Zinc is an essential micronutrient for organisms but is also toxic when present in excess. The yeast Zrt1, a zinc uptake transporter, is a member of ZIP family of transporters that are involved in metal uptake in bacteria, fungi, plants, and mammals. During the transition from zinc-limiting to zinc-replete growth conditions, the Zrt1 transporter is inactivated by endocytosis for preventing zinc accumulation to a toxic level. Zrt1 is ubiquitinated shortly after zinc treatment and prior to endocytosis. A region of Zrt1 required for ubiquitination and endocytosis in response to zinc was mapped and was designated as a potential ‘‘metal response domain’’ (MRD). The aim of this study is to understand how cells sense zinc concentration and transduce the signal to ubiquitinate Zrt1. I propose three hypotheses regarding the molecular mechanism: Zrt1 senses zinc as it’s being transported Zinc binds to MRD and its binding status controls ubiquitination An intra-or extra-cellular zinc binding protein (zinc sensor) transduces the signal directly or indirectly to MRD by protein-protein interaction. I will test these hypotheses by directed approaches and also by identification and characterization of novel proteins required for the mechanism. This study will contribute for understanding of the molecular mechanisms of substrate-dependent endocytosis of various transporters, important processes for nutrient homeostasis in various organisms.

Negative feedback is found all over biology and is crucial to suppress fluctuations and keep systems away from undesired states. However, because it always acts indirectly, either after a delay or through an intermediate molecular species, negative feedback could also increase fluctuations. It is in fact mathematically impossible for any control loop to completely overcome the problems associated with indirectness. This places general lower bounds on variances and creates frustration trade-offs where suppressing one noise source instead amplifies another, whether in transcriptional autorepression or the control of body temperature. The aim of my proposal is to achieve a more quantitative understanding of negative feedback acting under biological constraints, illustrated by replication control of bacterial plasmids. Bacterial plasmids are simple, tractable, and well characterized, and use negative feedback to suppress fluctuations and reduce extinction rates. I will develop methods to accurately count plasmids copy number in single cells, and to separate between different origins of fluctuations. I will then test and illustrate the mathematically predicted trade-offs and limits, and finally go back to refine the theory. The project and host laboratory were chosen to make my transition from synthetic organic chemistry to systems biology as interesting as possible.

My research interest is to determine how cell polarity mechanisms influence the fate of dividing progenitors and the morphology of their differentiating progeny during mammalian neurogenesis. To approach both questions, I propose to exploit the simplicity and the accessibility of the neural retina as a model system. By coordinating cell polarity with mitotic spindle alignment, the Inscuteable (Insc) adapter protein is central to the mechanism of asymmetric cell division. In Drosophila, this process ensures unequal inheritance of polarized determinants during mitosis, hence implementing distinct fates in the two daughter cells. We hypothesize that during retina development, Insc is a key regulator of the asymmetric divisions that generate a dividing progenitor and a post-mitotic neuron, or two neurons of different types. In addition, we will address a potential implication of Insc in neuronal morphology by testing the functional relevance of its polarized accumulation in the axons of retinal ganglion cells, the projection neurons which transmit the visual input to the cortex. To gain insights into subcellular mechanisms and their impact on whole retina architecture, the experimental approach will combine cell biology and live microscopy with mouse transgenesis. Questionings in cellular neurobiology and all analytical tools involved in this project are new to me. Previously, I applied a strictly genetic approach where analysis of mutant phenotypes lacked the cellular resolution important for addressing many biological questions today. This new training will thus provide me with the multidisciplinary skills important for succeeding as an independent investigator.

In vertebrates and many other metazoans, Hox genes specify segments along the developing body axis (and along other structures such as limbs) by following a spatial and temporal colinear activation which reflects their genomic order. By using a genetic approach in mice, our lab has brought the main studies throwing light on the cis-acting control mechanisms of Hox genes’ colinearity during limb development. They have shown that intergenic regulatory sequences are globally sufficient for the spatial and temporal colinear activation (with occasionally tissue-specific functions). Nevertheless, some specific distant regulatory elements are necessary for limb, external genitalia and intestinal hernia development. These overall data suggest that each developing structure shares common and specific regulation mechanisms of Hoxd genes’ colinearity. Here, I propose to address one of the major questions of vertebrate Developmental Biology since the discovery of the colinear Hox expression in 1988 : what are the mechanistic bases of the Hox colinear activation along the developing body axis ? In order to modify the topography of this cluster, I shall use series of mice carrying simple/combined deletions or duplications of Hoxd genes that have been generated in the lab. In each genetic configuration, the expression analysis of genes neighboring the mutation will be informative about how colinearity is implemented during the developing body axis. We hope that few of these configurations will reveal those unexpected features that are often the keys to understand complex processes.

Integral membrane proteins (IMPs) constitute up to 30% of cellular proteomes, and they represent targets for the majority of currently employed drugs. Thus, detailed knowledge of their structure, function and biogenesis is of major importance for biology and medicine. Membrane insertion of most IMPs occurs co-translationally by ribosomes bound to the protein conducting channel (PCC) of the Sec machinery. In order to obtain mechanistic insights into IMP biogenesis we propose to analyze the exact biochemical composition and the three dimensional structure of membrane protein insertion intermediates. Rather than using an in vitro system containing purified components, we propose to generate the insertion intermediates in vivo by making use of the well characterized SecM translation arrest sequence C-terminally fused to IMPs at specific positions. Subsequent affinity purification via tags on the arrested IMP and/or components of the PCC in combination with established protocols for isolation of cytoplasmic membranes and ribosomes should allow us to purify amounts that are sufficient for biochemical studies and cryo-EM. The components that remain attached to the insertion intermediate, such as cytoplasmic chaperones, targeting factors or the membrane protein integrase YidC, will be identified by mass spectrometry and immunodetection. Single particle analysis in combination with 3D reconstruction should result in structures at sufficiently high resolution to be interpreted on a molecular level. This novel approach will provide a highly versatile method that can be used to study the insertion mechanism of membrane proteins of various lengths and topologies.

Multiple forms of plasticity have been shown to co-exist in mammalian neural circuits using in vitro preparations. The relative contribution of different forms of plasticity to experience-dependent plasticity of sensory processing has however yet to be determined in vivo. The goal of this proposal is to study interactions between synaptic plasticity and plasticity of intrinsic excitability in inhibitory networks of the cerebellar cortex in live animals using a combination of electrophysiological, molecular and 2-photon imaging techniques. Given that interneurons control the activity of Purkinje neurons, which are the sole output neurons of the cerebellar cortex, plasticity in interneurons and at the interneuron-Purkinje cell synapse could have important consequences for cerebellar processing and output. I will perform whole-cell targeted in vivo recordings from identified inhibitory neurons, combined with dendritic and population Ca imaging, to study the localization and consequences of plasticity in interneurons. Plasticity will be induced by stimulation of afferents or by sensory stimuli, and I will dissect and modulate the contribution of different plasticity pathways using biophysical, pharmacological and molecular tools. Finally, I will study plasticity triggered by behavioral conditioning in awake, head-fixed mice. These experiments will reveal the contribution of different forms of cellular plasticity to sensory processing and behavioral learning in the living animal. The proposal builds on my previous research, but will take this research to a new level and will prepare me to run a lab in which sensory processing is studied from the level of molecules to behavior.

Robustness is defined as the capacity to buffer fluctuations in gene dosage or environmental conditions. Nodal signaling plays an important role in the patterning of the vertebrate embryo and must therefore be precisely regulated under all circumstances. The goal of my postdoctoral research is to study Nodal signaling in zebrafish as a model system to determine how miRNAs and other genes confer robustness to complex regulatory networks during vertebrate development. To this end, I will use two approaches: (i) I will test the hypothesis that regulation by miRNAs is a novel facet of controlling the Nodal pathway by dampening the expression of Nodal signaling components and (ii) I will perform a mutant screen to identify additional genetic modifiers of Nodal signaling. The former approach will add to the understanding of robustness of Nodal signaling and provide insights in the biological functions of miRNAs in complex regulatory networks. The latter approach has the potential to identify novel modulators of the pathway.

RNA interference (RNAi) can control chromatin modifications in fungi, plants and vertebrates. In S.pombe, small interfering RNAs (siRNAs) have been proposed to serve as guides to target the RNA-Induced Transcriptional Silencing (RITS) complex to specific chromatin regions. Once recruited to a genomic region, RITS triggers formation of heterochromatin. Although it is clear that siRNAs are required for RITS-driven heterochromatin assembly, many fundamental aspects of this process remain to be elucidated. This project addresses several of these aspects in S.pombe. 1) Among the proteins required for formation of heterochromatin evidences suggest that some might act very early in the process and might even be necessary for RITS recruitment to chromatin. Through the combination of biochemical purifications, genetics and chromatin techniques we intend to reveal the network of interactions between RITS and these proteins. 2) RITS recruitment to chromatin potentially involves direct interactions between its subunits and RNA or DNA. It is challenging to detect such interactions in vivo using classical methodologies. We will use an innovative UV laser crosslinking technique that will allow us to study these interactions both in vivo and in vitro. 3) Interestingly, unlike S.pombe and the vast majority of eukaryotes, the yeast S.cerevisiae does not possess RNAi. We will take advantage of this singularity to develop a bioinformatic screen to identify new proteins involved in RITS-mediated heterochromatin assembly pathway. HFSP career development award will help these projects to meet the highest standards and will greatly contribute to the successful development of my team.

Plants have a unique capacity for continuous postembryonic development linked to the existence of permanent stem cell niches, called meristems. The plant hormone auxin is an essential developmental signal and is a key factor in the control of cell fate in the shoot apical meristem. However hardly anything is known on how the auxin signal is perceived in the meristem and how it is translated into changes in cell fate and cell behavior. The aim of this project is to understand how the auxin signal is perceived and transduced in the meristem and how this generates the specific developmental response of organ initiation. To this end, I propose a systems biology approach combining genomics, genetics and modeling. Specifically I will identify the components of the auxin signal transduction pathway in the meristem and characterize the downstream gene network controlled by auxin during organ initiation. In addition I will use a simple suppressor screen of pin1 to identify new co-factors involved in auxin-mediated organ initiation. In collaboration, we will also develop a tridimensional model for auxin-mediated organ initiation starting with the data on auxin signal transduction. In the long term my goal is to generate a predictive model explaining how the auxin signal functions in the meristem. This project requires constant interactions with mathematicians and computer scientists. The CDA will be essential to consolidate existing collaborations and to stimulate new international collaborations. The CDA will also be crucial to secure my position as an independent researcher and to help me building a competitive team.

Iron is an essential element for all living organisms. Iron uptake is a strictly tuned process relying mostly on the control of iron transporters, at the transcriptional and post-transcriptional levels in yeast and mammals respectively. Plants were long thought to regulate their iron uptake machinery dependent on the IRT1 root iron transporter at the transcriptional level. However, recent evidences suggest a post-transcriptional regulation likely affecting IRT1 protein localization and stability, through iron-regulated endocytosis. Only a few examples of regulated endocytosis exist in plants and our understanding of the plant endocytic pathway is scarce. This proposal aims at : (i) elucidating how iron controls its own uptake through regulated endocytosis and degradation of IRT1 in the model plant Arabidopsis thaliana, and (ii) at establishing a system to study the poorly described plant endocytic pathway by specifically defining the route taken by IRT1 on its way to its degradation site. To this end, I will use an integrated approach of cell biology, forward genetic, molecular and biochemical methods. I will first thoroughly characterize the regulation of IRT1 localization and stability in plants by biochemistry and confocal microscopy. Second, I will develop genetic screens in yeast and plants to identify factors involved in the trafficking and destabilization of IRT1. Third, in collaboration with Dr. N. Geldner, IRT1 will be co-localized with a set of hundreds of marker lines for endomembrane system to provide a spatial and temporal analysis of membrane protein trafficking and to better characterize the plant endocytic pathway.

The discovery of eisosomes in yeast raises many intriguing questions, which I will address in my group at the Max Institute of Biochemistry, Martinsried/Germany. Among the most important are: How do eisosomes regulate endocytosis and integrate different signals to spatially distribute endocytosis? How are they targeted to specific sites at the plasma membrane and assembled to a specific size? Are eisosomes conserved in higher eukaryotes? In my laboratory, I propose to answer these questions to gain insight into the general principles regulating plasma membrane homeostasis. My goals are Aim 1. To determine the molecular functions of eisosomes 1.1. To identify all eisosome components and functional interactors 1.2. To define the mechanism how eisosomes organize and trigger endocytosis Aim 2. To determine the mechanism of eisosome biogenesis Aim 3. To determine if eisosomes or eisosome-like structures exist and function in endocytosis of higher eukaryotes. These studies are likely to provide new insights into the spatial regulation of endocytosis, the coordination between plasma membrane lipid composition and endocytosis, and more generally into plasma membrane homeostasis.

Although we move our eyes several times each second we are generally not aware of the retinal image created by this very fast motion. A suppression of the visual response to this image is assumed by many researchers however this model has not received support from animal studies and has not been formally tested in humans. The objective of this project is to identify the processing that occurs in the visual system that leads to a loss of visual sensitivity during a saccade. The first specific goal is to psychophysically and computationally investigate the system that causes lowered visual sensitivity during saccadic eye movements in humans and suggest the most appropriate model responsible for the perceptual effect of saccadic suppression. The second goal is to verify physiologically and localize, in the human brain, the site of saccadic suppression using functional magnetic resonance imaging based on the model proposed through the psychophysical and computational investigation. This research will use a multidisciplinary approach achieved by the integration of behavioural, computational and neuroimaging techniques, to elucidate a fundamental aspect of vision. It represents a departure from my previous research in terms of both technique (addition of computational and imaging approaches) and area of study within the visual system. By identifying the mechanism that operates every time we move our eyes this work will be addressing an as yet unanswered but often assumed proposition in eye movement research. It will be illuminating a process fundamental to successful visual interaction with the environment and will be central to our understanding of vision as a whole.

In eukaryotes the endoplasmic reticulum (ER) is responsible for the folding of secreted proteins. When folding fails, proteins are eliminated by a highly conserved process termed ER-associated degradation (ERAD). Remarkably, misfolded proteins are first retrotranslocated across the ER membrane into the cytosol and then degraded by the ubiquitin-proteasome system. While the field has seen rapid progress in identifying factors involved in ERAD, our mechanistic understanding of ERAD, including how substrates are transported across the membrane, is far less developed. Here I will address this deficiency in our knowledge. Specifically, I will exploit recent success in purification of the central ERAD machinery to establish an in vitro ERAD assay from purified components, focusing on the E3 ligase Doa10-associated complex that mediates degradation of membrane proteins. Simultaneously, I will identify mutants of Doa10 expressing specific defects in ERAD through an in vivo approach that monitors protein stability of fluorescently labeled model substrates by flow cytometry. Affected Doa10 variants will then be characterized using the biochemical reconstitution assay to allow an in depth mechanistic dissection of the ERAD pathway. These innovative approaches will provide powerful tools to address the functional contribution made by individual ERAD players and thus can be expected to dramatically advance our understanding of this fundamental cellular process.

Accurate positioning of the mitotic spindle is essential for genetic fidelity and the inheritance of cell fate determinants. The yeast Saccharomyces cerevisiae divides asymmetrically, and requires spindle alignment along the polarity axis of division to ensure accurate chromosome segregation. This proposal centers on the primary spindle positioning pathway, which relies on the microtubule (MT)-bound protein Kar9 and the cyclin-dependent kinase Cdc28/Cdk1. Kar9 specifically localizes to one spindle pole body (SPB) and its astral MTs, directing these elements into the daughter cell. The spatial asymmetry of Kar9 requires phosphorylation by its negative regulator Cdc28/Cdk1, a central element of the cell cycle machinery. Recent genetic studies in the host laboratory of Prof. Barral have suggested that Kar9 also binds and negatively regulates Cdc28, leading to the hypothesis that Kar9 and Cdc28 antagonize each other to ensure the proper spatial restriction of their activities. This spatial restriction, in turn, results in the correct placement of the mitotic spindle. In order to understand the mechanism of Kar9 and Cdc28 regulation, I propose to investigate the interactions between these two proteins using biochemical and structural assays, focusing particularly on the proposed role of posttranslational modifications in modulating the interactions between Kar9 and Cdc28 as well as the mechanism by which these two proteins regulate each other during mitosis. These studies will provide detailed picture of how restriction of protein activity can be used to establish the exquisite spatial control required during cell division.

In contrast to the large body of work on glutamatergic synaptic plasticity, relatively little is known about plasticity of GABAergic synapses, which have important roles in the function of neural circuits. This proposal aims to uncover how chronic changes in neuronal activity alters GABAergic function in the hippocampus. Through the use of organotypic slice cultures of hippocampal neurons, I plan to conduct electophysiological recordings in subpopulations of GABAergic neurons identified through the use of transgenic mice with targeted expression of fluorescent proteins. In addition, specific neuronal populations will be investigated through the adaptation of an elegant new method involving channelrhodopsin, a recently discovered algal protein which enables the activation of subpopulations of neurons using light. Finally, using electrophysiological, imaging, and molecular biological methods that will permit characterization of individual synapses, I will investigate potential presynaptic mechanisms underlying the reduction in GABAergic synaptic transmission. Combined with my previous experience, I am confident that the novel methodological approaches I will learn as well as the exiting exposure to new neurobiological questions during this project will qualify me to perform my own independent neurobiological research in my future career.