Abstract
In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Agui N, Granger NA, Gilbert LI, Bollenbacher WE (1979) Cellular localization of the insect prothoracicotropic hormone: in vitro assay of a single neurosecretory cell. Proc Natl Acad Sci USA 76(11):5694–5698
Barclay JW, Morgan A, Burgoyne RD (2012) Neurotransmitter release mechanisms studied in Caenorhabditis elegans. Cell Calcium 52(3–4):289–295. doi:10.1016/j.ceca.2012.03.005
Barr FA (2013) Review series: Rab GTPases and membrane identity: causal or inconsequential? J Cell Biol 202(2):191–199. doi:10.1083/jcb.201306010
Bhuin T, Roy JK (2014) Rab proteins: the key regulators of intracellular vesicle transport. Exp Cell Res 328(1):1–19. doi:10.1016/j.yexcr.2014.07.027
Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B (2000) Rab7: a key to lysosome biogenesis. Mol Biol Cell 11(2):467–480
Chan CC, Scoggin S, Wang D, Cherry S, Dembo T, Greenberg B, Jin EJ, Kuey C, Lopez A, Mehta SQ, Perkins TJ, Brankatschk M, Rothenfluh A, Buszczak M, Hiesinger PR (2011) Systematic discovery of Rab GTPases with synaptic functions in Drosophila. Curr Biol 21(20):1704–1715. doi:10.1016/j.cub.2011.08.058
Coutelis JB, Ephrussi A (2007) Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis. Development 134(7):1419–1430. doi:10.1242/dev.02821
Echard A, Jollivet F, Martinez O, Lacapere JJ, Rousselet A, Janoueix-Lerosey I, Goud B (1998) Interaction of a Golgi-associated kinesin-like protein with Rab6. Science 279(5350):580–585
Graf ER, Daniels RW, Burgess RW, Schwarz TL, DiAntonio A (2009) Rab3 dynamically controls protein composition at active zones. Neuron 64(5):663–677. doi:10.1016/j.neuron.2009.11.002
Grigoriev I, Splinter D, Keijzer N, Wulf PS, Demmers J, Ohtsuka T, Modesti M, Maly IV, Grosveld F, Hoogenraad CC, Akhmanova A (2007) Rab6 regulates transport and targeting of exocytotic carriers. Dev Cell 13(2):305–314. doi:10.1016/j.devcel.2007.06.010
Grigoriev I, Yu KL, Martinez-Sanchez E, Serra-Marques A, Smal I, Meijering E, Demmers J, Peranen J, Pasterkamp RJ, van der Sluijs P, Hoogenraad CC, Akhmanova A (2011) Rab6, Rab8, and MICAL3 cooperate in controlling docking and fusion of exocytotic carriers. Curr Biol 21(11):967–974. doi:10.1016/j.cub.2011.04.030
Harris E, Cardelli J (2002) RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion. J Cell Sci 115(Pt 18):3703–3713
Hartenstein V (2006) The neuroendocrine system of invertebrates: a developmental and evolutionary perspective. J Endocrinol 190(3):555–570. doi:10.1677/joe.1.06964
Hou L, Wang JX, Zhao XF (2011) Rab32 and the remodeling of the imaginal midgut in Helicoverpa armigera. Amino Acids 40(3):953–961. doi:10.1007/s00726-010-0720-2
Hoyer D, Bartfai T (2012) Neuropeptides and neuropeptide receptors: drug targets, and peptide and non-peptide ligands: a tribute to Prof. Dieter Seebach. Chem Biodivers 9(11):2367–2387. doi:10.1002/cbdv.201200288
Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91(1):119–149. doi:10.1152/physrev.00059.2009
Iwami M, Furuya I, Kataoka H (1996) Bombyxin-related peptides: cDNA structure and expression in the brain of the hornworm Agrius convolvuli [corrected]. Insect Biochem Mol Biol 26(1):25–32
Januschke J, Nicolas E, Compagnon J, Formstecher E, Goud B, Guichet A (2007) Rab6 and the secretory pathway affect oocyte polarity in Drosophila. Development 134(19):3419–3425. doi:10.1242/dev.008078
Junutula JR, De Maziere AM, Peden AA, Ervin KE, Advani RJ, van Dijk SM, Klumperman J, Scheller RH (2004) Rab14 is involved in membrane trafficking between the Golgi complex and endosomes. Mol Biol Cell 15(5):2218–2229. doi:10.1091/mbc.E03-10-0777
Kimura K, Kimura A (2012) Rab6 is required for the exocytosis of cortical granules and the recruitment of separase to the granules during the oocyte-to-embryo transition in Caenorhabditis elegans. J Cell Sci 125(Pt 23):5897–5905. doi:10.1242/jcs.116400
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Li J, Song CX, Li YP, Li L, Wei XH, Wang JL, Liu XS (2015) Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera. Dev Comp Immunol 50(2):78–86. doi:10.1016/j.dci.2015.01.008
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Masumura M, Satake S, Saegusa H, Mizoguchi A (2000) Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Gen Comp Endocrinol 118(3):393–399. doi:10.1006/gcen.1999.7438
Mizoguchi A, Okamoto N (2013) Insulin-like and IGF-like peptides in the silkmoth Bombyx mori: discovery, structure, secretion, and function. Front Physiol 4:217. doi:10.3389/fphys.2013.00217
Mizoguchi A, Ishizaki H, Nagasawa H, Kataoka H, Isogai A, Tamura S, Suzuki A, Fujino M, Kitada C (1987) A monoclonal antibody against a synthetic fragment of bombyxin (4K-prothoracicotropic hormone) from the silkmoth, Bombyx mori: characterization and immunohistochemistry. Mol Cell Endocrinol 51(3):227–235
Nagasawa H, Kataoka H, Isogai A, Tamura S, Suzuki A, Mizoguchi A, Fujiwara Y, Takahashi SY, Ishizaki H (1986) Amino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx mori. Proc Natl Acad Sci USA 83(16):5840–5843
Nagata K, Maruyama K, Nagasawa H, Urushibata I, Isogai A, Ishizaki H, Suzuki A (1992) Bombyxin-II and its disulfide bond isomers: synthesis and activity. Peptides 13(4):653–662
Nagata S, Kataoka H, Suzuki A (2005) Silk moth neuropeptide hormones: prothoracicotropic hormone and others. Ann N Y Acad Sci 1040:38–52. doi:10.1196/annals.1327.004
Nassel DR, Homberg U (2006) Neuropeptides in interneurons of the insect brain. Cell Tissue Res 326(1):1–24. doi:10.1007/s00441-006-0210-8
Nassel DR, Winther AM (2010) Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 92(1):42–104. doi:10.1016/j.pneurobio.2010.04.010
Ng EL, Tang BL (2008) Rab GTPases and their roles in brain neurons and glia. Brain Res Rev 58(1):236–246. doi:10.1016/j.brainresrev.2008.04.006
Nijhout HF, Grunert LW (2002) Bombyxin is a growth factor for wing imaginal disks in Lepidoptera. Proc Natl Acad Sci USA 99(24):15446–15450. doi:10.1073/pnas.242548399
Nijhout HF, Smith WA, Schachar I, Subramanian S, Tobler A, Grunert LW (2007) The control of growth and differentiation of the wing imaginal disks of Manduca sexta. Dev Biol 302(2):569–576. doi:10.1016/j.ydbio.2006.10.023
O’Brien MA, Katahira EJ, Flanagan TR, Arnold LW, Haughton G, Bollenbacher WE (1988) A monoclonal antibody to the insect prothoracicotropic hormone. J Neurosci 8(9):3247–3257
Pavlos NJ, Jahn R (2011) Distinct yet overlapping roles of Rab GTPases on synaptic vesicles. Small GTPases 2(2):77–81. doi:10.4161/sgtp.2.2.15201
Plutner H, Schwaninger R, Pind S, Balch WE (1990) Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J 9(8):2375–2383
Plutner H, Cox AD, Pind S, Khosravi-Far R, Bourne JR, Schwaninger R, Der CJ, Balch WE (1991) Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J Cell Biol 115(1):31–43
Proikas-Cezanne T, Gaugel A, Frickey T, Nordheim A (2006) Rab14 is part of the early endosomal clathrin-coated TGN microdomain. FEBS Lett 580(22):5241–5246. doi:10.1016/j.febslet.2006.08.053
Roller L, Yamanaka N, Watanabe K, Daubnerova I, Zitnan D, Kataoka H, Tanaka Y (2008) The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem Mol Biol 38(12):1147–1157
Sannerud R, Marie M, Nizak C, Dale HA, Pernet-Gallay K, Perez F, Goud B, Saraste J (2006) Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery. Mol Biol Cell 17(4):1514–1526. doi:10.1091/mbc.E05-08-0792
Schwartz SL, Cao C, Pylypenko O, Rak A, Wandinger-Ness A (2007) Rab GTPases at a glance. J Cell Sci 120(Pt 22):3905–3910. doi:10.1242/jcs.015909
Shin OH (2014) Exocytosis and synaptic vesicle function. Compr Physiol 4(1):149–175. doi:10.1002/cphy.c130021
Singh CO, Xin HH, Chen RT, Wang MX, Liang S, Lu Y, Cai ZZ, Zhang DP, Miao YG (2015) RNAi knockdown of BmRab3 led to larva and pupa lethality in silkworm Bombyx mori L. Arch Insect Biochem Physiol 89(2):98–110. doi:10.1002/arch.21228
Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10(8):513–525. doi:10.1038/nrm2728
Takamori S, Holt M, Stenius K, Lemke EA, Gronborg M, Riedel D, Urlaub H, Schenck S, Brugger B, Ringler P, Muller SA, Rammner B, Grater F, Hub JS, De Groot BL, Mieskes G, Moriyama Y, Klingauf J, Grubmuller H, Heuser J, Wieland F, Jahn R (2006) Molecular anatomy of a trafficking organelle. Cell 127(4):831–846. doi:10.1016/j.cell.2006.10.030
Thummel CS (2002) Ecdysone-regulated puff genes 2000. Insect Biochem Mol Biol 32(2):113–120
Tobe SS, Pratt GE (1974) Dependence of juvenile hormone release from corpus allatum on intraglandular content. Nature 252(5483):474–476
Tobler A, Nijhout HF (2010) A switch in the control of growth of the wing imaginal disks of Manduca sexta. PLoS ONE 5(5):e10723. doi:10.1371/journal.pone.0010723
Tong C, Ohyama T, Tien AC, Rajan A, Haueter CM, Bellen HJ (2011) Rich regulates target specificity of photoreceptor cells and N-cadherin trafficking in the Drosophila visual system via Rab6. Neuron 71(3):447–459. doi:10.1016/j.neuron.2011.06.040
Uno T, Nakada T, Okamaoto S, Nakamura M, Matsubara M, Imaishi H, Yamagata H, Kanamaru K, Takagi M (2007) Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori. Arch Insect Biochem Physiol 66(2):89–97. doi:10.1002/arch.20201
Uno T, Sakamoto K, Isoyama Y, Hiragaki S, Uno Y, Kanamaru K, Yamagata H, Takagi M, Mizoguchi A, Takeda M (2013) Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori. Histochem Cell Biol 139(2):299–308. doi:10.1007/s00418-012-1021-5
Vafopoulou X, Steel CG (2012) Insulin-like and testis ecdysiotropin neuropeptides are regulated by the circadian timing system in the brain during larval-adult development in the insect Rhodnius prolixus (Hemiptera). Gen Comp Endocrinol 179(2):277–288. doi:10.1016/j.ygcen.2012.08.018
Van de Velde S, Badisco L, Claeys I, Verleyen P, Chen X, Vanden Bosch L, Vanden Broeck J, Smagghe G (2007) Insulin-like peptides in Spodoptera littoralis (Lepidoptera): detection, localization and identification. Gen Comp Endocrinol 153(1–3):72–79. doi:10.1016/j.ygcen.2007.05.001
Vullings HG, Diederen JH, Veelaert D, Van der Horst DJ (1999) Multifactorial control of the release of hormones from the locust retrocerebral complex. Microsc Res Tech 45(3):142–153. doi:10.1002/(SICI)1097-0029(19990501)45:3<142:AID-JEMT2>3.0.CO;2-D
Wang T, Ming Z, Xiaochun W, Hong W (2011) Rab7: role of its protein interaction cascades in endo-lysosomal traffic. Cell Signal 23(3):516–521. doi:10.1016/j.cellsig.2010.09.012
Wang C, Liu Z, Huang X (2012) Rab32 is important for autophagy and lipid storage in Drosophila. PLoS ONE 7(2):e32086. doi:10.1371/journal.pone.0032086
Zavodska R, Sauman I, Sehnal F (2003) Distribution of PER protein, pigment-dispersing hormone, prothoracicotropic hormone, and eclosion hormone in the cephalic nervous system of insects. J Biol Rhythms 18(2):106–122
Zhao S, Torii S, Yokota-Hashimoto H, Takeuchi T, Izumi T (2002) Involvement of Rab27b in the regulated secretion of pituitary hormones. Endocrinology 143(5):1817–1824
Acknowledgments
This work was supported in part by a Grant-in-Aid for Research (No.26450468) in Priority Areas, from the Ministry of Education Science, Sports and Culture of Japan.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Uno, T., Furutani, M., Watanabe, C. et al. Rab proteins in the brain and corpus allatum of Bombyx mori . Histochem Cell Biol 146, 59–69 (2016). https://doi.org/10.1007/s00418-016-1422-y
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-016-1422-y