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.

Accurate segregation of the genetic material during mitosis is crucial for the maintenance of life. Chromosome segregation requires the formation of sturdy attachments between chromosomes and a microtubule-based structure named the mitotic spindle. The site of attachment of spindle microtubules onto chromosomes is named the kinetochore. Kinetochores are composed of 60-80 evolutionarily conserved proteins that form a structure up to 500 nm in diameter in metazoan cells. This is an order of magnitude larger than the largest macromolecular complexes such as RNA polymerase and the ribosome that are currently amenable to structural and enzymatic studies.

The characterization of the organization of kinetochores and their microtubule-binding activity is hampered by the inability to purify functional kinetochores from cells. The goal of our multidisciplinary international collaboration, therefore, is to reconstitute, from recombinant proteins, a functional kinetochore-microtubule interface. The microtubule-binding activity is provided by the so-called KNL1-Mis12 complex-Ndc80 complex (KMN) network, which contains ten proteins in three distinct complexes. Specifically, we propose to use an array of synthetic and analytic approaches to (i) rebuild the KMN network from purified proteins; (ii) evaluate how the immobilization of KMN network complexes on different nano-patterned substrates influences the ability to bind microtubules; (iii) study the dynamics of these arrangements using single-molecule fluorescence resonance energy transfer, total-internal-reflection fluorescence microscopy, and real-time atomic force microscopy; (iv) study the structural organization of the KMN networks by X-ray crystallography, electron microscopy and atomic force microscopy; and (v) test the significance of our conclusions for kinetochore-microtubule attachment in the genetically tractable yeast Saccharomyces cerevisiae.

We believe that our synthetic kinetochore-microtubule interface will eventually become accessible to the type of structural and enzymatic studies that continue to provide so much new understanding of the workings of other smaller biomolecular machines such as polymerases and the ribosome. Our studies will illuminate the process of kinetochore-microtubule attachment and will greatly advance the comprehension of the molecular bases of chromosome segregation.

Textbooks present motor memory as monolithic: once acquired, never forgotten. They dissociate it from declarative memory by suggesting that motor memory does not have a short-term form. However, recent theoretical and experimental results have overturned this view, suggesting that motor memory is supported by processes that have multiple timescales: fast processes that learn quickly but decay rapidly with time, and slow processes that are less sensitive to movement errors but show better retention. Indeed, it appears that formation of motor memory may be a gradual transformation from the fast to the slow processes, and this transformation may depend on time and the statistical properties of the learner’s performance errors. We propose to use this idea to shed new light on one of the fundamental problems in neuroscience: the distinct functions of the cortical motor structures vs. the cerebellum in learning and retention of skilled movements.

The unsolved problem is illustrated by two examples: whereas neurophysiological and neuroimaging experiments have found correlates of adaptation in the primary motor cortex (M1), functional disruption of this area by transcranial magnetic stimulation (TMS) appears to produce no effects on acquisition, yet produces post-adaptation amnesia, i.e., rapid forgetting. In contrast, while the cerebellum is crucial for adaptation, damage to this structure appears to spare some skills that were acquired before the damage. Our basic hypothesis is that the faster timescales of memory are dependent on the cerebellum, effectively learning to predict the consequences of motor commands and correcting the motor commands via internal feedback, and the slower timescales are dependent on the cerebral cortex, effectively learning to produce the motor commands appropriate for the specific conditions of the task.

Our project combines computational, psychophysical, neuropsychological, and neurophysiological approaches to investigate how time and statistics of performance. We will use common eye and arm movement paradigms in behavioral and neurophysiological experiments to explore how controlled changes in sensory and motor noise effect motor control and learning. This will enable us to directly relate high level behavior to neural mechanisms

Inositol has been exploited by evolution to generate a multitude of inositol polyphosphates structures (either soluble or lipid-bound species) that represents important class of signaling molecules controlling any aspects of cell physiology. The intrinsic inability of the inositol ring to be detected using standard spectrophotometric techniques means that, to date, it has not been successfully imaged (localized) within cells. Direct biochemical analysis (but not imaging) of inositides uses radioactive 3H-inositol labeling, followed by high performance liquid chromatographic separation of the 3H-inositol-containing species. Indirect imaging of lipid inositides species has been possible using specific fluorescent probes: the specificity of such probes, however, and their correct use is controversial and the literature harbors several examples of their misuse. We intend to use multi-isotope imaging mass spectrometry (MIMS) to analyze inositides signaling in cells. MIMS, which is the combination of a novel type of secondary ion mass spectrometer with tracer methods and intensive quantitative image analysis, allows the imaging and quantification of intracellular molecules labeled with stable isotopes. MIMS will allow high resolution direct quantitative imaging of inositol molecules once labeled with stable isotopes such 13C-inositol or 18O-inositol. This novel imaging approach should provide fundamental information about how cells handle inositol: the intracellular sites and concentration of its accumulation, the speed and location of lipid inositides synthesis. Additionally, further development of MIMS should allow the discrimination of the different inositol polyphosphates species. Consequently, multi-isotope imaging mass spectrometry may shed light on how the dynamic network of signaling inositides is maintained and regulated inside the cell.

This project aims to investigate how the embryonic brain develops its circuitry. We have evidence that electrical activity during development helps to define the way new neuronal circuits are formed, but we don’t know any of the detailed cellular mechanisms of the origin of these activities and of how they may control additional wiring patterns. The key problem is that standard electrode based measurements cannot provide data from multiple sites in the nervous system with cellular resolution. To solve this problem we will utilize new voltage sensitive dyes and non-linear optics to permit 4D (time plus 3 spatial dimensions) mapping of the system with resolutions that can permit the recording of activity down to the cellular and sub-cellular levels. The team of Drs. Sato and Momose-Sato has worked for many years to map electrical connectivity in the developing central nervous system. They have used optical recording as their primary method. But their previous instrumentation did not allow them to attain the necessary spatial resolution to understand the development of the circuitry at the cellular level. Dr. Pavone is an expert in biophotonics and has developed a unique non-linear optical random access microscope that will be ideal for this project. He will initially use this instrument to investigate the propagation of electrical activity at different stages of embryonic development. He will then enhance his non-linear optical microscope to permit its use with specialized voltage sensitive dyes in 1µm3 volumes within the tissue so as to investigate the cellular and subcellular mechanism electrical propagation. Dr. Loew has worked for many years to synthesize voltage sensitive dyes for optical recording of electrical activity. His laboratory will be responsible for the synthesis of the voltage sensitive dyes that will be required in this project. He will also characterize these dyes in vitro and in vivo. Additionally, the Loew lab pioneered the use of second harmonic generation (SHG) to image changes in membrane potential; Dr. Pavone’s microscope was designed explicitly to exploit the promise of SHG for 3D imaging of electrical activity in neuronal tissue. Importantly, the new science and the new methods developed in this proposed research will broadly impact the study of neuronal circuitry in both the developing and adult brain.

Fast orientation in complex, unknown environments is one of the most challenging sensory tasks for mammals. While most mammals meet this challenge with stereoscopic vision, one large group has pursued an alternative and highly successful strategy: Bats probe their surroundings via a biological sonar system, by analyzing the echoes of their ultrasonic vocalizations. In visual animals, including humans, the perceptual distortions caused by mirrors allow studying fundamental questions in brain processing of 3D objects in 3D space. We propose to bring these concepts into the auditory domain by conducting a set of novel studies on the effects of acoustic mirrors in bats. Water surfaces act as acoustic mirrors and represent a dramatic distortion of physical space, a common problem facing bats in the wild. We will study the question of how animals perceive 3D objects and 3D space, and how 3D environments are represented in the brain, by using acoustic mirrors as a novel experimental tool – and we will approach this problem by combining the expertise of three labs, located in Israel and Germany. Methods span from behavioral experiments in the field and in large flight rooms with naïve bats, via formal psychophysical experiments with trained bats looking at the effects of acoustic mirrors on the sonar perception of 3D objects and their cortical neural representation, and ending with the effect of mirrors on the neural representation of 3D space itself at the level of the bat hippocampus.

The polymerization of actin into filamentous networks provides the driving force for various biological processes involving cell motion such as wound repair, immune response, cancer metastasis and the establishment of neuronal connections. For a cell to migrate, it must first polarize orienting itself along the gradient of chemoatractants and guidance cues. At the leading edge of cells, the actin filaments organize into extended lamellipodial and filopodial networks that span several microns. A large number of techniques including electron microscopy and fluorescence microscopic techniques such as photobleaching recovery analysis and single-molecule observations have been used to monitor the dynamics of structural components of the actin network. Accumulating evidence indicates that the actin network undergoes constant remodeling throughout the lamellipodium. These remodeling processes likely involve actin filament severing into short oligomers, oligomer diffusion, depolymerization of oligomers into monomers, and reassembly by polymerization and filament annealing. However, little is known about the dynamics and the gradient of diffusible actin species in live cells. This project is aimed to elucidate the role of diffusible actin and its regulators in actin reorganization at the leading edge of motile cells. Development of novel image analysis methods will extend single molecule fluorescence microscopy to the study of diffusion and concentration of proteins within live cells. Single-molecule fluorescence observations of formin constructs (FH1-FH2 and FH2 alone that differentially respond to profilin-actin concentrations) will also be used to measure local nucleation and elongation rates of F-actin, and the concentrations of G-actin and profilin-actin. Single-molecule observations of Arp2/3 complex, cofilin and AIP1 will be used to probe actin assembly and disassembly. The results of the analysis of experiments will be used to develop 2D and 3D analytical and computational models involving reaction, diffusion, binding-dissociation, fragmentation and annealing. Using the feedback between experiment and theory, the goal of this work is to reveal the role of concentration gradients and local fluctuations of soluble actin regulators during cell polarization in response to administration of drugs, ligands and physical forces.