The success of a cell's survival under different growth conditions depends on its ability to control the coordinated transcription of selected genes in response to external stimuli. In bacteria, transcriptional regulation of genes depends on the binding of proteins to DNA, but also on the physical configurations of the resulting mesoscopic protein/DNA complex, the nucleoid. Particular nucleoid-shaping transcription factors (NSTFs) have the task of modulating the nucleoid's dynamic structure in response to changes in environmental conditions. NSTFs may thus affect the expression of many genes - some of which code for the same NSTFs. The current transcription network view of regulation considers a graph of all genome interactions, but disregards the effects due to changes of the nucleoid. What role does the nucleoid's structure play in the hierarchy of events leading to the large scale transcriptional response to changing growth conditions? Can this be incorporated into a generalized network description? To address these questions, we will develop a predictive integrated account of transcription in bacteria, including the thermodynamic state of the nucleoid as dictated by the local and global interplay of NSTFs and supercoiling, and apply it to the study of the cell's global response to a nutritional shift. The interdisciplinary nature of the team will help us build a theoretical generalized network model, make quantifiable predictions, and test them experimentally. In order to obtain quantitative systematic measurements of nucleoid structure we will (i) study a library of specifically designed nucleoid structure-sensitive reporter gene constructs at different chromosomal loci, (ii) develop new optical/microfluidic technologies allowing for time-resolved single cell measurements in liquid medium, (iii) perform in situ nucleoid purification and subsequent biophysical characterization, and (iv) develop new modeling approaches to characterize the biophysics of the nucleoid as a "complex polymer". These results will allow, for the first time, a complete description of the mechanisms of cellular adaptation to specific environmental changes through transcription, integrating the interplay of global nucleoid organization/structure with the classical network model. This project lies at the frontier of biology and statistical/soft-matter physics.

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.

How do we suppress inappropriate responses? One influential theory proposes that the anterior cingulate cortex works in conjunction with the lateral prefrontal cortex (PFC) to signal and suppress inappropriate actions. Despite a wealth of studies investigating the neuroelectric signatures of response conflict monitoring, almost nothing is known about how these different brain regions interact to form a cognitive control network. The proposed research combines existing theory with cutting-edge analysis techniques to test the novel hypothesis that oscillatory phase synchrony—a millisecond-resolution measure of transient functional interactions among brain regions—underlies the PFC’s ability to monitor and adjust inappropriate actions. The research comprises 3 Specific Aims. Aim 1: To examine how neural oscillatory synchrony links the medial and lateral PFC, and motor cortex, into a functional network. This will be done by measuring EEG while human participants engage in difficult reaction time (RT) tasks, and examining dynamic changes in phase synchrony before and after fast vs. slow RTs. Aim 2: To use transcranial magnetic stimulation (TMS) to understand how patterns of PFC oscillations and oscillatory coupling are altered by a TMS pulse. Aim 3: To test whether oscillations play a causal role in the cognitive control network by examining the effects of TMS during different oscillation cycles on response selection. In addition to combining different psychological and neurobiological theories in one unified framework, the proposed research has the potential to provide a novel neurobiological mechanism by which we can monitor our behavior and control inappropriate responses.

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.

Alpha-synuclein (AS) is the major constituent of Lewy bodies, a pathological hallmark of Parkinson’s disease (PD). Amyloid-like deposits of AS are generated by toxic oligomeric species of the protein, which are now considered to be a major cause of cell death in PD. The mechanisms by which the monomeric protein suffers structural rearrangements that lead to oligomerization are not yet known and are a subject of intense research in the field. The most appealing hypothesis is the one of oxidation-mediated oligomerization of AS, causing the formation of highly toxic early amyloidogenic species. Such intermediates appear to be scarcely populated, likely involving important structural rearrangements, which paves the way towards an effective therapeutic strategy against PD based on the stabilization of the native monomeric protein. The study of the early stages of fibrillogenesis requires the development and application of new methods. I propose tackling this challenge in a novel way, through exciting multidisciplinary research by scaling up the NMR techniques combined with methods of molecular dynamics simulations, together with the use of the more advanced single-molecule fluorescence techniques. The impact of metal-catalyzed oxidation in the structure of monomeric and oligomeric AS by means of low and high resolution structural techniques will be evaluated, shedding light into the role of oxidative damage as an effector in protein aggregation. The information obtained will be paramount in order to validate the use of pharmacological chaperones of AS as new therapeutic agents to treat PD.

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.

Members of the dynamin superfamily are mechano-chemical enzymes which oligomerise around lipid templates and use the energy of nucleotide binding and hydrolysis to remodel membranes. The underlying mechanisms of membrane deformation and the molecular basis for the mechano-chemical function are not understood and will be addressed in this proposal. My group will explore structure, function and mechanism of the dynamin-related Mx GTPases which mediate cellular resistance against several RNA viruses. The structure of a full-length Mx member will be solved by X‑ray crystallography to reveal the general architecture of this family. The catalytic mechanism of assembly-stimulated GTP hydrolysis and the subsequent conformational changes essential for the mechanical function will be biochemically and structurally characterised. Cryo-electron microscopy reconstructions will clarify how Mx proteins oligomerise around lipid and viral templates. Furthermore, the molecular basis of antiviral activity will be elucidated by characterising the interaction of Mx proteins with lipids and viral structures. Results obtained for the Mx family will be compared to other membrane-remodelling GTPases to reveal general principles governing oligomerisation and membrane remodelling in this superfamily.

Increasing evidence indicates that small RNAs participate in a vast number of biological processes. This is also of vital importance to the nervous system which relies on complex gene expression regulatory mechanisms during development and adult functions. While several studies have found non-coding RNA to participate in nervous system development and function, a detailed analysis of the identity, abundance and targets of non-coding RNAs during these processes is missing. We will search for new candidate non-coding RNAs that may affect differentiation of embryonic stem cells towards neurons. We will profile small RNAs and search for candidate miRNA targets based on biochemical purification of RISC bound mRNAs. By using special culture conditions for differentiated neurons we will separate dendrites from cell bodies to profile dendrite-localized small RNAs and its possible targets. Our hope is to identify miRNAs or other small RNAs that regulate local protein synthesis at dendrites which is postulated to mediate synaptic tagging in the most persistent forms of synaptic plasticity and memory. Next, we will develop new strategies to measure rapid induction of dendritic translation for studying the role of miRNAs in local derepression of translation upon synaptic stimulation.
As a postdoctoral fellow I hope to learn from multidisciplinary interactions offered in the Friedrich Miescher Institute (FMI). This project will allow me to interact with researchers from different fields such as bioinformatics, neuroscience and cell biology. My bet is that learning to interact with specialists from different fields will enable me to pursue high quality research in the future.

Animal development is characterized by the amplification and differentiation of progenitor cells into tissues of characteristic shape and function. The expression of many differentiation factors is concomitant with- and required for- any series of morphological changes, giving rise to final tissue and organ shape. The mechanisms whereby these factors lead to a stereotyped remodeling of cell- and tissue architecture are largely unknown. The goal of this project is to understand how cells reorganize their content in response to the expression of key developmental transcription factors. Changes in cell shape and behavior must be regulated by remodeling the extensive network of protein-protein interactions underlying both cytoskeleton as well as membrane dynamics. Structural and regulatory proteins must be transported and localized to their site of action following specific routes. This project focuses on how tissue specific gene expression drives specific patterns of protein and membrane trafficking and how this trafficking regulates cell fate and behavior.

Schizophrenia is a devasting mental disease causing psychotic episodes and social and cognitive impairments. Dopamine dependent brain circuits between prefrontal cortex and striatum are known to play a key role but their complexity prevented the disentanglement of their contributions to the disorder. The proposed project is aimed at examining the functional relationship between the elements of these circuits and schizophrenia-associated symptoms. The study will be based on optical control of the excitability of dopaminergic neurons. I will functionally integrate the fast light-responsive microbial opsins halorhodopsins (NpHR) to suppress spiking, and channelrhodopsins (ChR2) to enhance spiking of dopaminergic neurons, thus, making the neurons controllable by light. A combination of transgenic founder selection, viral delivery methods and the usage of the selective impact of fibers travelling between brain areas will be used to restrict the expression of the opsins to populations of dopaminergic neurons in the mice and monkey brain. I will use behavioral studies, optogenetic manipulations, fiber optic approaches, voltametry, and single-unit recordings to investigate the relationship among alterations in the dopaminergic circuits and the elucidation of putative schizophrenia-like endophenotypes. In contrast to existing pharmacological monkey models [1, 2] and knockout mouse models [3, 4] of schizophrenia, the optogenetic models will allow for locally highly restricted circuit manipulations on a millisecond time scale. This promises a detailed understanding of the causal dopamine-dependent factors of the disorder with clinical relevance particularly due to the monkey model.

Calcium-calmodulin-dependent protein kinase II (CaMKII) and the NMDA receptor are both necessary components of the cellular machinery underlying learning and memory. Growing evidence suggests that the interaction between theses two proteins is essential for Long-Term-Potentiation (LTP), a prominent cellular model of memory formation. The goal of this research project is to study the dynamics of the interaction between CaMKII and the NMDA receptor in the context of synaptic plasticity. To address this question, we will use FRET-FLIM imaging in living hippocampal neurons expressing GFP-tagged NMDARs and mCherry-tagged CaMKIIs. We will induce and measure LTP in the spines of theses organotypic slices by the means of theta-burst stimulation, electrophysiology recordings and confocal imaging of a diffusible volume marker. During this summer, I will learn several techniques associated with living slices, as well as doing experiments that will allow us to better understand the molecular basis of memory. The short-term fellowship will help to make this project possible; which will be a great opportunity for my scientific career.

Temperature and nutrient availability are crucial environmental parameters that restrict bacterial growth. To locate the sites of optimal nutrient availability and optimal temperature, bacteria perform chemotaxis (following the gradient of nutrient concentration) and thermotaxis (directing movement according to temperature). In E. coli, the chemotactic response is virtually temperature-independent. In contrast, thermotaxis is explicitly temperature-dependent. Despite these strongly contrasting functions, both chemotaxis and thermotaxis exploit the same signaling network. Our work aims at a quantitative characterization of the thermal properties of this network that enable both robust chemotaxis and thermotaxis over a wide temperature range.

We propose to exploit single-tethered-cell measurements and fluorescence resonance energy transfer (FRET) to elucidate the mechanisms underlying robust chemotaxis of bacteria. To this end, experimental data on the temperature influence on the network is being collected by means of FRET and tethered-cell measurements, that will serve as inputs for our theoretical modeling. Our study shall address the following questions. How does one signaling network achieve both thermotaxis and chemotaxis despite their apparently contrasting requirements? What are the mechanisms, that underlie the robustness of chemotaxis over a broad temperature range? How do multiple sensory stimuli such as attractants/repellents and temperature changes interact?

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.

How cells control the mutually exclusive expression of a single member of multigene families is a fascinating but unresolved question. For example, individual sensory neurons express a single odorant receptor out of a repertoire of about 1300 receptor genes. Similarly, the major variant surface antigen of the human malaria parasite Plasmodium falciparum, PfEMP1 (P. falciparum erythrocytic membrane protein 1), is encoded by the large, polymorphic var gene family whose ca. 60 members are expressed in a mutually exclusive manner. Monoallelic expression of odorant receptors is regulated in parts by a unique enhancer element that activates a single odorant receptor promoter via interchromosomal interactions. This project aims to determine whether a similar mechanism controls singular var gene activation. Specifically, I will use chromosome conformation capture techniques and large-scale sequencing (Solexa) to identify chromosomal elements associating with active var genes and analyse their role in mutually exclusive var gene expression through knock-in and knock-out studies. Furthermore, I aim to develop in vivo DNA pull-down techniques to identify proteins associating with var regulatory elements. These studies should provide new insights into the regulation of expression of a major virulence factor of this important human pathogen.

Intrinsically disordered proteins are most important for the eukaryotic cell, but many aspects of this recently discovered “class” of proteins are yet to be investigated. The main objectives of this proposal is to define a new single-molecule nanoanalytical technology which will enable the efficient detection of non-covalent molecular interactions with microsecond resolution in order to answer fundamental questions about these proteins, for example, what comes first, folding or binding? Importantly, we also aim to build a strong interdisciplinary collaboration based on our diverse backgrounds. This will hopefully lead to further collaborations not only within our team but with other fellow scientists. The fundamental role intrinsically unstructured proteins play in the cell has therefore only recently been appreciated. These proteins are not obscure peculiarities; up to 30% of the different proteins expressed in eukaryotic cells may be partly or completely unstructured in absence of their ligand. A number of reasons have been suggested as to why proteins are intrinsically unstructured which are as follows: 1. It is a way of decoupling affinity and specificity; i.e., a high specificity can be combined with a low affinity. 2. It increases the association rate with the ligand. 3. It allows for increased plasticity with regard to the ligand in the cell. 4. A large interaction surface area is provided in a short amino acid sequence as the protein folds around its ligand. (A short protein requires less energy when it is synthesized.) 5. Rapid turnover in the cell as unfolded proteins are normally short-lived. Numbers one and two are perhaps contradictory, as an increased association rate would lead to higher affinity, and not lower. The contradiction reflects the scarcity of data in the field and highlights the need for more experiments to be performed. What is clear is that intrinsically disordered proteins play key roles in many different cellular events such as transcription.

The function of the mammalian target of rapamycin (mTOR) protein is altered in metabolic diseases and cancer. mTOR coordinates nutrients availability with the complex signalling network necessary for cell growth and cell division. mTOR is activated by the presence of certain growth factors, insulin, high levels of ATP and amino acids. Interestingly, the molecular mediators of amino acid singling, the Rag family of GTPases, have only recently been characterized.Aberrant activation of mTOR by the insulin-dependent branch is known to take place in metabolic disorders as diabetes. However, the relevance of the amino acid-dependent mTOR activation is still not clear in vivo. The project I propose consists in defining the role of Rag GTPases in vivo, by means of using a knock-in (KI) mouse model for RagB GTPase. The endogenous RagB gene will be replaced by an artificial gene encoding a permanently-active mutant protein by substitution of the glutamine in amino acidic position 128 with leucine, under endogenous regulation. The KI will also contain a translation STOP cassette flanked by loxP sites, so we will be able to restrict temporally and spatially the expression of the transgene by means of different Cre recombinase expression systems. We hypothesize that permanent mTOR activation by RagB KI will lead to severe diabetic-like metabolic alterations and that these alterations play an important role in the clinic.My research expertise is based on cancer studies centred on the p53 gene. For my work as a postdoctoral fellow I will turn my research focus to the study of mTORC1 signalling in vivo and its consequences in metabolic diseases as diabetes.

Cytokinesis, the final step of cell division, has two crucial processes to achieve: equal sorting of cellular components between the daughter cells, and membrane ingression

and closure. Both processes strongly rely on membrane components and properties. It is known that some lipids localize to the cleavage site and that membrane transport is required to relieve cortical tension during cytokinesis, necessary for the physical separation of cells. However, the precise role and mechanism of this essential membrane retrieval are unknown, as is the nature of the lipids and proteins involved, in part because it has been difficult to study the role of lipids in membrane organization due to a lack of appropriate tools. The goal of this multi-disciplinary proposal is to understand which lipids participate in cytokinesis, how they are involved in membrane rupture, and how cells regulate their participation. Specifically, we aim to i) synthesize and validate probes to detect different lipids, ii) investigate which lipids are associated with cytokinesis and how they are spatially and temporally regulated during the completion of cytokinesis and iii) understand how mechanical parameters can influence the completion of cytokinesis.Unifying the scientific expertise of our three teams, we will use chemistry and chemical biology to design lipid probes, cell biology to use these probes to investigate the origin and role of lipids involved in cytokinesis and biophysics/modeling to address the functional importance of membranes during the mechanism of cytokinesis. The innovation of this proposal does not only lie in its unique question, but also in the combination of diverse approaches. Our comprehensive and systematic investigation will provide new insights into the important question of how cells divide, which will not only be important for basic biology, but also for understanding how cell division is mis-regulated in cancer.

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.).

Spatial regulation of translation within the cytoplasm results in the accumulation of newly synthesized proteins in discrete locations of the cell. mRNA localization mechanism is one aspect of the post-transcriptional control of gene expression fundamental to many cell types. The Zipcode Binding protein 1 (ZBP1) is a pivotal molecule in cellular morphology, physiology, development and cancer progression. ZBP1 is essential for localizing and regulating the translation of ß-actin mRNA near the leading edge in several cell types such as fibroblasts, or the growth cone of neurons. It binds to a short sequence in the ß-actin 3’UTR, the zipcode motif. Due to other mRNAs encoding for cell-contact related proteins also contain putative zipcode motif in their 3’UTRs, I propose that they may be coordinately localized and translated in a mechanism similar to ß-actin mRNA. Accordingly, I hypothesize that cell-contact sites represent a cellular microdomain where ZBP1 mediates localization and translation of these specific mRNAs in order to locally produce the factors required for the consolidation of these structures. Therefore, the cell may respond to extra-cellular stimuli regulating translation initiation of functionally related mRNAs at the correct time and place.