Plants, either directly or as fodder for livestock, feed the world and are increasingly important sources of fuel and fiber. Unfortunately, approximately half of the global crop yield each year is lost as a result of adverse environmental conditions. Drought is the abiotic stress with the greatest impact on crop yield worldwide and is expected to be exacerbated by global climate change. Plant physiology is affected by a number of other stressors including salinity, heavy metal ions, and reactive oxygen species. During times of stress, osmolytes and potassium ions accumulate to high concentrations inside of plants. These species are known to generally affect the folding of RNA, which often alters gene expression. Our hypothesis is that plants use certain RNA and DNA switches as a way to sense stressors and their indicators, which subsequently alters gene expression. Several plants have been fully sequenced, including the model plant species Arabidopsis thaliana, which allows us to apply a full complement of bioinformatic, biochemical, biophysical, and biological approaches. Our overall approach includes discovery and characterization of RNA/DNA switches in plants, including new and novel classes of stress-responsive switches, using a combination of biologically and chemically motivated searches driven by bioinformatics and experiments. We will then characterize the structures and physical and chemical properties of these RNA/DNA elements both in vitro and in planta. Techniques to be applied include enzymatic and chemical structure mapping of the nucleic acid, mutagenesis, thermodynamics, single molecule studies, small angle X-ray scattering (SAXS), fluorescence resonance energy transfer (FRET), and computation modeling. In addition, transgenic approaches will be used to assess the impact of RNA/DNA folding events on plant drought tolerance. Feasibility of this research plan is supported by initial bioinformatics searches and biochemical studies. These studies, if successful, will elucidate key mechanisms for how plants respond to stress at the cellular and molecular levels, and will likely help define mechanisms for gene regulation in response to stress in other eukaryotes.

Folding of epithelial sheets is one of the most ancient mechanisms leading to the formation of three-dimensional structures in developing tissues. In a highly simplified picture, epithelial morphogenesis can be separated into two steps. First, inductive signals establish two-dimensional patterns of gene expression across the epithelia. At the next step, these patterns are converted into spatial patterns of force generation and mechanical properties of cells, thus controlling the folding of a sheet into a target morphology. Previous studies of epithelial morphogenesis focused on single genes and small networks, but a systems-level model of morphogenesis in any given experimental context is yet to be developed. In developing such a picture, the key questions are related to the number and identities of involved genes, diversity and dynamics of their expression patterns, mechanisms of pattern formation, and connection between patterning and morphogenesis. We will investigate these questions in the context of the formation of the Drosophila eggshell, an established genetic model of epithelial morphogenesis.

This will be the first time that genetics, systems biology, cell biology, nonlinear dynamics, and continuum mechanics will be brought together to study patterning and morphogenesis in a system highly amenable to genetic manipulations. We will establish a two-dimensional atlas for the expression of dozens of genes involved in eggshell morphogenesis and formulate quantitative models for the formation of these patterns by signaling pathways. Based on live imaging and genetic experiments, we will formulate the hypotheses regarding the connection between patterns of gene expression and the mechanical properties of patterned epithelia. We will explore these hypotheses computationally, using a continuum mechanics approach, and experimentally, using live imaging. The result of this integrative approach will be the first experimentally validated systems-level model for two-dimensional epithelial patterning and resulting folding into a target three-dimensional morphology.

It is largely unknown how specific connections are established among the hundreds of cell types of the vertebrate CNS. Previous work has implicated cell adhesion molecules of the immunoglobulin, cadherin, and other families as mediators of this process. We have formed a collaborative network to explore a new gene family encoding leucine-rich repeat transmembrane (Lrrtm) cell-surface receptors. Individual members of the Lrrtm family are expressed in mutually exclusive patterns in the retina and tectum of zebrafish. The specific aims are 1) to establish a molecular interaction network for the Lrrtm family using biochemistry and biophysics (G. Wright), 2) to characterize the expression patterns of Lrrtm receptor/ligand pairs in the visual system of zebrafish using two-color fluorescent RNA in situ hybridzations and monoclonal antibodies (Wright & Baier), 3) to test if Lrrtms are necessary and/or sufficient for synaptic specificity by creating loss-of-function mutants, taking advantage of the Wellcome Trust Sanger Institute mutation resource, and by generating transgenic lines able to drive expression of Lrrtms and their genetically engineered variants in subsets of neurons (Baier & Wright), and finally 4) to identify the exact developmental step at which Lrrtms act (Trauner, Baier, & Wright). For this last experiment, we will genetically engineer an Lrrtm that can be masked with a photolabile, chemical compound and “unmasked” with ultraviolet light. We will remove the mask at defined time points before, during, and after synapse formation to determine if Lrrtms are required for neurite guidance, cell-cell recognition, synapse formation, and/or synapse consolidation. Together, these experiments will reveal the function of a novel candidate gene family in neuronal connectivity and will devise a broadly applicable paradigm for the study of other signalling systems in the developing nervous system and beyond.

Every day humans and animals are exposed to harmful micro-organisms, yet do not become ill. If you think about it, this is remarkable. We generally consider the absence of disease as not very exciting, and often research in infection biology is done with the aim to cure a disease or to prevent disease by development of specific vaccines. We propose to study the efficient systems that become involved to protect the host at the most vulnerable sites of the body. Mechanical barriers, innate immune receptors, and the adaptive immune system play a vital role in protecting the host from pathogenic invaders that attack the skin, lungs or gastro-intestinal tract. However, before the protection of the innate immune receptors kicks in, another “line of defence” seems to provide immediate protection. This protective system consists of peptides and proteins (“Innate Immune Effector Proteins”, IIEP), that are either soluble or loosely attached to mucus and other extracellular structures, and that exert direct effects (aggregation, killing) on a variety of pathogens ranging from viruses to fungi. Among these fascinating molecules, which appear to have largely physical mechanisms of action, are small antimicrobial peptides (AMPs) as well as large “pattern recognition” proteins such as the collectins. Our aim is to observe (for the first time) and subsequently analyze and understand the first encounters of pathogens with host proteins in the ARENA, a microfluidic model system that will mimic the interface between host and outside world. Our model system will mimic the air-water interface of mammalian lungs, and will contain bioluminescent bacteria that will encounter the lung collectins SP-A and SP-D plus the defensin hBD-1 and the cathelicidin LL-37. The ARENA chip device will permit carefully controlled on-chip dilutions of protein mixtures, bacteria (and host cells) by merging a stream carrying bacteria with a stream of IIEP to form a dynamic interface. Time lapse video microscopy is used to track aggregation and death of bacteria by fluorescent markers. Multiple tests per chip under multiple conditions can be run simultaneously.

Hypotheses: 1. The assembly state of collectins determines the propensity to quickly immobilize invading pathogens. 2. Concerted actions of collectins with AMPs kill bacteria effectively and protect the airways from biofilm formation.

A challenge in elucidating the signaling networks that control the organization of mammalian cells into tissues lies in the complexity of the whole organism, which limits analysis at molecular resolution. We propose to bridge this gap in scale between cell signaling networks and collective cell behaviour during tissue morphogenesis, by studying in experimental models of increasing complexity EphB2/ephrinB1-controlled cell positioning during vasculogenesis. Specifically, we will achieve this goal by integrated use of:

- novel phosphoproteomics strategies for unbiased elucidation of phosphotyrosine (PY)/

protein tyrosine phosphatase (PTP)-signaling networks

- novel strategies for analyzing PTPs, and high-resolution imaging of biosensor-tagged

Eph/ephrin signaling nodes during vasculogenesis.

Functional phosphoproteomics of metabolic (SILAC) labeled Eph+ or ephrin+ cells, and identification of ROS-oxidised PTP’s, will survey PTP-controlled PY signaling during Eph/ephrin-facilitated cell navigation, while distinguishing between distinct cell populations. Genetically encoded optical sensors for Eph/ephrin and PTP signaling activities will allow recording the correlation between bidirectional Eph/ephrin signaling activity within cells and the assembly of tissue during vasculogenesis. The underlying experimental models, with a common concept of confronting Eph-expressing with ephrin-expressing cells, will increase in complexity from simple 2-D co-culture and in-vitro capillary tube formation, to ‘confrontation’ cultures between tumour- and ES cell- spheroids to trigger in-vitro neoangiogenesis. Finally, modification of selected endogenous proteins in ES cells with fluorescent tags and biosensors by homologous recombination will allow assessment of emerging endogenous signalling clusters during ES cell differentiation.

Overall, these strategies will elucidate the molecular concepts underlying self-organized tissue assembly and will provide the experimental platform and knowledge base for our long-term goal - to assess at molecular resolution, but in the context of a whole animal, a signaling circuit controlling tissue patterning. Eph RTKs, as one of the principal signaling systems that control developmental cell navigation through feed-back controlled bi-directional signaling circuits, provide an ideal paradigm and experimental model system for this strategy.

Freezing avoidance in Antarctic fishes is enabled by the presence of antifreeze glycoproteins (AFGPs) in most of their body fluids, but how they deal with circulating ice remains unresolved. We have shown that AFGPs are synthesized in the exocrine pancreas and the anterior stomach and secreted into the gut, where they serve in defence against imbibed ice. We have also identified high levels of AFGPs in the blood, where they defend against ice crystal growth in the circulation. In this investigation we will combine biology with synthetic organic chemistry to address how AFGPs get from the gut into the blood and how fishes remove circulating ice.

To determine how AFGPs traffic from the gut to the blood, we will synthesise fluorescently-labelled AFGPs and use confocal microscopy to visualize the pathway of uptake following oral intubation. Mass spectrometry of blood samples obtained after the introduction of isotopically-labelled AFGPs will be undertaken to confirm translocation from the gut to the circulation.

We postulate that removal of ice from the circulation occurs in the spleen via a receptor-dependent interaction involving ice-bound antifreeze. Recently, we have identified a second antifreeze protein, termed antifreeze potentiating protein (AFPP), which also adsorbs to ice. This new observation leads us to hypothesize that the adsorption of both types of antifreeze is required for the recognition and removal of circulating ice.

To identify the AFGP receptor and the cell involved, we will prepare dual function orthogonally-labelled AFGP probes containing a UV-activatable cross-linker that will covalently bind to the putative receptor on photoactivation. An azide chemical reporter group will also be incorporated into the AFGP probe to enable the use of “click chemistry” to attach a biotin-conjugate to the cross-linked putative AFGP receptor. The receptor will be identified following streptavidin affinity chromatography of cell membrane extracts prepared following the culture of spleen cells in the presence of excess probe. Such probes, in conjunction with routine cytological analysis, will also enable us to identify the cell type involved. Following complete characterization of AFPP we will attempt synthesis and similar modifications to identify its receptor. Taken together with knowledge of the pathway of AFGPs to the blood, we will finally have a complete picture of the mechanism of organismal freezing avoidance.

The activity of nerve cells in behaving animals correlates with sensory processing, decisions and motor control. Such correlations are necessary, but not sufficient, to link specific aspects of brain activity to behaviour. In order to obtain causal evidence it is critical to manipulate identified neuronal circuits in the brain during behaviour. This demanding goal is the objective of this research program, where as a team we will develop, test and apply genetic technology for highly specific control of neuronal networks in the mammalian brain which will be quantitatively related to behavioural report of sensory perception. Edward Callaway of the Salk Institute in La Jolla, California will develop genetic tools for specifically controlling the activity of well-defined populations of cells in the rodent brain. Carl Petersen of the Brain Mind Institute in Lausanne, Switzerland and Mathew Diamond of the International School for Advanced Studies in Trieste, Italy will use these genetic tools to analyse the neuronal basis of perception through simultaneous measurement and manipulation of cortical activity during performance of tactile detection and discrimination tasks. Carlos Brody of Princeton University will carry out information theoretic analysis and neuronal network simulation necessary for quantitative evaluation of causal relationships between neuronal activity and behavioural report of perception. Together this team of investigators aims to provide pioneering evidence for different functional roles of the diverse types of neurons found in the neocortex. Furthermore, the rigorous testing of such genetic technology in vivo in the mammalian brain will provide an important step towards the many potential clinical applications for treating brain disorders.

Living cells must be highly organized, therefore many of their components are transported to particular places and anchored there. Disruptions in this transport and localization cause disease, particularly neuropathologies. Many copies of proteins are made from each working copy of a gene (messenger RNAs, mRNAs). Proteins are moved to their correct location or, more efficiently, made there from mRNAs that have been moved. Although the mechanisms that move proteins to their destinations have been intensively investigated, those that carry mRNAs have been much less studied. Indeed, RNAs have recently been discovered to participate in a growing number of new roles and unexpected qualities in cells.

The molecular “zip codes” hidden in RNAs that specify destinations have only been deciphered for a few RNAs. An important goal of this project is to create intelligent methods that can find these codes. Here we will address fundamental unanswered biological questions- are many or just a few key RNAs localized? How many different zip codes exist? Have these codes been conserved through evolution? Are transport codes and anchoring codes identical or separable? Does localization change on insult or in disease?

Through an international collaboration we now have a unique opportunity to answer these questions. In mammalian cells, genome-wide screening has provided us with a large set of mRNAs that localize to cell protrusions and axonal growth cones. In yeast, we have identified a set of mRNAs unexpectedly bound to membranes. These data offer an unrivaled opportunity to develop new computer programs for zip code identification, and to refine these methods by testing predictions with experiment. We will also investigate the evolutionary conservation of the zip codes from yeast to man. By combining the diverse expertise of a cell biologist, a yeast geneticist, and a computer scientist, we are in a unique position to uncover fundamental principles of mRNA localization. Our ultimate goal is to unravel a fundamental dictionary of RNA zip codes.

Axons of retinal ganglion neurons traverse molecularly distinct territories and choice points on their way to the optic tectum. The navigational behavior of their growth cones is governed by extracellular guidance factors that elicit chemotropic responses through local protein synthesis and degradation. Cyclic nucleotide signaling modulates these processes to allow response switching. Little is known about the orchestration of molecular mechanisms in axonal navigation, mainly due to a lack of intracellular real-time assays. In isolated neurons and the Xenopus brain, we wish to study how guidance cues are interpreted and integrated by the growth cone to produce the desired behavior. For this, we will apply a novel optical sensor paradigm that can offer a detailed quantitative view of the molecular navigation reactions in the growing tip, while minimizing possible deleterious effects of the introduction of sensor constructs. These approaches will be used to generate uniquely high-content information on the key reactions of local protein translation, folding, and proteasomal degradation. These powerful tools and highly interdisciplinary approaches will allow the dynamic dissection in vitro and in vivo of molecular events elicited by extracellular signals, and the molecular functional definition of their interplay at key choice points in the visual pathway where axons change their behavior in response to changes in guidance cues.

We propose to bring together an international and intercontinental team of biophysicists, cell biologists and neuroscientists to investigate the cellular and molecular mechanisms by which large proteinaceous aggregates can cross biological membranes. We propose three specific research aims. First, we will use state-of-the-art technology to understand the properties of protein aggregates that endow them with membrane-penetrating properties. The second aim seeks to determine the extent to which internalized aggregates can move between cells within a tissue and the role of endocytic, autophagic and cytoskeletal processes. Third, we propose to assess the extent to which protein aggregates can nucleate the aggregation of endogenous proteins within the brains of specially engineered mouse lines, and determine the role of motor-based axonal transport in this process. Together, this highly collaborative project seeks to understand the fundamental cellular and molecular basis for a newly discovered phenomenon that may play a critical role in the pathogenesis of diverse conformational diseases.

The control of cerebral vessel diameter is of fundamental importance in maintaining healthy brain function because it is critical to match cerebral blood flow to the metabolic demand of active neurons. The hypothesis that we will test is that astrocytes are essential communicators between blood vessels and neurons to regulate cerebral blood flow. Although there are numerous data indicating that astrocytes have critical roles in these processes there are still outstanding questions and critical gaps in our knowledge. A key shortcoming in previous studies is the difficulty to selectively activate neurons versus astrocytes. This grant will fund research to develop and apply the necessary tools for two photon uncaging and imaging in vivo. This team will apply novel two photon uncaging approaches in the cortex in vivo to address fundamental questions of how astrocytes signaling can alter cerebral blood vessels. A major question concerning the roles of astrocytes in the regulation of cerebral blood flow is the mechanism by which astrocytes are induced to cause either constrictions or dilations as a result of calcium transients. It is clear that astrocytes have the ability to induce either response in adjacent blood vessels. A recent study from MacVicar’s lab using a new two photon sensitive caged calcium (DMNPE-4) from Ellis-Davis’ lab has shown that the metabolic state of the surrounding tissue determines the polarity of the astrocyte influence on blood vessel diameter. This was determined in studies in brain slices in which oxygen levels and metabolic controls could be rigorously controlled and manipulated. The major pressing issue is to test these mechanisms described in brain slice studies using in vivo experiments. This collaborative effort will provide the expertise to do this. The Charpak lab has years of experience examining two photon signals in the olfactory bulb in vivo. The MacVicar and Charpak labs will combine efforts to examine astrocyte effects on blood vessel dynamics in the cortex and olfactory bulb in vivo. They will use new caged probes developed by the Ellis-Davis’ lab. New probes are necessary to provide the sensitivity that is required for in vivo photolysis and to provide new compounds to be uncaged for these investigations.

70% of mankind’s current energy needs are met by burning fossil fuels. This is already problematic since oil and gas supplies are limited and because of the adverse environmental effects of rising levels of carbon dioxide in the atmosphere. Moreover this situation is set to get worse as current predictions estimate that our energy needs will double by 2050. Mankind is therefore facing a major challenge to find new sources of clean renewable fuels. Photosynthesis is a biological process able to use solar energy to produce such fuels. The essence of photosynthesis can be described in terms of the following three partial reactions: light harvesting, charge separation, and electron transfer to take electrons from water in order to reduce carbon dioxide to carbohydrate (a fuel).

Since solar energy at the earth’s surface is rather diffuse, any artificial system designed to convert it into a fuel will need to incorporate a light-harvesting or light-concentration step. The first question any engineer would ask is: what is the design tolerance of such a system? How much architectural flexibility is possible while still retaining both high efficiency light-harvesting and energy delivery to the ‘sink’, where that energy can be productively utilized? The project addresses this problem by interrogating natural light-harvesting systems with sophisticated spectroscopic and microscopic techniques, which can tell us how their supra-molecular, meso-scale architecture relates to their overall light-harvesting efficiency.

We use as model complexes purple bacteria, which are very simple photosynthetic organisms, for which the structure and function of individual native light-harvesting complexes have been identified. We will first investigate native membranes of purple bacteria by the combined use of time- and space-resolved spectroscopies, in order to correlate their local organization with their light-harvesting function. We will then use this information to begin to construct arrays of artificial light-harvesting complexes, based on synthetic analogues of natural pigments, on surfaces where we can control supra-molecular architecture. Such arrays can, in the long term, be used in devices for producing solar fuels.

During mammalian embryogenesis, mesenchymal cells differentiate into stromal lymph node organizer cells (LNo). In the course of a continuous differentiation program, LNo specialize into stromal cells that specifically accommodating either T or B cell clustering, this process forms the characteristic cellular architecture of secondary lymphoid organs. Gene-targeted mice have provided insight into the hematopoietic cells interacting with stromal cells during their differentiation, and several of the molecules essential for this process have been elucidated. However, in vivo studies are intrinsically limited in their ability to allow for the simultaneous and tightly controlled manipulation of multiple cells and signals. In this project, we will develop artificial lymph node (ALN) environments in vitro that will allow us to study the complex cellular and molecular networks underlying LN organogenesis with unprecedented control. Using the ALN environments, we will unravel 1) Minimal cellular and molecular requirements for LN development; 2) Organization of stromal cells into distinct T and B cell specific areas and 3) A role for non-paracrine forces including mechanical stresses, oxygen tension, cell movement and extracellular matrix remodeling in development of LNs. To achieve this, 3D remoldable collagen scaffolds will be microfabricated that capture the anatomy of the developing LN microenvironment. The scaffolds will be seeded with appropriate human or mouse cells and connected to externally controlled fluidics to allow for direct modulation of the cellular (LTi, stroma, lymphocytes) and chemical (signalling molecules, inhibitors) microenvironments within the scaffold at any given time. Additionally, this approach will allow for direct modulation of physiological forces (oxygen, shear stress and wall tension). Seeded cells will be allowed to differentiate and organize while these parameters are varied. Stromal cells, LTi cells, lymphocytes and the extracellular matrix will be monitored in situ by real time 4D multicolour imaging and cells will be harvested from the scaffolds such that gene expression signatures between different conditions can be measured and compared and correlated with imaging results. From this data we will develop both physiological and genomic maps of LN formation and generate fully functional ALNs, providing a unique platform to study LN development and function in real time and allow visualization and modulation of biological and pathological processes.

Serotonin (5HT), like dopamine (DA), has long been implicated in the control of behavior, including decision-making and reinforcement learning (RL). Its relevance for behavior is illustrated by its implication in a variety of neuropsychiatric disorders ranging from affective disorders such as anxiety and depression to impulse control disorders. However, while the two neuromodulators are tightly related and have a similar degree of functional and clinical importance, compared to DA, we have a much less specific understanding about the mechanisms by which 5HT affects behavior. We address this issue by leveraging existing understanding of DA and a constellation of methods that has shed light on DA function: integrating functional magnetic resonance imaging (fMRI), psychopharmacology and neuronal recordings in behaving nonhuman and human primates, all guided by computational theory. We will test, and ultimately revise, a hypothesis that 5HT biases decision-making through an interaction with dopaminergic RL mechanisms.

Specifically, we will test the hypothesis that 5HT and DA have coordinated effects that serve to couple multiple axes that might otherwise be expected to have independent effects on decision making. We will manipulate different axes in behavior independently in a learned decision-making task, using functional neuroimaging in humans and neuronal recordings from DA and 5HT nuclei in monkeys to study correlations between neural activity and behavioral context. We will also assess the neuromodulators’ causal roles, by manipulating DA and 5HT in the same task and studying the effects on behavior and neural activity.

To maximize the comparability of the data, we will use experimental procedures as close to identical as practical between species, and use computational models in data analysis to bridge different levels of measurement. Experiments will be grounded in and quantitatively analyzed using current theoretical RL models, and will in turn inform the revision of these models.

Gene expression can be regulated through alterations in nuclear architecture providing control of genome function. Mechanical loading induces both nuclear distortion and alteration in gene expression in a variety of cell types. One putative transduction mechanism for this phenomenon involves alterations to nuclear architecture, resulting from the mechanical perturbation to the cell, to induce altered gene expression. Moreover, remodeling of the nucleus occurs during stem cell differentiation, causing alterations in nuclear stiffness and potentially influencing nuclear architecture-mediated mechano-regulation of transcription. These putative mechanotransduction processes have not been studied in detail before and form the basis of the current proposal. Accordingly the aims of this project are to test the following hypotheses:

1. Mechanically-induced nuclear distortion causes alterations to the nuclear architecture and chromosomal territories sufficient to affect gene transcription

2. Stem cell differentiation modulates the process set out in hypothesis 1, via differentiation-induced nuclear remodeling that affects the mechanical properties of the nucleus.

The successful realization of the aims necessitates collaboration between researchers with complementary expertise in bioengineering, biophysics and cell/molecular biology. Accordingly, we plan an ambitious and innovative program of research that benefits from a highly interdisciplinary approach, building on the existing strengths of the collaborators. The studies will utilize model systems involving human mesenchymal stem cells (MSC) and induced pluripotent stem cells (IPSC) in conjunction with state-of-the-art techniques for the mechanical perturbation of cells, functional analysis of nuclear organization and biomechanical analysis. Ultimately a mechanistic model for functional mechanoregulation of nuclear architecture will be developed to incorporate elements of the modeling of the mechanical properties of the nucleus in conjunction with functional genomic analysis.

Genes, the DNA sequences that are copied into RNA to specify proteins, carry the primary genetic information. The long DNA molecules in cells carry many genes and these are interspersed with other DNA sequences that provide a critical second level of information, controlling whether or not each gene is expressed within a specific cell at a specific time. These gene control sequences are recognized and bound by proteins. A gene's expression is controlled not only by protein-DNA complexes near its start (the promoter) but also by the specific pairing of that complex with proteins bound to other control sequences that are often far away on the same DNA molecule (an enhancer), causing the DNA in between to form a loop. These long-range DNA-looping interactions are not well understood and it is a mystery how the promoters of different genes on the DNA link up with the correct enhancers in order to control the genes properly. This integrated multidisciplinary project aims to test the idea that different protein-mediated DNA loops can interfere with each other, thus restricting this web of possible interactions. To do this, we will set up a model system that uses two well-understood proteins, each known to form simple DNA loops. Using different arrangements of the protein binding sites on the DNA, we will use a range of techniques to measure how readily each loop forms in the presence or absence of the other loop. Measurements will be made on DNA inside living bacterial cells by assaying the effect of the loops on gene expression. Outside cells, protein-induced DNA looping will also be measured with instruments that can hold, stretch and twist individual molecules of DNA, and will be visualised with specialised microscopes. Computer simulations that reproduce the movement, bending and twisting of naked DNA and DNA bound by proteins will be used to try to reproduce the experimental results. This combination of approaches will show just how strongly DNA loops interfere with each other and help us understand how.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that eliminates transcripts lacking complete open reading frames, thereby ensuring that potentially toxic polypeptide fragments do not accumulate. NMD is triggered by premature translation termination, a response that stands in striking contrast to stop codon recognition by the ribosome at the end of each round of normal translation. In this proposal, we seek to define the mechanistic differences between premature and normal translation termination, and to use that information to understand how only the former leads to NMD. Our plan considers four important aspects of this problem, namely that: the “core” components of termination and NMD have been identified (the eRFs, Pab1, the Upf proteins, and the ribosome); structural information is available for some components, but not for most of their complexes; the precise roles of the different factors and complexes in both termination and NMD is unknown; and there is a high likelihood that there is a large set of hitherto unidentified and/or uncharacterized regulators of termination and NMD that must play important roles in these processes. Accordingly, we have organized a France-Sweden-U.S. collaboration that brings to bear complementary sets of expertise in structural biology, biochemistry, and molecular genetics in pursuit of three aims. Specifically, it is our intent to: i) Determine 3D structures for the interactions of the Pab1, eRF, and Upf proteins; ii) Define the kinetics of the interactions between these proteins and the ribosome; and iii) Identify and characterize additional regulators of NMD and termination. To address these aims, we have planned a highly synergistic work flow, where the success of any one group will facilitate and enhance the success of the other two. The three complementary groups will provide each other with purified proteins and high resolution structures (France), detailed and dynamic biochemical parameters (Sweden), and mutants defective in specific steps of termination/NMD and new regulators of both processes (U.S.).

Myosin molecules are proteins that function as motors inside cells. These molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in myosins, like DFNA22 syndrome, which is characterized by deafness. Myosins transport other biomolecules by hauling them along the actin cytoskeleton. This transport consumes energy at the ‘head’ domain of the motor, while its ‘tail’ domain is traditionally considered more structural and mainly required for dimerisation, since it is rather rigid. Until recently, coiled coils in mechano-sensory proteins like myosins were thought to be relatively straight and geometrically ideal. Detailed investigations of the implications of structural deviations from a strong coiled coil motif on biological function of myosins are just emerging. For instance, myosin VI in both Drosophila and humans contains a peculiar distribution of charged residues in the tail that Jim Spudich’s group has shown, by biophysical approaches, to be a ~70-residue long stable, fairly rigid single α-helix, not the coiled coil that was predicted earlier. Ramanathan Sowdhamini’s lab has developed computer algorithms for the analysis of coiled coil interfaces through energy calculations. Jim Spudich’s lab has a long-standing expertise in biophysical and cellular experiments on myosins. Henrik Flyvbjerg’s lab leads efforts to properly analyze stochastic processes and data in biophysics, such as those manifested in molecular motor studies and in motility assays. The three groups together will make a unique team by uniting their complementary tools of bioinformatics, biophysical studies, and mathematical modeling to obtain important structural and functional understandings of the relatively ignored, but vital, tail domains of the myosin family of molecular motors.

The process of myelination, in which myelin sheaths are formed around axons, represents one of the most spectacular processes in biology. Myelination of axons is required for the efficient transmission of nerve impulses and maintainance of axonal integrity in the central nervous system. As well as being of intrinsic biological interest, this process is relevant to our understanding of the regenerative process of remyelination following myelin diseases such as multiple sclerosis. Critical to this process is the mechanism by which a population of neural stem/precursor cells called oligodendrocyte precursor cells (OPCs) differentiates into myelin-forming oligodendrocytes, cells that migrate to and wrap their processes around axons to form the multiple layers of compacted membrane that constitute myelin. Thus far, only the biochemical stimulation of OPC differentiation has been studied. However, OPCs are also subjected to various kinds of mechanical strain during the processes of cell migration, axon engagement, and neuron growth, and it remains entirely unknown how such physical stimuli may regulate or contribute to OPC differentiation. In this project we seek to clarify the importance and modes of mechanical stimuli that impact OPC differentiation, by integrating international expertise in OPC biology, novel biophysical experiments, and material mechanics. Using well-established protocols for growing, differentiating, and characterizing OPCs in vitro used in the Franklin lab, we will systematically consider effects of global, local, and receptor-mediated strain on OPC morphology, molecular markers, and myelination potential. These strains will be applied using optical, scanning-nanoprobe, and substrate-based cell deformation techniques being developed by Guck and Van Vliet, in order to identify the key modes and magnitudes of mechanical stimuli that contribute to OPC differentiation. The conversion of effective mechanical stimulus to intracellular biochemical signaling pathways will then be identified by RNA profiling (including microarray profiling and post-hoc bioinformatic pathway analyis tools) and validated by ICC and Western blotting. In summary, the proposed research will develop and apply innovative experimental approaches to improve our understanding of the process and stimulation of axon myelination, and to identify potential new targets for treating demyelinating diseases. More generally, these systematic studies on a key precursor cell type will serve as a model to clarify the general mechanisms involved in mechanotransduction in the maintenance and differentiation of stem and precursor cells.

The genes in our DNA encode messenger RNA molecules, which, in turn, encode proteins. Proteins are the molecules in cells that perform most of the tasks that make life possible; they are the catalysts that make chemical reactions happen, they act as structural molecules that hold cells together, and they help other molecules such as nutrients move into and out of cells. Machines called ribosomes inside of cells read, or “translate,” the information in messenger RNAs to make the encoded proteins. When this process is not done correctly diseases such as cancer can result. In addition, when we are infected with a virus, the virus hijacks our cells’ ribosomes and associated protein synthesis machinery to make copies of itself. A deeper understanding of how protein synthesis works will facilitate attempts to cure diseases such as these. Our project aims to elucidate the structure of the protein synthesis machinery as it begins to assemble on an mRNA in preparation for making the corresponding protein. This information will help us in the same way that an automobile mechanic is helped by knowing the details of how an engine is put together; it will tell us which pieces go where, what they look like, and what each one does. These pictures will dramatically advance our understanding of this central biological process.