FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2138105049> ?p ?o ?g. }

• W2138105049 endingPage "33729" @default.
• W2138105049 startingPage "33719" @default.
• W2138105049 abstract "Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic β-cells. This process is mediated by secretory granule exocytosis. However, how β-cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds β2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from β2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing β-cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes. Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic β-cells. This process is mediated by secretory granule exocytosis. However, how β-cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds β2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from β2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing β-cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes. Type 2 diabetes mellitus is a common metabolic disorder, the prevalence of which is rapidly increasing worldwide (1Zimmet P. Alberti K.G. Shaw J. Nature. 2001; 414: 782-787Crossref PubMed Scopus (4498) Google Scholar, 2Diamond J. Nature. 2003; 423: 599-602Crossref PubMed Scopus (335) Google Scholar). Excessive food intake and reduced physical activity are mainly responsible for the inability of insulin secretion to meet metabolic demand (3Saltiel A.R. Cell. 2001; 104: 517-529Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Discovering ways to ameliorate insulin release is therefore a major goal of diabetes research.Each β-cell stores ∼104 insulin secretory granules (4Bratanova-Tochkova T.K. Cheng H. Daniel S. Gunawardana S. Liu Y.J. Mulvaney-Musa J. Schermerhorn T. Straub S.G. Yajima H. Sharp G.W. Diabetes. 2002; 51 (Suppl. 1,): 83-90Crossref PubMed Google Scholar, 5Rorsman P. Renstrom E. Diabetologia. 2003; 46: 1029-1045Crossref PubMed Scopus (586) Google Scholar). Less than 1% of the granules are immediately releasable, whereas the remaining must be primed and recruited to membranes before they can undergo exocytosis. This process requires several ATP-, Ca2+-, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent 3The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; TMF, transmembrane fragment; CCF, cleaved cytosolic fragment; GST, glutathione S-transferase; GFP, green fluorescent protein; CgB, chromogranin B; HA, hemagglutinin; HG, high glucose; HK, high KCl; HGHK, high glucose and high KCl; SI, insulin stimulation index. 3The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; TMF, transmembrane fragment; CCF, cleaved cytosolic fragment; GST, glutathione S-transferase; GFP, green fluorescent protein; CgB, chromogranin B; HA, hemagglutinin; HG, high glucose; HK, high KCl; HGHK, high glucose and high KCl; SI, insulin stimulation index. steps (6Klenchin V.A. Martin T.F. Biochimie (Paris). 2000; 82: 399-407Crossref PubMed Scopus (98) Google Scholar, 7Waselle L. Gerona R.R. Vitale N. Martin T.F. Bader M.F. Regazzi R. Mol. Endocrinol. 2005; 19: 3097-3106Crossref PubMed Scopus (70) Google Scholar). Kinesin and myosin Va drive the ATP-dependent transport of insulin granules toward the cell surface on microtubules and cortical actin microfilaments, respectively (8Donelan M.J. Morfini G. Julyan R. Sommers S. Hays L. Kajio H. Briaud I. Easom R.A. Molkentin J.D. Brady S.T. Rhodes C.J. J. Biol. Chem. 2002; 277: 24232-24242Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 9Ivarsson R. Jing X. Waselle L. Regazzi R. Renstrom E. Traffic. 2005; 6: 1027-1035Crossref PubMed Scopus (76) Google Scholar, 10Varadi A. Ainscow E.K. Allan V.J. Rutter G.A. J. Cell Sci. 2002; 115: 4177-4189Crossref PubMed Scopus (114) Google Scholar, 11Varadi A. Tsuboi T. Rutter G.A. Mol. Biol. Cell. 2005; 16: 2670-2680Crossref PubMed Scopus (131) Google Scholar). The meshwork of cortical actin filaments, on the other hand, can inhibit insulin secretion by restricting granule mobility and their access to the plasma membrane (12Bruun T.Z. Hoy M. Gromada J. Eur. J. Pharmacol. 2000; 403: 221-224Crossref PubMed Scopus (19) Google Scholar, 13Li G. Rungger-Brandle E. Just I. Jonas J.C. Aktories K. Wollheim C.B. Mol. Biol. Cell. 1994; 5: 1199-1213Crossref PubMed Scopus (119) Google Scholar, 14Tomas A. Yermen B. Min L. Pessin J.E. Halban P.A. J. Cell Sci. 2006; 119: 2156-2167Crossref PubMed Scopus (126) Google Scholar). Despite progress in this area (15Waselle L. Coppola T. Fukuda M. Iezzi M. El-Amraoui A. Petit C. Regazzi R. Mol. Biol. Cell. 2003; 14: 4103-4113Crossref PubMed Scopus (133) Google Scholar), questions remain about how granules dynamically interact with the actin cytoskeleton.We recently proposed that ICA512 (islet cell autoantigen 512/IA-2) tethers insulin granules to actin microfilaments (16Ort T. Maksimova E. Dirkx R. Kachinsky A.M. Berghs S. Froehner S.C. Solimena M. Eur. J. Cell Biol. 2000; 79: 621-630Crossref PubMed Scopus (58) Google Scholar, 17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 18Solimena M. Gerdes H.H. Trends Cell Biol. 2003; 13: 399-402Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). ICA512 is a receptor protein-tyrosine phosphatase family member that lacks phosphatase activity and is mostly expressed in neuroendocrine cells (19Drake P.G. Peters G.H. Andersen H.S. Hendriks W. Moller N.P. Biochem. J. 2003; 373: 393-401Crossref PubMed Google Scholar, 20Lan M.S. Lu J. Goto Y. Notkins A.L. DNA Cell Biol. 1994; 13: 505-514Crossref PubMed Scopus (316) Google Scholar, 21Rabin D.U. Pleasic S.M. Shapiro J.A. Yoo-Warren H. Oles J. Hicks J.M. Goldstein D.E. Rae P.M. J. Immunol. 1994; 152: 3183-3188PubMed Google Scholar, 22Solimena M. Dirkx Jr., R. Hermel J.M. Pleasic-Williams S. Shapiro J.A. Caron L. Rabin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (228) Google Scholar, 23Zahn T.R. Macmorris M.A. Dong W. Day R. Hutton J.C. J. Comp. Neurol. 2001; 429: 127-143Crossref PubMed Scopus (61) Google Scholar). Its pro-form is a glycoprotein of 110 kDa that is processed by furin-like convertases during granule maturation (22Solimena M. Dirkx Jr., R. Hermel J.M. Pleasic-Williams S. Shapiro J.A. Caron L. Rabin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (228) Google Scholar). This cleavage generates a 65-kDa transmembrane fragment (ICA512-TMF) that in human ICA512 encompasses residues 449-979 (supplemental Fig. 1A). The cytoplasmic tail of ICA512-TMF binds to the PDZ domain of β2-syntrophin (16Ort T. Maksimova E. Dirkx R. Kachinsky A.M. Berghs S. Froehner S.C. Solimena M. Eur. J. Cell Biol. 2000; 79: 621-630Crossref PubMed Scopus (58) Google Scholar), which in turn interacts with actin microfilaments through utrophin (24Albrecht D.E. Froehner S.C. Neurosignals. 2002; 11: 123-129Crossref PubMed Scopus (69) Google Scholar). Glucose stimulation may induce the dissociation of ICA512-TMF from β2-syntrophin by affecting the phosphorylation of the latter, hence increasing granule mobility (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar).Ca2+-dependent exocytosis of secretory granules triggers the transient insertion of ICA512-TMF into the plasma membrane (22Solimena M. Dirkx Jr., R. Hermel J.M. Pleasic-Williams S. Shapiro J.A. Caron L. Rabin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (228) Google Scholar) and its intracellular cleavage by the Ca2+-activated protease calpain-1 (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar). The resulting cleaved cytosolic fragment (ICA512-CCF; residues 659-979) contains the decoy protein-tyrosine phosphatase module flanked by two regions that bind to the PDZ domain of β2-syntrophin (supplemental Fig. 1A). ICA512-CCF is targeted to the nucleus, where it enhances the transcription of insulin and other granule genes as well as β-cell proliferation (25Mziaut H. Kersting S. Knoch K.P. Fan W.H. Trajkovski M. Erdmann K. Bergert H. Ehehalt F. Saeger H.D. Solimena M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 674-679Crossref PubMed Scopus (48) Google Scholar, 26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). Accordingly, ICA512-/- mice display reduced insulin secretion, glucose intolerance, and impaired regeneration of β-cells (25Mziaut H. Kersting S. Knoch K.P. Fan W.H. Trajkovski M. Erdmann K. Bergert H. Ehehalt F. Saeger H.D. Solimena M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 674-679Crossref PubMed Scopus (48) Google Scholar, 28Saeki K. Zhu M. Kubosaki A. Xie J. Lan M.S. Notkins A.L. Diabetes. 2002; 51: 1842-1850Crossref PubMed Scopus (100) Google Scholar). Moreover, overexpression of ICA512 in mouse insulinoma MIN6 cells increases the number of granules as well as the content, half-life, and stimulated secretion of insulin (29Harashima S. Clark A. Christie M.R. Notkins A.L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8704-8709Crossref PubMed Scopus (58) Google Scholar). A mechanistic explanation for these findings, however, is lacking. Here we demonstrate that ICA512-CCF, in addition to granule biogenesis, enhances insulin secretion.EXPERIMENTAL PROCEDURESIslet Isolations and Cell Cultures—Pancreatic islets were isolated from 15-17-week-old ICA512-/- mice (26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar) and wild type littermates, as described (30Gotoh M. Maki T. Kiyoizumi T. Satomi S. Monaco A.P. Transplantation. 1985; 40: 437-438Crossref PubMed Scopus (535) Google Scholar), and then kept for 24 h in culture prior to the experiment. INS-1-derived INS-1E and tetracycline-inducible INS-r3 cells (31Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (470) Google Scholar, 32Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (115) Google Scholar) were gifts from C. Wollheim (University of Geneva, Geneva, Switzerland) and were grown as described (33Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar). Wild type and transfected INS-1E cells and pancreatic islets were incubated for 60 min in resting buffer (0 mm glucose and 5 mm KCl) and then for 105 min in fresh resting or stimulating buffer (25 mm glucose and 55 mm KCl), as described (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). Calpeptin and cycloheximide (Calbiochem, Gibbstown, NJ) were added in the last 15 min of the preincubation and left until cells were harvested.cDNA Constructs—The following constructs were described: human ICA512-GFP, ICA512-CCF-GFP, and ICA512-HA3 (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar); GST-ICA512-(601-979), GST-ICA512-(663-979), GST-ICA512-(700-959), GST-ICA512-(700-979), GST-ICA512extracellular, and GST-ICA512-(800-979) (16Ort T. Maksimova E. Dirkx R. Kachinsky A.M. Berghs S. Froehner S.C. Solimena M. Eur. J. Cell Biol. 2000; 79: 621-630Crossref PubMed Scopus (58) Google Scholar); ICA512-(601-979)-His (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar); ICA512-CCFAD/DA-GFP (26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar); and mouse GFP-β2-syntrophin (34Kachinsky A.M. Froehner S.C. Milgram S.L. J. Cell Biol. 1999; 145: 391-402Crossref PubMed Scopus (97) Google Scholar). ICA512-CCF-HA3 and HA3-ICA512-CCF were generated by subcloning the cDNA encoding residues 659-979 of human ICA512 as a KpnI-XbaI insert into pMoHa3 (a gift from M. Suchanek and C. Thiele, Max Planck Institute of Cell Biology and Genetics, Dresden, Germany) or as a BglII-EcoRI insert into pN3HA (Clontech, Mountain View, CA), respectively. For tetracycline-inducible expression of ICA512-CCF-GFP, the corresponding cDNA (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar) was subcloned as a BamHI/XbaI insert between the tetracycline-operator-tk-minimal promoter and the SV40 poly(A) in a vector derived from the tetracycline-tk-luc plasmid (35Anastassiadis K. Kim J. Daigle N. Sprengel R. Scholer H.R. Stewart A.F. Gene (Amst.). 2002; 298: 159-172Crossref PubMed Scopus (37) Google Scholar). Subsequently, the tetracycline-tk-ICA512-CCF-GFP-pA cassette was cloned between chicken β-globin insulators in a vector kindly provided by K. Anastassiadis (Biotec, TUD, Dresden, Germany). The mouse cDNA of the granule cargo chromogranin B (CgB; a gift from W. Huttner, Max Planck Institute of Cell Biology and Genetics) was fused in frame at the 5′-end of the cDNA for monomeric Red Fluorescent Protein 1 (36Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (1987) Google Scholar) (mRFP1; Planetgene, Menlo Park, CA). All clones were generated using conventional procedures and verified by DNA sequencing.Cell Transfections—INS-1E cells were electroporated using the Amaxa nucleoporator with a transfection efficiency of ∼60-70%, as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). Generation of stable GFP- and ICA512-CCF-GFP INS-1 cell clones was described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). INS-r3 cells expressing the reverse tetracycline-dependent trans-activator were transiently transfected with ICA512-CCF-GFP in the tetracycline-tk-luc-derived plasmid. Expression of ICA512-CCF-GFP was induced by exposure to 500 ng/ml doxycycline for 16, 24, 36, or 48 h. CgB-mRFP1 INS-1 clones were selected for puromycin resistance and further screened by fluorescence microscopy and immunoblotting for expression of the transgene.Cell Extracts and Western Blots—Purified mouse islets and INS-1E cells were washed with ice-cold PBS and extracted in lysis buffer (10 mm Tris-HCl, pH 8.0, 140 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% phosphatase inhibitors (Calbiochem), and 1% protease inhibitor mixture (Sigma, St. Louis, MO) at 4 °C. Protein measurements and immunoblottings were performed as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar) using the following primary antibodies: mouse monoclonal antibodies mICA512 (37Hermel J.M. Dirkx Jr., R. Solimena M. Eur. J. Neurosci. 1999; 11: 2609-2620Crossref PubMed Scopus (42) Google Scholar), mICA512-HM1 (26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar), anti-γ-tubulin (Sigma), and anti-GFP (Clontech); anti-ICA512ecto (22Solimena M. Dirkx Jr., R. Hermel J.M. Pleasic-Williams S. Shapiro J.A. Caron L. Rabin D.U. EMBO J. 1996; 15: 2102-2114Crossref PubMed Scopus (228) Google Scholar), anti-carboxypeptidase E/H (CPE) (Chemicon, Temecula, CA), and goat anti-GFP (a gift from D. Drechsel, Max Planck Institute of Cell Biology and Genetics). Chemiluminescence was developed and quantified as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar).Immunoprecipitations—INS-1E cells were transiently or stably transfected with one or more of the following constructs: GFP, ICA512-CCF-GFP, GFP-β2-syntrophin, ICA512-CCF-HA3, and HA3-ICA512-CCF. Protein extracts in 20 mm Tris-HCl at pH 8.0, 200 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% phosphatase inhibitors (Calbiochem), and 1% protease inhibitor mixture (Sigma) were incubated overnight at 4 °C with one of the following antibodies: goat anti-GFP, rabbit anti-ICA512ecto, goat or rabbit IgGs (Sigma), followed by incubation with protein G-Sepharose (GE Healthcare, Piscataway, NJ) for 90 min. The beads were then washed 10 times and loaded on 8-10% SDS-PAGE followed by Western blot with one of the following mouse antibodies: anti-ICA512, anti-GFP (Clontech), anti-β2-syntrophin (a gift from S. Froehner and M. Adams, University of Washington, Seattle), anti-HA, and anti-γ-tubulin.Pull-down Assays—ICA512-(601-979)-His or β2-syntrophin-His in pET28a (16Ort T. Maksimova E. Dirkx R. Kachinsky A.M. Berghs S. Froehner S.C. Solimena M. Eur. J. Cell Biol. 2000; 79: 621-630Crossref PubMed Scopus (58) Google Scholar) were in vitro transcribed and translated with the T7/coupled transcription-translation kit (Promega, Madison, WI). GST-ICA512-(601-979) or GST were expressed in bacteria as described (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar). [35S]Methionine-β2-syntrophin-His was incubated overnight at 4 °C in GST binding buffer (240 mm NaCl, 50 mm Tris, pH 8, 0.25% Nonidet P-40, 0.15% SDS, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) with GST fusion proteins or GST alone coupled to glutathione-Sepharose beads (GE Healthcare). Beads were washed 10 times with binding buffer and eluted with 10 mm reduced glutathione. Eluted proteins were subjected to SDS-PAGE and analyzed by autoradiography.Immunocytochemistry—Labeling of transfected or nontransfected INS-1E cells was performed as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). The following primary and secondary antibodies were used: mouse anti-insulin (Sigma), rabbit anti-TGN38 (BD Transduction Laboratories, Franklin Lakes, NJ), Alexa568-conjugated goat anti-mouse or Alexa488-conjugated goat-anti-rabbit IgGs and phalloidin-rhodamine (Molecular Probes, Inc., Eugene, OR). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Sigma), and coverslips were mounted with Mowiol (Calbiochem). Images of 0.5-μm optical sections were acquired at room temperature with an inverted confocal microscope Zeiss Axiovert 200M equipped with a Plan-Apochromat ×63 oil objective, numerical aperture 1.4, a Zeiss LSM510 scan head with photomultiplier tubes, and the software Zeiss LSM 510 AIM version 4 (Zeiss, Göttingen, Germany).RNA Interference—For knockdown of rat ICA512 and β2-syntrophin, the following oligonucleotides were used to generate silencing hairpin in the pGENE-CLIP U1 cassette system (Promega) according to the manufacturer's instructions: ICA512 short hairpin RNA oligo1, 5′-TCTCGCGCCATCATTCGAAACAATTCAAGAGATTGTTTCGAATGATGGCGCCT-3′; ICA512 short hairpin RNA oligo2, 5′-TCTCGCAGTACAAGCAGATGTAATTCAAGAGATTACATCTGCTTGTACTGCCT-3′, as previously described (25Mziaut H. Kersting S. Knoch K.P. Fan W.H. Trajkovski M. Erdmann K. Bergert H. Ehehalt F. Saeger H.D. Solimena M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 674-679Crossref PubMed Scopus (48) Google Scholar); β2-syntrophin short hairpin RNA oligo1, 5′-TCTCGTAGCTATCCACACCAACATATTCAAGAGATATGTTGGTGTGGATAGCTACCT-3′. Single or double transfections of INS-1E cells with pGENE-ICA512 and pGENE-β2-syntrophin by electroporation were performed as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar), using 4 or 3 μg of each plasmid for single or double transfection, respectively. Cells were stimulated and harvested 4 days after transfection, and knockdown of ICA512 and β2-syntrophin was determined by real time PCR and Western blot, as described (27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar).Insulin Radioimmune Assay—Insulin secretion was assessed as a ratio between insulin in the medium and the cell insulin content. After stimulation, 25-30 purified pancreatic islets were resuspended in fresh resting or stimulating medium and sonicated for 4 min. INS-1E cells were extracted overnight in 3:1:0.06 volumes of ethanol, H2O, and 37% HCl at -20 °C. Following centrifugation, insulin in islet cells and in the medium was measured with the Sensitive Rat Insulin RIA kit (Linco Research, St. Charles, MO). The insulin stimulation index was calculated as follows: secreted/total insulin in stimulated conditions versus secreted/total insulin in resting conditions.Total Internal Reflection Fluorescence Microscopy (TIRFM)—Images of INS-1 cells stably expressing mouse CgB-mRFP1 were acquired with a Roper Scientific MicroMAX512BFT charge-coupled device camera with a Zeiss Axiovert 200M microscope equipped with an argon laser and a Zeiss ×100/1.45 numerical aperture Plan-FLUAR objective and a ×1.6 optovar. The microscope was outfitted with a dual port total internal reflection fluorescence condenser (Till Photonics, Gräfelfing, Germany). Prior to imaging, cells grown in an open chamber were incubated in resting media and transferred onto a thermostat-controlled (37 °C) stage. CgB-mRFP1+ granules in GFP+ cells were visualized by excitation of mRFP1 at 488 nm using filter sets for tetramethylrhodamine isothiocyanate emission (Chroma Technology Corp., Rockingham, VT). Images were collected using the software MetaMorph (Molecular Devices, Sunnyvale, CA) at the speed of 2 frames/s with an exposure time of 100 ms, for a total of 4 min (480 frames) for each movie (supplemental Movies 1 and 2). Automated image analysis was performed with the MotionTracking/Kalaimoscope software (Transinsight GmbH, Dresden, Germany), as described (38Rink J. Ghigo E. Kalaidzidis Y. Zerial M. Cell. 2005; 122: 735-749Abstract Full Text Full Text PDF PubMed Scopus (1177) Google Scholar). The background of nonvesicular fluorescence was subtracted, and the granules were fitted by analytical function as described (38Rink J. Ghigo E. Kalaidzidis Y. Zerial M. Cell. 2005; 122: 735-749Abstract Full Text Full Text PDF PubMed Scopus (1177) Google Scholar). The accuracy of granule center definition was estimated from the mean square displacement (i.e. the average of squared displacements) and found equal to 60 nm. The jitter of center definition was suppressed by smoothing the tracks with a sliding window (±2 frames). Statistical data for granule density were obtained from 10 films. The total number of tracked CgB-mRFP1+ granules in GFP+ and ICA512-CCF-GFP+ cells was 779 and 888, respectively. Speed was measured between every two sequential frames, and the total number of speed measurements in GFP+ and ICA512-CCF-GFP+ cells was 13,332 and 14,828, respectively.Statistics and Graphics—Statistical analyses were performed as previously described (26Mziaut H. Trajkovski M. Kersting S. Ehninger A. Altkruger A. Lemaitre R.P. Schmidt D. Saeger H.D. Lee M.S. Drechsel D.N. Muller S. Solimena M. Nat. Cell Biol. 2006; 8: 435-445Crossref PubMed Scopus (70) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar). The error bars show S.D. values from at least three independent experiments. Histograms were prepared with Microsoft Excel (Microsoft, Redmond, WA).RESULTSICA512 Cleavage Regulates Exocytosis—We used several approaches to test whether the calpain-mediated cleavage of ICA512-TMF affects insulin secretion from INS-1E cells stimulated with 25 mm glucose (HG) and 55 mm KCl (HK). INS-1E cells were used, since they respond better to glucose than the parental INS-1 cells (31Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (470) Google Scholar). First, ICA512-TMF cleavage was prevented with the calpain inhibitor calpeptin. As shown for INS-1 cells, calpeptin reduced the cleavage of ICA512-TMF in INS-1E cells by ∼70% (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar) (supplemental Fig. 1B). Calpeptin also inhibited insulin release from HGHK-stimulated mouse pancreatic islets and INS-1E cells (Fig. 1, A and B).To test if this reduction resulted from the diminished cleavage of ICA512-TMF, we analyzed insulin secretion from ICA512-/- islets. Consistent with previous studies, the insulin stimulation index (SI) of untreated islets from ICA512-/- mice was 58 ± 8.7% lower than in ICA512+/+ islets (28Saeki K. Zhu M. Kubosaki A. Xie J. Lan M.S. Notkins A.L. Diabetes. 2002; 51: 1842-1850Crossref PubMed Scopus (100) Google Scholar) (Fig. 1A). Strikingly, calpeptin did not further decrease insulin secretion from ICA512-/- islets, indicating that calpain-1-mediated cleavage of ICA512-TMF regulates insulin secretion (Fig. 1A). Accordingly, both transient and stable overexpression of ICA512-GFP in INS-1E cells enhanced insulin release in a dose-dependent and calpeptin-sensitive fashion (Fig. 1B).Next, we investigated whether cleavage of ICA512-TMF affects insulin secretion by promoting insulin gene expression and granule biogenesis. Block of protein synthesis with 100 μm cycloheximide did not prevent calpeptin from inhibiting stimulated insulin release (Fig. 1C). Depolarization of β-cells with HK alone triggers Ca2+ entry, granule exocytosis, and cleavage of ICA512-TMF (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar) but not the rapid biosynthesis of preproinsulin and pro-ICA512 (17Ort T. Voronov S. Guo J. Zawalich K. Froehner S.C. Zawalich W. Solimena M. EMBO J. 2001; 20: 4013-4023Crossref PubMed Scopus (100) Google Scholar, 27Trajkovski M. Mziaut H. Altkruger A. Ouwendijk J. Knoch K.P. Muller S. Solimena M. J. Cell Biol. 2004; 167: 1063-1074Crossref PubMed Scopus (66) Google Scholar, 39Steiner, D. F., Chan, S. J., and Rubenstein, A. H. (2001) in Handbook of Physiology, Vol. II (Jefferson, L. S., Cherrington, A. D., and Goodman, H. M., eds) pp. 49–78, Oxford University Press, In" @default.
• W2138105049 created "2016-06-24" @default.
• W2138105049 creator A5007570063 @default.
• W2138105049 creator A5021760848 @default.
• W2138105049 creator A5037314002 @default.
• W2138105049 creator A5039754910 @default.
• W2138105049 creator A5044044576 @default.
• W2138105049 creator A5078646809 @default.
• W2138105049 date "2008-11-01" @default.
• W2138105049 modified "2023-10-12" @default.
• W2138105049 title "Regulation of Insulin Granule Turnover in Pancreatic β-Cells by Cleaved ICA512" @default.
• W2138105049 cites W1482452174 @default.
• W2138105049 cites W1877006252 @default.
• W2138105049 cites W1963181827 @default.
• W2138105049 cites W1967108735 @default.
• W2138105049 cites W1970723166 @default.
• W2138105049 cites W1975650380 @default.
• W2138105049 cites W1976819469 @default.
• W2138105049 cites W1979889913 @default.
• W2138105049 cites W1980800186 @default.
• W2138105049 cites W1987969991 @default.
• W2138105049 cites W1994157750 @default.
• W2138105049 cites W1994826393 @default.
• W2138105049 cites W1997010909 @default.
• W2138105049 cites W1999929698 @default.
• W2138105049 cites W2000461533 @default.
• W2138105049 cites W2006062639 @default.
• W2138105049 cites W2027409303 @default.
• W2138105049 cites W2028212562 @default.
• W2138105049 cites W2028482190 @default.
• W2138105049 cites W2032260752 @default.
• W2138105049 cites W2038240008 @default.
• W2138105049 cites W2041777802 @default.
• W2138105049 cites W2044710476 @default.
• W2138105049 cites W2045464110 @default.
• W2138105049 cites W2046766433 @default.
• W2138105049 cites W2053920298 @default.
• W2138105049 cites W2060505010 @default.
• W2138105049 cites W2063720970 @default.
• W2138105049 cites W2073392026 @default.
• W2138105049 cites W2074367229 @default.
• W2138105049 cites W2076923427 @default.
• W2138105049 cites W2080730085 @default.
• W2138105049 cites W2084026150 @default.
• W2138105049 cites W2087724572 @default.
• W2138105049 cites W2089041548 @default.
• W2138105049 cites W2089560759 @default.
• W2138105049 cites W2093160716 @default.
• W2138105049 cites W2099351017 @default.
• W2138105049 cites W2104726963 @default.
• W2138105049 cites W2105955034 @default.
• W2138105049 cites W2107913200 @default.
• W2138105049 cites W2113914274 @default.
• W2138105049 cites W2115622859 @default.
• W2138105049 cites W2126032145 @default.
• W2138105049 cites W2127402092 @default.
• W2138105049 cites W2144924187 @default.
• W2138105049 cites W2146166730 @default.
• W2138105049 cites W2149502850 @default.
• W2138105049 cites W2156189210 @default.
• W2138105049 cites W2157032387 @default.
• W2138105049 cites W2158788992 @default.
• W2138105049 cites W2166155213 @default.
• W2138105049 cites W2201616568 @default.
• W2138105049 cites W2339049224 @default.
• W2138105049 cites W4242695539 @default.
• W2138105049 doi "https://doi.org/10.1074/jbc.m804928200" @default.
• W2138105049 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2662281" @default.
• W2138105049 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18824546" @default.
• W2138105049 hasPublicationYear "2008" @default.
• W2138105049 type Work @default.
• W2138105049 sameAs 2138105049 @default.
• W2138105049 citedByCount "33" @default.
• W2138105049 countsByYear W21381050492012 @default.
• W2138105049 countsByYear W21381050492014 @default.
• W2138105049 countsByYear W21381050492015 @default.
• W2138105049 countsByYear W21381050492016 @default.
• W2138105049 countsByYear W21381050492017 @default.
• W2138105049 countsByYear W21381050492018 @default.
• W2138105049 countsByYear W21381050492019 @default.
• W2138105049 countsByYear W21381050492020 @default.
• W2138105049 countsByYear W21381050492021 @default.
• W2138105049 countsByYear W21381050492023 @default.
• W2138105049 crossrefType "journal-article" @default.
• W2138105049 hasAuthorship W2138105049A5007570063 @default.
• W2138105049 hasAuthorship W2138105049A5021760848 @default.
• W2138105049 hasAuthorship W2138105049A5037314002 @default.
• W2138105049 hasAuthorship W2138105049A5039754910 @default.
• W2138105049 hasAuthorship W2138105049A5044044576 @default.
• W2138105049 hasAuthorship W2138105049A5078646809 @default.
• W2138105049 hasBestOaLocation W21381050491 @default.
• W2138105049 hasConcept C126322002 @default.
• W2138105049 hasConcept C134018914 @default.
• W2138105049 hasConcept C151730666 @default.
• W2138105049 hasConcept C185592680 @default.
• W2138105049 hasConcept C2779306644 @default.
• W2138105049 hasConcept C49760210 @default.
• W2138105049 hasConcept C55493867 @default.

• next