Cells are constantly exposed to factors that damage DNA, either from exogenous sources, such as UV light, or endogenous sources, such as oxidative stress. Moreover, DNA replication itself poses a problem to genome integrity. Not surprisingly, a huge number of proteins work together to faithfully replicate DNA and to detect, signal and repair DNA damage. Failure to do so results in genome instability.

Over the last years, a considerable number of proteins involved in DNA metabolism have been identified to bind to an iron-sulphur (FeS) cluster as a cofactor. Considering that - upon FeS cluster oxidation - free iron atoms can generate reactive oxygen species and damage DNA, the abundance of FeS proteins in DNA replication and repair has come as a surprise, and the function of FeS clusters in these processes has remained elusive to date.

At the same time, their redox sensitivity makes FeS clusters particularly interesting and versatile cofactors that would be uniquely suited e.g. to sense oxidative stress conditions and allow adaption to suboptimal conditions of DNA replication.

I propose to exploit a combination of complementary experimental approaches and systems, such as proteomics, biochemistry, molecular and cellular biology and yeast genetics in order (1.) to identify new FeS proteins, (2.) to characterise new FeS proteins involved in DNA metabolism, and (3.) to gain insight into the role of FeS clusters in DNA replication and repair.

One of the most abundant proteins in that structures cell shape and guides its movement by forming filaments, moonlights in the nucleus. This protein, actin, has been studied for years yet precisely because of its dominant life as a filament, little is known how it acts in the cell nucleus. Recent evidence points to it serving in a non-filamentous or globular (G) form in the nucleus. Globular actin forms a complex with other proteins to help remodel chromatin and binds transcription factors to modulate gene expression. Recent data suggest that it directly influences the integrity of the genome once single and double strand damage is incurred. There has been no specific way to visualize or distinguish the filamentous from globular form of actin. Novel chemistry allows selection of circular peptidyl structures that are highly specifc for different forms of protein structure. These bicyclic peptides can be used to study the location, binding partners and function of actin in the nucleus. This program combines the expertise of a chemist, an electron microscopist, a yeast geneticist and a vertebrate protein expert in actin and actin related proteins, to try to solve this problem.

This proposal seeks to answer fundamental questions about cytoskeletal filaments, which are central regulators of cell biology. Our current understanding of bacteria is that these organisms use dynamic cytoskeletal systems to organize and regulate essential cellular processes. The results of our previous HFSP grant broke new ground by demonstrating that the number of these systems extends beyond homologs of the three primary eukaryotic cytoskeletons (i.e., actin, tubulin, and intermediate filaments). Two representative examples are our discovery of bactofilins, which are a bacteria-specific family of proteins, and of CTP synthase, an enzyme that is widely conserved across all kingdoms of life. These cytoskeletal proteins play key roles in regulating cell shape, metabolism, and intracellular compartmentalization, and their identification has had a transformative impact on the field of bacterial cell biology.
Building on our successful previous approaches, our renewal proposal is designed to characterize these novel cytoskeletons and, in addition, identify new cytoskeletal proteins. Apart from using a wide range of genetic, biochemical, and biophysical techniques, a central theme in this proposal is the identification, characterization, and application of small molecules to analyze protein function in vivo and in vitro. Small molecules can be powerful tools for studying bacterial and eukaryotic cytoskeletons, as they enable the rapid perturbation of protein function in vivo. Nevertheless, only few small molecules are currently available for specifically targeting cytoskeletal elements in bacteria. The discovery of chemical probes is poised to further our understanding of bacterial cell biology, while new classes of small molecules that emerge from these studies may provide starting points for developing new classes of antibacterial agents. Compounds that regulate widely conserved proteins may also inspire the development of therapeutic compounds for cancer and immune research and treatment.
In the first three years of the HFSP grant, we established a successful formula for productive collaboration between our labs. For the renewal, we have modified this formula to match the goals of the next phase of this project by focusing the expertise and capabilities of an interdisciplinary group of young scientists: Zemer Gitai uses high-throughput methods to study the bacterial cytoskeleton, Justin Kollman uses electron microscopy to study the biophysics of cytoskeletal assembly, Martin Thanbichler studies the biochemistry and genetics of the bacterial cytoskeleton, and Doug Weibel uses chemistry and engineering to study bacterial physiology. The salient features of this collaborative and interdisciplinary project will facilitate intellectual and cultural cross-pollination, and the scope and high-risk-high-reward merit of this proposal are designed specifically for an international funding mechanism such as the HFSP.

Given their biomedical significance, transmembrane (TM) proteins are especially desirable, but difficult targets for structure determination. In the past decade advancements in cryo-electron-microscopy (cryo-EM) have resulted in an increased number of low- and intermediate-resolution density maps of TM and soluble proteins. Yet, the number of cryo-EM-derived protein models is considerably lower due to the lack of a general powerful method. Elaboration of such methodology would resolve a bottleneck in proteins structure determination from cryo-EM maps. I will develop a method for proteins structure determination from density maps employing the deformable elastic network method and evolutionary conservation information. Recent studies have demonstrated the power of evolutionary conservation information in structure prediction of TM proteins in ab-initio, template-free modeling. Integration of this information into the interpretation process of low and intermediate-resolution cryo-EM maps will make the latter more accurate and fast. I will apply the resultant method on three systems: the 18 kDa translocator protein TspO, the cystic fibrosis transmembrane conductance regulator CFTR and a V-ATPase. Targets TspO and CFTR are appealing due to their involvement in brain pathological conditions and cystic fibrosis, respectively. ATPases are complex, not yet understood machineries coupling between the membrane electrochemical potential and the synthesis of the main energy carrier ATP, structure of which will shed light on their action mechanism. This proposal paves the way towards a powerful method for structure determination from cryo-EM maps and applies it to fascinating examples.

Bacterial biofilms are integrated communities of cells that adhere to surfaces and are fundamental to the ecology and biology of bacteria. The accommodation of a free-swimming cell to a solid surface is more complex than passive cell adhesion. We propose a multi-disciplinary study to investigate the interplay between motility appendages, molecular motors, exopolysaccharide (EPS) production, and hydrodynamics near the surface environment using tools not usually used by bacteriologists. We address two questions in P. aeruginosa.
How do different bacterial motor systems and EPS interact with solid surfaces? How do flagella/TFP adapt and respond to an interaction with a surface, and how do they couple to near-surface hydrodynamics and EPS? We will examine the transition from swimming to attaching for the two principal motor systems, flagella and type IV pili (TFP), using direct force measurements on single motors and correlate with multicell behavior using single cell tracking techniques. In all cases, the different roles of solid versus viscous friction in this low Reynolds number regime will be examined. With these anchoring measurements it will be possible to examine this important cdiGMP signaling network in a new light.
How do bacterial motors, exopolysaccharides, and guiding signals influence early biofilm formation? We track the entire motility history of every cell and make inferences about biofilm initiation at single cell resolution. Single molecule TFP force measurements will be used to examine the possibility of a TFP and flagella sensing function for EPS and EPS-mediated interactions between cells. Results will be interpreted using theoretical techniques developed to study nonlinear feedback mechanisms and ‘jamming’ transitions. We will perform new experiments exploiting cdiGMP signaling mutants that impact motility and/or EPS. Our physical measurements will be combined with techniques to measure cdiGMP levels at the single cell and sub-cellular levels, so that spatiotemporal phenomena in signaling and motility can be directly connected. Thus, the initial events in the association of a bacterium with a surface will be investigated in unprecedented detail by combining genetic techniques with single cell measurements, providing novel mechanistic insights into the earliest steps in biofilm formation.

Most neurons in the mammalian brain are born during embryogenesis and persist throughout the life of the animal. The mammalian olfactory bulb is a notable exception in that it receives many thousands of newly-born inhibitory granule cells (GCs) throughout adulthood. GCs provide recurrent and lateral inhibition to mitral cells, principal cells in the olfactory bulb, but the precise functional role of adult neurogenesis is unknown. Here we propose a collaborative and innovative project to test the hypothesis that adult-born GCs play a unique and essential role in odor learning, combining optogenetics, intersectional genetics, in vivo two-photon calcium imaging and behavioral tasks.
To enable selective characterization and manipulation of adult-born GCs, we will develop a genetic method to selectively target adult-born GCs. This strategy combines two independent recombinases for intersectional specificity and an inducible recombination system. With this genetic method, we will express a genetically encoded calcium indicator selectively in adult-born GCs and use in vivo two-photon calcium imaging to characterize their odor response properties in awake, head-fixed mice. Next, to test the effect of suppressing the activity of adult-born GCs, we will develop optogenetic tools that suppress neuronal activity acutely and reversibly. The strategy includes modifications of pre-existing opsins to develop novel, inhibitory step-function opsins (iSFOs). iSFO-expressing neurons will be suppressed upon the delivery of a brief pulse of activating wavelength of light for extended periods of time even in the absence of sustained illumination until the delivery of an inactivating wavelength of light. We will express iSFO selectively in adult-born GCs using the genetic method described above. We will use two-photon calcium imaging to monitor in vivo the odor responses of mitral cells when adult-born GCs are suppressed and unsuppressed on alternating trials with iSFO in awake, head-fixed mice. We will determine relative contributions of local dendritic activity and global, action potential-dependent activity of adult-born GCs by targeting iSFO in specific subcellular compartments. Finally, we will test the role of adult-born GCs in odor learning, by training mice for odor discrimination tasks and determining if the suppression of adult-born GC activity by iSFO affects the animal’s learning.
Together, this collaboration combines complementary expertise and cutting-edge techniques of three co-PIs to understand the functional role of adult neurogenesis, one of the most dramatic forms of plasticity in the adult brain.

Humans and animals respond to novel stimuli with distinct orienting and exploratory behaviors. From an evolutionary perspective, these responses seem highly adaptive, since they increase an animal’s chance of finding new resources or mating partners. Novelty-mediated orienting responses have been used extensively in developmental psychology as a non-verbal measure for object recognition in infants. Moreover, disturbances in novelty processing have been linked to human pathological conditions including schizophrenia and autism as well as human personality traits associated with maladaptive behaviors such as addiction. Together, these findings highlight the importance of novelty processing for human cognitive function. Although, previous studies have identified key brain areas involved in novelty processing, the neural mechanisms for how novel stimuli are detected and learned remain unresolved. Furthermore, a mechanistic description of how behavioral responses are initiated is currently lacking. Thus, I propose here to investigate the neural mechanisms for novelty processing using a combination of multielectrode recording and optogenetic techniques in awake, behaving mice. By genetically tagging specific neuronal subpopulations for electrophysiological identification and manipulation, this approach allows for the precise characterization of the firing properties of defined neuron types and for probing their causal involvement in specific aspects of novelty behavior.

Many medically important viruses have RNA genomes. These viruses replicate with an extremely high mutation rate, giving rise to highly diverse populations, and enabling rapid adaptation to changing environments. However, numerous mutations are deleterious as they destabilize the structure and compromise the function of viral proteins. It is not known how many viral variants in any given population are defective and how this affects its infectivity. Here, we propose to determine the relationship between the physical outcome of protein mutations and virus fitness. Through a systematic mutagenesis of the poliovirus genome, we plan to assess the effects of numerous mutations on protein thermodynamics and on viral infectivity. The link between protein thermodynamics and viral fitness will provide understanding of the biophysical origins of the majority of mutations and their effects on fitness.
This analysis will enable us to map the mutational composition of natural viral populations in terms of protein biophysics, and to estimate which variants are defective. Furthermore, we will analyze how the population composition changes in response to varying factors, such as availability of host factors. Thus, our study aims to give a quantitative understanding of viral population dynamics and evolution in light of the biophysical properties of its proteins. It will open avenues to addressing questions about virus pathogenesis, adaptation and molecular evolution.

How do animals process, represent and use multimodal sensory information from the natural environment? Our project will address this fundamental question by mounting ultra-lightweight sensors on free-flying bats to capture auditory, visual and chemical signals received by animals engaged in natural behaviors. The sensor data will lay the foundation for novel perceptual experiments, computational modeling, and biorobotic platforms. Our work will focus on the Egyptian fruit bat, a Neotropical frog-eating bat, a European trawling insectivorous bat, and a North American aerial insectivorous bat. The diversity of bat species and their adaptations to different environments and behavioral tasks make them especially well-suited for this line of inquiry.
Research on sensory processing in bats has, up until now, largely emphasized a single modality, typically echolocation. We will launch a major research effort aimed at uncovering the contribution of multisensory processing to the natural behaviors of free-flying bats. The use of novel on-board sensors will give us access to a bat’s view of the world as it moves in time and space. Sensor recordings will be taken in the field, where bats commute over long distances, negotiate obstacles and find food, and in the laboratory, where animals can be trained to perform tasks in experimentally controlled settings. Lab experiments will include creation of a visual-acoustic virtual reality environment.
Our project is directed at understanding the conditions that give rise to cross-modal enhancement of information, differential weighting of sensory information through separate channels, and behavioral decisions in the face of sensory conflicts. In addition, we propose studies that elucidate the role of spatial memory in a variety of tasks and determine the conditions in which bats switch from sensing modes to memory modes. We will investigate and develop mathematical representations of sensory processing and computational architectures for spatial memory, navigation, learning and adaptive behaviors with a view towards exploiting such architectures in complex robotic navigation systems.
The overarching goal of our project is to advance critical new insights to the multisensory representations and cognitive maps of the environment animals use to make decisions for goal-directed actions. Through integrated field and lab studies, computer modeling and biomimetic robotics, our project will contribute to a deeper understanding of multisensory integration, scene analysis, and spatial navigation in natural settings.

Our research team will investigate how pathogenic bacteria use molecular pumps as part of a mechanism to evade antibacterial drugs. When the drugs enter the bacteria, they can be rapidly expelled again by the action of molecular pumps, before the drugs have an opportunity to harm the organism. The pumps we will study are composed of three different types of proteins, two of which span the protective bacterial membrane, and the third helps to draw the other two together into a functional unit. It is not presently understood how these components of the pump assemble together to make a functioning unit, and how the unit works to recognise and displace the drug to the outside of the cell. The team will attempt to elucidate the three dimensional structure of representative molecular pumps to address these questions. The team will examine how the components interact and how the pump uses energy to displace drugs from the interior of the bacterium out into the medium. We will also examine how the components come together to form a functioning unit in a living bacterium. The information obtained from this work may help us to understand how to combat the growing problem of multidrug resistance in bacterial infections.

My overarching goal is to identify the defined set of factors that can rejuvenate an aged cell. Several lines of evidence indicate this might be possible (e.g. by manipulation of signaling pathways, reactivation of embryonic genes etc’); however, in most cases, the rejuvenation process is either incomplete or increases the risk of cancer. My idea is to adopt some of the concepts of reprogramming toward rejuvenation and to identify the set of factors that can efficiently rejuvenate aged cells, without de-differentiation or increased risk of malignant transformation.

Neural stem cells (NSCs) represent a paradigm for adult stem cells and provide an ideal model for studies on rejuvenation. They can be easily isolated and cultured from their native niche in vivo. Furthermore, NSC deterioration with time might underlie some of the age-related cognitive decline. How can we convert ‘old’ NSCs into ‘young’ ones? To identify candidates enriched in the young state, I will compare young and old cells from the neural lineage, by focusing on microRNAs and transcription factors. Next, I will perform a detailed functional characterization, by conducting gain-of-function screens in NSCs, combined with novel readouts of youthfulness (e.g. fluorescent reporters) to understand how the identified factors exert their functions. Finally, to address the physiological relevance of my findings, I will evaluate the in vivo rejuvenating potential in mice.

Identifying the circuitry that determines the biological age of a cell will unravel a fundamental mystery in biology - why do cells age? This will have a major impact for advancing basic research in stem cell biology, regeneration, and aging.

Persistent structural and functional modifications of excitatory synapses are suggested to represent the cellular substrate of learning and memory. Dendritic spine morphology, like synaptic function, is dynamically regulated by neuronal activity. Changes in both spine morphology and synaptic efficacy are primarily influenced by actin remodeling. However, how dynamic actin filaments are anchored within dendritic spines and control their morphology and synaptic function remains unknown.
Our working hypothesis is that anchoring of actin filaments in dendritic spines represents a critical determinant of their dynamic and plastic behavior. Anchoring to the plasma membrane (‘peripheral anchor’) likely contributes to shape spine head and hinder the local diffusion of transmembrane proteins whereas anchoring at the base of the spine head (‘central anchor’) is required to generate enlargement forces upon actin polymerization. Elucidating the role of actin anchoring in dendritic spine structure, function and learning-related plasticity therefore represents the scientific aim of this proposal. To this end, we will develop and implement a highly multi-disciplinary approach based on the complementary expertise of the participants and a combination of i) advanced in vitro and in vivo electrophysiology, ii) cutting-edge imaging techniques, molecular tracking and super-resolution microscopy, iii) innovative photo-chemical tools for protein tagging, acute photoinactivation and crosslinking and iv) analysis of network activities and specific cognitive performances in behaving animals.
We will establish the role of peripheral and central anchors of F-actin in dendritic spines. We will combine single cell electrophysiology and molecular imaging techniques to explore the functional consequences of suppressing these anchors either genetically or acutely using protein photo-inactivation (CALI) techniques. Next, we will directly visualize the 3D network of spine actin, its modulation upon long term plasticity induction as well as its alterations upon selectively suppressing peripheral or central anchoring. We will also adapt molecular tagging to reveal the molecular organization of F-actin and its anchors within dendritic spines. We will then further identify the molecular components of peripheral and central anchors of F-actin in spine using new photo-crosslinking strategies to identify the molecular partners of peripheral and central actin anchors. Finally, we will combine our efforts to explore the importance of actin anchoring regulation in learning and memory processes by combining in vivo electrophysiology, photoinactivation and Ca imaging while monitoring cognitive performances measured in behavioral assays.

Age-associated cognition decline poses increasing social problems to the aging population highlighting the importance of a better understanding of age-dependent learning and memory impairment. Epigenetic modifications in the brain play a major role in the consolidation and long-term storage of memory but the underlying mechanisms, however, are poorly understood.
I propose to develop and apply novel epigenome perturbation technologies to study the role of neuronal circuit-specific DNA methylation changes in learning and memory, with the ultimate objective to identify genome-wide DNA methylation changes contributing to age-associated cognition decline. To accomplish the challenging but transformative goal I plan to develop and deliver novel light-inducible transcriptional effectors (LITEs) into the mouse brain to selectively turning on or off enzymes involved in DNA methylation. I further plan to develop and apply designer epigenetic modification enzymes (epiTALEs) to precisely manipulate specific DNA methylation targets. I will integrate these innovative technologies with state-of-the-art circuit and epigenetic analysis methods, mouse behavior and optogenetics. By using these tools I will be able to systematically probe different brain regions in a highly temporal and spatial resolution to identify both the DNA methylation targets and the brain circuits involved in normal learning and age-dependent memory impairment. This research plan will help to identify the epigenetic mechanisms that are causally involved in learning and memory and age-associated cognitive decline. It will further provide new therapeutic targets for cognitive and memory-related neurological disorders.

The ubiquitous small signaling molecule cyclic di-guanosinemonophosphate (c-di-GMP) is a key cellular regulator of biofilm formation and globally controls the motile-sessile switch of pathogenic and non-pathogenic bacteria. Despite of the broad importance of c-di-GMP for bacterial behavior and virulence, the underlying molecular and cellular processes remain poorly understood. C-di-GMP is a highly versatile signaling molecule regulating cellular processes at the transcriptional, translational and post-translational level. I propose to use the simple model system Caulobacter crescentus to study the effect of c-di-GMP on transcriptional regulation and on the timing of gene expression during the cell cycle. I will focus on three members of the Ros/MucR family of transcription repressors, candidates for c-di-GMP effectors and known regulators of exopolysaccharide biogenesis and motility in Sinorhizobium meliloti and of virulence determinants in Agrobacterium tumefaciens. I will elucidate the exact role and molecular mechanisms of the coupling of the MucR-c-di-GMP transcriptional regulation. Therefore, I will utilize a broad spectrum of techniques including RNASeq and ChipSeq to define the c-di-GMP-dependent MucR gene regulation network. Then, I will study the c-di-GMP-MucR- and MucR-DNA-binding kinetics in vitro, which forms the basis for the structural analysis of c-di-GMP-mediated DNA binding using crystallography. Finally, I will dissect the spatio-temporal timing of MucR transcriptional regulatory activities in single cells using live cell microscopy to integrate c-di-GMP-mediated differential gene expression into the asymmetric cell development program of C. crescentus.

Calcium is an extremely versatile second messenger, which controls almost all kind of cellular biological events.
As a single atom, calcium regulates biological function by changing its concentration, which is driven by the ~20,000 fold concentration difference between the extracellular environment and the cytoplasm. To avoid catastrophic cytoplasmic calcium induction, cells strictly limit calcium influxes. Activated channels are rapidly shut down and intruding cytoplasmic calcium is swiftly removed to the extracellular space or transferred into inner cellular membrane organelles. Accordingly, the combination of acute ion influx, point source ionic diffusion, and its immediate removal creates an ideal environment for calcium signal oscillations. Here, cardiomyocytes serve as a prime example where a wide range of cellular function is controlled by calcium signaling. Cardiomyocytes develop unique membrane micro domains termed “couplons” which structurally control calcium diffusion. These couplons work to not only couple membrane excitation to cell contractions, but also function as an electric synapse, through which calcium reciprocally controls membrane potentials. Nonetheless, our current understanding of cardiac couplon architecture and its dynamics is significantly lacking. Thus, by using cardiomyocytes as a model system, the goal of this project is to clarify how eukaryotic cells assemble junctional micro membrane domains that are effectively used to generate oscillating calcium signals, and define how these structures spatiotemporally control the information carried by this signal. This goal will be achieved by integrating the diverse expertise of our international team members. We will use 3-dimensional electron microscopy to determine the complete structure of couplons and their clusters, and paint the geometric models with distributions of nodal couplon molecules, determined both by diffraction limited and super-resolution light microscopy. Minimum molecular requirements to synthesize membrane junctions will also be determined and local membrane dynamics will be monitored by the use of newly designed fluorescent bio-reporters. In summary, successful completion of the current project is expected to provide fundamental knowledge of how micro-domain structures and their dynamics control the oscillatory behavior of membrane signaling.

In higher eukaryotes, the centrosome consists of a pair of centrioles and plays two distinct roles during cellular development. In cycling cells, centrosomes organize the interphase microtubule network and the mitotic spindle. In quiescent cells, one centriole forms the basal body and the primary cilium, which acts as an important platform for growth factor signaling. Important, unresolved questions are how the centriole switches between these distinct functions, and the role of growth factor signaling in this decision. Interestingly, many centriolar proteins are agonists (ß-catenin) and antagonists (Chibby, ß-catenin destruction complex) of canonical Wnt signaling, and they may function both as upstream regulators of ciliogenesis as well as downstream regulators of Wnt signaling. Furthermore, the kinase Nek2 targets ß-catenin and thereby promotes cell division. These observations suggest a role for Wnt in controlling the transition from a cycling centrosome to a basal body. In this project, I will use cell-based assays to test whether canonical Wnt signaling regulates centriole functions and antagonizes the centriole-basal body switch. I will investigate the behavior of centriolar Wnt-related factors during the centriole-basal body switch and the importance of Wnt-mediated transcription in ciliogenesis. Finally, I will address how centriolar ß-catenin is regulated by GSK3ß and Nek2, and how this relates to ciliogenesis. Together, these studies have great potential to provide us with insights on how the primary cilium is regulated, novel roles of centriolar factors in ciliogenesis and a better understanding of the Wnt pathway in the context of development and disease.

Identifying genetic risk variants for human diseases using genome-wide association studies is the essential first step towards establishing personalized genetic profiles. However, building predictive models of complex phenotypes that are useful in a medical setting has proved extremely challenging. Although many statistical methods have been developed in recent years, we know little about how the risk loci combine to determine the total genetic risk for an individual because of our limited understanding of the genetic architecture of human diseases. Yet, the predicted risk scores can be useful to identify individuals at high genetic risk of disease. In this project, we propose to use genome-wide prediction methods for a large number of cases from over a dozen of common disorders to explore the covariation of individual genetic risks among these conditions. We hypothesize that individuals at high risk for multiple disorders are likely to carry risk loci involved in gene-gene interactions resulting in covariation among phenotypes. Based on their predicted genetic risk scores, we will select subjects at high-risk for multiple diseases and examine the risk alleles they carry. Using integration methods that use prior biological knowledge, we expect to identify candidates loci for biologically relevant gene-gene interactions. These potential interactions will be further tested in cohorts of cases for specific diseases. Finally, we intend to incorporate information about biological interactions in prediction models to improve individual genetic profiling. The goal is to provide a better understanding of molecular disease phenotypes to perform reliable personalized medicine.

The centrosome is the major microtubule organizing center in animals and fungi, being implicated in several human diseases. The control of centrosome number, structure and localization is poorly understood. I will take advantage of comparative genomic and synthetic biology approaches to study how those structures are assembled. Recent studies suggest that the common ancestor of animals and fungi had, similarly to extant animals, a centrosome composed of two centrioles. Fission yeast lost the centriole and the genes needed to assemble it, having a centrosome equivalent, the spindle pole body (SPB). Despite their difference in structure, animal and yeast centrosomes share components, and both assemble only once per cell cycle and close to an already existing centrosome. This suggests a conserved ancestral program regulates centrosome assembly. I will use the fission yeast system as a “test tube” to study centrosome assembly, by expressing and studying the behavior of Drosophila centriole proteins in fission yeast. I will subsequently validate my findings in Drosophila. Preliminary experiments performed by the host laboratory show that Drosophila centriole proteins interact with the SPB machinery and with each other, validating the premises of this project. This project combines my expertise on the fission yeast cell cycle with the host laboratory expertise on centrosomes and cytoskeleton. This approach will identify the minimum conditions to regulate and assemble the unique structure of centrosomes.

Gene expression is a stochastic process controlled at multiple levels ranging from DNA promoter states through transcription and degradation rates to translation rates. The low numbers of molecules involved in many of the biochemical processes in the cell invariably lead to stochastic fluctuations in the amount of proteins expressed, but cells seem to have developed regulatory mechanisms to either reduce or amplify these fluctuations. Several studies characterized the extent and roles of stochastic gene expression in single cell organisms ranging from bacteria trough yeast to mammalian cells as well as in the development of multi-cellular organisms. However the extent and roles of noise and single-cell variability in cells operating within an intact adult mammalian tissue have not been studied. In this proposed interdisciplinary project we aim to use novel single molecule imaging methods to characterize multiples facets of gene expression in single cells in the intact mouse intestine. Specifically we aim to: 1) Develop a single molecule imaging technique to quantify transcriptional bursts and RNA degradation in intact mammalian tissues. 2) Study the roles of stochastic gene expression in modulating differentiation decisions of intestinal stem cells. 3) Study the roles of RNA degradation in rapidly switching gene expression programs in the mouse intestine. The HFSP fellowship was essential for the emergence of my current research project, and the CDA will provide the financial support and scientific independence to develop it.

A central goal of modern neurobiology is to understand the neural mechanisms that convert sensory inputs from the outside world into behavioral outputs that generate specific actions. While a great deal is known about the general organization of the circuits underlying olfaction -- the main sensory modality used by most animals to interact with their environment -- the functional architecture of the wiring that transforms olfactory inputs into behavioral outputs has not been described. In addition, no mechanism has been described to explain why certain odors, such as those from food, predators and mates, evoke profound and innate behavioral changes, while most odors lack behavioral meaning without prior learning.
Here we propose experiments testing the hypothesis that innate behavioral responses to odors are generated by the interplay between the amygdala, a part of the brain involved in emotional processing, and the ventral striatum, which is critical for linking sensory inputs to motor outputs. This hypothesis has, to date, been untestable because of limitations imposed by the deep location of the ventral striatum. By taking advantage of a newly-developed surgical preparation that exposes the olfactory tubercle, a subregion of the ventral striatum, in living animals we will directly address this hypothesis. We will use a combination of in vivo whole-cell recordings, functional calcium imaging, optogenetic manipulations and behavioral analysis to reveal how olfactory circuits transform signals about the environment to lead to appropriate actions.