The dynamics of hippocampal place cells changes dramatically according to the behavior of the animal. During a run through a cell’s place field, spikes are emitted at progressively earlier phases of the underlying theta rhythm. During awake immobility and slow-wave sleep, rapid orderly activation of place cells expresses a possible or experienced trajectory of the animal (replay). When the animal transiently stops at turning points in a T-maze, sweeps of place cells activity probes all possible routes from that point. Moreover, non-spatial context experienced during running contributes to the encoding and long-term storage of newly acquired memories following sleep. Common mechanisms underlying these dynamical properties are poorly understood and cannot be accommodated by existing network models. We propose that such a diverse range of behaviors can be explained by endowing a network, storing a map of the environment in its recurrent collaterals, with short-term synaptic depression. We shall combine theoretical analysis, model-based experiments and data analysis to (i) test the hypothesis that short-term synaptic plasticity plays a fundamental role in the diverse hippocampal regimes of activity (ii) investigate how non-spatial cues (e.g. auditory or olfactory) triggers and affects the replay events in coordination with cortical inputs to the hippocampus. The interaction between this novel theory of hippocampal dynamics and experiments will provide a deeper understanding of spatial coding and memory mechanisms in the hippocampus.

A long standing problem in evolutionary biology is determining of the mechanisms that produce developmental divergence and genetic incompatibilities [1]. Several approaches have been established to address this problem: a) identification of loci responsible for performance differences in F1 offspring [2-4], b) expression analysis in F1 hybrids to quantify regulatory additivity [5-6] or the location of regulatory differences (cis/trans) [7-8], and c) comparative analysis of conserved traits in distantly related species which often shows divergence on gene function [9-10]. Although these three approaches focus on understanding species differences at various levels, it is unclear how they relate to one another. Here a novel integrated approach is proposed, covering the complexity of phenotypic levels (transcriptome and morphology) and comparing micro and macro evolutionary time scales. We study fruit development, a trait that can be related to fitness in plants. In a microevolutionary context, the fruit transcriptome will be studied in F1 hybrids of closely related lineages and analyzed for cis/trans regulatory differences and additivity, while fruit features will be quantified using morphometrics. This will be done both within Arabidopsis accessions (conserved fruit morphology), as well as tomato cultivars (divergent morphology). This data will allow a functional macroevolutionary comparison in the context of gene expression additivity and cis/trans regulatory differences within their known developmental networks. The proposed study will provide insights into the evolution of traits and their underlying developmental networks between closely and distantly related lineages.

Multipotent stem cells employ unique histone modification mechanisms to confer their ability for rapid and divergent cell fate decisions upon environmental cues. However, it remains unclear how histone epigenetic modifications work in largely quiescent tissue stem cells to modulate fate determination. Recently, Dr. Tumbar’s laboratory found that quiescence is a state in which hair follicle stem cells (HFSCs) are assigned a self-renewal or differentiation/cell death fate. Co-incidentally, at this stage histone repressive marks such as H3K27me3 are globally erased, and histone methylases mRNAs, such as Ezh2, are down-regulated. Here I am hypothesizing that the low level of epigenetic marks of tissue stem cells in quiescence confers them plasticity for subsequent flexibility in fate acquisition. To test this, I will transiently induce Ezh2 expression and block removal of epigenetic marks at two stages: (1) in quiescence, prior to fate decisions; (2) in proliferation, when hair follicle stem cells self-renew their pool. If erasure of the H3K27me3 marks were important at the quiescent but not the proliferative stage, the first but not the second Ezh2 induction would impair stem cell differentiation. In addition, ChIP sequencing analysis will reveal gene loci regulated in this process, with predicted impairment of de-methylation in differentiations specific genes upon expression of Ezh2 in quiescence. These approaches will test a model in which the recognized quiescence of tissue stem cells is functionally coupled with tissue stem cell plasticity and fate acquisition in tissue homeostasis.

AMPA, kainate and NMDA are members of the glutamate receptor family of ligand gated ion channels, these receptors are activated by glutamate the mayor excitatory neurotransmitter in the central nervous system and the presence of this receptors in the brain is essential for normal brain function, and higher order nervous system process including learning, memory and pain perception. Basically the members of this family form tetramers and each subunit is formed by four transmembrane domains, intracellular caboxy-termini and a extracellular amino-termini which form the ligand binding domain (LBD). The LBD are divided in in two lobes, the D1 and D2, and the movement of this lobes during the gating allows the channels to transit between active and desensitized state. The rapid kinetics of glutamate receptors enable them to follow the release of glutamate at rates approaching several kilohertz, but the transition between the active and desensitized states of the channel, limits their temporal sensitivity and underlies some forms of short-term depression. This type of synaptic depression contributes to neuronal computation at some synapses. However, these receptors have complex structures and differences on the conformations of activated and desensitized states is unknown. We hypothesize that there exist structural differences between those receptors on the activated and desensitized states who determine this differences on kinetics. In this application we propose a series of chemical, electrophysiological and structural experiments to find the structural determinants of activation and desensitization on AMPA and kainate receptors.

MicroRNAs (miRNAs) act as regulators of development and physiology through their ability to regulate the expression of most cellular genes. Although individual miRNAs can bind to up to a hundred of target genes, it can only bind to one RNA target at time. Thus, miRNAs that have a larger repertoire of target genes will down-regulate each individual target gene to a lesser extent than miRNAs with fewer targets. Since a single miRNA’s effectiveness is influenced by the concentrations of its target genes, it follows that the altering the expression levels of individual targets will have repercussions on all the other targets of that miRNA. Such alterations are exemplified by those commonly observed in cancer where oncogenes and tumor suppressor genes are frequently up-regulated and down-regulated, respectively.
In this proposal, it is hypothesized that any given RNA transcript, through its ability to compete for miRNA binding, can regulate the expression of other RNAs, in trans. Moreover, these non-coding functions of endogenous RNA transcripts have evolved as physiologically and pathologically relevant factors in multicellular organisms. The goal of this research proposal is to continue the characterization of the cellular relevance and physiological importance of RNA:miRNA networks by using complementary in vitro, in vivo and rational experimental or bioinformatic approaches. The investigation of these processes will shed light on novel and untapped regulatory mechanisms within the cell. Knowledge of such processes will permit the development of new means and methods to alter gene expression for therapeutic purpose and/or scientific discovery.

Ischemic heart disease associated with acute loss of cardiomyocytes is a major cause of heart failure. While an ideal cell-based therapy to replace the lost cardiac muscle has not yet emerged, recent reports on factors able to stimulate the proliferation of mature cardiomyocytes, which normally have little self-renewal capacity, has stirred hope for new therapeutic approaches. An inflammatory microenvironment is deleterious to cardiac repair by affecting cardiomyocyte survival. Macrophages in particular play a major role in the inflammatory response. However, macrophages constitute a heterogeneous population and their roles in cardiac repair are not fully understood. Here, I propose experiments that will define the function of distinct macrophage subtypes in cardiac regeneration at cellular and molecular levels by exploiting the zebrafish, a model organism that has a well-developed immune system and can regenerate its myocardium. First, I will characterize the heterogeneity of fish macrophages with a high-resolution expression profiling technique. Second, I will generate new transgenic lines that will allow monitoring of the recruitment and phenotypic alterations of each macrophage subtype in the injured heart. Third, I will employ cell-type specific inducible ablation and gene expression approaches to address the requirement of each macrophage subtype during the multistage process of cardiac regeneration. Finally, I will generate differential expression profiles of macrophages in healthy and injured heart to identify candidate molecules that modulate the regenerative ability of cardiomyocytes, and I will analyze their contribution using loss-of-function approaches.

Intrinsically disordered proteins play crucial roles in many biological processes. In paramyxoviruses such as Sendai and measles virus, transcription and replication require an interaction between the disordered tail of the nucleoprotein (N) which encapsidates the genomic RNA and the C-terminus of the phosphoprotein (P), a cofactor of the core polymerase L. The molecular mechanisms of protein recognition and interaction in the absence of a folded structure are not well understood, and it is not clear how interaction signals can be transmitted to distant effector domains via disordered sequences. To shed light on these phenomena, a multidisciplinary structural biology approach will be employed, applying solution-state NMR in conjunction with small-angle X-ray and neutron scattering, solid-state NMR and molecular modeling to the protein domains in isolation as well as in complex and in the context of the full-length nucleocapsid. The aim of this project is to obtain a complete picture of structure and dynamics in the intrinsically disordered regions of the N and P proteins of Sendai and measles virus. Their highly dynamic nature will be accounted for by providing a description in terms of explicit and statistical ensemble properties.

The vast majority of studies in systems neuroscience focus on the brain’s response to a stimulus or task. However, the brain is very active even in the absence of explicit input or output. This intrinsically generated activity accounts for most of the brain’s energy usage and displays a highly coherent spatio-temporal structure that is altered in a number of brain diseases, yet its role in brain function remains largely unknown. In particular, its impact on the neural processing of stimuli and its importance for behaviour are not yet understood. I intend to study this spontaneous brain activity measuring local field potentials from large multi-electrode arrays in combination with single-unit recordings in awake monkeys. The research revolves around 4 key goals. First, I will analyse the spatio-temporal structure of spontaneous activity at the scale of the entire brain, while the monkeys are at rest. Second, I will determine the precise nature of the interaction between spontaneous activity and activity evoked by stimuli, while the monkeys are performing a simple task in which stimuli are repeatedly presented. Third, by manipulating the monkey’s task expectations, I will test the extent to which the structure of spontaneous activity reflects expectations about future stimulus events. Lastly, I will complicate the monkey’s task even further to study the role of spontaneous activity in determining the monkey’s behaviour. For the data analysis of these studies I will apply newly developed computational methods, in collaboration with a computational neuroscientist. Taken together, these studies will yield important insights into how and why the brain is active spontaneously.

Angiogenesis is a critical process in vascular development and diseases such as cancer and ischemic coronary artery disease. Angiogeneic growth factors stimulate proangiogenic signals of endothelia cells (ECs) via ligand-induced dimerizations of receptor-tyrosine kinases (RTKs) and modulate several key processes in ECs including proliferation, migration, and assembly. Despite remarkable advances in this field, however, it is still unclear how these biomolecular regulators coordinate highly complex phenotypic events such as vascular sprouting, migration, branching, and network formation. To understand molecular mechanisms of angiogenesis with high spatio-temporal complexity, it is required to monitor angiogenesis in real-time at the single molecule level with multivariate analytical capability. Here, I propose a research program that investigates molecular mechanisms of angiogenesis at the single molecule level via in situ surface plasmon dark field microscopy. Unlike conventional microscopy that only provides a static assessment of cell status, the proposed surface plasmon microscopy allows us to visualize single molecule events of the signaling molecules continuously over the entire angiogenesis process as well as morphogenetic development. With this new technique, specifically, I will challenge the following topics: 1. In situ monitoring RTK dimerization of individual ECs and its correlation analysis with distinct morphogenesis (migration, branching, and proliferation). 2. Focused angiogenic signal activation of a single cell and its communication with surrounding cells. 3. Establishment of an artificial magnetic tweezer system for directed neovascular development.

Coral reefs are ecologically and economically important marine ecosystems that are currently under threat as a consequence of environmental degradation, global climate change and emerging diseases. An important element in determining the health of corals is the stability and function of the coral 'holobiont', the complex and diverse community of microorganisms living in partnership with the coral host. Shifts in the balance of the holobiont community composition can lead to coral disease and bleaching. However, studying the mechanisms behind these shifts is hindered by the physical and biochemical complexities of the coral holobiont. To overcome these obstacles we will for the first time apply microfluidic technology to create a "Microfabricated Coral Holobiont" (MCH). This simulated microenvironment will replicate the key physical, chemical and biological features of the coral holobiont and will represent a powerful new experimental platform to study the ecology of the coral holobiont within a controlled setting in the laboratory. We will couple this system with videomicroscopic measurements of microbial behavior and a suite of specific cell stress response probes to examine the hypothesis that specific chemical cues produced by different members of the coral holobiont can favor or impede the colonization of pathogenic bacteria involved in coral disease. We will use a second novel microfluidic-based tool, the "In Situ Chemotaxis Assay" (ISCA), to investigate the chemical ecology of natural coral-associated microbial communities within two important coral reef ecosystems, the Great Barrier Reef (Australia) and the Red Sea (Eilat, Israel). Coupled with modern molecular tools (metagenomics), the ISCA will allow for the first in situ measurement of microbial behavioral responses to chemical cues released by different compartments of the holobiont. The interdisciplinary research team is uniquely positioned to tackle this challenge, as it brings together expertise in nanotechnology, engineering, fluid dynamics, biochemistry, molecular biology, microbial and chemical ecology and oceanography. The new approach proposed here will deliver novel insights into the cellular mechanisms behind coral disease, which will ultimately aid in the management of coral reefs and in the preservation of their ecological, economic and societal value.

Decision making spans a vast range of types and complexity, from choosing your partner to deciding whether to dive left or right to save a goal. Progress in a variety of fields has led to an understanding of the mechanisms that evaluate evidence and reach a decision. Studies of simple decisions have provided insight into the neurobiological mechanisms responsible for decision making in both monkeys and humans. These studies often require a choice between two possibilities, such as leftward or rightward motion of dots moving on a screen. Human and nonhuman primate experiments support a mechanism in which a decision is made when the accumulated evidence reaches a criterion level. Such an accumulation process explains both the accuracy of the decisions made over a range of difficulty levels as well as the time required to make each decision. However, a decision in the absence of an action (or the withholding of an action) has little value, and in recent years there has been substantial progress in understanding how a task goal determines the actions that will be made. In this new framework, the movement system sets up a feedback rule that determines how the commands we send to our muscles should depend on sensory inputs we receive, so as to optimizes certain criteria such as a tradeoff between effort and accuracy.

Traditionally, decision making and movement-control were believed to operate separately and sequentially, but recent evidence from cognitive psychology and neuroscience suggests a more intimate connection. A fascinating implication of this connection is that it can explain why, in the absence of additional information, a decision-maker might change his or her mind. We have recently developed a task in which we can monitor confidence in a decision and also changes-of-mind, showing that once the brain reaches a decision and commits to a choice, it can continue to analyze evidence that was in the pipeline but unavailable to impact the initial decision. Our project extends and unifies the fields of decision making and movement control with a combined behavioral, neurophysiological and theoretical approach within a common conceptual framework. Our hypothesis is that a common framework applies to the revision of both actions and ideas, and we will develop a theory of decision making and control that extends to the acts of vacillation and self-correction.

Antigen-specific B-cells form germinal centers (GC) in the lymph node follicles 1-2 weeks after antigen challenge. GCs function to generate B-cells bearing higher affinity antigen receptors, which subsequently differentiate into memory B-cells and plasma-cells. This process, termed affinity maturation, includes random hypermutation of the BCR genes that results in altered affinity for the BCR. The precise mechanism by which high affinity B cells get selected among a pool of cells bearing a range of affinities is still unclear. In GCs B-cells engage and communicate with other cells like T-helper and follicular dendritic cells (FDCs). FDCs present antigen to B-cells in form of immune complexes, whereas bone marrow derived dentritic cells may present antigen to follicular T-cells. GC is an open structure which ensures that rare high affinity B-cells will continuously enter and participate in antibody responses. The goal of the proposed work is to elucidate the immune cellular communications that govern the formation of GCs. We will use imaging in conjunction with molecular genetic and cellular immunology technologies to investigate the cellular interactions during maturation of GCs in vivo. We will use models based on knock-in mice expressing low and high affinity BCR specific for nitrophenol haptens. This antigen can be attached to ovalbumin to elicit T-cell help from endogenous Ova-specific T-cells. Cellular interactions and antigen uptake will be analyzed and tested for their role in GC formation and maintenance.

Cell differentiation during development is driven by the action of signaling molecules. Although the molecular components necessary for transduction of the differentiating signals have been described extensively, the way these signaling cascades allow the cell to integrate multiple, sometimes contradictory signals remains largely unknown. The TGF-beta pathway is central in development and composed of two antagonistic branches (BMP and TGF-beta). It controls the differentiation of the two cell lines we will study (cultured myoblast C2C12 and human Embryonic Stem Cells RUES 1 and 2 lines). We will use a microfluidic automated cell culture chip allowing precise temporal control of extracellular environment, combined with time-lapse fluorescence microscopy to follow the real time response to stimulation by TGF-beta ligands, by recording both the nuclear-translocation of the Smad transcription factors and subsequent gene expression. We will monitor the real-time pathway response, as a function of parameters like the intensity or the time course of signaling and the timing and relative proportions of antagonistic signaling (BMP vs TGF-beta). We will also consider the importance of cell-cell communication and pathway induced inhibitors. We expect that these experiments, combined with mathematical modeling of the pathway, will teach us how cells encode differentiating signals.

The functions of immune cells are governed by the intracellular calcium (Ca[2+]) levels in conjunction with other signals. Ca[2+] entry into cells is accomplished through specialized calcium release activated Ca[2+] channels (CRAC channels) which consist of two membrane proteins, namely, stromal interaction molecule 1 (STIM1) and CRAC modulator 1 (CRACM1 or ORAI1). STIM1 is an endoplasmic reticulum (ER) resident protein with a calcium sensor gating Ca[2+] entry, and ORAI1 is a plasma membrane (PM) channel protein. Cellular studies have shown that regulation of the CRAC channel is dictated by the dynamics of STIM1 and ORAI1 movements to sites of ER-PM apposition and by direct interaction of STIM and ORAI proteins. However, several important parameters have not been accessible for quantitative study in cells. The proposed research aims to address this biological question by modeling the close apposition between PM and ER using lipid vesicles tethered to a surface or a supported lipid bilayer. The dynamics of STIM1 and ORAI1 movements and CRAC channel activation in this rigorously controlled environment will be fluorescently visualized with an approach using microfabrication technology, protein engineering, and optics. The influence of critical parameters such as the concentrations of STIM1, ORAI1, tethering proteins, Ca[2+] ions and the constituents of the lipid bilayers on the kinetics of STIM-ORAI complex assembly and channel activation will be examined in detail. The study will provide a detailed accounting of factors controlling store-operated Ca[2+] entry under physiologically relevant conditions.

The first lineage specification in mammals occurs at blastocyst stage, when cells are set aside from pluripotent embryonic cells to make placenta and other supporting tissues for the fetus. In the mouse, trophoblast stem (TS) cells are derived from blastocyst and later give rise to the majority of the placenta. By understanding the genetic regulation of TS cells, we can gain greater insights into the causes behind pregnancy issues resulting from trophoblast deficiencies. Furthermore, as TS cells and embryonic stem (ES) cells are both derived from the blastocyst, understanding the genetic network preventing ES cells from becoming TS cells would allow better control of pluripotency, and improve efficiency during induced pluripotent stem cells production. Building on previous studies of genes necessary for TS formation and maintenance, we will undertake genome-wide approaches to identify the core TS regulatory networks and their interactions with the pluripotency networks. We will mine this dataset for putative novel regulators of TS cell development and differentiation, and test their functions in vitro and in vivo. Mouse TS cells is currently uniquely positioned to provide new insights relevant to human stem cell development and reprogramming, since lineage decisions in different blastocyst cell lines can only be compared and contrasted in the mouse. Furthermore, we can generate transgenic animals to assess in vivo functions of novel regulators to address their importance during trophoblast and placental development. These studies will provide initial data on which to build interaction networks that drive lineage development in both mouse and human development.

In the proposed project, I aim to identify novel determinants for cell polarity and understand the mechanisms regulating polar localization of plasma membrane proteins in plants.
PIN proteins are polar localized transporters for a plant hormone auxin. PIN polarity changes dynamically according to endogenous and external positional cues, and triggers developmental responses. New components regulating polar targeting of PIN proteins will be identified by molecular genetic approach using Arabidopsis mutants with defects in PIN protein endocytosis or exocytosis.
In the aerial tissues, cuticle is specifically deposited on the surface or organs. Recently identified cuticle-precursor transporter PEL1 localizes to the outer side of the plasma membrane in epidermal cells. I aim to identify sequence-based determinants on PEL1 protein of ‘outer’ polarity by expressing series of mutated PEL1 in plants. To identify novel components regulating polar localization of PEL1, mutations and chemicals which inhibit localization of PEL1-GFP will be identified by screening. Identified mutants and compounds will be further used to identify target proteins. In addition, positional cues for cell polarity in shoot epidermis and cell-type specific trafficking regulators will be identified.
By these proposed approaches, I expect to identify new cell polarity determinants that are involved in polar localization along different axis (apical-basal, and outer-inner, respectively), gain insight into their developmental control and mutual regulation.

Over the past decade, noncoding RNA has been recognized as an important regulator in many developmental and essential processes. Preliminary indications suggest that especially long noncoding RNAs (lncRNAs) may have a prominent role in stem cell function and axial organization, two processes involved in regeneration.
In this project we will address the role of lncRNAs in adult regeneration using the emerging model planaria. Planarians are famous for their ability to regenerate missing body parts, and the process of regeneration is readily accessible to experimentation in vivo.
We will use an enriched cDNA library approach to identify planarian lncRNAs, and we will analyze their expression patterns with regard to regeneration. Next, we will use RNAi-mediated knockdown of lncRNAs in combination with several in vivo assays to determine the effect of lncRNA on regeneration potential. Based on their importance in the various regenerative processes, we will select several lncRNAs for detailed analysis of their mode of action using RNA pull-downs and ChIP-based techniques that will be developed for this purpose.
As planarians combine in vivo regeneration -which most other invertebrate models lack- with the availability of RNAi-screening technology to identify lncRNA function, they will be a powerful model to analyze the role of lncRNA in adult stem cell biology, and will provide us with new insights both in the regulation of regeneration and in lncRNA function.

The PRC1 and PRC2 complexes maintain Hox gene repression during development and also influence tumorigenesis. PRC1/2-mediated gene repression involves PRC2-mediated trimethylation of histone H3 at Lys 27 (H3K27me3) followed by PRC1-mediated chromatin condensation. In contrast, the MLL methyltransferases antagonize gene silencing and promote gene activation by trimethylating H3 at Lys 4 (H3K4me3). Recently, the H3K27-specific demethylase UTX was shown to be an integral subunit of the MLL3/4 complexes, suggesting a possible linkage between H3K4 methylation and H3K27 demethylation. However, the mechanisms underlying the potentially synergistic effects of MLL3/4 and UTX in countering the PRC1/2-mediated chromatin compaction and gene repression are poorly understood. To investigate this problem, this proposal will focus on the RARb2 (and HoxA2) gene locus that is induced by retinoic acid (RA) during cell differentiation. I will use a human EC cell line as a model in conjunction with powerful biochemical assay systems reconstituted with purified factors and recombinant chromatin templates. The specific aims are (i) to analyze dynamic changes in H3K4 methylation and H3K27 demethylation, along with recruitment of related factors, on the RA-induced RARb2 gene, (ii) to assess the function and regulation of factors in RA-induced RARb2 gene activation, (iii) to assemble a highly compacted chromatin template containing the natural RARb2 promoter, and (iv) to use the template in defined cell free systems to analyze the action mechanism of the MLL3/4-UTX complex in countering the H3K27me3- and PRC1/2-mediated RARb2 gene repression and in effecting activation of RARb2 transcription.

Protein synthesis in neurons is spatially and developmentally confined, to control processes as diverse as axon guidance, learning and regeneration. While neuronal RNA localization is extensively studied, little is known about mechanisms regulating the spatial and temporal distribution of ribosomes in neurons. Ribosomes are readily detected in cell bodies, dendrites and growing axons, yet are sparse in mature axons. Upon injury, ribosomes are again detected in axons. Hence, mechanisms should exist to promote or inhibit ribosomal trafficking to axons in response to developmental and external cues. The dynamics of neuronal ribosomes has hardly been explored in-vivo.
I will develop tools to monitor the distribution of ribosomes in the PVD neuron of C.elegans, which grows distinct processes at developmental stages that allow continuous imaging. I will then characterize axonal and dendritic ribosome dynamics in growing and mature processes. Special attention will be paid to the transition from growth to mature stages of the axon, when ribosomes are expected to be eliminated from the process. Ribosomal distribution will also be monitored in a model of axon injury, where localized translation plays a vital role in regeneration.
To understand the molecular mechanisms regulating ribosomal distribution in neurons, I will test mutations in candidate genes for an effect on ribosomal localization, and perform a forward visual genetic screen to identify new mutants. Subsequent mutant analysis will enable to delineate signaling pathways and the underlying cellular machinery that regulate the dynamic distribution of ribosomes in developing or regenerating axons.

Chloroplasts (Plastids) function as the main suppliers of cellular energy in plants. Recent studies have provided significant insights into the evolutionary origin of the plastid-dividing (PD) machinery: 1) The PD machinery is constructed by bacterial-derived and eukaryote-specific proteins, 2) the PD ring, the main structure of the PD machinery, is composed of PDR1-associated poly-glucan. However, the conserved chloroplast division mechanism is not fully understood throughout the plant kingdom. Moreover, some components of chloroplast division in higher plants are different from those in primitive eukaryotes. I would like to investigate which dividing mechanisms have been conserved throughout evolution and how these dividing mechanisms have evolved and changed in different lineages. I will mainly analyze the Arabidopsis PDR1 proteins and conserved unknown proteins identified by proteomic analysis of isolated PD machineries from Cyanidioschyzon merolae. I will analyze these proteins using the chloroplast division analytical methods established in Osteryoung’s lab. In addition, mitochondrial division is similar to plastid division in primitive eukaryotes; therefore, the results generated by my proposal could lead to an understanding of the conserved mitochondrial division mechanism. Further analyses might reveal how eukaryotic host cells engulfed free-living cyanobacteria and a-proteobacteria, which subsequently changed into chloroplasts and mitochondria, respectively. Thus, the results will shed light on the long-standing puzzle of how eukaryotes were established. Successful completion of this study will be indispensable for my career development.