著者

Shinji C. Nagasaki Tomonori D. Fukuda Mayumi Yamada Yusuke III Suzuki Ryo Kakutani Adam T. Guy Itaru Imayoshi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.22074, (Released:2022-12-16)

被引用文献数

1

---

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid

著者

Kenju Kobachi Sota Kuno Shinya Sato Kenta Sumiyama Michiyuki Matsuda Kenta Terai

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.20010, (Released:2020-06-25)

被引用文献数

2 13

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Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools. Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool

著者

Kei Nishida Kenta Tsuchiya Hiroyuki Obinata Shizuka Onodera Yu Honda Yen-Cheng Lai Nami Haruta Asako Sugimoto

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.21022, (Released:2021-05-08)

被引用文献数

12

---

Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes—α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2—were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans

著者

Faryal Ijaz Koji Ikegami

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.21002, (Released:2021-01-26)

被引用文献数

9

---

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5’UTR of the endogenous gene loci. With this 5’UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5’UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.Key words: CRISPR/Cas9, NHEJ, Knock-in, Primary Cilium, UTR, Tubulin

著者

Md. Imrul Hasan Chowdhury Tomoki Nishioka Noriko Mishima Toshihisa Ohtsuka Kozo Kaibuchi Daisuke Tsuboi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.45, no.2, pp.143-154, 2020 (Released:2020-09-01)

参考文献数

46

被引用文献数

2

---

Prickle2 has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. Here, because Igsf9 family proteins are associated with psychiatric diseases and seizures, we studied the physiological interaction between Prickle2 and Igsf9b. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.Key words: Prickle2, Igsf9b, axon initial segment, neuronal excitability, ASD

著者

Yoshihiro Miyake Hideki Tada Masafumi Yano Hiroshi Shimizu

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.19, no.6, pp.363-370, 1994 (Released:2006-03-27)

参考文献数

18

被引用文献数

10 21

---

The relationship between intracellular period modulation and external environment change was investigated from the viewpoint of internal information coding in Physarum plasmodium. For the external conditions, concentration changes of attractant (galactose) and repellent (KC1) were used, and the internal responses were measured as the thickness oscillation of the plasmodium. (i) Period of the intracellular oscillation decreased when the concentration of attractant was increased and when the concentration of repellent was decreased, (ii) The period increased when the attractant was decreased and when the repellent was increased, (iii) The larger concentration change induced the larger period modulation, (iv) These responses were observed when the change of concentration was greater than a threshold value. From these results, it was clarified that the relative change in environmental condition is encoded on the relative period modulation in intracellular oscillation. This means that the period change does not directly represent the environment itself but represents the change of its condition. Thus, it is further suggested that the plasmodium estimates the environmental condition based on the relationship between the previous external condition and the present one.

著者

Karin Tanabe Rie Awane Tsuyoshi Shoda Kanta Yamazoe Yoshihiro H. Inoue

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.19022, (Released:2019-09-05)

被引用文献数

8

---

Drosophila Mxc protein is a component of the histone locus body (HLB), which is required for the expression of canonical histone genes, and severe mxc mutations generate tumors in larval hematopoietic tissues. A common characteristic of cancer cells is chromosomal instability (CIN), but whether mxc mutants exhibit this feature is unknown. Here, examination of post-meiotic spermatids created after male meiosis revealed that a fraction of the spermatids in hypomorphic mxcG46 mutants contained extra micronuclei or abnormally sized nuclei, corresponding to CIN. Moreover, we observed that the so-called lagging chromosomes retained between chromosomal masses separated toward spindle poles at telophase I. Time-lapse recordings show that micronuclei were generated from lagging chromosomes, and the abnormal chromosomes in mxcG46 mutants lacked centromeres. In normal spermatocyte nuclei, the HLB component FLASH colocalized with Mxc, whereas FLASH was dispersed in mxcG46 spermatocyte nuclei. Furthermore, we observed genetic interactions between Mxc and other HLB components in meiotic chromosome segregation, which suggests that inhibition of HLB formation is responsible for aberrant chromosome segregation in mxcG46. Quantitative real-time PCR revealed that canonical histone mRNA levels were decreased in mxcG46. Lastly, similar meiotic phenotypes appeared in the spermatids of histone H4 mutants and in the spermatids in testes depleted for chromosome-construction factors. Considering these genetic data, we propose that abnormal chromosome segregation leading to CIN development results from a loss of chromosome integrity caused by diminished canonical histone levels in mxc mutants. Key words: Chromosome instability, Drosophila, meiosis, tumor-suppressor gene

著者

Asuka Hamamoto Natsuki Kita Siddabasave Gowda B Gowda Hiroyuki Takatsu Kazuhisa Nakayama Makoto Arita Shu-Ping Hui Hye-Won Shin

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.23066, (Released:2023-12-09)

---

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent

著者

Keisuke Ikawa Souta Hiro Shu Kondo Shizue Ohsawa Kaoru Sugimura

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.23049, (Released:2023-11-30)

---

Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis.Key words: Morphogenesis, Cell rearrangement, Actin binding proteins (ABPs)

著者

Ashuei Sogawa Ryota Komori Kota Yanagitani Miku Ohfurudono Akio Tsuru Koji Kadoi Yukio Kimata Hiderou Yoshida Kenji Kohno

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.23072, (Released:2023-09-28)

---

Secretory pathway proteins are cotranslationally translocated into the endoplasmic reticulum (ER) of metazoan cells through the protein channel, translocon. Given that there are far fewer translocons than ribosomes in a cell, it is essential that secretory protein-translating ribosomes only occupy translocons transiently. Therefore, if translocons are obstructed by ribosomes stalled or slowed in translational elongation, it possibly results in deleterious consequences to cellular function. Hence, we investigated how translocon clogging by stalled ribosomes affects mammalian cells. First, we constructed ER-destined translational arrest proteins (ER-TAP) as an artificial protein that clogged the translocon in the ER membrane. Here, we show that the translocon clogging by ER-TAP expression activates triage of signal sequences (SS) in which secretory pathway proteins harboring highly efficient SS are preferentially translocated into the ER lumen. Interestingly, the translocon obstructed status specifically activates inositol requiring enzyme 1α (IRE1α) but not protein kinase R-like ER kinase (PERK). Given that the IRE1α–XBP1 pathway mainly induces the translocon components, our discovery implies that lowered availability of translocon activates IRE1α, which induces translocon itself. This results in rebalance between protein influx into the ER and the cellular translocation capacity.Keywords: endoplasmic reticulum, translocation capacity, translocon clogging, IRE1, signal sequence

著者

Yusuke Kondo Tomoka Ogawa Emiri Kanno Masafumi Hirono Takako Kato-Minoura Ritsu Kamiya Toshiki Yagi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.2, pp.175-185, 2023 (Released:2023-09-23)

参考文献数

54

---

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 μm/sec (WT) and ~4.3 μm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the β HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.Key words: cilia, axoneme, dynein heavy chain, cooperativity

著者

Da Young Shin Hiroaki Takagi Michio Hiroshima Satomi Matsuoka Masahiro Ueda

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.2, pp.145-160, 2023 (Released:2023-08-31)

参考文献数

58

被引用文献数

2

---

In eukaryotic motile cells, the active Ras (Ras-GTP)-enriched domain is generated in an asymmetric manner on the cell membrane through the excitable dynamics of an intracellular signaling network. This asymmetric Ras signaling regulates pseudopod formation for both spontaneous random migration and chemoattractant-induced directional migration. While membrane lipids, such as sphingomyelin and phosphatidylserine, contribute to Ras signaling in various cell types, whether they are involved in the Ras excitability for cell motility is unknown. Here we report that functional Ras excitability requires the normal metabolism of sphingomyelin for efficient cell motility and chemotaxis. The pharmacological blockade of sphingomyelin metabolism by an acid-sphingomyelinase inhibitor, fendiline, and other inhibitors suppressed the excitable generation of the stable Ras-GTP-enriched domain. The suppressed excitability failed to invoke enough basal motility to achieve directed migration under shallow chemoattractant gradients. The fendiline-induced defects in Ras excitability, motility and stimulation-elicited directionality were due to an accumulation of sphingomyelin on the membrane, which could be recovered by exogenous sphingomyelinase or phosphatidylserine without changing the expression of Ras. These results indicate a novel regulatory mechanism of the excitable system by membrane lipids, in which sphingomyelin metabolism provides a membrane environment to ensure Ras excitation for efficient cellular motility and chemotaxis.Key words: cell polarity, cell migration, Ras, excitability, sphingomyelin

著者

Tomoyo Ikeda Tokiro Ishikawa Satoshi Ninagawa Tetsuya Okada Masaya Ono Kazutoshi Mori

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.2, pp.123-133, 2023 (Released:2023-07-19)

参考文献数

49

---

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or β-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and β-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, β-oxidation, triacylglycerol, cholesterol, export

著者

Yukinari Haraoka Mai Miyake Tohru Ishitani

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.1, pp.113-121, 2023 (Released:2023-06-16)

参考文献数

61

---

Oncogenic mutations drive tumorigenesis, and single cells with oncogenic mutations act as the tumor seeds that gradually evolve into fully transformed tumors. However, oncogenic cell behavior and communication with neighboring cells during primary tumorigenesis remain poorly understood. We used the zebrafish, a small vertebrate model suitable for in vivo cell biology, to address these issues. We describe the cooperative and competitive communication between oncogenic cells and neighboring cells, as revealed by our recent zebrafish imaging studies. Newly generated oncogenic cells are actively eliminated by neighboring cells in healthy epithelia, whereas oncogenic cells cooperate with their neighbors to prime tumorigenesis in unhealthy epithelia via additional mutations or inflammation. In addition, we discuss the potential of zebrafish in vivo imaging to determine the initial steps of human tumorigenesis.Key words: zebrafish, imaging, cell-cell communication, cell competition, EDAC, senescence, primary tumorigenesis

著者

Sung Hoon Back

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.45, no.1, pp.65-76, 2020 (Released:2020-06-04)

参考文献数

71

被引用文献数

4 8

---

It is often assumed that α-subunit phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) complex is just a mechanism to control protein synthesis. However, eIF2α phosphorylation induced by multiple kinases can recognize various intracellular and extracellular stress conditions, and it is involved in various other cellular processes beyond protein synthesis. This review introduces the roles of eIF2α phosphorylation in translational regulation, the generation of reactive oxygen species, changes in mitochondria structure and shape, and mitochondrial retrograde signaling pathways in response to diverse stress conditions.Key words: eIF2α phosphorylation, Translation, Unfolded Protein Response, Reactive Oxygen Species, Mitochondria

著者

Md Shajedul Haque Yoshikazu Emi Masao Sakaguchi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.1, pp.71-82, 2023 (Released:2023-03-09)

参考文献数

38

---

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl– excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41–60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4

著者

Yuka Takeda Shuji Matsuguchi Sae Nozaki Taisei Mihara Junya Abe Yohei Hirai

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.1, pp.49-57, 2023 (Released:2023-02-08)

参考文献数

28

被引用文献数

4

---

In embryonic stem (ES) cell colonies, a small subpopulation that changes cell shape and loses pluripotency often appears in two-dimensional (2D) cultures, even in the presence of a stemness factor. We have previously shown that membrane translocation of the syntaxin4, t-SNARE protein contributes to this phenomenon. Here, we show that ES cells in three-dimensional (3D) aggregates do not succumb to extruded syntaxin4 owing to suppressed expression of P-cadherin protein. While extracellular expression of syntaxin4 led to the striking upregulation of P-cadherin mRNA in both 2D and 3D-ES cells, morphological changes and appreciable expression of P-cadherin protein were detected only in 2D-ES cells. Importantly, the introduction of an expression cassette for P-cadherin practically reproduced the effects induced by extracellular syntaxin4, where the transgene product was clearly detected in 2D-, but not 3D-ES cells. An expression construct for P-cadherin-Venus harboring an in-frame insertion of the P2A sequence at the joint region gave fluorescent signals only in the cytoplasm of 2D-ES cells, demonstrating translational regulation of P-cadherin. These results provide the mechanistic insight into the uncontrollable differentiation in 2D-ES cells and shed light on the validity of the “embryoid body protocol commonly used for ES cell handling” for directional differentiation.Key words: differentiation, embryoid body, ES cells, P-cadherin, syntaxin4

著者

Shinji C. Nagasaki Tomonori D. Fukuda Mayumi Yamada Yusuke III Suzuki Ryo Kakutani Adam T. Guy Itaru Imayoshi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.1, pp.31-47, 2023 (Released:2023-02-08)

参考文献数

77

被引用文献数

1

---

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid

著者

Ryutaro Kashikuma Makoto Nagano Hiroki Shimamura Kouya Nukaga Ikumi Katsumata Junko Y. Toshima Jiro Toshima

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.48, no.1, pp.19-30, 2023 (Released:2023-01-28)

参考文献数

54

被引用文献数

1

---

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood. In this study, to clarify the role of PS in the endocytic pathway, we analyzed the effect of PS depletion on endocytic internalization and post-endocytic transport. We demonstrated that in cell lacking the PS synthase Cho1p (cho1Δ cell), binding and internalization of mating pheromone α-factor into the cell was severely impaired. Interestingly, the processes of endocytosis were mostly unaffected, but protein transport from the trans-Golgi network (TGN) to the PM was defective and localization of cell surface proteins was severely impaired in cho1Δ cells. We also showed that PS accumulated in intracellular compartments in cells lacking Rcy1p and Vps52p, both of which are implicated in endosome-to-PM transport via the TGN, and that the number of Snx4p-residing endosomes was increased in cho1Δ cells. These results suggest that PS plays a crucial role in the transport and localization of cell surface membrane proteins.Key words: phosphatidylserine, endocytosis, recycling, vesicle transport

著者

Kentaro Matsumoto Shenwei Ni Hiroyuki Arai Takashi Toyama Yoshiro Saito Takehiro Suzuki Naoshi Dohmae Kojiro Mukai Tomohiko Taguchi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.22085, (Released:2022-12-28)

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Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2’,3’-cyclic guanosine monophosphate&endash;adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist.Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide