Biology subjects

Leu, J.-Y.

Publications and source records attributed to Leu, J.-Y..

11 recordsLinked to original sources

bioRxiv2026

Cellular remodeling during photosymbiosis establishment revealed by single-cell dual proteomics

Endosymbiosis of phytoplankton in heterotrophic hosts is ecologically important and has led to key evolutionary innovations. However, the dynamic molecular processes underlying endosymbiosis establishment remain poorly understood. Here, using large-particle sorting and liquid chromatography-tandem mass spectrometry, we unravel heterogeneous changes in proteomes of the cosmopolitan ciliate Paramecium and algal endosymbiont Chlorella from engulfment to stable endosymbiosis. The initial digestion sees a sharp decline of intracellular Chlorella cells, along with host cellular reorganization involving a reduction of the cortex-localized defensive organelles, trichocysts, and proteins for intracellular transport and recycling. The remaining Chlorella cells enter a bottleneck stage characterized by energy production and cell cycle commitment before active proliferation. Comparison of Paramecium with successful and failed endosymbiosis further identifies a solute carrier transporter that potentially mediates metabolic homeostasis of the endosymbiotic system. Our study reveals inter-organismal coordination during the transition from predator-prey to host-endosymbiont relationships. The approach of time-course single-cell dual proteomics can be useful for investigating diverse interactions between microbial eukaryotes.

microbiology↗

bioRxiv2026

Endosymbiotic algal photosynthesis shapes diel transcriptome architecture in its ciliate host Paramecium bursaria

How photosynthetic endosymbionts reorganize host daily regulation remains unclear. Paramecium bursaria displays pronounced day-night behaviors, but whether its algal symbionts drive host temporal programs has been unresolved. We compared host gene expressions across a 24-hour light-dark cycle in symbiotic and aposymbiotic cells. Symbiotic cells exhibit an expanded and highly temporally ordered diel transcriptome compared with aposymbiotic cells. These rhythmic programs encompass motility, signaling, metabolism, and growth regulation, consistent with observed behaviors. Symbiosis-associated rhythmic programs recruit gene families encoding post-translational regulatory domains, including kinases, ubiquitin-related factors, WD40 scaffolds, and calcium-binding proteins, despite lacking recognizable canonical clock genes. Disrupting photosynthesis with paraquat altered these temporal profiles, shifting them toward an aposymbiotic-like state. A distantly related ciliate, Tetrahymena utriculariae, with an independently evolved symbiosis, showed similar symbiosis-associated daily programs, suggesting that photosynthetic endosymbionts can act as important organizers of host daily gene regulation in endosymbiotic protists.

evolutionary biology↗

bioRxiv2025

Host-derived lipid transfer and metabolic reprogramming in a ciliate-algal symbiosis

Symbiotic associations enable species to integrate complementary traits and adapt to novel environments. However, how the host and endosymbiont exchange nutrients remains poorly understood in most cases. Previously Paramecium bursaria cells were shown to reorganize lipid droplets to accommodate endosymbiotic Chlorella cells and interfering with lipid metabolism reduced the endosymbiont number. Here, we combined transcriptomics, lipidomics, imaging mass spectrometry, and stable-isotope tracing to investigate organic nutrient exchange in this symbiotic system. Our results reveal that endosymbiotic algae undergo extensive reprogramming of lipid metabolic pathways and accumulate markedly higher levels of triglycerides than free-living algae. Isotope-labeling experiments demonstrate that at least some of these lipids originate from the host, providing direct evidence for organic carbon transfer from Paramecium to its algal endosymbionts. Together, our results show that the establishment of symbiosis fundamentally reshapes algal lipid metabolism and uncover an unexpected host-to-symbiont lipid provisioning mechanism--opposite to the canonical direction of carbon flow observed in most photosynthetic symbioses. This work provides new insight into the metabolic principles that sustain and stabilize endosymbiotic partnerships.

microbiology↗

bioRxiv2025

Splicing regulation and intron evolution in the short-intron ciliate model of endosymbiosis Paramecium bursaria

The integration of symbionts into host cells during endosymbiosis significantly alters gene expression and cell physiology. Though alternative splicing facilitates cellular adaptation through rapid modulation of gene expression and protein isoform diversity, its regulatory role during endosymbiosis remains poorly understood. Paramecium bursaria, which harbors hundreds of Chlorella variabilis algae within its cytoplasm, offers a powerful model to study splicing during endosymbiosis, especially given its exceptionally short introns (median [~]24 nt). Using time-course RNA sequencing of symbiotic and aposymbiotic cells, we found that splicing, especially of 5 proximal introns, enhances gene expression. Moreover, we identified 883 genes with differentially spliced introns, particularly enriched in transmembrane transporters essential for establishing nutrient exchange between a host cell and algal symbionts. Splicing regulation correlated with expression changes in conserved spliceosome components, implicating that these factors act as splicing enhancers or repressors during symbiosis. By exploring intron orthology across ciliates, we found that conserved introns exhibited more efficient splicing, characterized by lower GC content and uniform length, suggesting that intron evolution favors features that optimize expression. Our study reveals how splicing contributes to host adaptation during endosymbiosis and highlights the evolutionary dynamics of short introns in eukaryotes. Key pointsParamecium retains conserved core spliceosome genes but employs unique splicing regulation. Alternative splicing regulates metabolism during endosymbiosis, especially for transporter genes. Older introns evolve toward [~]24 nt length and low GC for efficient splicing and expression.

evolutionary biology↗

bioRxiv2025

An early evolutionary stage of mutualistic endosymbiosis reveals a parasitic aspect of endosymbionts

Mutualistic endosymbiosis is a cornerstone of evolutionary innovation, enabling organisms to exploit diverse niches unavailable to individual species. However, our knowledge about the early evolutionary stage of this relationship remains limited. The association between the ciliate Tetrahymena utriculariae and its algal endosymbiont Micractinium tetrahymenae indicates an incipient stage of endosymbiosis. Although T. utriculariae cells rely on endosymbiotic algae to grow in low-oxygen conditions, they gradually lose the endosymbionts in aerobic conditions. Our comparative genomics reveals that the mitochondria-related genes of T. utriculariae are fast-evolving. Symbiotic cells display elongated mitochondria, which interact intimately with endosymbionts. Moreover, inhibiting mitochondrial fatty acid oxidation reduces host fitness but increases the endosymbiont population. Time-series transcriptomics reveal physiological fine-tuning of the host during day and night, underscoring adaptations for nutrient exchange and regulation. Notably, endosymbiotic algae downregulate photosynthesis-related genes compared with free-living cells, correlated with a substantially reduced chlorophyll content. These findings indicate that the endosymbionts exploit host metabolites to supplement reduced photosynthesis. Consistently, symbiotic Tetrahymena cells exhibit lower fitness under aerobic conditions than aposymbiotic cells. Our results support that mutualistic and parasitic relationships between symbiotic organisms are condition-dependent, especially at an early evolutionary stage.

evolutionary biology↗

bioRxiv2025

Spatial proteomics reveals lipid droplet reorganization in symbiotic Paramecium bursaria cells

Endosymbiosis is an important adaptive mechanism allowing organisms to exploit novel niches. The flexible relationship between the ciliate Paramecium bursaria and green algae represents a model system for studying early endosymbiosis evolution. However, the mechanisms underlying how P. bursaria cells maintain this endosymbiotic relationship remain unclear. Here, we use mass spectrometry-based proteomics to generate a spatial proteomic atlas of the host P. bursaria cells with and without endosymbionts. This atlas defines the protein composition of the endosymbiont-containing compartment (perialgal vacuole) and reveals pronounced remodeling of host lipid droplets upon symbiosis. Imaging analyses confirm that lipid droplets change in size, morphology, and positioning, accumulating near intracellular algae. Perturbing symbiotic cells with chemical inhibitors of lipid metabolism reduces endosymbiotic algal numbers, revealing a role for lipid droplets in the host-endosymbiont interaction. Our data provide a comprehensive resource for protein localization in P. bursaria cells and elucidate how those cells remodel an existing cellular compartment to maintain endosymbiosis.

biochemistry↗

bioRxiv2025

Orthologous transcription factor replacement reveals that stable TFIIIC complexes are required for proper mitotic chromosome segregation

Transcription factors are speculated to play crucial roles in adaptive evolution. Using ortholog replacement of essential transcription factors (eTFs) from other yeast species, we investigated how eTFs can change. Several orthologs could not fully complement Saccharomyces cerevisiae mutants, indicating that functions or interactions of these eTFs have changed, rendering them incompatible. We further characterized TFIIIC, a fast-evolving protein complex that assists RNA polymerase III-mediated transcription, which exhibited complete or partial incompatibility in several subunits. In the orthologous Tfc7-replacement line, binding of TFIIIC to tRNA genes was reduced, yet tRNA abundance was not severely affected. However, the chromosomes of Tfc7-replacement cells were often mis-segregated during mitosis and their fitness was further reduced in a spindle checkpoint mutant. Our chromatin-immunoprecipitation experiments uncovered that unstable TFIIIC binding results in defective cohesion loading, leading to chromosome mis-segregation. Swapping the highly divergent C-terminal domain of Tfc7 orthologs rescued its interaction with Tfc1 and cell fitness, supporting that incompatibility is caused by altered interactions between complex subunits. Our results reveal distinct essential functions of a well-studied protein complex.

evolutionary biology↗

bioRxiv2022

Rapid compensatory evolution by secondary perturbation of a primary disrupted transcriptional network

The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection over long time-scales, especially when a regulon is being transferred from one transcription factor (TF) to another. Cooperative DNA binding between redundant regulatory components at the intermediate transition stage is believed to mediate this process, enabling a conflict-free switch between two TFs without a disruptive change in gene expression. Here, we have performed an evolutionary repair experiment on the Lachancea kluyveri yeast sef1{Delta} mutant by means of a suppressor development strategy. Complete loss of SEF1 forced cells to activate a rewiring process to compensate for the pleiotropic defects arising from misexpression of multiple TCA cycle genes. Using different selective conditions, we identified one generalist and one specialist suppressive loss-of-function mutation of IRA1 and AZF1, respectively. Our subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for both the compensatory and trade-off phenotypes. Our results indicate that the pleiotropic effects of dual perturbation of transcriptional networks are a potential mechanism for rapid adaptive compensation, facilitating the process of incipient transcriptional rewiring, and formation of complex traits.

evolutionary biology↗

bioRxiv2020

Differential Hsp90-dependent gene expression is strain-specific and common among yeast strains

Enhanced phenotypic diversity increases the likelihood of a population surviving catastrophic conditions. It has been suggested that Hsp90, an essential molecular chaperone in eukaryotes, can suppress (i.e., buffer) or enhance (i.e., potentiate) the effects of genetic variation, enabling organisms to adjust their levels of phenotypic diversity in response to environmental cues. Many Hsp90-interacting proteins are involved in signaling transduction pathways and transcriptional regulation. However, it remains unclear if Hsp90-dependent differential gene expression is common in natural populations. By examining the gene expression profiles of five diverse yeast strains, we identified many genes exhibiting Hsp90-dependent strain-specific differential expression. We employed an analysis pipeline to identify transcription factors (TFs) potentially contributing to variable expression. We found that upon Hsp90 inhibition or heat stress, activities or abundances of Hsp90-dependent TFs varied among strains, resulting in differential strain-specific expression of their target genes, which consequently led to phenotypic diversity. We provide evidence that individual populations can readily display specific Hsp90-dependent gene expression, suggesting that the evolutionary impacts of Hsp90 are widespread in nature. HighlightsO_LIHsp90-dependent gene expression varies among different yeast strains. C_LIO_LIHsp90 differentially influences transcriptional activity or protein abundances of transcription factors among yeast strains. C_LIO_LIDifferential strain-specific gene expression is correlated with phenotypic variations upon Hsp90 inhibition. C_LIO_LIHsp90-mediated strain-specific regulation manifests under environmental stress. C_LI

evolutionary biology↗

bioRxiv2019

Engineering recombination between diverged yeast species reveals genetic incompatibilities

The major cause of the sterility of F1 hybrids formed between Saccharomyces cerevisiae and Saccharomyces paradoxus is anti-recombination. The failure of homologous chromosomes from the different species to recombine causes them to mis-segregate, resulting in aneuploid gametes, most of which are inviable. These effects of anti-recombination have previously impeded the search for other forms of incompatibility, such as negative genetic interactions (Bateson-Dobzhoansky-Muller incompatibilities). By suppressing the meiotic expression of MSH2 and SGS1, we could increase recombination and improve hybrid fertility seventy-fold. This allowed us to recover meiotic tetrads in which all four gametes were viable, ensuring that segregation had occurred properly to produce perfectly haploid, not aneuploid, recombinant hybrid gametes. We sequenced the genomes of 84 such tetrads, and discovered that some combinations of alleles from different species were significantly under-represented, indicating that there are incompatible genes contributing to reproductive isolation.

evolutionary biology↗

bioRxiv2019

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and noise buffering is primarily achieved via two Hmt1 methylation targets. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.\n\nAuthor SummaryCell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise and identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 is a central hub protein that is involved in multiple basic cellular pathways, including chromatin remodeling/transcription, translation, ribosome biogenesis, and post-transcriptional regulation. Our results show that Hmt1 constrains the noise level of multiple cellular pathways under normal conditions, so the physiology of individual cells in a population will not deviate too much from optimal peak fitness. However, when cells encounter environmental stresses, HMT1 is quickly down-regulated and expression noise is enhanced to increase the likelihood of population survival. Moreover, the noise buffering function of Hmt1 is conserved in Schizosaccharomyces pombe that diverged from the common ancestor of Saccharomyces cerevisiae more than 400 million years ago. Since the Hmt1 network is conserved from yeast cells to human, it is quite possible that Hmt1-mediated noise buffering also operates in multicellular organisms.

molecular biology↗