In eukaryotes, endocytosis and the anterograde and retrograde secretory pathways, are mediated by inter-connected endomembrane systems to maintain cellular homeostasis. In budding yeast, viral K28 toxin protein exerts its killing action via clathrin mediated endocytosis and subsequent retrograde transport. Here I propose to generate fluorescent K28 derivatives to study its trafficking mechanisms. Using such a unique tool I propose to determine whether different clathrin coated pits/vesicles and endocytic proteins are used for distinct cargos with distinct cellular destinations, and how proper targeting is achieved. I also propose to identify the endomembrane compartment in which K28 is diverted to the reverse secretory pathway. Furthermore, I will identify the poorly understood molecular mechanisms by which the retrograde transport pathway is regulated. This question will be answered via the dynamic tracing of fluorescent K28 in a variety of yeast mutants, recently identified in the host lab, to be either resistant or super-sensitive to K28 toxin. My proposed research will provide novel understanding of fundamental mechanisms of endocytosis and intracellular protein trafficking.

The formation of silent chromatin or heterochromatin plays important roles in gene regulation, as well as in the maintenance of genome stability from yeast to man. In budding yeast, silent information regulators, consisting of Sir2, Sir3 and Sir4, nucleate transcriptionally inactive chromatin at silent mating-type loci and telomeres. The molecular mechanisms by which silencing represses transcription are poorly understood. The aim of the proposed research is to understand the mechanism of transcriptional silencing in terms of chromatin structure. Using budding yeast as a model organism, I will isolate silent chromatin formed in vivo for study in vitro. In addition to biochemical characterization of the chromatin, the isolated silent chromatin will be analyzed by cryo-electron microscopy (cryo-EM) and 3D image reconstruction. Moreover, to examine the functions of Sir complex and histone modifications in the formation of silent chromatin, cryo-EM analysis will be performed with various mutant strains. The proposed research represents a new approach to the investigation of higher order chromatin structure, and will give insights into the connection between chromatin structure and function. Together with my previous research experience, which was based on genetics and fluorescent microscopy, these additional methods will provide me with the best possible basis for exploration of chromosome biology in the future.

Cell differentiation is a very stable process, yet it can be reversed when a nucleus is transferred into an enucleated egg. My project consists in the identification and characterization of the genes involved in nuclear reprogramming, using nuclear transfer as a model system. Hundreds of differentiated mammalian nuclei can be reprogrammed upon their transfer into a frog oocyte, such that these same nuclei begin to express pluripotency genes. This system is perfectly suited for this analysis as it allows to combine the power of mouse genetics with the cell biological advantages of the frog oocyte. To identify these genes, I will perform a random, phenotype-driven mutagenesis screen and identify genes that affect the predisposition of a differentiated mouse nucleus to be reprogrammed by nuclear transfer into a frog oocyte. I will use the Blm mutant mouse embryonic stem cells, combined with the revertible gene trap insertion method to create a series of homozygous mutant cell lines. These will be differentiated by retinoic acid treatment and their nuclei will be prepared and injected into frog oocytes. The efficiency of nuclear reprogramming will be determined by monitoring the timing and intensity of pluripotency gene re-expression in the mouse nuclei after the nuclear transfer. Understanding nuclear reprogramming will allow the derivation of pluripotent cells from a diseased individual and their administration as replacement stem cells. Such therapy could greatly improve treatments against many degenerative diseases. In addition, it will help to understand how differentiated cells can acquire stem cell-like properties through mutations and give rise to cancer stem cells.

Senescence is a state of stable cell cycle arrest in response to cellular stress, such as genotoxic or oncogenic stress. A series of the recent studies have identified multiple effector mechanisms, highlighting the very complex and heterogeneous nature of senescence. To gain a better understanding of the diversity of mechanisms involved we propose a new concept, that senescence is a process of re-constructing pre-existing programs using parallel regulatory networks for a different purpose. Our aims are to provide a proof of this concept and to characterise a new senescence effector program. To identify such new senescence effector programs, we have been using p53 as a tool. While the p53 transcriptional targets that are associated with DNA damage checkpoints and apoptosis are well documented, ‘senescence specific p53-targets’ are unknown. Human diploid fibroblasts (HDFs) are a flexible system in which we can induce a range of p53-associated phenotypes in the same cell type with a defined genetic background. Our preliminary microarray data identifies a gene set that is downregulated by p53 knockdown during senescence, but not during apoptosis. Among the ‘senescence-specific p53 targets’, we will identify genes that are originally involved in physiological programs, such as tissue differentiation where they are regulated by a distinct transcription factor. We will use these genes as a model to test our new concept of senescence and to extend the concept to more general stress responsive programs. The projects will utilize two emerging technologies, next generation DNA sequencing and chromosome-engineering, in addition to conventional cell biology and mouse genetics, to provide fundamental insights into this multilayer transcriptional network - the default and stress responsive regulations - and its functional outcome in stress conditions both in vitro and in vivo.

Human cells contain two copies of all 23 chromosomes—one copy inherited from the mother and one copy from the father. Passing on this genetic information accurately is essential to the health and survival of each subsequent generation. Importantly, to prevent offspring having twice the number of chromosomes as their parents, gamete cells (oocytes and sperm) must contain only one copy of each chromosome. The chromosome copy number is halved during meiosis. Meiosis includes the formation and repair of chromosome breaks used to align, and then separate apart, pairs of chromosomes—thereby halving their number.
Some chromosomal breaks create physical connections between the chromosomes (crossovers), others do not (noncrossovers). Crossovers reassort large regions of the paired chromosomes. As a result, when the chromosomes separate, they will have an altered assortment of genes. Because crossovers occur at different chromosome positions in every meiosis, they generate a great source of genetic variability. It is this process that causes siblings to appear similar but non-identical even though they have the same parents.
Little is known about the relationship between the distribution of chromosome breaks and crossovers. This project will test the idea that crossovers and noncrossovers are initiated by different events: DNA breaks or DNA gaps. We will test this relationship by mapping the chromosomal position of these different events. Understanding this fundamental relationship has wide-ranging implications for our understanding of genetic variability, infertility, and for the agricultural industry, where the assortment of desired characteristics is of great value.

The nucleolus, as the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly, is at the core of all cellular functions and alterations in nucleolar function have been linked to various human diseases. Nucleolar hypertrophy has been reported in cancers with poor prognosis and proto-oncogenes, as c-myc, have been described to regulate nucleolar functional output. The cellular pathways that feed-in and regulate nucleolar size during development and tumor formation are poorly understood. Here, I propose to combine complementary cell culture based screening, computational and biochemistry approaches in combination with in vivo studies to characterize the genetic networks that underlie nucleologenesis and nucleolar size regulation.
First, I will generate tools that will allow me to study nucleolar assembly and morphology in Drosophila cell lines and set up a genome wide RNAi screen to identify genes required for nucleolar biogenesis and size regulation. Second, I will analyze the nucleolar proteome by mass spectrometry and combine these results with the RNAi data to identify the core set of nucleolar organizer proteins. Finally, I will test the contribution of nucleolar hyper- and hypotrophy to tumor formation in vivo, using Drosophila as a model system.
Altogether, these studies aim to decipher the regulatory networks that underlie nucleologenesis and nucleolar size regulation and aim to determine the contribution of nucleolar hypertrophy in tumorigenesis.

Kinetochore is a conserved multi-protein structure required for segregation of sister chromatids during mitosis. It functions as an interface with spindle microtubule. Without a functional kinetochore, chromosome segregation fails and results in aneuploidy or even cancer. Recent proteomic analyses have identified components of the vertebrate kinetochore that now constitutes approximately 80 proteins. These proteins assemble on centromere that is marked by centromere specific histone H3 variant (CENP-A) containing nucleosomes. However, the targeting of CENP-A alone is not enough to form a functional kinetochore, and there seems to be a complex regulation. One of the key components is the CENP-T/W complex that binds histone H3 containing nucleosome adjacent to the CENP-A nucleosome. Many complexes assemble on CENP-T/W complex but how and when this assembly takes place is still unknown. Moreover, CENP-T/W targeting to centromere requires its histone fold but the structural basis of this recognition and the specificity remain elusive. To unravel the kinetochore structure and function, we will combine cell biology, biochemistry, and structural biology analyses. Native kinetochore sub-complexes will be purified from various cell cycle stages. The constituents and overall structure will be analyzed by mass spectrometry and fast scanning AFM, respectively. The structural basis of kinetochore complexes including CENP-T/W will be analyzed by X-ray crystallography and NMR. Domain structure and solution dynamics will be analyzed by fast scanning AFM. CDA award will allow me to challenge into the new field of chromosome biology based on my structural biology background.

Visual search is a common task in our everyday life. We search for our child in a crowd, or a chocolate bar out of several different brands. The ease of the search depends on how distinguishable the target is from the background and how different it is from other surrounding objects. Attention can improve our search. Visual search, attention, and the neuronal circuitry underlying them are very dynamic fields in human psychophysics. But to understand the minimum neuronal hardware necessary to solve such tasks, we here turn to an animal that is becoming a model for analysing how simple nervous systems solve complex cognitive tasks: the bumblebee.
Interestingly, the common tests run by psychologists often seem more appropriate to a bee's world than to a human's. Subjects are asked to search a display for one or several targets (e.g. a yellow square). The targets are commonly mixed with other stimuli (distractors), which differ from the targets. They may differ in one stimulus dimension only (e.g. color), or in several dimensions (e.g. color and shape). Performance of subjects is evaluated in terms of reaction time and accuracy, and in terms of individual strategy used to optimize the speed-accuracy tradeoff.
We will use a novel technique, the virtual bee meadow, to ask a range of questions dealing with visual search and attention in bumblebees. How do searches vary if the targets and distractors differ in more than one dimension? How does attention affect the perception of 'irrelevant' details? How do bees cope with multiple visual targets? Is attention more influenced by the nature of the stimuli or by mental processes in the bee's brain?

mRNA localization coupled with regulated translation is a fundamental mechanism for regulation of gene expression in eukaryotic cells. Local protein expression requires translocation of mRNAs to specific sub-cytoplasmic loci. In neurons, mRNAs are transported along with RNA binding proteins (RNPs) and ribosomes in multi-megadalton complexes called RNA granules. These RNA granules move from the cell body to dendrites along microtubules with the help of motor proteins. Efforts at imaging dendritically localized mRNAs and purifying mRNA granules have revealed key components of granules. However, the mRNA composition of individual neuronal RNA granules remains elusive. In particular, it is completely unknown whether mRNAs randomly associate to form granules, or whether specific mRNAs (e.g., those encoding the individual subunits of a multiprotein complex) are intentionally packaged together to allow for highly coordinated coexpression. Here I propose to develop a new method to elucidate the mRNA composition of neuronal granules. This method involves highly specific pull-down of an individual mRNA, followed by deep sequencing to identify physically associated mRNAs. Detected associations will be confirmed by fluorescence in situ hybridization (FISH), which should also yield information as to where in the cell the mRNAs first associate. Bioinformatics will be used to predict the cis-acting elements leading to co-association of specific mRNA components. Together, these studies should provide significant insight into both the mRNA composition of neuronal RNA granules and the more general processes of mRNA localization and local translation.

Recent genome-wide expression analyses have led to the unexpected finding that most of our genome is transcribed, generating a large heterogeneous pool of non-coding RNAs (ncRNAs). While the indispensability of small ncRNAs is unquestioned, very little is known about the biological functions of long ncRNAs (lncRNAs). The goal of my postdoctoral research is to functionally characterize lncRNAs during early vertebrate development, using zebrafish embryogenesis as in vivo model. Zebrafish lncRNAs will be identified in collaboration with Drs. John Rinn and Aviv Regev (Broad Institute, Cambridge, MA), using a combination of bioinformatic and experimental approaches. This strategy, employed in a candidate approach, has already allowed me to discover two novel, putative lncRNAs that are genomically linked to Nodal signaling pathway components. A detailed functional characterization (gain- and loss-of-function experiments) of these two candidates promises not only to uncover a previously unknown role of lncRNAs in regulating Nodal signaling and embryogenesis, but will also serve as proof-of-principle for my subsequent genome-wide analysis of lncRNA function during zebrafish embryogenesis. Selection of interesting candidates for in-depth functional characterization will be based on multiple criteria, including conservation, genomic context and early embryonic/Nodal-induced expression. This project is significant and innovative because it will comprise the to date first systematic analysis of lncRNA function during vertebrate embryogenesis. As such it has the potential to provide novel insights into RNA-mediated regulation during development.

Drug resistance is a major problem in diseases such as cancer, malaria and HIV and despite strategies to avoid resistance (such as combination therapy) resistance still occurs and many questions remain. One question is why non-adherence (when a patient doesn't takes his drugs as prescribed) sometimes increases and sometimes decreases the risk of evolution of resistance. In principle, non-adherence can both slow down the evolution of resistance, because selection pressure decreases, or it can speed up the evolution of resistance, because the evolving population (e.g., the virus population) will grow, leading to more genetic variation on which selection can act. Whether the net result of non-adherence is slower or faster evolution must therefore depend on factors such as drug or virus characteristics, but it is not yet understood how.
HIV is an ideal model to study the effect of the level of adherence on the evolution of drug resistance for two reasons: 1) because a patient usually gets infected with HIV once and patients stay infected for life. An HIV strain therefore evolves within a patient for many years, independently from HIV strains in other patients. Every patient therefore represents an independent evolutionary history of a viral population. 2) The second reason for studying HIV is that there are good data available on patient adherence, on drug characteristics, and resistance mutations. I will combine stochastic modeling approaches and data-analysis to reach a better understanding of adherence-resistance relationships. The results will allow for design of better treatment strategies.

The phosphoinositides PI(3,4)P2 and PI(3,4,5)P3 regulate apical-basal and front-rear cell polarity during chemotactic migration, respectively. Cells migrating collectively within cohesive tissues fine-tune both types of polarity in response to chemical and mechanical signals. While it is likely that dynamic relocalization of phosphoinosites modulate polarity changes within migrating epithelial tissues, this topic has yet been addressed. The aim of this proposal is to evaluate the distribution and function of PI(3,4)P2 and PI(3,4,5)P3 in the zebrafish lateral line primordium, a migratory tissue composed of epithelial cells. The cells are heterogenous in their polarity with cells becoming more apicobasally polarized the farther they are from the leading edge. In our proposal, we plan to localize PI(3,4)P2 and PI(3,4,5)P3 in CldnB::lynGFP zebrafish by using fluorescent-tagged proteins or probes. The dynamics of the lipids will be detected by digital scanned light sheet microscopy, which will provide 3-dimensional distribution of the lipids during the migration and differentiation of the primordium. Furthermore, we will evaluate the function of these lipids in regulating cell polarity by mislocalizing their expression, chemically inhibiting PI3K or PTEN or introducing exogenous PIPs. Finally, we will investigate whether lipid rafts have a role in restricting PIP localisation during the development of the primordium.

Alzheimer's disease (AD) is a form of senile dementia, featuring the formation of fibrillar amyloid deposits as the main neuropathological factor. The proteinaceous constituents of AD plaques are fibrils formed by the amyloid-beta-peptide (Abeta). The neural dysfunction correlates poorly with the amount of fibrillar deposits, whereas it correlates better with the amount of soluble diffusible Abeta oligomers, hence the latter were suggested to play a special role in the onset of AD. So far the oligomers were characterized mainly morphologically using microscopy, while the amount of molecular level information, giving insight into the oligomer structure, is limited. Such information is essential for understanding their role in fibrils assembly, their toxic activity and pathological significance in the onset of AD. In this work we propose to study the structure of the amyloid-beta peptide oligomers and their pathway to fibrils assembly using solid-state NMR enhanced by dynamical nuclear polarization at low temperatures. Such an approach will allow us to take advantage of the enhanced NMR signals, and to study thereby the intact oligomers without necessity of lyophilization. First, we plan to follow the progress of a monomeric Abeta aggregation, by identifying and characterizing the intermediates. Also we plan to study the isolated oligomers and correlate their structures to those found in time-resolved experiment. The suggested approach will help to determine the stage when structural features of Abeta fibrils are formed, define off-pathway intermediates, and correlate between toxicity and specific structural features.

Survival depends on generating appropriate responses to the barrage of continually changing sensory stimulation from the world. To accurately and efficiently encode the world, populations of sensory neurons could dynamically adapt, matching their sensitivity to prevailing environmental conditions. Concentrating neural sensitivity on recent stimuli should enhance behaviors that depend on detecting or identifying stimuli in the prevailing range. A classic example is luminance adaptation, which matches the sensitivity of retinal cells to recent light levels, facilitating vision under lighting conditions ranging from starlight to sunshine.
This project addresses 2 major gaps in our understanding of neural and perceptual adaptation. First, I will study the timescales and stimulus-dependence of changes in neural tuning and perceptual sensitivity: it is unclear if they are affected by the full distribution of stimulus statistics over many minutes, or only the mean stimulus over recent seconds. Second, I will attempt to reconcile neural and perceptual adaptation: it is poorly understood what aspects of neural tuning underlie perception and over what timescales neural activity is informative. I hypothesize that neural tuning optimally incorporates recent stimulus statistics, maximizing sensitivity and improving perceptual performance. This work has important implications for understanding how sensory neurons represent the world and how their responses are decoded to inform perception and guide behavior. Further, it examines the broad issue of how a brain can accurately represent a changing world when the underlying neural sensitivity is continually changing.

Two key challenges in biology are to understand how biological forms are generated, and to elucidate the basis for their diversity. We propose to address these problems by studying the development and evolutionary diversification of leaf forms. To this end, we will integrate developmental genetics and computational modeling to compare, contrast, and put in an evolutionary context the mechanisms that shape the simple leaf of the model plant Arabidopsis thaliana and the compound subdivided leaf of its close relative Cardamine hirsuta. In addition to being taxonomically related, these plants show exceptional genetic tractability, thus providing excellent opportunities for comparative studies of leaf development and diversity.

We will characterize the spatiotemporal expression of key leaf-shape regulating genes and the activity of auxin during leaf development, and the influence of these genes on cell division, tissue growth and vein patterning. On this basis, we propose to devise models of A. thaliana and C. hirsuta leaf development. These models will integrate molecular-level signaling, the pattern of growth and division of individual cells, mechanical interactions between cells, and vein pattern formation. We will iteratively test, improve and validate the models by comparing them with experimental data for A. thaliana, C. hirsuta, and their mutants and transgenic variants. To conceptualize the diversity and evolution of leaf forms we will construct a morphospace with coordinates reflecting the activity of selected genes.

We expect to gain an understanding of the development, diversification, and evolution of leaf shapes in mechanistic terms including genetic regulation, developmental signaling, cell division and differentiation, and tissue growth. We will also advance the state of the art in plant modeling by introducing the propagating-boundary method to simulate development of organs, devising a representation of cellular structures developing in 3D, and modifying finite-element methods to efficiently capture the dynamics of growing 2D and 3D tissues and structures. All aspects of the proposed research have a collaborative, interdisciplinary character, bridging developmental biology and computational modeling.

One striking feature of the mammalian heart is that its form is dictated to a large degree by function. To connect functional need with ongoing patterning, chamber and conduction system formation and growth, cardiac developmental pathways engage complex feedback loops and cross-regulation. My project aims at utilizing Systems Biology tools to reconstruct the mammalian cardiac gene regulatory network (GRN) to unravel these complex interactions leading to proper heart formation.
First, I will obtain the network components by mining datasets from genome-wide interrogation experiments performed in the laboratory and from publicly available data. From this information, I will model the heart GRN and its dynamics and will extract network motifs to unravel the role of novel players involved in cardiac development. This model will be used to predict the network behavior upon alteration of key components and these predictions will be experimentally verified. I will compare these alterations to known mutations associated with congenital heart disease. Finally, I will tackle heart GRN evolution by cross-species comparison of this network in mouse, fish and flies.
This work will provide a significant input into deciphering the mammalian heart GRN and will pioneer systems level approaches to the understanding of heart diseases.

Recognition by T-cell receptors (TCR) of peptide fragments (p) presented on the surface of a target cell by major histocompatibility complexes (MHC) is a critical step in the immune response of higher organisms. Formation of a TCR-pMHC complex results ultimately in killing of the cell and the molecular recognition events taking place upon complex formation is of great interest in immunology and medicine, in particular, autoimmune and cancer therapy. However, TCR-pMHC complexes are not readily accessible to structural analysis by most experimental techniques due to strict sample requirements. The proposed project will design a novel computational modelling procedure that uses hydrogen/deuterium exchange (HX) data to refine molecular model building to accurately determine three-dimensional structures of unknown TCR-pMHC complexes. Such HX data can be rapidly generated with very modest sample requirements by a newly introduced technology. The developed computational algorithm will be used to generate much needed accurate structural models of TCR-pMHC complexes. Binding patterns that govern recognition of specific epitopes by selected TCRs will be revealed to enable a general molecular understanding of the formation of TCR-pMHC complexes that are critically important for the immune response during normal function, autoimmune disease or cancer. Furthermore, the applications of the developed protocol should extend to a wide range of other biologically important proteins and protein complexes similarly not readily compatible with traditional techniques for protein structure determination.

A major question in developmental neurobiology research is the understanding of the mechanisms that instruct the wiring of the circuits that perform specific functions. Direction selective ganglion cells (DSGC) are sensitive to object motion in one direction, giving larger responses to motion in the preferred direction versus motion in the opposite, or null, direction. The mechanisms that guide the establishment of direction-selective circuits are unknown. We aim to reveal the age at which the circuits are first established in mice, and whether their maturation requires neural activity. Prior to the maturation of visual responses, the retina is spontaneously active, exhibiting retinal waves, which are bursts of correlated activity that propagate across the retina. Using transgenic mice in which single subtypes of DSGC express GFP, we will determine whether DSGC exhibit directional responses to retinal waves, firing more robustly to waves propagating in the preferred direction over others. This finding would indicate the age at which the first directional responses emerge. Next, we will determine whether the directional preference of DSGC in juvenile mice can be "trained" to respond to a new highly overrepresented directional stimulus. This finding would indicate that visual experience plays a role in establishing the direction-selective circuit. The results will provide important insights into the role of neural activity in the establishment of functional visual neural circuits.

Adaptation from standing genetic variation is central to the evolution of phenotypes, yet its dependence on the molecular details that connect genotype, phenotype, and fitness is a major unsolved problem in genetics. The effect of recombination, in particular, is expected to depend on the extent of genetic interaction among alleles. While higher rate of recombination facilitates combinatorial exploration of the genotypic space, in the presence of interaction it also has a detrimental effect of breaking up synergistic combinations of alleles. We propose a multidisciplinary investigation of the roles of recombination and genetic interactions. We will study laboratory adaptation of C. elegans populations that start with high genetic variability and are engineered to have different rates of inbreeding and outcrossing. The project integrates experimentally determined genome-wide genotype distributions, multidimensional phenotype (gene expression) measurements, and fitness data across 100 generations of culture in laboratory populations that differ in their rates of outcrossing. This integration forms the empirical test bed for theoretical models of the role of genomic architecture in adaptation. In particular, we will test the predicted positive correlation among non-additive genetic effects and levels of linkage disequilibrium across the genome. The proposed work combines population genetics and high-throughput molecular biology approaches with statistical physics modeling methods, and aims to bridge population genetics with systems biology.

The heat shock response (HSR) is a powerful transcriptional program which acts genome-wide, through the induction of specific heat shock proteins (HSP) such as HSP70 and HSP90, as well as by re-shaping global cellular pathways controlling survival, growth and metabolism. In mammals, this ancient response is regulated primarily by the evolutionary conserved Heat Shock Factor 1 (HSF1) transcription factor. Surprisingly, recent work from the Lindquist lab has shown that the beneficial effects of HSF1 known to enhance the survival of organisms under stress come at the cost of facilitating the initiation and maintenance of cancer. Acting globally, HSF1 function permits cancer cells to survive the drastic imbalances in signaling and profound alterations in DNA, protein and energy metabolism that occur during transformation. Indeed, ablation of HSF1 expression using genetic techniques, while well tolerated in normal cells and even in whole animals under physiological conditions, is detrimental to malignant cells. Data from the Lindquist lab indicates that modulation of the activity of HSF1 affects not only the HSR, but also the redox state of the cell, and the antioxidative stress response. I intend to exploit my strong background and expertise in redox biology, biochemistry and imaging, as well as my understanding of transcriptional regulation, to understand the connection between HSR, reactive oxygen species and cancer. I plan to: 1) explore the role of redox regulation of HSPs, particularily HSP60, in the proliferation of human cancer cells, and 2) dissect the transcriptional components of redox regulatory responses driven by HSF1 in cancer.