In eukaryotes, translation is initiated by a complex mechanism in which the 40S ribosomal subunit is recruited to the mRNA 5’-end cap structure, recognizes the start codon by a scanning mechanism before association with the 60S ribosomal subunit to form the active 80S ribosome. In an alternative pathway used by viruses such as the human pathogenic hepatitis C virus (HCV), an RNA structure called internal ribosomal entry site (IRES) located in the 5’ untranslated region recruits, properly positions, and activates the ribosome in a cap- and end-independent process which involves only a small subset of the initiation factors (eIFs) needed for canonical translation initiation. The aim of this proposal is to investigate the molecular basis of HCV IRES translation initiation using X-ray crystallography complemented by biochemical methods. Specifically, (i) the crystal structure of the human 80S ribosome bound to the HCV IRES shall be solved; (ii) essential IRES residues and their function in the 80S assembly process will be determined; (iii) the mechanism of IRES dissociation from the ribosome after translation has been initiated will be investigated, and (iv) whether IRES can reassociate with the ribosome after translation initiation thereby possibly enhancing multiple rounds of translation. The goal of the project is to provide new insights into both the function of the eukaryotic ribosome and the IRES-mediated translation initiation pathway. Insight from the proposed research may help designing new anti-viral drugs.

ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transfer. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which ~3Mbp of chromosomal DNA are transported across a division septum by the SpoIIIE ATPase. The goal of this project is to elucidate the molecular mechanisms of DNA transfer machines by developing single-molecule and microscopy methods and applying them to the study of DNA translocation by SpoIIIE. We recently showed that SpoIIIE translocates DNA at ~5kbp/s while specifically interacting with highly skewed chromosomal sequences (SRS) that guide its directional motion. In this project, we will develop and use hybrid transverse magnetic tweezers, high-speed atomic force microscopy and a novel assay for monitoring DNA translocation in vivo to understand the molecular mechanism by which SpoIIIE/SRS interactions impart translocation directionality. Central to the understanding of this mechanism is the architecture of the membrane-bound SpoIIIE complex. Novel super-resolution microscopy methods will allow us to directly visualize the architecture and the assembly dynamics of this complex. Our studies showed that SpoIIIE can wirestrip DNA of nucleo-protein complexes and thus reset the cellular epigenetic program. We will use our new technologies to shed light on the molecular mechanism of DNA wirestripping and its biological relevance. The CDA will be key at this early stage in my career by helping me establish my first independent research project.

Helper and regulatory CD4+ T cells are major determinants and modulators of immune responses.
To date, well-defined helper CD4+ T cell subsets are Th1, Th2, Th17 and immunosuppressive regulatory T cells(Treg). These subsets can be characterized by the high expression of “lineage-specific” transcription factors (TFs), namely, T-bet/Runx3, GATA-3/c-Maf, RORgt, and FoxP3, in Th1, Th2, Th17 and Treg, respectively. However, it remains to be determined what signals control the expression of these lineage-specific TFs and other undefined key genes, and which genes are the targets of these TFs during T cell differentiation. In order to understand the dynamics of gene regulation during T cell development, I will combine mathematical modelling with genome-wide analysis of transcriptional regulation by microarray, and molecular and immunological experiments. With help of mathematical modelling, I will try to identify key target genes of the lineage-specific TFs with statistically ranked data. I will also use mathematical models of the “lineage-specific” TF interactions, most of which are in fact expressed by other T cell lineages, to identify the factors that determine T cell fate and assess the models by immunological experiments. Finally, I will predict signaling pathways that control the development of these T cell subsets by mathematical modelling and assess the prediction by molecular and immunological experiments including retroviral gene transduction and RNA interference. This will establish an iterative approach between mathematical modelling and wet experiments in this field.

nimals display remarkable diversity in body plans. However, the origins of this diversity and the mechanisms by which it evolved remain largely unresolved. Recent efforts to understand the evolution of body form have focused on the role of changes in the establishment of body axes and organization early in development arising from differences in gene expression during development. Research into the molecular basis for such changes in gene regulation have largely focused on single morphological traits and target genes from isolated genomic regions, yielding data that is too sparse and scattered to lead to general mechanistic principles. Systematic integrated studies of sequence evolution and the effect of sequence changes on the molecular events that underlie gene regulation are needed to infer general principles governing the evolution of genetic regulatory networks and thus of body shapes. I propose to apply new experimental genomic technologies to study the evolution of gene regulation and gene expression in multiple species from the fruit fly genus Drosophila, placing experimental analysis of gene regulation into an evolutionary framework. Genomes of both closely and distantly related Drosophila species (spanning dozens of million years) have been sequenced, and many genetic and developmental tools are available. This unique combination of developmental and genomic tools on a wide evolutionary range for the first time allows studying the evolution of the regulation of gene expression on a large scale, and may pave the way toward general laws governing body shape evolution.

Gene therapy approaches are based on the replacement of a mutated gene with a correct allele. Homologous recombination promoted by a specific DNA break in the proximity of the gene of interest is the most effective way to precisely insert the therapeutic gene. Homing endonucleases are employed in nature for similar tasks, but their sequence requirements do not necessarily match the target genes for therapy. The focus of my work will be, thus, the design of new homing endonucleases to attain the specificity required for gene therapy. Homing endonucleases will be computationally designed to target genes responsible for severe combined immunodeficiencies (X-SCID and ADA-SCID). The designed endonucleases will be further refined using directed evolution techniques. Therefore, the designed proteins will be of immediate impact as potential therapy. Moreover, the methods developed will represent a general approach for the generation of homing endonucleases with new specificities and for the design of new DNA binding proteins and nucleases with potential applications in the fields of genome engineering and gene therapy.

The maintenance of an undifferentiated state or the commitment to a specific lineage are fundamental transitions in the cell fate that involve nuclear reprogramming and changes in transcription. Alternative splicing affects the majority of mammalian genes and it can greatly expand proteomic complexity and regulate key biological processes. However, its role in cell fate determination during early embryogenesis is largely unknown.
We will use high-throughput analyses of gene expression, including whole genome splicing-sensitive microarray and deep sequencing, to explore changes in alternative splicing during the transition from totipotency to pluripotency in mouse preimplantation development, and from stem cell pluripotency to lineage differentiation in embryoid bodies derived from embryonic stem cells.
In addition, we aim to correlate these transitions with changes in the expression or activity of splicing factors, with the goal of understanding the logic of the regulatory programs behind these changes in posttranscriptional regulation.
Hypotheses derived from the transcriptome data analyses will be tested in vivo by monitoring the effect of downregulation of selected splicing factors on these developmental transitions. In addition, we will employ bichromatic fluorescent reporters that recapitulate specific splicing events to follow changes in splicing regulation in space and time at single-cell resolution during mouse preimplantation development.
The results of this research project should shed light on posttranscriptional mechanisms of genomic regulation, at exon-level resolution, during the acquisition of crucial cell fate decisions at the onset of embryogenesis.

Understanding how neuronal circuits are formed during the development of the nervous system has been a major challenge for neuroscientists. The role of genetic programs in this process is increasingly well understood. However, our understanding of the way in which electrical activity affects the structure and function of developing circuits is quite limited.
This research will develop a coherent understanding of where electrical activity is important, when it is important, and what it is important for, using a simple three-neuron microcircuit that develops within a 10 hr period. The long-term goal of the project is to identify the role(s) of electrical activity in the assembly of microcircuits during the development of the nervous system. For this purpose we will use state-of-the-art techniques in genetics, molecular biology, imaging and neurophysiology: we will use a recently developed Gal4/UAS system in a vertebrate embryo to track the development of specific neurons and manipulate their activity selectively.

Cell division and migration are frequently recognized as isolated processes but their interdepence has been largely neglected in developmental biology. The roundworm C. elegans provides a unique opportunity to study the crosstalk of both processes during embryogenesis. The complete lineage and the divisions of all cells can be followed and the exact time of each cell division is known. Together with a recently established automated microscopy system for lineage tracing and the ease of genetic manipulation, a quantitative analysis of cell cycle timing at a level that exceeds the possibilities in any other model system can be achieved. This project will therefore exploit the invariant developmental timing of C. elegans to provide insight into how cell cycle regulation is linked to migration.

Brains evolution is the consequence of many years of selective pressure within specific environments, enriching genotypes and neural phenotypes that result in adaptive behaviors. The driving mechanisms of this critical process are largely unexplored. My long-term goal is to discover how environmental and biological mechanisms direct neural network changes, accomplishing behavioral adaptation. The objective of this proposal is to examine these adaptive changes in robotic and Drosophila olfactory networks during experimental evolution in controlled environments. It has been hypothesized that 1) the convergence of early olfactory networks across species towards discrete, glomerular topologies reflects an adaptive response to common features of olfactory environments and 2) that the divergence of odorant receptor repertoires and glomerular identity across species indicates pliability and natural diversity of olfactory networks within species. These hypotheses will be tested in three specific aims: 1) measure the impact of odorant spatial covariance on the topology of emergent robotic olfactory networks, 2) test the function of novel connections in the Drosophila olfactory system, and 3) identify natural biological strategies that modify the Drosophila olfactory system to enhance innate chemosensory behaviors. This approach innovates by leveraging the distinct advantages of robotics and Drosophila and exploring the dynamics of emergent and pre-existing olfactory networks, respectively. I expect to reveal the mechanistic principles underlying olfactory network evolution, an important step in understanding why brains are the way they are and how they can change.

Cells can respond strongly and specifically to mechanical cues in their environment, including the viscoelastic properties of the extracellular matrix. Remarkably, the lineage distributions of adult neural stem cells (NSCs) can be directed in vitro by tuning the elasticity (stiffness) of their environment.

We propose to develop a phase diagram of stem cell mechanoregulation in three steps: First, we will identify the viscoelastic parameters of the extracellular matrix substrate cells sense. Second, we will determine which parts of the signal transduction machinery must be present in an NSC for it to sense its mechanical environment. To this end, we will make use of lentiviral vectors to precisely modulate expression of targets in the mechanosening pathway. This will allow us to generate a “phase diagram” of cell response to stiffness of the material, in dependence of signal transduction machinery. Third, we will use lentiviral expression systems to address the question of whether the expression of cytoskeletal proteins characteristic for one lineage is simply correlated with differentiation, or whether it is a cause of differentiation. This is likely to have particularly high impact on the field, as the expression of these cytoskeletal proteins is frequently used as “gold standard” marker of cell fate.

The proposed project requires expertise in materials science, mechanics, cell biophysics, and stem cell biology. We propose to use the support of the HFSP fellowship to combine my expertise in the former two areas with new training in the latter two to tackle the problem of neural stem cell mechanoregulation.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease. A major pathological hallmark of PD is the presence of cytoplasmic inclusions called Lewy bodies in the substantia nigra. Recently, several mutations in the leucine-rich repeat kinase, LRRK2, were found to be associated with autosomal dominantly inherited, late-onset PD. Mutations in LRRK2 is thought to cause disease by a toxic gain-of-function. LRRK2 is a multi-domain protein with dual enzymatic activity. LRRK2 has been shown to accumulate along with a-synuclein, the major constituent of Lewy bodies. The exact function of wild-type LRRK2 is however unclear. C. elegans is being increasingly used as a model organism to study human neurodegeneration. I will aim to develop and characterise a transgenic C. elegans model expressing either wild-type human LRRK2 or the most common PD mutation G2019S LRRK2. I will also attempt to study the role of C. elegans LRRK2 homolog, LRK1 on autophagy and whether autophagy contributes to LRRK2 accumulation and disease.

This HFSP short-term fellowship will be an excellent opportunity to establish in a new but related area of research and get some pilot data to apply for career development fellowships in the UK. Working at the forefront in the areas of autophagy and protein conformation diseases together with excellent support from my current supervisor, I strongly believe that this short-term fellowship would immensely help in this crucial stage of my career. This will be a stepping-stone towards establishing an independent and successful group in the coming years.

Hypochlorous acid (HOCl), the active ingredient of household bleach, is used as powerful antimicrobial strategy by the mammalian host defense. HOCl apparently kills bacteria by causing oxidative unfolding and aggregation of essential bacterial proteins. As defense mechanism, bacteria employ the redox-regulated chaperone Hsp33. Hsp33 uses the oxidative unfolding activity of HOCl to convert from a fully folded yet inactive protein to a partially unfolded, highly active chaperone, which protects cellular proteins against stress-induced unfolding and bacteria against HOCl-stress. In my postdoctoral research, I will H/D exchange in combination with mass spectrometry to visualize the structural dynamics of Hsp33 activation and inactivation process. These studies will provide me with a detailed mechanistic picture about how protein conformation regulates substrate binding and release in ATP-independent chaperones. Based on these results and the analysis of Hsp33’s substrate binding specificity using high affinity peptides, I will conduct computational modeling of Hsp33-substrate interactions. These studies will guide mutational studies, which will provide insights into the general mechanism of chaperone-substrate interactions and the central role Hsp33 plays in protecting proteins against HOCl-stress. Finally, I will exploit the modular character of Hsp33, which makes this protein uniquely suited for a directed evolution approach to generate oxidant-specific redox modules. These experiments have the clear potential to generate sensor proteins that detect specific oxidants in living cells, and to elucidate how distinct oxidative stress sensing mechanisms evolved in proteins.

Circadian clocks enable organisms to anticipate daily recurring physiological and environmental changes by modulating a plethora of body functions according to the time of day. In peripheral organs of mammals, metabolism and the circadian clock are strongly coupled, and a severe disruption of circadian rhythmicity can evoke diseases such as metabolic syndrome. It is therefore of high interest to unveil the mechanisms that connect metabolic and circadian regulation on the molecular level. Here I propose to study a novel regulator I identified in the host lab of my HFSP fellowship as a paradigm for metabolic sensor proteins that regulate the circadian clock. A number of projects will be initiated in order to analyze the function of this regulator, heat shock factor 1 (HSF1), in the circadian network. Specifically, I want to address the question of how HSF1 is mechanistically linked to the circadian clock by employing various hypothesis-driven and unbiased approaches. Furthermore, I want to identify the cues that regulate the activity of HSF1 under physiological conditions in order to better understand the metabolic parameters the body has to adjust to under normal and disease conditions. Finally, in a collaborative effort an attempt will be made to identify novel metabolic regulators of the circadian clock in a large scale loss-of-function screen. The CDA would help me to make a successful transition to an independent group leader by giving me the financial means to set up a competitive chronobiology lab. This would greatly help me to pursue my long term goal of creating a lab with a research focus at the interface between metabolic and circadian regulatory networks.

The goal of this project is to develop a computational system for displaying gene regulatory network information in an evolutionary context. It is envisaged that the preliminary version of the new system, at the end of the short research stay funded by this grant, will provide the relational schema to hold and query gene regulatory information and putative orthologues of every gene in the network. This will be presented in a text-based form in the PlnTFDB (http://plntfdb.bio.uni-potsdam.de/), GabiPD (http://www.gabipd.org/) and GRASSIUS databases, and will provide the basis for graphical display using e.g., Cytoscape (YEUNG et al., 2008). The understanding of the the evolution of gene regulatory networks (GRN) and their conservation patterns is being hindered by the lack of a computational system that displays such networks in an evolutionary context. A system encompassing a broad phylogenetic range of plant species, as the proposed in this application, will provide insights into the evolution and control of GRNs in the plant lineage.

Regeneration of tissues and organs is an enigmatic field in biology, with fundamental unsolved questions. Humans may regenerate finger and toe tips in a process which involves the integrated regeneration of many tissues, including nail matrix, nail bed, finger pulp, sensory organs, dermis and epidermis, all of which reform to a normal cosmetic and functional state. Mouse (newborn & adult) exhibit similar inherent capacities and may regenerate digit tips of a forelimb and hind limb within 4-5 weeks providing an experimental animal model on which to study this phenomenon. Both cellular and molecular mechanisms of digit tip regeneration are poorly defined. The specific research aims are: 1. To trace the cell and germ layer origin of digit tip regeneration and explore their somatic fates 2. To elucidate the molecular mechanism of digit tip regeneration and to study the functions of key genes in early regeneration events. My graduate studies involved in-depth familiarization with principle mechanisms and processes of tissue and organ development and regeneration. My post doctorate training will focus on the questions of the molecular mechanisms and the ‘cell of origin’ during mammalian digit tip regeneration. Integrating a single-cell level of understanding of the biology of stem cells within this regeneration processes. I plan to conduct this post doctoral activity in one of the leading stem cell biology laboratories, under the supervision of Irving L. Weissman at Stanford.

The overall goal of this project is to analyze the kinetic profile of protein sequences as they are synthesized, one at a time, by detecting the pattern and timing of optical signals emitted from labeled single ribosomes. The ribosomes and tRNAs will be labeled by FRET pairs and will be extensively characterized in a cell-free system. Various coding mRNAs will be utilized in order to follow the translation process in real time using total internal reflectance fluorescent (TIRF) microscopy.
The ribosomal protein L11 will be tagged with a FRET acceptor whereas the cognate FRET donor will label a specific tRNA. The signal will be in the form of temporal FRET pulses, reporting the accommodation of specific tRNAs. Knowing the mRNA sequence will allow real-time analysis of rhythm and pauses that modulate the translation rate. Factors that govern the kinetic profile of protein synthesis will be inspected; these include rare codon usage, mRNA secondary structure, and nascent chain sequence.
Single-molecule recording of protein synthesis directly reports the kinetic trajectory of that individual ribosome. Tabulation of the pausing times will enable statistical inferences concerning the kinetic mechanism of translation. In contrast to single molecule investigation, ensemble techniques suffer from lack of synchronization. Although bulk measurements are very informative, the mentioned disadvantage became apparent during my PhD studies. Acquiring the methodologies in my host labs will enable me to combine single molecule spectroscopy and imaging with bulk measurements in my future career. This will lead to a deep mechanistic understanding of dynamical processes at the molecular level.

Understanding how histone post-translational modifications regulate chromatin structure and a range of chromatin-related processes is fundamental to our understanding of, epigenetics, differentiation, stem cell biology, and human pathologies. While the importance of histone acetylation and methylation is well established, the role of histone ubiquitylation is only beginning to unravel, and insights have been largely restricted to monoubiquitylation of H2A on lysine 119 and H2B on lysine 120. Given the dynamic and transient nature of protein ubiquitylation and chromatin modifications, it is very likely that additional histone ubiquitylation sites exist that play important roles in chromatin-related processes. I provide herein a comprehensive strategy for the analysis of histone ubiquitylation states and functions. By combining yeast genetics, mass spectrometry, modification-specific antibodies, “designer chromatin”, and in vitro transcription assays I intend to identify novel histone ubiquitylation sites, study their function, and explore the biological machineries that deposit and interpret them. The newly identified modifications and the underlying enzymatic machineries will be verified in higher organisms, and their function will be studied in relevant experimental systems such as mammalian stem cells. This work has the potential to shed important light on the biological significance of novel and previously known histone ubiquitylation, and improve our understanding of chromatin-based processes. Mechanistic insight into these fundamental issues lies in the heart of chromatin biology with far-reaching implications for human biology and human diseases.

Fission yeast mating-type switching occurs in one of two sister cells based on the inheritance of older ‘Watson’ vs. ‘Crick’ DNA chains at mat1 locus. Possibly this mechanism of asymmetric cell division also functions in diploid organisms. Here, epigenetically differentiated sister chromatids, based on the replication history of their DNA chains, are selectively segregated to daughter cells. Analysis of mouse chromosome 7 segregation patterns - which is biased in embryonic stem cells (ESCs), ESC-derived endoderm and ectoderm, but not in mesoderm cells - supports this theory. Biased segregation is dependent on a dynein motor (Dnahc11) previously implicated in embryonic left/right body axis specification.

I propose to study segregation patterns of mouse chromosomes 7, 11 and X in vivo, by generating mice harbouring chimeric transgenes, consisting of part of the coding sequences of GFP and RFP, separated by a loxp site. Upon Cre-recombination, chromatids acquire either functional GFP or RFP. Depending on segregation pattern, either red and green or yellow (red+green) and colorless cell clones develop. I will investigate if segregation pattern is affected in Dnahc11 mutant mice in vivo. In addition, I will test predictions of the strand segregation model regarding embryonic lethality rates. Moreover, biochemical and cell biological studies with FLAG and GFP tagged Dnahc11 protein in ESCs will address mechanistic aspects of selective chromatid segregation.

One central aim in neuroscience is to relate the cortical anatomical structure to its function with the expectation to find answers to fundamental questions about neural processing, learning, memory, and even cognition. A major gap in the understanding of cortical circuit function arises from the difficulty to integrate the knowledge about the functional properties of synapses and neurons, because on one hand experiments usually include only small cell/synapse numbers, and on the other hand the knowledge of the circuit anatomy lacks fine detail required for biologically realistic models. The goal of my proposal is to bridge this gap. I will use channelrhodopsin assisted stimulation of axons together with postsynaptic calcium imaging to explore the properties and dendritic locations of the synapses from a large number of inputs simultaneously. This will provide crucial information for understanding synaptic integration and how the cortex samples for appropriate synaptic connections. Furthermore, I will use 2-photon glutamate uncaging to stimulate single synapses in vivo, in order to analyze the impact of the activity of a single synapse on its parent neuron, which is crucial to understand how structural and functional plasticity at the synapse level relates to circuit performance. The Career Development Award will help me accomplishing these goals by providing the necessary funds to establish a strong, productive team applying state of the art techniques to fundamental scientific questions at the Max Planck Institute for Neurobiology.

All higher animals use a complex muscle - tendon network to execute their daily movements. For these to happen properly all muscles have to form at the correct positions, grow to the appropriate size and produce just enough force. Muscle development is a multi-step process; myoblasts proliferate, migrate and fuse with each other at particular locations within the body depending on their identity. The forming myotubes search for appropriate tendon cells for a stable connection. My aim is to understand how the complex muscle network of an adult fly is built. What are the molecular mechanisms creating diversity and instructing the different muscles to target to specific tendon cells? As an entry point we developed and applied genome-wide, inducible RNAi in Drosophila and systematically surveyed the entire genome for genes with a potential role in adult muscle morphogenesis. 1) From this screen I aim to focus on two types of molecules for a phenotypic characterisation: transcriptions factors to investigate muscle diversity and cell surface molecules to study muscle targeting. 2) For a detailed phenotypic and mechanistic characterisation of these genes I will apply in vivo imaging. This will enable me to directly follow the entire process from muscle fusion to targeting in the intact animal. 3) Complementary to the above efforts I wish to compare the myoblast proteome of wild type to mutants in identified transcriptions factors. This should result in a quantitative measure of muscle diversity. I envision to unravel developmental principles instructing muscle morphogenesis that will be conserved and directly applicable to vertebrates and hence be of wide general interest.