Comparative genomics has proven a valuable tool to identify non-coding elements on the genome that are highly conserved outside the human lineage but have rapidly evolved since we diverged from our common ancestor with chimpanzees. The molecular evolution of such elements may have contributed to the emergence of human-specific features. However, the increasing availability of genome-wide epigenomic data, reflecting the biochemical activity of genomic DNA, exposed a discrepancy between evolutionary DNA-sequence constraint and actual annotated functionality of DNA elements. Therefore, one may expect that a large number of yet unidentified non-coding functional elements (such as enhancers) exists, showing more moderate evolutionary constraint. Through a functional approach, complementing comparative genomics strategies, I will aim to identify functional elements on the genome, disregarding evolutionary constraint as primary selection criteria. With the goal of defining a molecular basis for human-specific brain features, I will perform genome-wide profiling of histone-methylation marks and transcription factor binding sites in human and mouse ES-cell derived neurons to identify genomic elements that display a unique epigenomic signature in the human lineage. These elements may act as human-specific transcriptional enhancers with regards to spatial, temporal or quantitative regulatory properties, which will be assayed in transgenic ES-cell lines. Finally, high confidence human-specific enhancers will be analyzed in transgenic mouse lines to determine their gene-regulatory roles in the brain and assess whether and how they may have contributed to human-specific brain features.

P-glycoprotein and BCRP (breast cancer resistance protein), belonging to ABC (ATP-binding cassette) multidrug transporters, are large membrane proteins that use the energy from ATP hydrolysis to transport substrates across biological membranes. Overexpression of P-glycoprotein and BCRP in tumor cells confers drug resistance by pumping the drugs out of the cells, one of the major barriers to cancer chemotheraphy. It is therefore important to understand the mechanism of ABC transporters at molecular detail. I will use both biochemical methods and x-ray crystallography to elucidate the mechanism of multidrug transporters. In particular, I plan to determine high resolution structures of P-glycoprotein and BCRP in complexes with anticancer drugs. To this end, the genes of P-glycoprotein and BCRP from 26 species have been already synthesized in the host institute. I will express these proteins in yeast and insect cell expression systems and carry out biochemical and structural studies. If successful, this project will provide a comprehensive view of how these transporters recognize and extrude drugs and how multidrug resistance is achieved. Structural information of P-glycoprotein and BCRP will directly facilitate drug design for effective treatment of cancer. It will be a significant change in my research direction, because my previous work was limited to soluble proteins. However, I am willing and prepared to take this challenge for independent research in the membrane protein field. I believe that my solid training in structural biology combined with the host supervisor’s expertise in ABC transporters places me in a competitive position to accomplish my research goals.

Despite the complexity of the retinal circuitry involving more than 50 discernible cell types, the prevailing view of the retina is that of a mere relay of visual information towards higher brain areas where all the intricate computations are performed. The main goal of my proposal is to challenge this view by assessing the diversity of computations performed within the retina and probing their significance for visually guided behavior. To this end I will take advantage of newly developed transgenic mouse lines that allow specific expression in distinct retinal cell types. I aim to characterize the response properties of targeted ganglion cells and uncover their presynaptic neuronal circuitry. Transgenic expression of photosensitive ion channels permits a reliable identification and manipulation of the genetically specified neuronal populations. Recording retinal responses during classic visual stimulation combined with light induced activation of these channels enables the excitation of targeted cells and the subsequent dissection of their retinal circuit function. I will perform such a mechanistic circuit analysis by electrode-array and patch clamp recordings in retinal explants. In particular, I aim to unravel the role of genetically specified bipolar cell types in the response properties of genetically targeted ganglion cells. In parallel, the described approach will be used to stimulate the same set of ganglion cells in vivo to examine their functional role in visually driven behavior. In summary, this approach will allow me to analyze mechanisms of neuronal processing within the retina and the role of different retinal output streams for vision and behavior.

The brain contains two main sub-cortical loops: the basal ganglia and the cerebellum. There are many similarities between them. Both are embedded in cortical computation and have similar micro circuitry; for example, in both structures the principal neurons are inhibitory. In addition, impairment to either loop leads to motor disorders. Nevertheless, very few studies have directly compared directly these networks. In my Ph.D., I examined the computational physiology of the basal ganglia and in my post-doctoral work I intend to research the cerebellum. Recent technological advances now make simultaneous multiple electrode recordings in cerebellum and brainstem possible. Using multiple electrode techniques I plan to investigate the interaction of the cerebellum with its brainstem targets during the control of eye movement. Achieving this goal will place me in a unique position in the future- one that will allow me to conduct experiments comparing these sub-cortical loops. Eye movements are at the core of human interactions with the environment and many human disorders are due to inappropriate ocular control. I hope that my future studies of the computational physiology of the cerebellum and basal ganglia will shed light on the normal and pathological aspects of these neural networks.

Stem cells (SCs) have the ability to self-renew long term and differentiate along one or more lineages. The hair follicle serves as a useful model in SC biology since it undergoes periodic cycles of growth, degeneration and rest, which is fueled by SCs at the base of the non-cycling part of the follicle (bulge). Sox9 is an essential bulge SC transcription factor, but how it governs SC function is unknown. I propose to determine how Sox9 regulates the maintenance of SCs during tissue homeostasis and in response to wounding. Using Sox9 as a marker, I plan to isolate and characterize early functional bulge SCs and determine how they differ molecularly from their adult counterparts and from their committed rapidly proliferating progeny. I will then elucidate how this SC expression program changes when Sox9 is absent. I will complement my transcriptional profiling with chromatin immunoprecipitation and deep sequencing to identify which genes in my SC signature bind Sox9 directly and whether they are in a chromatin active or repressed state. Taking advantage of the ability to culture bulge SCs under conditions that maintains Sox9 expression and stemness, I will select the most interesting putative Sox9 targets and investigate their roles in SC proliferation, survival, suppression of differentiation and migration. I will then use in vivo lentiviral infection strategies in mouse embryos to test the hypotheses emerging from my in vitro studies, and dissect the physiological significance of Sox9 and its targets. Together, these studies will provide valuable insights about the regulation of SC maintenance and provide us useful information about the consequences of its malfunction.

Glucose is a fundamental source of energy for all cells. The cellular uptake of glucose and related molecules is mediated by membrane transporters GLUTs. Fourteen members of the GLUT family have been identified to date, and although GLUTs show several common features, each isoform has distinct tissue distribution and substrate specificity. The roles played by some of the GLUTs have been thoroughly characterized, and their malfunction linked to several diseases well established. However, the molecular principles that underpin the functions of GLUTs and their transport mechanisms remain unknown. I propose to understand the workings of GLUTs by determining and characterizing their 3-dimensional structure using X-ray crystallography. Specifically I aim to obtain GLUT structures in different stages of their transport cycle. For a variety of reasons producing membrane proteins in large quantities can be challenging. To overcome these hurdles, key parameters will be varied to optimize both the expression and detergent-solubilized stability of GLUT proteins. In particular, I will focus on stabilizing GLUT proteins by developing a generic randomized mutagenisis approach for each GLUT isoform, and functional GLUTs will be subject to crystallization trials in various formats. The microfocus beamline at the Diamond Synchrotron, which is suitable for often fragile membrane protein crystals, will be used for collecting X-ray data. The outcome of this research will greatly increase our understandings of GLUTs and other membrane transporters. The new skills and knowledge that I learn during this project will help advance my research career.

The extremely high brightness and photo-stability of quantum dots make them ideal fluorophores for single molecule imaging in living cells. However, since they do not cross the cell membrane, they can not be used for fluorescent labeling inside living cells. In this research project, I propose novel methods for selectively silencing quantum dot fluorescence in the endosome of living cells and detaching cell-penetrating peptides, which deliver quantum dots into the cell, from quantum dot cargo inside the cytosol of living cells. Whereas delivery of quantum dots into endosome occurs easily, their escape from endosome into the cytosol are extremely inefficient. To suppress the background fluorescence of endosomally trapped quantum dots, I will use quantum dot conjugated with pH-sensitive CypHer5 dye which take advantage of the endogenous pH differences between the cytosol and the endosome. Since CypHer5 has strong absorption in the acidic condition but no absorption in the neutral condition, quantum dot-CypHer5 conjugate shows strong fluorescence in the neutral cytosol but no fluorescence in the acidic endosome. For the deliver of quantum dots into cells, I capitalize on the poly-cationic cell-penetrating peptide-based delivery method. To detach the interruptive peptide inside cytosol from quantum dots, I will use quantum dots modified with cell-penetrating peptides via disulfide linkers. Whereas the cytosol maintains a reducing environment, the endosomal compartment is oxidizing. Thus, the disulfide linker should be cleaved only inside the cytoplasm, and quantum dots will be released from a poly-cationic cell-penetrating peptide.

Typically studies of visual processing are performed in anaesthetized, head-fixed, passive animals. Recent studies on the auditory system, however, have shown that responses of neurons in auditory cortices are strongly dependent on motor output. During self-initiated vocalizations predictions of auditory feedback in the form of corollary discharges modulate sensory responses to amplify unexpected feedback. Based on these findings, I propose to investigate visual processing in visual cortex of the awake, behaving mouse. To disentangle signals coding for actual visual feedback from signals coding for predictions of visual feedback, I will compare cortical responses during self-motion induced visual feedback to cortical responses during passive visual stimulation, and I will investigate the effects of unexpected alterations of visual feedback.

Plants successfully repel most infections dependent on perceiving and responding to two classes of elicitors that activate two “branches” of the plant immune system. These branches share many common outputs, but little is known about the signaling and executor mechanisms linking them. Arabidopsis RIN4 is a 23 kD peripheral plasma membrane protein that regulates each branch during immune responses against the Gram-negative bacteria P. syringae. The mechanism(s) of RIN4 action remain obscure. RIN4 associates in a yeast two-hybrid system with two Arabidopsis Exo70 family proteins, which are putative components of the plant exocyst. In yeast and animal cells, the exocyst tethers vesicles to target sites at the plasma membrane during the last steps of exocytosis. Polarized vesicle secretion is a key aspect of plant immune defenses, but a role of the exocyst is unknown. The RIN4-Exo70 association leads us to hypothesize that RIN4 interacts with the exocyst to facilitate plasma membrane targeting of defense-associated vesicles. The first objective is to examine the RIN4-Exo70 interaction in yeast and in planta. The second objective is to test plants with perturbed Exo70 expression for effects on RIN4-regulated immune responses. The proposed studies may begin to define molecular mechanisms of RIN4 function and establish a role for the exocyst in plant innate immunity.

The ribosome is a large ribonucleoprotein complex with the unique ability to translate the genetic information encoded in mRNA into polypeptide chains. In prokaryotes it is composed of a small subunit (30S), which decodes the genetic information, and a large subunit (50S), which catalyses peptide bond formation. Together these two subunits form the 70S ribosome. Over the last decade a number of crystal structures of these three complexes have been solved, which have shed light on prokaryotic translation and its regulation.
Eukaryotic ribosomes are larger than their prokaryotic homologues. The small (40S) and large (60S) subunit form the 80S eukaryotic ribosome. This assembly contains many additional proteins and RNA segments, which are absent in prokaryotic ribosomes. Our understanding of the eukaryotic ribosome and its regulation is currently limited by a lack of structural information.
The aim of this proposal is to structurally characterize a complex between the 60S ribosomal subunit and the eukaryotic initiation factor 6 (eIF6). The expertise obtained from this study will be employed at a later stage to study the architecture of the 80S ribosome with substrates or regulatory GTPases. Structures of any of these complexes will significantly further our understanding of eukaryotic translation and its regulation.

Natural variation of phenotype between related individuals is one of the most prevalent, yet least understood characteristics of biological systems. Such variations served as a catalyst to generate the theory of evolution, are likely the building blocks that lead to differentiation and speciation, and are rapidly becoming the focus of extensive medical research in humans. Despite its importance, the means by which natural variation is generated and maintained are far from clear. To this end, we propose to characterize recently derived phenotypic diversity in the plant C. rubella. C. rubella has recently diverged from its ancestral population to radiate throughout the European continent. In accordance with this radiation, C. rubella collections demonstrate spectacular phenotypic diversity, presumably as adaptation to colonize new habitats. We will analyze the genetic divergence of twenty C. rubella ecotypes at the whole genome level and use this information to delineate the evolutionary history of the organism. In parallel, we will assay the transcriptome of each accession in several tissue types, allowing us to combine intergenic polymorphism data and transcript level variation. Finally, this large data set will be used to examine natural variation in the adaptive trait of flowering time. Identification of candidate polymorphisms influencing this trait will be confirmed through development and application of new, rapid QTL mapping techniques. This approach will aid in the establishment of C. rubella as a model system for evolutionary and genetic studies and is expected to significantly expand our understanding of phenotypic divergence and evolutionary processes.

For any protein to function properly, the polypeptide chain produced by the ribosome, the protein-synthesizing factory of the cell, has to fold into the correct three-dimensional structure. Protein folding in the cell starts while the growing peptide chain is still bound to the ribosome. The information on the sequence of amino acids of the protein is stored in the sequence of codons in the messenger RNA (mRNA). The ribosome reads the mRNA codons one by one and translates them into the sequence of amino acids of the protein. Not all codons are read with the same speed: periods of rapid translation are separated by pauses. The role of such pauses is not known, but interfering with a fine-tuned rhythm of translation causes incorrect folding of proteins which can lead to disease. Translation rates may be changed by synonymous mutations in the mRNA, i.e. mutations that do not change the meaning of a codon in terms of which amino acid to be incorporated, but affect the speed by which the codon is translated. The goal of this project is to understand how nascent proteins fold while being synthesized on the ribosome and how the speed of translation affects their folding. Fluorescent reporters introduced at defined positions in the nascent protein will allow us to distinguish the unfolded, partially folded, and fully folded protein as it is produced by the ribosome. By determining, in real time, the hierarchy by which the structural elements in the protein are formed, we will delineate the protein´s co-translational folding pathway and compare it to the folding properties of the completed protein when it is released from the ribosome. By introducing synonymous mutations in the mRNA, we will examine how translational pausing affects folding. We will also determine the structure and dynamics of independent folding units in the growing peptide. The work requires an interdisciplinary approach that combines the expertise in molecular biology, biochemistry, biophysics, and structural biology. The results will provide important insight into the mechanism of protein folding in the cell. Furthermore, the work will contribute to better understanding the origin of human diseases caused by incorrect protein folding and give a tool to upscale the production of functional proteins for medical and biotechnological purposes.

Many clinically important antibiotics are peptide natural products of great structural complexity made by bacteria or fungi. The biosynthesis is achieved by nonribosomal peptide synthetases (NRPS), in which catalytic subunits and carrier domains form an assembly line-like mega-enzyme and interact in a coordinated way to achieve a directed product assembly via covalently bound intermediates. As such, a fundamental mechanistic understanding of these remarkable molecular machines will have direct implications in the rational design of novel synthetic pathways by biocombinatorial approaches. Large-amplitude conformational transitions have been identified and are likely to play a central role in the apparent directionality of the stepwise peptide assembly within the NRPS. Two conceptually distinct hypotheses have been proposed for triggering the conformational changes: They could be driven by chemical-reaction directed interactions between the individual domains with well-defined protein conformations or, alternatively, they could be promoted by Brownian random movements that eventually lead to productive interactions. Using these two still highly speculative models as guide, this proposal is intended to dissect the multi-step enzymatic reactions and clarify the mechanistic roles of large-amplitude conformational transitions. Specifically, we aim (1) to monitor the sub-domain dynamics in the adenylation domain, (2) to monitor the domain-domain interactions between the adenylation and the peptidyl carrier domain, and (3) to monitor the dynamical domain coordination of a minimal NRPS in real time. We will apply single-molecule FRET spectroscopy to this kind of multi-domain mega-synthetases for the first time. We will also develop new techniques for site-specific protein labeling and new data-driven statistical analysis methods to interpret the data. An international research consortium consisting of leading research groups in chemical biology (Germany), single-molecule spectroscopy (US), and mathematical physics (Japan) is formed to tackle this challenging yet fundamentally important problem. This will be a new research direction for all three participating labs and it will be impossible for any one of the groups to achieve the aims working alone.

FtsK is the fastest known molecular motor, moving along DNA at up to 6.7 kilobases per second. In E. coli, translocation by FtsK aligns the two newly-replicated chromosomes, so that recombination by XerCD can allow efficient chromosome segregation at cell division. There are still many questions about the molecular mechanism of FtsK translocation and the coordination of its activity. Analysis of biological systems at the level of single molecules avoids ensemble averaging and allows observation of rapid dynamics and small movements. This project proposal is to observe single molecules of FtsK translocating on DNA, to determine quantitatively how the motor moves on DNA and how FtsK is stopped by encountering XerCD. The first part of the proposal involves functionalization of FtsK with monovalent quantum dots (QDs) and careful evaluation that FtsK function is not perturbed. These monovalent QDs will allow stable binding to FtsK through a biotin-streptavidin linkage without cross-linking, while their brightness and photostability will allow imaging with high precision for such a rapidly translocating motor. We devote the second part to single-molecule measurements of FtsK translocation along DNA, looking at speed, pausing, reversal of translocation, and the rotation of FtsK as it moves along this track. In the final part of the project, we will investigate how XerCD arrests FtsK translocation, observing whether FtsK ever translocates through the XerCD roadblock and if FtsK motion ceases or partially reverses. The single molecule imaging of FtsK translocation and arrest on DNA that I propose here should illuminate one of the key steps for bacterial cell division.

Maintenance, replication and expression of genetic information require intricate molecular mechanisms applied by a variety of molecular motors. Helicases are a large group of motors that catalyze the separation of nucleic acid strands, which is essential during replication, transcription, recombination and DNA repair. The physical basis of helicase activity is a fundamental yet poorly explored problem in biology, which we will address through a synergistic combination of complementary and unconventional rapid kinetic (temperature-jump/stopped-flow), force spectroscopic (single-molecule manipulation) and high-resolution AFM techniques. Based on their essential biological roles and wide-ranging mechanochemical activities, RecQ-family helicases (RFHs) are ideal models for studying helicase activity. RFHs are an important component in the maintenance of genomic integrity in organisms ranging from bacteria to humans: their mutations are characterized by severe chromosomal abnormalities. Five RFHs have been identified in humans: WRN, BLM, RECQ4, RECQ1 and RECQ5. Mutations in WRN, BLM and RECQ4 lead respectively to the heritable Werner’s, Bloom’s and Rothmund-Thomson syndromes, which are associated with chromosomal instability and cancer predisposition. In addition to unwinding duplex DNA, RFHs resolve multi-stranded DNA structures, e.g., displacement-loops and Holliday junctions, and interact with Topoisomerase III and single-stranded DNA binding protein to form an evolutionarily conserved complex with the ability to resolve double Holliday junctions (DHJs). The molecular details underlying these processes have yet to be elucidated and we lack a basic understanding of the physical basis of RFH activity, how this is activity is adapted to process complex DNA structures, and how these activities are modified and possibly regulated by specific binding partners. In Aim 1 we will determine the mechanochemical mechanism of DNA unwinding by human BLM and E. coli RecQ helicases. These studies will provide the basis for Aim 2 in which we will determine the changes in helicase activity required for processing of more complex structures. In Aim 3 we will determine how the interaction of RFHs with binding partners results in the complex activity of DHJ resolution. Completion of these aims will significantly advance our understanding of the mechanisms of RFHs and nucleic acid motors in general.

One fundamental question in biology is how the exact same genome gets partitioned into distinct gene expression profiles. This epigenetic mechanism is regulated by histone modifications and DNA methylation marks, which are established by enzymatic complexes. However, what attracts these complexes to distinct and specific sites and under different cellular contexts is not understood. It has long been suspected that non-coding RNA molecules provide site specificity to guide these complexes to their site of action.
Studies by our laboratory and others have discovered several examples of large non-coding RNA molecules that guide chromatin formation by physically associating with chromatin remodeling complexes (1-3). Thus, I hypothesize: (a) the physical association of long RNA molecules with epigenetic complexes may be a general mechanism for the establishment and maintenance of epigenetic states in development; (b) long RNAs play a key and global role that imparts specificity to chromatin modifying complexes.

In this proposal, I would like to address these issues by elucidating the mechanisms by which large non-coding RNAs are involved in regulating the epigenetic landscapes of the mammalian genome. I intend to focus on ES cells as model system with reliable readouts of differentiation to monitor how large non-coding RNAs influence cell fate decisions by undergoing different epigenetic conformations.

Vision plays as an essential role in controlling behaviors in a variety of animals. During this process, visual information is processed and transformed to specific motor outputs. Orienting behavior in zebrafish is an excellent model system in which to study such visuomotor transformation. The orienting behavior in zebrafish is known to require an intact optic tectum, a visual processing center, which receives visual input from the retina, and sends information to premotor network in the midbrain and hindbrain, called reticulospinal neurons. However, it remains to be elucidated how tectal neurons project to the premotor reticulospinal system to produce coordinated orienting behavior: how is each tectal neuron connected to reticulospinal neurons and how does it contribute to the production of orienting behavior?
To understand the neural bases of the visuomotor circuits controlling orienting behavior, I first plan to map neuronal connectivities of tectal neurons onto reticulospinal neurons using genetic labeling methods in zebrafish larvae, aiming at comprehensive analyses of their wiring patterns. Secondly, the morphological connectivities observed between tectal and reticulospinal neurons will be further tested for their physiological connectivities by calcium imaging. Finally, to examine the role of tectal neuron projections in visuomotor transformation, I will manipulate neural activities of genetically defined small subset of tectal neurons using optogenetic tools and simultaneously monitor their behaviors in living zebrafish. These approaches should give novel insights into neural mechanisms underlying sensorimotor coordination during animal behaviors.

Voltage-sensitive dye imaging in vivo provides unique insight into the correlated electrical activity of the neocortex offering millisecond temporal resolution and subcolumnar spatial resolution. Voltage-sensitive dye imaging has revealed propagating waves of activity and specific sequences of interactions between sensory and motor areas. Both spontaneous and sensory-evoked activity imaged with voltage-sensitive dye are profoundly regulated by different spontaneous behavioural states in mice. However, there have not yet been any reports of voltage-sensitive dye imaging in mice performing a learned task. Here, I therefore propose to image the cortical spatiotemporal dynamics of mice carrying out simple motor actions motivated by reward expectation predicted by a tactile whisker stimulus. During a training period, mice will learn to associate a whisker deflection with a reward that is delivered if the mouse licks or presses a lever within a few hundred milliseconds of the whisker stimulus. After mice have learned such a whisker detection paradigm, I will image the sensorimotor cortex and analyse trial-by-trial correlations of voltage-sensitive imaging data investigating events linked to the brain states, sensory processing and motor control. These measurements will be combined with electrophysiology to provide measures of spiking activity. In addition, I will carry out highly specific manipulations of cortical activity using optogenetic approaches through viral expression of channelrhodopsin 2 and halorhodopsin. My goal is to define the contributions of different interacting cortical areas during execution of a simple well-defined behavioural task.

In many different cell types, ranging from yeast to human epithelial cells, proper polarization is essential for cell function. Yeast needs to polarize for proliferation; epithelial cells that lose their polarization can become cancer cells. The polarization mechanisms differ in different cell types. Even closely related species use a variety of polarization machines. I propose to study how adaptable the polarization machine is. How many mutations are necessary to switch from one mechanism to another and how much do different mechanisms differ between species? I will address these questions in fungi, which are classical model systems to study cell polarization. I will destroy the polarization machinery in budding yeast and subsequently restore polarization by evolution experiments. By evolving different parallel lineages with different initial growth morphologies, my aim will be to evolve simultaneously different mechanisms. Next I will analyze the evolved cells to determine which new polarization mechanisms have evolved and compare these new mechanisms and the types of polarization they produce to fission yeast and Ashbya gossypii, which are related fungi. These evolution experiments will shed more light on the mechanism of polarization but also on how easy it is to switch from one polarization mechanism to another.

Our body is constantly exposed to microorganisms present in the environment and breaks in our skin integrity (wounds, burns or insect bites) can open doors for microbes and result in skin infection. Innate immune cells represent the front line of host immune defense and ensure that most infections are repelled without any symptoms of disease. Resident keratinocytes, macrophages and mast cells in the skin react to tissue damage and pathogen entry by secreting a cocktail of cytokines, chemokines, and soluble mediators. Among other things, these released factors initiate neutrophil recruitment from blood vessels and directed migration to the site of inflammation, an essential step in clearing infections. By employing advanced immunohistochemical methods, two-photon intravital microscopy, computational modeling, and genetic manipulation, I will investigate the crosstalk between innate immune cells and the sequence of in vivo events leading to neutrophil recruitment in the course of a sterile wound, as well as during bacterial and parasitic infections of the skin.