory process involves detecting and following plumes of volatile host emissions, which include skin odor and carbon dioxide (CO2). In mosquitoes, CO2 is sensed by populations of olfactory sensory neurons in the maxillary palps, and functions as a powerful attractant. In A. aegypti, CO2 additionally acts to sensitize the olfactory system of mosquitoes alerting them to the presence of human skin odor. Despite the importance of carbon dioxide reception to host seeking, the molecular and cellular basis of this olfactory process in mosquitoes remains virtually unexplored. I propose to apply molecular genetic approaches to A. aegypti to gain insight into the mechanistic basis of CO2 reception and its functional significance to mosquito host-seeking behavior.