Abstract
ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we comprehensively examined the cellular and subcellular localization of Arf6 in adult mouse peripheral tissues by immunofluorescence and immunoelectron microscopy using the heat-induced antigen retrieval method with Tris–EDTA buffer (pH 9.0). Marked immunolabeling of Arf6 was observed particularly in epithelial cells of several tissues including the esophagus, stomach, small and large intestines, trachea, kidney, epididymis, oviduct, and uterus. In most epithelial cells of simple or pseudostratified epithelia, Arf6 exhibited predominant localization to the basolateral membrane and a subpopulation of endosomes. At an electron microscopic level, Arf6 was localized along the basolateral membrane, with dense accumulation at interdigitating processes and infoldings. Arf6 was present in a ring-like appearance at intercellular bridges in spermatogonia and spermatocytes in the testis and at the Flemming body of cytokinetic somatic cells in the ovarian follicle, thymus, and spleen. The present study provides anatomical clues to help understand the physiological roles of Arf6 at the whole animal level.
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Akiyama M, Zhou M, Sugimoto R, Hongu T, Furuya M, Funakoshi Y, Kato M, Hasegawa H, Kanaho Y (2010) Tissue- and development-dependent expression of the small GTPase Arf6 in mice. Dev Dyn 239(12):3416–3435. doi:10.1002/dvdy.22481
Akiyama M, Hasegawa H, Hongu T, Frohman MA, Harada A, Sakagami H, Kanaho Y (2014) Trans-regulation of oligodendrocyte myelination by neurons through small GTPase Arf6-regulated secretion of fibroblast growth factor-2. Nat Commun 5:4744. doi:10.1038/ncomms5744
Altschuler Y, Liu S, Katz L, Tang K, Hardy S, Brodsky F, Apodaca G, Mostov K (1999) ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells. J Cell Biol 147(1):7–12
Belge H, Gailly P, Schwaller B, Loffing J, Debaix H, Riveira-Munoz E, Beauwens R, Devogelaer JP, Hoenderop JG, Bindels RJ, Devuyst O (2007) Renal expression of parvalbumin is critical for NaCl handling and response to diuretics. Proc Natl Acad Sci USA 104(37):14849–14854. doi:10.1073/pnas.0702810104
Caumont AS, Galas MC, Vitale N, Aunis D, Bader MF (1998) Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D. J Biol Chem 273(3):1373–1379
Cavenagh MM, Whitney JA, Carroll K, Zhang C, Boman AL, Rosenwald AG, Mellman I, Kahn RA (1996) Intracellular distribution of Arf proteins in mammalian cells. Arf6 is uniquely localized to the plasma membrane. J Biol Chem 271(36):21767–21774
Choi S, Ko J, Lee JR, Lee HW, Kim K, Chung HS, Kim H, Kim E (2006) ARF6 and EFA6A regulate the development and maintenance of dendritic spines. J Neurosci 26(18):4811–4819. doi:10.1523/JNEUROSCI.4182-05.2006
Claing A (2004) Regulation of G protein-coupled receptor endocytosis by ARF6 GTP-binding proteins. Biochem Cell Biol 82(6):610–617. doi:10.1139/o04-113
Claing A, Chen W, Miller WE, Vitale N, Moss J, Premont RT, Lefkowitz RJ (2001) beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis. J Biol Chem 276(45):42509–42513. doi:10.1074/jbc.M108399200
Donaldson JG, Jackson CL (2011) ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol 12(6):362–375. doi:10.1038/nrm3117
D’Souza-Schorey C, Chavrier P (2006) ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7(5):347–358. doi:10.1038/nrm1910
D’Souza-Schorey C, Li G, Colombo MI, Stahl PD (1995) A regulatory role for ARF6 in receptor-mediated endocytosis. Science 267(5201):1175–1178
Dyer N, Rebollo E, Dominguez P, Elkhatib N, Chavrier P, Daviet L, Gonzalez C, Gonzalez-Gaitan M (2007) Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes. Development 134(24):4437–4447. doi:10.1242/dev.010983
El-Annan J, Brown D, Breton S, Bourgoin S, Ausiello DA, Marshansky V (2004) Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ. Am J Physiol Cell Physiol 286(4):C768–C778. doi:10.1152/ajpcell.00250.2003
Fielding AB, Schonteich E, Matheson J, Wilson G, Yu X, Hickson GR, Srivastava S, Baldwin SA, Prekeris R, Gould GW (2005) Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis. EMBO J 24(19):3389–3399. doi:10.1038/sj.emboj.7600803
Fukaya M, Fukushima D, Hara Y, Sakagami H (2014) EFA6A, a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin-1 and regulates neurite outgrowth. J Neurochem 129(1):21–36. doi:10.1111/jnc.12524
Gaschet J, Hsu VW (1999) Distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle. J Biol Chem 274(28):20040–20045
Gershon E, Plaks V, Dekel N (2008) Gap junctions in the ovary: expression, localization and function. Mol Cell Endocrinol 282(1–2):18–25. doi:10.1016/j.mce.2007.11.001
Gillingham AK, Munro S (2007) The small G proteins of the Arf family and their regulators. Annu Rev Cell Dev Biol 23:579–611. doi:10.1146/annurev.cellbio.23.090506.123209
Greenbaum MP, Yan W, Wu MH, Lin YN, Agno JE, Sharma M, Braun RE, Rajkovic A, Matzuk MM (2006) TEX14 is essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci USA 103(13):4982–4987. doi:10.1073/pnas.0505123103
Greenbaum MP, Ma L, Matzuk MM (2007) Conversion of midbodies into germ cell intercellular bridges. Dev Biol 305(2):389–396. doi:10.1016/j.ydbio.2007.02.025
Hagen SJ, Trier JS (1988) Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy. J Histochem Cytochem 36(7):717–727. doi:10.1177/36.7.3290330
Hara Y, Fukaya M, Hayashi K, Kawauchi T, Nakajima K, Sakagami H (2016) ADP ribosylation factor 6 regulates neuronal migration in the developing cerebral cortex through FIP3/Arfophilin-1-dependent endosomal trafficking of N-cadherin. eNeuro. doi:10.1523/ENEURO.0148-16.2016
Hashimoto S, Onodera Y, Hashimoto A, Tanaka M, Hamaguchi M, Yamada A, Sabe H (2004) Requirement for Arf6 in breast cancer invasive activities. Proc Natl Acad Sci USA 101(17):6647–6652. doi:10.1073/pnas.0401753101
Hernandez-Deviez DJ, Casanova JE, Wilson JM (2002) Regulation of dendritic development by the ARF exchange factor ARNO. Nat Neurosci 5(7):623–624. doi:10.1038/nn865
Hernandez-Deviez DJ, Roth MG, Casanova JE, Wilson JM (2004) ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha. Mol Biol Cell 15(1):111–120. doi:10.1091/mbc.E03-06-0410
Hiroi T, Someya A, Thompson W, Moss J, Vaughan M (2006) GEP100/BRAG2: activator of ADP-ribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via E-cadherin and alpha-catenin. Proc Natl Acad Sci USA 103(28):10672–10677. doi:10.1073/pnas.0604091103
Hu CK, Coughlin M, Mitchison TJ (2012) Midbody assembly and its regulation during cytokinesis. Mol Biol Cell 23(6):1024–1034. doi:10.1091/mbc.E11-08-0721
Huang Y, Joshi S, Xiang B, Kanaho Y, Li Z, Bouchard BA, Moncman CL, Whiteheart SW (2016) Arf6 controls platelet spreading and clot retraction via integrin alphaIIbbeta3 trafficking. Blood 127(11):1459–1467. doi:10.1182/blood-2015-05-648550
Hunzicker-Dunn M, Gurevich VV, Casanova JE, Mukherjee S (2002) ARF6: a newly appreciated player in G protein-coupled receptor desensitization. FEBS Lett 521(1–3):3–8
Ikenouchi J, Umeda M (2010) FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex. Proc Natl Acad Sci USA 107(2):748–753. doi:10.1073/pnas.0908423107
Joseph N, Hutterer A, Poser I, Mishima M (2012) ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration. EMBO J 31(11):2604–2614. doi:10.1038/emboj.2012.139
Klein S, Partisani M, Franco M, Luton F (2008) EFA6 facilitates the assembly of the tight junction by coordinating an Arf6-dependent and -independent pathway. J Biol Chem 283(44):30129–30138. doi:10.1074/jbc.M803375200
Lahuna O, Quellari M, Achard C, Nola S, Meduri G, Navarro C, Vitale N, Borg JP, Misrahi M (2005) Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway. EMBO J 24(7):1364–1374. doi:10.1038/sj.emboj.7600616
Lanzetti L (2007) Actin in membrane trafficking. Curr Opin Cell Biol 19(4):453–458. doi:10.1016/j.ceb.2007.04.017
Lawrence JT, Birnbaum MJ (2003) ADP-ribosylation factor 6 regulates insulin secretion through plasma membrane phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA 100(23):13320–13325. doi:10.1073/pnas.2232129100
Le TL, Yap AS, Stow JL (1999) Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol 146(1):219–232
Londono I, Marshansky V, Bourgoin S, Vinay P, Bendayan M (1999) Expression and distribution of adenosine diphosphate-ribosylation factors in the rat kidney. Kidney Int 55(4):1407–1416. doi:10.1046/j.1523-1755.1999.00365.x
Lugo-Martinez VH, Petit CS, Fouquet S, Le Beyec J, Chambaz J, Pincon-Raymond M, Cardot P, Thenet S (2009) Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions. Am J Physiol Gastrointest Liver Physiol 296(2):G235–G244. doi:10.1152/ajpgi.90313.2008
Macia E, Luton F, Partisani M, Cherfils J, Chardin P, Franco M (2004) The GDP-bound form of Arf6 is located at the plasma membrane. J Cell Sci 117(Pt 11):2389–2398. doi:10.1242/jcs.01090
Macia E, Partisani M, Paleotti O, Luton F, Franco M (2012) Arf6 negatively controls the rapid recycling of the beta2 adrenergic receptor. J Cell Sci 125(Pt 17):4026–4035. doi:10.1242/jcs.102343
Madara JL (1990) Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions. J Membr Biol 116(2):177–184
Majoul IV, Onichtchouk D, Butkevich E, Wenzel D, Chailakhyan LM, Duden R (2009) Limiting transport steps and novel interactions of Connexin-43 along the secretory pathway. Histochem Cell Biol 132(3):263–280. doi:10.1007/s00418-009-0617-x
Makyio H, Ohgi M, Takei T, Takahashi S, Takatsu H, Katoh Y, Hanai A, Ueda T, Kanaho Y, Xie Y, Shin HW, Kamikubo H, Kataoka M, Kawasaki M, Kato R, Wakatsuki S, Nakayama K (2012) Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis. EMBO J 31(11):2590–2603. doi:10.1038/emboj.2012.89
Matsukawa J, Nakayama K, Nagao T, Ichijo H, Urushidani T (2003) Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion. J Biol Chem 278(38):36470–36475. doi:10.1074/jbc.M305444200
Miura Y, Ngo Thai Bich V, Furuya M, Hasegawa H, Takahashi S, Katagiri N, Hongu T, Funakoshi Y, Ohbayashi N, Kanaho Y (2017) The small G protein Arf6 expressed in keratinocytes by HGF stimulation is a regulator for skin wound healing. Sci Rep 7:46649. doi:10.1038/srep46649
Miyazaki H, Yamazaki M, Watanabe H, Maehama T, Yokozeki T, Kanaho Y (2005) The small GTPase ADP-ribosylation factor 6 negatively regulates dendritic spine formation. FEBS Lett 579(30):6834–6838. doi:10.1016/j.febslet.2005.11.022
Montagnac G, Sibarita JB, Loubery S, Daviet L, Romao M, Raposo G, Chavrier P (2009) ARF6 Interacts with JIP4 to control a motor switch mechanism regulating endosome traffic in cytokinesis. Curr Biol 19(3):184–195. doi:10.1016/j.cub.2008.12.043
Mukherjee S, Gurevich VV, Jones JC, Casanova JE, Frank SR, Maizels ET, Bader MF, Kahn RA, Palczewski K, Aktories K, Hunzicker-Dunn M (2000) The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization. Proc Natl Acad Sci USA 97(11):5901–5906. doi:10.1073/pnas.100127097
Nakayama K (2016) Regulation of cytokinesis by membrane trafficking involving small GTPases and the ESCRT machinery. Crit Rev Biochem Mol Biol 51(1):1–6. doi:10.3109/10409238.2015.1085827
Palacios F, Price L, Schweitzer J, Collard JG, D’Souza-Schorey C (2001) An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration. EMBO J 20(17):4973–4986. doi:10.1093/emboj/20.17.4973
Palacios F, Schweitzer JK, Boshans RL, D’Souza-Schorey C (2002) ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly. Nat Cell Biol 4(12):929–936. doi:10.1038/ncb881
Peters PJ, Hsu VW, Ooi CE, Finazzi D, Teal SB, Oorschot V, Donaldson JG, Klausner RD (1995) Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J Cell Biol 128(6):1003–1017
Qualmann B, Kessels MM, Kelly RB (2000) Molecular links between endocytosis and the actin cytoskeleton. J Cell Biol 150(5):F111–F116
Rait VK, Xu L, O’Leary TJ, Mason JT (2004) Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Lab Investig 84(3):300–306. doi:10.1038/labinvest.3700041
Santy LC, Casanova JE (2001) Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J Cell Biol 154(3):599–610. doi:10.1083/jcb.200104019
Schweitzer JK, D’Souza-Schorey C (2002) Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis. J Biol Chem 277(30):27210–27216. doi:10.1074/jbc.M201569200
Schweitzer JK, Sedgwick AE, D’Souza-Schorey C (2011) ARF6-mediated endocytic recycling impacts cell movement, cell division and lipid homeostasis. Semin Cell Dev Biol 22(1):39–47. doi:10.1016/j.semcdb.2010.09.002
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741–748. doi:10.1177/39.6.1709656
Shi SR, Shi Y, Taylor CR (2011) Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem 59(1):13–32. doi:10.1369/jhc.2010.957191
Su V, Lau AF (2014) Connexins: mechanisms regulating protein levels and intercellular communication. FEBS Lett 588(8):1212–1220. doi:10.1016/j.febslet.2014.01.013
Suzuki T, Kanai Y, Hara T, Sasaki J, Sasaki T, Kohara M, Maehama T, Taya C, Shitara H, Yonekawa H, Frohman MA, Yokozeki T, Kanaho Y (2006) Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development. Mol Cell Biol 26(16):6149–6156. doi:10.1128/MCB.00298-06
Tachow A, Thoungseabyoun W, Phuapittayalert L, Petcharat K, Sakagami H, Kondo H, Hipkaeo W (2017) Co-localization of endogenous Arf6 and its activator EFA6D in the granular convoluted tubule cells of mouse submandibular glands under normal conditions and when stimulated by isoproterenol, noradrenaline and carbachol. Arch Oral Biol 82:153–159. doi:10.1016/j.archoralbio.2017.06.007
Takahashi S, Takei T, Koga H, Takatsu H, Shin HW, Nakayama K (2011) Distinct roles of Rab11 and Arf6 in the regulation of Rab11-FIP3/arfophilin-1 localization in mitotic cells. Genes Cells 16(9):938–950. doi:10.1111/j.1365-2443.2011.01538.x
Theard D, Labarrade F, Partisani M, Milanini J, Sakagami H, Fon EA, Wood SA, Franco M, Luton F (2010) USP9x-mediated deubiquitination of EFA6 regulates de novo tight junction assembly. EMBO J 29(9):1499–1509. doi:10.1038/emboj.2010.46
Tushir JS, D’Souza-Schorey C (2007) ARF6-dependent activation of ERK and Rac1 modulates epithelial tubule development. EMBO J 26(7):1806–1819. doi:10.1038/sj.emboj.7601644
Williams JM, Duckworth CA, Burkitt MD, Watson AJ, Campbell BJ, Pritchard DM (2015) Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip. Vet Pathol 52(3):445–455. doi:10.1177/0300985814559404
Winterhager E, Kidder GM (2015) Gap junction connexins in female reproductive organs: implications for women’s reproductive health. Hum Reprod Update 21(3):340–352. doi:10.1093/humupd/dmv007
Yamaoka M, Ando T, Terabayashi T, Okamoto M, Takei M, Nishioka T, Kaibuchi K, Matsunaga K, Ishizaki R, Izumi T, Niki I, Ishizaki T, Kimura T (2016) PI3 K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a. J Cell Sci 129(3):637–649. doi:10.1242/jcs.180141
Yang CZ, Heimberg H, D’Souza-Schorey C, Mueckler MM, Stahl PD (1998) Subcellular distribution and differential expression of endogenous ADP-ribosylation factor 6 in mammalian cells. J Biol Chem 273(7):4006–4011
Zhang X, Kim KM (2017) Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis. Biomol Ther (Seoul) 25(1):26–43. doi:10.4062/biomolther.2016.186
Zhang Q, Cox D, Tseng CC, Donaldson JG, Greenberg S (1998) A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages. J Biol Chem 273(32):19977–19981
Acknowledgements
The authors thank staff members of the Bioimaging center at Kitasato University School of Medicine for their technical support in electron microscopic analyses. This study was supported by KAKENHI Grant no. 15H04672 to H.S. from the Japan Society for the Promotion of Science.
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Katsumata, O., Mori, M., Sawane, Y. et al. Cellular and subcellular localization of ADP-ribosylation factor 6 in mouse peripheral tissues. Histochem Cell Biol 148, 577–596 (2017). https://doi.org/10.1007/s00418-017-1599-8
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DOI: https://doi.org/10.1007/s00418-017-1599-8