The Golgi apparatus is a key compartment for post-translational modification, sorting and transport of proteins. It has a particular structure in mammalian cells: the stacks are linked to form a Golgi ribbon and this ribbon is maintained in the vicinity of the centrosome. Several proteins were shown to be involved in the stacking and the ribbon formation like the Golgi matrix proteins, GTPases, motors, and kinases. Surprisingly, several of these proteins were also shown to be involved in cell cycle regulation. However to date we do not understand the relationship between cell cycle regulation, Golgi activity and centrosome organisation. A recent screen revealed a new role for twenty Golgi proteins in cell cycle progression. Around ten of these proteins are indeed unknown. During the course of my postdoctoral research, I will define the function of the twenty Golgi proteins identified in the screen in mitosis using RNAi and localisation experiments, colocalisation by immunofluorescence and electron microscopy. The phenotypes observed after knockdown and the observations reported in the literature will be combined to understand the interactions between these proteins leading to the identification of the different pathways of mitosis regulation by these Golgi proteins.
The characterisation of Golgi fragmentation and the proteins involved in this process may improve our understanding of the complex relationship between Golgi proteins and cell cycle regulation and provide us tool to manipulate this process.

Our goal is to examine deliberative decision-making in rats from neurophysiological, cognitive, and mathematical perspectives. Deliberative decision-making entails behaviors in which agents explicitly consider multiple possibilities before acting. Early experiments identified behaviors in which a rat paused, looking back and forth, before acting (Tolman1948). This behavior occurred during learning and after changes in experimental contingencies, and was suggested to create expectancies of the consequences of the available choices, but the computational and experimental techniques needed to address this question were not available at the time. An interest in this behavior has been revived in the past few years based on new computational models of decision-making systems (Daw2005, Niv2006, Redish2007a, Redish2008), on the availability of new behavioral measures (e.g. Lauwereyns2006), and on recent discoveries of prospective encoding in the hippocampus (Wood2000; Johnson2007a) and other structures (Ramus2007, vanderMeer2009, vanDuuren2009). Our team brings together expertise in behavioral neuroscience, neurophysiology, computational neuroscience, cognitive psychology, and mathematics. Experiments will examine prediction, evaluation, and action-selection mechanisms in rats making deliberative decisions. Computational projects will examine mechanisms by which these systems can shift suddenly from consideration of one possibility to the other, based on models of chaotic attractors (Tsuda2001a).

Centromeres are key regions of eukaryotic chromosomes that ensure proper chromosome segregation at cell division. In most eukaryotes, centromere identity is defined epigenetically by the presence of a centromere-specific histone variant CenH3 (CENP-A in humans or CID in flies). How CenH3 is incorporated and reproducibly propagated during the cell cycle is key to understanding this essential epigenetic mechanism. Much is known about the CenH3 function and assembly in mitosis, but its roles and regulation in meiosis are largely unknown. Meiosis is an essential part of the reproductive cycle in most eukaryotes and segregation defects result in aneuploid eggs, sperm and the resulting zygotes. In addition, much information about CenH3 has been obtained from single cell eukaryotes or cultured cells in multi-cellular eukaryotes, but the dynamics of CenH3 during development are unknown. Recently, the cell cycle timing of new CenH3 deposition at centromeres has emerged as a key aspect of centromere specification and function. In human cultured cells, newly synthesized CENP-A is deposited at centromeres at late telophase/early G1, whereas Drosophila CID loading occurs earlier, in mitosis. Thus, the regulation of CenH3 loading may differ between organisms, tissue types and developmental stages. Here we propose to investigate the function and timing of CenH3 deposition during development and meiosis using the multi-cellular Drosophila melanogaster as a model system. This study aims to further our understanding of the dynamics of centromeric chromatin and the maintenance of genome stability in meiotic and mitotic cells in the animal.

Spontaneous and evoked neuronal activity in the cortex of awake behaving animals is characterized by complex dynamics that remain largely unexplained. This research project will investigate how the ongoing activity, the cognitive state of the animal, and the learning phase of a behavioral task affect information coding and the dynamics of neuronal networks. This requires combining behavioral assays with single-unit electrophysiological recording from dozens to a hundred cells simultaneously.
We will use two complementary approaches, one measuring neuronal activity patterns in response to specific sensory inputs, and one manipulating the relation between activity and motor output, allowing direct tests of the information content of neuronal discharge. The first study will be performed on the whisker system of freely behaving rats trained in a vibrissal discrimination task. The cognitive context and attentional demands of the task will be varied in order to study their influence on cortical processing and on its plasticity during learning. The second approach will be to use the firing of a subset of neurons to drive a neuroprosthetic device. In such an operant conditioning framework, neurons adapt rapidly their firing characteristics so that the rat successfully retrieves rewards. Performance of the brain-machine interface can be tested for different combinations of neurons and decoding algorithms.
The Career Development Award will be used to build the experimental rig, to develop the research group and to establish international collaborations. This interdisciplinary project should help unveil cellular mechanisms of neuronal coding during perception and action.

Probabilistic differentiation - the formation of multiple cell type within a given environemnt - is common to both metazoan development and bacterial physiological and developmental responses. In bacteria, Probabilistic differentiation was suggested to increase the survival of a clonal population by enabling bet-hedging and division of labor. Probabilistic differentiation in bacteria arises by intercellular feedbacks that lead to multi-stability, but the frequency of each state and the dynamics of transition between states can be affected by interactions with other cells, environmental conditions and genetic background. The soil bacteria B. subtilis has a multitude of stress responses, including the highly characterized sporulation pathway. These are expressed probabilistically under diverse conditions. During my post-doc I studied the development and evolution of probabilistic differentiation within the sporulation program and showed how an evolutionary transition between two modes of sporulation can be attributed to network changes that affect their relative frequency. The present research plan aims to elucidate three aspects of probabilistic differentiation and sporulation in bacteria. My specific aims are to (a) Study spatial interaction between cell types during B. subtilis biofilm formation. (b) Study micro-evolutionary variation in probabilistic sporulation initiation and its relation with physiological variation. (c) Study macro-evolution of probabilistic symmetry breaking during sporulation. The HFSP fellowship was essential for the emergence of my current research project, and the CDA will provide the financial support and scientific independence to develop it.

The computations performed by single neurons in a system can be seen as the transformation of an ensemble of inputs into an output signal, mediated by the connectivity of the circuit. In that sense, the connectivity of inputs to a single cell and the changes it undergoes define the substrate of computability and system plasticity in the brain. How the brain is wired at the cellular level, and how changes in this wiring underlie emergent properties of the system, are therefore crucial in order to understand the principles of computation and self-organization in neural systems. But in the plastic, individually unique brains of mammals, questions of connectivity present a special challenge.
Using and developing a novel combination of imaging tools and a newly available trans-synaptic labeling virus, we will characterize the morphology and sensory responses of the entire input ensemble of single neurons in the mature and developing brain. Focusing on coding in visual cortex, we will explore how the underlying connectivity relates to the visual properties computed and coded by single cells, in the context of the representation of visual stimuli in the system. By comparing connectivity patterns before and after eye-opening in normal and dark-reared animals and driving direction selectivity only in specific directions, we will ask how connectivity changes during the experience-dependent emergence of direction selectivity in the developing brain.
We believe that our proposed approach will create a unique bridge between the cellular and the systemic in sensory neuroscience, thus enabling future research to access many open questions about the connectivity of neuronal circuits.

Life is governed a multitude of rhythms. These cover daily (circadian) periods, but also shorter and longer cycles. Many of these rhythms depend on light, and govern the timing of essential biological processes, such as growth, cell divisions and sexual reproduction. The sea, as the cradle of evolution, harbors the greatest diversity of life, and possesses an unparalleled and diverse set of light conditions. For example, spectral light qualities and light intensities vary significantly depending on depth. Despite their fundamental significance, molecular ‘marine’ rhythms have so far been poorly investigated.

We hypothesize that marine organisms have evolved specific molecular mechanisms to integrate the diversity in light signals and periodicity present in the sea. We focus our study on circadian rhythms in three marine species – diatoms, sea urchins, and annelids – and their environmental significance and modulation. Our reference species cover different branches of eukaryote evolution, as well as diverse marine habitats with different light properties, life styles and rhythms. Each of them has unique experimental advantages, which allow exploring different aspects of marine rhythms. Sea urchins will be used for advanced circadian regulatory network studies, annelids to uncover the interplay between circadian and lunar cycles, and diatoms to explore the adaptive value of rhythms in complex environmental conditions.

As an interdisciplinary team, we will integrate molecular, biochemical and biophysical analyses to assemble and compare circadian regulatory networks. These circadian models will furthermore serve as a reference to study how the species integrate non-circadian light input (in particular lunar light) as well as different environmental cues. For our molecular, evolutionary and ecological studies, cryptochrome (cry) genes will serve as a common entry point, as these molecules are shared across kingdoms and appear to occupy a central role in several known molecular clocks.

We expect that our study of light-regulated marine rhythms will identify novel molecular players of marine rhythms and reveal how organisms integrate different light sources and cycles. The study will provide new insights to the field of rhythm biology, and, more generally, will help to assess how organisms adapt to a complex environment.

Membrane shape affects the distribution and activity of signalling molecules: Inward bending of the plasma membrane allows forming endocytic vesicles that remove signalling molecules from the cell surface. Conversely bending the plasmalemma away from the soma yields protrusions to probe the environment. My research uses developmental genetics, cell biological tools and novel live imaging approaches in Drosophila and zebrafish to study the biogenesis and function of two cellular signalling interfaces:
1. Cilia are protrusions of the cell surface that allow cells to communicate with their environment by chemosensation, mechanosensation and the creation of fluid flows. I use zebrafish to study the role of an evolutionarily conserved membrane-bending machinery, the ESCRT complex, in vertebrate development. While ESCRTs are known for their role in endosomal membrane remodelling, I have uncovered an unexpected function of these proteins in ciliogenesis. The first aim of my project is to characterize this novel function of the ESCRT machinery.
2. Endosomal membranes are essential for intracellular signal processing. Additionally, endosomes contribute to plasma membrane remodelling during mitosis. This raises the question whether conversely cell division also affects the function of endosomal signalling compartments. I have shown that during the asymmetric division of fly sensory precursor cells, multivesicular endosomes carrying internalized Delta/Notch promote Notch activation while simultaneously undergoing marked membrane remodelling. The second aim of my project is to study how mitosis and the associated remodelling of endosomal membranes affect developmental signalling.

Biological processes like the regulation of gene expression often involve interacting proteins with low copy numbers on a few locations within the cell. Due to the inherently stochastic nature of the movement and action of biomolecules, these processes cannot be synchronized between different cells. In order to study the fundamental dynamics and mechanisms of these processes and the participating molecules in their physiological context, it is therefore desirable to monitor individual molecules in single living cells. Recent developments in single fluorophore detection enabled investigating principles of gene regulation in live prokaryotic cells. In this postdoctoral study, I want to establish the capability to detect single molecules in living eukaryotic cells using detection by localization. Due to the large size and fast intracellular motions of eukaryotes, epi-fluorescence illumination as for prokaryotes is not sufficient to monitor single fluorescent molecules. Instead, I plan to use multiphoton excitation to reduce backround fluorescence and multifocal scanning illumination to increase image acquisition speed. Following the very recent development of non fluorescent chromophore detection by stimulated emission microscopy, I want to additionally implement the capability to detect single chromophores into the setup, increasing the repertoire of reporter molecules to study live cell processes. With this instrument, I want to study the target site binding kinetics of single transcription factors as well as gene silencing mechanisms in living cells. These experiments will yield new insights into gene regulation mechanisms in eukaryotes.

GABA is the major neurotransmitter for inhibitory synaptic transmission in the mammalian brain. Following its release from presynaptic terminals, it diffuses across the synaptic cleft and binds to GABAA receptors to cause rapid inhibition. These GABA-gated ion channels form pentameric heteromers whereby the extracellular domains of each subunit interact to form a ring.
GABAA receptors display considerable heterogeneity due to multiple subunit families (alpha, beta, gamma, delta, epsilon, theta, pi), some of which contain multiple members (alpha, beta, gamma). However, the most prevalent subtypes found at synapses in the brain are composed of alpha/beta/gamma subunits. These receptors are not only activated by GABA binding at the alpha/beta extracellular interface, but are also sensitive to benzodiazepines, which bind to the alpha/gamma extracellular interface. Moreover, GABAA receptors are potently modulated by endogenous neurosteroids.
While structure-function studies have identified key residues for the binding of agonists and modulators, we do not understand what structural rearrangements occur to couple the binding of these molecules to the opening of the channel, and how the different binding sites structurally interact with each other.
Toward these goals, I aim to use simultaneous electrophysiological recordings and fluorescence spectroscopy of GABAA receptors. The spectroscopic method will reveal conformational rearrangements in the receptor by reporting changes in hydrophobicity around an environmentally sensitive fluorophore attached to defined domains. I thus hope to provide mechanistic insight into the operating principles of this important inhibitory receptor.

The aim of this project is to use mouse Embryonic Stem (ES) cells and preimplantation embryos, from which ES cells are derived, to understand how interactions between signal transduction pathways and gene regulatory networks regulate processes of cell fate assignment. We propose to take advantage of the operational simplicity of these developmental systems to test whether fate transitions are guided by digital additive genetic systems or by analog dynamical integrative devices. Specifically we shall focus on the emergence of the pluripotent epiblast (EPI) from a precursor population, the Inner Cell Mass (ICM). Ultimately we shall try to recreate this process in culture and thus determine the parameters that influence the decision to be one or the other cell type.

Understanding the brain computational principles requires detailed understanding of network architectures, the ability to measure and perturb the activity of its components and the ability to relate these neural activities to the resulting behavior. In light of these requirements, the rodent olfactory system provides an ideal model system because of the simpler neural circuits and the availability of advanced genetic and imaging techniques. Moreover, newly developed “optogenetic” methods using the light-gated ion channel, channelrhodopsin (ChR2) allows us to directly control olfactory neural activity.
The olfactory system transforms the physicochemical information from the olfactory epithelium, the olfactory bulb and to the olfactory cortex. In the olfactory bulb, odors are encoded by a complex spatial and temporal pattern of activation across glomeruli. However, the functional relevance of these codes and their relation to behavior is unknown.
In this project I will explore the neural coding strategies used during odors behavioral tasks. Optically activating specific olfactory neural circuits and monitoring the neural activity of higher brain regions using extracellular recording I will be able to resolve cortical odor coding principles and resolve how odor information is transformed and represented in the different neural layers. Moreover, by monitoring sniffing behavior while optically activating different olfactory neurons I will explore neural coding principles during odor perceptual tasks. These suggested projects will reveal brain computational principles underlying object perception.

The brain is a constantly adapting organ, with connections between brain cells and the physiology of individual cells changing to reflect learning and life events. This dynamic reshaping is a critical element of human behavior and disease, but it has been difficult to study at the molecular level because, for most important ‘signaling’ molecules in brain cells, there are no tools to alter molecular dynamics in real time as changes in brain shape and interactions are taking place. In a recent development, Hahn lab demonstrated a new molecular engineering approach capable of rendering the activity of signaling molecules sensitive to light, and therefore subject to study during dynamic events. A protein fragment used by plants to turn their leaves towards the sun was engineered into a completely unrelated protein, a ‘small GTPase’ used by cells to control shape and movement. The fragment of light-sensitive plant protein could be used to control the activity of the GTPase. This enabled photomanipulation of protein activity in living cells, and was quite valuable in studies of metastasis and cell movement. Perhaps more importantly it promises a new general approach that can potentially make light sensitive versions of many molecules for their study in living cells. In this proposal, we combine the forces of a theoretician focused on understanding how protein structures can be altered to generate new function, Dr. Kuhlman, with the cell biologist and protein biochemist who developed the photoactivatable GTPase, Dr. Hahn. These two will devlope new caged proteins capable of shedding light on important and previously inaccessible questions in neurobiology. This will be accomplished via the development of generally applicable methods that can be used by other researchers to control protein activity with light. The selection of target proteins was guided by Dr. Kasai, a pioneer in the role of neuronal dynamics in brain function. He will use the new photoactivatable proteins to study brain tissues both in vitro and in vivo. The new effective PA-probes produced by our collaboration will have wide applications in various fields of biology, as these small GTPase are key to diverse cellular functions and diseases.

Synapses are highly complex and dynamic sites of intercellular communication, playing a pivotal role for learning and memory formation in the mammalian brain. As they are very small - usually smaller than what diffraction-limited light microscopy can resolve - and densely packed in light-scattering brain tissue, it has been extremely difficult to study their physiology in mechanistic terms. As a result, we still lack a clear understanding of the basic dynamic organization of neurotransmitter receptors and their molecular partners at mammalian synapses. While electron microscopy provided us with detailed snapshots of where glutamate receptors are located inside synapses, it does not convey dynamic information about their function. Therefore, a functional assay is required that non-invasively probes receptor localization and activation with nanoscale resolution. Since existing optical approaches such as 2-photon glutamate uncaging do not have sufficient spatial resolution, progress in this area relies on fundamental breakthroughs in live-cell-compatible techniques relying on focused visible light.

We seek to establish a new paradigm for controlling physiological processes, which reconciles instant non-invasive molecular control with nanoscale resolution in arbitrary locations inside a cell. Specifically, we will (1) engineer photosensitive compounds for superresolution-based photo-activation, (2) advance microscopy technology to concurrently activate and image beyond the diffraction limit, and (3) use this new methodology to probe synaptic physiology in brain slices with unprecedented resolution, addressing timely questions about the dynamic behavior of neurotransmitter receptors in synapses.

After breaking the diffraction barrier in imaging, diffraction-unlimited photomanipulation will be the next paradigmatic step in neuroscience methodology. Substantially departing from the state of the art, the project brings together three research groups working at the cutting edge of probe development, superresolution optics, and neuroscience in order to reach a goal that offers a huge potential for scientific breakthroughs. Beyond addressing key questions about the molecular physiology of synapses, the project will have a substantial impact in many other areas where the arbitrary release of molecules with nanometer spatial resolution will provide a paradigmatic shift.

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.