Chemical synapses represent the close apposition between the presynaptic terminal of an axon and the dendrite of another neuron and is essential for proper neurotransmission i.e. for brain function. Despite decades of research, the molecular mechanisms underlying presynaptic differentiation of growing axons are still poorly understood especially in mammalian neurons. Synapse formation is the ultimate expression of neuronal polarization during which axons and dendrites acquire a set of distinct molecular and functional specialization. On the presynaptic side, axons accumulate through directed trafficking the specialized neurotransmitter-filled vesicles and the proteins required for fast presynaptic release. What are the signaling mechanisms regulating this last step of axonal development? We hypothesized that the kinase LKB1 which is critical for the establishment of an axon during neuronal polarization [1,2] as well as axon growth and terminal branching (Courchet and Polleux, in preparation) might also be involved in presynaptic differentiation. We have accumulated preliminary results showing that genetic deletion of LKB1 in cortical neurons after axon formation critically impairs presynaptic vesicle trafficking and results in severe behavioral locomotor deficits. We therefore propose to test (1) the exact molecular mechanisms underlying the function of LKB1 in presynaptic differentiation, (2) the upstream and downstream mechanisms regulating LKB1 function during presynaptic development and (3) the electrophysiological and behavioral evaluation of the consequences of inhibiting LKB1 on presynaptic function.

During development, cells differentiate and form specific adhesive contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities and metastatic cancers. The strength and mechanical properties of cell-cell adhesions govern the mechanical strength and integrity of tissues subject to mechanical stress, such as epithelia. Despite these important physiological outcomes, mechanisms underlying the response of cells to external mechanical forces remain poorly studied. Gaining insight into these mechanisms is crucial to our understanding of normal development, wound healing, disease processes and tissue engineering therapies.The proposed project integrates the expertise of 4 teams, on 3 continents to examine how mechanical forces influence the formation and properties of cell-cell adhesions.
This collaborative project brings together expertise in the design of: (i) devices to accurately measure forces at the molecular and cellular scale (Lim & Ladoux); (ii) nano and micro-fabricated environments (Ladoux); (iii) molecular and cellular tools and (iv) biochemical characterization of molecules in cell-cell contacts (Nelson and Mège). Additionally, the interface between these groups provides a novel training platform for postdoctoral fellows, and undergraduate and graduate students in team building and multi-disciplinary research that crosses boundaries between engineering, biophysics, biochemistry and cell biology. The ability to examine the effects of forces on both cellular and molecular scales offers a powerful way to define the relations between molecular and structural intracellular organization, cell mechanical properties and cell behavior.
The specific goals of the proposal are: 1. Design and validate new tools that enable experimental procedures to be more quantitative in the context of mechanics of cell-cell junctions in epithelial tissues, and develop well-defined microenvironments to control cell adhesion and obtain quantitative measurements of cell behaviors ; 2. Investigate how cells regulate cell-cell junctions in coordination with cytoskeleton dynamics; 3. Investigate the mechano-sensitivity of cadherin-associated proteins at the single-molecule level. These studies will define the mechanisms of tissue cohesion at molecular, subcellular and multicellular scales, and provide a mechanistic framework of the effects of external mechanical forces on epithelial cell-cell contacts.

Chemical communication by means of pheromones is central to the mating systems of a wide range of organisms, from bacteria to vertebrates. Because mate choice and reproductive isolation are often based on pheromone differences, understanding how pheromone systems diverge is necessary for a complete comprehension of the evolutionary process of speciation. During the last decades, our understanding of the molecular bases of odorant and pheromone reception has greatly advanced, with key findings such as the identification of odorant receptors resulting from studies using Mus musculus mice as model. Because the chemoreceptors are the first elements in the cascade of events ultimately modulating behaviours, changes that affect their biochemical properties or expression pattern have the potential to modulate specific behavioural responses. My aim is to (i) take advantage of the recent advances made in our understanding of the molecular bases of olfaction in mammals, and in particular pheromone detection in laboratory mouse; (ii) apply that knowledge to understand the evolution of pheromone systems in wild populations, using Peromyscus deer mice as model, a relative of the lab mouse; (iii) understand the molecular changes that affect pheromone detection and their consequences on the evolution of mating systems. To achieve these goals, I will benefit from the interactions with experts at Harvard University and use a combination of approaches including RNA-Sequencing, immunocytochemical studies and calcium imaging. This work will be critical to forge a link between the laboratory and the field and begin to address key issues about the evolution of chemical communication in mammals

How the brain stores memories is a fundamental unanswered question, but new insights are emerging with the discovery that the persistent enzymatic activity of the kinase PKMzeta (PKMz) is a crucial molecular mechanism for memory maintenance. Whereas the interfering effects of stress on other memory processes has been extensively studied, its effect on memory maintenance has never been explored. A recent study showed that acute stress can enhance stable (‘consolidated’) memory through a phenomenon named “out-of-context activation of memory” (OCAM). We propose that the memory-strengthening effect of OCAM corresponds to the impact of stress on the PKMz-mediated mechanism of memory maintenance, specifically by increasing PKMz levels at the specific synapses that store memory information. To test this hypothesis, we will first identify the pattern of PKMz expression in the brain network that stores a left/right discrimination memory and determine how OCAM alters this region-specific PKMz activity. We will then analyze the OCAM-associated changes in synaptic localization and levels of PKMz mRNA and protein in these regions, as well as one of the main consequences of PKMz activity, the trafficking of the GluA2 subunit of the AMPA-receptor to potentiated synapses. By using a newly generated transgenic mouse that allows the persistent tagging of neurons recruited by memory acquisition, we will finally determine whether stress specifically exerts its effect on PKMz levels within the particular neuronal population that participated to the formation of the L/R memory.

The animal body is made up of hundreds of different cell types, specialized in various functions. In order to follow and understand the fate of cells during development and disease progression, it is essential to identify and characterize specific cellular subpopulations and ultimately individual cells. Here, I propose to take advantage of the fact that certain cells express a unique combination of transcription factors. Specifically, I propose to develop a synthetic genetic circuit that can sense the concurrent expression of several endogenous transcription factors. First, I will design a set of artificial promoters that each responds to expression of a single endogenous cardiac transcription factor. Second, I will design transcription-based logic gates that sense the simultaneous activity of two promoters. By combining these gate modules with the artificial promoters I will engineer circuits that can sense the simultaneous expression of two endogenous cardiac transcription factors. Third, I will make use of these circuits to improve the efficiency of in vitro differentiation of embryonic stem cells into cardiomyocytes. These experiments will serve as a proof of principle that such devices can be employed to specifically label cellular subpopulations. Finally, I will expand the set of artificial promoters and thereby generate sensors for additional transcription factors. Together these experiments aim at establishing a novel device that will contribute to the synthetic biology field by advancing mammalian bioengineering techniques. Furthermore, such a genetic circuit will have the potential to provide a valuable tool for characterization of many distinct cell states.

Cyanobacteria rely on photosynthesis for metabolism, hence they must respond to changes in light levels that occur over the day-night cycle, as well as to other environmental changes that occur on shorter timescales. As the only bacteria to evolve a circadian clock, Cyanobacteria are able to anticipate some, but not all, of these fluctuations. Thus, Cyanobacteria face a complex signal integration problem, of analagous complexity to that faced by higher plants. They provide an ideal model system to address the question of how cells respond to diverse, dynamically varying inputs over multiple timescales. Previous research has studied specific pathways in Cyanobacteria, especially the circadian clock, but mainly done so in isolation from other regulatory pathways using traditional methods based on analysis of cell populations. These methods can ‘average’ away dynamical behavior.

Here, I aim to comprehensively analyse the dynamic regulatory behaviours of individual Cyanobacteria cells. First, I will systematically identify genes that are regulated dynamically and use mathematical modelling and synthetic biology approaches to test the mechanism of the observed dynamics. Second, I will elucidate the regulatory coupling between distinct pathways by constructing a ‘matrix’ of reporter strains to correlate dynamics between pathways. Finally, I will specifically investigate how the circadian clock couples to other dynamic pathways in the cell by analysing how the activation of regulatory pathways effects clock phase. The work will lead to a comprehensive understanding of how Cyanobacteria integrate a range of environmental signals to control gene expression dynamics.

Next to Bacteria and Eukarya, Archaea represent the third domain in the tree of life. Cells from the eukaryotic domain use a cell division machinery, in which actin and myosin play a central role for constriction, while most bacterial and archaeal species use a mechanism based on the tubulin-homologue FtsZ. Some members of a lineage within the archaeal domain, however lack both, an actin/myosin as well as FtsZ-based system. Instead, these species use a protein system closely related to the eukaryotic ESCRT (endosomal sorting complex required for transport) apparatus to perform cytokinesis.
In the proposed project, I will reconstitute archaeal cell division using purified proteins of the ESCRT system from Sulfolobus acidocaldarius and biomimetic membranes prepared from purified archaeal lipids. Using this reconstituted system and advanced fluorescence microscopy, I will be able to get insight into the molecular processes required for archaeal cell division, which will also facilitate our understanding of the mechanistic details of eukaryotic ESCRT mediated vesicle formation and viral budding. Furthermore, these results will shed light on the evolution of binary fission and the relationship between the different domains in the tree of life.

Ubiquitylation of target proteins has been recognized as an eminent regulator of diverse biological processes including apoptosis, the inflammation response, and selective autophagy. The TNF signaling pathway is a signal transduction cascades that, upon specific stimulation of cognate receptors, can stimulate the expression of pro-survival and pro-inflammation factors. Paradoxically, the same stimulus can also lead to caspase-dependent apoptosis. Thus, intricate systems of networks function to switch between different patterns of responses. Failure to properly regulate these networks can lead to sustained inflammation, auto-immunity or aberrant cell death. Autophagy has recently emerged as an ubiquitin-dependent catabolic process that can degrade proteins non-specifically by engulfing cytosolic material during starvation conditions. Selective autophagy, on the other hand, can specifically degrade large protein aggregates, organelles and bacteria. Analogous to proteasomal receptors that bring ubiquitylated proteins to the proteasome for degradation, autophagy receptors recognize ubiquitylated cargo and deliver them to the lysosome for degradation. In this study, I propose to investigate ABIN-1, a protein that functions as a pro-survival factor in NF-kappaB signaling, as a novel autophagy receptor that may function to degrade death-inducing complexes to enable the inhibition of apoptosis.

This project assembles an international team of biologists, mathematicians and engineers to uncover the secrets of the world’s most spectacular animal-built structure: the mound built by the southern African termite Macrotermes. These spectacular structures, long known as the famous “air-conditioned termite mound”, has many new secrets to reveal. The mound does not appear to “air condition” the nest, but is in fact a remarkable organ of extended physiology, essentially the lung for the underground colony. Among the secrets revealed are a novel mechanism for capturing turbulent wind energy to do useful work, and building behavior that is far more sophisticated and complicated than ever realized.
This project seeks to push these new findings into new scientific frontiers, including the physics of using turbulent wind, the largely unexplored biology of the termite brain, and the nature of the collective cognition of the termite colony. From this, we hope to learn new methods for analyzing the adaptation of organisms to their environment, the process of building coherent organs of physiology, new ways to program robot swarms, and perhaps novel ways to exploit wind in climate control of our own buildings.

Cryo-electron tomography is an advanced technique for exploring the structure of cells and sub-cellular compartments at the molecular level in life-like conditions. Here it will be applied to a first in situ ultrastructural analysis of centrosomes in C. elegans embryos. This project is undertaken in a collaborative effort between the groups of Prof. Baumeister at the MPIB in Martinsried and Prof. Hyman at the MPI-CBG in Dresden. State of the art cryo-electron microscopy will be combined with focused ion-beam milling, a unique preparation technique which has only recently been adapted for thinning of biological frozen samples. Correlative cryo-fluorescence microscopy will be essential to localize the centrosome within the large C. elegans single-cell embryos. Cryo-electron tomography on these preparations will provide a high resolution 3-dimensional view of centrosome supramolecular organization. Single particle analysis of expressed and purified structural components of the yet poorly characterized pericentriolar matrix will complement the imaging approaches. Advanced optical microscopy, including super-resolution fluorescence microscopy, will be used to explore localization of functional centrosomal domains and to gain insight into the physico-chemical behaviour of centrosomes as non-membrane bound organelles. This project will provide unprecedented structural data on functional centrosomes and the pericentriolar matrix in particular, in situ and at the nano-scale. The employed interdisciplinary approach represents cutting edge research in both the fields of electron tomography and of basic cell biology.

Modifications in gene regulation play a central role in generating phenotypic diversity. Changes in the timing and conditions under which genes are expressed, and not only modifications in their composition, lead to new phenotypes. Extensive differences in the way genes are regulated have been observed even between closely related species. However, little is known about the intermediary steps that have led to such regulatory diversity and whether these steps are adaptive. The aim of my project is to explore the changes in gene regulation that underlie the diversity in biofilm formation, and thus pathogenesis, of Candida species. The latter as a proxi to understand general principles of regulatory evolution. Biofilm formation in Candida species is an ideal evolutionary model since these species span a wide evolutionary range and show gradual phenotypic differences in pathogenicity. Genome-wide approaches will be used to identify and characterize the regulatory circuits that are responsible for biofilm formation in each species. Interspecific comparison of the transcription factors, their binding affinities and target genes will reveal the evolutionary changes that occurred in each linage. Finally, phenotypic profiling of the regulatory changes will be used to evaluate their adaptive value. Overall the project will shed light on the evolutionary pathways that have led to the diversity observed in gene regulation and on the relative importance of natural selection in these pathways.

The nervous and vascular systems of vertebrates display stereotypical organization of their networks, which are often closely aligned. Abnormalities in these precisely patterned networks lead to a variety of tissue dysfunction and disease. Thus, understanding how these two systems establish their highly patterned networks is important. The central aim of this proposal is to investigate whether and how these two systems cooperate to establish their networks during development and regeneration, a question that remains largely unclear. Here I propose a set of experiments that exploit many attributes of the zebrafish system to define the roles of neurovascular interplay in development and regeneration. First, I will simultaneously monitor the dynamic elongation of blood vessels and different types of nerves during development and regeneration using in vivo time-lapse imaging. Second, I will compare these dynamic behaviors in wild-type and mutant zebrafish, focusing on mutants that lack blood vessels or specific types of nerves. Finally, I will analyze these mutant animals to examine the consequences of the loss of each cell type in the development and regeneration of the other network.

Circadian rhythms are essential for many aspects of human physiology including sleep/wake times, body temperature, hormone secretion, metabolism, glucose homeostasis, and cell cycle progression. The canonical mammalian clock is based on transcriptional activators and repressors intertwined in interlocking feedback loops. Recent work has shown a different type of biological rhythm occurs in mammalian red blood cells, which lack a nucleus and many core clock genes. Specifically, the oxidation state of an antioxidant protein peroxiredoxin oscillates over a 24-hour period. These oscillations do not depend on transcription and are conserved in an ancient eukaryotic alga, which suggests a fundamental role for non-transcriptional oscillators in cells.

During my post-doctoral research, I plan to measure these oscillations using redox-sensitive GFP as a real-time reporter. This will allow me to directly compare how redox oscillations compare to transcription of classical clock genes. By employing RNAi, genetic, and chemical perturbation strategies, I aim to discover components involved in this new oxidation clock and probe how oxidation is coupled to canonical clock components. Furthermore, because red blood cells are an ideal lysate system, I plan to biochemically identify and purify components of the oxidation clock with the ultimate goal of reconstituting an oxidation cycle in vitro.

Circadian rhythms are fundamental to many physiological and behavior processes in wide variety of organisms, so it of interest to understand the mechanisms of this novel oxidation clock and how oxidation couples to classical clock components.

Cancer stem cells (CSCs), like normal somatic stem cells, are defined as cells that can self-renew and produce the progeny. Therefore, if CSC population is survived even after optimal therapy, patients will have relapses. That is why CSCs are considered to be a good target for novel cancer therapies. However, as shown in many reports about CSCs, the important factors such as frequency or specific markers are not consistent. And the mechanisms to cause variation remain obscure. To understand the mechanisms of variation seen in CSCs, it is more reasonable to identify the cells of origin of CSCs and analyze when or how this variation is induced than to isolate cancer stem cells from individual tumors and analyze their variation.
In this study, I will focus on epithelial ovarian cancers (EOCs) which are most common subtype in ovarian cancers and thought to be derived from ovarian surface epithelium (OSE). Morphological and pathological diversity in EOCs is a notable characteristic. Although there is increasing evidence that EOCs contain CSC population like other types of cancer, ovarian CSCs are also heterogeneous and inconsistent. To address this issue, I will reveal a lineage hierarchy within normal OSE and what cells in the hierarchy are responsible for tumor formation showing similar morphology to human ovarian cancers by using a model mouse of human ovarian cancer. Furthermore, to figure out the morphological diversity, I will compare the gene expression profile with the other epithelium in the female reproductive tissues which share the common origin of OSE and identify the responsible genes to give rise to the diversity and heterogeneity in morphology and pathology.

I am interested in exploring the mechanism by which the DNA damaging function of AID is naturally controlled and whether regulation of this mutagenic process can be achieved at the protein level instead of gene manipulation. Currently, no work has been conducted in aiming at directed regulation of AID’s mutagenic potential. In the first part of my work, I would like to investigate the contribution of AID’s self-interacting ability in its functional regulation. I plan to analyze the interaction dynamics in living cells so that I can evaluate the impact of an interaction directly in certain events, such as CSR, SHM or DNA damage condition. The second part of my work aims to isolate synthetic RNA ligand(s) that can bind to AID or its specific domain with high affinity, followed by detailed analysis of the characteristic features and cellular counterpart prediction. I will then examine the most promising candidates for their potential to interfere with the function of AID, including their ability to disrupt AID-AID self-interaction. Successful outcome of this work will open up new dimension in AID research field and will give rise to pioneering work in designing future regulatable molecular device for RNA/DNA editing enzymes.

The emergent morphological properties of metazoan tissues result from the interactions of individual cells, which are animated by intracellular molecular events. A common design in epithelial organs is that of interconnected networks made of a series of tubes. The combination of molecular genetics and live imaging techniques has led to descriptions of the cellular processes involved and the molecular players required for tube morphogenesis (a developmental program also known as branching morphogenesis). The aim of my project is to understand how these processes interact and self-organize to give rise to the ordered structure of branched organs. I will use an innovative approach to develop a predictive model of a paradigmatic example of branching morphogenesis: the HGF induced branching of MDCK cells cysts. To study operatively the program induced by HGF and to follow the information transfer from a cell to the neighbors, I will activate single cells of the cysts by using laser-switchable protein-protein interaction domains that allow the activation of signaling molecules with fine spatial resolution. Thanks to these new kind of perturbative experiments, I will improve current computational models with the intracellular layer to generate multiscale models. Then, I will use the model to build synthetic circuits that will be able to simulate autonomously the endogenous program of morphogenesis; this will reveal the relevance of the components of the program. In sum, this project will use a combination of perturbative, computational, and synthetic approaches with the aim of a predictive understanding of the branching morphogenesis developmental program.

For decades, conscious events and brain states were considered fundamentally different (albeit related): while the former are subjectively experienced, the latter can only be later inferred from external data (e.g., fMRI, EEG). Yet recent neuroscientific developments open new possibilities of making brain states accessible for human subjects, by using a unique online system that intracranially records neural activity and presents it in real time as sensory feedback. To examine the effect of such feedback on subjects' behavior I will use Binocular rivalry, in which subjects' perception alternates between two stimuli presented simultaneously but separately to each eye. Subjects will be asked to focus on one of the images (i.e., try to exert volitional control over rivalry). I hypothesize that sensory feedback about the otherwise unconscious neural activity correlated with the processing of either the dominant or the suppressed image (both typically change towards an alternation), will enhance subjects' ability to delay the alternation. This promises to yield new insights about the widely debated role of consciousness in voluntary control. In addition, I will hold a preparatory experiment to investigate the relations between the content of consciousness and Local Field Potentials (LFPs) evoked by neurons in the Medial Temporal Lobe (MTL). As opposed to spiking activity in the MTL that accompanies conscious percepts only, LFPs might be sensitive also to suppressed images. Such finding will open new venues of research, enabling the acquisition of intracranial information during unconscious processing.

A 14-year old high school student once asked us “Why are microtubules hollow?”. We could have elaborated at length on the central roles played by microtubules in segregating the genetic material and in building tracks for long-range motor movement. Truth to be told, none of us could provide the beginning of a cogent answer to this tremendously fundamental question. In fact, the notion that important events take place inside the lumen of microtubules seemed entirely without basis. To this day, the only known molecular event that takes place inside the microtubule lumen is acetylation of alpha-tubulin on lysine 40, a major post-translational modification whose functional consequences remain elusive. While a correlation between tubulin acetylation and microtubule stability is well-accepted, the causal relationship between stability and acetylation is still being debated and a function for microtubule acetylation remain speculative. In this context, our recent identification of the tubulin acetyltransferase TAT1 provides a much-needed molecular handle on tubulin acetylation and on the biology of the microtubule lumen.
We will now combine our expertise in microtubule biochemistry (Nachury) and in nano-engineering (Théry) to test the interplay between acetylation and the mechanical behavior of microtubules. The proposed research is highly collaborative with most experiments involving reagents from one team and techniques from another team. An important theoretical component will provide an overarching model of microtubule mechanics and a vigorous back-and-forth between modeling and experimental work will ensure that we arrive at an accurate physical understanding of how acetylation influences microtubule mechanics and vice-versa. In the end, we hope to uncover novel molecular paradigms for how events taking place inside the microtubule lumen influence microtubule biology.

Plant cell proliferation and morphogenesis rely on successful and correctly oriented cell division. Eukaryotic cell division is carried out by microtubule-based mechanisms. In plant cells several distinct arrays of microtubules are created and function over the course of the cell cycle: the most obvious are the interphase cortical array, preprophase band, spindle and phragmoplast. All of these arrays are created without benefit of central microtubule organizing body such as a centrosome. As a cell progresses through the cell cycle, microtubule structures are gradually replaced or rearranged from array to the next, but we have little knowledge of the mechanisms that carry out these transitions in microtubule array organization.
Recent studies indicate that gamma tubulin complexes and katanin are required for plant cell division and localized at plant cell spindle poles in a late anaphase. While concentration of nucleation and severing complexes at the spindle poles is expected, the specific functions of these proteins in spindle formation, phragmoplast organization and the reorganization/transition of mitotic arrays are largely unexplored.
We hypothesize the physical or functional interaction between microtubule nucleation factors and microtubule severing factors are of particularly importance for mitotic microtubule array reorganization. The subject of this research is to investigate the spatiotemporal function and relationship of microtubule nucleation and severing during mitotic cells. Specifically, I will apply a new cell biological technique to understand spatiotemporal function of proteins, focusing on microtubule severing function and mechanisms during mitosis.

RNA polymerases are very large enzymes with 0.5-0.7 MDa, which is consisting of 12-17 subunits. These subunit conformational changes enable the interaction with many additional factors, thereby transcribing different classes of genes. Precise regulation mechanism of transcription by multi-subunit RNA polymerases assembly is crucial to all organisms. However, much less is known about the biosynthesis of these enzymes themselves and the nuclear transport pathway remains elusive. This proposal focuses on Iwr1, which is one of a few examples, known to be involved in Pol nuclear import mechanism. The first aim of this proposal is the structure determination of the free form of Iwr1 and Pol II-Iwr1 complex, using several structural method including X-ray crystallography and MD simulation. Such structures would reveal the conformational change of Iwr1 and the interaction surface of Iwr1 on Pol II. In addition, to verify this observation, structure-based mutational analysis of this complex will be carried out. Confocal microscopy of Pol II localization and quantitative immuno-blotting of each Pol II subunits in ?IWR1 strain will be used to obtain functional insight of Iwr1 in vivo. The second aim of this proposal addresses identification and structural characterization of other nuclear transport factors for Pol I and Pol III. To find potential candidates, I will start with a bioinformatics search of NLS containing proteins from the protein interactome data of Pol I and III. To detect the cytoplasmic accumulation of Pol I and Pol III caused by siRNA-mediated depletion of candidate protein, I will identify new nuclear transport factors.