During embryonic development, both biochemical and mechanical cues establish the cell differentiation patterns that shape tissues and organs. While the role of signaling molecules has been widely studied, how mechanical forces affect progenitor cell differentiation/renewal in developing organs remains largely unknown, mainly because of a lack of technologies allowing quantitative measurements of tissue mechanics in vivo. Using novel force transducers that we have recently developed to measure cell-generated mechanical forces within living embryonic tissues, we propose to unveil the role of cellular forces during the progressive formation of the vertebrate limb skeleton. To do so, we will both measure and perturb the differential forces within the differentiation and self-renewal progenitor zones of developing limb cartilages. Moreover, we will test the role of Fibroblast Growth Factor (FGF) signaling pathway (known to be necessary for limb progenitor cell maintenance) in modulating the endogenous distribution of cellular forces within this tissue. Finally, we plan to apply ectopic compressive forces on developing limbs using a custom-made microindenter while simultaneously monitoring the effect of such mechanical disturbance on chondrogenic differentiation in developing limbs. The combination of physics and developmental biology approaches promises to reveal how signaling pathways and mechanics interplay to control the progenitor cell maintenance/differentiation balance during development. We believe that the results from this project will establish a framework to understand the role of mechanical signals in development and set the stage for future experiments in many other organs.

Sensory percepts are believed to arise from the spiking activity of populations of cortical neurons. However, a mechanistic understanding of the underlying cellular mechanisms is still missing. One prominent feature of cortical activity is a significant amount of variability. Yet, its impact on perception remains unclear. On the behavioral side, multiple repetitions of the same perceptual task lead to different responses. To what extent does neural noise in sensory cortices account for behavioral variability? While these questions have been intensively discussed by theoreticians during the last decades, technical challenges have made them impossible to tackle experimentally. Here we argue that recent technological advances on microscopy and genetics can be used to address these fundamental issues. We propose to use two-photon calcium imaging to record hundreds of neurons simultaneously while mice perform an auditory perceptual decision-making task. We will apply machine learning tools to optimally decode stimulus- and decision-related variables from neural activity in auditory cortex in real time. This will provide conclusive evidence on how much information about the external stimulus can be extracted from sensory cortices and how this compares with the animal's psychophysical performance. Further, we will quantify to what extent neural variability can account for the animal's perceptual errors in the task. Finally, we will optogenetically manipulate specific subsets of neurons in order to bias the animal's behavior in a predictable manner. These experiments will causally demonstrate the links between neural noise, perception and behavior.

The autonomous nervous system (ANS) includes the sympathetic nervous system (SNS), the parasympathetic (PNS) and the enteric nervous system (ENS). Catecholamines and adrenergic receptors have been shown to promote the recruitment of leukocytes within tissues and therefore modulate tissue integrity and innate immunity [1]. The Merad’s group has also shown that in steady-state conditions macrophages located in the intestinal muscle layer localize in close apposition with intestinal neurons and produce neuromodulators, which promote the peristaltic activity of enteric neurons thereby regulating intestinal transit time [2]. Macrophages represent one of the major population of the tumor and in an effort to promote tissue healing, are thought to lead to immune suppression and progression of many tumors [3]. Importantly, the autonomic nerve fibers present in the prostate gland were found to regulate prostate cancer development and dissemination in mouse models [4]. Based on the observation that macrophages and neurons colocalize and modulate their reciprocal function, we raise the fundamental question of whether macrophages recruited into the tumor via adrenergic signals further communicate with emerging neural networks to promote tumor progression, and propose to study the nature and function of this communication within the tumor environment.
1.Scheiermann, C., et al. Immunity,2012.37(2).
2.Muller, P.A., et al. Cell,2014.158(2).
3.Lavin, Y. and Merad, M. Cancer Immunol Res, 2013. 1(4).
4.Magnon, C., et al. Science, 2013. 341(6142).

In variable environments (either natural or altered by humans) organisms can adjust to new conditions for better success in producing offspring. In many cases, organisms do this through cognitive abilities: their ability to gather information from their surroundings, process that information, and make decisions to improve their lot. We now realize that there is extensive variation in cognitive capacity both between species and within species suggesting that cognitive abilities evolve, yet we still know very little about how cognition evolves. Most organisms have a number of different cognitive abilities (memory, attention, learning, etc…) and comparative research suggests that certain environments select for improvement in some, but not all, of these abilities, but this has not been demonstrated directly. Furthermore, while our understanding of the neural basis of cognition has improved in model species, we still know very little about the ecological importance of most cognitive capacities and their underlying neural structure. Our project will investigate the evolution of cognitive abilities and neural structure in a wild great tits, and thus provide a big step forward in our understanding of cognitive evolution. To do this, we will study 6 populations in the French Pyrenees that differ in ecology (altitude and urbanization), and thus likely favor different cognitive traits. Our team, specialists in cognition, brain imaging, computer science, and evolution will develop new tools (touchscreens) and analytical approaches (automated video analysis, MRI of non-model species) to examine cognitive traits that could present a clear advantage under some, but not all, ecological contexts. We will measure cognitive abilities of birds in these contrasted populations and quantify their fitness to understand what cognitive traits and neural structures are favored in different ecological settings. In doing so, we can measure natural selection on cognitive abilities and neural structure, understand what factors in the environment drive this selection, and ultimately gain a much better understanding of how evolution shapes minds in the wild. This interdisciplinary project provides advances to a number of different fields of research while together contributing to an important stride forward in our understanding of the neural basis of cognition and how cognitive evolves.

Atmospheric carbon dioxide levels are increasing at an accelerating rate and broke past 400 parts per million (ppm) in 2013, having exceeded the 350 ppm safety limit since 1988. Cyanobacteria are responsible for over half of the carbon fixed by phytoplankton in the oceans, which in turn constitute half of the global annual organismal carbon fixation. Carbon fixation is performed by protein organelles in cyanobacteria called carboxysomes that concentrate the carbon-fixing enzyme RuBisCo. Yet, despite their significant role in the global carbon cycle, factors regulating the formation and organization of carboxysomes are poorly characterized. I will investigate the initial nucleation of RuBisCo required for carboxysome assembly through in vivo fluorescence microscopy and in vitro high resolution imaging. I will characterize carboxysome shell permeability and the internal environment using fluorescent pH and redox biosensors. Based on these findings, I will use RNA scaffolds to precisely organize RuBisCo and generate a conducive internal environment to assess the efficiency of carboxysome formation and carbon fixation. Ultimately, using a novel approach comprised of computational modeling, synthetic biochemical reconstitution, genetic engineering and quantitative live-cell microscopy, I will study the spatial-temporal dynamics and metabolic consequences of carboxysome biogenesis. The proposed research will answer many important questions about microbial carbon fixation and organelle formation, as well as provide a foundation for the improvement and adaptation of these processes in the context of bioremediation and energy production.

Successful acclimation of photosynthetic organisms to variable environments requires a tight management of source (light harvesting) and sink (CO2 assimilation) relationships. Photosynthetic organisms optimize photosynthesis in low light, where excitation energy is limiting CO2 fixation and minimize photo-oxidative damage in high light, dissipating excess photons. Despite extensive studies of these phenomena, the mechanism governing light utilization in plants is still poorly understood. Our results pinpointing a K+ channel (TPK3) as an essential component of light-biomass conversion have pioneered a new research field (photosynthesis optimization by ion flux). By counterbalancing proton pumping with potassium flux, TPK3 modulates specific functions of the proton motive force in the light (non-photochemical dissipation of excess light, electron flow efficiency), ultimately improving plant fitness. Very recently, other channels/exchangers have been suggested to play similar roles. Here, we propose to establish the rules governing ion modulation of photosynthesis using a comprehensive and integrative approach indifferent model organisms. Complementary expertise in our team (electrophysiology, spectroscopy, bioinorganic chemistry and molecular genetics) will allow to identify chloroplast ion channels, generate down-regulated mutants, characterize their function in vitro (electrophysiology) and in vivo (using Stark spectroscopy) and collate their phenotype to ion dynamics in vivo (developing new fluorescent sensors for ion flux imaging in living cells). This will allow the complex regulation of light conversion into chemical energy to be dissected. Focusing on multiple phylogenetically relevant organisms (green algae, diatoms, mosses and vascular plants) is feasible due to expertise in their physiology and molecular biology in this consortium, and will provide essential insights into the evolution of photosynthesis regulation. Gathering these four investigators in a structured network will enable a cutting-edge, interdisciplinary and integrative approach to answer fundamental biological questions, which no individual lab could solve on its own.

Obesity is a global epidemic and major risk factor for leading killers including type 2 diabetes, cardiovascular disease, and cancers. Visceral adiposity - or intra-abdominal fat - is established to correlate strongly with these sequelae of obesity and increased mortality compared to subcutaneous (SubQ) adiposity, which is predictive of better health outcomes. While reprogramming of adipocyte metabolism is central to the distinct pathophysiology of visceral adiposity, the underlying molecular features and mechanisms remain largely uncharacterized. However, hallmarks of visceralized adipocytes have emerged in recent years that implicate altered metabolism and reactive oxygen species (ROS) production within mitochondria as upstream pathophysiological drivers.

I propose that reprogramming of adipocyte mitochondrial metabolism and ROS production are critical uncharacterized upstream drivers particular to the pathophysiology of visceral adiposity. To test this hypothesis, the first comprehensive determination of the mitochondrial proteomic, metabolomic, and ROS signatures unique to expanding visceral adipocytes in mice will be performed. I will employ comparative mass spectrometric approaches recently developed by my host laboratory that facilitate identification of unique pathway features in distinct adipocyte depots. I expect to identify and manipulate mitochondrial protein factors and metabolic ROS mechanisms that drive the distinct pathophysiology of visceral adipose expansion. These findings will set the stage for a new class of targeted therapeutic strategies for this major pathophysiological driver of obesity.

The aim of this research is to understand how visual processing is influenced by an animal’s behavioral state, intentions and actions. Activity in sensory areas of the cortex not only reflects the occurrence of a sensory stimulus, but also contains information about the stimulus context, its behavioral relevance, as well as goals, expectations, actions, or past experience. For example, even in primary visual cortex (V1), responses are boosted by attention, locomotion, and by mismatch between actual and expected visual input. These and other ‘internal state' variables must originate in part within specialized cortical areas, and be carried to the visual cortex by an extensive network of intracortical long-range projections.
I propose to image the flow of visual information as it is processed by multiple cortical areas during behavior. By coupling hemisphere-wide imaging to dynamical systems methods that identify 'internal state' variables in population activity, we will elucidate how the information conveyed by these pathways relates to behavior of the animals and how it influences responses in visual cortex. We hypothesize that different visually-guided behaviors will change the structure of intracortical feedback, and thus modify population dynamics in V1. We’ll identify higher cortical areas involved in complex visual tasks and target them for recordings to correlate the activity of specific neural populations with behavior. Likely, trial-to-trial fluctuations in activity between different cortical areas will influence the trial-to-trial differences in V1 responses, which may contribute to, or predict, the animal’s ability to detect or discriminate visual stimuli.

Fundamental to sexual reproduction in eukaryotes is the generation of haploid gametes from diploid germ cells during meiosis, errors in which lead to chromosome gain or loss (aneuploidy), a major cause of miscarriage and developmental disorders such as Down Syndrome. Orderly reduction of chromosome numbers requires that homologous chromosomes (homologs) pair and become physically linked through DNA recombination events called crossovers, prior to the first meiotic division. Crossover formation entails the introduction of DNA double strand breaks, which are repaired by a specialized homologous recombination pathway. To protect genome integrity and avoid aneuploidy, meiotic recombination is tightly regulated and meiotic progression is prevented by checkpoints until all homologs have paired and all DNA breaks have been repaired. Recently, a conserved meiosis-specific structure called the chromosome axis has been identified as a vital regulator of recombination: prior to homolog pairing, the axis is believed to promote DNA break formation and block meiotic progression. Upon successful homolog pairing, the axis is incorporated into the conserved synaptonemal complex, which is proposed to suppress DNA breaks and alleviate the block to meiotic progression.
We aim to uncover the molecular mechanisms of axis- and synaptonemal complex-mediated control of meiotic recombination, using a combination of biochemistry, structural biology, and genetics. We will use chemical cross-linking and mass spectrometry to first define the components and overall architecture of chromosome axes and synaptonemal complexes purified from mouse spermatocytes. Based on these data, we will reconstitute defined sub-complexes, biochemically dissect important interactions, and determine subunit and sub-complex structures using x-ray crystallography. These combined data will enable us to build detailed mechanical models of the chromosome axis and synaptonemal complex. We will validate these models using targeted mutations in in vitro-reconstituted complexes, and test functional predictions in vivo using genetic manipulations in mice. This full-circle approach will achieve a comprehensive mechanistic understanding of meiotic regulation by the chromosome axis and synaptonemal complex, and will aid the understanding and prevention of human aneuploidies and their associated developmental disorders.

The intermolecular association of misfolded proteins into fibrillar oligomers is a phenomenon of increasing interest due to its connection to disease pathology. For example, neurodegenerative diseases, such as Alzheimer’s, have been linked to the accumulation of such species in the brain. Despite the great amount of research devoted to the study of disease-related protein aggregation, a microscopic understanding of this process remains lacking. Herein, we propose to employ novel NMR methods to provide insights about the initial stages of aggregation of two proteins whose oligomerization is related to the development of ALS: SOD1 and TDP-43.
ALS is a disease that leads to the degradation of motor neurons in individuals, and is fatal within 2-5 years of onset. The progression of ALS has been related to TDP-43, an RNA-binding protein involved in transcription, and SOD1, a redox enzyme that metabolizes free radical-containing oxygen, via the discovery of aggregated forms of these proteins in the neurons of deceased ALS patients. In the case of both proteins, the native structural states are not believed to be responsible for aggregation; instead, transiently populated states (<5%), so-called conformationally excited states, are believed to be responsible. The examination of these species is possible via NMR techniques such as relaxation dispersion and chemical exchange saturation transfer. Here, we aim to study conformationally excited states of both of these proteins to reveal potential aggregation-prone structures and pathways, with the ultimate goal of potentially contributing to ALS disease prevention and/or treatment.

hERG, encoded by the human ether-a-go-go-related gene, is a voltage gated potassium channel and mainly expressed in heart, neural tissues and certain cancer cells. In heart, hERG can conduct potassium ions out of the cardiac myocytes and this current is critical in correctly timing the return to the resting state (repolarization) of the cell membrane during the cardiac action potential. Thus, in heart, hERG coordinates heart’s beating. Malfunction of hERG caused by inherited mutations in the HERG gene or inhibited by pharmacological compounds would cause potentially fatal disorder and even lead to sudden death. Thus, hERG is an important antidrug target in preclinical safety trials of all drugs. Compared to its important biological functions, detailed structural information of hERG is still unknown. Traditional crystallographic approach requires formation of highly ordered crystals, which has so far unsuccessful. I propose to determine atomic structure of hERG by single particle electron cryo-microscopy (cryo-EM), which does not require formation of any crystals, and to unravel the gating mechanism of hERG ion channel. The final goal of this project is to find the fundamental mechanism of drug induced long QT syndrome by determining a series of structures of hERG, and provide guidance for new drug design in pharmaceutical field avoiding side effect and therapeutic intervention of hERG related disease.

Bacterial biofilms are antibiotic-resistant, sessile communities that live on most moist surfaces on Earth, and cause chronic and medical device-associated infections. The central objective of the proposed research is to discover fundamental mechanisms that govern biofilm development. Previous studies have primarily focused on identifying essential genes for biofilm formation, and on understanding triggers that initiate biofilm formation. However, how cells dynamically arrange themselves within a biofilm, which cells are dividing, which genes are important in particular spatial and temporal domains, and how all of these features coordinate the self-organization of millions of cells into intricate multicellular morphologies, are central questions that remain unanswered. In this project we will use recent advances in microscopy technology to image Vibrio cholerae biofilm development at the single cell level, and to track the transcriptional dynamics of every promoter in the genome. These data will be complemented by mathematical models of biofilm growth, which are aimed at identifying minimal conditions for achieving the observed developmental complexity.

Since Weismann formulated the distinction between innate and acquired characteristics in the 19th century, the debate about the inheritance of acquired traits has raised many controversies in the scientific community. Following convincing arguments against, e.g. by William Bateson, this debate was then largely set aside. However, a number of phenomena and epigenetic mechanisms involving RNA, histone modification or DNA methylation have renewed interest in this area. Transgenerational epigenetic effects likely have wide-ranging implications for human health, biological adaptation and evolution, however their mechanisms remain poorly understood. Already, a number of human diseases including metabolic syndromes, cancer and heart disease have been shown to be mediated by transgenerational effects.
We have identified the recent discovery of RNA-mediated multigenerational stable inheritance in the nematode C. elegans as a unique opportunity to dissect the systems biology and mechanism as well as the organismal and species impact of transgenerational epigenetic inheritance (TEI). C. elegans is an ideally suited animal for this undertaking due to its short generation time of three days (organismal studies), small genome size (genomics and functional genomics), transparency (imaging) and genetic tool kit (genomic engineering).
To advance this area of biomedical research an integrated multi-disciplinary approach will be essential. Our team collectively has the skill set to have a transformative impact. We will integrate genetic, genomic, dynamic imaging, biochemistry and proteomic technologies into a quantitative description of RNAi-mediated TEI. This work will provide a mechanistic understanding of the expression of TEI in an animal. Furthermore, this project will test if and how TEI provides an environmental memory (“Lamarckism”) and examine its implications for the adaption of individuals and the evolution of a population. We anticipate that this work will help guide the analysis of TEI in other species including humans.

Ribosomes, the cellular protein production machines, are efficiently assembled from a large number of RNA and protein components in a short time. Although in vitro work has provided great insight into the mechanism of ribosome assembly, there are critical limitations to the in vitro approach. One major difference is that in vivo the RNA is co-transcribed in the presence of the ribosomal proteins. Another caveat in the past ribosome assembly work, is that almost all mechanistic findings are based on ensemble biophysical methods in which binding of each ribosomal protein is detected as the average state of all populations in which it is present. In the proposed project, I will investigate ribosome assembly at the single-molecule level using a scheme where the rRNA is transcribed in the presence of the r-proteins. I will develop the methodology for studying the co-transcriptional assembly of the 30S ribosomal subunit, first using total internal reflection single molecule microscopy, and then adapting it to the unique and more powerful zero mode waveguide technology. The latter method allows detection of weak, and so far inaccessible physiological relevant interactions, and allows observation of up to four differently fluorescently labeled macromolecules at a time. Once the fundamental technique is established for this model system, I can easily extend it to answer other key biological questions such as the influence of RNA modifying enzymes, ribosome assembly factors or even potential small molecule drugs on ribosome assembly in real time.
Overall, this new experimental approach will alter the mechanistic understanding of ribosome and ribonucleoprotein assembly in general.

The germinal center (GC) is a specialized tissue microenvironment of the immune system in which T, B and stromal cells cooperate in the evolution and production of high affinity antibodies and immunological memory. While GCs are a prominent feature of lymphoid tissues, there is a limited understanding of information transfer between the component cells and recent evidence for communication between different GCs further enlarges the spectrum of information processing and evolution of optimal functionality.
The main aim of this project is to develop an experimentally validated, predictive, and scale-bridging theory of the GC reaction, i.e. a theory that connects experiments on different spatial scales. Bringing together experts for quantitative GC experimentation at the molecular, cellular, and organismal level with multi-scale mathematical modelling, we expect to uncover the molecular mechanisms important for the development of optimized antibodies in the fight against invading pathogens. Research will unravel principles of the three-dimensional organization of the immunological synapse at cellular interfaces, of the evolution of high affinity clones at the cellular level of individual GCs, of the exchange of cells between different GCs within the same lymphoid organ, and of the coordination of system-wide humoral immune responses. With the resulting scale-bridging GC modeling platform we will explore a new degree of complexity in multi-scale modeling, which will rely on the exchange of sophisticated and innovative experimental approaches between the partner labs. All partners will have to go beyond their established research themes in order to tackle this challenge because the research will be focused on the interface of the different spatial scales.
Human GCs have not been investigated so far for technical reasons. With a highly innovative approach combining the expertise of all partners, the project will explore the possibilities and limits of extending GC research from the murine to the human organism. This will only be possible based on a team effort and transforms the research program of all partners.
The results of GC research in mouse and human will provide a fundamental understanding of information processing in a complex biological system, in which evolution of new functionality can be assessed on a timescale of weeks.

Mammalian skin develops from an ectodermal monolayer into a self-renewing, stratified epithelium to create a fluid-retaining barrier against pathogens. Employing live imaging and molecular genetics, I’ll explore this essential but poorly understood process. The major aims are to first characterize actomyosin and intercellular adhesion dynamics during epidermal stratification and differentiation in WT embryos. Then I’ll establish the degree to which the observed behaviors are stereotyped. Notch and Wnt signaling reporters will also be used to determine how cytoskeleton/adhesion dynamics may be coupled with changes in these pathways to drive differentiation. Finally, to provide insight into the molecular mechanisms governing coordination between actomyosin and cell adhesion networks, I’ll derive a list of potential regulators that are expressed by embryonic epidermis as it undergoes these developmental dynamics, and use lentiviral-mediated RNAi transduction to screen for candidates that are critical to epidermal stratification/differentiation. By repeating live imaging analyses in embryos mutant for these regulators, and combining this information with biochemical and whole-mount immunofluorescence techniques, I’ll assess how each component contributes to the formation of a stratified epithelium through spatiotemporally-regulated cytoskeleton and adhesion dynamics. The proposed work will shed light into the cell behaviors that contribute to and result from a switch between symmetric and asymmetric cell division, a conserved phenomenon that underlies not only stratification/differentiation of embryonic epidermis but also stem cell maintenance and homeostasis in adult skin.

In order to understand the brain, tools need to be developed to allow the investigation of interactions between many individual neurons. Noninvasive tools (MRI, EEG) provide structural and functional information, but at low spatial resolution. Invasive recording methods (patch-clamp, multi-electrode array) provide high temporal resolution data from single neurons, but are bulky and lack spatial resolution and throughput. For this reason, considerable efforts have been invested in developing optical detection methods including the utilization of voltage-sensitive dyes (VSDs) and genetically-encoded fluorescent voltage-sensor proteins (VSPs). Despite these advances, VSDs and VSPs are not yet preforming at the needed level of detection.
We therefore propose to develop solid-state nanoscale voltage sensors nanoparticles (vsNPs) that report on action potentials (APs) on the nanoscale. These devices could dramatically change the way we image and study nervous system activities, and how we interface these systems to provide novel therapeutic approaches and prosthetic strategies. The proposed vsNPs are based on the quantum confined Stark effect. They will be designed to be injectable, targetable, and to be self-inserted into the membrane. They will optically record, non-invasively, APs at the single particle level at the nanoscale, at multiple sites and across a large field-of-view. They will display high voltage sensitivity, high brightness, single-particle detection sensitivity, large spectral shift, fast temporal response, minimal photobleaching, large Stokes shifts, large two-photon cross sections, excellent NIR performance, and compatibility with lifetime imaging.
We will pursue the following aims: (i) synthesis of heterostructure vsNPs (Weiss & Oron) and characterization of their performance (Enderlein, Weiss & Oron); (ii) development of functionalization methods based on ligand exchange with transmembrane (amphiphilic) peptides by self-assembly (Weiss) and optimization of vsNPs insertion into the membrane (Weiss and Enderlein); (iii) development of optical vsNPS recording methodologies and benchmarking of vsNPS performance (Enderlein, Oron, & Weiss); (iv) demonstration of vsNPs utility in model neuronal systems (Triller), exploration of their use in studying synapses and neuronal integration of excitatory and inhibitory inputs (Triller).

Our recent sensations affect our current expectations and perceptions of the environment. Neural correlates of this process exist throughout the brain, and are loosely termed adaptation. In this proposal, we will address adaptation in the cerebral cortex. Sensory adaptation potentially carries a severe price of increasing the ambiguity of neural coding, since the same stimulus can give rise to different responses depending on the context and adaptation state. However, the ubiquity of adaptation suggests that it has considerable advantages, possibly serving similar functions across sensory modalities. More than 40 years ago Barlow suggested that adaptation reduces the dynamic range required to encode natural stimuli by emphasizing changes in intensity rather than absolute values. Later studies emphasized other roles, including predictive coding, optimizing neural coding and detection of unexpected stimuli. However, these ideas were developed using different modalities and different stimulation paradigms. As a consequence, we lack a theoretical framework that affords a general understanding of sensory adaptation in cortex. The goal of this project is to develop such a unified theory. Given the similarity in circuitry across neocortex, we suggest that neural responses adapt to ongoing statistical changes in input, independent of modality. We hypothesize that the diverse adaptation properties of specific subtypes of cortical neurons serve similar functions across sensory modalities.
Toward testing these ideas, we will use stimuli with similar time course to study adaptation in the three modalities (somatosensation, audition, and vision) and successively vary the statistical complexity of the stimulus to probe the context dependence of dynamical representations across sensory cortex; using the same recording techniques and using the same analysis methods. This approach will enable us to discover the common roles that neuronal subclasses play across sensory areas in adjusting cortical responses according to statistical properties of stimulus history. This research will be conducted in tight collaboration with theory, that will inform both the construction of the stimulus sets we will use and the analysis of our neural data. We believe that this approach will allow us to build a ‘grand unified theory’ of cortical adaptation.

Membrane proteins constitute roughly 25% of the proteomes in all living organisms, yet their production and folding in cells is poorly understood. One critical step for these proteins is attaining the correct topology, which comprises the insertion and orientations of all their transmembrane-helices (TMs). TMs are believed to insert into the membrane strictly during translation, yet recent studies predict that nearly half of the TMs may not be efficiently inserted co-translationally, and their insertion remains enigmatic. Recently, studies on a few proteins have shown that the transmembrane topology may sometimes change after translation. Here, I propose that these cases might be much more common than currently believed. In the proposed research, I will investigate how post-translational processes affect the topology of membrane proteins. I will generate detailed quantitative assays to map the topology of several multi-spanning membrane proteins. Using these assays, I will examine how two common post-translational processes affect topology: protein folding and protein oligomerization. Using genetic and mutational tools, I will perturb the folding or oligimerization of proteins and thoroughly examine how these perturbations impact their topology. Collectively, these studies will establish the potential link between membrane protein folding and topology. Ultimately, my studies will provide a holistic view on how membrane proteins fold, and answer unresolved energetic issues in membrane protein biology.

Spatial and temporal control of transcription activity is fundamental in animal development and disease. Establishment of precise on/off pattern of gene expression is regulated by the interaction between enhancers and promoters. Enhancers provide binding platform for a variety of sequence-specific transcriptional factors, thereby facilitating assembly of active transcriptional machinery at the promoters. Enhancers are often located at thousands base pairs away from upstream or downstream of target promoters. By looping out intervening sequences, enhancers directly interact with promoters during gene activation. However, the dynamics of this process has yet to be determined.
To explore the kinetics of enhancer looping, I will develop novel live imaging and visualize enhancer-promoter interaction in a living cell. Recently, real-time monitoring of transcription activity in a developing fly embryo has been developed. This relies on fluorescent labeling of nascent transcript by using specific interaction between GFP-tagged bacteriophage MS2 coat protein and its cognate MS2 stem loops. By introducing RFP-tagged PP7 coat protein and PP7 stem loops into this system, I will develop two-color live imaging. By creating series of transgenes that contain a single enhancer located between MS2 and PP7 reporter genes, I will analyze the kinetics of enhancer looping among two target promoters. Furthermore, by combining BAC transgenesis and live imaging, I will directly visualize interaction between distal enhancer and target promoter in cis and trans. This research project will allow first quantitative measurement of enhancer looping in a living cell.