A key challenge in biology is to understand the significance of genome duplication for adaptation and speciation. We propose to address this issue by analyzing gene network function following genome duplication in polyploid species of the plant genus Cardamine, which is related to the model species Arabidopsis thaliana. To this end, we will integrate bioinformatics and plant ecology to infer polyploid gene networks from transcriptome datasets of homeologous gene pairs. To functionally test network inferences we will generate synthetic ‘partial polyploid’ lines by transforming genes from one parent species into the other parent.

Network merging occurs in many diverse contexts. For example, in the successful merger and acquisition of companies, a strong leader reorganizes the social network in a top-down approach by removing redundant sections and generating new and enhanced functions. In contrast to this, little is known about how polyploid species acquire adaptive traits via the merging of parental gene networks. We hypothesize that adaptations for a new niche may arise via novel network reconfigurations. We further expect that sub-networks for stress responses that are unique to each parent may be safeguarded in the polyploid species, thus allowing them to exploit fluctuating habitats. To test each hypothesis we will infer and test nodes in the polyploid gene network that may have adaptive significance.

The merge of species living in wet and dry habitats occurred recurrently in Cardamine, and the resultant polyploid species occupy new ecological niches with fluctuating water levels. We can exploit transgenic methodologies developed in C. hirsuta to test network inferences and gene functions at critical nodes in polyploid gene networks. Moreover, two polyploids arose during the last century, providing a unique opportunity to study recent network merging in its native environment.

We will develop new algorithms in order to infer gene networks in merged polyploid genomes. We will design next generation sequencing and custom microarray approaches to distinguish homeologous gene expression, and we will develop novel network comparison algorithms to infer critical genes in the polyploid networks. Thus, we will advance the state of the art in network analysis and reveal principles underpinning successful network mergers in the formation of polyploid species.

Collective migration of endothelial cells is responsible for the formation and maintenance of the vasculature, for healing following injury, stroke or cardiac infarct as well as for cancer formation. While necessary for migration, the role and molecular regulators of membrane trafficking during the migration process are not yet understood. Here I propose to investigate the molecular mechanisms and physiological role of membrane trafficking during collective endothelial cell migration following three main experimental strategies. First, during planar cell migration in vitro, by validating a focused siRNA screen targeting known trafficking pathways. Second, during endothelial tube formation, where endothelial cells growing on beads form vascular sprouts in a 3D environment. Third, in zebrafish, during regenerative angiogenesis following caudal fin amputation. This combination of in vitro and in vivo methodologies will allow us to rapidly evaluate the physiological relevance of identified key regulators. Detailed biochemical and cell biological analyses involving transport assays, high-resolution imaging of localized signaling activities by confocal and TIRF microscopy, and real-time analysis of vascular sprouting, will provide the basis for the development of mechanistic models of how membrane trafficking orchestrates migration and vascular morphogenesis. Together, the proposed research will provide mechanistic insight of broad interest, as collective cell migration is a widespread phenomenon in animal physiology, while also identifying potential targets for anti-angiogenic therapies.

Polycomb group (PcG) proteins are transcriptional repressors that have been implicated in various developmental and disease processes in a broad spectrum of animals, from nematode worms to humans. PcG proteins mediate chromatin remodelling and are required for the stable silencing of homeotic (Hox) genes, which control patterning along the anteroposterior body axis. Despite intensive efforts in a variety of systems, the exact mechanisms of PcG function have remained elusive.

Recent studies suggest that PcG proteins interfere with the transcriptional state of RNA Polymerase II (Pol II) at paused Hox genes in the Drosophila embryo. We propose to experimentally validate this interaction and show that it eliminates plasticity in the expression of Hox genes, and therefore represents a mechanism that ensures the robustness of embryonic patterning and maintenance of organ identity.

Our model will be tested using a combination of whole-genome assays and functional genetics methods. We will analyse the transcriptional state of Pol II in contexts where PcG function is impaired in the absence of a phenotype. Our aim is to reveal the existence of a 'poised' state in Drosophila: although silent, paused genes contain fully activated Pol II and are ready for rapid activation by inducing signals. We will test the hypothesis that PcG enzymatic complexes mediate a conversion from a 'poised' state into a 'stalled' state at Hox gene promoters and that this interaction contributes to homeotic silencing. Finally, we aim to demonstrate that a poised transcriptional state introduces plasticity in Hox expression and reduces gene pattern accuracy in a fluctuating environment.

How structural novelties arise is a key question in evolutionary developmental biology. Land plants evolved several morphological innovations such as roots, leaves and vascular system. Vascular cambium is a proliferative tissue essential for radial thickening of plant body, which is found in seed plants: angiosperms and gymnosperms. In this tissue, cambial stem cells continue to produce daughter cells that will differentiate into specific types of vascular cells. To address how the cambial stem cells and therefore vascular cambium originate in the ancestor of seed plants, I propose to explore the functional evolution of TDIF peptide signaling, which regulates cambial stem cell maintenance in the angiosperm Arabidopsis thaliana. Because the ligand and receptor genes for TDIF signaling are conserved in the lycophyte (non-seed plant) Selaginella moellendorffii, these genes provide a clue to investigate this issue. Specific aims of this project are: 1. To identify TDIF signaling genes from species in the major taxa of vascular plants including lycophytes, ferns, gymnosperms and angiosperms. 2. To compare the expression pattern, physiological activity and molecular function of the identified genes/proteins to those in Arabidopsis. From the analyses, I expect to clarify how TDIF signaling is incorporated into cambial stem cell maintenance and whether the vascular cambia of seed plants share a common origin as suggested in previous anatomical studies. Experimental methods developed in this project will contribute greatly to investigate evolution of other traits in land plants.

The catacombs of ancient Egypt contain literally millions of well-preserved Sacred Ibis (Threskiornis aethiopica) mummies. These birds were revered by the Egyptians from the Predynastic period (7,500 – 5,100yrBP), by which time their relationship to the god Thoth had been well established. Ibis have been a focus of worship ever since and by the Late Period (~2600yrBP) the numbers being mummified had reached the millions. At one site alone in a single year 10,000 were estimated to have been interned. These mummies are common in many museums around the world and large numbers are still preserved in catacombs from at least five sites in Egypt. Using archaeological and Optically Stimulated Luminescence methods we can precisely age these mummies. The research team aims to use ancient DNA and genomic methods to study both rates and modes of DNA evolution using this remarkable resource. In recent years, a number of ancient DNA studies have made significant contributions to our understanding of the processes that govern DNA change. However, generally these studies have been limited by the lack of large numbers of known-age remains from multiple time points. Specifically, we will recover more than 200 nuclear microsatellite DNA loci from samples of different ages and use these data to test mutational models that have been formulated to explain the evolution of these DNA repeat loci. In order to develop powerful methods of analysis we need to have a clear understanding of the factors affecting the development and evolution of microsatellite DNA alleles in vertebrates. The proposed research will directly contribute to this improved understanding. In addition, we will sequence more than 100 mitochondrial genomes from ancient Ibis. With the aid of new mathematical and evolutionary models we can greatly improve our estimation of molecular rates. These mitochondrial data will also help us to understand the Egyptian traditions relating to animal mummification. In preparation for this work, we have shown that we can recover both mitochondrial and single copy nuclear DNA sequences from Ibis mummies. The large number of known-age mummified Sacred Ibis in Egypt, together with the multidisciplinary nature of the team (ancient genomics, evolutionary biology, mathematics and Egyptology), provides a unique opportunity to study DNA changes over time.

Cellular bistable systems draw increasing attention because of their roles in epigenetics, differentiation and cellular memory. It remains unclear what parameters are tuned by the cells in order to improve cellular memory or stabilize cellular differentiation states. We will explore the strategies evolution employs for adjusting parameters in bistable systems to optimize their function. In Saccharomyces cerevisiae, the galactose regulatory network induces ON and OFF cells with stable, high and low expression states, respectively. The state of a cell population does not only depend on the present galactose concentration but also on the past condition. This bistable memory requires positive feedback loops. After whole genome duplication, homologous GAL1 and GAL3 subfunctionalized and assumed negative and positive regulatory roles, enclosing negative and positive feedback loops, respectively. K. lactis, a relative of S. cerevisiae, maintains also a positive feedback loop through Gal4, the transcriptional activator of the network. We will investigate the evolution of the GAL network by measuring the system parameters. In addition, we will apply synthetic networks to mimic the natural network for the flexibility to tune the parameters and observe the responses. This project will focus on the evolutionary reparametrization of the network and how nonlinear responses and stochastic gene expression contribute to the development of cellular memory. Combining experimental and computational approaches, this project will lead to a better understanding of the stability of cellular differentiation states and the roles and origins of the cellular memory.

During neuronal differentiation, precursor cells must acquire their definitive functional transcriptomes. Regulation of transcriptional expression of several genes as well as of post-transcriptional processing, especially alternative splicing (AS), is crucial to this cellular process. Importantly, these layers of transcriptomic regulation are interconnected. On the one hand, changes in the transcription of neuron-specific splicing factors have great effect in modulating many alternative exons. On the other hand, several studies point to a regulation of transcription factor (TF) function by AS, which may have a profound impact in the regulation of the associated TF target networks. However, despite some examples, the extent and architecture of this crosstalk between layers largely remain unknown. The goal of this proposal is thus to assess the impact of AS of TFs in the process of neuronal differentiation, by pursuing the following specific aims: (1) Build a comprehensive database of alternatively spliced exons in TFs; (2) Study the regulation of TF’s alternative exons during neuronal differentiation, using RNA-seq; (3) Investigate the specific functions of 10-15 regulated alternative exons, using isoform-specific knockdowns in several neuronal differentiation assays; and (4) Elucidate isoform-specific networks of regulated targets, using isoform-specific ChIP-seq and microarray quantifications. We will focus on cases in which AS is predicted to affect TF DNA-binding specificity/affinity or cofactor interactions. These are expected to have a profound impact in which targets each isoform regulates, challenging the way we think of the regulation exerted by a single TF gene.

Bone marrow is an organ where various kinds of hematopoietic cells reside and undergo maturation or differentiation into specific lineages, processes that occur in specialized locations called 'niches'. Whereas other hematopoietic and lymphoid organs, such as lymph nodes and spleen, have definite inner substructures that are easily identifiable because of the distinct histological architecture, such substructures are not as obvious in the bone marrow space. However, the requirements regarding process specificity in bone marrow are at least as high as in the other hematopoietic and lymphoid organs. Even though there is ample indirect evidence of the existence of such specific substructures that operate as functional niches, it is still not possible to clearly distinguish which areas of the bone marrow correspond to, for instance, the "B cell niche", the "osteoclast niche" or the "hematopoietic stem cell (HSC) niche". Previous studies have been interpreted as indicating that the HSC niche is located along endosteum close to osteoblasts, or around the vasculature. However, these concepts derive only from static analyses using conventional immunohistochemistry and/or adoptive transfer experiments and thus do not allow for a comprehensive investigation of the complex dynamic biological processes required for the functional characterization and understanding of the different niches. The purpose of this study therefore is to visualize and identify the distinct niches in bone marrow cavities by performing cross-disciplinary studies that combine advanced imaging technology with novel computational techniques. Imaging mass spectrometry will be exploited to identify molecules specific for certain niches, whereas the dynamics of bone marrow cell behavior in the respective niches will be analyzed in detail by using intravital two-photon bone imaging that the applicant has originally pioneered. By combining these advanced imaging approaches with novel high-throughput computational image analysis of cellular migration and interaction dynamics within the native tissue context, and then integrating all the results in multicellular simulation models, we believe our team will be able to anatomically and functionally define the different "niche" substructures in bone marrow cavities in vivo with greater resolution and assurance than previously possible.

Cortical interneurons are remarkably diverse in their properties. Despite a growing appreciation of the role played by sonic hedgehog (Shh) and fibroblast growth factor (FGF) signaling in patterning the ventral telencephalon, little is known about how these pathways and their downstream effectors influence the emergence of interneuron diversity later during telencephalic development. The proposal aims to further elucidate the transcriptional cascade involved in specifying different interneuron subtypes focusing on genes acting downstream of FGF. I propose to study two Ets-domain transcription factors (TF), Ets1 and Ets2, known to act downstream of FGF signaling upon activation through MAP kinases, and recently identified as highly expressed in both caudal and medial ganglionic eminences (CGE, MGE). I speculate that the Ets-TFs might contribute to interneuron subtypes specification. I intend to investigate the regulation of Ets-TFs in vivo in both GE through FGF signaling-dependant phosphorylation, as well as their role in the specification of cortical interneurons using the Ets1 and Ets2 null mice, through electrophysiology and morphological analyzes. Finally, I intend to explore the consequences of Ets1 or -2 genetic inactivation on mice behavior and locomotor activity. This transversal project will attempt to better understand the molecular basis of interneuron development and specification as well as the contribution of specific interneuron subtypes to behavior, to further increase our knowledge of forms of neurological and neuropsychiatric disorders (epilepsy, autism spectrum disorders, schizophrenia) in which specific populations of interneurons have been implicated.

Type 2 diabetes mellitus (T2DM) and obesity are leading causes of mortality and are associated with the Western lifestyle, which is characterized by excessive nutritional intake and lack of exercise. A central player in the pathophysiology of these diseases is the nuclear hormone receptor PPARgamma (PPARg), a lipid sensor and master regulator of adipogenesis. PPARg is also the molecular target for the Thiazolidinedione (TZD)-class of insulin sensitizers, which command a large share of the current oral anti-diabetic drug market. Despite their efficacy in glycemic control, TZDs are associated with various adverse side effects, including weight gain, edema, and liver and cardiovascular toxicity, leaving room for novel therapeutic approaches that target this pathway.
During the course of my HFSP long-term fellowship I have identified gamma activated peptide 1 (GAP1) as a novel PPARg target. In experiments with GAP1 knockout mice, we further demonstrated that these mice develop high-fat diet induced insulin resistance and hepatic steatosis, underscoring its significance in the development of T2DM.
Basis for the current proposal is our finding that administration of GAP1 to diabetic mice has potent and long lasting beneficial metabolic effects, including lowering of blood glucose and insulin, similar to TZDs. The studies proposed here aim to mechanistically understand the pharmacologic effects of GAP1 on glucose homeostasis and the development of T2DM. Therapeutic targeting of GAP1 could serve as an improved strategy for the treatment of T2DM, eliminating some of the adverse effects of TZDs that are mediated through direct activation of PPARg.

Circadian clocks keep temporal order and help organism to anticipate daily changes in the environment. In mammals, most, if not all, differentiated cell types possess a circadian clock, with over 10% of their transcriptome changing throughout the day. This is achieved through a network of activators and repressors that form interconnected feedback loops. Cells likely acquire circadian rhythms only upon differentiation, since the only cell systems in which circadian rhythms have not been found are embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). Thus, pluripotent cells either cannot sustain a circadian clock, or the clock they possess neither depends nor causes transcriptional oscillations. As a result, pluripotent cells may offer an ideal system to develop a model for the emergence and maintenance of the circadian clock, and to study how key properties of these timing devices – such as robustness, plasticity, and temperature compensation -- are implemented into the system.

Here, we aim to decipher the molecular and cellular mechanisms behind the generation of the circadian clock. We will use innovative and interdisciplinary experimental and analytical approaches to dissect the activation of the transcriptional circadian cycle during ESC differentiation, as well as its deactivation during somatic cell reprogramming to iPSCs. We will build on our team’s expertise in circadian rhythms and RNA biology (SK), ESCs, differentiation and reprogramming (EM) and systems biology (AR). We will analyze circadian gene expression during the development or suppression of the circadian system using both live imaging-based single cell assays and genome-wide transcriptome sequencing (RNA-Seq). We will use computational algorithms to integrate the data into a system scale mechanistic model of the initiation and shut down of the circadian clock. We will further validate and refine the model by assessing these processes under genetic perturbation of key system components as well as by biochemical experiments (e.g. ChIP-Seq) in differentiated and pluripotent cells to test the regulatory connections in our model. Our work will provide a comprehensive view of a clock as a biological system, and will shed light on the initiation of the circadian clock during differentiation and on the role of this timing device during the exit from pluripotency. Our approach harnesses the power of two distinct facets of systems biology – single cell studies of system function and genomic studies of systems organization – to tackle a fundamental question in circadian biology, and can serve as a paradigm for similar studies of other biological systems.

Species remain unaltered over millions of years yet can rapidly adapt to changing conditions. Compelling evidence from prokaryotic biology, vertebrate immunoglobulin diversification, and tumorigenesis has demonstrated that compromising genome integrity can be of adaptive value. Although stress-inducible genome instability has been described in many organisms, the molecular mechanisms underlying these phenomena in eukaryotes remain unknown. Given the links between the heat-shock protein (Hsp) Hsp90 and genome maintenance, I hypothesize that Hsp90 can act as a “molecular rheostat” to adjust the fidelity of DNA transactions in response to environmental cues, with the net effect that when an organism is not well adapted to its environment new traits will appear at higher rates that when it is well adapted. This project seeks to elucidate the mechanistic roles of Hsp90 in genome maintenance in S.cerevisiae, by characterizing key signal transduction cascades that act in coordination with Hsp90 upon stress. For this purpose, I will first undertake a systematic survey for DNA damage sensitivities and signatures of genomic instability that are released upon Hsp90 inhibition. Secondly, I will employ quantitative high-throughput biochemical approaches to dissect the effects of DNA damage signaling on key Hsp90-client physical interactions. Finally, I will characterize the signaling cascades and posttranslational modifications involved and evaluate their impact on genome integrity. This work will provide insights into the biological mechanisms that drive evolution and tumorigenesis.

Every day we are assaulted by ionizing radiation (IR), in addition to clinical exposure, both of which induce significant pro-inflammatory cytokine responses. IR induction of pro-inflammatory responses can be quite severe and compromise radiation therapy, necessitating use of anti-inflammatory cortico-steroids, often with limited effectiveness. The AUF1 protein is a major attenuator of these responses through its targeted rapid destruction of pro-inflammatory cytokine mRNAs. Loss of AUF1 in mice promotes acute psoriasis, loss of follicular B lymphocytes, septic shock, and surprisingly, premature aging, cellular senescence and telomere loss, all also linked to effects of IR. Unpublished studies demonstrate that AUF1 is a key target inactivated by IR, providing an exciting molecular link between IR and inflammatory disorders. It is important to understand how IR promotes a pathological inflammatory response, for little is known about the mechanism by which this occurs. This application is directed at understanding the molecular and mechanistic basis by which AUF1 is targeted for inactivation by IR, and its role in pathological inflammatory responses. I will use immortalized wild type and AUF1-/- macrophages from the AUF1 knockout mouse and mass spectrometry, to identify and functionally characterize IR-inducible AUF1 interacting proteins. Studies have already found an exciting connection to the microRNA processing machinery and AUF1. I will also identify the major inflammatory cytokine mRNA targets regulated by IR through control of AUF1 activity. These studies forge an entirely novel area of research in understanding inflammatory disorders and environmental interactions.

At the molecular level no cell is like any other. Differences between genetically identical cells can in fact be quite pronounced, and these differences can have profound biological and biomedical implications. Stem cells divide and differentiate in response to physiological and environmental cues but exhibit considerable differences in how they do so; equally, tumour cells respond differently to drug treatment or radiation therapy. The mechanisms driving such variability among to all intents and purposes seemingly identical cells are only poorly understood. In order to understand these fundamental processes – which also have numerous biomedical and pharmacological implications – we have to overcome experimental and mathematical modelling challenges.

Here we develop the necessary experimental and theoretical frameworks, which allow us to probe single cells, measure the abundances of key signalling molecules, and infer mathematical mechanistic models for signal transduction. We will then analyze these mathematical models in order to understand better how the structure of the signal transduction network influences cell-to-cell variability. We will then test these models and their resulting predictions in further experiments. Most importantly, however, we will develop novel strategies which will allow us to guide cellular decision making processes and determine patterns of differences between cells. If we manage to reduce or otherwise affect such variability then we gain a novel way to guide the behaviour of cells. The scope for applying such techniques is enormous and could, for example, include novel therapeutic interventions, progress in regenerative medicine, as well as better understanding of stem cells and their behaviour.

At the same time we will also investigate what determines the behaviour of cells more generally. Often cells exhibit high levels of robustness against external perturbations. We will use the mathematical models inferred for single cells in order to generate hypotheses as to which molecules, molecular processes and interactions affect such robustness and how this can be interfered with.

Among the various lipid species, fatty acyls represents the major lipid building block of complex lipids, such as a component of membrane phospholipid and a precursor of lipid mediator. Phospholipase A hydrolyzes the fatty acyls at the sn-1 and sn-2 positions of phospholipids, while acyltransferase incorporates the fatty acyls into the lysophospholipids. The dynamic equilibrium between deacylation and reacylation by these enzymes, known as phospholipid remodeling, maintains the membrane stability and the cellular function normally. Under the pathophysiological conditions, eicosanoids, the lipid mediators derived from arachidonic acids, are produced by a sequential actions of eicosanoid synthesizing enzymes. However, it is still unclear the molecular basis of the interconnectedness between eicosanoid production and phospholipid remodeling in inflammation. I will address the question by examining the kinetics of their metabolism and exposing the relationship between signaling pathway and lipid metabolism. Focusing on inflammatory cells like macrophages and mast cells may allow us to address the question because of the existence of a series of enzymes and their important roles in inflammation. I plan to a) apply the deuterium and radioisotope-labeled lipid species to monitor the dynamics of lipids in the cells, b) measure the lipid levels by a liquid chromatography-mass spectrometry system, and c) use computational modeling to understand the underlying mechanism of lipid metabolism. This study will create a bridge between lipid metabolism and inflammation. The results may also contribute towards developing an innovative drugs against inflammation and related diseases.

A fundamental aspect of cell division is the proper segregation of chromosomes in a spatially oriented manner. While the essential mechanisms of cell division are conserved in eukaryotes, additional mechanisms may exist in human cells to achieve proper differentiation, for example in asymmetrically dividing cells.

The orientation of the cell division axis is determined by forces generated at the cell cortex, where astral microtubules (MTs) emanating from the mitotic spindle pole are anchored to the plasma membrane. The minus end directed microtubule-based motor cytoplasmic dynein is a key player in generating force at the cell cortex by pulling on astral MTs. Cortical dynein functions for spindle positioning, orientation, and anaphase spindle elongation. However, how cortical dynein is targeted and regulated to achieve these different tasks remains unclear. Cortical dynein must function during both symmetric and asymmetric cell division to generate cell type diversity. However, whether cortical dynein is differently regulated between these different types of cell division and how cortical dynein functions during differentiation in human cells are unknown.

For this proposal, I will:
1: Identify dynein interacting partners at the cell cortex
2: Define the regulation of dynein localization and function at the cell cortex
3: Analyze cortical dynein localization and binding partners during differentiation in human ES cells

This project is completely distinct from my previous work on kinetochore function and will allow me to learn new ideas, techniques, and systems, including mass spectrometry, in vitro biochemistry, and ES cells, which will drive my future research.

Sensation of mechanical forces is crucial for all living organisms from bacteria to vertebrates. In metazoans, the sensation of external forces is important for hearing, touch sensitivity and pain response. The nematode Caenorhabditis elegans is an excellent model system to study mechanotransduction during touch sensation because many genes involved therein have been identified. Despite the knowledge of the molecular players participating in the touch reception, it is not known how mechanical forces lead to mechanosensory transduction (MeT) channel activation and depolarization of the membrane. This lack of knowledge is mainly due to the absence of appropriate tools to exert well-controlled forces and to study their influence on touch receptor neurons (TRNs).
Using a combination of Micro-Electro-Mechanical-Systems and elastic substrates to exert forces onto cultured TRNs, confocal imaging and patch clamp electrophysiology to study their consequences I propose to characterize how mechanical force activates electrical signal in isolated touch receptor neurons. Different scenarios of MeT channel opening will be revisited and based on these findings a mechanical model of MeT channel gating will be proposed. These results will contribute to our understanding on cellular mechanotransduction in general and how touch is sensed in particular and might also help to understand neuropathies such as allodynia and chronic pain.

Plants regulate intake of CO2 and transpirational water loss by a highly dynamic and specific cell type, the guard cell. Since fresh water has become a globally limited resource scientific attention is focusing on understanding signal transduction mechanisms and pathways in guard cells in recent years.
To adjust the water evaporation through the stomatal pore, the surrounding guard cells regulate their turgor. This is achieved by the production or degradation of the main anionic osmolyte, malate. While an astonishing scientific progress has been made in understanding abscisic acid and Ca2+ driven signal transduction, in the identification of ion channels and initial malate transporters, very little is known about the genetics of malate metabolism mechanism. This gap of knowledge underlines the need for direct genetic identification and characterizations, to understand the molecular processes of malate metabolism and regulation in guard cells.
In my project I propose to identify the source of malate and its metabolic rate limiting enzymatic steps. Beyond that I will discover the mechanisms by which the physiological stimuli abscisic acid and CO2 control malate metabolism in guard cells. Newly developed tools of Prof. Julian Schroeder and his group open now the possibility to perform guard cell specific gene silencing or expression. Together with the highly-developed tools for guard cell signaling available in the Schroeder Lab I will identify the mechanisms mediating the essential control of malate metabolism during stomatal movements. These research discoveries may also lead to new avenues for future enhancement of water use efficiency and drought tolerance of plants.

Ribbon synapses are specialized for transmission of signals in sensory systems such as the retina. They are distinguished by ribbon-like structures at the active zone. The function, assembly and targeting of ribbons to active zones are not yet well understood. As such, our broad aim is to investigate the structure, assembly and function of ribbon synapses in the mature and developing mouse retina. We will focus on ribbon synapses of photoreceptors and bipolar cells which collectively are essential for relaying light information from the retina to the brain. We propose a highly interdisciplinary approach, combining the technical expertise of the investigators which include optical imaging, electrophysiology and molecular analysis. We will specifically address three key areas:

The first area concerns the assembly and maintenance of ribbons at synaptic sites. To date, is not known how RIBEYE, an essential component of ribbons, is dynamically transported and assembled into ribbon-like structures at active zones during development. Moreover, the cellular and molecular mechanisms underlying the maintenance of synaptic ribbons in mature circuits have yet to be determined. Thus, monitoring RIBEYE trafficking in real-time and in situ is important not only for increasing our basic understanding of the normal assembly and maintenance of ribbons, but knowledge gained from such dynamic views will help interpret the effects of perturbing RIBEYE-associated molecular interactions at synapses. In Aim 1, we propose to investigate how ribbons are trafficked, targeted and maintained at retinal synapses by generating novel transgenic animals expressing fluorescently-tagged RIBEYE and using live-cell imaging approaches (RW,FS,LL).

Second, some retinal synapses are functional during development prior to the assembly of ribbons at the active zones. How synaptic transmission changes before and after ribbons form is unclear. Knowledge obtained from physiological experiments addressing this question will advance our understanding of the function of ribbon-mediated transmission at sensory synapses. Thus, in Aim 2, we seek to understand how properties of synaptic transmission alter as ribbon synapses form and mature at inner and outer retinal synapses. Synaptic function will be assessed at different structural stages by electrophysiology (LL,RW,FS) and by two-photon imaging of synaptically-localized reporters of neurotransmission expressed in new transgenic mouse lines (LL,RW).

Third, ribbon function in neurotransmission has thus far been largely studied in mutants with altered transmitter release due to perturbed exocytosis. The Schmitz lab has discovered a novel function for the B-domain of RIBEYE itself, potentially affecting endocytosis. In Aim 3, we will determine the role of the enzymatic activity of RIBEYE(B) in the formation, maintenance and function of ribbon synapses using a knockout mouse (FS,RW & LL).

Increasing evidences indicate prolyl isomerase Pin1 regulates key proteins in phosphorylation signaling especially related to proliferation and transformation. In many cancers, Pin1 is overexpressed and correlates with poor clinical outcome. Transgenic mice studies demonstrate Pin1 knockout effectively prevents oncogene-induced breast cancer, and MMTV-Pin1 transgenic mice develop hyperplasia of mammary epithelial cells and breast cancer. A small subset of cells referred as cancer stem cells have been implicated to be responsible for tumor initiation, distant metastasis and drug resistance. However, the role of Pin1 in neoplastic stem cell is not known. My objectives are to determine the function and regulation of Pin1 in normal and neoplastic mammary stem cells. Aim 1 is to investigate the role of Pin1 in self-renewal and lineage-specific differentiation of normal mammary stem cells in vitro and in vivo using Pin1 knockout mice and MMTV-Pin1 transgenic mice that we generated. Aim 2 is to investigate role of Pin1 in breast cancer stem cells in vitro using breast cancer cell models, and in vivo by monitoring breast cancer stem cell population and the breast cancer development using Neu mouse model with Pin1 knockout or overexpression. Aim 3 is to determine the role of miRNAs in regulating Pin1 function in stem cells. This is especially exciting given miRNA-200c downregulation links breast cancer stem cell with normal stem cell and that I found several microRNAs including miR-200c target Pin1 mRNA. Results from proposed study would provide significant novel insight into the turmorigenesis of breast cancer and might help design more effective therapies against breast cancer.