Abstract
Rab proteins are low-molecular weight (20–25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori.
Similar content being viewed by others
References
Asahina K, Watanabe K, Duistermars BJ, Hoopfer E, Gonzalez CR, Eyjolfsdottir EA, Perona P, Anderson DJ (2014) Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156(1–2):221–235. https://doi.org/10.1016/j.cell.2013.11.045
Baron RA, Seabra MC (2008) Rab geranylgeranylation occurs preferentially via the pre-formed REP-RGGT complex and is regulated by geranylgeranyl pyrophosphate. Biochem J 415(1):67–75. https://doi.org/10.1042/bj20080662
Bednar B, Roller L, Cizmar D, Mitrova D, Zitnan D (2017) Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori. Cell Tissue Res 368(2):259–275. https://doi.org/10.1007/s00441-016-2556-x
Bhuin T, Roy JK (2014) Rab proteins: the key regulators of intracellular vesicle transport. Exp Cell Res 328(1):1–19. https://doi.org/10.1016/j.yexcr.2014.07.027
Bustos MA, Lucchesi O, Ruete MC, Mayorga LS, Tomes CN (2012) Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis. Proc Natl Acad Sci USA 109(30):E2057–E2066. https://doi.org/10.1073/pnas.1121173109
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. https://doi.org/10.1016/j.cub.2011.08.058
Coutelis JB, Ephrussi A (2007) Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis. Development (Camb Engl) 134(7):1419–1430. https://doi.org/10.1242/dev.02821
Dunst S, Kazimiers T, von Zadow F, Jambor H, Sagner A, Brankatschk B, Mahmoud A, Spannl S, Tomancak P, Eaton S, Brankatschk M (2015) Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila. Dev Cell 33(3):351–365. https://doi.org/10.1016/j.devcel.2015.03.022
Foley AJ (2017) An RNAi screen to identify components of a polyamine transport system. Honors in the major thesis 187. http://stars.library.ucf.edu/honorstheses/187
Fu Y, Zhu JY, Zhang F, Richman A, Zhao Z, Han Z (2017) Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes. Cell Tissue Res 368(3):615–627. https://doi.org/10.1007/s00441-017-2575-2
Giansanti MG, Fuller MT (2012) What Drosophila spermatocytes tell us about the mechanisms underlying cytokinesis. Cytoskeleton (Hoboken NJ) 69(11):869–881. https://doi.org/10.1002/cm.21063
Grosshans BL, Ortiz D, Novick P (2006) Rabs and their effectors: achieving specificity in membrane traffic. Proc Natl Acad Sci USA 103(32):11821–11827. https://doi.org/10.1073/pnas.0601617103
Hou L, Wang JX, Zhao XF (2011) Rab32 and the remodeling of the imaginal midgut in Helicoverpa armigera. Amino Acids 40(3):953–961. https://doi.org/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. https://doi.org/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. https://doi.org/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
Jin EJ, Chan CC, Agi E, Cherry S, Hanacik E, Buszczak M, Hiesinger PR (2012) Similarities of Drosophila rab GTPases based on expression profiling: completion and analysis of the rab-Gal4 kit. PLoS One 7(7):e40912. https://doi.org/10.1371/journal.pone.0040912
Joti P, Ghosh-Roy A, Ray K (2011) Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila. Sci Rep 1:173. https://doi.org/10.1038/srep00173
Kataoka H, Toschi A, Li JP, Carney RL, Schooley DA, Kramer SJ (1989) Identification of an allatotropin from adult manduca sexta. Science 243(4897):1481–1483. https://doi.org/10.1126/science.243.4897.1481
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Lau AS, Mruk DD (2003) Rab8B GTPase and junction dynamics in the testis. Endocrinology 144(4):1549–1563. https://doi.org/10.1210/en.2002-220893
Lee G, Bahn JH, Park JH (2006) Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila. Proc Natl Acad Sci USA 103(33):12580–12585. https://doi.org/10.1073/pnas.0601171103
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. https://doi.org/10.1016/j.dci.2015.01.008
Liang S, Chen RT, Zhang DP, Xin HH, Lu Y, Wang MX, Miao YG (2015) Hedgehog signaling pathway regulated the target genes for adipogenesis in silkworm Bombyx mori. Insect Sci 22(5):587–596. https://doi.org/10.1111/1744-7917.12164
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
Mountjoy JR, Xu W, McLeod D, Hyndman D, Oko R (2008) RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis. Biol Reprod 79(2):223–232. https://doi.org/10.1095/biolreprod.107.065060
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
Nassel DR, Homberg U (2006) Neuropeptides in interneurons of the insect brain. Cell Tissue Res 326(1):1–24. https://doi.org/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. https://doi.org/10.1016/j.pneurobio.2010.04.010
Nyberg KG, Carthew RW (2017) Out of the testis: biological impacts of new genes. Genes Dev 31(18):1825–1826. https://doi.org/10.1101/gad.307496.117
Okamoto N, Yamanaka N, Endo Y, Kataoka H, Mizoguchi A (2011) Spatiotemporal patterns of IGF-like peptide expression in the silkmoth Bombyx mori predict its pleiotropic actions. Gen Comp Endocrinol 173(1):171–182. https://doi.org/10.1016/j.ygcen.2011.05.009
Orville Singh C, Xin HH, Chen RT, Wang MX, Liang S, Lu Y, Cai ZZ, Miao YG (2016) BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx mori. Insect Sci 23(1):28–36. https://doi.org/10.1111/1744-7917.12189
Pereira-Leal JB, Seabra MC (2001) Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313(4):889–901. https://doi.org/10.1006/jmbi.2001.5072
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
Satoh A, Tokunaga F, Kawamura S, Ozaki K (1997) In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila. J Cell Sci 110(Pt 23):2943–2953
Schoofs L, De Loof A, Van Hiel MB (2017) Neuropeptides as regulators of behavior in insects. Annu Rev Entomol 62:35–52. https://doi.org/10.1146/annurev-ento-031616-035500
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. https://doi.org/10.1242/jcs.015909
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. https://doi.org/10.1002/arch.21228
Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10(8):513–525. https://doi.org/10.1038/nrm2728
Sugahara R, Jouraku A, Nakakura T, Kusakabe T, Yamamoto T, Shinohara Y, Miyoshi H, Shiotsuki T (2015) Two adenine nucleotide translocase paralogues involved in cell proliferation and spermatogenesis in the silkworm Bombyx mori. PLoS One 10(3):e0119429. https://doi.org/10.1371/journal.pone.0119429
Tayler TD, Pacheco DA, Hergarden AC, Murthy M, Anderson DJ (2012) A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila. Proc Natl Acad Sci USA 109(50):20697–20702. https://doi.org/10.1073/pnas.1218246109
Tiwari AK, Alone DP, Roy JK (2008) Rab11 is essential for fertility in Drosophila. Cell Biol Int 32(9):1158–1168. https://doi.org/10.1016/j.cellbi.2008.04.002
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. https://doi.org/10.1016/j.neuron.2011.06.040
Uno T, Nakao A, Katsurauma C (2004) Phosphorylation of Rab proteins from the brain of Bombyx mori. Arch Insect Biochem Physiol 57(2):68–77. https://doi.org/10.1002/arch.20014
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. https://doi.org/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. https://doi.org/10.1007/s00418-012-1021-5
Uno T, Isoyama Y, Sakamoto K, Uno Y, Sakamoto K, Kanamaru K, Yamagata H, Takagi M, Mizoguchi A, Takeda M (2014) Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori. Histochem Cell Biol 141(3):311–320. https://doi.org/10.1007/s00418-013-1160-3
Uno T, Furutani M, Watanabe C, Sakamoto K, Uno Y, Kanamaru K, Yamagata H, Mizoguchi A, Takeda M (2016) Rab proteins in the brain and corpus allatum of Bombyx mori. Histochem Cell Biol. https://doi.org/10.1007/s00418-016-1422-y
Uno T, Furutani M, Sakamoto K, Uno Y, Kanamaru K, Mizoguchi A, Hiragaki S, Takeda M (2017) Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori. Arch Insect Biochem Physiol. https://doi.org/10.1002/arch.21404
Wang C, Liu Z, Huang X (2012) Rab32 is important for autophagy and lipid storage in Drosophila. PLoS One 7(2):e32086. https://doi.org/10.1371/journal.pone.0032086
White-Cooper H (2010) Molecular mechanisms of gene regulation during Drosophila spermatogenesis. Reproduction 139(1):11–21. https://doi.org/10.1530/rep-09-0083
Yang C, Lin Y, Liu H, Shen G, Luo J, Zhang H, Peng Z, Chen E, Xing R, Han C, Xia Q (2014) The broad complex isoform 2 (BrC-Z2) transcriptional factor plays a critical role in vitellogenin transcription in the silkworm Bombyx mori. Biochim Biophys Acta 1840(9):2674–2684. https://doi.org/10.1016/j.bbagen.2014.05.013
Yang XZ, Li XX, Zhang YJ, Rodriguez-Rodriguez L, Xiang MQ, Wang HY, Zheng XF (2016) Rab1 in cell signaling, cancer and other diseases. Oncogene 35(44):5699–5704. https://doi.org/10.1038/onc.2016.81
Zhang P, Cao G, Sheng J, Xue R, Gong C (2012) BmTGIF, a Bombyx mori homolog of Drosophila DmTGIF, regulates progression of spermatogenesis. PLoS One 7(11):e47861. https://doi.org/10.1371/journal.pone.0047861
Zhang P, Zhong J, Cao G, Xue R, Gong C (2014a) BmAly is an important factor in meiotic progression and spermatid differentiation in Bombyx mori (Lepidoptera: Bombycidae). J Insect Sci (Online) 14:188. https://doi.org/10.1093/jisesa/ieu050
Zhang Z, Teng X, Chen M, Li F (2014b) Orthologs of human disease associated genes and RNAi analysis of silencing insulin receptor gene in Bombyx mori. Int J Mol Sci 15(10):18102–18116. https://doi.org/10.3390/ijms151018102
Zhen Y, Stenmark H (2015) Cellular functions of Rab GTPases at a glance. J Cell Sci 128(17):3171–3176. https://doi.org/10.1242/jcs.166074
Acknowledgements
We thank Edanz Group (http://www.edanzediting.com/ac) for editing a draft of this manuscript. 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., Ozakiya, Y., Furutani, M. et al. Functional characterization of insect-specific RabX6 of Bombyx mori. Histochem Cell Biol 151, 187–198 (2019). https://doi.org/10.1007/s00418-018-1710-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-018-1710-9