Biology subjects

Lee, W.-J.

Publications and source records attributed to Lee, W.-J..

11 recordsLinked to original sources

bioRxiv2026

Systematic analysis reveals an Mln1-dependent trade-off between metabolic flexibility and thermotolerance in Cryptococcus neoformans

During infection, fungal pathogens must adapt to multiple environmental challenges, including changes in temperature, nutrient availability, and immune stress. Although transcriptional regulators are central to these adaptations, how individual regulators contribute to distinct environmental responses and how these responses are functionally interconnected remain poorly understood. Here, we investigated the functions of infection-associated transcription factors under distinct host-relevant conditions in Cryptococcus neoformans. Integrating in vivo expression profiling with infectivity data from signature-tagged mutagenesis, we prioritized 24 transcription factors, 12 of which were induced under host-mimicking conditions. Initial functional analyses revealed distinct roles for these transcription factors in adaptation to temperature, carbon, and nitrogen availability. Further characterization of Mln1, a fungal basic helix-loop-helix transcription factor, uncovered an unexpected connection between carbon source adaptation and thermotolerance. Mig1-dependent repression limited MLN1 expression in the presence of glucose, whereas Mln1 accumulated in the nucleus in the presence of trehalose or ethanol, but not glucose. Loss of MLN1 impaired carbon-starvation gene expression and growth on non-preferred carbon sources, particularly trehalose. Conversely, mln1{Delta} cells accumulated more intracellular trehalose and exhibited enhanced thermotolerance, yet showed reduced melanization, titan cell formation, virulence in Drosophila, and brain fungal burden in mice. Together, these results suggest that Mln1 contributes to metabolic flexibility and fungal virulence while differentially influencing thermotolerance. More broadly, these findings highlight a potential physiological trade-off between nutrient utilization and stress resistance, suggesting that these adaptive processes may be interconnected in fungal pathogens.

microbiology↗

bioRxiv2026

A convergent proline biosynthetic step couples cell wall integrity to virulence in Cryptococcus neoformans

Metabolic adaptation is essential for the pathogenicity of the opportunistic fungus Cryptococcus neoformans. Although amino acid biosynthesis pathways are critical fitness determinants in fungal pathogens, the physiological contributions of proline biosynthesis remain incompletely understood, with most studies focusing on proline catabolism. Here, we characterized the proline biosynthetic enzymes Pro1, Pro2, and Pro3 in C. neoformans. Proline is produced by two routes that converge on {Delta}1-pyrroline-5-carboxylate (P5C): a de novo pathway from glutamate via Pro1 and Pro2, and an arginine-derived bypass via Car1 and Car2--both requiring Pro3 for the final reduction of P5C to proline. Although all biosynthetic mutants were strictly auxotrophic for proline, their requirements for virulence differed markedly: in a murine infection model, pro1{Delta} and pro2{Delta} mutants retained full virulence, whereas pro3{Delta} exhibited significantly attenuated virulence accompanied by growth defects and impaired cell wall integrity. Genetic disruption of P5C-generating routes partially restored distinct pro3{Delta} phenotypes, with PRO2 deletion alleviating delayed growth and combined PRO2 and CAR2 deletion partially restoring cell size, while cell wall defects remained unresolved. This dissociation indicates that growth requires proper balance of P5C-generating metabolic flux, whereas cell wall integrity depends on a distinct mechanism that is not resolved by blocking P5C production. Together, these findings identify Pro3 as a metabolic gatekeeper coupling proline biosynthesis to cell wall homeostasis, establishing the biosynthetic branch of proline metabolism as a previously unappreciated determinant of fungal pathobiology.

microbiology↗

bioRxiv2026

The protein phosphatase 2A catalytic subunit Pph22 partners with Tap42 and Rrd2 to control viability and virulence in Cryptococcus neoformans

Protein phosphatase 2A regulates eukaryotic signaling, but the function of its associated regulators remains poorly understood in the human fungal pathogen Cryptococcus neoformans. Here, we used affinity purification coupled with mass spectrometry and identified 81 candidate proteins associated with the catalytic subunit Pph22, including the scaffold subunit Tpd3, the regulator Tap42, and the protein phosphatase methylesterase 1. Conditional-expression and meiotic spore analyses supported an essential role for TAP42 in viability. We identified CNAG_05505 as the Rrd2 ortholog and detected an interaction between Rrd2 and Pph22 in a yeast two-hybrid assay. Deletion of RRD2 impaired thermotolerance, altered responses to multiple stresses and antifungal agents, reduced melanin and capsule production, delayed sexual development, and inhibited titan cell formation. Mutants also displayed altered cell wall staining and reduced expression of genes involved in chitin synthesis and deacetylation. RRD2 deletion increased basal Hog1 phosphorylation, whereas PPH22 overexpression reduced it. Double-mutant analysis revealed phenotype-dependent genetic interactions between RRD2 and HOG1. Although RRD2 deletion did not significantly alter overall survival in the Drosophila infection assay, it markedly attenuated virulence in mice: all mutant-infected mice survived the 70-day observation period, and fungal burdens were reduced across multiple organs at 21 days post-infection. These findings distinguish the essential contribution of Tap42 from the broad roles of Rrd2 in stress adaptation, morphogenesis, cell surface organization, and mammalian virulence, extending the functional analysis of the cryptococcal protein phosphatase 2A regulatory network.

microbiology↗

bioRxiv2026

Systematic profiling of WD40 proteins reveals Wcp1, a cyclophilin linking CO2/heat tolerance to acidic pH adaptation in Cryptococcus neoformans

WD40 domains are major protein-protein interaction (PPI) scaffolds, yet their contributions to fungal pathogenicity remain poorly defined. We systematically analysed 94 canonical WD40 proteins in Cryptococcus neoformans. Conditional knockdown and sporulation identified 36 essential WD40 proteins, while in vitro and in vivo profiling of 103 signature-tagged deletion strains spanning 52 genes uncovered 31 pathogenicity-related WD40 proteins, including epigenetic and post-transcriptional regulators. We identified Wcp1, a dual-domain protein whose WD40-repeat and cyclophilin domains are required for growth at 37{degrees}C under 5% CO2. Its WD40 scaffold and PPIase domain supported CO2/heat tolerance and virulence. Notably, Wcp1 couples these functions to acidic pH adaptation: wcp1{Delta} failed to grow under elevated temperature and CO2 at acidic pH, exhibited enhanced intracellular acidification, reduced macrophage survival and attenuated virulence in Drosophila and mice. Integrated transcriptomic and proteomic analyses place Wcp1 at the centre of intracellular pH homeostasis, coordinating proton transport, metabolic adaptation and stress-buffering networks.

microbiology↗

bioRxiv2026

Modeling echolocation as an active pursuit of information via infotaxis

Echolocation is a closed-loop active sensing modality in which animals not only choose how they move to acquire information, but also actively modulate incoming sensory (echo) information by shaping the acoustic signals they emit to probe the environment. While many models describe how echolocating animals react to prior echoes by adjusting subsequent behavior, few explicitly model how they cognitively reason about information embedded in echoes when determining future actions. Here, we extend "infotaxis," an information-greedy algorithm originally developed for olfactory search, to sonar sensing by formulating an echolocating agent searching for a single target under sensory uncertainty characterized by probabilities of miss and false alarm. Through analytical and computational analyses, we show that the characteristic exploration-exploitation balance of infotaxis also emerges in echolocation, and that the efficiency and reliability of infotaxis search depend strongly on sensory information quality. Compared with a maximum a posteriori agent that always directs the beam to the most probable target location, the infotaxis agent consistently completes searches with fewer pings and greater robustness to sensory uncertainty. These results highlight information as a powerful concept for understanding active sensing and developing models for sonar-guided autonomy in both biological and engineered systems.

animal behavior and cognition↗

bioRxiv2025

Head-related transfer function predictions reveal dominant sound transmission mechanisms in a dolphin head

Toothed whales possess specialized anatomical structures in the head, including thin, excavated lower mandible embedded in mandibular fat bodies, complex skull morphology fused with the upper jaw, and extensive air spaces surrounding the middle ears and beneath the skull. In this study, we use finite element modeling to investigate how these structures influence the transmission of water-borne sounds to the ears. The models are based on volumetric representations derived from computed tomography (CT) scans of a live bottlenose dolphin (Tursiops truncatus). We iteratively modify the anatomical structures included in the model and use the predicted head-related transfer functions (HRTFs) as a proxy for comparison. Our results show that the mandibular fat bodies, which support a lower sound speed than the surrounding tissues, significantly enhance the forward receiving directionality at echolocation frequencies through refraction, in a manner similar to the melon in shaping the dolphins highly directional transmission beams. Additionally, we show that, in the frequencies encompassing dolphin communication signals, the air volumes help block the otherwise complex sound transmission through the bones. These findings highlight convergent evolutionary solutions in toothed whale anatomy to create strong directionality in both sound emission and reception governed by the same physical principles.

biophysics↗

bioRxiv2025

Influence of duty-cycle recording on measuring bat activity in passive acoustic monitoring

Echolocating bats provide vital ecosystem services and can be monitored effectively using passive acoustic monitoring (PAM) techniques. Duty-cycle subsampling is widely used to collect PAM data at regular ON/OFF cycles to circumvent battery and storage capacity constraints for long-term monitoring. However, the impact of duty-cycle subsampling and potential detector errors on estimating bat activity has not been systematically investigated for bats. Here, we simulate the influence of duty-cycle subsampling in measuring bat activity via three metrics-Call Rate (CR), Activity Index (AI), and Bout-Time Percentage (BTP)-using three months of continuous recordings spanning summer to fall in a temperate urban natural area. Our simulations show that subsampled bat activity estimates more accurately track true values when the listening ratio is high and the cycle length is low, when the true call activity is high, or when recorded calls have lower frequency content. Generally, among the three metrics, AI provides the best subsampling estimates and is robust against false negatives but sensitive to false positives, whereas BTP provides better temporal resolution compared to AI and is robust against both false positives and false negatives. Our results offer important insights into selecting sampling parameters and measurement metrics for long-term bat PAM.

ecology↗

bioRxiv2025

Real-time spike sorting with 3D neural probe and triangulation localization

A key challenge in correlating neuronal activity with brain function is the limited sampling probability of neuronal activity in real time. It is crucial to increase the sampling probability substantially and in real time. We hypothesized that 3-dimensional (3D) neural probes offer faster and stronger prospects for cell yield than 2D electrode arrays. We simulated a 1000-neuron neuronal network, mimicking the granular layer of the barrel cortical column, recorded signals from inserted 384 electrodes (organized in 3D or 2D), and sorted units using Kilosrt or triangulation localization. We demonstrated that 3D electrode arrays converge more space for triangulation spike sorting than 2D probes do. 3D neural probes, together with triangulation, could isolate up to 80% of the simulated 1000 neurons (as ground truth) and have a cell yield of up to 5, which is, to the best of our knowledge, significantly higher than standard 2D electrodes with Kilosort or triangulation. With a signal-to-noise ratio (SNR) of 10, which is close to the real world, the simulation data suggest that 3D electrode arrays in a face-centric cubic (FCC) arrangement provide a better cell yield. However, larger background noise (e.g. an SNR of 1, which can be improved with lower electrode impedance) has a stronger impact on the triangulation spike sorting. Since only the peak value of spikes are required for triangulation localization, the computing loading is much less than spike waveform-based spike sorting approach. Thus, combining 3D electrode arrays with triangulation localization is ideal for real-time spike sorting. Thus, we demonstrated that adding one more dimension in designing neural probes can dramatically increase cell yield and speed up isolating neuronal unit activity. We, for the first time, provide a tool for utilizing computer simulations to optimize the design of neural electrode arrays before time-consuming probe fabrication.

neuroscience↗

bioRxiv2025

The calcineurin pathway regulates extreme thermotolerance, cell membrane and wall integrity, antifungal resistance, and virulence in Candida auris

Candida auris, an emerging fungal pathogen characterized by its multidrug resistance and high mortality rates, poses a significant public health challenge. Despite its importance, the signaling pathways governing virulence and antifungal resistance in C. auris remain poorly understood. This study investigates the calcineurin pathway in C. auris, critical for virulence and antifungal resistance in other fungal pathogens. Calcineurin, a calcium/calmodulin-dependent protein phosphatase, comprises a catalytic subunit (Cna1) and a regulatory subunit (Cnb1) in C. auris. Our findings reveal that deletion of CNA1 or CNB1 disrupts extreme thermotolerance and cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins. Moreover, we identified a downstream transcription factor, Crz1, which plays a central role in this pathway in other fungal species. Deletion of CRZ1 resulted in similar membrane integrity defects observed in the cna1{Delta} and cnb1{Delta} mutants and increased susceptibility to azole drugs. Supporting it, fluconazole treatment induced Crz1 nuclear translocation in a Cna1-dependent manner. However, unlike cna1{Delta} and cnb1{Delta} mutants, the crz1{Delta} mutant displayed increased resistance to echinocandins, suggesting the opposing roles for Crz1 in regulating cell wall integrity. Nevertheless, echinocandins also promoted Crz1 nuclear translocation via Cna1, underscoring the complex regulatory mechanisms at play. Cna1 was found to be required for virulence in both the Drosophila systemic infection model and the murine skin infection model. However, in a systemic murine infection model, both calcineurin and Crz1 appeared dispensable for C. auris virulence. Our findings highlight that the evolutionarily conserved calcineurin pathway employs distinct regulatory mechanisms to perform divergent roles in regulating cell wall and membrane integrity, antifungal drug resistance, and virulence in C. auris. Author SummaryThe fungal pathogen Candida auris presents a global health threat due to its multidrug resistance and high mortality rates. Despite its clinical significance, the molecular mechanisms underlying its virulence and antifungal resistance remain poorly understood. This study investigates the complex role of the calcineurin signaling pathway in C. auris pathogenicity. Deletion of the calcineurin complex impairs extreme thermotolerance and compromises cell membrane and wall integrity, leading to increased susceptibility to azoles and echinocandins, antifungal agents targeting the cell membrane and wall, respectively. We also identified the Crz1 transcription factor as a downstream target of calcineurin signaling. Interestingly, unlike calcineurin mutants, Crz1 mutants are susceptible only to cell-membrane-targeting azoles but surprisingly exhibit increased resistance to cell-wall-targeting echinocandins, suggesting that the calcineurin pathway may regulate multiple transcription factors, including Crz1. Additionally, calcineurin was found to be essential for virulence in vivo, using two different animal infection models, Drosophila and mice. These findings highlight the essential role of the calcineurin pathway in C. auris virulence, offering novel insights into its role in antifungal resistance and virulence, and paving the way for targeted therapies.

microbiology↗

bioRxiv2019

Coordination between stabilizing circuits and updating circuits in spatial orientation working memory

Spatial orientation memory plays a crucial role in animal navigation. Recent studies of tethered Drosophila melanogaster (fruit fly) in a virtual reality setting showed that the head direction is encoded in the form of an activity bump, i.e. localized neural activity, in the torus-shaped ellipsoid body (EB). However, how this system is involved in orientation working memory is not well understood. We investigated this question using free moving flies (Drosophila melanogaster) in a spatial orientation memory task by manipulating two EB subsystems, C and P circuits, which are hypothesized for stabilizing and updating the activity bump, respectively. To this end, we suppressed or activated two types of inhibitory ring neurons (EIP and P) which innervate EB, and we discovered that manipulating the two inhibitory neuron types produced distinct behavioral deficits, suggesting specific roles of the inhibitory neurons in coordinating the stabilization and updating functions of the EB circuits. We further elucidate the neural mechanisms underlying such control circuits using a connectome-constrained spiking neural network model. Significance statementHead-direction (HD) system has been discovered in rodents for decades. But the detailed neural circuit mechanisms underlying the HD system were only described recently by studies of fruit flies on the similar HD system. However, how this fruit fly HD system involves in orientation memory was not well investigated. The present study addresses this question by investigating free moving flies in a spatial orientation working memory task. By combining neural functional experiments and neural circuit modelling, the study shows how disrupting either of the two subcircuits, one stabilizing and the other updating the neural activity, in the HD system leads to different behavioral impairments. The result suggests specific roles of the HD subcircuits in the spatial orientation working memory. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/819185v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@101cb04org.highwire.dtl.DTLVardef@b7ac8borg.highwire.dtl.DTLVardef@a10a41org.highwire.dtl.DTLVardef@a8691e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2018

Naa10p promotes metastasis by stabilizing matrix metalloproteinase-2 protein in human osteosarcomas

N--Acetyltransferase 10 protein (Naa10p) mediates N-terminal acetylation of nascent proteins. Oncogenic or tumor suppressive roles of Naa10p were reported in cancers. Here, we report an oncogenic role of Naa10p in promoting metastasis of osteosarcomas. Higher NAA10 transcripts were observed in metastatic osteosarcoma tissues compared to non-metastatic tissues and were also correlated with a worse prognosis of patients. Knockdown and overexpression of Naa10p in osteosarcoma cells respectively led to decreased and increased cell migratory/invasive abilities. Re-expression of Naa10p, but not an enzymatically inactive mutant, relieved suppression of the invasive ability in vitro and metastasis in vivo imposed by Naa10p-knockdown. According to protease array screening, we identified that matrix metalloproteinase (MMP)-2 was responsible for the Naa10p-induced invasive phenotype. Naa10p was directly associated with MMP-2 protein through its acetyltransferase domain and maintained MMP-2 protein stability via NatA complex activity. MMP-2 expression levels were also significantly correlated with Naa10p levels in osteosarcoma tissues. These results reveal a novel function of Naa10p in the regulation of cell invasiveness by preventing MMP-2 protein degradation that is crucial during osteosarcoma metastasis.

cancer biology↗