A biological neural network is formed by living neurons with massive communication links between them. Neurons communicate by delivering electrical signals through insulated axonal fibers, providing the communication speed of up to 100 m/s. Although this biological neural network enables powerful human computation, the more powerful and efficient computation can be made if the neural network equips a faster mode of communication. Inspired by the revolution in the modern communication industry from electrical cables to optical fibers, we aim to explore whether a living neural network can be engineered to communicate with optical signals. Compared to electrical signals, optical communication is faster by orders-of-magnitude and selective by wavelength, which could significantly improve both communication speed and bandwidth. Specifically, we will first construct light-emitting neurons using a ‘cell laser’ technique and light-receiving neurons constructed by optogenetics. Based on these living optical elements, we will develop optical logical circuits on a chip for logical computational operations. We will further incorporate the time-varying property of living neurons by using the light-controlled expressions of photoactive proteins. To successfully achieve the research goal, we have assembled a multidisciplinary team of a physicist (Dr. Humar) for microlasing and optical configuration, a biomedical engineer (Dr. Choi) for neural engineering, and an electrical engineer (Dr. Im) for on-chip integration and analysis. Successful completion of this project will bring a new concept of optically communicating neural circuits that could realize the fastest communication mode in biology.

Protein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. We will use our expertise of single-molecule protein detection and material sciences to develop novel sequencing tools. In particular, we will use graphene mass sensors to measure the mass of proteins with sub-Dalton sensitivity. Utilizing this high sensitivity, we will measure the mass of protein fragments and identify the sequence of the fragments. We will also apply this method for detecting post-translational modifications of single proteins. Ultimately we aim to achieve sequencing of full-length proteins. This proof of concept will open the door to single-molecule protein sequencing and pave the road toward the development of a new, fast, and reliable diagnostic tool.

Optical nanostructures are highly organized composites of materials with varying refractive indices (e.g. keratin, melanin and air) that produce some of the brightest colors found in nature through coherent light scattering. How these tissues organise themselves at the nanometer scale to produce colors is poorly understood, despite its fundamental significance to developmental and evolutionary biology and potential to spark advances in the biomimetic design and "green" commercial manufacture of self-assembling optical materials.
We thus propose to use both transcriptomic, laser diffraction and microscopy-based tools of developmental biology to elucidate the mechanisms by which these nanostructures self-assemble in a subsample of birds (Class Aves), a group with incredibly diverse structural colors and mechanisms. Our working hypothesis is that iridescent colors form through depletion-attraction, phase separation and other self-assembly mechanisms. Because most developmental biology is done at larger size scales, testing these hypotheses will require the use and development of methods such as wet cell TEM and in situ laser diffraction analysis to adequately resolve nanometer-scale changes in developing tissue. We will then test these proposed mechanisms using biomimetic approaches that replicate natural conditions as closely as possible (e.g. at room temperature,at biological pH) using natural or semi-natural materials. Use of optical techniques including angle-resolved spectrophotometry and microspectrophotometry will enable us to compare these properties between the natural and synthetic versions. This approach will enable us to not only experimentally test modes of development but also generate and test new materials and/or processes to produce them.
There are three highly innovative aspects to this proposal. First, it attempts to unlock the developmental pathways producing nanostructured tissues. This is a long-standing question with few answers thus far. Second, it uses biomimicry in novel ways to test developmental hypotheses and pushes the technical boundaries of developmental biology by focusing on nanometer-scale organisation of tissues. Finally, the use of biologically realistic chemistry in our biomimetic approaches is a huge leap forward in this field where most work is done at high temperature or with non-biocompatible materials. This work will therefore significantly advance both our fundamental understanding of these materials and the tools to study them and other nanoscale materials.

Cancer evolves through the continuous acquisition of DNA mutations. As a result, multiple diverse tumor subclones can arise during the course or tumor progression from benign to malignant carcinoma. These diverse cancer subclones have different traits, such as different levels of therapy resistance or metastatic potential. Intriguingly, cancers can obtain a hypermutator phenotype that leads to accelerated acquisition of DNA mutations. In gastric cancers, a medical burden especially in East Asia, multiple types of mutations are observed, suggestive of pronounced impact of hypermutator phenotypes on the development of these cancers. However, how hypermutator action facilitates the evolutionary trajectory of cancers is not fully understood.
In this HFSP project, we intend to use organoid technology to study the impact of diverse hypermutator phenotypes in gastric cancers. Organoid technology is a state-of-the-art culture technique for human mini-organs in a dish from both normal tissues as well as cancers, and mimics the in vivo scenario to great extent.
To understand the role of hypermutator action in human gastric cancer, three research teams integrate their expertise into a novel experimental pipeline: 1) the establishment and analysis of patient-derived tumor organoids with natural occurring hypermutator phenotypes, in parallel with engineered tumor organoids with introduced hypermutator phenotypes. 2) Monitoring mutational accumulation within these tumor organoids using DNA sequencing at every intermediate step along their progression towards malignant carcinoma and 3) filming the diverging cellular behaviors between different tumor subclones using advanced microscopy. Ultimately, we aim to map the dynamic mutation landscape during the evolutionary trajectory of diverging tumor subclones in comparison to the phenotype of the changing tumor cells.
This project will provide a unique opportunity to obtain a comprehensive understanding on the role of hypermutator phenotypes in cancer evolution. Moreover, we expect that our improved insights on the emergence of genetic subclones in gastric cancer can guide us to understand to other cancer subtypes and will help us to fight against chemo resistance and metastasis of cancer.

Animals must constantly integrate information from the environment with prior experience to maximize reward and avoid danger. Such goal-directed functions require the prefrontal cortex (PFC), a brain structure that is extensively interconnected with sensory regions, limbic structures and motor systems. The PFC circuit is thought to orchestrate fear, anxiety, motivation, attention and other behavioral processes that are crucial for survival in a constantly changing environment, and impaired prefrontal functions have been associated with diverse psychiatric conditions including schizophrenia, major depression, attention and anxiety disorders. The PFC interacts with multiple cortical and sub-cortical brain regions through its long-range output projections, originating from defined populations of excitatory PFC output neurons. These neurons send their axons to a wide range of downstream brain regions, including the amygdala, the hypothalamus, the basal ganglia and other structures known to directly modulate the animal’s behavioral state and its responses to the environment. How do these neurons route information to one downstream target or another? What are the genetic, structural and functional changes through which they facilitate the acquisition of goal-directed behavior? Previous work has explored these questions, revealing unique roles for distinct types of PFC neurons in complex behaviors such as working memory function, attentional modulation, learning and memory. However, these studies typically focused on single populations of PFC neurons, while the state of the network as a whole is known to be dependent on the joint function of all of its components. We will combine advanced single-cell genomic analysis (Amit lab) with optogenetic functional experiments, electrophysiological approaches (Yizhar lab), innovative tissue processing and volumetric fluorescence imaging techniques (Kim lab), to understand the organization and function of PFC output neurons and their learning-related plasticity. Our results will reveal the unique molecular, structural and functional properties of defined prefrontal cortical output pathways, and allow for the first time a system-level understanding of the functional dynamics supporting the regulation of goal-directed behavior by this complex circuit.

Sleep deprivation disrupts memory consolidation, and alters gene transcription in the cortex and hippocampus. We hypothesize that sleep deprivation-induced deficits in memory processes are caused in part by misregulation of the molecular circadian (~24hr) clock. Until now, experimentally testing this hypothesis has been a major challenge. Specifically, the molecular clock controls sleep and wake behavior through effects on the suprachiasmatic nucleus and other sleep-regulatory circuits. Furthermore, sleep and wake cause widespread, simultaneous changes in transcription, translation, neural activity, neuromodulation, and hormone release. Thus ascribing functional causality to one specific state-dependent variable (and in particular, clock gene expression) has proven difficult. Here, we propose to develop and use novel optogenetic tools to rapidly up- or downregulate individual clock genes in vivo, in a circuit-specific manner, independent of the animal’s behavioral state. We will use cutting-edge computational models and bioinformatic analyses to optimize optogenetic regulation of clock genes in the brain. We will test our hypotheses using both traditional and novel optogenetic tools, computational modeling, bioinformatics, behavioral analysis and electrophysiology. This can only be achieved by a synergistic collaborative intercontinental, interdisciplinary research team: a group at the University of Düsseldorf (Germany) will develop optogenetic tools, a group at the Korea Advanced Institute of Science and Technology (South Korea) will carry out mathematical modeling and bioinformatics analyses of CCGs, and groups at the University of Michigan (United States) will study effects on cortical plasticity, and a group at the University of Groningen (Netherlands) will examine effects on hippocampus-mediated memory.

Neuroscience research using rodents as an animal model relies on the assumption that results should generalize across species to primates and ultimately to humans. However, many brain areas, including the neocortex, have species specific functional organizations. The mouse lemur (Microcebus murinus), the World’s smallest primate, has the potential to become an ideal animal model bridging the gap between rodents and primates. It has most the advantages of the rodent model (small brain size, quick reproduction, relatively short life cycle), but additionally offers the evolutionary closeness of primates. Therefore, the mouse lemur promises to revolutionize the transferability of experimental results from small sized animal models to human applications. In this project, three labs will combine their expertise in primate behavior, in-vivo optical imaging, and cutting edge histology and molecular biology to explore the functional organization of the mouse lemur cortex, as well its behavioral and cognitive capacity. This collaborative project will lay the groundwork to establish the mouse lemur as a novel neuroscience model system.

Lymph nodes (LNs) are situated at junctures of the blood vascular and the lymphatic system where antigens drain from peripheral tissues via afferent lymphatics. During the development of malignant breast cancer, new LNs emerge within the glandular tissues that are normally devoid of LNs. However, we do not understand the mechanism of development of these LNs and their role in antitumor immunity. We hypothesize that de novo LNs form in the vicinity of mammary carcinomas as an accessory “base camp” for the initiation and maintenance of antitumor immunity. Here, we combine and leverage our expertise in immunology, vascular biology, and bioengineering to address the hypothesis in three specific aims: (1) To molecularly dissect the pathways of tumor-induced development of accessory LNs with a particular emphasis on lymphovasculokines. (2) To assess to which extent accessory LNs support antitumor immunity. (3) To stimulate formation of accessory LNs in mammary tissues to foster antitumor immunity. The potential impact of this project is high as a deeper understanding of the interplay between accessory LNs and tumors may lead to a novel cancer treatment approach.

How does the brain encode sensory perceptions? Although we know approximately how sensory input is routed through different brain areas the “language” used by the brain to represent sensory experiences remains elusive. Brain cells are known to translate external sensory stimuli into electrical pulses called “spikes” to process sensory information. The patterns of spikes contain a code, the neural code, that is used for communication. Theoretically, if we can “crack” the neural code then we will be able to read and write sensory information to the brain. This project aims to combine approaches from cellular, systems, behavioural and computational neuroscience in order to demonstrate that the brain uses two distinct neural codes simultaneously to represent the sensation of touch. To do this, we will study different types of inhibitory cells in the brain that we hypothesize are responsible for carrying the two different codes. Using electrical recordings of individual cells, computer models of neural circuits, and optical recordings of electrical activity in the circuits of mice feeling objects with their whiskers, we will study how these two types of cells integrate information encoded in different ways, and examine how they affect activity in the brain. Using what we learn about these two types of inhibitory cells we will then develop and a novel, optical approach to bias the codes in a mouse’s brain as it performs sensory discrimination tasks with its whiskers. By shifting the mouse’s brain from one code to the other we hope to be able to generate illusory percepts in the mice—making them think they are feeling something with their whiskers that they are not actually feeling. If we can do this for both of the codes that we hypothesize are used by the brain, then we will have demonstrated for the first time that the brain uses two codes simultaneously.

Are roots in the dark? Darwin proposed that the “brain” of a plant resides in its roots. Subsequent research has shown that roots integrate environmental information about resource availability, competitors, etc., to determine critical life history traits of the shoot, such as the timing of resource allocations to reproduction, growth, defense and senescence. During domestication, this environmentally sensitive “brain” is commonly disabled to make crops less environmentally sensitive, easier to transplant, and better synchronized to facilitate harvesting. We propose to explore the importance of a new sensory modality of roots, that of light-perception, in enhancing the ecological performance of a native plant in its native habitat.
Plants use light as both an energy source and a source of signals to alter its physiology to adapt to current or anticipated environmental changes. Forward and reverse genetics have identified photoreceptors and their associated signaling cascades. Surprisingly, some photoreceptors are highly and specifically expressed in roots, which are generally thought to develop in the total darkness of the soil. Light penetration in most soils is limited to a few millimeters close to the soil surface but roots that express a plethora of photoreceptors grow deep in the soil. Since natural selection rapidly winnows non-functional genes from genomes, these root- expressed photoreceptors are likely being maintained in the genomes of higher plants because they play an important role; the goal of this interdisciplinary proposal is to uncover the function of these root-expressed photoreceptors.
While the idea of light-piping through vascular bundle was suggested three decades ago, theoretical and empirical examinations of ‘light-piping’ remains largely unexplored and the question of its relevance for plant function has simply been out of the grasp of single experts working alone or in loose collaborations with other experts. By combining state-of-the-art optical physics and molecular biology, we propose to characterize this transmitted light, uncover how plants respond to this light, and determine if the responses are relevant for the optimization of ecological fitness during the plant’s entire life in the field. Building on the impressive knowledge of light signaling in the model plant, Arabidopsis thaliana community, we will use a wild tobacco, Nicotiana attenuata to uncover the ecological relevance of having “sighted roots” in the plant’s natural habitat. We will employ the versatile molecular and biochemical tools available in Arabidopsis and incorporate the relevant components into an ecological model plant, N. attenuata. We have developed analytical, molecular and ecological platform in N. attenuata to study its rich ecological interaction in the Great Basin Desert of Utah. We propose that the intense light of the desert will illuminate the ecological function of light signaling in roots.

Are roots in the dark? Darwin proposed that the “brain” of a plant resides in its roots. Subsequent research has shown that roots integrate environmental information about resource availability, competitors, etc., to determine critical life history traits of the shoot, such as the timing of resource allocations to reproduction, growth, defense and senescence. During domestication, this environmentally sensitive “brain” is commonly disabled to make crops less environmentally sensitive, easier to transplant, and better synchronized to facilitate harvesting. We propose to explore the importance of a new sensory modality of roots, that of light-perception, in enhancing the ecological performance of a native plant in its native habitat.
Plants use light as both an energy source and a source of signals to alter its physiology to adapt to current or anticipated environmental changes. Forward and reverse genetics have identified photoreceptors and their associated signaling cascades. Surprisingly, some photoreceptors are highly and specifically expressed in roots, which are generally thought to develop in the total darkness of the soil. Light penetration in most soils is limited to a few millimeters close to the soil surface but roots that express a plethora of photoreceptors grow deep in the soil. Since natural selection rapidly winnows non-functional genes from genomes, these root- expressed photoreceptors are likely being maintained in the genomes of higher plants because they play an important role; the goal of this interdisciplinary proposal is to uncover the function of these root-expressed photoreceptors.
While the idea of light-piping through vascular bundle was suggested three decades ago, theoretical and empirical examinations of ‘light-piping’ remains largely unexplored and the question of its relevance for plant function has simply been out of the grasp of single experts working alone or in loose collaborations with other experts. By combining state-of-the-art optical physics and molecular biology, we propose to characterize this transmitted light, uncover how plants respond to this light, and determine if the responses are relevant for the optimization of ecological fitness during the plant’s entire life in the field. Building on the impressive knowledge of light signaling in the model plant, Arabidopsis thaliana community, we will use a wild tobacco, Nicotiana attenuata to uncover the ecological relevance of having “sighted roots” in the plant’s natural habitat. We will employ the versatile molecular and biochemical tools available in Arabidopsis and incorporate the relevant components into an ecological model plant, N. attenuata. We have developed analytical, molecular and ecological platform in N. attenuata to study its rich ecological interaction in the Great Basin Desert of Utah. We propose that the intense light of the desert will illuminate the ecological function of light signaling in roots.

Our ability to behave meaningfully in our environment depends critically on having a transient memory buffer in which to upload and hold on-line past experiences, goals or aspects about the current context, so that they can be integrated in choosing our course of action. This buffer is referred to as working memory and is critical to cognition because it allows behavior to be guided by internal representations acquired in the past, rather than by the immediate contingencies of our surroundings. Without it, our behavior would not amount to much more than a series of reflexes. Experiments with patients with brain lesions together with measurements of the activity of neurons from behaving animals show that the frontal part of the brain, in particular a brain area called the prefrontal cortex, is critically implicated in working memory.
In the laboratory, working memory is studied using simple tasks. For instance, a particular image displayed for 1 s might require pressing a particular key, but the subject is instructed to only perform the action after a ‘delay period’ of 5 s subsequent to the image presentation has elapsed. Animal experiments have shown that particular neurons in the prefrontal cortex are activated during the delay period while animals remember certain images but not others. These neurons might, thus, be part of a neuronal representation of the memory of the image.
Despite decades of working memory research, a number of fundamental questions remain unanswered: What properties of neurons and circuits allow their activity to persist in time without sustaining inputs from the senses? How robust are neuronal representations in working memory? This last point is crucial because the brain is constantly active, so the neurons participating in the memory representations are always receiving unrelated information which must be filtered out. Without stability against these perturbations, working memory function would not be possible. Because networks of neurons are very complex dynamical systems, answering these questions will ultimately require, not only new experimental approaches, but also a quantitative conceptual framework, in order to guide the design of experiments and to interpret the experimental data.
In this proposal, we will use a parallel experimental-theoretical approach to address these questions. Recently developed techniques for measuring and manipulating brain activity in mice performing simple working memory tasks will allow us, for the first time, to measure directly the stability of neural memory representations and to elucidate which properties of prefrontal neurons and circuits are responsible for generating them. At the same time, the data from our experiments will be used to assess the validity of current theoretical frameworks of working memory, to refine them, and to generate novel hypothesis regarding the circuit basis of working memory function in the prefrontal cortex.

Neurons have to perform a wide variety of complex morphogenetic events in order to functionally wire the developing brain. In the adult brain, this process continues with changes in morphology that alters their functional connectivity upon learning and experience, or during the repair of injured neural tissues. Specific signaling events to the cytoskeleton are essential for the control of these processes and are likely to be highly regulated at fine spatiotemporal scales. Accordingly, novel cell biological tools such as fluorescent biosensors that enable dissection of these signaling events in time and space, have given novel insights in signaling complexity. The aim of this proposal is to visualize the dynamics of the activation of specific signaling molecules in a panel of prototypical neuronal behaviors with a strong emphasis on the axon. We will study: axonal specification (the establishment of an axon and a dendrite), axonal guidance (the response of a growth cone to attractive and repulsive cues) and inhibition of axonal regeneration after injury (the response of the axon to a glial scar). Our hypothesis is that these different morphogenetic behaviors involve distinct signaling programs with a tight crosstalk between the signaling molecules of interest. Comparing these in the prototypical cell behaviors should give essential insights in how a neuron can perform this diverse set of tasks using the same signaling machinery. We propose to engineer a new generation of highly sensitive fluorescent probes to monitor the spatio-temporal activation profiles of the specific signaling molecules in primary E18 embryonal hippocampal neurons. Microfluidic technology will be used to engineer highly defined extracellular environments to induce and orient the different axonal behaviors. This will evoke the asymmetric nature of extracellular cues observed in vivo, and recapitulate the associated signal amplification mechanisms that will lead to robust signaling. Finally, computer vision techniques will be devised that allow the pooling of measurements from many cells into statistically significant datasets for identification of the spatiotemporal relationships among different signaling programs and cell behavior. Multivariate time-series analysis methods will be devised to inform the model on the cross-coordination between the activation of the signaling molecules of interest and neuronal morphogenesis.

Development of functional neurons from embryonic neuroepithelium is mediated by series of events including specification into multipotent neural precursor cells, commitment to differentiated neurons, and maturation into functional neurons composing of neural network. These processes are tightly controlled by intrinsic genetic programs, but also affected by environmental condition. However, the extent of the external signals that can modify the intrinsic programs is not clearly addressed yet. My previous studies identified an intrinsic program involves in glial fate specification against default neural fate of optic neuroepithelium. Vax homeotic transcription factors were critical player for glial fate acquisition of optic neuroepithelium under reciprocal antagonism between Shh and protein kinase A (PKA) signaling pathways. My current researches are aiming to the understanding roles of intracellular signaling pathways such as PKA and phosphoinositide-3 kinase (PI3K) pathways, which are cross-talking to many external developmental signaling pathways in neurogenesis of vertebrate retina. The strength of the signaling pathways will be genetically modified in mouse retinal progenitor cells (RPCs) to investigate the roles of intracellular signaling pathways as central modulators of intrinsic genetic programs upon the changes of external cues.

The goal of this proposal is to understand the mechanism of IL-2 mRNA stabilization in response to extracellular stimuli. In response to T cell activation signals, the half life of IL-2 mRNA is greatly extended. The 3'UTR of IL-2 mRNA contains AU rich elements (AREs) that mediate rapid IL-2 mRNA degradation. In order to study these cellular processes that modulate the IL-2 mRNA turnover during T cell activation, I propose to find transacting factors involved in the control of lL-2 mRNA stability in activated T cells. In conection with this, I have purified NF90 protein as one of the IL-2 ARE binding proteins in stimulated T cells, suggesting that NF90 modulates the activity of the degradation machinery that assembles on the 3'UTR stabilizes an mRNA containing the IL-2 ARE. To gain a better understanding how these trans-acting proteins including NF90 regulate gene expression at the post-transcriptional level, I will identify the nature of these cytoplasmic protein complex that control IL-2 mRNA stability using biochemical and genetic approaches. This project will contribute to obtain both unberstanding of general mechanism in the signal-regulated mRNA stabilization and methodology of defining the RNA binding protein related mRNA stability. Furthermore, understanding the regulation of IL-2 expression may provide us with new targets for screening for novel agents. Such drugs by inhibiting or promoting the expression of IL-2 should prove useful in the prevention of T-cell leukemia and may improve IL-2 mediated immunotheraphy for leukemia. Personally, this project provides me the progress in growing as an independent researcher in this important area.