研究者詳細 - 鈴木　健史

論文 - 鈴木　健史

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Caveolae-mediated endocytosis pathway regulates endothelial fenestra homeostasis in the rat pituitary.

Takashi Nakakura, Hideyuki Tanaka, Takeshi Suzuki

Biochemical and biophysical research communications 675 177 - 183 2023年07月 [国際誌]

Endothelial fenestrae are transcellular pores separated by diaphragms formed by plasmalemma vesicle-associated proteins (PLVAP) and function as channels for peptide hormones and other substances. Caveola, a key regulator of clathrin-independent endocytosis, may be involved in the invagination and fusion of plasma membranes, which are essential for fenestra formation. In this study, we first found that caveolin-1 and -2, the major components of caveolae, was localized in fenestrated endothelial cells in the anterior lobe of the rat pituitary by immunohistochemistry. As we also observed caveolae in the endothelial cells of the anterior lobe of the rat pituitary by transmission electron microscopy, we studied the relationship between the caveolae-mediated endocytosis pathway and fenestrae structure in cultured endothelial cells isolated from the anterior lobe of the rat pituitary (CECAL) by immunofluorescence staining and scanning electron microscopy. The inhibition of caveolae-mediated endocytosis by genistein enlarged the PLVAP-positive oval-shaped structure that represented the sieve plate and induced the formation of a doughnut-shaped bulge around the fenestra in CECAL. In contrast, the acceleration of caveolae-mediated endocytosis by okadaic acid induced the diffusion of PLVAP-positive signals in the cytoplasm and reduced the number of fenestrae in CECAL. These results indicate that the caveolae-mediated endocytosis pathway is involved in the fenestra homeostasis in the fenestrated endothelial cells of the rat pituitary.

DOI PubMed

Regulation of fenestra formation via actin-dynamin2 interaction in rat pituitary endothelial cells

Takashi Nakakura, Hideyuki Tanaka, Takeshi Suzuki

Cell and Tissue Research ( Springer Science and Business Media LLC ) 2022年09月

DOI

Fibronectin-integrin signaling regulates PLVAP localization at endothelial fenestrae by microtubule stabilization.

Takashi Nakakura, Takeshi Suzuki, Kotaro Horiguchi, Hideyuki Tanaka, Kenjiro Arisawa, Toshio Miyashita, Yoko Nekooki-Machida, Haruo Hagiwara

Cell and tissue research 384 ( 2 ) 449 - 463 2021年05月 [国際誌]

Endothelial fenestrae are the transcellular pores existing on the capillary walls which are organized in clusters referred to as sieve plates. They are also divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). In this study, we examined the involvement of fibronectin signaling in the formation of fenestra and diaphragm in endothelial cells. Results showed that Itga5 and Itgb1 were expressed in PECAM1-positive endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, and integrin α5 was localized at the fenestrated capillaries of the rat pituitary and cultured PECAM1-positive endothelial cells isolated from AL (CECAL). Inhibition of both integrin α5β1 and FAK, a key molecule for integrin-microtubule signaling, respectively, by ATN-161 and FAK inhibitor 14, caused the delocalization of PLVAP at the sieve plates and depolymerization of microtubules in CECAL. Paclitaxel prevented the delocalization of PLVAP by the inhibition of integrin α5β1. Microtubule depolymerization induced by colcemid also caused the delocalization of PLVAP. Treatment of CECAL with ATN-161 and colcemid caused PLVAP localization at the Golgi apparatus. The localization of PLVAP at the sieve plates was inhibited by BFA treatment in a time-dependent manner and spread diffusely to the cytoplasm. These results indicate that a constant supply of PLVAP proteins by the endomembrane system via the Golgi apparatus is essential for the localization of PLVAP at sieve plates. In conclusion, the endomembrane transport pathway from the Golgi apparatus to sieve plates requires microtubule cytoskeletons, which are regulated by fibronectin-integrin α5β1 signaling.

DOI PubMed

Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary.

Takashi Nakakura, Takeshi Suzuki, Hideyuki Tanaka, Kenjiro Arisawa, Toshio Miyashita, Yoko Nekooki-Machida, Toshiki Kurosawa, Yuma Tega, Yoshiharu Deguchi, Haruo Hagiwara

Cell and tissue research 383 ( 2 ) 823 - 833 2021年02月 [国際誌]

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle-associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary.

DOI PubMed

ATAT1 is essential for regulation of homeostasis-retaining cellular responses in corticotrophs along hypothalamic-pituitary-adrenal axis

Takashi Nakakura, Takeshi Suzuki, Seiji Torii, Anshin Asano-Hoshino, Yoko Nekooki-Machida, Hideyuki Tanaka, Kenjiro Arisawa, Yoshimi Nishijima, Takao Susa, Tomoki Okazaki, Yoshiko Kiuchi, Haruo Hagiwara

CELL AND TISSUE RESEARCH ( SPRINGER ) 370 ( 1 ) 169 - 178 2017年10月 [査読有り]

The production and secretion of adrenocorticotropin, a proopiomelanocortin (POMC)-derived hormone, by corticotrophs in the anterior pituitary, is regulated by corticotrophin-releasing hormone (CRH) and glucocorticoids. We have previously demonstrated that adrenalectomy induces a-tubulin N-acetyltransferase 1 (ATAT1) expression and atubulin acetylation in corticotrophs. However, the regulatory mechanism of ATAT1 expression and the function of acetylated microtubules in corticotrophs are unclear. Here, we analyze the effect of CRH or dexamethasone on Atat1 expression in the mouse corticotroph AtT20 cell line. The expression of Atat1 was increased by CRH and decreased by dexamethasone in AtT20 cells. We examined the effect of Atat1 knockdown on the expression of POMC-associated genes and the dexamethasone-induced nuclear translocation of glucocorticoid receptor (GR) by real-time polymerase chain reaction and Western blot analysis, respectively. Atat1 knockdown resulted in a significant increase in the expression of ACTH-producing genes and decreased the dexamethasone-induced nuclear translocation of GR accompanied with a reduction in a-tubulin acetylation. Atat1 overexpression resulted in a significant increase in a-tubulin acetylation and the dexamethasone-induced nuclear translocation of GR. These results suggest that the acetylated microtubules function as the rail-line for the transportation of GR into the nucleus. We conclude that ATAT1 finely tunes the cellular responses of corticotrophs to hormonal stimulation through an intracellular feedback circuit.

DOI PubMed

Expression and localization of forkhead box protein FOXJ1 in S100β-positive multiciliated cells of the rat pituitary.

Nakakura T, orresponding author, Suzuki T, Horiguchi K, Fujiwara K, Tsukada T, Asano-Hoshino A, Tanaka H, Arisawa K, Nishijima Y, Nekooki-Machida Y, Kiuchi Y, Hagiwara H

Medical molecular morphology 50 ( 2 ) 59 - 67 2017年06月 [査読有り] [国内誌]

S100β-positive cells exist in the marginal cell layer (MCL) of the adenohypophysis and follicle structure in the parenchyma of anterior lobe (ALFS) in pituitary. They have multiple functions as phagocytes or cells that regulate hormone secretion. Majority of S100β-positive cells in the adenohypophysis express sex determining region Y-box 2 protein (SOX2), a stem cell marker; therefore, S100β/SOX2 double positive cells are also considered as one type of stem/progenitor cells. MCL and ALFS are consisting of morphologically two types of cells, i.e., multiciliated cells and non-ciliated cells. However, the relationship between the S100β-positive cells and multiciliated cells in the pituitary is largely unknown. In the present study, we first immunohistochemically verified the feature of multiciliated cells in MCL and ALFS. We then examined the expression patterns of FOXJ1, an essential expression factor for multiciliated cell-differentiation, and SOX2 in the S100β-positive multiciliated cells by in situ hybridization and immunohistochemistry. We identified anew the S100β/SOX2/FOXJ1 triple positive multiciliated cells, and revealed that they were dispersed throughout the MCL and ALFS. These results indicate that the MCL and ALFS are consisting of morphologically and functionally distinct two types of cells, i.e., S100β/SOX2 double positive non-ciliated cells and S100β/SOX2/FOXJ1 triple positive multiciliated cells.

DOI PubMed

Adrenalectomy facilitates ATAT1 expression and α-tubulin acetylation in ACTH-producing corticotrophs.

Nakakura T, orresponding author, Nemoto T, Suzuki T, Asano-Hoshino A, Tanaka H, Arisawa K, Nishijima Y, Kiuchi Y, Hagiwara H

Cell and tissue research ( SPRINGER ) 366 ( 2 ) 363 - 370 2016年11月 [査読有り]

Microtubules play an important role in the intracellular transport of secretory granules in endocrine cells and in mitosis and the maintenance of cell morphology and are composed of heterodimers of alpha- and beta-tubulin. alpha-Tubulin N-acetyltransferase 1 (ATAT1), which acetylates the lysine residue at position 40 of alpha-tubulin, functions not only in stabilizing microtubule structures and forming the primary cilium assembly but also in vesicular trafficking in neurons. However, the localization of ATAT1 and the role of alpha-tubulin acetylation in endocrine cells in the pituitary are still poorly understood. Corticotrophs in the anterior lobe of the pituitary produce and secrete adrenocorticotropin (ACTH). Although removal of the adrenal gland, a target organ of ACTH, is reported to promote the synthesis and secretion of ACTH in corticotrophs and to induce structural alterations in their organelles, uncertainty remains as to whether the acetylation of alpha-tubulin is involved in such intracellular events of corticotrophs. We investigate the expression and localization of ATAT1 and the acetylation of alpha-tubulin in the pituitary of normal and adrenalectomized rats. We find that ATAT1 is localized to the Golgi apparatus of endocrine cells in the anterior lobe of normal pituitary and that the expression levels of ATAT1 and acetylation levels of alpha-tubulin increase following adrenalectomy. These results agree with the hypothesis that the acetylation of alpha-tubulin by ATAT1 regulates the intracellular transport of secretory granules in corticotrophs.

DOI PubMed

Intracellular localization of α-tubulin acetyltransferase ATAT1 in rat ciliated cells.

Nakakura T, orresponding author, Suzuki T, Nemoto T, Tanaka H, Asano-Hoshino A, Arisawa K, Nishijima Y, Kiuchi Y, Hagiwara H

Medical molecular morphology ( SPRINGER JAPAN KK ) 49 ( 3 ) 133 - 143 2016年09月 [査読有り]

Cilia are microtubule-based hair-like organelles on basal bodies located beneath the cell membrane in various tissues of multicellular animals, and are usually classified into motile cilia and primary cilia. Microtubules are assembled from the heterodimers of alpha- and beta-tubulin. The lysine residue at position 40 (K40) of alpha-tubulin is an important site for acetylation, and this site is acetylated in the cilium. alpha-Tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to the K40 residue of alpha-tubulin; however, its intracellular distribution in mammalian tissues remains unclear. In this study, we analyzed ATAT1 localization in rat trachea, oviduct, kidney, retina, testis and the third ventricle of the brain by immunohistochemical techniques using a specific antibody against ATAT1. ATAT1 was distributed to the motile cilia of multiciliated cells of the trachea, third ventricle of the brain and oviduct, and in the primary cilia of the renal medullary collecting duct. ATAT1 also localized to the primary cilia, inner and outer segments of retinal photoreceptor cells, and at the Golgi apparatus of spermatocytes and spermatids of testis. These results indicated that alpha-tubulin acetylation by ATAT1 at distinct subcellular positions may influence the functional regulation of microtubules and cilia in a variety of ciliated cells.

DOI PubMed

The elongation of primary cilia via the acetylation of α-tubulin by the treatment with lithium chloride in human fibroblast KD cells.

Nakakura T, Asano-Hoshino A, Suzuki T, Arisawa K, Tanaka H, Sekino Y, Kiuchi Y, Kawai K, Hagiwara H

Medical molecular morphology ( SPRINGER JAPAN KK ) 48 ( 1 ) 44 - 53 2015年03月 [査読有り]

Primary cilium, an organelle found on nearly every cell in the human body, typically serves as the mechanical sensor of the cell. Lithium ion is known to promote the elongation of primary cilia in a variety of cell types, but it is unknown whether lithium is involved in the acetylation of alpha-tubulin which is essential for the assembly of primary cilia. In order to reveal the relationship between the elongation of primary cilia with lithium and the acetylation of alpha-tubulin, we first observed the formation and structure of primary cilia in KD cells, a cell line deriving fibroblasts in human labium. Subsequently, by immunohistochemical and western blot analysis we elucidated that the length of primary cilia and acetylation of alpha-tubulin are regulated by lithium chloride (LiCl) in the medium in a time- and concentration-dependent manner. We next performed the RT-PCR, RNAi-based experiments and biochemical study using an inhibitor of glycogen synthase kinase-3 beta GSK-3 beta). We found that LiCl mobilizes the alpha-tubulin N-acetyltransferase 1 (alpha TAT1) in the signaling pathway mediating GSK-3 beta and adenylate cyclase III. In conclusion, our results suggested that LiCl treatments activate alpha TAT1 by the inhibition of GSK-3 beta and promote the alpha-tubulin acetylation, and then elongate the primary cilia.

DOI PubMed

遺伝子組み換え実験計画時に有用なMicrosoft Word用マクロプログラム

鈴木健史, 田中憲一

札幌医科大学医療人育成センター紀要 ( 札幌医科大学医療人育成センター ) 5 ( 5 ) 5 - 10 2014年 [査読有り]

CiNii

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates release of somatolactin (SL)-α and SL-β from cultured goldfish pituitary cells via the PAC₁ receptor-signaling pathway, and affects the expression of SL-α and SL-β mRNAs.

Azuma M, Suzuki T, Mochida H, Tanaka S, Matsuda K

Peptides 43 40 - 47 2013年05月 [査読有り]

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10-10-10-7 M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10 -9 M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC1R) antagonist, PACAP (6-38) (10-6 M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10-9 and 10-8 M) for 6 h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10-8 M) on SL-β mRNA expression was blocked by treatment with PACAP(6-38) (10-6 M), whereas PACAP(6-38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC1R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC1R signaling. © 2013 Elsevier Inc.

DOI PubMed

哺乳動物細胞におけるチューブリンアセチル化酵素の細胞内局在動態の可視化

鈴木健史, 中倉敬, 萩原治夫

札幌医科大学医療人育成センター紀要 ( 札幌医科大学医療人育成センター ) 4 ( 4 ) 11 - 15 2013年 [査読有り]

CiNii

Close Association of Aquaporin-2 Internalization with Caveolin-1

Takeo Aoki, Takeshi Suzuki, Haruo Hagiwara, Michio Kuwahara, Sei Sasaki, Kuniaki Takata, Toshiyuki Matsuzaki

ACTA HISTOCHEMICA ET CYTOCHEMICA ( JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY ) 45 ( 2 ) 139 - 146 2012年 [査読有り]

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively. Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.

DOI

Function of the Membrane Water Channel Aquaporin-5 in the Salivary Gland

Toshiyuki Matsuzaki, Taketo Susa, Kinue Shimizu, Nobuhiko Sawai, Takeshi Suzuki, Takeo Aoki, Satoshi Yokoo, Kuniaki Takata

ACTA HISTOCHEMICA ET CYTOCHEMICA ( JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY ) 45 ( 5 ) 251 - 259 2012年 [査読有り]

The process of saliva production in the salivary glands requires transepithelial water transfer from the interstitium to the acinar lumen. There are two transepithelial pathways: the transcellular and paracellular. In the transcellular pathway, the aquaporin water channels induce passive water diffusion across the membrane lipid bilayer. It is well known that aquaporin-5 (AQP5) is expressed in the salivary glands, in which it is mainly localized at the apical membrane of the acinar cells. This suggests the physiological importance of AQP5 in transcellular water transfer. Reduced saliva secretion under pilocarpine stimulation in AQP5-null mice compared with normal mice further indicates the importance of AQP5 in this process, at least in stimulated saliva secretion. Questions remain therefore regarding the role and importance of AQP5 in basal saliva secretion. It has been speculated that there would be some short-term regulation of AQP5 such as a trafficking mechanism to regulate saliva secretion. However, no histochemical evidence of AQP5-trafficking has been found, although some of biochemical analyses suggested that it may occur. There are no reports of human disease caused by AQP5 mutations, but some studies have revealed an abnormal subcellular distribution of AQP5 in patients or animals with xerostomia caused by Sjogren's syndrome and X-irradiation. These findings suggest the possible pathophysiological importance of AQP5 in the salivary glands.

DOI

Nonlinear phase imaging using two-beam interferometry in SPE microscopy.

Masatomo Yamagiwa, Yasuyuki Ozeki, Gen Omura, Takeshi Suzuki, Shin’ichiro Kajiyama, Kiichi Fukui, Kazuyoshi Itoh

Jpn J Appl Phys 48 062501 2009年 [査読有り]

DOI

The primary cilia of secretory cells in the human oviduct mucosa

Haruo Hagiwara, Nobuo Ohwada, Takeo Aoki, Takeshi Suzuki, Kuniaki Takata

MEDICAL MOLECULAR MORPHOLOGY ( SPRINGER TOKYO ) 41 ( 4 ) 193 - 198 2008年12月 [査読有り]

The human oviduct is lined with a simple columnar epithelium composed of ciliated cells and secretory cells. Primary cilia or solitary cilia usually extend from the apical surface of the secretory cells. The axoneme of the primary cilia is composed of nine peripheral microtubule doublets (9 + 0 pattern) that lack dynein arms and nexin links. Displacement of peripheral doublets to the central region, which is suggested to be attributable to the lack of nexin links, is one of the distinctive features of oviductal primary cilia. The basal body that extends the primary cilium connects to its paired centriole by the striated connector. The basal body is associated with the accessory structures, such as alar sheets, basal feet, and striated rootlets. Several basal feet project laterally from the basal body. The cap of the basal foot serves as the microtubule organizing center. Several striated rootlets radiate from the basal body toward the nucleus. The basal body, the paired centriole, and the basal body-associated structures are considered to play important roles in the stabilization and fixing of the cilium in the proper position on the apical cell surface.

DOI PubMed CiNii

Immunohistochemical and electron microscopic observations of stromal cells in the human oviduct mucosa

Haruo Hagiwara, Nobuo Ohwada, Takeo Aoki, Takeshi Suzuki, Kuniaki Takata

MEDICAL MOLECULAR MORPHOLOGY ( SPRINGER TOKYO ) 41 ( 4 ) 221 - 226 2008年12月 [査読有り]

Stromal cells in the lamina propria of the human oviduct mucosa are unique cells that can differentiate into decidual cells during ectopic pregnancy in the oviduct. The nature of stromal cells is still unknown. In the present study, we investigated human oviductal stromal cells with transmission electron microscopy and immunohistochemistry and revealed that they had ultrastructural features similar to myofibroblasts and expressed alpha-smooth muscle actin, a marker used to identify myofibroblasts. Primary cilia were also one of the characteristic profiles of the stromal cells. These findings showed that the connective tissue-stromal cells in the human oviduct mucosa are myofibroblasts. They are considered to play an important role in the transport of oocytes by bringing about contraction of the mucosal folds.

DOI PubMed CiNii

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: Expression of AQP4 in neuronal sensory cells.

Abduxukur Ablimit, Takeo Aoki, Toshiyuki Matsuzaki, Takeshi Suzuki, Haruo Hagiwara, Shigeru Takami, Kuniaki Takata

Chem Senses 33 ( 5 ) 481?488 - 488 2008年 [査読有り]

DOI PubMed CiNii

Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells

Takahiro Hasegawa, Toshiyuki Matsuzaki, Yuki Tajika, Abduxukur Ablimit, Takeshi Suzuki, Takeo Aoki, Haruo Hagiwara, Kuniaki Takata

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 127 ( 3 ) 233 - 241 2007年03月 [査読有り]

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4-EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells.

DOI

Recent advances in fluorescent labeling techniques for fluorescence microscopy

Takeshi Suzuki, Toshiyuki Matsuzaki, Haruo Hagiwara, Takeo Aoki, Kuniaki Takata

ACTA HISTOCHEMICA ET CYTOCHEMICA ( JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY ) 40 ( 5 ) 131 - 137 2007年 [査読有り]

Tremendous progress in recent computer- controlled systems for fluorescence and laserconfocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.

DOI CiNii

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