The applicant wants to expand his research towards biology; more specifically in the area of cell motility, since his ambition is to obtain a permanent academic position in the future. This fellowship would be the perfect start to achieve this goal. The host group of Prof. Grzybowski has recently shown a revolutionary new way of biasing the direction of various cancerous cell types (i.e. cell motility) using asymmetric (ratchet) surface micro geometries. This approach has great potential in cancer therapy because of the difference in motility of healthy versus cancerous cells. Another reason for choosing this group is the strong multidisciplinary character (math, physics, chemistry and biological backgrounds) of the researchers. Their research is supported with appropriate mathematical models (including the research on cell motility), which enables them to extract more information of the underlying fundamental principles. Inspired by the recent cell motility research, we propose to combine not only surface patterning but also gradients of known stimuli (e.g. signaling molecules and/or temperature) to influence the motility of human breast cancer MDA-MD-231 cells (i.e. telotaxis). Moreover, by this new approach we hope to induce synergy (1+1>2) in the cell movement by using multiple stimuli simultaneously. We expect this new approach to have a major impact in the field of cancerous metastasis, since the understanding of the fundamentals of cell motility can potentially provide new ways of preventing this phenomenon.

Glutamate receptors are key players in the fast excitatory synaptic transmission throughout the human brain. They are involved in processes like memory and learning as well as in neurodegenerative diseases. N-Methyl-D-Aspartate receptors (NMDAR) belong to this family of glutamate receptors and show a very complex behavior. Their intriguing pharmacological, biochemical and physiological properties have been studied extensively by different experimental techniques, but the mechanisms underlying gating and ion permeation are not fully understood. So far crystal structures of the extracellular ligand binding domain have permitted insight into agonist recognition and allow speculation about the mechanisms underlying the gating process.
The goal of my proposed research project is to solve the first high-resolution structure of a full-length NMDAR. NMDARs are eukaryotic proteins that are assembled from two homologous subunits. Even if NMDARs have been successfully expressed in mammalian cell lines, for structural studies high amounts of homogeneous protein are required. The main challenge in this project is to obtain a well-folded, functional NMDAR in sufficient amounts, which can subsequently be used in broad crystallization screening and determination of the structure. To assess the functional state of the receptor and characterize it further, I will apply electrophysiological techniques in parallel to the biochemical studies. A high-resolution crystal structure of an NMDAR would represent a leap forward for the field and disclose the way to new therapeutic approaches.

Once considered the dark matter of the protein topology universe, topological knots have recently been identified in many protein structures. Despite the apparent complexity that a polypeptide chain needs to thread itself through a loop in order to form a knotted structure, emerging experimental evidence has shown that knotted proteins can unfold and refold efficiently in vitro without auxiliary factors, and that even under highly denatured conditions, the knotted structures are present. How and when these knots are formed during folding, and how they may be retained in the unfolded state remain poorly understood. It is likely that such structural elements are formed on the ribosome co-translationally or post-translationally during de novo folding, and that specific chaperones are required to overcome kinetic barriers for knotting. If a global solution to the protein folding problem is to be found, the puzzle of how such knotted structures form must be solved. The proposed project aims to devise an experimental strategy based on solution state NMR spectroscopy to probe the structures and dynamics of a variety of knotted proteins along their folding pathways in order to monitor knot formation and to identify potentially common key elements that are responsible for promoting the knotting events. An integrated approach combining biophysical and computational methods will be employed to generate structural models of protein folding intermediates. The ability to reconstitute a de novo folding system, comprised of the target protein, the ribosome and associated chaperones, for NMR studies will bring us closer to understating protein folding in a cellular environment.

Especially during embryonic development, angiogenesis and vasculogenesis are highly dynamic processes that play a crucial role in the formation and maintenance of the vasculature. Even though developmental processes have been studied in certain parts of the zebrafish such as the intersomitic vessels or branching arches, a dynamic reconstruction of the interconnected web of vessels is currently missing. The proposed project aims to create a detailed, dynamic atlas of the zebrafish vasculature as a first step to describe and quantify wildtype development through high-resolution timelapse image acquisition, image processing, segmentation and quantification. The digital data is automatically annotated with blood flow direction and speed, vessel diameter and their hierarchy, etc. In a following step this knowledge will be used to identify and numerically describe novel vascular mutants and quantify the effects of drugs. The zebra?sh can thereby act as a model of human diseases and their treatment. Tumor angiogenesis and regeneration can be followed in vivo using advanced light microscopy techniques, photo-manipulation tools, and small molecule screens. At the same time the data taken from individual embryos is used to find common underlying mechanisms of vessel formation and to describe how each embryo differs from the “average embryo”.

Polycomb group (PcG) and trithorax group (trxG) proteins are key transcriptional regulators of major developmental genes thereby coordinating cell differentiation and proliferation. PcG proteins contribute to gene silencing, while trxG proteins alleviate silencing in appropriate cell types. Recently it has been shown that polycomb complexes are required in maintaining ES cell self-renewal and plasticity during embryonic development.
Drosophila germline stem cells (GSCs) are one of the most accessible stem cell populations for in vivo study. Therefore, GSCs represent an ideal cell type to further study the role of PcG proteins in stem cell biology. The GSCs undergo asymmetric divisions, whereby one of the daughter cells loses contact with the niche and begin differentiation into a mature egg. Understanding the choice between self renewal and differentiation will help clarify the special properties of stem cells that are important in tumor formation, aging, and therapeutic applications. By characterizing the chromosomal distribution of PcG proteins in GSCs and documenting the effects of PcG protein mutants on stem cell function I will significantly advance our understanding of the role of Polycomb group genes in maintaining stemness and plasticity.
Since Polycomb group proteins are evolutionary conserved from fly to human, this study in Drosophila will help understanding some of their roles in human.

One of the design principles of multi-cellular organisms is the hierarchical division of epithelial tissues into stem cells, non-differentiated cells with unlimited proliferative potential, and differentiated progenies with a more defined phenotype and limited life-span. In recent years epithelial tumors have been shown to display a similar hierarchy of cells with distinct gene expression profiles and tumorigenic capacities, an observation that led to the cancer stem cell hypothesis. Unraveling the details of the stem cell differentiation program in epithelia can address outstanding questions in stem cell and cancer research, including: How do stem cells decide which fate to undertake, what is the contribution of the stem cell niche to the differentiation process, what is the variability in gene expression between different stem cell lineages, what is the cell of origin of a neoplastic clonal expansion and to what extent is the normal hierarchical gene-expression program maintained in different stages of tumorigenesis. My research will aim at characterizing the single-cell gene expression program associated with the stem-cell hierarchy in crypts of the normal instestine, and at studying how this program changes in colon adenocarcinomas. By simultaneously measuring transcripts, expressed by either intestinal stem cells or their differentiated progenies, in stained frozen tissues, we will seek a quantitative description of this differentiation program and its perturbation in cancer, thus gaining important insights into the somatic evolution of tumors.

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.