記述言語：日本語   出版者・発行元：(株)ニュー・サイエンス社  

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記述言語：日本語   出版者・発行元：(公社)日本薬学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

"Trim-Away" technology enables rapid degradation of endogenous proteins without prior modification of protein-coding genes or mRNAs through delivery of antibodies that target proteins of interest. Although this approach can be readily applied to almost any cytosolic protein, strategies for cytosolic antibody delivery have been limited to microinjection or electroporation, which require skill-dependent operation or specialized equipment. Thus, the development of antibody delivery methods that are convenient, scalable, and preferably do not require detachment of adherent cells is required to extend the versatility of the Trim-Away method. Here, we developed a cell resealing technique optimized for Trim-Away degradation, which uses the pore-forming toxin streptolysin O (SLO) to permeabilize the cell membrane and delivered the antibodies of interest into HEK293T, HeLa, and HK-2 cell lines. We demonstrated the ability of Trim-Away protein degradation using IKKα and mTOR as targets, and we showed the availability of the developed system in antibody screening for the Trim-Away method. Furthermore, we effectively coupled Trim-Away with cyclic immunofluorescence and microscopic image-based analysis, which enables single-cell multiplexed imaging analysis. Taking advantage of this new analysis strategy, we were able to compensate for low signal-to-noise due to cell-to-cell variation, which occurs in the Trim-Away method because of the heterogenous contents of the introduced antibody, target protein, and TRIM21 in individual cells. Therefore, the reported cell resealing technique coupled with microscopic image analysis enables Trim-Away users to elucidate target protein function and the effects of target protein degradation on various cellular functions in a more quantitative and precise manner.

DOI： 10.3389/fcell.2022.1027043

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier {BV}  

DOI： 10.1016/j.isci.2021.102724

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell-cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.

DOI： 10.1038/s41598-021-82452-5

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

DOI： 10.3390/ijms21176278

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier {BV}  

DOI： 10.1016/j.bbagen.2019.03.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0223300

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.

DOI： 10.1016/j.bbamcr.2019.02.005

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

ATP-binding cassette A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL) and preventing atherosclerosis. ABCA1 exports cholesterol and phospholipid to apolipoprotein A-I (apoA-I) in serum to generate HDL. We found that streptolysin O (SLO), a cholesterol-dependent pore-forming toxin, barely formed pores in ABCA1-expressing cells, even in the absence of apoA-I. Neither cholesterol content in cell membranes nor the amount of SLO bound to cells was affected by ABCA1. On the other hand, binding of the D4 domain of perfringolysin O (PFO) to ABCA1-expressing cells increased, suggesting that the amount of cholesterol in the outer leaflet of the plasma membrane (PM) increased and that the cholesterol dependences of these two toxins differ. Addition of cholesterol to the PM by the MβCD-cholesterol complex dramatically restored SLO pore formation in ABCA1-expressing cells. Therefore, exogenous expression of ABCA1 causes reduction in the cholesterol level in the inner leaflet, thereby suppressing SLO pore formation.

DOI： 10.1038/s41598-019-39973-x

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.

DOI： 10.1007/978-3-030-23173-6_10

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.11648/j.bs.20180402.11

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.

DOI： 10.1038/s41598-018-29128-9

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Royal Society of Chemistry  

DOI： 10.1039/c7cp05188c

Scopus

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a "cell-resealing technique" that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.

DOI： 10.1038/s41598-018-20482-2

PubMed

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japanese Biochemical Society  

DOI： 10.14952/SEIKAGAKU.2018.900433

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

<jats:title>Abstract</jats:title><jats:p>Cell-based assays are growing in importance for screening drugs and investigating their mechanisms of action. Most of the assays use so-called “normal” cell strain because it is difficult to produce cell lines in which the disease conditions are reproduced. In this study, we used a cell-resealing technique, which reversibly permeabilizes the plasma membrane, to develop diabetic (Db) model hepatocytes into which cytosol from diabetic mouse liver had been introduced. Db model hepatocytes showed several disease-specific phenotypes, namely disturbance of insulin-induced repression of gluconeogenic gene expression and glucose secretion. Quantitative image analysis and principal component analysis revealed that the ratio of phosphorylated Akt (pAkt) to Akt was the best index to describe the difference between wild-type and Db model hepatocytes. By performing image-based drug screening, we found pioglitazone, a PPARγ agonist, increased the pAkt/Akt ratio, which in turn ameliorated the insulin-induced transcriptional repression of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1. The disease-specific model cells coupled with image-based quantitative analysis should be useful for drug development, enabling the reconstitution of disease conditions at the cellular level and the discovery of disease-specific markers.</jats:p>

DOI： 10.1038/s41598-017-15443-0

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その他リンク： http://www.nature.com/articles/s41598-017-15443-0

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Apoptotic death of pancreatic β cells is a major cause of type 2 diabetes mellitus (T2D) progression. Two isoforms of pyruvate kinase, PKM1 and PKM2, have been reported to participate in cell death in several cell types; however, little is known about their causal pathways in pancreatic β-cell death. We examined whether the suppression of PKM1 or PKM2 affects endoplasmic reticulum (ER) stress-induced apoptosis in a pancreatic β-cell line, MIN6, and Beta-TC-6 and found that knockdown of PKM1, but not of PKM2, leads to the induction of ER stress-induced apoptosis in these cells. We also investigated the mechanism by which PKM1 inhibits ER stress-induced apoptosis. We confirmed that PKM1 interacts with A-Raf, an upstream regulator of the MEK/ERK pathway, and that this interaction contributes to MEK1 phosphorylation by A-Raf. PKM1 knockdown suppresses the phosphorylation of MEK, ERK, and caspase-9 (Thr125), which is phosphorylated by the MEK/ERK pathway, thereby inhibiting the cleavage and activation of caspase-9. Thus, PKM1 knockdown activates the caspase-9/caspase-3 pathway under ER stress conditions and leads to apoptosis.

DOI： 10.1016/j.cellsig.2017.07.017

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

DOI： 10.1038/cddis.2017.147

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbamcr.2015.05.005

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Forkhead box O1 (FOXO1) is an important target for insulin. It is widely accepted that insulin-induced phosphorylation of FOXO1 by Akt leads to its nuclear exclusion and results in the inhibition of FOXO1-mediated transcription of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1 (PCK1) in hepatocytes. However, many results that contradict this model have accumulated. Here, we provide a new mechanism for insulin-dependent repression of FOXO1-mediated transcription. We showed insulin-induced translocation of endogenous Ser256-phosphorylated FOXO1, which is essential for regulation of FOXO1-mediated transcription, from nuclear speckles to the nuclear periphery. This insulin-dependent translocation of FOXO1 regulated transcriptional repression of PCK1 concomitant with the formation of the FOXO1-euchromatic histone-lysine N-methyltransferase2 (EHMT2) complex and histone modifications of the PCK1 promoter region. Notably, our results suggest that FOXO1 uses nucleoporin 98 kDa NUP98 for this transcriptional regulation. These results provide a new insight into various FOXO1-mediated transcriptional regulation and FOXO1-mediated essential biological pathways.

DOI： 10.1111/gtc.12226

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

DOI： 10.1073/pnas.1417197112

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.ppat.1004747

Web of Science

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Rab2A, a small GTPase localizing to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), regulates COPI-dependent vesicular transport from the ERGIC. Rab2A knockdown inhibited glucose-stimulated insulin secretion and concomitantly enlarged the ERGIC in insulin-secreting cells. Large aggregates of polyubiquitinated proinsulin accumulated in the cytoplasmic vicinity of a unique large spheroidal ERGIC, designated the LUb-ERGIC. Well-known components of ER-associated degradation (ERAD) also accumulated at the LUb-ERGIC, creating a suitable site for ERAD-mediated protein quality control. Moreover, chronically high glucose levels, which induced the enlargement of the LUb-ERGIC and ubiquitinated protein aggregates, impaired Rab2A activity by promoting dissociation from its effector, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in response to poly (ADP-ribosyl)ation of GAPDH. The inactivation of Rab2A relieved glucose-induced ER stress and inhibited ER stress-induced apoptosis. Collectively, these results suggest that Rab2A is a pivotal switch that controls whether insulin should be secreted or degraded at the LUb-ERGIC and Rab2A inactivation ensures alleviation of ER stress and cell survival under chronic glucotoxicity.

DOI： 10.1038/srep06952

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/gtc.12158

Web of Science

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Biophysical Society of Japan  

DOI： 10.2142/biophys.54.206

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：Nova Science Publishers, Inc.  

Scopus

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent studies have shown that DNA demethylation goes through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by Tet proteins. However, it is still unclear how the target regions for demethylation are distinguished within their genomic context. Here we show that the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has the ability to direct local demethylation around its binding sites, the PPAR response elements (PPREs), during adipocyte differentiation. PPARγ is a key regulator of the differentiation process that forms a PPARγ co-activator complex on PPREs and activates the expression of adipocyte-specific genes. The complex is poly(ADP-ribosyl)ated (PARylated) on PPREs, and Tet proteins catalyse the conversion of 5mC to 5hmC locally by their ability to bind to the PAR polymer, thereby inducing region-specific demethylation. Our study demonstrates that a sequence-dependent transcription factor complex can, through its post-translational modification, serve for Tet proteins as a landmark to identify sites of DNA demethylation.

DOI： 10.1038/ncomms3262

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.53.S210_1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT(1A)R in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons.

DOI： 10.1523/JNEUROSCI.6329-11.2012

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0044127

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記述言語：日本語   出版者・発行元：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

正常および病態の網羅的マイクロアレイ解析やプロテオーム解析が進み，多数の疾患関連遺伝子やタンパク質が抽出され，その細胞内機能や病態との関連が遺伝子改変マウスなどを用い盛んに研究されようとしている．しかし，その網羅的解析によって抽出される遺伝子・タンパク質の数は膨大であり，創薬・診断研究の現場では，動物個体を使用した前臨床研究におけるコストと時間のパフォーマンスの増大のため，リード化合物のスクリーニングやその副作用・作用機序研究における「細胞アッセイ」の重要性はますます増大してきている．しかし，現行の細胞アッセイの多くは「正常細胞」を使用したものであり，「病態環境」における細胞機能のかく乱の分子解析や，病態状態を示す細胞を利用した創薬スクリーニングを実践することが難しいのが現状である．本総説では，この「病態環境」を再現した細胞アッセイ系として，セミインタクト細胞リシール法を用いて作製した「病態モデル細胞」とその利用法を紹介するとともに，この誕生したばかりのツールの利点と，その克服すべき問題点と今後の展望について概説する．

DOI： 10.1271/kagakutoseibutsu.50.510

CiNii Books

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その他リンク： https://jlc.jst.go.jp/DN/JALC/10006703508?from=CiNii

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.

DOI： 10.1016/j.bbamcr.2012.01.007

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

DOI： 10.1016/j.bbamcr.2011.01.023

PubMed

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.51.S5_4

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.51.S145_5

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記述言語：英語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/jcs.063941

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PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/IAI.01022-09

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.50.S119_1

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.50.S55_1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbamcr.2009.08.010

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.m109.038869

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/jcs.043414

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Membrane trafficking is an important cellular process that enables the precise localization of membrane proteins. The disturbance of membrane trafficking results in various disease states. To explore systematically the defects in trafficking pathways that cause these disturbances or disease states, we developed an automated high-throughput fluorescence-based imaging system and carried out visual screening for kinase-regulated trafficking pathways of the cation-independent mannose 6-phosphate receptor (CI-M6PR) in HeLa cells. As the result of our visual screening, which examined the effect of kinase inhibitors and a kinase siRNA library, we identified five kinases (CDC42BPB, PRKACA, PRKACG, GSK3 beta and CSNK2A1) that regulate CI-M6PR trafficking. Moreover, we focused on Alzheimer's disease (AD) to study the relationship between the five kinases and a disease state. Notably, two trafficking pathways, which were regulated by PRKACG and GSK3 beta, respectively, induced high levels of secretion of Abeta, the hallmark of AD. In addition, we found that the modulation of GSK3 beta activity affected the microtubule plus end tracking function of cytoplasmic linker protein-associating protein 2 and resulted in the perturbation of BACE1 localization/trafficking and extensive A beta secretion. Our systems provide new approaches for the analysis of spatially-regulated membrane trafficking and related disease states.

DOI： 10.1111/j.1365-2443.2008.01274.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1365-2443.2008.01213.x

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J-GLOBAL

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2008.03.018

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J-GLOBAL

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent advances in microscopic techniques have shed light on the roles of specific subcellular structures in the regulation of gene expression. One such structure is the stress granule (SG), which is engaged in stress-triggered translational arrest by sequestering pre-initiation complexes of translation. Recent studies revealed the spatial, compositional, and functional linkage of the SG to the processing body (P-body), another cytoplasmic structure that has been implicated in mRNA degradation and siRNA- or miRNA-mediated gene silencing. In this study, we report that PCBP2, a facilitator of IRES (Internal Ribosomal Entry Site)-mediated translation, is a novel constituent of the SG and P-body. Immunofluorescence studies revealed that while PCBP2 is diffusely distributed throughout the nucleoplasm and the cytoplasm, the protein is enriched in a subset of P-bodies under normal conditions. Upon exposure to heat and arsenic stress, PCBP2 became predominantly accumulated at the SG, but was still present in Dcp1a-positive P-bodies. Live-cell imaging revealed the dynamic association of PCBP2-enriched P-bodies and the SG, and FRAP experiments demonstrated that PCBP2 actively moves in and out of the SG and P-body. Taken together, these results suggest that PCBP2 shuttles between the cytoplasm and the two structures under stress. We propose that PCBP2 may be involved in stress-induced remodeling of mRNP complexes and that it may also play a role in the rapid transition of certain silenced mRNAs into a translationally active state. Additionally, given the property of PCBP2 as a nuclear-cytoplasmic shuttling protein, PCBP2 may play a role in directly targeting nascent mRNPs to specific P-bodies for storage.

DOI： 10.1261/rna.780708

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The mRNA-binding protein CUGBP-1 is a multi-faceted factor, involved in a wide range of biological processes including splicing, translation initiation and mRNA degradation. Here we show that CUGBP-1 is a novel constituent of stress granule (SG), the translational silencing machinery assembled in response to environmental stress. CUGBP-1 was rapidly routed to SGs upon exposure to a variety of environmental stress, and actively shuttles between the nucleus and SGs. The linker domain located between the second and third RNA recognition motifs (RRMs) was found to be essential for the recruitment of CUGBP-1 to SGs. Importantly, we discovered that the linker domain is also required to direct CUGBP-1 to another subcellular structure, perinucleolar compartment (PNC). These results demonstrate the dynamic behavior of CUGBP-1 during stress response and that the linker region, in concert with RRMs, plays a significant role in defining its subcellular localization and dynamics.

DOI： 10.1016/j.yexcr.2007.10.024

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.yexcr.2007.10.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/978-1-59745-000-3_25

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PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1111/j.1365-2443.2005.00837.x

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1091/mbc.E03-11-0822

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

NSF and p97 are ATPases required for the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of organelles. NSF uses ATP hydrolysis to dissociate NSF/SNAPs/SNAREs complexes, separating the v- and t-SNAREs, which are then primed for subsequent rounds of fusion. In contrast, p97 does not dissociate the p97/p47/SNARE complex even in the presence of ATP. Now we have identified a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), and show that it binds to the p97/p47/syntaxin5 complex and dissociates it via p97 catalyzed ATP hydrolysis. In living cells, VCIP135 and p47 are shown to function in Golgi and ER assembly.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1083/jcb.149.2.357

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J-GLOBAL

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CiNii Books

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記述言語：日本語   出版者・発行元：日本生化学会  

CiNii Books

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その他リンク： http://search.jamas.or.jp/link/ui/2016293344

記述言語：日本語   出版者・発行元：(一社)日本細胞生物学会  

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記述言語：日本語   出版者・発行元：(一社)日本細胞生物学会  

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記述言語：日本語   出版者・発行元：(一社)日本細胞生物学会  

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記述言語：英語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.49.S8_2

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記述言語：英語  

DOI： 10.1186/1741-7007-7-38

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記述言語：英語  

DOI： 10.1016/j.bbamcr.2009.02.002

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記述言語：日本語   出版者・発行元：日本膜学会  

Organelles are connected and exchange proteins and lipids each other by vesicular transport, while maintaining their specific morphology and components during interphase. At the onset of mitosis, the balance of vesicular transport between organelles is disturbed, resulting in the drastic change of organelle morphology. To understand the coupling mechanisms between organelle morphology and vesicular transport, we developed the reconstitution systems for the Golgi disassembly, the disruption and reformation of the endoplasmic reticulum (ER), mitotic disruption of ER exit sites, and vesicular ...

DOI： 10.5360/membrane.33.24

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記述言語：英語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.47.S164_3

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記述言語：英語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.47.S164_2

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記述言語：日本語   出版者・発行元：共立出版  

CiNii Books

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出版者・発行元：日本顕微鏡学会  

DOI： 10.11410/kenbikyo2004.42.143

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.45.S4_1

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.45.153

CiNii Books

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.44.S157_4

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.44.S157_3

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.44.S157_4

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記述言語：日本語   出版者・発行元：シーエムシー出版  

CiNii Books

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記述言語：日本語   出版者・発行元：バイオテクノロジ-開発技術研究組合  

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記述言語：日本語   出版者・発行元：日本生物物理学会  

At the onset of mitosis, each organelle in a mammalian cell must be inherited accurately by two daughter cells. Recent advances in GFP visualization techniques and in genetic approaches have made it possible to understand the dynamic behavior of organelle during their inheritance and to identify the intracellular machinery required for accurate inheritance. Extensive studies have revealed that the inheritance strategies used by each organelle are not stochastic and passive but are rather controlled by the molecules, which are needed for membrane fusion/vesiculation in intracellular membrane...

DOI： 10.2142/biophys.42.116

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.41.S212_1

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.41.S211_4

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記述言語：日本語   出版者・発行元：金原一郎記念医学医療振興財団  

DOI： 10.11477/mf.2425902527

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記述言語：日本語   出版者・発行元：公益社団法人日本生物工学会  

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記述言語：日本語   出版者・発行元：秀潤社  

CiNii Books

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記述言語：英語  

DOI： 10.1016/S0301-4622(00)00133-2

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.39.S47_4

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記述言語：日本語   出版者・発行元：日本生物物理学会  

In mammalian cells, especially in polarized cells, the Golgi apparatus plays a crucial role in the intracellular sorting and directional-transport of proteins and lipids. The dynamic nature of the apparatus is underscored by the cell cycle-dependent redistribustion and coalesce near the restricted pericentriolar and apical locarlzation in polarized cells. Cytoskeletons might be involved in the dynamic nature, the characteristic shape and the intracellular position of the Golgi apparatus. However, the precise mechanisms of the Golgi-positioning and of the maintenance of its shape in polarize...

DOI： 10.2142/biophys.39.29

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語  

CiNii Books

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記述言語：日本語  

CiNii Books

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記述言語：日本語   出版者・発行元：日本生物物理学会  

CiNii Books

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記述言語：日本語   出版者・発行元：日本生物物理学会  

CiNii Books

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記述言語：日本語   出版者・発行元：秀潤社  

CiNii Books

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