Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 397 records · Page 22Linked to original sources

bioRxiv2024

Protein Kinases MpkA and SepH Transduce Crosstalk Between CWI and SIN Pathways to Activate Protective Hyphal Septation Under Echinocandin Cell Wall Stress

This study investigates a previously unreported stress signal transduced as crosstalk between the Cell Wall Integrity (CWI) pathway and the Septation Initiation Network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific; our findings suggest this is due largely to CWI-SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of {beta}-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery, of previously unknown crosstalk between the CWI and SIN pathways, not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also unveil, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. Hence, this work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin resistant species.

microbiology↗

bioRxiv2024

Structural diversity of mitochondria in the neuromuscular system across development

As an animal matures, its neural circuit undergoes alterations, yet the developmental changes in intracellular organelles to facilitate these changes is less understood. Using 3D electron microscopy and deep learning, we developed semi-automated methods for reconstructing mitochondria in C. elegans and collected mitochondria reconstructions from normal reproductive stages and dauer, enabling comparative study on mitochondria structure within the neuromuscular system. We found that various mitochondria structural properties in neurons correlate with synaptic connections and these properties are preserved across development in different neural circuits. To test the necessity of these universal mitochondria properties, we examined the behavior in drp-1 mutants with impaired mitochondria fission and discovered that it caused behavioral deficits. Moreover, we observed that dauer neurons display distinctive mitochondrial features, and mitochondria in dauer muscles exhibit unique reticulum-like structure. We propose that this specialized mitochondria structure may serve as an adaptive mechanism to support stage-specific behavioral and physiological needs.

neuroscience↗

bioRxiv2024

Computationally designed mRNA-launched protein nanoparticle vaccines

Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1-6. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7-10, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.

immunology↗

bioRxiv2024

Highly ordered clustering of TNFα and BAFF ligand-receptor-adaptor complexes bound to lipid membranes

We report the cryo-EM structures of clusters of TNF receptor family proteins, TNFR1 and BAFFR. The receptor-ligand complexes were anchored to a flat lipid layer to mimic the membrane-bound state. We observed that the TNF-TNFR1 complex forms highly ordered binary, bent, trigonal, and linear quadruple clusters of trimers on the lipid membrane. A non-competitive antagonist of TNFR1 disrupted these clusters without interfering with ligand binding. Moreover, we found that the BAFF-BAFFR complex forms pentagonal, double-pentagonal, or half-spherical clusters of trimers. Mutations in BAFF that inhibit BAFFR receptor activation prevented ordered clustering without disrupting receptor binding. TRAF3 induced a structural shift in the BAFF-BAFFR cluster, resulting in a flat hexagonal cluster. Our data demonstrated that precisely structured clustering is essential for the activation of these receptors. The lipid monolayer method will aid in studying the clusters of other transmembrane proteins and facilitate the discovery of therapeutic agents that regulate their clustering.

biochemistry↗

bioRxiv2024

Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice

Genetic changes and epigenetic modifications are associated with neuronal dysfunction in the pathogenesis of neurodegenerative disorders. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear. Here, we identified an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element, using convolutional neural network. The enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits. Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.

neuroscience↗

bioRxiv2024

Transient TELC and transmembrane potential in a laser flashed bacteriorhodopsin purple membrane open flat sheet

The transmembrane-electrostatically localized protons/cations charges (TELC, also known as TELP) model may serve as a unified framework to explain a wide range of bioenergetic phenomenon. Transient TELC and transmembrane potential in a laser flash-energized bacteriorhodopsin (bR) purple membrane (PM) open flat sheet are now better analyzed. Under the Heberle et al. 1994 experimental conditions, the number of bR molecules is now calculated to be 8200 per PM open flat sheet with a diameter of 600 nm. With a single-turnover laser flash intensity of 3 mJ/cm2 to photoexcite 10% of the bR molecules, the number of laser flash-induced peak TELC density is calculated to be 2900 per {micro}m2 of PM, which translates to a peak transient transmembrane potential of 50 mV. The bR protonic outlet protrudes into the liquid phase outside the putative "potential well/barrier". The observation is in line with the TELP model; but does not support the "potential well/barrier" model. The author encourages research on more relevant protonic cell systems that have transmembrane potential with TELC comprising excess positive charges at one side and excess anions at the other side of the membrane.

biophysics↗

bioRxiv2024

Robustness with Limited Specificity of Telomere Binding Proteins Underlies Telomere Evolution in Nematoda

Telomeres, the nucleoprotein complexes that protect the ends of linear chromosomes, are essential for maintaining the stability of eukaryotic genomes. As telomeres generally consist of repetitive DNA associated with specifically bound proteins, telomeric repeat motifs (TRMs) are thought to be difficult to evolve. However, a recent study identified nematodes with telomeric repeats distinct from the canonical TTAGGC motif. Here, we investigated how telomere repeats could have evolved despite the challenge posed by the specificity of telomere-binding proteins (TBPs) to the telomeric DNA in Nematoda. We performed a phylogenetic analysis and electrophoresis mobility shift assays to assess the binding affinities of two TBPs, which displayed different conservation patterns. Our results revealed that the well-conserved protein CEH-37 exhibits limited specificity, unable to distinguish telomeric repeats found in nematodes except for the TTAGGG motif, while the less conserved POT proteins displayed rigid specificity. These findings suggest that the emergence of novel telomeric repeat motifs correlated with the characteristics and evolutionary outcomes of TBPs in Nematoda. Our study not only revealed the dynamics of telomere evolution but also enhanced the understanding of the evolutionary relationship between proteins and DNAs. Lay summaryTelomeres are chromosomal end structures composed of repetitive DNA where telomere-binding proteins (TBPs) specifically bind. This nucleoprotein complex is crucial for eukaryotes to maintain their genome integrity, making it difficult to evolve. However, a recent study identified nematodes with non- canonical telomeric repeat motifs (TRMs). Through an analysis of TBP conservation in Nematoda and assessments of their binding affinities, we observed that the binding capabilities of TBPs align with their conservation patterns. This correlation is also linked to the emergence of novel TRMs, explaining the evolution progress of TRMs in Nematoda. The evolutionary process discovered in this research is not limited to telomeres, providing further insights into evolutionary relationships between proteins and DNA.

cell biology↗

bioRxiv2024

Sarm1-Dependent Metabolic Reprogramming of Schwann Cells Following Nerve Injury

Schwann cells (SCs) transition into a Repair state after peripheral nerve injury; however, the early SC injury response preceding this transition remains poorly understood. We demonstrate that Sarm1, a key regulator of axon degeneration, is expressed and upregulated in SCs after nerve injury. Cell-type-specific Sarm1 knockout SCs exhibit enhanced axon protection in vitro, and SC- and glia-specific Sarm1 deletion confers axon protection in mouse sciatic nerve and Drosophila wing injury models. Single-nucleus RNA sequencing revealed that Sarm1-deficient SCs are enriched in a distinct cluster expressing genes with developmental roles in axon and myelin protection, with increased oxidative phosphorylation gene expression across all injured SC states. We propose that Sarm1 gates the transition from a Protection-Associated Schwann Cell (PASC) state to a Repair SC state, establishing Sarm1 as a multi-functional regulator with implications for peripheral neuropathies and neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Biomolecular condensates can function as inherent catalysts

We report the discovery that chemical reactions can be catalyzed by condensates formed by intrinsically disordered proteins (IDPs). The proteins themselves lack any catalytic activities. Catalytic functions of condensates emerge as a consequence of sequence-dependent mesoscale electrochemical microenvironments created by phase separation. Stimulated Raman spectroscopy suggests that the catalytic behaviors of condensates are attributable to the spatial variations of water activities across condensate interiors and interfaces. We show that condensates are capable of catalyzing diverse cellularly relevant hydrolysis reactions. Through sequence design, the electrochemical properties of condensates can be programmed to exert control over catalytic behaviors. Incorporation of synthetic condensates into live cells alters transcription profiles and enables the activation of gene circuits that depend on products of hydrolysis reactions catalyzed by condensates. Our discovery of suggests that condensates, depending on their composition-dependent electrochemical properties, can be "Condenzymes", which contribute unexpected emergent chemical functions in cells.

biophysics↗

bioRxiv2024

Genetic architecture of the tomato fruit lipidome; new insights link lipid and volatile compounds

Tomato (Solanum lycopersicum L.) fruit flavor is determined by a combination of multiple volatile compounds, including several derived from lipids and fatty acids. Although fruit flavor has been intensively studied, the linkage between lipid metabolism and flavor remains largely undefined. Here, we performed a genome-wide association study (GWAS) and QTL mapping for the fruit lipid content from 550 tomato accessions and 107 backcross inbred lines (BILs) in two consecutive seasons. Over 130 lipid compounds were identified and mapped, allowing for the identification of over 600 metabolic QTL (mQTL). We further described and validated candidate genes associated with lipid content. Among them is a lipase-like protein (TomLLP) whose function was validated in vivo using overexpression lines in tomato and knockout mutants in Arabidopsis. We also identified functions for three enzymes: a class III lipase (Sl-LIP8), a cyclopropane-fatty-acyl-phospholipid synthase (CFAPS1), and Lipoxygenase C (TomLoxC). By utilizing knockout lines for CFAPS1 and CRISPR-Cas9 loss-of-function lines for Sl-LIP8 and TomLoxC, we demonstrated the functional importance of these enzymes in fruit lipid metabolism. Our study provides a comprehensive analysis of the tomato fruit lipidome and insights into key genes that shaped the natural variation in tomato lipid content and their links to flavor-associated volatile compounds.

molecular biology↗

bioRxiv2024

Diversification of gene expression across extremophytes and stress-sensitive species in the Brassicaceae

Plants are composed of diverse cell types that facilitate adaptations to the environment, yet cross-species comparisons of such response programs at single-cell resolution remain scarce. To explore this diversity, here we profiled >200,000 root cells from five Brassicaceae species, including stress-sensitive species (Arabidopsis thaliana, Sisymbrium irio), extremophytes (Eutrema salsugineum, Schrenkiella parvula), and the polyploid crop Camelina sativa under control, NaCl, and abscisic acid (ABA) treatments. We developed a computational pipeline to characterize the conservation and divergence of cell-type gene expression across the Brassicaceae, revealing that approximately half of previously defined Arabidopsis cell-type markers fail to maintain conserved expression in one or more non-Arabidopsis species. Therefore, we curated a refined set of pan-Brassicaceae markers and identified orthologs whose expression profiles have diverged across lineages. Using in situ hybridization, we mapped distinct cortex subpopulations to specific cortical layers across species, and found flavonoid biosynthesis programs preferentially localized to the inner cortex layer, linking cortex-layer specification to metabolic specialization. Cell-type contributions to stress responses differed among species and across treatments, with lineage-specific losses of responsiveness occurring less frequently but evolutionarily more favored than lineage-specific gains. In C. sativa, sub-genomes contributed equally to stress responses, and homeologs with divergent responses typically lacked other signatures of functional divergence. Together, this work establishes a foundational root single-cell atlas and an analytical framework for multi-species comparative transcriptomics, providing insights into how stress responses diversify across cell types, stress-sensitive to stress-adapted species, and in crop lineages.

plant biology↗

bioRxiv2024

Chronic Social Defeat Stress Gives Rise to Social Avoidance Through Fear Learning

Chronic social defeat stress (CSDS), a widely used rodent model of stress, reliably leads to decreased social interaction in stress susceptible animals. Here, we investigate a role for fear learning in this response using 129Sv/Ev mice, a strain that is more vulnerable to CSDS than the commonly used C57BL/6 strain. We first demonstrate that defeated 129Sv/Ev mice avoid a CD-1 mouse, but not a conspecific, indicating that motivation to socialize is intact in this strain. CD-1 avoidance is characterized by approach behavior that results in running in the opposite direction, activity that is consistent with a threat response. We next test whether CD-1 avoidance is subject to the same behavioral changes found in traditional models of Pavlovian fear conditioning. We find that associative learning occurs across 10 days CSDS, with defeated mice learning to associate the color of the CD-1 coat with threat. This leads to the gradual acquisition of avoidance behavior, a conditioned response that can be extinguished with 7 days of repeated social interaction testing (5 tests/day). Pairing a CD-1 with a tone leads to second-order conditioning, resulting in avoidance of an enclosure without a social target. Finally, we show that social interaction with a conspecific is a highly variable response in defeated mice that may reflect individual differences in generalization of fear to other social targets. Our data indicate that fear conditioning to a social target is a key component of CSDS, implicating the involvement of fear circuits in social avoidance.

animal behavior and cognition↗

bioRxiv2024

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

developmental biology↗

bioRxiv2024

Quantum-classical hybrid approach for codon optimizationand its practical applications

Codon optimization is crucial for gene expression in heterologous hosts with varying genetic codes and codon usage, potentially resulting in enhanced protein expression and stability. Traditionally, the codon optimization problem has been solved using classical numerical techniques; however, with recent advancements, quantum algorithms deployed on quantum computers have been adopted for this purpose. This study proposes a codon sequence search protocol tailored to host preferences. Specifically, codon optimization is formulated as a constrained quadratic binary problem and solved using a quantum-classical hybrid approach, integrating quantum annealing with the Lagrange multiplier method. The proposed methodology is then applied to two real-world scenarios: optimizing the codon sequence of the severe respiratory syndrome coronavirus 2 spike protein in human hosts and insulin in Escherichia coli (E. coli) hosts. Finally, evaluations of several biological metrics demonstrate the effectiveness of our protocol, offering insights into the codon usage patterns governing translational efficiency and adaptation to the genetic code preferences of the host organisms.

bioinformatics↗

bioRxiv2024

Determination of the absolute concentration of Rayleigh particles via scattering microscopy

Nanoparticles are crucial in diverse fields such as healthcare, electronics, and energy. Their small size allows them to cross biological barriers, enhancing drug delivery but also posing health risks. Accurate characterization of nanoparticles is essential for assessing their safety and efficacy, particularly in medical applications. Traditional methods such as dynamic light scattering and mass spectrometry have limitations in sensitivity and range of application. To address these challenges, we introduce the interferometric Concentration Analyzer and Ultrasmall Nanoparticle Tracker (iCAUNT), a technique for detecting and quantifying nanoparticles smaller than one-tenth of the imaging light wavelength. As a non-invasive method, iCAUNT provides precise size and concentration measurements of biological and synthetic nanoparticles, offering significant potential for diagnostics, therapeutics, and broader nanoscience applications.

biophysics↗

bioRxiv2024

Molecular tension authenticates apoptotic cells being phagocytosed.

Profound cytoskeletal reorganization and plasma membrane deformation in phagocytes is indispensable for phagocytosis of massive apoptotic cells, but whether these dynamics accompany a mechanical signal modulating signaling during efferocytosis remains largely unexplored. Here, we report that tension between phosphatidylserine (PS) and PS receptors (PSR) generated at the phagocytic synapse serves as a signal to determine whether PS-exposing cells should be phagocytosed. Mechanistically, increased membrane tension of phagocytes via Rac1-dependent actin polymerization and membrane stiffness of apoptotic cells caused tension between PS and PSRs, leading to phosphoinositide 3-kinase recruitment to PSRs, which resulted in Rac1 inactivation and myosin II phosphorylation required for phagocytic cup closure. Our observations imply that tension between PS and PSRs acts as a mechanical fail-safe to prevent removal of all PS-exposing cells for the integrity of efferocytosis. One-Sentence SummaryTension between phosphatidylserine and its receptors acts as a final decision-maker determining phagocytosis of apoptotic cells.

cell biology↗

bioRxiv2024

Mechanical Rejuvenation of Mesenchymal Stem Cells from Aged Patients

Mesenchymal stem cells (MSC) are an appealing therapeutic cell type for many diseases. However, patients with poor health or advanced age often have MSCs with poor regenerative properties. A major limiter of MSC therapies is cellular senescence, which is marked by limited proliferation capability, diminished multipotency, and reduced regenerative properties. In this work, we explored the ability of applied mechanical forces to reduce cellular senescence in MSCs. Our studies revealed that mechanical conditioning caused a lasting enhancement in proliferation, overall cell culture expansion potential, multipotency, and a reduction of senescence in MSCs from aged donors. Mechanistic studies suggested that these functional enhancements were mediated by oxidative stress and DNA damage repair signaling with mechanical load altering the expression of proteins of the sirtuin pathway, the DNA damage repair protein ATM, and antioxidant proteins. In addition, our results suggest a biophysical mechanism in which mechanical stretch leads to improved recognition of damaged DNA in the nucleus. Analysis of the cells through RNA-seq and ATAC-seq, demonstrated that mechanical loading alters the cells genetic landscape to cause broad shifts in transcriptomic patterns that related to senescence. Overall, our results demonstrate that mechanical conditioning can rejuvenate mesenchymal stem cells derived from aged patients and improve their potential as a therapeutic cell type. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/597781v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@a479d4org.highwire.dtl.DTLVardef@3a728eorg.highwire.dtl.DTLVardef@136dc3dorg.highwire.dtl.DTLVardef@b54d51_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

UNC-6/Netrin promotes both adhesion and directed growth within a single axon

During development axons undergo long-distance migrations as instructed by guidance molecules and their receptors, such as UNC-6/Netrin and UNC-40/DCC. Guidance cues act through long-range diffusive gradients (chemotaxis) or local adhesion (haptotaxis). However, how these discrete modes of action guide axons in vivo is poorly understood. Using time-lapse imaging of axon guidance in C. elegans, we demonstrate that UNC-6 and UNC-40 are required for local adhesion to an intermediate target and subsequent directional growth. Exogenous membrane-tethered UNC-6 is sufficient to mediate adhesion but not directional growth, demonstrating the separability of haptotaxis and chemotaxis. This conclusion is further supported by the endogenous UNC-6 distribution along the axons route. The intermediate and final targets are enriched in UNC-6 and separated by a ventrodorsal UNC-6 gradient. Continuous growth through the gradient requires UNC-40, which recruits UNC-6 to the growth cone tip. Overall, these data suggest that UNC-6 stimulates stepwise haptotaxis and chemotaxis in vivo.

developmental biology↗