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• W2097788259 abstract "Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca2+-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtβγ heterodimers. High affinity binding to Gtβγ and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gβγ of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions. Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca2+-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtβγ heterodimers. High affinity binding to Gtβγ and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gβγ of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions. Vertebrate rod and cone photoreceptor cells are highly polarized neurons that consist of morphologically and functionally distinct cellular compartments. Light-sensitive outer segments are linked via a non-motile connecting cilium with inner segments that contain the organelles typical for the metabolism of eukaryotic cells (see Fig. 6A). The outer segments are characterized by specialized disklike membranes where one of the best studied examples of a G-protein transduction cascade is arranged (1Pugh Jr., E.N. Lamb T. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. 3. Elsevier Science Publisher B.V., Amsterdam2000: 183-255Google Scholar, 2Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Photoexcitation leads to photoisomerization of the visual pigment rhodopsin (Rh*), 1The abbreviations used are: Rh, rhodoposin; Rh*, photoactivated rhodopsin; Gt, retinal G-protein, transducin; MmCen1-4, mouse centrin isoforms 1-4; Cen1-4, centrin isoforms 1-4; Cen1p-4p, centrin isoform 1-4 proteins; pMmC1-4: polyclonal antibody against mouse centrins 1-4; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; GST, glutathione S-transferase; RT, reverse transcription; GTPγS, guanosine 5′-3-O-(thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: Rh, rhodoposin; Rh*, photoactivated rhodopsin; Gt, retinal G-protein, transducin; MmCen1-4, mouse centrin isoforms 1-4; Cen1-4, centrin isoforms 1-4; Cen1p-4p, centrin isoform 1-4 proteins; pMmC1-4: polyclonal antibody against mouse centrins 1-4; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; GST, glutathione S-transferase; RT, reverse transcription; GTPγS, guanosine 5′-3-O-(thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid. which catalyzes GDP/GTP exchange on the heterotrimeric holo G-protein transducin (Gtholo). This releases the α-subunit of transducin (Gtα), which in turn activates a phosphodiesterase, catalyzing cGMP hydrolysis in the cytoplasm and closure of cGMP-gated channels localized in the plasma membrane (2Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 3Heck M. Hofmann K.P. Biochemistry. 1993; 32: 8220-8227Crossref PubMed Scopus (55) Google Scholar). The closure of these channels leads to a drop of the circulating cationic current, resulting in the hyperpolarization of the cell membrane (4Molday R.S. Kaupp U.B. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. 3. Elsevier Science Publishers B.V., Amsterdam2000: 143-182Google Scholar). The recovery phase of the enzymatic machinery of visual transduction and rapid light adaptation of photoreceptor cells (time scale of subseconds) rely on a feedback mechanism. This depends on changes in the intracellular Ca2+ concentration [Ca2+]i, affecting the phototransduction cascade through Ca2+-binding proteins (5Palczewski K. Polans A.S. Baehr W. Ames J.B. Bioessays. 2000; 22: 337-350Crossref PubMed Scopus (134) Google Scholar). However, massive bidirectional translocation of transduction cascade components between the functional compartments of photoreceptor cells can also contribute to a much slower adaptation of rod photoreceptor cells (6Sokolov M. Lyubarsky A.L. Strissel K.J. Savchenko A.B. Govardovskii V.I. Pugh E.N. Arshavsky V.Y. Neuron. 2002; 34: 95-106Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 7Sokolov M. Strissel K.J. Leskov I.B. Michaud N.A. Govardovskii V.I. Arshavsky V.Y. J. Biol. Chem. 2004; 279: 19149-19156Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).Light-induced exchanges of signal cascade components were first noted about a decade ago (8Philp N.J. Chang W. Long K. FEBS Lett. 1987; 225: 127-132Crossref PubMed Scopus (158) Google Scholar, 9Brann M.R. Cohen L.V. Science. 1987; 235: 585-587Crossref PubMed Scopus (159) Google Scholar, 10Whelan J.P. McGinnis J.F. J. Neurosci. Res. 1988; 20: 263-270Crossref PubMed Scopus (180) Google Scholar) and are currently of prominent interest in the field (e.g. Hardie (11Hardie R. Neuron. 2002; 34: 3-5Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), and see current review by Giessl et al. (12Giessl A. Trojan P. Pulvermüller A. Wolfrum U. Williams D.S. Cell Biology and Related Disease of the Outer Retina. World Scientific Publishing Company Pte. Ltd., Singapore2004: 195-222Google Scholar)): upon illumination, 80% of Gtα and Gtβγ move in minutes from the outer segment to the inner segment and the cell body of rod photoreceptor cells. A recent study indicates that binding of the photoreceptor-specific protein phosducin to Gtβγ is not essential for this movement but facilitates light-driven Gtβγ translocation to the inner segment (7Sokolov M. Strissel K.J. Leskov I.B. Michaud N.A. Govardovskii V.I. Arshavsky V.Y. J. Biol. Chem. 2004; 279: 19149-19156Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The G-protein subunits return to the outer segments in the dark in a more leisurely time course of hours. In contrast, arrestin translocates under these light conditions in an exactly reciprocal way (8Philp N.J. Chang W. Long K. FEBS Lett. 1987; 225: 127-132Crossref PubMed Scopus (158) Google Scholar, 10Whelan J.P. McGinnis J.F. J. Neurosci. Res. 1988; 20: 263-270Crossref PubMed Scopus (180) Google Scholar). Since any intracellular exchange between the inner and outer segmental compartments of photoreceptor cells should occur through the slender non-motile connecting cilium (13Besharse J.C. Horst C.J. Bloodgood R.A. Ciliary and Flagellar Membranes. Plenum Press, New York1990: 389-417Crossref Google Scholar), this represents a suitable domain for potential regulation of intersegmental molecular exchange (14Wolfrum U. Salisbury J.L. Exp. Cell Res. 1998; 242: 10-17Crossref PubMed Scopus (59) Google Scholar). Our initial studies revealed that transducin is translocated through the photoreceptor connecting cilium and further indicated that the Ca2+-induced assembly of a protein-protein complex of the G-protein transducin and the cytoskeletal protein centrin 1 regulates ciliary G-protein translocation (12Giessl A. Trojan P. Pulvermüller A. Wolfrum U. Williams D.S. Cell Biology and Related Disease of the Outer Retina. World Scientific Publishing Company Pte. Ltd., Singapore2004: 195-222Google Scholar, 15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 16Wolfrum U. Giessl A. Pulvermüller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref PubMed Scopus (36) Google Scholar). The assembly of this centrin 1-G-protein complex strictly depends on Ca2+ and is mediated by the Gtβγ complex.Centrin 1 is a member of the centrin protein family, a subfamily of the parvalbumin superfamily of Ca2+-binding proteins (17Salisbury J.L. Curr. Opin. Cell Biol. 1995; 7: 39-45Crossref PubMed Scopus (311) Google Scholar, 18Schiebel E. Bornens M. Trends Cell Biol. 1995; 5: 197-201Abstract Full Text PDF PubMed Scopus (150) Google Scholar). Centrins were first described in unicellular green algae where they form filamentous structures that contract in response to an increase of [Ca2+]i (17Salisbury J.L. Curr. Opin. Cell Biol. 1995; 7: 39-45Crossref PubMed Scopus (311) Google Scholar, 18Schiebel E. Bornens M. Trends Cell Biol. 1995; 5: 197-201Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 19Salisbury J.L. Baron A. Surek B. Melkonian M. J. Cell Biol. 1984; 99: 962-970Crossref PubMed Scopus (288) Google Scholar). In vertebrates, centrins are ubiquitously expressed and commonly associated with centrosome-related structures such as spindle poles of dividing cells or centrioles in centrosomes and basal bodies (17Salisbury J.L. Curr. Opin. Cell Biol. 1995; 7: 39-45Crossref PubMed Scopus (311) Google Scholar, 18Schiebel E. Bornens M. Trends Cell Biol. 1995; 5: 197-201Abstract Full Text PDF PubMed Scopus (150) Google Scholar). At least four different centrin genes are expressed in mammals (20Lee V.D. Huang B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11039-11043Crossref PubMed Scopus (116) Google Scholar, 21Errabolu R. Sanders M.A. Salisbury J.L. J. Cell Sci. 1994; 107: 9-16Crossref PubMed Google Scholar, 22Levy Y.Y. Lai E.Y. Remillard S.P. Heintzelman M.B. Fulton C. Cell Motil. Cytoskelet. 1996; 33: 298-323Crossref PubMed Scopus (110) Google Scholar, 23Madeddu L. Klotz C. Le Caer J.P. Beisson J. Eur. J. Biochem. 1996; 238: 121-128Crossref PubMed Scopus (62) Google Scholar, 24Meng T.C. Aley S.B. Svard S.G. Smith M.W. Huang B. Kim J. Gillin F.D. Mol. Biochem. Parasitol. 1996; 79: 103-108Crossref PubMed Scopus (37) Google Scholar, 25Middendorp S. Paoletti A. Schiebel E. Bornens M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9141-9146Crossref PubMed Scopus (127) Google Scholar, 26Wottrich R. Cloning and Computer-based Structural Analyses of Centrin Isoforms in the Rat (Rattus norvegicus). Diploma, Universität Karlsruhe, Karlsruhe, Germany1998Google Scholar, 27Gavet O. Alvarez C. Gaspar P. Bornens M. Mol. Biol. Cell. 2003; 14: 1818-1834Crossref PubMed Scopus (58) Google Scholar). As a consequence of the isoform diversity in the mammalian genome, the four mammalian centrins should exhibit differences in their subcellular localization as well as in their cellular function. Little is known about the specific subcellular localization of the different centrin isoforms in diverse cell types and tissues. Most studies on the localization of centrins in mammalian cells and tissues have been performed with polyclonal and monoclonal antibodies raised against green algae centrins that do not discriminate between the mammalian centrin isoforms. Using these antibodies, centrins were detected in the centrioles of centrosomes and in the pericentriolar matrix (28Salisbury J.L. Baron A.T. Sanders M.A. J. Cell Biol. 1988; 107: 635-641Crossref PubMed Scopus (188) Google Scholar, 29Baron A.T. Greenwood T.M. Salisbury J.L. Cell Motil. Cytoskelet. 1991; 18: 1-14Crossref PubMed Scopus (28) Google Scholar, 30Baron A.T. Greenwood T.M. Bazinet C.W. Salisbury J.L. Biol. Cell. 1992; 76: 383-388Crossref PubMed Scopus (76) Google Scholar). In previous studies on the mammalian retina, centrins were localized in two basically distinct subcellular domains (12Giessl A. Trojan P. Pulvermüller A. Wolfrum U. Williams D.S. Cell Biology and Related Disease of the Outer Retina. World Scientific Publishing Company Pte. Ltd., Singapore2004: 195-222Google Scholar, 14Wolfrum U. Salisbury J.L. Exp. Cell Res. 1998; 242: 10-17Crossref PubMed Scopus (59) Google Scholar, 16Wolfrum U. Giessl A. Pulvermüller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref PubMed Scopus (36) Google Scholar). As in other animal cells, centrins are components of centrosomes and basal bodies in retinal neurons but were also found to be present in the connecting cilium of photoreceptor cells (14Wolfrum U. Salisbury J.L. Exp. Cell Res. 1998; 242: 10-17Crossref PubMed Scopus (59) Google Scholar, 15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 16Wolfrum U. Giessl A. Pulvermüller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref PubMed Scopus (36) Google Scholar, 32Wolfrum U. Cell Motil. Cytoskelet. 1995; 32: 55-64Crossref PubMed Scopus (53) Google Scholar). Although our recent studies provided evidence that isoform Cen1 is localized in the connecting cilium, a ciliary expression of other centrin isoforms remained elusive (16Wolfrum U. Giessl A. Pulvermüller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref PubMed Scopus (36) Google Scholar).Here we show by glutathione S-transferase (GST) pull-down assays, size exclusion chromatography, and kinetic light-scattering experiments that all four centrin isoforms bind to the Gtβγ complex with different affinities. In the present study, we also demonstrated retinal expression of the four centrin isoforms. Furthermore we were able to show for the first time that the centrins are co-expressed in the same cell type, particularly in highly specialized photoreceptor cells. Nevertheless there they are localized in different subcellular domains. The localization of the centrin isoforms Cen1 to Cen3 in the photoreceptor connecting cilium suggests that these centrins can be part of the centrin-transducin complex. In contrast, the centriolar localization of centrin isoforms 2-4 indicates an additional function of these centrin isoforms.EXPERIMENTAL PROCEDURESAnimals and Tissue Preparation—All experiments described herein conform to the statement by the Association for Research in Vision and Ophthalmology as to the care and use of animals in research. Adult Sprague-Dawley albino rats and C57BL76 mice were maintained on a 12/12-h light/dark cycle with lights on at 6 a.m. with food and water ad libitum. After sacrifice of the animals in CO2, retinas were removed through a slit in the cornea prior to fixation and embedding for microscopy or further molecular biological and biochemical analysis. Bovine eyes were obtained from the local slaughter houses and were kept on ice in the dark until further processing.Antibodies—Affinity-purified polyclonal rabbit antibodies against the α- and β-subunit of G-proteins were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA), and a second affinity-purified polyclonal rabbit antibody against the β-subunit of G-proteins (T-20) was purchased from Molecular Probes (Eugene, OR). Monoclonal antibody against centrin (clone 20H5) and a monoclonal antibody against HsCen2p (clone hCetn2.4) have been characterized previously (30Baron A.T. Greenwood T.M. Bazinet C.W. Salisbury J.L. Biol. Cell. 1992; 76: 383-388Crossref PubMed Scopus (76) Google Scholar, 33Hart P.E. Poynter G.M. Whitehead C.M. Orth J.D. Glantz J.N. Busby R.C. Barrett S.L. Salisbury J.L. Gene (Amst.). 2001; 264: 205-213Crossref PubMed Scopus (17) Google Scholar). Polyclonal antisera from rabbit or goat against recombinantly expressed mouse centrins 1-4 (MmCen1 to MmCen4) were generated and affinity-purified on high trap N-hydroxysuccinimide columns (Amersham Biosciences).SDS-PAGE and Western Blot—For Western blots, isolated retinas or GST pull-down complexes were homogenized and placed in SDS-PAGE sample buffer (62.5 mm Tris-HCl (pH 6.8) containing 10% glycerol, 2% SDS, 5% mercaptoethanol, 1 mm EDTA, and 0.025% bromphenol blue). Proteins were separated by SDS-PAGE (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205874) Google Scholar) using 15% polyacrylamide gels, transferred electrophoretically to polyvinylidene difluoride membranes (Bio-Rad), and probed with primary and secondary antibodies (32Wolfrum U. Cell Motil. Cytoskelet. 1995; 32: 55-64Crossref PubMed Scopus (53) Google Scholar).Recombinant Expression of Centrin Isoforms—Cloning of a mouse centrin 1 cDNA into the pGEX-4T3 expression vector (Amersham Biosciences) was described previously by Pulvermüller et al. (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar). Mouse centrin 2, 3, and 4 cDNAs were cloned from reverse transcription (RT)-PCR products into the pGEX-4T3 expression vector (Amersham Biosciences) using BamHI and XhoI restriction sites. Expression and purification of the GST fusion protein was performed according to the manufacturer's instructions (Amersham Biosciences). After cleavage of the fusion protein with thrombin on the column, centrin was eluted in 20 mm BTP (pH 7.5) containing 130 mm NaCl and 1 mm MgCl2 and passed over a benzamidine-Sepharose 6B (Amersham Biosciences) column to remove thrombin.Membrane and Protein Preparations—Rod outer segments were prepared from frozen bovine retinas using a sucrose gradient procedure as described previously (35Papermaster D.S. Methods Enzymol. 1982; 81: 48-52Crossref PubMed Scopus (253) Google Scholar). Hypotonically stripped disk membranes were prepared from rod outer segments by the Ficoll floating procedure similar to the procedure described previously (36Smith Jr., H.G. Stubbs G.W. Litman B.J. Exp. Eye Res. 1975; 20: 211-217Crossref PubMed Scopus (202) Google Scholar) except that 2% (w/v) Ficoll instead of 5% was used. This method yielded osmotically intact disk vesicles with a vesicle size >400 nm. Contamination by vesicle aggregates was removed by a 2-μm filter (Roth, Karlsruhe, Germany). Membranes were either kept on ice and used within 4 days without any loss of activity or stored at -80 °C until use. Rhodopsin concentration was determined from its absorption spectrum using ϵ500 = 40,000 m-1 cm-1. Transducin (Gtholo) was isolated from frozen dark-adapted bovine retinas according to Ref. 3Heck M. Hofmann K.P. Biochemistry. 1993; 32: 8220-8227Crossref PubMed Scopus (55) Google Scholar. Subunits were further purified on Blue Sepharose (1 ml of HiTrap Blue, Amersham Biosciences) using a salt gradient (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar). Gtholo, Gtα, and Gtβγ concentrations were determined by the Bradford assay (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (212995) Google Scholar) using bovine serum albumin as the standard. The amount of intact, activable Gtα was determined precisely by fluorometric titration with GTPγS (38Ernst O.P. Bieri C. Vogel H. Hofmann K.P. Methods Enzymol. 2000; 315: 471-489Crossref PubMed Google Scholar).GST-Centrin Pull-down Assay—Bacteria expressing GST-centrin fusion proteins were resuspended in PBS (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4 (pH 7.3)). Cells were lysed by lysozyme (0.2 mg/ml) and sonicated. Cleared lysates were incubated for 2 h at 4 °C with 50 μl of glutathione-S-Sepharose 4B (Amersham Biosciences) in NETN buffer (20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) in a final volume of 500 μl. Sepharose beads with fusion proteins were washed with NETN buffer and buffer F (20 mm Na-Hepes (pH 8.0), 2 mm EDTA, 10 mm CaCl2, 100 mm NaCl, 11 mm CHAPS, 1 mm dithiothreitol) and incubated with retina extracts in buffer F for 2 h at 4 °C (final volume, 600 μl). Beads were washed three times with buffer F, and proteins were eluted from the beads by incubation for 20-30 min at 25 °C in 50 mm Tris-HCl (pH 8.0) containing 15 mm glutathione and 11 mm CHAPS.Size Exclusion Chromatography—Size exclusion chromatography is a very useful tool to determine protein-protein interaction (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 39Schröder K. Pulvermüller A. Hofmann K.P. J. Biol. Chem. 2002; 277: 43987-43996Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and was used in this study to characterize direct complex formation between the four different mouse centrins (MmCen1 to MmCen4), transducin (Gt), and its subunits. To determine the binding of the centrins to transducin the molecular weight shift of the complex was used. 10 μg of each recombinant centrin isoform and 10 μg of Gtholo (or Gt subunits Gtα and Gtβγ) were incubated in 50 mm BTP (pH 7.0) containing 80 mm NaCl, 1 mm MgCl2, and either 100 μm CaCl2 or 1 mm EGTA for 5 min at room temperature. As controls, all samples (MmCen1 to MmCen4, Gt, and Gt subunits) were incubated alone. The reaction mixtures were loaded on a Superose™ 12 column (Amersham Biosciences) equilibrated with the same buffer using the Smart System (Amersham Biosciences; flow rate, 40 μl/min). Elution was monitored by absorbance at 280 nm, and 40-μl fractions were collected for the subsequent SDS-PAGE analysis.Kinetic Light Scattering—The gain or loss of membrane-bound protein mass can be readily measured by light-scattering changes using a setup described in detail in Heck et al. (40Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar). All measurements were performed in 10-mm path cuvettes with 300-μl volumes in 50 mm BTP (pH 7.5) containing 80 mm NaCl, 5 mm MgCl2, and either 100 μm CaCl2 or 1 mm EGTA at 20 °C (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar). Reactions were triggered by flash photolysis of rhodopsin with a green (500 ± 20 nm) flash attenuated by appropriate neutral density filters. The flash intensity was quantified photometrically by the amount of rhodopsin bleached and expressed in the mole fraction of photoexcited rhodopsin (Rh*/Rh = 32%). The scattering signal is interpreted as a gain of protein mass bound to disk membranes and quantified as described previously (40Heck M. Pulvermüller A. Hofmann K.P. Methods Enzymol. 2000; 315: 329-347Crossref PubMed Google Scholar, 41Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Light-scattering binding signals were corrected by a reference signal (N-signal) measured on a sample without added Gtholo as described previously (42Pulvermüller A. Palczewski K. Hofmann K.P. Biochemistry. 1993; 32: 14082-14088Crossref PubMed Scopus (85) Google Scholar).RT-PCR—Total RNA was isolated from mouse and rat retinas using TRIzol reagent (Invitrogen). Samples of purified total RNA were treated with DNase I (Sigma) for 15 min to remove genetic DNA. To stop the reaction the samples were treated for 15 min at 75 °C. Poly(dT)-primed cDNA synthesis (reverse transcriptase reaction) was performed using the Invitrogen cDNA Cycle™ kit and 5 μg of total RNA according to the directions. In control preparations, total RNA (DNase-treated or -untreated) was amplified by PCR without prior reverse transcriptase reactions using the MmCen1 primers. PCR was performed in a volume of 50 μl using 2.5 μl of prepared cDNA according to directions and 0.25 μg of each primer/reaction. Cycling conditions were 39 cycles at 94 °C for 1 min, 59 °C for 30 s, and 72 °C for 3 min followed by a 10-min 72 °C extension. PCR product lengths were determined on 0.8% agarose gels. As DNA markers, a 1-kb DNA ladder (Invitrogen) was used. Sequencing of PCR products was performed by Genterprise (Mainz, Germany). For sequence comparisons and oligonucleotide generation the computer program Omiga™ Version 2.0 (Oxford Molecular Ltd., Oxford, UK) was used.PCR Primers Used for RT-PCR and DNA Sequencing—Primers specific for mouse centrin isoforms were as follows: MmCen1 primers, the forward primer MmCen1-forw (5′-GTACGGATC CATGGCGTCCACCTTCAGGAAG-3′) and the reverse primer MmCen1-EF3,4-rev (5′-GCGGCTCGAGTTAATCTTTCTCGGCCATCTT-3′); MmCen2 primers, the forward primer MmCen2-EF1,2-forw (5′-GTACGGATCCACTAAAGAAGAAATCCTGAAA-3′) and the reverse primer MmCen2-rev (5′-GCGGCTCGAGTTACAGACAAGCTGTGACCGT-3′); MmCen3 primers: the forward primer MmCen3-forw (5′-GTACGGAT CCGAGAACTGTCTGAGGAACAGA-3′) and the reverse primer MmCen3-rev (5′-GCGG CTCGAGTATGTCACCAGTCATAATAGC-3′); MmCen4 primers, the forward MmCen4-Nterm-forw (5′-GTACGGATCCCAAGAAGTTCGGGAAGCCTTT-3′) and the reverse primer MmCen4-rev (5′-GCGGCTCGAGCTAATAAAGGCTGGTCTTCTT-3′).Peptide Preadsorption of pMmC Centrin Antibodies with Recombinant Expressed Centrins—To increase antibody specificity, the polyclonal antibodies pMmC1 to pMmC4 were preincubated with the appropriate recombinant centrin isoform proteins. For this purpose centrin isoform proteins were immobilized on polyvinylidene difluoride membranes (Bio-Rad) and incubated with the affinity-purified antibodies for 12 h at 4 °C in blocking solution (0.5% cold-water fish gelatin (Sigma) plus 0.1% ovalbumin (Sigma) in PBS). The following protein amounts were used: pMmC1: 200 μg of MmCen2, 350 μg of MmCen3, and 150 μg of MmCen4; pMmC2: 400 μg of MmCen1, 300 μg of MmCen3, and 200 μg of MmCen4; pMmC3: 100 μg of MmCen1 and 30 μg of MmCen4; pMmC4: 300 μg of MmCen1, 200 μg of MmCen2, and 400 μg of MmCen3. The supernatants containing the “preabsorbed” antibodies were subsequently used in Western blots or immunocytochemical experiments, respectively.Fluorescence Staining of Retinal Cryosections—Immunofluorescence studies were essentially performed as described previously (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 43Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Briefly eyes from adult mice were prefixed in 4% paraformaldehyde in PBS for 1 h at room temperature, washed, soaked with 30% sucrose in PBS overnight, and cryofixed in melting isopentane. Cryosections were placed on poly-l-lysine-precoated coverslips (44Wolfrum U. Cell Tissue Res. 1991; 266: 231-238Crossref Scopus (47) Google Scholar, 45Wolfrum U. Schmitt A. Cell Motil. Cytoskelet. 2000; 46: 95-107Crossref PubMed Scopus (132) Google Scholar). Specimens were incubated with 50 mm NH4Cl and 0.1% Tween 20 in PBS and blocked with blocking solution (0.5% cold-water fish gelatin (Sigma) plus 0.1% ovalbumin (Sigma) in PBS). The sections were incubated with antibodies or, in the case of double labeling, with a mixture of antibodies in blocking solution overnight at 4 °C. The specimens were washed and subsequently incubated with secondary antibodies conjugated to Alexa® 488 or Alexa 546 (Molecular Probes) in blocking solution for 1 h at room temperature in the dark. Washed sections were mounted in Mowiol 4.88 (Hoechst, Frankfurt, Germany) containing 2% n-propyl gallate and, in the case of triple staining, 1 μg/ml 4,6-diamidino-2-phenylindole. Mounted retinal sections were examined with a Leica DMRP microscope. Images were obtained with a Hamamatsu Orca ER CCD camera (Hamamatsu City, Japan) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA).Immunoelectron Microscopy—After 12-h dark or light adaptation, respectively, isolated rat or mouse retinas were fixed, embedded in LR White resin, and further processed for immunoelectron microscopy as described previously (45Wolfrum U. Schmitt A. Cell Motil. Cytoskelet. 2000; 46: 95-107Crossref PubMed Scopus (132) Google Scholar). Nanogold™ labeling (Nanoprobes, Yaphank, NY) was silver-enhanced according to Ref. 46Danscher G. Histochemistry. 1981; 71: 81-88Crossref PubMed Scopus (387) Google Scholar. Counterstained sections were analyzed in an FEI Tecnai 12 Biotwin electron microscope.RESULTSAssembly of Transducin-Centrin Complexes—In the search for centrin 1-interacting proteins in photoreceptor cells, we have previously identified the βγ subunit of the visual G-protein transducin as a potent interacting partner for MmCen1 (15Pulvermüller A. Giessl A. Heck M. Wottrich R. Schmitt A. Ernst O.P. Choe H.W. Hofmann K.P. Wolfrum U. Mol. Cell. Biol. 2002; 22: 2194-2203Crossref PubMed Scopus (55) Google Scholar, 16Wolfrum U. Giessl A. Pulvermüller A. Adv. Exp. Med. Biol. 2002; 514: 155-178Crossref Pu" @default.
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