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

pp.22060, (Released:2022-12-28)

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

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

vol.46, no.1, pp.51-64, 2021 (Released:2021-06-30)

参考文献数

61

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

Yoichiro Fujioka Sayaka Kashiwagi Aiko Yoshida Aya O. Satoh Mari Fujioka Maho Amano Yohei Yamauchi Yusuke Ohba

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.21047, (Released:2022-04-28)

被引用文献数

4

---

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Keywords: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein

著者

Haruka Kemmoku Yoshihiko Kuchitsu 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.21080, (Released:2022-02-05)

被引用文献数

13

---

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA or self‐DNA leaked from mitochondria/nuclei. In response to the emergence of such DNAs in the cytosol, STING relocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK‐binding kinase 1 (TBK1), a cytosolic kinase essential for the activation of STING-dependent downstream signalling. To understand at which subcellular compartments TBK1 becomes associated with STING, we generated cells stably expressing fluorescent protein-tagged STING (mNeonGreen-STING) and TBK1 (TBK1-mScarletI). We found that after STING stimulation, TBK1 became associated with the trans‐Golgi network (TGN), not the other parts of the Golgi. STING variants that constitutively induce the type I interferon response have been identified in patients with autoinflammatory diseases named “STING-associated vasculopathy with onset in infancy (SAVI)”. Even in cells expressing these constitutively active STING variants, TBK1 was found to be associated with TGN, not the other parts of the Golgi. These results suggest that TGN acts as a specific platform where STING associates with and activates TBK1.Key words: the Golgi, membrane traffic, innate immunity, STING

著者

Ayako Imanishi Tomokazu Murata Masaya Sato Kazuhiro Hotta Itaru Imayoshi 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

vol.43, no.2, pp.129-140, 2018 (Released:2018-08-10)

参考文献数

49

被引用文献数

3 10

---

For more than a century, hematoxylin and eosin (H&E) staining has been the de facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.Key words: in vivo imaging, histology, machine learning, molecular activity

著者

Justine Renauld Nicolas Thelen Odile Bartholomé Brigitte Malgrange Marc Thiry

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.20057, (Released:2021-01-20)

被引用文献数

1

---

The development of hearing in mammals requires the formation and maturation of a highly organized and specialized epithelium known as the organ of Corti. This epithelium contains two types of cells, the sensory cells, which are the true receptors of auditory information, and the surrounding supporting cells, which are composed of a highly developed cytoskeleton essential to the architecture of the mature organ of Corti. The supporting cells are the only mammalian cells reported to contain the unusual 15-protofilament microtubules. In this paper, we show that 15-protofilament microtubules appear between the second and fourth day after birth in the pillar cells of the organ of Corti in mice. We also show that contrary to what has been described in the nematode worm Caenorhabiditis. Elegans, microtubule acetylation is not essential for the formation of 15-protofilament microtubules in mice but is required for fine-tuning of their diameter. Key words: Acetylation, cytoskeleton, microtubule, inner ear, supporting cells

著者

Tatsuaki Mizutani Yusuke Ohba Satoshi Mizuta Jiro Yasuda Shuzo Urata

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.20030, (Released:2020-11-13)

被引用文献数

1

---

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z’-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl) -pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.Key words: Arenavirus, Förster resonance energy transfer, anti-viral drugs, Z protein

著者

Yuto Matsui Yukihiro Hirata Ikuo Wada Nobuko Hosokawa

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.20025, (Released:2020-06-18)

被引用文献数

14

---

Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60–80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α1-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes. Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC

著者

Mitsuyuki Kuroki Naoki Kamo Yonosuke Kobatake Yukichi Abe Shin-ichi Ishii

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.11, no.2, pp.199-204, 1986 (Released:2008-04-18)

参考文献数

22

被引用文献数

2 2

---

Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (α-ConA) induces membrane depolarization preceding the onset of superoxide anion (O2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O2-production and divalent derivatives were less active than native a-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA.

著者

Hiroyuki Aizawa Masazumi Sameshima Ichiro Yahara

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.22, no.3, pp.335-345, 1997 (Released:2006-03-27)

参考文献数

34

被引用文献数

40 59

---

We transformed Dictyostelium discoideum cells by a vector for expression of a chimerical fusion protein consisting of Aequorea Victoria green fluorescent protein (GFP) and D. discoideum actin at its aminoand carboxy-terminal, respectively. The amount of expressed GFP-actin was about 3% of total actin molecules in the transformed cells. The expression of GFP-actin in D. discoideum completely inhibited cytokinesis in suspension culture. The expression decreased the rate of random cell locomotion to about a half of that of control cells. The expression also caused the cells to round up. These phenotypic observations suggested that GFP-actin acts as a dominant negative form of actin in the cells. The rounding up by expression of GFP-actin was suppressed by genetical elimination of myosin II heavy chain. This result suggested that myosin II is necessary for the rounding up of GFP-actin expressing cells. GFP-actin constructed cortical actin filament architectures together with intrinsic actin in the cells. Purified GFP-actin polymerized and de-polymerized repetitively according to the solution conditions in vitro. The critical concentration of GFP-actin for polymerization is the same as that of actin. The GFP-actin filaments was able to bind to coverglass surfaces coated with myosin head fragments. However, the GFP-actin filaments did not slide at all on the coverglass by addition of ATP. This indicates that the GFP-actin filaments form rigor complex with myosin II in vitro even in the presence of ATP. The formation of rigor complex may cause the cells to round up.

著者

Kyohei Umebayashi

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.28, no.5, pp.443-453, 2003 (Released:2004-01-26)

参考文献数

84

被引用文献数

21 27

---

After cell surface receptors are internalized for endocytosis, they are accurately sorted in endosomes. Some are recycled to the plasma membrane and others are downregulated by delivery to lysosomes. Evidence is rapidly accumulating that ubiquitination of cargo proteins acts as a sorting signal during endocytosis. Sorting devices that recognize ubiquitin are distributed to various compartments, probably acting in a concerted manner. Cholesterol is enriched in the plasma membrane and endosomes, and is involved in protein sorting by forming microdomains called lipid rafts. Ubiquitin and cholesterol hold the key to control the endocytic sorting, and they are likely acting cooperatively.

著者

Kotaro Horikawa Tomohiro Yorimitsu Chie Kodera Ken Sato

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.19019, (Released:2019-08-09)

被引用文献数

3

---

The coat protein complex II (COPII) generates transport carriers that deliver newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The small GTPase Sar1 is a well-known regulator of the assembly of the COPII coat. In the present study, we demonstrate that, besides its well-established role in ER-to-Golgi trafficking, the nuclear localization of Sar1 is essential for the viability of Saccharomyces cerevisiae. Inhibition of either the nuclear entry or retention of Sar1 leads to a severe growth defect. Additionally, in vivo deletion of Sar1, by using conditional genetic depletion, further demonstrates that the loss of nuclear localization of Sar1 results in an abnormal nuclear envelope shape. Our findings highlighted a possible novel role of Sar1 within the nucleus, which may relate to the proper formation of the nuclear envelope. Keywords: Sar1, COPII, small GTPase, nuclear envelope, membrane traffic

著者

Masakiyo Sakaguchi Tadashi Kondo Hong Pu Masayoshi Namba

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.23, no.2, pp.69-72, 1998 (Released:2006-03-27)

参考文献数

15

被引用文献数

3 4

---

In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present; one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

著者

Kanae Sasaki Ryota Komori Mai Taniguchi Akie Shimaoka Sachiko Midori Mayu Yamamoto Chiho Okuda Ryuya Tanaka Miyu Sakamoto Sadao Wakabayashi Hiderou Yoshida

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.18031, (Released:2018-11-28)

被引用文献数

15

---

The Golgi stress response is a homeostatic mechanism that augments the functional capacity of the Golgi apparatus when Golgi function becomes insufficient (Golgi stress). Three response pathways of the Golgi stress response have been identified in mammalian cells, the TFE3, HSP47 and CREB3 pathways, which augment the capacity of specific Golgi functions such as N-glycosylation, anti-apoptotic activity and pro-apoptotic activity, respectively. On the contrary, glycosylation of proteoglycans (PGs) is another important function of the Golgi, although the response pathway upregulating expression of glycosylation enzymes for PGs in response to Golgi stress remains unknown. Here, we found that expression of glycosylation enzymes for PGs was induced upon insufficiency of PG glycosylation capacity in the Golgi (PG-Golgi stress), and that transcriptional induction of genes encoding glycosylation enzymes for PGs was independent of the known Golgi stress response pathways and ER stress response. Promoter analyses of genes encoding these glycosylation enzymes revealed the novel enhancer elements PGSE-A and PGSE-B (the consensus sequences are CCGGGGCGGGGCG and TTTTACAATTGGTC, respectively), which regulates their transcriptional induction upon PG-Golgi stress. From these observations, the response pathway we discovered is a novel Golgi stress response pathway, which we have named the PG pathway. Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation

著者

Hideki Gotoh Toshifumi Takenaka Masanori Shozushima

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.8, no.1, pp.11-18, 1983 (Released:2008-04-18)

参考文献数

23

被引用文献数

1 1

---

Skinned nerve fiber was prepared by treating a giant axon of squid with saponin. Filamentous networks and membranous organelles such as vacuoles, smooth membranes and mitochondria were present after the extraction. Ruthenium red staining of the extracted tissue showed dense fibrillar networks in the cytoplasm that correspond to the "microtrabeculae" or "cytoskeleton" reported by other investigators. Our method of preparation, thus, gives further evidence that these structures are not the result of artifactual condensation or precipitation. The present results also indicated that the networks are composed of acid mucopolysaccharides and/or mucoproteins. The density of microtubules was not changed by the extraction per se, but the number of microtubules remaining after extraction decreased by alteration of environmental conditions such as low temperature (4°C), or the presence of Ca++ (1 mM), Mg++ free medium or colchicine (1 mM). These conditions are known to inhibit axonal transport. The usefulness of our extracted cytoplasm as a model of microtubule-related physiological functions is discussed.

著者

Gillian L. Ryan Naoki Watanabe Dimitrios Vavylonis

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.38, no.1, pp.1-7, 2013 (Released:2013-01-19)

参考文献数

30

被引用文献数

5 12

---

We present a set of flexible image analysis tools to analyze dynamics of cell shape and protein concentrations near the leading edge of cells adhered to glass coverslips. Plugins for ImageJ streamline common analyses of microscopic images of cells, including the calculation of leading edge speeds, total and average intensities of fluorescent markers, and retrograde flow rate measurements of fluorescent single-molecule speckles. We also provide automated calculations of auto- and cross-correlation functions between velocity and intensity measurements. The application of the methods is illustrated on images of XTC cells.

著者

Koichiro Suzuki Takahiro Yamada Keiko Yamazaki Masato Hirota Narumi Ishihara Mizuki Sakamoto Daisuke Takahashi Hideki Iijima Koji Hase

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.17022, (Released:2018-01-18)

被引用文献数

9

---

Inflammatory bowel disease (IBD) is a refractory disease of the gastrointestinal tract that is believed to develop in genetically susceptible individuals. Glycosylation, a type of post-translational modification, is involved in the development of a wide range of diseases, including IBD, by modulating the function of various glycoproteins. To identify novel genes contributing to the development of IBD, we analyzed single nucleotide polymorphisms (SNPs) of glycosylation-related genes in IBD patients and identified MAN2A1, encoding alpha-mannosidase II (α-MII), as a candidate gene. α-MII plays a crucial, but not exclusive, role in the maturation of N-glycans. We also observed that intestinal epithelial cells (IECs), which establish the first-line barrier and regulate gut immunity, selectively expressed α-MII with minimal expression of its isozyme, alpha-mannosidase IIx (α-MIIx). This led us to hypothesize that IEC-intrinsic α-MII is implicated in the pathogenesis of IBD. To test this hypothesis, we generated IEC-specific α-MII-deficient (α-MIIΔIEC) mice. Although α-MII deficiency has been shown to have a minimal effect on N-glycan maturation in most cell types due to the compensation by α-MIIx, ablation of α-MII impaired the maturation of N-glycans in IECs. α-MIIΔIEC mice were less susceptible to dextran sulfate sodium-induced colitis compared with control littermates. In accordance with this, neutrophil infiltration in the colonic mucosa was attenuated in α-MIIΔIEC mice. Furthermore, gene expression levels of neutrophil-attracting chemokines were downregulated in the colonic tissue. These results suggest that IEC-intrinsic α-MII promotes intestinal inflammation by facilitating chemokine expression. We propose SNPs in MAN2A1 as a novel genetic factor for IBD. Key words: inflammatory bowel disease, alpha-mannosidase II, intestinal epithelial cell, N-glycosylation

著者

Yoko Takata Hiroe Kishine Takefumi Sone Taichi Andoh Masami Nozaki Michael Poderycki Jonathan D. Chesnut Fumio Imamoto

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.36, no.2, pp.209-222, 2011 (Released:2011-11-17)

参考文献数

56

被引用文献数

16 19

---

Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64–98%. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15–24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.

著者

Yoko Takata Hiroe Kishine Takefumi Sone Taichi Andoh Masami Nozaki Michael Poderycki Jonathan D. Chesnut Fumio Imamoto

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

pp.1109300086, (Released:2011-10-06)

被引用文献数

16 19

---

Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64-98 %. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15-24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.

著者

Kenta Saito Kentaro Kobayashi Tomomi Tani Takeharu Nagai

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.33, no.1, pp.133-141, 2008 (Released:2008-09-05)

参考文献数

21

被引用文献数

4 4

---

Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca2+ propagation, and the multi-color imaging of Ca2+ and PKC-γ dynamics in living cells.