Our goal-directed behaviors critically depend on top-down control, as represented by working memory to hold the relevant information in mind while ignoring the irrelevant. The prefrontal cortex (PFC) is known to play a major role in top-down control of sensory processing. The basal forebrain (BF) also contributes to the modulation of sensory processing through its cholinergic projections to the entire cortices. Since the PFC uniquely sends direct projections to the BF, it is hypothesized that this pathway may constitute a critical top-down control network for sensory processing. However, this hypothesis has not been tested because targeting specific neural pathways was technically difficult. I propose a new paradigm to selectively perturb the pathway from the PFC to the BF using optogenetics in rats. I will express Halorhodopsin (NpHR) and Channelrhodopsin-2 (ChR2) selectively in the PFC cells projecting to the BF, through two virus injections: one virus encoding a fusion protein WGA (wheat germ agglutinin)-Cre into the BF, and another virus encoding Cre-dependent NpHR and ChR2 into the PFC. As a result, only PFC cells projecting to the BF will express NpHR and ChR2. While the rat performs a visual working memory task, I will selectively inhibit and activate these PFC cells by optical stimulation, and simultaneously monitor the activities in the visual cortex and the BF using silicon polytrodes. This study can test how selective perturbation of the neural pathway affects working memory performance and neuronal processing in sensory cortical areas. It will provide a novel paradigm to assess the causal relationship between specific neural pathways and cognitive operations.

It is an open question how tumor cell heterogeneity is generated in vivo. The longitudinal (over time) imaging of the tumor development process in living animals is the most direct and straightforward approach to the problem. I propose to use the zebrafish system to perform long-term in vivo imaging, and to apply a novel optogenetic strategy for non-invasive and conditional gene induction. Combined, these tools offer a means to make endogenous single cells become oncogenic and traceable in a heritable manner. My experiments will extract quantitative parameters associated with cell behavior, morphology, and localization, providing novel insights into the properties of individual tumor cells descended from single defined cells. My experiments will introduce selected oncogenic signals into distinct subsets of epithelial cells, and the follow them over time to systematically evaluate the influence of oncogenic signals and initial cell types on heterogeneity. Cells showing distinct properties will be photoconverted during in vivo imaging, extracted ex vivo, and characterized by single cell-based gene expression analysis. The genes selectively regulated in the photoconverted cells will be further investigated by generating reporter models and light-induced over-expression (or suppression) models for revealing the expression dynamics and the molecular functions, respectively, of the target genes during tumor development process. With these new experimental tools, I will define tumor cell heterogeneity in terms of cell dynamics, and study the molecular mechanisms that play pivotal roles in individual tumor cells acquiring their specialized abilities.

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

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

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

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

Protein degradation has two basic functions: (i) to eliminate abnormal proteins and (ii) to limit the lifetime of specific proteins and thus make them sensitive to regulation that can rapidly alter their activity levels. The latter function is often utilized during development. While previous studies have shown that the ubiquitin-proteasome system (UPS) is required for proper development, little is known about mechanism that controls UPS in response to developmental cues. To understand how these machineries are assembled and recognize specific substrates during development, I will utilize C. elegans oocyte-to-embryo transition system, in which the degradation of meiosis specific protein MEI-1 occurs in response to oocyte maturation. E3 ubiquitin ligase (E3) and posttranslational modification of Cullin regulates MEI-1 degradation. Interestingly, some of these components have both nuclear localization and export signals (NLS and NES), suggesting that they shuttle between the nucleus and cytoplasm. Moreover, the subcellular localization of E3 and MEI-1 are distinct, suggesting that the compartmentalization of these components is developmentally regulated. To analyze E3 assembly and substrate recognition, I will visualize these complexes and modified Cullins using Proximity Ligation Assay (PLA) to obtain spatial and temporal information that is not obtainable with methods used in previous studies. Concurrently, I will analyze the effects of NLS and/or NES depleted subunits on E3 assembly and function.

Analyzing the molecular mechanism of timely regulated E3 assembly and target recognition will contribute to a broader understanding of the developmental control of UPS.

X-ray crystallography is one of the most powerful methods to reveal the structure and mechanism of biomacromolecules. However, some molecules, in particular many RNAs and ribonucleoproteins have properties unsuitable for crystallization: multiple conformation, structural flexibility as well as vulnerability to thermal or chemical denaturation. Engineering them for higher stability can improve their chances to be crystallized, but the engineering process is time-consuming, because it requires iterative expression and analysis of individual clones. We propose a synthetic biological approach to build a quick screen for crystallizable mutants. In this approach, the genotype and phenotype of a variant will be linked by co-segregation of its gene, encoded enzyme, and reaction system in a water-in-oil emulsion. This method would allow quick and efficient selection of variants with desired, high stability phenotype, which would then be iteratively screened for crystallization using high-throughput methods.

We will apply this approach to targets which contain RNA and have proven largely resistant to crystallization, 1) the RNA polymerase ribozyme, and 2) a ribonucleoprotein, telomerase. An artificial RNA polymerase ribozyme has been engineered by synthetic biological approach and is thought as a modern equivalent to the central ribozyme in the RNA world. Structural information of this ribozyme would greatly help to engineer it further, and to understand its function in mechanistic detail. Telomerase is a key enzyme of genome maintenance. A crystal structure of full-length human telomerase would serve as a basis for functional understanding of telomere synthesis and drug discovery

In the mammalian brain, dopamine (DA) transmission in the prefrontal cortex (PFC) is known to play an essential role in mediating executive functions such as decision making and working memory. How the DA signal exerts its influence differentially on distinct executive functions in the PFC, however, remains to be understood. In this project, I will tackle this question by applying optogenetic perturbation methods to mice performing a delayed response task that engages executive functions in the PFC. Although numerous studies have shed light on DA’s role in executive function in the PFC, the precise mechanisms by which DA modulates these functions remains poorly understood. By exploiting the high cell-type specificity and temporal/spatial precision of optogenetic methods, I will be able to avoid the issues such as poor cell-type specificity and/or the decreased temporal and spatial control of perturbations encountered in previous studies. In this project, I will first exploit the mouce’s superb capacity for olfaction by using a delayed-response task with olfactory cues. Second, I will be able to control neurons in the PFC and the ventral tegmental area (VTA) using channelrhodopsin-2 and halorhodopsin. Third, I will record and analyze the neuronal responses in the PFC during specific periods of the task, thus enabling direct comparison of the effect of DA signals on neuronal activity and on animal behavior under specific cognitive demands. By integrating these strategies, I will elucidate the differential effects of dopaminergic modulation on prefrontal executive functions such as working memory and decision making.

Faithful chromosome segregation is essential for the proliferation of all organisms. Although studies in model eukaryotic organisms have found that the kinetochore complex formed on centromeres facilitates their chromosome segregation, it is not known if this mechanism applies to all eukaryotes. To uncover novel principles of the molecular machinery that drives segregation, I will study chromosome segregation in Trypanosoma brucei because this protozoan parasite branched early in eukaryotic evolution and has various unusual features. Trypanosomes lack a centromeric histone H3 variant that is essential for kinetochore assembly in all other studied eukaryotes. Trypanosoma brucei possesses large chromosomes that have centromeres and small chromosomes that lack centromeres, yet both types of chromosomes undergo accurate segregation during mitosis. Very little is known about the molecular machinery that segregates these chromosomes. I propose to identify associated factors by PICh (Proteomics of Isolated Chromatin segments) and yeast one-hybrid assays. Identified proteins will be systematically characterized by GFP tagging and RNAi depletion analyses to test whether they have roles in chromosome segregation. I will also identify domains that confer mitotic stability of small chromosomes that lack centromeres. These efforts should lead to an understanding of the segregation machinery in this evolutionary distant eukaryote.

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.

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.

Gene alterations by translocation is a hallmark of most hematopoietic malignancies, and other aberrations in microRNAs and their target sites, or in promoter sequences have recently emerged as additional factors in hematopoietic carcinogenesis. However, we still know little about the temporal process by which carcinognesis progresses in hematopoietic cells. Here, I will combine will combine experimental and computational approaches to construct and test a model of carcinogenesis in B-cell chronic lymphocytic leukemia (CLL). First, I will use RNA-Seq to measure expression levels for coding and noncoding RNAs, and to identify any aberrant transcripts in samples from B-cell CLL. Combining those with a larger number of microarray and genetic profiles for CLL (available in our lab), I will construct a provisional model to identify key candidate regulators and their putative targets. I will then use RNAi libraries and overexpression systems – combined with a novel delivery approach and signature based profiling – to test the role of these regulators in affecting the CLL transcriptional program. Finally, I will perform transformation assays and FACS analysis of markers indicating carcinogenesis to confirm whether the changing expression level of the regulators induce carcinogenesis in B cells. My model will provide a detailed view of the regulatory program controlling carcinogenesis in B-cell CLL, as well as a deeper understanding of the complex networks controlling fundamental biological processes in mammalian cells.

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.

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.

The extremely high brightness and photo-stability of quantum dots make them ideal fluorophores for single molecule imaging in living cells. However, since they do not cross the cell membrane, they can not be used for fluorescent labeling inside living cells. In this research project, I propose novel methods for selectively silencing quantum dot fluorescence in the endosome of living cells and detaching cell-penetrating peptides, which deliver quantum dots into the cell, from quantum dot cargo inside the cytosol of living cells. Whereas delivery of quantum dots into endosome occurs easily, their escape from endosome into the cytosol are extremely inefficient. To suppress the background fluorescence of endosomally trapped quantum dots, I will use quantum dot conjugated with pH-sensitive CypHer5 dye which take advantage of the endogenous pH differences between the cytosol and the endosome. Since CypHer5 has strong absorption in the acidic condition but no absorption in the neutral condition, quantum dot-CypHer5 conjugate shows strong fluorescence in the neutral cytosol but no fluorescence in the acidic endosome. For the deliver of quantum dots into cells, I capitalize on the poly-cationic cell-penetrating peptide-based delivery method. To detach the interruptive peptide inside cytosol from quantum dots, I will use quantum dots modified with cell-penetrating peptides via disulfide linkers. Whereas the cytosol maintains a reducing environment, the endosomal compartment is oxidizing. Thus, the disulfide linker should be cleaved only inside the cytoplasm, and quantum dots will be released from a poly-cationic cell-penetrating peptide.

Vision plays as an essential role in controlling behaviors in a variety of animals. During this process, visual information is processed and transformed to specific motor outputs. Orienting behavior in zebrafish is an excellent model system in which to study such visuomotor transformation. The orienting behavior in zebrafish is known to require an intact optic tectum, a visual processing center, which receives visual input from the retina, and sends information to premotor network in the midbrain and hindbrain, called reticulospinal neurons. However, it remains to be elucidated how tectal neurons project to the premotor reticulospinal system to produce coordinated orienting behavior: how is each tectal neuron connected to reticulospinal neurons and how does it contribute to the production of orienting behavior?
To understand the neural bases of the visuomotor circuits controlling orienting behavior, I first plan to map neuronal connectivities of tectal neurons onto reticulospinal neurons using genetic labeling methods in zebrafish larvae, aiming at comprehensive analyses of their wiring patterns. Secondly, the morphological connectivities observed between tectal and reticulospinal neurons will be further tested for their physiological connectivities by calcium imaging. Finally, to examine the role of tectal neuron projections in visuomotor transformation, I will manipulate neural activities of genetically defined small subset of tectal neurons using optogenetic tools and simultaneously monitor their behaviors in living zebrafish. These approaches should give novel insights into neural mechanisms underlying sensorimotor coordination during animal behaviors.

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.

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.