An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells
Tóm tắt
Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.
Tài liệu tham khảo
Ahras M, Otto GP, Tooze SA (2006) Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells. J Cell Biol 173:241–251
Bock JB, Lin RC, Scheller RH (1996) A new syntaxin family member implicated in targeting of intracellular transport vesicles. J Biol Chem 271:17961–17965
Borgonovo B, Ouwendijk J, Solimena M (2006) Biogenesis of secretory granules. Curr Opin Cell Biol 18:365–370
Brunner Y, Coute Y, Iezzi M, Foti M, Fukuda M, Hochstrasser DF, Wollheim CB, Sanchez JC (2007) Proteomics analysis of insulin secretory granules. Mol Cell Proteomics 6:1007–1017
Colomer V, Kicska GA, Rindler MJ (1996) Secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acidic pH. J Biol Chem 271:48–55
D’Amico F, Skarmoutsou E, Imbesi RM, Sanfilippo S (2001) Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study. Eur J Histochem 45:169–175
Davidson HW (2004) (Pro)insulin processing—a historical perspective. Cell Biochem Biophys 35(1):143–157
Dittie AS, Tooze SA (1995) Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells. Biochem J 310(Pt 3):777–787
Docherty K, Hutton JC (1983) Carboxypeptidase activity in the insulin secretory granule. FEBS Lett 162:137–141
Hendershot LM, Valentine VA, Lee AS, Morris SW, Shapiro DN (1994) Localization of the gene encoding human Bip/Grp78, the endoplasmic-reticulum cognate of the Hsp70 family, to chromosome-9q34. Genomics 20:281–284
Huber LA, Pfaller K, Vietor I (2003) Organelle proteomics—implications for subcellular fractionation in proteomics. Circ Res 92:962–968
Hutton JC, Hansen F, Peshavaria M (1985) Beta-granins: 21 kDa co-secreted peptides of the insulin granule closely related to adrenal medullary chromogranin A. FEBS Lett 188:336–340
Iezzi M, Escher G, Meda P, Charollais A, Baldini G, Darchen F, Wollheim CB, Regazzi R (1999) Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells. Mol Endocrinol 13:202–212
Kim T, Gondre-Lewis MC, Arnaoutova I, Loh YP (2006) Dense-core secretory granule biogenesis. Physiology 21:124–133
Klumperman J, Kuliawat R, Griffith JM, Geuze HJ, Arvan P (1998) Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles. J Cell Biol 141:359–371
Konrad RJ, Young RA, Record RD, Smith RM, Butkerait P, Manning D, Jarett L, Wolf BA (1995) The heterotrimeric G-Protein G(I) is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis. J Biol Chem 270:12869–12876
Mast S, Peng L, Jordan TW, Flint H, Phillips L, Donaldson L, Strabala TJ, Wagner A (2010) Proteomic analysis of membrane preparations from developing Pinus radiata compression wood. Tree Physiol 30:1456–1468
McCrossan M, Windsor M, Ponnambalam S, Armstrong J, Wileman T (2001) The trans Golgi network is lost from cells infected with African swine fever virus. J Virol 75:11755–11765
Mundy DI, Warren G (1992) Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein. J Cell Biol 116:135–146
Nakagawa M, Miranda AF (1987) A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria. Exp Cell Res 168:44–52
Orci L, Ravazzola M, Amherdt M, Madsen O, Perrelet A, Vassalli JD, Anderson RGW (1986) Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J Cell Biol 103:2273–2281
Rhodes C (2000) Processing of the insulin molecule. In: Diabetes Mellitus: a fundamental and clinical text, p 27–50
Schvartz D, Brunner Y, Coute Y, Foti M, Wollheim CB, Sanchez JC (2012) Improved characterization of the insulin secretory granule proteomes. J Proteomics 75:4620–4631
Smeekens SP, Montag AG, Thomas G, Albigesrizo C, Carroll R, Benig M, Phillips LA, Martin S, Ohagi S, Gardner P et al (1992) Proinsulin processing by the subtilisin-related proprotein convertases furin, Pc2, and Pc3. Proc Natl Acad Sci USA 89:8822–8826
Steiner DF, Park SY, Stoy J, Philipson LH, Bell GI (2009) A brief perspective on insulin production. Diabetes Obes Metab 11:189–196
Tooze SA, Stinchcombe JC (1992) Biogenesis of secretory granules. Semin Cell Biol 3:357–366
Tooze SA, Flatmark T, Tooze J, Huttner WB (1991) Characterization of the immature secretory granule, an intermediate in granule biogenesis. J Cell Biol 115:1491–1503
Wasmeier C, Hutton JC (1996) Molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes. J Biol Chem 271:18161–18170
Weir GC, Laybutt DR, Kaneto H, Bonner-Weir S, Sharma A (2001) Beta-cell adaptation and decompensation during the progression of diabetes. Diabetes 50(Suppl 1):S154–S159
Wendler F, Page L, Urbe S, Tooze SA (2001) Homotypic fusion of immature secretory granules during maturation requires syntaxin 6. Mol Biol Cell 12:1699–1709