We propose to unravel a long-standing mystery in neuroscience: the origin and functionality of endogenous extracellular electric field in the brain. While neuroscientists have heavily relied on extracellular recordings to study mechanisms of brain processing very few have addressed their identity. We focus on the local field potential (LFP), the slow portion of the recorded extracellular voltage (Ve), thought to reflect neural circuit computations. We seek to quantitatively understand the origin of the LFP in neocortical and hippocampal structures and to manipulate such fields to characterize the extent to which these can directly affect the state and function of neurons and neural populations through field (ephaptic) effects. We do so in a highly interdisciplinary manner allowing us to study these effects in multiple scales. We will use novel multi-patch setups, silicon probe recordings, optical stimulation protocols to obtain intra- and extracellular recordings at multiple locations in- and outside neurons both in the hippocampus and the neocortex to understand the origin of extracellular fields induced by different neural types and the contribution of compartments such as the soma or dendrites. Concurrently, optical stimulation, transcranial electric stimulation and massively parallel recordings will be conducted to study Ve and the endogenous field in anesthetized and behaving rodents. In addition to the experiments, we plan to carry out biophysical simulations of individual neurons as well as neocortical and hippocampal circuits to understand the origin of Ve (and the LFP in particular) by examining various Ve-contributors such as postsynaptic currents from pyramidal neurons and other neural types. Finally, we seek to provide a theoretical framework to address the extent to which endogenous electric fields can alter or entrain the activity of a neural circuit via ephaptic coupling both in terms of subthreshold and spiking activity.

The kidneys are essential organs required for excretion of waste products and the regulation of fluid balance, blood pressure, blood volume, ion balance and bone density. The functional units of the kidney are the epithelial nephrons which can filter the entire blood volume every minute, creating 200 litres of filtrate each day and then recapturing 95% of this volume. All of the nephrons form during fetal development from two key stem/progenitor populations that interact with each other to form the intricately branched plumbing of the kidney as well as the filtering tubules. In humans, the number of nephrons per kidney varies 9 fold between individuals and there is an inverse correlation between nephron number and the likelihood of hypertension and kidney disease later in life. While much of kidney development has been described anatomically, due to a lack of quantitative and spatial modeling of this complex process across the fetal development, there is little understanding of how such variation in kidney size and nephron number can come about. The purpose of this application is to bring together mathematical modeling, advanced imaging and developmental genetics in order to develop a comprehensive model of the process of kidney development. Having created a framework that predicts the interactions between different cell types, we will test and refine this model by genetically altering the size of different cell populations to see how this affects development and whether the model holds. This will ultimately allow us to predict the outcome of perturbations in different cell compartments over space and time and potentially guide us with respect to understanding why nephron formation ends and what parameters might need to be altered to prolong the process.

The three-dimensional organization of the genome plays important and yet poorly understood roles in its regulation including control of gene expression and genome segregation. Control of gene expression by distantly located regulatory elements involves formation of chromatin loops driven by physical promoter-element interactions. Moreover, active and inactive segments of the genome appear spatially separated, which may contribute to their coordinated expression or silencing. Finally, formation of complex higher-order chromosome structures plays critical roles in chromosome condensation and segregation during mitosis. Thus, chromosomes assume multiple distinct conformations in relation to the expression status of resident genes, and undergo dramatic alterations in higher-order structure throughput the cell cycle. Detailed insights into chromosome conformation will greatly contribute to a more complete characterization of genome regulation.

Our aim is to combine light microscopy, cell and molecular biology with physics and statistics to determine a probabilistic map of the organization of chromatin domains in intact cells during cell cycle and in relation to gene expression. This project breaks important new ground by deriving high-resolution three-dimensional structures of higher-order chromatin organization and by discovering how these structures change dynamically during the cell cycle. This work will allow the characterization of the dynamics of higher-order chromatin folding and the identification of specific roles of genomic elements in these processes.

Birds are among the most colorful of vertebrate species, and most birds live in visually rich habitats. Not surprisingly, the avian retina is endowed with one of the most sophisticated cone photoreceptor systems of any vertebrate. Yet, despite this richness, the various optical adaptations of avian cones remain poorly understood. Our lack of knowledge stems, in part, from the fact that most studies of the visual system have focused on mammalian retina. In addition, studies of avian cone function have been hampered by the lack of model systems amenable to molecular/genetic manipulation. For this reason, almost all studies of bird vision have been restricted to wild-type animals. The overarching goal of this proposal is to deconstruct the optical adaptations of avian cone photoreceptors via an interdisciplinary combination of molecular genetics, optical physics, computer modeling and vision-guided behavioral assays in chickens. We plan to co-opt molecular/genetic techniques originally developed in the chicken to study embryogenesis, and use them to engineer post-hatch birds with modified cone photoreceptors. We will then apply cutting-edge optical analysis and modeling to both wild-type and genetically modified photoreceptors to elucidate their adaptations for light handling. Lastly, we will implement innovative vision-guided behavioral assays in newly hatched birds to link the genetic modifications and their optical effects to the resultant functional changes in vision. These studies will shed light on a range of amazing optical adaptations found in avian cones which are either absent or poorly developed in mammals and will thus reveal new insights into the visual world of birds.

Cyclic expression of genes (i.e. oscillation) is involved in basic processes such as cell-cycle and circadian clock. The molecular mechanisms behind the generation of these oscillations are being elucidated and have led to the identification of “biological clocks” in different biological processes. Ultradian oscillations, i.e. with periods much less than 24 hours, at the molecular level have been observed also in the major signaling pathways, but their relevance is still unclear. We hypothesise that one of the roles of cell signaling is to allow "mutual synchronisation" and "entrainment" of these oscillations across a tissue for robust coordination of biological processes at the tissue-level. Mutual synchronisation of oscillations happens when different “biological clocks” start “beating in time” by spontaneously synchronizing their oscillations via some form of cell-cell coupling, which we hypothesise may be mediated by cell-cell signaling. Entrainment refers to the existence of an external “pace-maker” signal that drives the individual clocks and forces them to beat in time. In this project we will investigate, via an integrated mathematical and experimental approach, the biological mechanisms of entrainment and mutual synchronization of cell-autonomous oscillators involved in signaling. Our objectives are: (1) to characterise, via mathematical modeling, entrainment and mutual synchronization of a population of cell-autonomous oscillators, using recent results from dynamical systems theory and numerical simulations; (2) to use synthetic biology to implement a synthetic cell-autonomous oscillator in mammalian cells to experimentally test entrainment and mutual synchronization, in the context of a well characterized oscillator mechanism; (3) to test the physiological relevance of entrainment and mutual synchronisaton of cyclic gene expression/protein localisation in mammalian cells. We will consider four examples: (i) the hes1-oscillator. We will investigate the 2-hour periodic expression of Notch signaling molecules in different cellular models, where these oscillations are known to occur. (ii) The p53-oscillator. We will focus on the tumor suppressor gene p53, which exhibits oscillations in its expression in individual cells. (iii) The Smad-oscillator. This refers to the cyclic nuclear shuttling of Smad proteins following stimulation by TGFb and serum. (iv) The Stat-oscillator. This is another example of cyclic nuclear shuttling following cytokine stimulation. The experimental protocol will require the determination of the fluorescence intensity in live cells as a function of time, at single cell resolution, and the ability to deliver periodic perturbations to the cells in the form of precisely controlled time-varying concentration of inducer molecules. To this end, we will develop innovative microfluidic ‘lab-on-a- chip’ devices in conjunction with time-lapse microscopy. These devices are able to trap single cells, or small populations of cells, for long-term data acquisition and to precisely control the extracellular environment. The output of this project will be to characterise oscillatory behaviour of genes involved in signaling in a cell population. By investigating different signaling pathways, where oscillations in expression or localization of proteins are known to occur, we will be able to understand whether different kinds of cell-autonomous oscillators make use of similar principle for their entrainment and synchronization, or on the contrary, they exhibit very different properties and rely on different mechanisms.

Glycogen, the glucose storage molecule, is considered an invaluable asset for cells. Glycogen Storage Diseases (GSDs) are pathologies linked to mutations of the genes in the glycogen metabolism pathway. Symptoms vary from mild metabolic defects to severe degenerative processes. The glycogen found in these disorders is abnormal in quantity, quality, or both. Our hypothesis is that these glycogen molecules harm the cell.

Wild-type glycogen branching enzyme (GBE1) deficiency (Andersen Disease, Adult Polyglucosan Body Disease (APBD)) results in the accumulation of aggregates of linear glycogen. We aim to identify the mechanisms by which such aggregates trigger cell dysfunction and death. To this end, we will model the impact of mutations found in patients on GBE1 structure and function. Using animal and in silico models, we will study the effects of abnormal glycogen accumulation on energy metabolism, mitochondrial function and cell viability. Involving techniques from biology, biophysics and chemistry, this project will reveal the deleterious effects of a molecule, glycogen, generally considered advantageous, and may offer new clues for the treatment of APBD and other GSDs.

Morphologically diverse networks of microtubules (MTs) underlie the regulation of key cellular processes such as chromosome segregation, division, morphogenesis, and motility of eukaryotic cells. In large part, the necessary flexibility in network architecture is made possible by the spatial and temporal regulation of sites from which MT growth is initiated. Although centrosomes are prominent sources of MTs, for many networks spatially dispersed non-centrosomal MT nucleation pathways are essential. Understanding how order arises out of an ensemble of non-centrosomal MTs requires the simultaneous investigation of nucleation and sorting mechanisms. Here, we will focus on the emergence of bipolarity in non-centrosomal spindles involved in chromosome segregation in mitotic and meiotic cells. Our aim is to understand at a mechanistic level how systems evolve to having two poles and how the distribution of MT lengths sets the size of the forming spindle. To this end we identify five conserved MT organizing activities that will be studied in an integrated manner. We propose to change system parameters and quantify the induced response using three complementary approaches. The Goshima lab will study bipolarity in the mitotic spindle of the moss Physcomitrella patens, a system that lacks centrosomal nucleation altogether. The Janson lab will reconstitute MT networks from selected purified components whilst controlling MT nucleation spatially. The Kruse group will develop physical descriptions of MT networks explicitly accounting for MT end dynamics. The integration of the three lines of research will be accomplished by quantitative comparison of protein distributions in spindles and by assessment of system responses to perturbations. The combined studies will allow us to characterize fundamental pathways generating bipolar MT arrangements. Beyond spindles, we expect that these mechanisms will significantly deepen our understanding of the vast variety of non-centrosomal MT network architectures observed throughout biology.

Cancer is a dynamic ecological and evolutionary community of cells where mutations in genes give rise to new clones of cells that either survive or become extinct. We propose that cancers can evolve by cooperation, whereby two or more mutations occur in separate cell clones, which can then cooperate to produce an emergent malignant phenotype. This contrasts with the conventional theory, in which cancer-causing mutations accumulate in one clone that has every property needed to be malignant. However, often more than one clone persist in cancers. If more than one clone persist, why should only one be active? And if more than one are active, how do they interact? Cooperation is possible, but does it occur? We will test this new idea of how cancers evolve using an integrated interdisciplinary approach of genetic analysis, biological assays and mathematical modeling. We will genotype microscopic parts of human tumours and use innovative mouse models to determine how often tumours arise from two or more clones. At the same time, we will use a mathematical multicellular simulation model of cancer to explore the biological consequences of clonal cooperation and to guide interpretation of the data obtained from the laboratory studies. The outcome will be a new genetic paradigm for cancer that could revolutionise cancer research and therapy.

The long-term health of all metazoan cells is inextricably linked to protein quality control. An imbalance in protein homeostasis, whether caused by the expression of mutant proteins or environmental and physiological stress, can result in severe molecular damage to the cell, dysregulation of tissues leading to pathology, and increased risk of protein conformational diseases including cancer, immunodeficiencies, metabolic disease, and neurodegeneration. Adaptation to stress and survival, when challenged by disease, requires an ability to sense damaged proteins and to coordinate induction of protective stress response pathways, chaperones and clearance networks.

This proposal brings together the complementary strengths of the Hartl lab at the Max Planck Institute of Biochemistry (Martinsried, Germany), the Nagata lab at Kyoto Sangyo University (Kyoto, Japan) and the Morimoto lab at Northwestern University (Evanston, USA) to decipher the mechanisms underlying the maintenance of protein homeostasis (proteostasis) at the biochemical, cellular, and organismal levels. We will employ quantitative proteomics to map the proteins that are sequestered by disease protein aggregates, identify different aggregation states and the toxic species that mediate aberrant protein interactions. Bioinformatic analysis of the physico-chemical properties of the candidate proteins will provide insight on function, localization and conservation and reveal features of proteins at-risk and the ability of proteostasis modulators to maintain functionality. Together, this represents a comprehensive analysis of the primary causes for proteostasis collapse in stress and aging and strategies to prevent or reduce damage and decline.

Key to the success of this proposal is the innovation described above, and that this can only be accomplished in a concerted effort by which we form new teams comprised of lab members from each group. We posit that only together will it be possible to address the ideas presented here. We will train talented investigators with a much broader scientific perspective and capabilities employing tools and approaches normally limited to cell biological, proteomics, or organismal approaches. Such efforts will be invaluable for the next generation of biomedical researchers as we work together to tackle such large-scale biological problems as proposed in this grant.

The catacombs of ancient Egypt contain literally millions of well-preserved Sacred Ibis (Threskiornis aethiopica) mummies. These birds were revered by the Egyptians from the Predynastic period (7,500 – 5,100yrBP), by which time their relationship to the god Thoth had been well established. Ibis have been a focus of worship ever since and by the Late Period (~2600yrBP) the numbers being mummified had reached the millions. At one site alone in a single year 10,000 were estimated to have been interned. These mummies are common in many museums around the world and large numbers are still preserved in catacombs from at least five sites in Egypt. Using archaeological and Optically Stimulated Luminescence methods we can precisely age these mummies. The research team aims to use ancient DNA and genomic methods to study both rates and modes of DNA evolution using this remarkable resource. In recent years, a number of ancient DNA studies have made significant contributions to our understanding of the processes that govern DNA change. However, generally these studies have been limited by the lack of large numbers of known-age remains from multiple time points. Specifically, we will recover more than 200 nuclear microsatellite DNA loci from samples of different ages and use these data to test mutational models that have been formulated to explain the evolution of these DNA repeat loci. In order to develop powerful methods of analysis we need to have a clear understanding of the factors affecting the development and evolution of microsatellite DNA alleles in vertebrates. The proposed research will directly contribute to this improved understanding. In addition, we will sequence more than 100 mitochondrial genomes from ancient Ibis. With the aid of new mathematical and evolutionary models we can greatly improve our estimation of molecular rates. These mitochondrial data will also help us to understand the Egyptian traditions relating to animal mummification. In preparation for this work, we have shown that we can recover both mitochondrial and single copy nuclear DNA sequences from Ibis mummies. The large number of known-age mummified Sacred Ibis in Egypt, together with the multidisciplinary nature of the team (ancient genomics, evolutionary biology, mathematics and Egyptology), provides a unique opportunity to study DNA changes over time.

Circadian clocks keep temporal order and help organism to anticipate daily changes in the environment. In mammals, most, if not all, differentiated cell types possess a circadian clock, with over 10% of their transcriptome changing throughout the day. This is achieved through a network of activators and repressors that form interconnected feedback loops. Cells likely acquire circadian rhythms only upon differentiation, since the only cell systems in which circadian rhythms have not been found are embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). Thus, pluripotent cells either cannot sustain a circadian clock, or the clock they possess neither depends nor causes transcriptional oscillations. As a result, pluripotent cells may offer an ideal system to develop a model for the emergence and maintenance of the circadian clock, and to study how key properties of these timing devices – such as robustness, plasticity, and temperature compensation -- are implemented into the system.

Here, we aim to decipher the molecular and cellular mechanisms behind the generation of the circadian clock. We will use innovative and interdisciplinary experimental and analytical approaches to dissect the activation of the transcriptional circadian cycle during ESC differentiation, as well as its deactivation during somatic cell reprogramming to iPSCs. We will build on our team’s expertise in circadian rhythms and RNA biology (SK), ESCs, differentiation and reprogramming (EM) and systems biology (AR). We will analyze circadian gene expression during the development or suppression of the circadian system using both live imaging-based single cell assays and genome-wide transcriptome sequencing (RNA-Seq). We will use computational algorithms to integrate the data into a system scale mechanistic model of the initiation and shut down of the circadian clock. We will further validate and refine the model by assessing these processes under genetic perturbation of key system components as well as by biochemical experiments (e.g. ChIP-Seq) in differentiated and pluripotent cells to test the regulatory connections in our model. Our work will provide a comprehensive view of a clock as a biological system, and will shed light on the initiation of the circadian clock during differentiation and on the role of this timing device during the exit from pluripotency. Our approach harnesses the power of two distinct facets of systems biology – single cell studies of system function and genomic studies of systems organization – to tackle a fundamental question in circadian biology, and can serve as a paradigm for similar studies of other biological systems.

At the molecular level no cell is like any other. Differences between genetically identical cells can in fact be quite pronounced, and these differences can have profound biological and biomedical implications. Stem cells divide and differentiate in response to physiological and environmental cues but exhibit considerable differences in how they do so; equally, tumour cells respond differently to drug treatment or radiation therapy. The mechanisms driving such variability among to all intents and purposes seemingly identical cells are only poorly understood. In order to understand these fundamental processes – which also have numerous biomedical and pharmacological implications – we have to overcome experimental and mathematical modelling challenges.

Here we develop the necessary experimental and theoretical frameworks, which allow us to probe single cells, measure the abundances of key signalling molecules, and infer mathematical mechanistic models for signal transduction. We will then analyze these mathematical models in order to understand better how the structure of the signal transduction network influences cell-to-cell variability. We will then test these models and their resulting predictions in further experiments. Most importantly, however, we will develop novel strategies which will allow us to guide cellular decision making processes and determine patterns of differences between cells. If we manage to reduce or otherwise affect such variability then we gain a novel way to guide the behaviour of cells. The scope for applying such techniques is enormous and could, for example, include novel therapeutic interventions, progress in regenerative medicine, as well as better understanding of stem cells and their behaviour.

At the same time we will also investigate what determines the behaviour of cells more generally. Often cells exhibit high levels of robustness against external perturbations. We will use the mathematical models inferred for single cells in order to generate hypotheses as to which molecules, molecular processes and interactions affect such robustness and how this can be interfered with.

Ribbon synapses are specialized for transmission of signals in sensory systems such as the retina. They are distinguished by ribbon-like structures at the active zone. The function, assembly and targeting of ribbons to active zones are not yet well understood. As such, our broad aim is to investigate the structure, assembly and function of ribbon synapses in the mature and developing mouse retina. We will focus on ribbon synapses of photoreceptors and bipolar cells which collectively are essential for relaying light information from the retina to the brain. We propose a highly interdisciplinary approach, combining the technical expertise of the investigators which include optical imaging, electrophysiology and molecular analysis. We will specifically address three key areas:

The first area concerns the assembly and maintenance of ribbons at synaptic sites. To date, is not known how RIBEYE, an essential component of ribbons, is dynamically transported and assembled into ribbon-like structures at active zones during development. Moreover, the cellular and molecular mechanisms underlying the maintenance of synaptic ribbons in mature circuits have yet to be determined. Thus, monitoring RIBEYE trafficking in real-time and in situ is important not only for increasing our basic understanding of the normal assembly and maintenance of ribbons, but knowledge gained from such dynamic views will help interpret the effects of perturbing RIBEYE-associated molecular interactions at synapses. In Aim 1, we propose to investigate how ribbons are trafficked, targeted and maintained at retinal synapses by generating novel transgenic animals expressing fluorescently-tagged RIBEYE and using live-cell imaging approaches (RW,FS,LL).

Second, some retinal synapses are functional during development prior to the assembly of ribbons at the active zones. How synaptic transmission changes before and after ribbons form is unclear. Knowledge obtained from physiological experiments addressing this question will advance our understanding of the function of ribbon-mediated transmission at sensory synapses. Thus, in Aim 2, we seek to understand how properties of synaptic transmission alter as ribbon synapses form and mature at inner and outer retinal synapses. Synaptic function will be assessed at different structural stages by electrophysiology (LL,RW,FS) and by two-photon imaging of synaptically-localized reporters of neurotransmission expressed in new transgenic mouse lines (LL,RW).

Third, ribbon function in neurotransmission has thus far been largely studied in mutants with altered transmitter release due to perturbed exocytosis. The Schmitz lab has discovered a novel function for the B-domain of RIBEYE itself, potentially affecting endocytosis. In Aim 3, we will determine the role of the enzymatic activity of RIBEYE(B) in the formation, maintenance and function of ribbon synapses using a knockout mouse (FS,RW & LL).

Haematopoietic stem cells (HSC) are the source for life-long regeneration of immune and blood cells thanks to their ability to balance proliferation and quiescence, differentiation and self-renewal. Much evidence has been accumulated about the role of extrinsic signals in regulating HSC and the scientific concept of the stem cell niche refers to the physical and functional environment responsible for the integration of such signals. Despite the long tradition of haematopoietic research, the organizational principles underlying the spatial arrangement of the niche components, e.g. cells, extracellular matrix, soluble factors, and their impact on HSC localization, function, and fate are still incompletely understood.

Due to its complexity, HSC–niche interactions cannot be fully tackled by a single methodology. An interdisciplinary, integrated approach is needed to obtain a systemic characterization of the spatio-temporal control of HSC localization within the bone marrow (BM) and its impact on stem cell fate. We propose an iterative strategy, implementing in vivo and in vitro data from specifically designed experiments into the development of mathematical models that investigate regulatory principles in silico. In particular, we will use 3D in vivo imaging and cell tracking in the murine calvarium to provide a realistic picture of the spatial arrangement of HSC and BM components. Furthermore, specific regulator molecules will be identified by a novel high-throughput PCR technology allowing for screening of HSC responses to cell–cell/cell–molecule interactions. Long-term single HSC tracking in bioengineered in vitro environments will add high-content data on HSC localization, migration, and function in response to different regulator gradients. Together, these data will provide the basis for the development of mathematical models of defined HSC–niche interactions that allow to quantitatively predict the dynamical properties of these interactions and the effect of spatial niche organization.

Our interdisciplinary approach will lead to the first quantitative model of the 4D dynamics (i.e., organization in space and time) of cellular and biochemical components determining HSC fate in the BM, opening the door to predictive targeting on the HSC niche for novel therapeutic applications.

Decision making spans a vast range of types and complexity, from choosing your partner to deciding whether to dive left or right to save a goal. Progress in a variety of fields has led to an understanding of the mechanisms that evaluate evidence and reach a decision. Studies of simple decisions have provided insight into the neurobiological mechanisms responsible for decision making in both monkeys and humans. These studies often require a choice between two possibilities, such as leftward or rightward motion of dots moving on a screen. Human and nonhuman primate experiments support a mechanism in which a decision is made when the accumulated evidence reaches a criterion level. Such an accumulation process explains both the accuracy of the decisions made over a range of difficulty levels as well as the time required to make each decision. However, a decision in the absence of an action (or the withholding of an action) has little value, and in recent years there has been substantial progress in understanding how a task goal determines the actions that will be made. In this new framework, the movement system sets up a feedback rule that determines how the commands we send to our muscles should depend on sensory inputs we receive, so as to optimizes certain criteria such as a tradeoff between effort and accuracy.

Traditionally, decision making and movement-control were believed to operate separately and sequentially, but recent evidence from cognitive psychology and neuroscience suggests a more intimate connection. A fascinating implication of this connection is that it can explain why, in the absence of additional information, a decision-maker might change his or her mind. We have recently developed a task in which we can monitor confidence in a decision and also changes-of-mind, showing that once the brain reaches a decision and commits to a choice, it can continue to analyze evidence that was in the pipeline but unavailable to impact the initial decision. Our project extends and unifies the fields of decision making and movement control with a combined behavioral, neurophysiological and theoretical approach within a common conceptual framework. Our hypothesis is that a common framework applies to the revision of both actions and ideas, and we will develop a theory of decision making and control that extends to the acts of vacillation and self-correction.

In recent years the use of advanced optical techniques has had a strong impact in neurobiology not only for visualizing neuronal structures and signaling processes, but also for controlling neuronal activity. This is possible thanks to a growing list of photosensitive tools that can modify their chemical or conformational structures upon illumination and precisely trigger the propagation of neuronal signals. In addition to the commonly used caged neuroactive compounds, genetically encoded light-sensitive proteins have recently been developed permitting neuronal activation and inactivation. In conjunction with spatiotemporally resolved photo-stimulation techniques, these photoswitchable proteins represent the most promising alternative to electrical stimulation, providing ways to control precisely in space and time the activity of specific types of brain cells.

These approaches require fast, flexible and precise illumination schemes, permitting the selective activation and imaging of sub-cellular regions or multi-cellular ensembles. The Emiliani group has recently developed the first one-photon scan-less holographic microscope where a liquid crystal spatial light modulator (LC-SLM) in the excitation path generates 2D and 3D multiple diffraction limited spots or shaped domains of excitation that can accurately cover a single cell or sub-cellular structures. They showed that shaped holographic illumination for large excitation areas has a significant higher axial resolution than a Gaussian beam, and concentrates all of the laser power on the desired region, thus providing the high intensity needed for fast photostimulation. They have also shown that further optical confinement can be achieved in two photon holographic illumination combined with temporal focusing.

The aim of this proposal is to use this innovative approach to study fundamental properties of central neurons. Holographic photolysis of caged compounds will be used as a novel means for mapping the subcellular distribution, modulation and function in neuronal signaling of ion channels in brain slices. Holographic photoactivation in vivo of the engineered light gated channel LiGluR will be used to elucidate the neural circuit basis of locomotion in zebrafish.

Neuroscientists have measured biophysical properties of individual nerve cells and monitored the activation of large brain areas in intact animals including humans. A coherent understanding of brain functions now requires quantitative insights into the interactions between individual neurons in order to understand how sophisticated computations are performed by neuronal circuits. This question raises a series of conceptual and technical challenges. First, connections between many or even all neurons in a circuit have to be determined by 3-dimensional imaging methods that achieve nanometer resolution throughout very large volumes. Second, activity patterns in the intact brain have to be measured with a resolution of individual nerve cells to quantitatively characterize neuronal computations. Combined measurements of activity and connectivity in the intact brain are then required to understand how the function of a circuit is determined by its structure. Third, activity and connectivity have to be manipulated in order to perturb a circuit and examine the effects onto neuronal computations and behaviors. Fourth, principles underlying neuronal circuit structure and function have to be extracted by mathematical approaches. To address these issues we will focus on the sense of smell in a small vertebrate, the zebrafish, which provides unique advantages. The connectivity among large numbers of neurons will be determined by novel 3-dimensional electron microscopy techniques and analysis methods based on machine learning. Activity patterns will be measured using non-linear optical methods that permit the visualization of activity across thousands of neurons in the intact brain. Advanced genetic methods will be used to manipulate the function of defined neurons in the intact brain. These approaches include optical techniques to precisely control the activity of individual neurons in space and time. Structure-function relationships of circuits will then be analyzed using mathematical methods and computational modelling. This interdisciplinary approach is expected to provide elementary insights into the mechanisms by which neuronal circuits process information and control behaviors, which is essential to understand how higher brain functions emerge from complex communications between large numbers of nerve cells.

A fascinating, unanswered question in biology is how some organisms respond to injury by regenerating the lost body structure. Two fundamental aspects of the problem are first, how does injury trigger a growth and patterning response? Second, how does the regenerating tissue “read” the overall size of the animal to produce an appropriately sized structure? While some molecular pathways have been implicated, a clear mechanism linking physical injury to cellular and molecular response is wholly lacking. Furthermore organ growth control remains a challenging problem in biology. Here we propose to couple the research efforts in three regeneration model organisms, Hydra, Axolotl (salamander) and Drosophila, to quantitative mathematical modeling as a means to address these issues in a novel way.

Drosophila has been a fertile system for identifying and analyzing molecular pathways involved in imaginal disc growth and patterning. We will therefore use the Drosophila imaginal disc as a starting point for identifying and analyzing mechanisms that link tissue injury to a growth and patterning response. Disadvantage: we cannot observe the regeneration in discs as it is happening. Therefore complementarily, we will use two classical regeneration models, Hydra and salamander (Axolotl) where regeneration can be exquisitely controlled and visualized in live animals to build on the physical link between injury and molecular signaling to cellular response. To harmonize the systems, we will all focus on the regeneration of epithelial structures where it is most likely that cellular responses may be similar: the Drosophila wing and leg discs are simple epithelia. In analogy, the Hydra body column consists of a bilayered myoepithelium, and finally, amputation of the Axolotl tail results in regeneration of the spinal cord through a neuroepithelium.

Therefore, we are not looking for molecules responsible for regenerating a particular body structure but for the general mechanisms used to control regeneration and size control of epithelial-based organs. Our specific aim is to come to a deeper mechanistic and integrative understanding of how signaling pathways respond to cell/tissue injury to initiate growth, patterning as well as size control response. For this reason mathematical modeling will help integrate the results obtained in the different phyla to identify shared mechanisms.

This project will investigate the forces between two double-stranded DNA molecules in a protein-free environment. Due to the chiral nature of DNA, these interactions are also chiral and affect DNA conformation and, in turn, are affected by topological constraints imposed on the molecule. Existing (Kornyshev-Leikin) electrostatic theory provides an explanation for the homology-promoted attraction. This theory also predicts a chiral torque that tends to make long DNA molecules wind around each other. The effect will be stronger if the DNA tracts in close juxtaposition are physically identical (i.e., homologous).

The double-stranded helix of DNA in cells is slightly (~30%) underwound relative to relaxed DNAs such as those used to test the homology interaction theory mentioned above. Additionally, cellular forces (in the pN ranges) cause transient extreme under- and overwinding of DNA. These topological states are essential for DNA transactions and without them cells die. The complex interplay between homology and supercoiling is the core subject of this project.

The project will determine: (i) the degree to which homologous tracts attract each other more strongly than non-homologous tracts; (ii) the force keeping two homologous tracts in juxtaposition; (iii) how homology recognition is influenced by supercoiling. These questions will be answered by performing a complementary series of single-molecule experiments and biochemical experiments with circular DNAs of varying degrees of supercoiling. Experiements will then be move to inside live cells. The experiments will be supported and rationalised by a theory, to be developed. This new theory requires the merger of the continuum theory of elastic braids and the theory of chiral electrostatic DNA-DNA interactions. The theory will distinguish juxtapositions of homologous and non-homologous DNA tracts and will allow us to investigate the dependence of supercoiled structures of DNA on structural and environmental parameters, including homology length and type and concentration of counterions.

We will build the minimal molecular engine required to initiate bacterial division by reconstituting its molecular machinery (the divisome) and reproduce its vital functions in vitro. Cytokinesis is a fundamental process that is known in bacteria in sufficient detail to provide a comprehensive list of its molecular effectors.

Our novel idea is to reconstruct and track the operation of the minimal functional protein set needed to initiate division (the proto-ring) and the two main positioning mechanisms (Min system and nucleoid occlusion) that select the constriction site.

We will use complementary biochemical, biophysical, genetic and imaging methodologies to reconstitute the proto-ring components (FtsZ, FtsA and ZipA) into artificial membrane-bounded compartments that mimic the divisome organization at the cell membrane. The use of biomimetic materials, vesicles and nanodiscs, as substrates on which to reconstitute the machine will be complemented using biovessels to assemble the proto-ring within structures that reproduce the cellular environment with minimal genetic information. Ideal conditions regarding the composition and shape of the membrane compartments will be designed for divisome reconstruction in nanodiscs, biomimetic bilayers, giant vesicles, and membrane tubes pulled to various geometries.

Research will then focus on the reconstruction and stability of proto-rings in biovessels, the interactions and dynamics of proteins within the division ring and how ring constriction is regulated. In vivo top-down strategies will be designed to minimize the components necessary for cytokinesis. The mechanism of proto-ring positioning by Min proteins and the membrane deformation imposed by proteins responsible for its constriction will finally be investigated.

Because of their relative simplicity, bacteria will probably be the first cells to be fully understood at a systems level, and our results will help usher in the new field of synthetic biology of cell division and will open new horizons for pharmacological applications.