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Park, S.

Publications and source records attributed to Park, S..

At least 235 records · Page 13Linked to original sources

The midbody and midbody remnant are assembly sites for RNA and active translation

The midbody (MB) is a transient structure at the spindle midzone that is required for cytokinesis, the terminal stage of cell division. Long ignored as a vestigial remnant of cytokinesis, we now know MBs are released post-abscission as extracellular vesicles called MB remnants (MBRs) and can modulate cell proliferation, fate decisions, tissue polarity, neuronal architecture, and tumorigenic behavior. Here, we demonstrate that the MB matrix--the structurally amorphous MB core of unknown composition--is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, that we are calling the MB granule. Using a quantitative transcriptomic approach, we identified a population of mRNAs enriched in mitotic MBs and confirmed their presence in signaling MBR vesicles released by abscission. The MB granule is unique in that it is translationally active, contains both small and large ribosomal subunits, and has both membrane-less and membrane-bound states. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. We demonstrate that the MB is the assembly site of an RNP granule. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III was necessary to maintain translation levels in the MB. Our data suggest a model in which the MB functions as a novel RNA-based organelle with a uniquely complex life cycle. We present a model in which the assembly and transfer of RNP complexes are central to post-mitotic MBR function and suggest the MBR serves as a novel mode of RNA-based intercellular communication with a defined biogenesis that is coupled to abscission, and inherently links cell division status with signaling capacity. To our knowledge, this is the first example of an autonomous extracellular vesicle with active translation activity. HighlightsO_LIThe MB, the center region of the intercellular bridge, is the assembly site of a ribonucleoprotein granule, we call the MB granule C_LIO_LIDistinct oncogenic and pluripotent transcription factor RNAs, including Jun/Fos and KLF4, are packaged in MBs and MBRs C_LIO_LIThe MB granule is coincident with the MB matrix, or dark zone, of the MB C_LIO_LIThe Kif23/MKLP1 kinesin is a core hexanediol-sensitive MB granule component C_LIO_LIThe MB and MBR are site of active translation that begins in early G1 and continues post-mitotically C_LIO_LIMKLP1 and ARC are necessary for RNA targeting/maintenance and translation at the MB C_LIO_LIDepletion of ESCRT-III increases the levels of translation during abscission C_LIO_LIAbscission releases MBRs as MB granule-harboring, translating extracellular vesicles C_LIO_LIMultiple cell types including cancer, stem, neural stem, all have actively translating MBRs C_LIO_LIMBRs are proposed as a novel mode of intercellular communication by extracellular vesicle-mediated direct transfer of RNA C_LI

cell biology↗

Intercellular crosstalk in adult dental pulp is mediated by heparin-binding growth factors Pleiotrophin and Midkine

In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis or regeneration are not well understood. To address these questions, we performed data analysis of publicly available single-cell transcriptomes of DP. This analysis revealed that DP resident fibroblasts have a unique gene expression profile when compared with fibroblasts from 5 other reference tissues: blood, bone marrow, adipose tissue, lung, and skin. Genes coded for heparin-binding growth-factors, pleiotrophin (PTN) and midkine (MDK), possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP cells, including Schwann cells, MSCs and odontoblasts. These findings suggest that fibroblast-derived growth factors regulate DP niches, and thus have a potential role as dental therapeutic targets.

cell biology↗

The Penultimate Step of Proteasomal ATPase Assembly is Mediated by a Nas2-Dependent Switch

The proteasome holoenzyme is a complex molecular machine that degrades most proteins. In the proteasome holoenzyme, six distinct ATPase subunits (Rpt1 through Rpt6) enable protein degradation by injecting protein substrates into it. Individual Rpt subunits assemble into a heterohexameric "Rpt ring" in a stepwise manner, by binding to their cognate chaperones. Completion of the heterohexameric Rpt ring correlates with release of a specific chaperone, Nas2, however it is unclear whether and how this event may ensure proper Rpt ring assembly. Here, we examined the action of Nas2 by capturing the poorly characterized, penultimate step of heterohexameric Rpt ring assembly. We show that Nas2 uses steric hindrance to block premature progression of the penultimate step into the final step of Rpt ring assembly. Importantly, Nas2 can activate an assembly checkpoint via its steric activity, when the last ATPase subunit, Rpt1, cannot be added in a timely manner. This checkpoint can be relieved via Nas2 release, when it recognizes proper addition of Rpt1 to one side of its cognate Rpt5, and ATP hydrolysis by Rpt4 on the other side of Rpt5, allowing completion of Rpt ring assembly. Our findings reveal dual criteria for Nas2 release, as a mechanism to ensure both the composition and functional competence of a newly assembled proteasomal ATPase, to generate the proteasome holoenzyme.

biochemistry↗

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of keratin-10 nascent RNA, which marks the onset of stem cell differentiation, we find that keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

genetics↗

Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans

The development of human adaptive immunity progresses faster than previously appreciated, with the emergence of memory CD4+ T cells alongside regulatory T (Treg) cells by the second trimester of pregnancy. We previously identified a prenatal-specific subset of PLZF+ CD4+ T cells with heightened effector potential that accounted for most memory T cells in the developing intestine and accumulated in the cord blood of infants exposed to prenatal inflammatory pathologies. However, the signals that drive their tissue distribution and effector maturation are unknown. In this report, we define the transcriptional and functional heterogeneity of prenatal PLZF+ CD4+ T cells and identify compartmentalization of Th-like effector function across the small intestine (SI) and mesenteric lymph nodes (MLN). We find that IL-7, which is more abundant in the SI relative to the MLN, drives the preferential expansion of naive PLZF+ CD4+ T cells via JAK/STAT and MEK/ERK signaling. Exposure to IL-7 induces a subset of PLZF+ CD4+ T cells to acquire a memory-phenotype and rapid effector function, identifying the human analogue of memory-phenotype CD4+ T cells. Further, IL-7 modulates the differentiation of Th1- and Th17-like PLZF+ CD4+ T cells, and thus likely contributes to the anatomic compartmentalization of prenatal CD4+ T cell effector function.

immunology↗

Safety switch optimization enhances antibody-mediated elimination of CAR T cells

Activation of a conditional safety switch has the potential to reverse serious toxicities arising from the administration of engineered cellular therapies, including chimeric antigen receptor (CAR) T cells. The functionally inert, non-immunogenic cell surface marker derived from human epidermal growth factor receptor (EGFRt) is a promising safety switch that has been used in multiple clinical constructs and can be targeted by cetuximab, a clinically available monoclonal antibody. However, this approach requires high and persistent cell surface expression of EGFRt to ensure that antibody mediated depletion of engineered cells is rapid and complete. Here we show that incorporating a short juxtamembrane sequence into the EGFRt polypeptide enhances its expression on the surface of T cells and their susceptibility to antibody-dependent cellular cytotoxicity (ADCC). Incorporating this optimized variant (EGFRopt) into bicistronic and tricistronic CAR designs results in more rapid in vivo elimination of CAR T cells and robust termination of their effector activity compared to EGFRt. These studies establish EGFRopt as a superior safety switch for the development of next-generation cell-based therapeutics.

bioengineering↗

Genetic architecture of creativity and extensive genetic overlap with psychiatric disorders revealed from genome-wide association analyses of 241,736 individuals

Creativity is heritable and exhibits familial aggregation with psychiatric disorders, but its genomic basis and genetic relationship with psychiatric disorders remain largely unknown. Here, we conducted a genome-wide association study (GWAS) using an expanded, machine learning-based definition of creativity in individuals of European ancestry from the UK Biobank (n = 241,736) and identified 25 creativity-associated loci. Extensive genetic overlap with psychiatric disorders, including schizophrenia, major depression, bipolar I disorder, attention deficit/hyperactivity disorder, and anorexia nervosa, was demonstrated by the genetic correlation, polygenic risk score, and MiXeR analyses. The condFDR and conjFDR analyses identified additional loci for creativity and psychiatric disorders, as well as shared genetic loci between creativity and psychiatric disorders. This GWAS showed significant correlations with GWASs using traditional definitions of creativity and GWASs adjusted for educational attainment. Our findings contribute to the understanding of the genetic architecture of creativity and reveal its polygenic relationships with psychiatric disorders.

genomics↗

Molecular and Anatomical Characterization of Parabrachial Neurons and Their Axonal Projections

The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, pain sensation, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns on dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are localized to distinct PBN subregions. We also describe two major ascending pathways that innervate distinct brain regions by analyzing axonal projections in 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.

neuroscience↗

Beyond winglets: evolution of flight-related morphology in stick insects (Phasmatodea)

The first winged insects evolved from a wingless ancestor, but details of the transition to a fully-winged morphology remain unclear. Studying extant pterygotes with partial wings, such as the stick insects (Order Phasmatodea), may help us to understand such a transition. Here, we address how a series of flight-related morphological parameters may correlate with flight evolution by studying different phasmids representing a volancy continuum ranging from miniaturized to full-sized wings. Variation in phasmid wing shape, venation, wing mass and the mass of flight muscle can be described by specific scaling laws referenced to wing length and wing loading. Also, the mass distribution of the body-leg system is conserved in spite of a wide range of variation in body shape. With reduced wing size and increased wing loading, the longitudinal position of the wing-bearing thoracic segments is shifted closer to the insects center of body mass. These results demonstrate complex reconfiguration of the flight system during wing morphological transitions in phasmids, with various anatomical features potentially correlated with reduced flight performance attained with partial wings. Although these data represent phasmid-specific features of the flight apparatus and body plan, the associated scaling relationships can provide insight into functionality of intermediate conditions between wingless and fully-winged insects more generally.

evolutionary biology↗

Highly accurate isoform identification for the human transcriptome

We explore a new hypothesis in genome annotation, namely whether computationally predicted protein structures can help to identify which of multiple possible gene isoforms represents a functional protein product. Guided by structure predictions, we evaluated over 140,000 isoforms of human protein-coding genes assembled from over 10,000 RNA sequencing experiments across many human tissues. We illustrate our new method with examples where structure provides a guide to function in combination with expression and evolutionary evidence. Additionally, we provide the complete set of structures as a resource to better understand the function of human genes and their isoforms. These results demonstrate the promise of protein structure prediction as a genome annotation tool, allowing us to refine even the most highly-curated catalog of human proteins. One-Sentence SummaryWe describe the use of 3D protein structures on a genome-wide scale to evaluate human protein isoforms for biological functionality.

genomics↗

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila

Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses. Author SummaryInsulin is an essential hormone that controls metabolism in all animals, by regulating energy use and growth of target tissues. Impaired insulin signaling ("resistance") in humans underlies development of type 2 diabetes, a pandemic disease causing significant morbidity and mortality. The genetic risk for insulin resistance is complex, and studies of diabetes are limited by a lack of tools to measure insulin resistance in a sensitive, quantitative, and tissue-specific way. Here, we describe a new technique to measure the strength of insulin signaling in fruit flies. By combining fruit fly genetics with antibody-based assays, we can quantify phosphorylated Akt, an evolutionarily conserved target of insulin signaling, in specific tissues of the adult fly. We show this technique can detect changes in insulin signaling after fasting and refeeding, addition of exogenous insulin, or genetic disruption of the insulin signaling pathway. We used this method to discover a new regulator of insulin signaling, a phosphatase enzyme encoded by Pp1-87B. This exciting new tool should advance our ability to study and discover additional regulators of insulin signaling and resistance.

genetics↗

Herd immunity on chip: recapitulating virus transmission in human society

Virus transmission is affected by population density, social distancing, and vaccination. This has been simulated only by mathematical models. Here, we report the first experimental model to mimic herd immunity to a human coronavirus using a microfluidic device filled with host cells. The device consists of 444 microchambers filled with susceptible (S0), infected (I0), and unsusceptible (U0) cells at specific ratios. The transmission rate and reproduction numbers were directly proportional to S0 and I0 and inversely proportional to U0. Herd immunity was achieved when the proportion of U0 was at 80% in a fixed number of uninfected (S0+U0) cells. These results were consistent with those from a mathematical model. The device can be used for predicting virus transmission. One-Sentence SummaryWe present the first experimental model enabling the simulation of herd immunity in a microfluidic device filled with host cells to human coronavirus.

microbiology↗

An iPSC library from a family with Creutzfeldt-Jakob disease caused by the E200K mutation

The most common subtype of genetic prion disease is caused by the E200K mutation of the prion protein. We have obtained samples from 22 members of a multi-generational Israeli family harboring this mutation, and generated a library of induced pluripotent stem cells (iPSCs) representing nine carriers and four non-carriers. Whole-exome sequencing was performed on all individuals. A comparison of neurons derived from E200K iPSCs to those from non-carriers revealed the presence of several disease-relevant phenotypes. Neurons from E200K carriers were found to contain thioflavin S-positive accumulations of PrP in their cell bodies. In addition, these neurons displayed disruptions of NMDA receptor/PSD95 co-localization at postsynaptic sites. Our study shows that iPSC-derived neurons, which express physiologically relevant levels of mutant PrP in a human neuronal context, can model certain aspects of human prion disease, offering a powerful platform for investigating pathological mechanisms and testing potential therapeutics.

neuroscience↗

Elevated arginine levels in liver tumors promote metabolic reprogramming and tumor growth

Arginine auxotropy, due to reduced expression of urea cycle genes, is common in cancer. However, little is known about the levels of arginine in these cancers. Here, we report that arginine levels are elevated in hepatocellular carcinoma (HCC) despite reduced expression of urea cycle enzymes. Liver tumors accumulate high levels specifically of arginine via increased uptake and, more importantly, via suppression of arginine-to-polyamine conversion due to reduced arginase 1 (ARG1) and agmatinase (AGMAT) expression. Furthermore, the high levels of arginine are required for tumor growth. Mechanistically, high levels of arginine promote tumorigenesis via transcriptional regulation of metabolic genes, including upregulation of asparagine synthetase (ASNS). ASNS-derived asparagine further enhances arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a novel second messenger-like molecule that reprograms metabolism to promote tumor growth.

cancer biology↗

Nucleosome sliding by the Chd1 chromatin remodeler relies on theintegrity of the DNA duplex

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers are biased by DNA sequences, suggesting that they may be sensitive to properties of the DNA duplex. Here, we present a high-throughput method for determining nucleosome positioning in vitro using site-specific DNA cleavage coupled with next-generation sequencing. This method allowed us to systematically test how the introduction of poly(dA:dT) tracts and other perturbations affected the distribution of nucleosomes remodeled by the Chd1 remodeler. We found that Chd1 is sensitive to poly(dA:dT) tracts as short as 3 bp, and that its nucleosome sliding activity is severely perturbed by DNA mismatches and single-nucleotide insertions. These results suggest that remodelers rely on the integrity of duplex DNA for nucleosome sliding. We also discovered that DNA on the nucleosome can shift in the absence of a remodeler when multiple mismatches are placed at superhelix location 2 (SHL2). This DNA movement in response to a disruption of the double helix may explain why SHL2 is the preferred site of engagement by most chromatin remodelers.

biophysics↗

A novel oral GyrB/ParE dual binding inhibitor effective against multidrug resistant Neisseria gonorrhoeae and other high-threat pathogens

Drug resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance, as well as limit development of new resistance. We describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6- fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2- oxa-8-azaspiro[4.5]decan-3-yl)methanol], which simultaneously binds to ATP binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae including drug-resistant strains. A phosphate prodrug JSF-2659 was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram positive organisms, and JSF-2659 was shown in a deep tissue model of VRSA and a model of C. difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

microbiology↗

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.

biophysics↗

BK channel properties correlate with neurobehavioral severity in three KCNMA1-linked channelopathy mouse models

KCNMA1 forms the pore of BK K+ channels, which regulate neuronal and muscle excitability. Recently, genetic screening identified heterozygous KCNMA1 variants in a subset of patients with debilitating paroxysmal non-kinesigenic dyskinesia, presenting with or without epilepsy (PNKD3). However, the relevance of KCNMA1 mutations and the basis for clinical heterogeneity in PNKD3 has not been established. Here we evaluate the relative severity of three KCNMA1 patient variants in BK channels, neurons, and mice. In heterologous cells, BKN999S and BKD434G channels displayed gain-of-function (GOF) properties, whereas BKH444Q channels showed loss-of-function (LOF) properties. The relative degree of channel activity was BKN999S > BKD434G > WT > BKH444Q. BK currents and action potential firing were increased, and seizure thresholds decreased, in Kcnma1N999S/WT and Kcnma1D434G/WT transgenic mice but not Kcnma1H444Q/WT mice. In a novel behavioral test for paroxysmal dyskinesia, the more severely affected Kcnma1N999S/WT mice became immobile after stress, consistent with stress-induced immobility episodes observed in PNKD3-affected individuals. Homozygous Kcnma1D434G/D434G mice showed similar immobility, but in contrast, homozygous Kcnma1H444Q/H444Q mice displayed hyperkinetic behavior. These data establish the relative pathogenic potential of patient alleles as N999S > D434G > H444Q and validate Kcnma1N999S/WT mice as a model for PNKD3 with increased seizure propensity.

neuroscience↗