Papers - Kimura, Shunsuke

Roles of a macrophage molecule M‐mod in type 2 diabetes

H Kitamura, M Ito, S Kimura, Y Shimamoto, T Miyamoto, H Watarai, ...

The FASEB Journal 26, 686.5-686.5  2012

The epithelia-specific membrane trafficking factor AP-1B controls gut immune homeostasis in mice.

Takahashi D, Hase K, Kimura S, Nakatsu F, Ohmae M, Mandai Y, Sato T, Date Y, Ebisawa M, Kato T, Obata Y, Fukuda S, Kawamura YI, Dohi T, Katsuno T, Yokosuka O, Waguri S, Ohno H

Gastroenterology (2)  141 ( 2 ) 621 - 632 2011.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0016-5085

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BACKGROUND & AIMS: Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. METHODS: The role of AP-1B in intestinal immunity was examined in AP-1B-deficient mice (Ap1m2(-/-)) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B-mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2(-/-) mice. RESULTS: Ap1m2(-/-) mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A-producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. CONCLUSIONS: AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B-mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

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M-Sec: Emerging secrets of tunneling nanotube formation.

Ohno H, Hase K, Kimura S

Communicative & integrative biology (3)  3 ( 3 ) 231 - 233 2010.05

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1942-0889

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Tunneling nanotubes (TNT) are the latest addition to the array of strategies used for intercellular signaling. TNTs are continuous conduits of the plasma membrane that allow direct physical connection of plasma membranes and cytosol among remote cells. They are important for intercellular communication by mediating exchange of cellular components as well as signal transduction molecules. Despite ample evidence suggesting the pathophysiological importance of TNTs, virtually nothing is known about the molecular basis for their formation. With the lack of specific TNT markers, their study has relied solely on morphological analyses, and the precise identity of TNT and TNTlike structures have been difficult to define. We have now shown that M-Sec is a TNT marker and a central factor for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton is involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels. © 2010 Landes Bioscience.

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Essential role of membrane trafficking factor AP-1B in gut immune homeostasis (90.4)

D Takahashi, K Hase, S Kimura, H Ohno

The Journal of Immunology 184 (1 Supplement), 90.4-90.4  2010

M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex.

Hase K, Kimura S, Takatsu H, Ohmae M, Kawano S, Kitamura H, Ito M, Watarai H, Hazelett CC, Yeaman C, Ohno H

Nature cell biology (12)  11 ( 12 ) 1427 - 1432 2009.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1465-7392

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Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells(1,2). These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

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Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, Kadokura K, Tobe T, Fujimura Y, Kawano S, Yabashi A, Waguri S, Nakato G, Kimura S, Murakami T, Iimura M, Hamura K, Fukuoka S, Lowe AW, Itoh K, Kiyono H, Ohno H

Nature (7270)  462 ( 7270 ) 226 - 230 2009.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0028-0836

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The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens(1-3). To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches(4). This function, called antigen transcytosis, is mediated by specialized epithelial M cells(5,6). The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases(7,8), the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

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Monitoring autophagy in mammalian cultured cells through the dynamics of LC3.

Kimura S, Fujita N, Noda T, Yoshimori T

Methods in enzymology 452 ( C ) 1 - 12 2009

Joint Work, Accepted,  ISSN  0076-6879

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In this chapter, we introduce several methods that rely on the analysis of LC3, a versatile marker protein of autophagic structures in mammalian cultured cells. The appearance of LC3-positive puncta is indicative of the induction of autophagy, and it is observed either by immunofluorescence or by GFP-based microscopy. The maturation process by which autophagosomes are converted into autolysosomes can be monitored by the GFP and RFP tandemly tagged LC3 (tfLC3) method. Lysosomal turnover of LC3 is a good index of the proceeding of autophagy and can be assessed by Western blotting. These methods will provide a relatively easy assessment of autophagy, and the details of the procedure will be described along with possible pitfalls. © 2009 Elsevier Inc. All rights reserved.

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Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response

K Hase, K Kawano, T Nochi, GS Pontes, S Fukuda, M Ebisawa, ...

Nature 462 (7270), 226-230  2009

Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes.

Kimura S, Noda T, Yoshimori T

Cell structure and function (1)  33 ( 1 ) 109 - 122 2008

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0386-7196|1347-3700

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Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.

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Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.

Kimura S, Noda T, Yoshimori T

Autophagy (5)  3 ( 5 ) 452 - 460 2007.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  1554-8627

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During the process of autophogy, autophagosomes undergo a maturation process consisting of multiple fusions with enclosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

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[Autophagic degradation of unfolding proteins].

Kimura S, Kamimoto T, Yoshimori T

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme (10 Suppl)  51 ( 10 Suppl ) 1494 - 1498 2006.08

Joint Work, Accepted,  ISSN  0039-9450

Autophagic degradation of unfolding proteins

S Kimura, T Kamimoto, T Yoshimori

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 (10 Suppl …  2006

The analysis of autophagosome movement

S Kimura, T Yoshimori

Autophagy 2 (4), 355-355  2006

Mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in gelatinous drop-like corneal dystrophy.

Murakami A, Kimura S, Fujiki K, Fujimaki T, Kanai A

Japanese journal of ophthalmology (4)  48 ( 4 ) 317 - 320 2004.07

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0021-5155

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Purpose: To report mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in two members of the same family who showed symptoms of gelatinous drop-like corneal dystrophy (GDLD).
Methods: DNA was extracted from leukocytes of peripheral blood of the two affected members of the family and from controls, and the coding region of M1S1 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing. Normal and mutant M1S1 expression vectors were constructed and transfected into CHO cells to identify the cellular location of the gene products.
Results: The affected members had compound heterozygous mutations consisting of a nonsense change at codon 84 (K84X) and a missense mutation resulting in a substitution of arginine for cysteine at codon 108 (C108R). Neither of these mutations was found in the 50 controls. Protein expression analysis showed that the C108R product was distributed diffusely in the cytoplasm, whereas the normal gene product accumulated at cell-to-cell adhesion borders.
Conclusion: These data indicate that the K84X and C108R mutations in M1S1 cause GDLD. (C) Japanese Ophthalmological Society 2004.

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