Gene alterations by translocation is a hallmark of most hematopoietic malignancies, and other aberrations in microRNAs and their target sites, or in promoter sequences have recently emerged as additional factors in hematopoietic carcinogenesis. However, we still know little about the temporal process by which carcinognesis progresses in hematopoietic cells. Here, I will combine will combine experimental and computational approaches to construct and test a model of carcinogenesis in B-cell chronic lymphocytic leukemia (CLL). First, I will use RNA-Seq to measure expression levels for coding and noncoding RNAs, and to identify any aberrant transcripts in samples from B-cell CLL. Combining those with a larger number of microarray and genetic profiles for CLL (available in our lab), I will construct a provisional model to identify key candidate regulators and their putative targets. I will then use RNAi libraries and overexpression systems – combined with a novel delivery approach and signature based profiling – to test the role of these regulators in affecting the CLL transcriptional program. Finally, I will perform transformation assays and FACS analysis of markers indicating carcinogenesis to confirm whether the changing expression level of the regulators induce carcinogenesis in B cells. My model will provide a detailed view of the regulatory program controlling carcinogenesis in B-cell CLL, as well as a deeper understanding of the complex networks controlling fundamental biological processes in mammalian cells.

Functions of polymeric cytoskeletal assemblies depend not only on their positional, but also directional organization. For example, axonal microtubules are always oriented with their (+)-ends outward toward the axon terminus. To achieve cell division, coordination of the polarity of actin bundles in the contractile ring is required. The structural basis of how these assemblies function remain to be revealed, because the methods of measuring the polarity of cytoskeletal filaments in live cells are inadequate. We aim to fill this gap. Our approach is to correlate the measured orientation of the polarized fluorescent tags (sparsely attached to the filaments) and the orientation of the birefringence axis of the native filaments. By using the fluorescent tags whose emission dipoles maintain a fixed angle with respect to the filament axis and by measuring their position at nanometer-scale precision with respect to the filament, one can estimate the polarity of the filaments across the assembly. The host laboratories will provide cutting-edge microscopy techniques capable of measuring polarized fluorescence of single molecules and axes of the weakly birefringent filaments in live specimens. I aim to combine these techniques with accurate quantitative analysis algorithms based on the imaging properties of the microscope. We will refine the methods by imaging biological model systems of increasing complexity, accessible at the host laboratory. Such significant advances in image acquisition and analysis are important for many structure-function inquiries in cell biology.

Pluripotent stem cells can give rise to all cell types in the body and have therefore enormous potential for regenerative medicine. They also provide a powerful tool for studies in developmental biology and pharmacology. Recent advances in transforming somatic cells directly into pluripotent (induced pluripotent stem: iPS) cells provide an attractive avenue for generating large numbers of customized stem cells. However, despite remarkable technical progress in recent years, very little is known about the mechanisms underlying the reprogramming process and its dynamics: How does a single somatic cell turn into many pluripotent cells? Why do so few (~1%) of the cells end up as iPS cells, and what states do the others adopt? How is the process influenced by the starting cell’s state and what are the possible paths that cells can take towards pluripotency? What roles do signaling pathways and specific reprogramming factors play in these paths? A better understanding of the key steps and mechanism would be instrumental in increasing the safety and efficiency of reprogramming, as well as our basic knowledge on the plasticity of cell states.

In this project we aim to advance our understanding of the dynamics and mechanisms of cell state transitions during mammalian cell reprogramming using a combined high-resolution live cell imaging and probabilistic modeling approach. To achieve these goals, we will need to build novel cell and molecular tools and develop advanced statistical analysis and modeling.

Our collaborative design will provide a look with unprecedented resolution on the cell state dynamics following the reprogramming signal. Elucidating the “black box” of reprogramming will greatly increase the potential therapeutic applications and have a substantial impact on many areas including developmental and regenerative biology. We also believe that these findings will shed light on cell state dynamics in general, including abnormal cell state transitions observed in cancer.

Selfish genetic elements such as transposable elements (TEs) make up large parts of eukaryotic genomes. To suppress their deleterious mobility, animals evolved a highly conserved genome defense mechanism that is active in gonads. At its core are PIWI proteins and bound 23-30 nt long RNAs (termed piRNAs). Via sequence complementarity, piRNAs guide PIWI proteins to TE transcripts, which triggers their silencing. Recent evidence suggests that Rhino, a Drosophila ovary-specific heterochromatin protein 1 (HP1) homolog, has a pivotal function in piRNA biology. There is also strong evidence that the classical HP1 family member Su(var)205 is involved in piRNA mediated TE silencing. Despite this, the precise role of chromatin and HP1 proteins in the piRNA pathway is elusive. Here, I propose a systematic in vivo analysis of all 5 Drosophila HP1 family members in the context of the piRNA pathway in order to dissect the role of chromatin in small RNA-mediated TE silencing. A strong focus will be to unravel the molecular function, binding partners and targets of Rhino (HP1d) and HP1e, which are specifically expressed in ovaries and testes, respectively. Both proteins evolve rapidly and are under strong positive selection, suggesting that they act at the center of the genetic conflict between genome and TEs.
The proposed research combines genome-wide studies of HP1 binding patterns with sophisticated Drosophila genetics. It will yield the first systematic survey of HP1 binding profiles in any cell type and will relate these to the piRNA pathway. Finally, it sheds light on the function of germline specific HP1 proteins and their involvement in defending the animal genome against TEs.

Degenerative diseases pose increasing medical problems to the aging population of developed countries highlighting the importance of a better understanding of regenerative processes. Monocytes and macrophages have been recognized as important mediator of tissue repair but the underlying mechanisms are poorly understood.
Therefore, we plan to examine the regenerative potential of monocytes and macrophages in the context of cardiac muscle injury, an experimentally accessible regeneration model of high medical relevance. We plan to define the subtype specific contribution of monocytes/macrophages to tissue regeneration, to analyze and modulate macrophage derived cytokine production in a pro-regenerative micro-environment and to explore the potential of monocytes/macrophages for direct linage reprogramming, either spontaneously in injured tissue or upon forced expression of an appropriate transcription factor cocktail. For these experiments we will take advantage of self-renewing Maf-DKO macrophages that have been developed by the Sieweke lab. These cells can be amplified in culture without tumorigenic transformation but maintain functional macrophage identity and integrate into tissue macrophage networks upon transplantation. They are thus ideally suited tools to genetically probe and manipulate macrophage function in tissue regeneration. They may also pioneer monocyte based cellular therapies that could eventually represent an attractive alternative to stem cell based cell replacement therapies in regenerative medicine.

Stem cell biology is one most rapidly developing fields in biomedical research and there is significant hope that stem cell therapies will be common clinical practice in the near future. My previous research into immunological tolerance during fetal development prompted an interest in stem cell biology. I now wish to shift my focus towards studying stem cell differentiation pathways in human beings. I chose to pursue my research in the laboratory of Dr. Frisén because he has demonstrated an ability to approach complex biological questions in human beings utilizing creative, innovative approaches with great success. In collaboration with current members of the Frisén lab, I intend to delineate the relationships between stem and progenitor cell populations in healthy humans by establishing phylogenetic trees of individual cells of the hematopoetic system. Somatic mutations occur in specific, defined genomic regions as a result of normal cell division. These mutations are inherited by each progeny cell and maintained in future generations. Because hematopoiesis is believed to occur in a hierarchical fashion, with a parental stem cell giving rise to downstream progenitors and finally mature hematopoietic lineages, it is possible to interrogate the relationship of these cells by determining the pattern of inherited mutations. By employing recent technological advances in single cell analysis and genomic sequencing, I intend to determine the identity the most primitive stem and progenitor cells in the human hematopoietic system and to define the hierarchical relationship between different hematopoietic lineages isolated from human tissues.

Neural stem cells (NSCs) are multipotent cells that are throughout life capable of creating diverse progeny such as neurons and glia through asymmetric division, or identical progeny through symmetric divisions. Notably, there is a dramatic age-dependent decline in neurogenesis. Current hypotheses suggest that this may be directly linked to stem cell “aging”, even though the underlying mechanisms remain largely unknown. Recently, it was shown that yeast budding leads to the development of a diffusion barrier, segregating specific elements between the mother and daughter cell during division. One function of this barrier is to keep aging factors, such as toxic DNA circles, within the mother, allowing the progeny to have a long lifespan. We here aim to analyze whether this type of diffusion barrier exists in mammalian NSCs during cell division and what cellular components may be retained in stem and daughter cells during asymmetric cell division. One candidate of asymmetric segregation may be extrachromosomal circular DNA that exists in mammalian cells, which not only increases with aging, but also leads to damaging effects on DNA. By observing both symmetric and asymmetric divisions within mammalian NSCs, I aim to determine the presence of a diffusion barrier during NSC division, to characterize the cellular components retained by this barrier, and to analyze its functional significance. Thus, the projects proposed here may identify a completely novel aspect of stem cell biology, underlying asymmetric cell division and stem cell aging.

The coevolution of host and pathogen is shaped and tempered by the selective pressures exerted in this interaction. Rarely is this interaction one to one, instead, hosts may harbour many pathogens; likewise, pathogens may infect several hosts. This latter case is especially true for the plant pathogen Puccinia striiformis f. sp. tritici, commonly known as wheat yellow rust, as evidenced by a host range that includes wheat, barley, rye, and several other grass species. Several resistance (R) genes have been identified in domesticated wheat, the principal host of wheat yellow rust, yet little is known about the mechanisms underlying resistance in wild and taxonomically distant hosts. In addition, it is unclear how the structural constituents that underlie resistance may shape the host range of the pathogen. To address this knowledge gap, I propose to clone R genes from wild and distant grass hosts using a technology-driven approach that leverages the strengths of association and linkage mapping. First, I will use next-generation sequencing and genotyping technologies to interrogate the genomic and genetic structure of wild and distant hosts of wheat yellow rust. Next, association mapping will be performed using de novo and a priori approaches based on our present understanding of R gene structure in plant-pathogen interactions. In parallel, this approach will be complemented with the linkage mapping of resistance and cloning of R genes. This investigation will impact our understanding of the molecular components involved in resistance recognition specificity and how this may limit the host range of plant pathogens.

Imitation is a very cost-effective way of learning and adding new actions to the motor repertoire of the imitator. For example, tying a shoe for the first time is much easier when imitating someone else perform the same action as opposed to verbal instruction in the absence of any sensory guidance. The underlying neural mechanism of this behavior, most highly developed in humans, is not clear. In monkeys, a subset of brain cells involved in the execution of specific actions has been shown to be engaged also during the perception of similar actions – thus demonstrating a functional overlap between perception and action at the neurophysiological level. During my post-doctoral studies, I have demonstrated the existence of neurons with similar properties in humans. This provides the exciting opportunity to explore the interactions and functional overlap between perceptual and motor systems in the human brain and examine their relevance to imitation and learning by observation. To that end, I intend to use a unique combination of techniques that will provide a wide range of spatio-temporal resolutions (from whole brain functional MRI to the extra-cellular activity of individual cells). By modulating parameters such as the amplitude of imitated actions, and measuring the effect on neural activity in various brain regions, I hope to gain insight into the mechanism underlying this important capacity in humans. This step will be crucial for devising optimal schemes for teaching and transferring motor and cognitive skills across individuals.

Successful neuronal migration is a prerequisite for proper brain development and function. How individual proteins influence migration in subsets of neurons has been studied, but a general overview of underlying kinetics and mechanics for different forms of migration that co-exist in vivo is lacking. This proposal aims to create a detailed understanding of the kinetics of migration and how migratory modes interact as well as the cell biological components involved in migration in the zebrafish retina. Although the retina features five different neuronal types it is a simpler system for understanding all migratory modes and their underlying mechanisms than other parts of the brain due to the shorter migratory distances and smaller number of overall neurons.
To compose a detailed picture of events on the cellular level we will perform 4D trajectory analysis to investigate the different migratory modes. These in the retina are mainly basal translocation and free migration. Additionally, cell biological studies to understand the exact roles of subcellular components and cytoskeletal elements will be performed. Cytoskeletal and organelle markers will be used to first assemble the “inventory” of the neurons at distinct developmental times. This will lead to hypotheses on how migration is achieved at varying developmental stages in different neurons. Hypothesises will then be tested by interfering with cytoskeletal elements and motor proteins which will help to understand migration on a mechanistic level. The outcome of this study should facilitate the understanding of neuronal migration in other parts of the brain that are less accessible.

Pentameric ligand-gated ion channels (pLGIC) mediate fast chemo-electrical signal transduction between cells in the central nervous system. This protein superfamily performs key functions in the brain and is of major relevance for drug design in the fields of smoke addiction, antidepressants and neurodegenerative diseases. Typically, each pLGIC is made up of 5 homologous subunits; subunit composition determines distribution and pharmacological properties of the pLGIC. Heterologous expression of several subunits cannot produce a predetermined, unique receptor population composed of five specific subunits in a specific order.
The project proposes to label and/or purify a unique, particular, heteropentameric LGIC from a mixed population. The project relies on the use of fusion proteins between pLGIC and bacterial toxins (B5 proteins) that display avidity, i.e. dramatically increased affinity for their ligands due to the oligomeric assembly. Each B subunit recognizes its ligand with milimolar affinity but a pentameric B5 protein can recognize multivalent ligands with nanomolar affinity. Here we will engineer the interfaces of B subunits such that the B5 fusions correctly assemble into pentamers only for one specific composition of pLGIC subunits. The engineered B5-pLGIC fusion will be characterized in heterologous cells and in reconstituted membranes using combined single molecule fluorescence and single channel patch clamp techniques. This project has a wide range of applications in studies ranging from the molecular scale (e.g. crystallography) to the cellular scale (e.g. imaging).

Eph receptors and their ephrin ligands are a family of membrane anchored tyrosine kinases that bind each other during cell-cell contact, triggering a bidirectional signal in neighboring cells. Eph-ephrin transduction can be regarded as a chemotactic guidance system that forms and stabilize patterns of cell organization during many developmental processes, such as neural crest cells migration, axonal guidance, or when mutated, during tumor invasiveness.

Here, we aim to analyze the bidirectional Eph-ephrin transduction by performing simultaneously single-cell time-lapse microscopy in neighbouring cells. We will decompose the overall signal processing behavior into functional modules that receive, transduce and execute the signal induced by the ligand in the Eph- or ephrin-expressing cell. Protein dynamics of EphB2 and ephrinB1 will be reported by fusion constructs harboring fluorescent proteins. The signal transduction will be recorded in real-time using time-lapse imaging of FRET probes that emit signal when adaptor tyrosin kinases or GTPases are activated. The cytoskeleton redistribution will be quantified by measuring paxillin activation with specific antibodies binding to its phosphorylated form. With this experimental approach, we aim to gather suitable data to perform quantitative 4D imaging (xyz+t) at single-cell level. Input-output mathematical models will help us elucidate the bidirectional dynamics of the pathway response to time or space variations of ligand concentrations.

Mitochondria form tight connections with a subfraction of the endoplasmic reticulum (ER). These contact sites are required for calcium and phospholipid exchange between both organelles, which is essential for the regulation of mitochondrial physiology and maintenance of the phospholipid composition of all cellular membranes, respectively. The mechanisms mediating and regulating the interaction between the two organelles remain poorly understood. Recently, a protein complex, named ERMES, has been identified to mediate tethering of mitochondria and the ER in yeast. Mutation of the evolutionary conserved rho GTPase Gem1 results in similar phenotypes compared to ERMES mutants as revealed by a systematic genetic analysis. Moreover, Gem1 and ERMES components physically interact with each other, strongly suggesting a role of Gem1 at mitochondria-ER contact sites.
In the proposed study, I will characterize the function of Gem1 at these interfaces and will determine the role of the EF hands and GTPase domains of Gem1 in this process. Furthermore, I will perform a systematic large scale analysis to comprehensively define the components impacting on the interaction of mitochondria and the ER in yeast. Mammalian cells lack ERMES counterparts, wherefore I will exploit the mammalian Gem1 homologs, Miro1 and Miro2, as starting points for the investigation of mitochondria-ER contact sites in human cells. Altogether, these analyses hold the promise to significantly enhance our understanding of the formation and regulation of mitochondria-ER contact sites and their importance for diverse cellular processes.

The JAK-STAT signaling plays critical roles in development, immune responses and stem cell maintenance, and its aberrant activation is associated with human malignancies. It has widely been accepted that the effects of JAK activation are mainly mediated by direct transcriptional activation of STAT. Recently, the Green lab has demonstrated that JAK2 phosphorylates histone H3Y41 to exclude HP1 from chromatin, which would enable expression of genes not necessarily under direct STAT transcriptional regulation. However, it is unclear how and whether these changes in chromatin structure influence JAK2-mediated haematopoietic malignancies. I hypothesize that HP1alpha constitutes a tumor suppression mechanism in mammalian haematopoietic systems and activation of JAK2 antagonizes the heterochromatin-mediated tumor suppression to induce haematological malignancies. To test this hypothesis, I propose to define the HP1alpha-mediated tumor suppressive mechanism by identifying HP1alpha target genes modulated by JAK2 activity. I expect to find that some genes are bound by HP1alpha and their expression is subsequently repressed in response to JAK2 inhibition. I plan to perform functional studies on the identified HP1alpha target genes. I will further determine whether decreased HP1alpha levels are associated with increased susceptibility to JAK2-induced haematopoietic malignancies. Defining this novel epigenetic mode of JAK/STAT signalling in the context of tumorigenesis is critical to the understanding of the mechanisms underlying JAK-mediated tumors. In addition, this will be the first study aimed at identifying genome-wide target genes of the epigenetic mode of JAK/STAT signaling.

The hippocampal formation is determinant in the establishment of episodic memories and in spatial navigation. The strong correlates of principal cells in this area with the position of the animal in its environment would be the neurophysiological substrate of such behavioral capabilities: in the hippocampus cells fire in one particular position whereas in the entorhinal cortex, the main output and input structure of the hippocampus, cells fire at the nodes of a grid covering the environment. However, both lesions and neurophysiological studies have failed so far to determine the hierarchy of integration of spatial information between the two structures. In this project, we propose to use recently developed optogenetic tools to selectively inhibit neurons from one structure that directly project to the other while network activity will be monitored with dense extracellular recordings. Moreover, the relationship between the two structures could depend on the remoteness of the spatial memory of the particular environment the animal is exploring. To test the effect of spatial learning on this interaction, we propose to compare the effect of selective inhibition in familiar environments and in new places previously unreachable. Thus, this project will shed new light on the neurophysiological substrates of spatial navigation and of the formation of long-term episodic memories.

The cerebral cortex is the latest structure to appear in the evolution of the nervous system, and its circuitry constitutes one of the most complex known arrangements. Studies of model circuits in various CNS regions have identified a handful of molecules that participate in basic guidance decisions that direct axons to their targets. To address the principles of axon pathfinding in the elaborate circuitry of the cerebral cortex, I propose to examine how callosal axons are guided to homotopic regions of the contralateral cortex after crossing the midline.

While the paths of homotopic projections display great heterogeneity, they follow a simple common rationale, backtracing their initial trajectory, from the soma towards the midline, to form a bilaterally symmetric projection. This simple architecture may allow the molecular dissection of the cell-surface mechanisms linking positional cues and cellular signaling.

Combining retrograde tracing with FACS purification, neurons projecting to distinct cortical areas can be isolated. Using surgical methods, neurons projecting to one area will be ectopically transplanted to assay commitment of projection route. In a complementary approach combining post-sort cell culture, membrane proteins differentially present in neurons projecting to different cortical areas will be identified using quantitative mass spectrometry. Candidate molecules arising from the proteomic screen will be validated using in vivo knockdown and tracing to examine the fidelity of homotopic axon projections. The aim of this study is to identify molecular mechanisms that allow axons to navigate the cortical plane and form circuitry within the cerebral cortex.

Phenotypic evolution results often from changes in gene expression patterns, which are caused, usually, by alterations in cis-regulatory regions. Cis-regulatory regions integrate inputs from multiple gene-regulatory networks by encoding transcription factor binding sites. The Drosophila shavenbaby (svb) gene provides a superb model for studying cis-regulatory structure, function, and evolution. The svb gene encodes a transcription factor that directs cuticular trichome development. Six enhancer modules located in the cis-regulatory region of svb together recapitulate the entire svb embryonic expression pattern. Multiple nucleotide changes in these enhancers have led to the evolutionary loss of many trichomes in D. sechellia, a sibling species to D. melanogaster. Surprisingly, none of these nucleotide changes map to known transcription factor binding motifs.
The central aim of this study is to identify the transcription factors that bind and regulate the D. melanogaster svb enhancers in vivo. I plan to extend existing high-throughput methods of chromatin immunoprecipitation and mass spectrometry to screen the svb enhancers in vivo for their associated proteins. I will complement these experiments with a genetic screen using lines carrying RNAi constructs targeting Drosophila transcription factors, to identify factors regulating svb enhancer activity.
My project will lead to a deeper understanding of the relationship between cis-regulatory structure and function. In addition, my work will identify the individual transcription factors that bind differentially to nucleotide sites in D. melanogaster and D. sechellia and that have contributed to morphological evolution.

When reaching for a cup of coffee, people are strikingly variable in their performance but almost never spill. This contrast leads to a fundamental question: why are inconsistent movements so consistently successful? Recent theories of sensorimotor control suggest that this inconsistency reflects an extremely sophisticated control process that preferentially corrects those errors that are relevant to succeeding at the task. The proposed research will test the merits of this concept, termed the minimum intervention principle, in the context of human object manipulation. We will determine whether the nervous system is more sensitive to some errors than others and explore whether the resulting corrections are consistent with the minimum intervention principle by comparing our results to biologically-realistic mathematical models. Critically, we will induce errors with a novel experimental approach whereby disturbances are injected directly into the sensorimotor apparatus via electrical muscle stimulation rather than the traditional approach of applying an external load to the hand. This approach provides several important advantages. First, we expect that internal perturbations will mimic sensorimotor noise and thus provide insight into how the nervous system compensates for its own disturbances. Second, since the subject will not be constrained by the experimental apparatus, we will be able to study many naturalistic object manipulation tasks. And last, since our approach is compatible with a range of physiological techniques including functional magnetic resonance imaging we will be able to elucidate the underlying neural mechanisms of the minimum intervention principle.

During development tissue remodeling leads to shape changes of organs and parts of the organism: for example tissues can invaginate, elongate, or stretch. Distinct signaling pathways regulate these behaviors through the control of the actin cytoskeleton and of myosin-based tension required for force generation and cell shape change, as well as the control of cell adhesion. Although some of the basic mechanisms responsible for shape changes are known, it is not understood how they are coordinated within groups of cells or tissues. What is the spatiotemporal pattern
of forces that orient tissue morphogenesis? What is the nature of the generated forces and how do they originate? I propose to use the ventral furrow formation of the Drosophila embryo as a model system to investigate how different tissues coordinate and interact to drive morphogenesis.

Number of biological processes (such as replication, splicing, transcription, translation, transport, degradation…) involve the generation of mechanical forces and torques. These functions are performed by molecular motors –enzymes that couple chemical energy sources such as nucleoside triphosphates or electrochemical gradients into mechanical work. Many bacteriophages and certain animal viruses actively package their genomes into small proteinaceous capsids, compressing the nucleic acid to near-crystalline densities and macroscopic pressures. At the heart of this process is a multimeric molecular complex known as the packaging motor. This machine catalyzes the hydrolysis of adenosine triphosphate (ATP) and converts the chemical energy released into the mechanical work necessary to surmount the large energetic barriers associated with genome compaction. Phi 29 presents the ideal system for the study of DNA packaging for several reasons: it is one of the simplest of the double-stranded DNA bacteriophages, it has been extensively characterized via bulk techniques and exhibits a slow and homogeneous translocation. In the frame of this HFSP fellowship, I will utilize the tools of single-molecule manipulation (such as optical tweezer) to study the fundamental mechanical processes that are at the core of enzymatic activity. A detailed understanding of structural and functional dynamics in the packaging motor of Phi 29 promises to provide insight into the general mechanism by which these crucial cellular ATPases convert the chemical energy released in ATP hydrolysis into the mechanical work necessary for their varied biological tasks.