Biology subjects

Hwang, J. Y.

Publications and source records attributed to Hwang, J. Y..

11 recordsLinked to original sources

bioRxiv2024

Extracellular domains of CATSPERε are essential for CatSper assembly during development and activity modulation in sperm capacitation

The flagellar-specific Ca2+ channel CatSper is a multiprotein complex that is critical for successful fertilization by controlling the sperm Ca2+ signaling in space and time. Large extracellular domains (ECDs) of four single-pass transmembrane subunits, CATSPER{beta}, {gamma}, {delta}, and {varepsilon}, form a unique canopy structure over the pore-forming channel. However, the molecular mechanisms of canopy assembly during development and its physiological function in mature sperm remain unknown. Here, using two genetic mouse models and the biochemical isolation of a bioactive CATSPER{varepsilon} fragment, we report that CATSPER{varepsilon} ECDs are essential for assembling the CatSper canopy, and thus the entire channel complex, and for modulating CatSper function for sperm hyperactivation and fertilization. CATSPER{varepsilon}-deficient males are sterile because their sperm fail to develop hyperactivated motility due to the absence of the entire channel. In transgenic mice overexpressing CATSPER{varepsilon} with truncated ECDs in testicular germ cells, truncated CATSPER{varepsilon} is unable to interact with native CatSper subunits and incorporate into the complex, thus failing to rescue the defective sperm hyperactivation and infertility of Catspere-null males. These findings provide insight into the underlying molecular and developmental mechanisms of CatSper complex assembly and how CatSper channels can be modulated in physiological settings and by therapeutic intervention.

cell biology↗

bioRxiv2023

LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.

cell biology↗

bioRxiv2022

Non-catastrophic collapse of VEEV by lethal mutagenesis

RNA viruses replicate at a high mutation rate, providing a highly adaptive capacity to the population as well as vulnerability to an extinction due to additional mutations, which is the conceptual basis for lethal mutagenesis. However, the mechanistic understanding of lethal mutagenesis has remained unclear due to the lack of RNA mutagens with potent antiviral activity and the inherent inability to distinguish between infectious and non-infectious sub-populations within a viral population. In our study, we investigated how the replication competency and mutation frequency of Venezuelan Equine Encephalitis Virus (VEEV) change following treatment with the RNA mutagen {beta}-d-N4-hydroxycytidine, a potent antiviral RNA mutagen. By pairing limiting dilution with a long-read sequencing approach, we specifically determined genomic sequences of replication-competent viral clones from the total population. We found that replication-competent VEEV population maintained itself within a narrow mutation spectrum with a significantly lower mutation frequency than the total population. We also found that treatment with an RNA mutagen did not induce catastrophic destruction of the infectious population even at a high exposure, allowing replication-competent subpopulations with increased variability in mutational fitness and enhanced adaptability to new selection pressures, such as antiviral treatment, to continue to propagate. Together our study provides new understanding of viral population landscape and suggests a careful consideration of lethal mutagenesis as an antiviral strategy for high-capacity replicating viruses such as alphaviruses.

microbiology↗

bioRxiv2022

Glia-mediated cerebellar pain modulation

Clinical studies have revealed that the cerebellum is activated by noxious stimuli or pathological pain, and its removal results in somatosensory dysfunction. However, the neural circuits and molecular mechanisms underlying the processing of noxious information in the cerebellum remain unknown. Using two-photon microscopy and optogenetics in mice, we found that the locus coeruleus (LC) terminals in the cerebellar cortex release noradrenaline (NA) in response to cutaneous noxious electrical stimuli. Most Bergmann glia (BG) accumulated this LC-NA noxious information by increasing intracellular calcium in an integrative manner. This global calcium activation of BG, referred to as "flare," was also elicited in response to an intraplantar capsaicin injection. Chemogenetic inactivation of LC terminals or BG in the cerebellar cortex suppressed BG flares and reduced licking, a nocifensive behavior associated with capsaicin-induced pain. BG-specific knockdown of -1 adrenergic receptors also suppressed capsaicin-induced BG flares and licking. Chemogenetic activation of BG or an intraplantar capsaicin injection reduced Purkinje cell firings, which disinhibited the output activity of the deep cerebellar nuclei. These results suggest that BG in the cerebellar cortex play an essential role in computing noxious information ascending from the LC and modulate pain-related behaviors by controlling the activity of the cerebellar neural circuits. One Sentence SummaryBergmann glia mediate noxious information processing in the cerebellum

neuroscience↗

bioRxiv2021

TXNRD3 supports male fertility via the redox control of spermatogenesis

Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

biochemistry↗

bioRxiv2021

C2cd6-encoded CatSper{tau} Targets Sperm Calcium Channel to Ca2+ Signaling Domains in the Flagellar Membrane

In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSper{tau}, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSper{tau} loss-of-function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSper{tau} targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSper{tau} interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting. HighlightsO_LICatSper{tau} encoded by C2cd6 is a C2 membrane-associating domain containing protein C_LIO_LICatSper{tau} loss-of-function impairs sperm hyperactivation and male fertility C_LIO_LICatSper{tau} adopts ciliary trafficking machineries for flagellar targeting via C2 domain C_LIO_LICatSper{tau} targets the CatSper channel into nanodomains of developing sperm flagella C_LI

cell biology↗

bioRxiv2021

3D structure and in situ arrangements of CatSper channel in the sperm flagellum

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSpers complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

biophysics↗

bioRxiv2021

The Global Protein-RNA Interaction map of Epithelial Splicing Regulatory Protein 1 defines a post-transcriptional program that is essential for epithelial cell function

The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3 and 5 untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.

molecular biology↗

bioRxiv2020

3D in situ imaging of female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice

Out of millions of ejaculated sperm, only a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here, using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that all spermatozoa past the UTJ possess intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

cell biology↗

bioRxiv2019

Neuropilin-1 regulates the secondary CD8 T cell response to virus infection

Neuropilin-1 plays important roles in axonal guidance in neurons, and in the growth of new blood vessels. There is also a growing appreciation for roles played by neuropilin-1 in the immune response. This molecule is important for the function of regulatory T cells, however roles in other T cell populations have not been identified. Here we show that neuropilin-1 is expressed during the peak of the antiviral CD8 T cell response during murine gammaherpesvirus infection. Using a conditional knockout model, we deleted Nrp1 either before infection, or after CD8 T cell memory had been established. We found deletion of Nrp1 skewed the acute CD8 T cell response toward a memory precursor-like phenotype, however the ensuing resting memory response was similar regardless of Nrp1 expression. Interestingly Nrp1 deletion had differing effects on the recall response depending on the timing of deletion. When deleted before infection, Nrp1 deficiency inhibited the secondary response. Deletion just prior to re-exposure to virus lead to an enhanced secondary response. Interestingly these effects were observed only in mice infected with a persistent strain of murine gammaherpesvirus, and not a non-persistent mutant strain. These data highlight a multifaceted role for neuropilin-1 in memory CD8 T cell differentiation, dependent upon the stage of the T cell response and characteristics of the infectious agent. Several therapeutic anti-cancer therapies focus on inhibition of Nrp1 to restrict tumor growth, so knowledge of how Nrp1 blockade may affect the CD8 T cell response will provide a better understanding of treatment consequences.\n\nImportanceCD8 T cell responses are critical to control both virus infections and tumors. The ability of these cells to persist for long periods of time can result in lifelong immunity, as relatively small populations of cell can expand rapidly to counter re-exposure to the same insult. Understanding the molecules necessary for this rapid secondary expansion is critical if we are to develop therapies that can provide lifelong protection. This report shows an important and complex role for the molecule neuropilin-1 in the secondary response. Several cancer therapies targeting neuropilin-1 are in development, and this work will lead to better understanding of the effect these therapies could have upon the protective CD8 T cell response.

immunology↗

bioRxiv2018

EFCAB9 is a pH-Dependent Ca2+ Sensor that Regulates CatSper Channel Activity and Sperm Motility

SummaryVarying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calciumbinding domain-containing protein 9 (EFCAB9) as a dual function, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CAT SPER{zeta}, is essential for pH-dependent and Ca2+ sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPER{zeta} in a Ca2+ dependent manner and dissociates at elevated pH.These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.\n\nHighlightsO_LIEfcab9 encodes an evolutionarily conserved, sperm-specific EF-hand domain protein\nC_LIO_LIEfcab9-deficient mice have sperm motility defects and reduced male fertility\nC_LIO_LIEFCAB9 is a pH-tuned Ca2+ sensor for flagellar CatSper Ca2+ channel\nC_LIO_LIEFCAB9 is a dual function machine in gatekeeping and domain organization of CatSper\nC_LI

cell biology↗