Dendritic spines are small dendritic protrusions (~0.5 µm in diameter) where most excitatory synapses reside. Spine enlargement is associated with, and essential for, induction of long-term synaptic potentiation (LTP), a cellular model for learning and memory. Following LTP induction, the spine volume increases rapidly by 3-4 times within 1 min and decays over several minutes, followed by a smaller sustained enlargement lasting more than 1 hour. This spine structural plasticity requires re-organization of the actin cytoskeleton, and exocytosis and endocytosis of vesicles. However, the detailed dynamics of membrane trafficking and cytoskeleton rearrangements during spine growth are unknown. This project proposes to develop a technique to image structural dynamics of spines with nanometer resolution using high-speed atomic force microscopy (AFM). By resolving the detailed structural changes including filopodia extension, membrane trafficking, and rearrangement of surface proteins during the spine growth, this project is expected to lead to a better understanding of the mechanisms underlying structural plasticity of the spine and LTP, and ultimately learning and memory.

This project seeks to understand how cells migrate in complex environments. While cell migration on flat substrates has been thoroughly studied, cells migrating in vivo are faced with substrates of varying geometry, molecular composition and mechanical properties. Here, the cells show a much richer phenotype and are known to adapt their internal machinery to the extracellular environment. Different cell types exhibit specific migration strategies such that some of them use predominantly actin polymerization-powered protrusions, while others use motility based on pressure-driven blebbing as a default. Furthermore, cells have been observed to switch between these two different migration strategies upon manipulation of the actin polymerization dynamics within the cell, but also upon modification of such substrate properties as rigidity and adhesiveness. We will employ zebrafish germ cells and mouse leukocytes as in vivo model systems that represent the “professionally” blebbing vs. the actin polymerization driven phenotype. In vitro, these and other model cell types will be subjected to genetic, pharmacological and most importantly environmental manipulations that will uncover their range of plasticity regarding force generation and transduction. We will study how the cellular protrusive and contractile activities respond to variations of the geometry, adhesiveness and stiffness of the external substrate and to the presence of external loads. Readouts will include the dynamic visualization of cytoskeletal components, morphological response of the cell and actual motility, while forces exerted by the cell will be recorded quantitatively. Our data will be analyzed in the light of physical models to understand the mechanisms by which internal and external factors affect the strategy of cell motility, and to identify the key physical parameters governing the efficiency of actin-powered protrusions and bleb-based motility. The proposed research will establish novel techniques to quantitatively measure forces and visualize cytokeletal dynamics in a wide range of engineered as well as physiological settings. The integration of experimental data and theoretical modeling should ultimately lead to the development of a universal phase diagram that allows to predict the migratory strategy of a cell as a function of internal and environmental conditions.

Certain types of memories, especially fear memories, are stored in collections of cells in a part of the brain called the amygdala, in a manner that depends on the activity of the transcription factor CREB.

Experiments in which CREB activity was altered in subsets of neurons in the amygdala revealed that enhancement of CREB activity can enhance memory formation in those neurons and inhibition of CREB can decrease it. However, these experiments controlled CREB levels on time scales of days to weeks, whereas a mouse can form memories as a result of a single event lasting a few seconds. Is CREB required precisely during this training event? Soon after? Just before?

Memories, once formed, are not static but may be reconsolidated or undergo extinction depending on the nature of further experience. These processes also depend on CREB, but again it is unclear when CREB is required.

Using a combination of computational protein design, genetic selection via phage display, and structural biochemistry we will create light-switchable versions of CREB (photo-CREB) and a dominant negative form of CREB (photo-DN-CREB). Photo-CREB will allow activation of CREB with blue light irradiation. Photo-DN-CREB will allow inhibition of CREB with blue light irradiation. Both processes will be reversible (i.e., activation or inhibition will cease after the light source is removed). These proteins will be chimeras of the basic leucine zipper (bZIP) DNA binding region of CREB and the light sensitive protein PYP (photoactive yellow protein). These designed and evolved proteins will be analyzed and optimized in vitro using a range of biophysical techniques (X-ray, NMR, DNA binding assays). Experiments with cells in culture will assay kinetics of photoswitching and control of transcription using CREB reporter assays. Light-switchable CREB proteins will then be expressed in freely moving mice with optical stimulation delivered with an optical fiber directly to the mouse’s brain.

This approach using light-switchable proteins will permit the processes of memory formation to be probed on a time scale of seconds to minutes - an unprecedented degree of temporal resolution – and will therefore enable fundamental questions about the role of CREB in memory formation to be addressed for the first time.

As mechanical systems, cells control their shape via tight coordination of the dynamic self-assembly of cytoskeletal polymers (e.g. actin filaments) and modulation of intracellular tension exerted among these filaments by molecular motor proteins (e.g. myosins). Highly dynamic and contractile actomyosin-based structures, such as stress fibers of non-muscle cells and sarcomeres of muscle tissue, lie at the heart of fundamental cellular processes including morphogenesis, establishment of polarity and overall motility. Although it is well established that stress fibers play central roles in cell biology, the chemical and physical principles driving the formation of these highly organized contractile structures are largely unknown. The identification of the biochemical mechanisms and biophysical laws that govern the macroscopic organization of the cytoskeleton in cells is rendered difficult by the complexity of the systems. Simplified experimental systems in which actin dynamics can be reconstituted in the presence of regulatory factors and motor proteins are needed to understand the mechanism of stress fiber formation and the consequences of myosin-dependent force generation on the organization and stability of complex actin structures. We will use a multi-disciplinary approach to reconstitute the assembly, turnover, and contractility of stress fibers in vitro and determine how the mechanical and contractile properties of intermediate and assembled structures depend on the individual protein components. Our approach will integrate a new versatile micropatterning method capable of imposing spatial boundary condition to the growth of actin filaments, the use of diverse molecular motors with unique biochemical properties, and the use of AFM and laser trap nanometry to obtain reliable determination of the generated contractile fiber structures and mechanical properties. Theoretical biophysical modeling and computer simulation will complement these experimental works. General principles regarding cell mechanics and the hierarchy of molecular components will emerge from this multi-disciplinary work.

Many Nobel Prize winning advances in biology were driven by x-ray studies of crystallized biomolecular systems. A recent revolution in x-ray sources, the x-ray free electron laser (XFEL), eliminates the radiation damage problem that plagues studies using x-rays and electrons. XFELs open a new horizon of atomic resolution study of non-periodic biological structures. The Linac Coherent Light Source, LCLS, at the SLAC National Accelerator Laboratory recently became the first operational hard x-ray FEL. Its x-rays are a billion times more brilliant than any other source. One of the most promising scientific applications of XFELs is coherent diffraction imaging (CDI) of non-crystalline biological samples at sub-nanometer resolution. The basis of this "diffract-and-destroy" method is the recent observation that a sufficiently brief x-ray pulse provides a useful diffraction pattern before significant radiation damage commences. Our international team is comprised of the requisite expertise to uncover new scientific discoveries in complex non-periodic biological systems using XFEL experiments. We have i) extensive experience in algorithms for CDI, ii) over six years of experience in XFEL experiments, including LCLS, iii) the requisite biological expertise for virus preparation and data interpretation, and iv) unique experimental apparatus. Our primary objective is to achieve the highest resolution structure of algal (PBCV-1) viruses. Little is known structurally about the algal virus PBCV-1. 3D reconstructions of PBCV-1 at 2.2 nm resolution exist but many questions remain open. These include, for example, how the genome is packed and the structure of the unique vertex involved in infection. Since single particle CDI is so new, significant effort will be directed toward method development. At present, there are at least two computational methods in the literature that we will use for determining the relative orientations of diffraction patterns, permitting a 3D data set of diffraction intensity to be assembled for phasing and image reconstruction. XFEL-CDI methodologies are now on the verge of contributing to the biological questions posed for many viruses, including PBCV-1. We were awarded LCLS beamtime to begin addressing these questions and will use HFSP support to help stimulate new biological frontiers by the x-ray imaging of viruses.

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.