The Experts below are selected from a list of 8106 Experts worldwide ranked by ideXlab platform
Yukio Kimata - One of the best experts on this subject based on the ideXlab platform.
-
The unfolded Protein response in Pichia pastoris without external stressing stimuli.
FEMS Yeast Research, 2020Co-Authors: Yasmin Nabilah Binti Mohd Fauzee, Hiroshi Takagi, Yuki Ishiwata-kimata, Naoki Taniguchi, Yukio KimataAbstract:Dysfunction or capacity shortage of the endoplasmic reticulum (ER) is cumulatively called ER stress and provokes the unfolded Protein response (UPR). In various yeast species, the ER-located transmembrane Protein IRE1 is activated upon ER stress and performs the splicing reaction of HAC1 mRNA, the mature form of which is translated into a transcription factor Protein that is responsible for the transcriptome change on the UPR. Here we carefully assessed the splicing of HAC1 mRNA in Pichia pastoris (Komagataella phaffii) cells. We found that, inconsistent with previous reports by others, the HAC1 mRNA was substantially, but partially, spliced even without ER-stressing stimuli. Unlike Saccharomyces cerevisiae, growth of P. pastoris was significantly retarded by the IRE1-gene knockout mutation. Moreover, P. pastoris cells seemed to push more abundant Proteins into the secretory pathway than S. cerevisiae cells. We also suggest that P. pastoris IRE1 has the ability to control its activity stringently in an ER stress-dependent manner. We thus propose that P. pastoris cells are highly ER-stressed possibly because of the high load of endogenous Proteins into the ER.
-
Categorization of endoplasmic reticulum stress as accumulation of unfolded Proteins or membrane lipid aberrancy using yeast IRE1 mutants.
Bioscience Biotechnology and Biochemistry, 2018Co-Authors: Duc Minh Tran, Hiroshi Takagi, Yukio KimataAbstract:Endoplasmic reticulum (ER)-located Protein IRE1 triggers the unfolded Protein response against ER-stressing stimuli, which are categorized as ER accumulation of unfolded Proteins or membrane lipid-related aberrancy. Here we demonstrate that by using yeast IRE1 mutants, we can distinguish the category to which a stress-inducing stimulus belongs. For instance, ethanol was found to activate IRE1 through both types of cellular damage.
-
A chimeric mutant analysis in yeast cells suggests BiP independent regulation of the mammalian endoplasmic reticulum-stress sensor IRE1α.
Bioscience Biotechnology and Biochemistry, 2018Co-Authors: Thanh Chi Mai, Takeo Munakata, Duc Minh Tran, Hiroshi Takagi, Yukio KimataAbstract:An endoplasmic reticulum (ER)-located transmembrane Protein, IRE1, triggers cytoprotective events upon ER stress. Chimeric yeast IRE1 carrying the luminal domain of the mammalian major IRE1 paralog...
-
Tight regulation of the unfolded Protein sensor IRE1 by its intramolecularly antagonizing subdomain
Journal of Cell Science, 2015Co-Authors: Rubwad Mathuranyanon, Yuki Ishiwata-kimata, Kenji Kohno, Tomoko Tsukamoto, Asumi Takeuchi, Yuichi Tsuchiya, Yukio KimataAbstract:Accumulation of unfolded Proteins in the endoplasmic reticulum (ER) accompanies ER stress and causes the type‐I transmembrane Protein IRE1 (also known as ERN1) to trigger the unfolded Protein response (UPR). When dimerized, the core stress‐sensing region (CSSR) of IRE1 directly captures unfolded Proteins and forms a high‐order oligomer, leading to clustering and activation of IRE1. The CSSR is N‐terminally flanked by an intrinsically disordered subdomain, which we previously named Subregion I, in Saccharomyces cerevisiae IRE1. In this study, we describe tight repression of IRE1 activity by Subregion I under conditions of no or weak stress. Weak hyperactivation of an IRE1 mutant lacking Subregion I slightly retarded growth of yeast cells cultured under unstressed conditions. Fungal IRE1 orthologs and the animal IRE1 family Protein PERK (also known as EIF2AK3) carry N‐terminal intrinsically disordered subdomains with a similar structure and function to that of Subregion I. Our observations presented here cumulatively indicate that Subregion I is captured by the CSSR as an unfolded Protein substrate. This intramolecular subdomain interaction is likely to compromise self‐association of the CSSR, explaining why Subregion I can suppress IRE1 activity when ER‐accumulated unfolded Proteins are not abundant.
-
Ethanol stress impairs Protein folding in the endoplasmic reticulum and activates IRE1 in Saccharomyces cerevisiae.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Ken-ichi Miyagawa, Kenji Kohno, Yuki Ishiwata-kimata, Yukio KimataAbstract:Impaired Protein folding in the endoplasmic reticulum (ER) evokes the unfolded Protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane Protein IRE1. Here, we report that ethanol stress damages Protein folding in the ER, causing activation of IRE1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.
Kenji Kohno - One of the best experts on this subject based on the ideXlab platform.
-
Tight regulation of the unfolded Protein sensor IRE1 by its intramolecularly antagonizing subdomain
Journal of Cell Science, 2015Co-Authors: Rubwad Mathuranyanon, Yuki Ishiwata-kimata, Kenji Kohno, Tomoko Tsukamoto, Asumi Takeuchi, Yuichi Tsuchiya, Yukio KimataAbstract:Accumulation of unfolded Proteins in the endoplasmic reticulum (ER) accompanies ER stress and causes the type‐I transmembrane Protein IRE1 (also known as ERN1) to trigger the unfolded Protein response (UPR). When dimerized, the core stress‐sensing region (CSSR) of IRE1 directly captures unfolded Proteins and forms a high‐order oligomer, leading to clustering and activation of IRE1. The CSSR is N‐terminally flanked by an intrinsically disordered subdomain, which we previously named Subregion I, in Saccharomyces cerevisiae IRE1. In this study, we describe tight repression of IRE1 activity by Subregion I under conditions of no or weak stress. Weak hyperactivation of an IRE1 mutant lacking Subregion I slightly retarded growth of yeast cells cultured under unstressed conditions. Fungal IRE1 orthologs and the animal IRE1 family Protein PERK (also known as EIF2AK3) carry N‐terminal intrinsically disordered subdomains with a similar structure and function to that of Subregion I. Our observations presented here cumulatively indicate that Subregion I is captured by the CSSR as an unfolded Protein substrate. This intramolecular subdomain interaction is likely to compromise self‐association of the CSSR, explaining why Subregion I can suppress IRE1 activity when ER‐accumulated unfolded Proteins are not abundant.
-
Ethanol stress impairs Protein folding in the endoplasmic reticulum and activates IRE1 in Saccharomyces cerevisiae.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Ken-ichi Miyagawa, Kenji Kohno, Yuki Ishiwata-kimata, Yukio KimataAbstract:Impaired Protein folding in the endoplasmic reticulum (ER) evokes the unfolded Protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane Protein IRE1. Here, we report that ethanol stress damages Protein folding in the ER, causing activation of IRE1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.
-
BiP-bound and nonclustered mode of IRE1 evokes a weak but sustained unfolded Protein response.
Genes to Cells, 2013Co-Authors: Yuki Ishiwata-kimata, Kenji Kohno, Thanyarat Promlek, Yukio KimataAbstract:In eukaryotic cells under nonstressed conditions, the endoplasmic reticulum (ER)-located molecular chaperone BiP is associated with an ER-membrane Protein IRE1 to inhibit its self-association. While ER stress leads IRE1 to form transiently BiP-unbound clusters, which strongly evoke the unfolded Protein response (UPR), here we propose an alternative activation status of IRE1. When yeast cells are physiologically ER-stressed by inositol depletion for a prolonged time, the UPR is weakly activated in a sustained manner after a transient peak of activation. During persistent stress, IRE1 foci disappear, while IRE1 continues to be self-associated. Under these conditions, IRE1 may be activated as a homo-dimer, as it shows considerable activity even when carrying the W426A mutation, which allows IRE1 to form homo-dimers but not clusters. Unlike the IRE1 clusters, the nonclustered active form seems to be associated with BiP. An IRE1 mutant not carrying the BiP-association site continued to form clusters and to be activated strongly even after long-term stress. Similar observations were obtained when cells were ER-stressed by dithiothreitol. We thus propose that upon persistent ER stress, IRE1 is weakly and continuously activated in a nonclustered form through its (re)association with BiP, which disperses the IRE1 clusters.
-
F-actin and a type-II myosin are required for efficient clustering of the ER stress sensor IRE1.
Cell Structure and Function, 2013Co-Authors: Yuki Ishiwata-kimata, Kenji Kohno, Yo-hei Yamamoto, Ken Takizawa, Yukio KimataAbstract:Endoplasmic reticulum (ER) stress causes the ER-resident transmembrane Protein IRE1 to self-associate, leading to the formation of large oligomeric clusters. In yeast cells, this induces strong unfolded Protein response (UPR) through splicing of HAC1 mRNA. Here, we demonstrate that highly ER-stressed yeast cells exhibited poor IRE1 clustering in the presence of the actin-disrupting agent latrunculin-A. Under these conditions, IRE1 may form smaller oligomers because latrunculin-A only partially diminished the IRE1-mediated splicing of HAC1 mRNA. IRE1 cluster formation was also impaired by deletion of the type-II myosin gene MYO1 or SAC6, which encodes the actin-bundling Protein fimbrin. Finally, we demonstrated that IRE1 clusters are predominantly located on or near actin filaments. Therefore, we propose that actin filaments play an important role in ER stress-induced clustering of IRE1.
-
Yeast unfolded Protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface Proteins.
Genes to Cells, 2005Co-Authors: Yukio Kimata, Yuki Ishiwata-kimata, Seiko Yamada, Kenji KohnoAbstract:The unfolded Protein response (UPR) is a cellular protective event against endoplasmic reticulum (ER) stress. In the yeast UPR signaling pathway, the ER-located transmembrane Protein IRE1 promotes splicing of the HAC1 premRNA (HAC1(u)) to produce the translatable transcription factor mRNA (HAC1i). We generated a HAC1i gene-bearing strain, in which the UPR pathway was constitutively activated, and compared its gene expression profile with that of a DeltaIRE1 HAC1u strain using cDNA microarray technology. Comparison of the gene expression profile was also performed between non-stressed wild-type cells and those exposed to ER stress. Genes for which the expression level was significantly changed in both of these experiments were categorized as targets of the IRE1-HAC1 signaling pathway. This analysis revealed that in addition to the previously known UPR targets, some anti-oxidative stress genes were up-regulated by the IRE1-HAC1 pathway, possibly in order to reduce reactive oxygen species produced during the cellular response to ER stress. Moreover, we categorized 15 genes as those down-regulated by the UPR, most of which seem to encode cell surface or extracellular Proteins. This UPR-mediated gene repression may alleviate the load of client Proteins targeted to the ER.
Yuki Ishiwata-kimata - One of the best experts on this subject based on the ideXlab platform.
-
The unfolded Protein response in Pichia pastoris without external stressing stimuli.
FEMS Yeast Research, 2020Co-Authors: Yasmin Nabilah Binti Mohd Fauzee, Hiroshi Takagi, Yuki Ishiwata-kimata, Naoki Taniguchi, Yukio KimataAbstract:Dysfunction or capacity shortage of the endoplasmic reticulum (ER) is cumulatively called ER stress and provokes the unfolded Protein response (UPR). In various yeast species, the ER-located transmembrane Protein IRE1 is activated upon ER stress and performs the splicing reaction of HAC1 mRNA, the mature form of which is translated into a transcription factor Protein that is responsible for the transcriptome change on the UPR. Here we carefully assessed the splicing of HAC1 mRNA in Pichia pastoris (Komagataella phaffii) cells. We found that, inconsistent with previous reports by others, the HAC1 mRNA was substantially, but partially, spliced even without ER-stressing stimuli. Unlike Saccharomyces cerevisiae, growth of P. pastoris was significantly retarded by the IRE1-gene knockout mutation. Moreover, P. pastoris cells seemed to push more abundant Proteins into the secretory pathway than S. cerevisiae cells. We also suggest that P. pastoris IRE1 has the ability to control its activity stringently in an ER stress-dependent manner. We thus propose that P. pastoris cells are highly ER-stressed possibly because of the high load of endogenous Proteins into the ER.
-
Tight regulation of the unfolded Protein sensor IRE1 by its intramolecularly antagonizing subdomain
Journal of Cell Science, 2015Co-Authors: Rubwad Mathuranyanon, Yuki Ishiwata-kimata, Kenji Kohno, Tomoko Tsukamoto, Asumi Takeuchi, Yuichi Tsuchiya, Yukio KimataAbstract:Accumulation of unfolded Proteins in the endoplasmic reticulum (ER) accompanies ER stress and causes the type‐I transmembrane Protein IRE1 (also known as ERN1) to trigger the unfolded Protein response (UPR). When dimerized, the core stress‐sensing region (CSSR) of IRE1 directly captures unfolded Proteins and forms a high‐order oligomer, leading to clustering and activation of IRE1. The CSSR is N‐terminally flanked by an intrinsically disordered subdomain, which we previously named Subregion I, in Saccharomyces cerevisiae IRE1. In this study, we describe tight repression of IRE1 activity by Subregion I under conditions of no or weak stress. Weak hyperactivation of an IRE1 mutant lacking Subregion I slightly retarded growth of yeast cells cultured under unstressed conditions. Fungal IRE1 orthologs and the animal IRE1 family Protein PERK (also known as EIF2AK3) carry N‐terminal intrinsically disordered subdomains with a similar structure and function to that of Subregion I. Our observations presented here cumulatively indicate that Subregion I is captured by the CSSR as an unfolded Protein substrate. This intramolecular subdomain interaction is likely to compromise self‐association of the CSSR, explaining why Subregion I can suppress IRE1 activity when ER‐accumulated unfolded Proteins are not abundant.
-
Ethanol stress impairs Protein folding in the endoplasmic reticulum and activates IRE1 in Saccharomyces cerevisiae.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Ken-ichi Miyagawa, Kenji Kohno, Yuki Ishiwata-kimata, Yukio KimataAbstract:Impaired Protein folding in the endoplasmic reticulum (ER) evokes the unfolded Protein response (UPR), which is triggered in budding yeast, Saccharomyces cerevisiae, by the ER-located transmembrane Protein IRE1. Here, we report that ethanol stress damages Protein folding in the ER, causing activation of IRE1 in yeast cells. The UPR likely contributes to the ethanol tolerance of yeast cells.
-
BiP-bound and nonclustered mode of IRE1 evokes a weak but sustained unfolded Protein response.
Genes to Cells, 2013Co-Authors: Yuki Ishiwata-kimata, Kenji Kohno, Thanyarat Promlek, Yukio KimataAbstract:In eukaryotic cells under nonstressed conditions, the endoplasmic reticulum (ER)-located molecular chaperone BiP is associated with an ER-membrane Protein IRE1 to inhibit its self-association. While ER stress leads IRE1 to form transiently BiP-unbound clusters, which strongly evoke the unfolded Protein response (UPR), here we propose an alternative activation status of IRE1. When yeast cells are physiologically ER-stressed by inositol depletion for a prolonged time, the UPR is weakly activated in a sustained manner after a transient peak of activation. During persistent stress, IRE1 foci disappear, while IRE1 continues to be self-associated. Under these conditions, IRE1 may be activated as a homo-dimer, as it shows considerable activity even when carrying the W426A mutation, which allows IRE1 to form homo-dimers but not clusters. Unlike the IRE1 clusters, the nonclustered active form seems to be associated with BiP. An IRE1 mutant not carrying the BiP-association site continued to form clusters and to be activated strongly even after long-term stress. Similar observations were obtained when cells were ER-stressed by dithiothreitol. We thus propose that upon persistent ER stress, IRE1 is weakly and continuously activated in a nonclustered form through its (re)association with BiP, which disperses the IRE1 clusters.
-
F-actin and a type-II myosin are required for efficient clustering of the ER stress sensor IRE1.
Cell Structure and Function, 2013Co-Authors: Yuki Ishiwata-kimata, Kenji Kohno, Yo-hei Yamamoto, Ken Takizawa, Yukio KimataAbstract:Endoplasmic reticulum (ER) stress causes the ER-resident transmembrane Protein IRE1 to self-associate, leading to the formation of large oligomeric clusters. In yeast cells, this induces strong unfolded Protein response (UPR) through splicing of HAC1 mRNA. Here, we demonstrate that highly ER-stressed yeast cells exhibited poor IRE1 clustering in the presence of the actin-disrupting agent latrunculin-A. Under these conditions, IRE1 may form smaller oligomers because latrunculin-A only partially diminished the IRE1-mediated splicing of HAC1 mRNA. IRE1 cluster formation was also impaired by deletion of the type-II myosin gene MYO1 or SAC6, which encodes the actin-bundling Protein fimbrin. Finally, we demonstrated that IRE1 clusters are predominantly located on or near actin filaments. Therefore, we propose that actin filaments play an important role in ER stress-induced clustering of IRE1.
Hyung Don Ryoo - One of the best experts on this subject based on the ideXlab platform.
-
A modified UPR stress sensing system reveals a novel tissue distribution of IRE1/XBP1 activity during normal Drosophila development
Cell Stress and Chaperones, 2013Co-Authors: Michio Sone, Xiaomei Zeng, Joseph Larese, Hyung Don RyooAbstract:Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded Proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded Protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor Protein, IRE1, ATF6, and PERK, respectively. In Drosophila , IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, we modified previously reported XBP1 stress sensing systems (Souid et al., Dev Genes Evol 217: 159–167, 2007 ; Ryoo et al., EMBO J 26: 242-252, 2007 ), based on the recent reports regarding the unconventional splicing of XBP1 / HAC1 mRNA (Aragon et al., Nature 457: 736–740, 2009 ; Yanagitani et al., Mol Cell 34: 191–200, 2009 ; Science 331: 586–589, 2011 ). The improved XBP1 stress sensing system allowed us to detect new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide us with novel insights into the brain homeostasis regulated by the UPR.
Elias M. Puchner - One of the best experts on this subject based on the ideXlab platform.
-
Precisely calibrated and spatially informed illumination for conventional fluorescence and improved PALM imaging applications.
Methods and Applications in Fluorescence, 2020Co-Authors: Angel Mancebo, Luke A. Demars, Christopher T Ertsgaard, Elias M. PuchnerAbstract:Spatial light modulation using cost efficient digital micromirror devices (DMD) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate Proteins in optogenetic experiments. However, precise illumination by DMDs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMD and camera coordinate systems caused by optical components in the excitation and emission path. Here we develop a fast and easy to implement calibration procedure that determines these distortions and matches the DMD and camera coordinate system with a precision below the optical diffraction limit. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to a rigid transformation allowed by manual alignment of the DMD. We first demonstrate the application of our precisely calibrated light modulation by performing a proof of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized Protein IRE1 fused to GFP in budding yeast (S. cerevisiae). Next, we develop a spatially informed photoactivation approach for SMLM in which only regions of the cell that contain photoactivatable fluorescent Proteins are selected for photoactivation. The reduced exposure of the cells to 405 nm light increased the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in cell morphology. As a result, the mean number of reliable single-molecule localizations was also significantly increased by 28%. Since the localization precision and the ability for single-molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatially informed photoactivation significantly improves the quality of SMLM images and single-molecule tracking data. Our precise calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.
-
Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications
2019Co-Authors: Angel Mancebo, Luke A. Demars, Elias M. PuchnerAbstract:Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate Proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized Protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent Proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cell9s morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.
