Biology subjects

Yamano, T.

Publications and source records attributed to Yamano, T..

11 recordsLinked to original sources

bioRxiv2026

A CO2-limitation-induced cytosolic repressor enables shutdown of the algal CO2-concentrating mechanism

Aquatic photosynthetic organisms face limited CO2 availability because CO2 diffuses slowly in water and most dissolved inorganic carbon (Ci) exists as HCO3- at physiological pH. To overcome this limitation, aquatic photoautotrophs operate CO2-concentrating mechanisms (CCMs) that elevate CO2 around Rubisco and sustain carbon fixation. Because CCM operation consumes energy, it must be suppressed when CO2 becomes abundant, but how this shutdown occurs remains poorly understood. In Chlamydomonas reinhardtii, the nuclear protein CBP1 was identified as a CCM repressor, but its loss causes only partial derepression under high CO2, indicating that an additional mechanism is required for complete shutdown. Here, we identify High-Affinity CCM Repressor 1 (HCR1), a cytosolic protein related to CBP1, as a second repressor. Under high CO2, hcr1 mutants retained high affinity for Ci and derepressed CCM and photoacclimation genes. Combined disruption of HCR1 and CBP1 further increased Ci affinity, approaching that of wild-type cells with a fully induced CCM under CO2 limitation, and promoted the accumulation of Ci transporters. HCR1 loss also prevented redistribution of the chloroplast regulator CAS away from the pyrenoid and was accompanied by retention of a pyrenoid starch sheath. In contrast, LCIB, a chloroplast CO2-recapture protein, relocated normally. Unexpectedly, HCR1 accumulated during CO2 limitation and declined after transfer to high CO2. These results show that CCM shutdown is an active transition rather than the passive reversal of induction. We propose that CBP1 restrains CCM1-dependent transcription, while HCR1 is preloaded during CO2 limitation to terminate the CAS-associated, starch-sheathed, high-affinity state when CO2 becomes replete.

plant biology↗

bioRxiv2026

Unsupervised machine-learning identifies latent pyrenoid states linked to mitotic remodeling defects and CO2-dependent growth

Biomolecular condensates that persist through cell division must be reorganized and inherited, yet it remains unclear whether subtle defects before division are associated with later organelle or growth phenotypes. We examined the Chlamydomonas reinhardtii pyrenoid, a liquid-like condensate that concentrates ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the photosynthetic CO2-fixing enzyme. As part of the algal CO2-concentrating mechanism, the pyrenoid raises CO2 availability around Rubisco. We generated an RBCS1-mGold Rubisco reporter and developed an unsupervised image-analysis pipeline combining a convolutional autoencoder and a one-class support vector machine. Using 4,905 wild-type single-cell images, augmented 22-fold to 107,910 image instances, we defined the range of normal pyrenoid morphology. A combined machine-learning and visual screen of approximately 21,000 insertional mutants yielded 17 pyrenoid integrity mutants (pim1-pim17). Differential reconstruction-error maps highlighted local deviations from the wild-type reference, including phenotypes difficult to classify by eye. Four-dimensional live imaging showed defects in matrix dispersal, partitioning of Rubisco-containing foci, or pyrenoid recondensation in multiple pim strains. Growth assays identified broad defects and phenotypes that became more apparent as CO2 supply decreased. Insertion-site mapping nominated candidate loci, including STT7, which encodes a chloroplast kinase best known for regulating photosynthetic light harvesting. Independent STT7-edited lines lacked detectable STT7 accumulation and showed pyrenoid-region reconstruction-error patterns, supporting an association between impaired STT7 function and altered pyrenoid morphology. These findings show that unsupervised image screening can extend forward genetics to subtle pyrenoid phenotypes accompanied by mitotic remodeling or growth defects.

cell biology↗

bioRxiv2026

Modular mRNA platform for in vivo antigen-specific immune tolerance

Immune-mediated diseases such as autoimmunity and allergy arise from dysregulated antigen-specific responses, yet current therapies largely rely on broad immunosuppression and rarely re-establish durable, antigen-selective tolerance. Here we introduce Tol-mRNA, a modular tolerogenic mRNA platform designed to deliver multiple immune-regulatory signals in vivo to program antigen-specific tolerance. Tol-mRNA co-encodes an antigen together with two complementary regulatory cues, PD-L1 and TGF-{beta}, enabling coordinated antigen presentation and tolerogenic signaling that robustly induces antigen-specific Tregs and suppresses pathogenic effector responses. In murine disease models, Tol-mRNA conferred therapeutic benefit across distinct immune pathologies. In experimental autoimmune encephalomyelitis, Tol-mRNA ameliorated clinical disease and reduced inflammatory immune activation. In an ovalbumin-driven food allergy model, Tol-mRNA prevented allergic symptoms and dampened type 2 inflammation. Extending these findings to humans, Tol-mRNA demonstrated potent, antigen-dependent immunosuppressive activity and a strong capacity to promote antigen-specific Treg induction in human immune settings. Collectively, these results establish Tol-mRNA as a scalable and versatile approach to reprogram antigen-specific immunity and support its development as a next-generation therapeutic modality for immune-mediated diseases.

immunology↗

bioRxiv2025

mRNA-Based Designer APCs Elicit Robust CD8⁺ and CD4⁺ T Cell Responses

mRNA-based therapeutics have demonstrated notable success in SARS-CoV-2 vaccines and are emerging in cancer immunotherapy. However, conventional mRNA cancer vaccines are limited by the low immunogenicity of tumor-associated and neoantigens. We addressed this limitation by formulating a modular, liposome-based mRNA cocktail comprising three distinct mRNAs encoding tumor antigen, the co-stimulatory molecule CD80, and membrane-tethered IL-2. Administration of this mRNA mixture transforms somatic cells into designer antigen-presenting cells (APCs) in vivo, which simultaneously express the antigen, a co-stimulatory molecule, and a cytokine. These designer APCs more effectively activated tumor antigen-specific CD8 T cells than mRNA encoding the antigen alone and elicited robust anti-tumor immune responses. In addition, substituting IL-2 in the mRNA mixtures with membrane-tethered IL-12 led to the expansion and differentiation of endogenous antigen-specific Th1 helper T cells in vivo. Importantly, this platform activated NY-ESO-1-specific CD8 T cells both in human PBMCs in vitro and in HLA-A*02:01-transgenic mice, highlighting its translational potential. This modular mRNA strategy reprograms somatic cells in situ into designer APCs, providing a flexible and translatable platform for precision immunotherapy.

immunology↗

bioRxiv2025

A nuclear CobW/WW-domain factor represses the CO2-concentrating mechanism in the green alga Chlamydomonas reinhardtii

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthesis when dissolved CO2 is limited, but how this energy-intensive system is suppressed when CO2 levels rise has remained unclear. The CCM consumes 15-30% of photosynthetically-generated ATP, making its regulation critical for cellular energy balance. Here, we identify a nuclear repressor of the CCM in the green alga Chlamydomonas reinhardtii. A pull-down screen for interacting partners of the master activator CCM1/CIA5 revealed an uncharacterized protein that co-purifies with CCM1 even after high-salt washes. This protein, designated CCM1-binding protein 1 (CBP1), combines a CobW/CobW_C GTP-binding metallochaperone module with a WW domain characteristic of protein-protein interactions. CBP1 colocalizes with CCM1 in the nucleus regardless of CO2 conditions, and the two proteins interact in vivo. CRISPR/Cas9-based disruption of CBP1 does not affect growth or CCM induction under CO2 limitation but derepresses 27 of 41 CCM1-dependent low-CO2 inducible genes under high-CO2 conditions. These include the periplasmic and intracellular carbonic anhydrases (CAH1, LCIB) and inorganic carbon transporters/channels (LCIA, LCI1, BST1, BST3). Consistently, cbp1 mutants accumulate higher levels of CAH1 and LCIB proteins and exhibit a 40% increase in inorganic carbon affinity under high-CO2 conditions; this phenotype is rescued by CBP1 complementation or by acetazolamide treatment. These results demonstrate that CBP1 prevents unnecessary CCM activity when CO2 is abundant, acting upstream of both transporter/channel and carbonic anhydrase modules. Our findings suggest a regulatory mechanism potentially linking zinc-dependent protein chemistry to CCM gene repression, providing insights into energy-efficient CO2 sensing in aquatic photosynthetic organisms. Significance statementAlgae flourish by activating a CO2-concentrating mechanism (CCM) when dissolved CO2 is limited but must deactivate it when CO2 levels increase to conserve energy. We have identified the nuclear protein that functions as this long sought "off switch" in the model green alga. Deletion of this protein causes cells to overproduce CCM transporters and enzymes, maintaining CCM activity even under high CO2 conditions, demonstrating its essential role in suppressing the system when carbon is abundant. This finding illuminates how algae balance energy consumption with carbon capture and offers a new target for engineering strains that fix CO2 more efficiently for biofuel production or climate-mitigation technologies.

plant biology↗

bioRxiv2024

Periplasmic carbonic anhydrase CAH1 contributes to high inorganic carbon affinity in Chlamydomonas reinhardtii

Carbonic anhydrase (CA), an enzyme conserved across species, is pivotal in the interconversion of inorganic carbon (Ci; CO2 and HCO3-). Compared to the well-studied intracellular CA, the specific role of extracellular CA in photosynthetic organisms is still not well understood. In the green alga Chlamydomonas reinhardtii, CAH1, located at the periplasmic space, is strongly induced under CO2-limiting conditions by the Myb transcription factor LCR1. While it has been observed that the lcr1 mutant shows decreased Ci-affinity, the detailed mechanisms behind this phenomenon are yet to be elucidated. In this study, we aimed to unravel the LCR1-dependent genes essential for maintaining high Ci-affinity. To achieve this, we identified a total of 12 LCR1-dependent inducible genes under CO2-limiting conditions, focusing specifically on the most prominent ones - CAH1, LCI1, LCI6, and Cre10.g426800. We then created mutants of these genes using the CRISPR-Cas9 system, all from the same parental strain, and compared their Ci-affinity. Contrary to earlier findings (Van and Spalding, 1999) that reported no reduction in Ci-affinity in the cah1 mutant, our newly created cah1-1 mutant exhibited a significant decrease in Ci-affinity under high HCO3-/CO2-ratio conditions. Additionally, when we treated wild-type cells with a CA inhibitor with low membrane permeability, a similar reduction in Ci-affinity was observed. Moreover, the addition of exogenous CA to the cah1 mutant restored the decreased Ci-affinity. These results, highlighting the crucial function of the periplasmic CAH1 in maintaining high Ci-affinity in Chlamydomonas cells, provide new insights into the functions of periplasmic CA in algal carbon assimilation. One-sentence summaryCAH1, a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii, plays a crucial role in maintaining a high affinity for inorganic carbon, particularly under CO2-limiting conditions.

plant biology↗

bioRxiv2023

Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy

Extracellular vesicles (EVs), including exosomes, are emerging as novel mediators of cell-cell communications, involved in various processes such as immune activation and immunosuppression. Despite the recent development of several EVs-based cancer immunotherapies, their clinical efficacy remained limited. Here, using fusion with tetraspanin as one of the EV engineering techniques, we created antigen-presenting extracellular vesicles (AP-EVs) to reproduce the functional characteristics of professional antigen-presenting cells (APCs). AP-EVs were also equipped with surface-bound IL-2, a feature not inherent to APCs, which facilitated selective delivery of IL-2 to antigen-specific CD8+ T cells. AP-EVs were engineered to express a peptide-major histocompatibility class I (pMHCI) complex, a costimulatory CD80 molecule, and IL-2, allowing the simultaneous presentation of multiple immune modulators to antigen-specific CD8+ T cells. This promoted the clonal expansion and differentiation of antigen-specific cytotoxic T lymphocytes, leading to potent anticancer immune responses. Combination therapy with AP-EVs and anti-PD-1 demonstrated enhanced anticancer immunity against established tumors compared with anti-PD-1 monotherapy. Our engineered EVs represent a novel effective strategy for cancer immunotherapy.

bioengineering↗

bioRxiv2022

Proton-pumping rhodopsins in marine diatoms

Diatoms are a major phytoplankton group responsible for about 20% of Earths primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO2-concentrating mechanism and assist CO2 fixation in diatom cells.

microbiology↗

bioRxiv2020

Chlamydomonas reinhardtii Tubulin-Gene Disruptants for Efficient Isolation of Strains Bearing Novel Tubulin Mutations

The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2) and two {beta}-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different -tubulin mutations and 24 strains bearing 12 different {beta}-tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants. IMPORTANCEChlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for - and {beta}-tubulin. Here, we obtained several strains in which one of the - or {beta}-tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure-function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

cell biology↗

bioRxiv2018

Inner lumen proteins stabilize doublet microtubules in cilia/flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

cell biology↗

bioRxiv2016

Engineered Cpf1 Enzymes with Altered PAM Specificities

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

synthetic biology↗