Biology subjects

Onishi, M.

Publications and source records attributed to Onishi, M..

24 records · Page 2Linked to original sources

bioRxiv2021

The GET pathway serves to activate Atg32-mediated mitophagy by ER targeting of the Ppg1-Far complex

Mitophagy removes defective or superfluous mitochondria via selective autophagy. In yeast, the pro-mitophagic protein Atg32 localizes to the mitochondrial surface and interacts with the scaffold protein Atg11 to promote degradation of mitochondria. Although Atg32-Atg11 interactions are thought to be stabilized by Atg32 phosphorylation, how this posttranslational modification is regulated remains obscure. Here we show that cells lacking the guided entry of tail-anchored proteins (GET) pathway exhibit reduced Atg32 phosphorylation and Atg32-Atg11 interactions, which can be rescued by additional loss of the ER-resident Ppg1-Far complex, a multi-subunit phosphatase negatively acting in mitophagy. In GET-deficient cells, Ppg1-Far is predominantly localized to mitochondria. An artificial ER anchoring of Ppg1-Far in GET-deficient cells significantly ameliorates defects in Atg32-Atg11 interactions and mitophagy. Moreover, disruption of GET and Msp1, an AAA-ATPase that extracts non-mitochondrial proteins localized to the mitochondrial surface, elicits synthetic defects in mitophagy. Collectively, we propose that the GET pathway mediates ER targeting of Ppg1-Far, thereby preventing dysregulated suppression of mitophagy activation.

cell biology↗

bioRxiv2021

Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ( CDK) CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen (EB1-NG) allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1-NG signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by comets of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC mitosis modulate microtubule dynamics while regulating mitotic progression.

cell biology↗

bioRxiv2020

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocyte accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), often causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR in HBV infection is poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFN causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFN mediated liver injury. Analyses of total gene expression and UPR biomarkers protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFN administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFN mediated liver injury. These results suggest that IFN induces ER stress-associated cell death by reducing UPR. IFN{gamma} uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism by which IFNs selectively induce cell death in virus-infected cells. This study also identifies UPR as a potential target for regulating ER stress-associated cell death. Author summaryHepatitis B virus (HBV) causes acute and chronic infections that kill over 600,000 people every year from severe hepatitis, liver cirrhosis, and cancer. Mechanisms of chronic liver injury remain largely unknown. Both type 1 and type 2 interferons (IFNs) have been implicated in hepatic flares during chronic HBV infection, although HBV per se is a poor IFN inducer. In addition, while IFN, a type 1 IFN, used to be the first-line treatment for chronic hepatitis B (CHB) patients, adverse side effects, including hepatic flares, severely limit their therapeutic effectiveness. These clinical observations suggest a pathogenic role of IFNs in HBV infection. Here, we demonstrate that IFN-1s cause severe and moderate hepatitis in transgenic mice expressing hepatitis B surface antigen (HBs-Tg mice) and human hepatocyte chimeric mice infected with HBV, respectively. HBsAg accumulation appears to cause ER stress because a counteractive response to ER stress, namely, unfolded protein response (UPR), was induced in both HBs-Tg mice and HBV infected chimeric mice. Our results indicate that IFN-1s suppress UPR before causing liver injury. UPR was also suppressed by IFN{gamma}. Induction of UPR in HBs-Tg mice before treatment with IFN and IFN{gamma} significantly alleviated liver injury. We suggest that IFNs exert cytotoxicity to ER stress accumulating cells by suppressing UPR.

microbiology↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2019

Cleavage-furrow formation without F-actin in Chlamydomonas

It is widely believed that cleavage-furrow formation during cell division is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii. We found that although F-actin is concentrated in the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cell division itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that at least the microtubule plus-ends are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.

cell biology↗

bioRxiv2019

Transfer of Septin Rings to Cytokinetic Remnants Directs Age-Sensitive ER stress Surveillance Cell Cycle Re-entry

During cell division, cells must actively pass on organelles. Previously, we discovered the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway that ensures the inheritance of functional ER. Activation of the ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we found that the septin ring mislocalizes to previously utilized cell division sites called cytokinetic remnants (CRMs). The transfer of the septin ring to CRMs requires Nba1, a negative polarity component that normally prevents septin ring formation at CRMs. Furthermore, septin ring movement to CRMs relies on the ERSU component Slt2, which is recruited by binding Bem1. During ER stress, Bem1 also binds the GTP exchange factor Cdc24, without activating Cdc42, a GTPase that normally establishes polarized growth. Failure to translocate septin rings to CRMs delays the cells ability to re-enter cell division when ER homeostasis is re-established. Thus, ER stress considers the history of previous cell cycle for future cell cycle re-entry upon ER stress recovery.

cell biology↗