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• W2078584735 abstract "The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, from fungi to mammals. We describe the isolation of the snc1, sso1, and sso2 genes encoding exocytic SNARE proteins from the filamentous fungus Trichoderma reesei. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in subapical but not apical hyphal regions. The vesicle SNARE SNCI also localized to the apical vesicle cluster within the Spitzenkoörper of the growing hyphal tips. Using fluorescence lifetime imaging microscopy and Foerster resonance energy transfer analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of ternary SNARE complex formation between SNCI and SSOI or SSOII, respectively, is spatially segregated. SNARE complex formation could be detected between SNCI and SSOI in subapical hyphal compartments along the plasma membrane, but surprisingly, not in growing hyphal tips, previously thought to be the main site of exocytosis. In contrast, SNCI·SSOII complexes were found exclusively in growing apical hyphal compartments. These findings demonstrate spatially distinct sites of plasma membrane SNARE complex formation in fungi and the existence of multiple exocytic SNAREs, which are functionally and spatially segregated. This is the first demonstration of spatially regulated SNARE interactions within the same membrane. The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, from fungi to mammals. We describe the isolation of the snc1, sso1, and sso2 genes encoding exocytic SNARE proteins from the filamentous fungus Trichoderma reesei. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in subapical but not apical hyphal regions. The vesicle SNARE SNCI also localized to the apical vesicle cluster within the Spitzenkoörper of the growing hyphal tips. Using fluorescence lifetime imaging microscopy and Foerster resonance energy transfer analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of ternary SNARE complex formation between SNCI and SSOI or SSOII, respectively, is spatially segregated. SNARE complex formation could be detected between SNCI and SSOI in subapical hyphal compartments along the plasma membrane, but surprisingly, not in growing hyphal tips, previously thought to be the main site of exocytosis. In contrast, SNCI·SSOII complexes were found exclusively in growing apical hyphal compartments. These findings demonstrate spatially distinct sites of plasma membrane SNARE complex formation in fungi and the existence of multiple exocytic SNAREs, which are functionally and spatially segregated. This is the first demonstration of spatially regulated SNARE interactions within the same membrane. Protein transport through the exocytic and endocytic pathways in eukaryotic cells occurs via vesicle trafficking between successive membrane-bounded compartments. The transport of vesicles from endoplasmic reticulum to the plasma membrane requires a series of events involving budding from donor compartments and docking and fusion with acceptor compartments (1Bennett M.K. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2559-2563Crossref PubMed Scopus (542) Google Scholar, 2Goötte M. von Mollard G.F. Trends Cell Biol. 1998; 8: 215-218Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Membrane-associated proteins called SNAREs 2The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; TCSPC, time-correlated single photon counting; FLIM, fluorescence lifetime imaging microscopy; FRET, Foerster resonance energy transfer; PDB, Protein Data Bank; TPE, two-photon excitation; FWHM, full width at half-maximum; SC, synthetic complete; MCP-PMT, multichannel plate-photomultiplier tube. 2The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; TCSPC, time-correlated single photon counting; FLIM, fluorescence lifetime imaging microscopy; FRET, Foerster resonance energy transfer; PDB, Protein Data Bank; TPE, two-photon excitation; FWHM, full width at half-maximum; SC, synthetic complete; MCP-PMT, multichannel plate-photomultiplier tube. (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) facilitate the fusion between vesicles and target membranes. SNAREs were originally divided into two classes according to their localization to vesicle (v-SNARE) or target (t-SNARE) membranes (3Soöllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2589) Google Scholar). Each SNARE protein contains a motif that is able to form a parallel α-helical structure with other SNARE motifs that brings the vesicle and target membranes into close proximity, allowing membrane fusion (3Soöllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2589) Google Scholar). Mammalian synaptic t-SNAREs can interact as a binary complex of one syntaxin and one SNAP-25 in cells (4Rickman C. Meunier F.A. Binz T. Davletov B. J. Biol. Chem. 2004; 279: 644-651Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), in the absence of associated v-SNARE. The subsequent interaction of the appropriate v-SNARE with this binary SNARE complex allows the formation of the SNARE ternary complex and thus defines the location of vesicle fusion (4Rickman C. Meunier F.A. Binz T. Davletov B. J. Biol. Chem. 2004; 279: 644-651Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It is unclear whether the stable ternary SNARE complex forms before the final fusion event is initiated. In addition to the mammalian exocytic SNAREs, the best characterized SNAREs are the yeast Saccharomyces cerevisiae membrane trafficking counterparts (5Gerst J.E. Biochim. Biophys. Acta. 2003; 1641: 99-110Crossref PubMed Scopus (75) Google Scholar, 6Jahn R. Lang T. Sudhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1200) Google Scholar). SNARE complex formation in the yeast secretory pathway has been shown to begin with the formation of binary complexes between two t-SNAREs, Ssop (a syntaxin homolog) and Sec9 (the SNAP-25 homolog) (7Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (172) Google Scholar), that are then capable of binding the v-SNARE Sncp. The Sso and Sec9 proteins have been shown to be located uniformly over the plasma membrane in these geometrically simple cells (8Brennwald P. Kearns B. Champion K. Keraönen S. Bankaitis V. Novick P. Cell. 1994; 79: 245-258Abstract Full Text PDF PubMed Scopus (311) Google Scholar). In contrast to yeast cells, filamentous fungi exhibit highly polarized growth at the hyphal tips. This involves the delivery of Golgi apparatus-derived secretory vesicles to the apical plasma membrane via an apical vesicle cluster within a multicomponent complex called the Spitzenkoörper (9Virag A. Harris S.D. Mycol. Res. 2006; 110: 4-13Crossref PubMed Scopus (82) Google Scholar). These secretory vesicles contain membrane proteins and lipids, enzymes involved in cell wall synthesis, and possibly cell wall precursors (10Grove S.N. Bracker C.E. J. Bacteriol. 1970; 104: 989-1009Crossref PubMed Google Scholar). Although the exocytosis of extracellular enzymes (i.e. in contrast to “constitutive” secretion at growth cones or buds) into the external medium is generally believed to occur mainly from hyphal tips, few studies have actually localized the subcellular sites of secretion. Immunogold and green fluorescent protein localization of glucoamylase in Aspergillus niger supported the hypothesis that the secretion of this enzyme primarily takes place from hyphal tips (11Gordon C.L. Archer D.B. Jeenes D.J. Doonan J.H. Wells B. Trinci A.P. Robson G.D. J. Microbiol. Meth. 2000; 42: 39-48Crossref PubMed Scopus (65) Google Scholar, 12Woösten H.A. Moukha S.M. Sietsma J.H. Wessels J.G. J. Gen. Microbiol. 1991; 137: 2017-2023Crossref PubMed Scopus (228) Google Scholar). Other studies have suggested that enzymes might also be secreted from subapical hyphal locations (13Nykaönen M. Saarelainen R. Raudaskoski M. Nevalainen H. Mikkonen A. Appl. Environ. Microbiol. 1997; 63: 4929-4937Crossref PubMed Google Scholar, 14Weber R. Pitt D. Mycol. Res. 1997; 101: 1431-1439Crossref Scopus (14) Google Scholar), but this has never been shown directly. Understanding of the fungal secretion pathway has significantly increased recently. Based on the analysis of sequenced fungal genomes, it is clear that the secretory machinery of filamentous fungi has similar features to that of other eukaryotes (15Borkovich K.A. Alex L.A. Yarden O. Freitag M. Turner G.E. Read N.D. Seiler S. Bell-Pedersen D. Paietta J. Plesofsky N. Plamann M. Goodrich-Tanrikulu M. Schulte U. Mannhaupt G. Nargang F.E. Radford A. Selitrennikoff C. Galagan J.E. Dunlap J.C. Loros J.J. Catcheside D. Inoue H. Aramayo R. Polymenis M. Selker E.U. Sachs M.S. Marzluf G.A. Paulsen I. Davis R. Ebbole D.J. Zelter A. Kalkman E.R. O'Rourke R. Bowring F. Yeadon J. Ishii C. Suzuki K. Sakai W. Pratt R. Microbiol. Mol. Biol. Rev. 2004; 68: 1-108Crossref PubMed Scopus (471) Google Scholar, 16Diener S.E. Dunn-Coleman N. Foreman P. Houfek T.D. Teunissen P.J.M. van Solingen P. Dankmeyer L. Mitchell T.K. Ward M. Dean R.A. FEMS Microbiol. Lett. 2004; 230: 275-282Crossref PubMed Scopus (19) Google Scholar), and some genes encoding the proteins involved in vesicle trafficking have been cloned and characterized (17Gupta G.D. Free S.J. Levina N.N. Keraönen S. Heath I.B. Fungal Genet. Biol. 2003; 40: 271-286Crossref PubMed Scopus (19) Google Scholar, 18Heintz K. Palme K. Diefenthal T. Russo V.E. Mol. Gen. Genet. 1992; 235: 413-421Crossref PubMed Scopus (17) Google Scholar, 19Punt P.J. Seiboth B. Weenink X.O. van Zeijl C. Lenders M. Konetschny C. Ram A.F. Montijn R. Kubicek C.P. van den Hondel C.A. Mol. Microbiol. 2001; 41: 513-525Crossref PubMed Scopus (51) Google Scholar, 20Saloheimo M. Lund M. Penttilaö M.E. Mol. Gen. Genet. 1999; 262: 35-45Crossref PubMed Scopus (64) Google Scholar, 21Saloheimo M. Wang H. Valkonen M. Vasara T. Huuskonen A. Riikonen M. Pakula T. Ward M. Penttilaö M. Appl. Environ. Microbiol. 2004; 70: 459-467Crossref PubMed Scopus (28) Google Scholar, 22Veldhuisen G. Saloheimo M. Fiers M.A. Punt P.J. Contreras R. Penttila M. van den Hondel C.A. Mol. Gen. Genet. 1997; 256: 446-455PubMed Google Scholar). Here, we have used a combination of in vitro biochemistry, in vivo complementation assays, and advanced live cell imaging techniques to quantify novel SNARE protein targeting, co-localization, and interactions in filamentous fungi. As the v-SNARE interacts with t-SNAREs after formation of the fungal SSO-Sec9 binary complex (7Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (172) Google Scholar), by quantifying the interaction of a vesicular v-SNARE with two plasma membrane t-SNAREs using fluorescence lifetime imaging microscopy (FLIM)/FRET analysis, we have identified different subcellular sites for exocytic SNARE ternary complex formation in living fungal hyphae. Interestingly, the interactions between a sole v-SNARE and two alternative t-SNAREs are spatially segregated. This is the first time that SNARE protein interactions have been demonstrated directly in living fungal cells; these data reveal contrasts with mammalian SNARE cluster regulation and also suggest an alternative exocytic pathway to the constitutive secretion known to occur via the Spitzenkoörper at hyphal tips. Importantly, this is the first time that spatially regulated SNARE interactions have been found on the plasma membrane. S. cerevisiae Suppression and Complementation Studies—A Trichoderma reesei cDNA expression library (23Margolles-Clark E. Tenkanen M. Nakari-Setaölaö T. Penttilaö M. Appl. Environ. Microbiol. 1996; 62: 3840-3846Crossref PubMed Google Scholar) in the vector pAJ401 (24Saloheimo A. Henrissat B. Hoffren A.M. Teleman O. Penttilaö M. Mol. Microbiol. 1994; 13: 219-228Crossref PubMed Scopus (150) Google Scholar) was transformed into the SSO2 temperature-sensitive S. cerevisiae strain H1152 (Mata, sso2-1, leu2-1, trp1-1, ura3-1, sso1::HIS3 (25Jaöntti J. Aalto M.K. Oyen M. Sundqvist L. Keraönen S. Ronne H. J. Cell Sci. 2002; 115: 409-420Crossref PubMed Google Scholar). Transformants were plated on synthetic complete (SC) medium (-Ura) with 2% galactose as the carbon source, and the plates were incubated at the restrictive temperature, 31 °C. Transformants that were able to grow on these plates were grown as streaks under the same conditions and then replicated for growth at 31, 33, 35, and 37 °C. Plasmids were isolated from clones that were able to grow at 37 °C and transferred into Escherichia coli for sequencing. The suppression of the SSO2 temperature-sensitive mutation was tested by transforming the plasmid pMS84 obtained in the original screening back to the H1152 strain. After transformation, the strain was grown on SC (-Ura) plates with 2% galactose as a carbon source. For the suppression studies, the transformants were streaked on the same medium and replicated to parallel plates that were incubated at 24, 31, 33, and 37 °C. Suppression of Sso1p and Sso2p depletion was tested in the H458 strain (Matα, ade2-1, his3-11, trp1-1, ura3-1, sso1-S1::LEU2, sso2-S1::leu2::(GAL1:SSOI, HIS3) (courtesy of Hans Ronne, Uppsala University, Sweden). The strain was grown after transformation on SC (-Ura) plates with 2% galactose as a carbon source, and the transformants were replicated on either YP (1% yeast extract, 2% peptone) or SC (-Ura) plates with 2% glucose as the carbon source and grown at 30 °C. For the analysis of the complementation of Snc1p and Snc2p depletion, the T. reesei snc1 cDNA was cloned as an EcoRI-XhoI fragment into a S. cerevisiae single copy plasmid pKKl with the PGK1 promoter and terminator and LEU2 selectable marker. The resulting plasmid was called pMV20, and it was transformed into the JG8 T15:85 (Mata his3 leu2 trp1 snc1::URA3 snc2::ADE8 pGAL-TSNCI) (26Protopopov V. Govindan B. Novick P. Gerst J.E. Cell. 1993; 74: 855-861Abstract Full Text PDF PubMed Scopus (250) Google Scholar) strain. Transformants were grown on SC (-Leu-Trp) plates with 2% galactose as the carbon source. For complementation testing, the transformants were grown on SC (-Leu-Trp) plates with 2% galactose as the carbon source for 3 days and then replicated on YPD (1% yeast extract, 2% peptone, 2% glucose) or SC (-Leu-Trp) plates with 2% galactose as the carbon source and grown at 30 °C for 4 days. To test the complementation of the temperature sensitivity defect, transformants were replicated on SC (-Leu-Trp) plates with 2% glucose as the carbon source and grown at 24, 30, and 37 °C for 4 days. For the analysis of the complementation of Sso1p and Sso2p depletion, the T. reesei sso1 cDNA was cloned into the S. cerevisiae expression vector pAJ401, either as a fusion with the mCerulean fluorescent protein (27Rizzo M.A. Springer G.H. Granada B. Piston D.W. Nat. Biotechnol. 2004; 22: 445-449Crossref PubMed Scopus (897) Google Scholar) or as a non-fusion, creating vectors pMV61 and pMV62, respectively. The T. reesei sso2 cDNA was cloned into the pYES-DEST53 Gateway vector (Invitrogen), creating the pMV72 plasmid. All constructs were transformed into the SSO2 temperature-sensitive S. cerevisiae strain H1152. Transformants were plated on SC (-Ura) with 2% galactose as the carbon source, and the plates were incubated at the permissive temperature, 24 °C. Transformants that were able to grow on these plates were incubated as streaks under the same conditions and then replicated for growth at 24, 31, 33, 35, and 37 °C. Invertase Assay—To study the ability of T. reesei SNCI to suppress the defect in invertase secretion in a strain depleted of both Snc proteins, an experiment was done with modifications as described by Ref. 26Protopopov V. Govindan B. Novick P. Gerst J.E. Cell. 1993; 74: 855-861Abstract Full Text PDF PubMed Scopus (250) Google Scholar. The invertase assay was done using the JG8 T15:85 strain transformed with the pMV20-construct or the expression vector pKKl alone and with the wild type strain NY15 (Matα, ura3-52, his4-619, courtesy of Peter Novick, Yale University, New Haven, CT). Two parallel cultures of the strains were grown in SC (-Leu-Trp) medium with 4% galactose as the carbon source at 30 °C for 2 days. The cultures were then diluted to an A600 of 0.2 in the same medium and grown at 30 °C until the A600 was 2.5. After this, the cultures were transferred to SC (-Leu-Trp) medium with 2% glucose as the carbon source and grown at 24 or 30 °C. This was done to deplete the cells of the endogenous Snc1 protein. The cultures were kept in the early logarithmic growth phase throughout the experiment by repeated dilutions in the same medium. After growth in glucose-containing medium for 12 h, samples of 4 × 108 cells were removed from each culture, centrifuged, and resuspended in 20 ml of SC-Leu-Trp medium with 0.05% glucose as the carbon source to derepress the invertase gene. The samples were incubated at 24 or 30 °C for 2 h. A sample of A600 2.5 was taken from each culture for the invertase assay. The invertase assay was performed as described in Ref. 28Goldstein A. Lampen J.O. Methods Enzymol. 1975; 42: 504-511Crossref PubMed Scopus (298) Google Scholar. Confocal Live Cell Imaging—For imaging, the yeast transformants with the pMV62 expression construct and the controls (transformants with pAJ401 vector alone) were grown in SC (-Ura) with 2% galactose for 2 days. 200-μl drops of diluted cell suspension were placed in an eight-well slide culture chamber (Nalgene Nunc International, Rochester, NY). For imaging T. reesei, the snc1 cDNA was cloned into the pContV vector (courtesy of Dr. N. Curach, Macquarie University, Sydney, Australia), where it was expressed as an N-terminal fusion to the fluorescent protein Venus (29Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2138) Google Scholar) under the endogenous cbh1 promoter. The resulting expression vector was called pMV63. For the construction of the mCer·SSOI and mCer·SSOII fusion proteins under the cbh1 promoter, the Venus in pContV vector was replaced with mCerulean. The sso1 and sso2 cDNAs were cloned into this vector, and the resulting vectors were called pMV67 and pMV77, respectively. The expression vectors were transformed into T. reesei RutC-30 (30Montenecourt B.S. Eveleigh D.E. Adv. Chem. Ser. 1979; 181: 289-301Crossref Google Scholar), creating pMV63, pMV67, and PMV77 single transformants or pMV63/67 and pMV63/77 double transformants. For imaging purposes, the strains were grown on plates on T. reesei minimal medium (31Penttilaö M. Nevalainen H. Rattoö M. Salminen E. Knowles J. Gene (Amst.). 1987; 61: 155-164Crossref PubMed Scopus (507) Google Scholar) for 5 days, and the inverted agar block method of sample preparation (32Hickey P.C. Swift S.R. Roca M.G. Read N.D. Savidge T. Pothoulakis C. Meth. Microbiol.2004: 63-87Google Scholar) was used. 10 or 30 μm FM4-64 (prepared from 1 m stock in Me2SO; Invitrogen-Molecular Probes) was used in combination with fluorescent protein labeling. The stain was added to the agar block containing the mycelial sample 10 min before imaging. Confocal laser scanning microscopy was performed using a Radiance 2100 confocal system equipped with blue diode and argon ion lasers (Bio-Rad Microscience) mounted on a Nikon TE2000U Eclipse inverted microscope. mCerulean was imaged on its own with excitation at 405 nm and fluorescence detection at 470/90 nm; Venus was imaged on its own with excitation at 514 nm and fluorescence detection at 530/50 nm; and Venus and FM4-64 were imaged simultaneously by excitation with the 514 nm laser line with fluorescence detected at 530/50 nm (for Venus) and >620 nm (for FM4-64). A ×60 (1.2 NA) plan apo water immersion objective lens was used for imaging. The laser intensity and laser scanning were kept to a minimum to reduce phototoxic effects. Simultaneous, bright-field images were captured with a transmitted light detector. Kalman filtering (n = 1) was used to improve the signal-to-noise ratio of individual images. Images were captured with a laser scan speed of 50 lines/s at a resolution of 1,024 by 1,024 pixels. Confocal images were captured with Lasersharp 2000 software (version 5.1; Bio-Rad Microscience) and were further processed using Image J 1.34S software (W. Rasband, National Institutes of Health, Rockville, MD). Two-photon Microscopy—All further imaging experiments were performed using a Zeiss LSM 510 Axiovert confocal laser scanning microscope, equipped with a pulsed excitation source (MIRA 900 Ti:Sapphire femtosecond pulsed laser, with a coupled VERDI 10-watt pump laser (Coherent, Ely, UK). The laser was tuned to provide a two-photon excitation (TPE) wavelength of 800 nm, which efficiently excited mCerulean, without any detectable excitation or emission from Venus in the absence of FRET from a donor. TPE data acquisition was performed using 1024 × 1024-pixel image sizes, with 4× frame averaging, using a Zeiss Plan NeoFLUAR ×63 (1.4 NA) oil immersion objective or a Zeiss C-Apochromat ×63 (1.2 NA) water immersion objective. Band pass and long pass emission filters were used, as detailed under “Results”, in conjunction with a Schott (New York, NY) BG39 IR filter to attenuate the TPE light. Image data acquired at Nyquist sampling rates were deconvoluted using Huygens Pro software (Scientific Volume Imaging, Hilversum, The Netherlands), and the resulting three-dimensional models were analyzed using Imaris software (Bitplane AG, Zuörich) on dual Opteron workstations. Co-localization was quantified as described previously by extracting all voxels in three dimensions containing both mCerulean and Venus data above background and reconstructing them into a new, co-localization channel. Co-localization maps were generated using the Manders approach, with automatic thresholding performed according to the method of Costes. Cell peripheries were determined using transmitted light imaging combined with confocal laser scanning microscopy data (33Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). TCSPC-FLIM—A number of different approaches to analyzing FRET have been described, using fluorescence intensities in image data and requiring arithmetical corrections to subtract background and to remove undesired spectral bleed-through between the donor and the acceptor. We found that intensity based approaches were not sufficiently sensitive to quantify FRET efficiency between the two fluorescent proteins with useful spatial resolution; also, the limited dynamic range reported an all-or-nothing signal. Thus, to further quantify FRET between labeled SNCI and the two SSO proteins, we employed multidimensional time-correlated single photon counting (TCSPC)-FLIM, essentially as described previously (33Rickman C. Medine C.N. Bergmann A. Duncan R.R. J. Biol. Chem. 2007; 282: 12097-12103Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Duncan R.R. Bergmann A. Cousin M.A. Apps D.K. Shipston M.J. J. Microsc. (Oxf.). 2004; 215: 1-12Crossref PubMed Scopus (116) Google Scholar). TCSPC measurements were made under 820 nm TPE, using a non-descanned detector (Hamamatsu R3809U-50; Hamamatsu Photonics UK Ltd., Herts, UK) multichannel plate-photomultiplier tube (MCP-PMT) or a fast photomultiplier tube (H7422; Hamamatsu Photonics) coupled directly to the rear port of the Axiovert microscope and protected from room light and other sources of overload using a Uniblitz shutter (Rochester, NY). Dark count rates were 102-103 photons/s. The MCP-PMT was operated at 3 kV, and signal pulses were preamplified using a Becker & Hickl HFAC-26 26 dB, 1.6-GHz preamplifier. TCSPC recording used the “reversed start stop” approach, with accurate laser synchronization using a Becker & Hickl SPC-830 card together with a PHD-400 reference photodiode, routinely at 79.4 MHz. Band pass and long pass filters were used to enable spectral separation of donor and acceptor FRET- and sensitized-emissions. 3-6-mm Schott BG39 filters were positioned directly in front of the MCP-PMT. TCSPC recordings were acquired routinely for between 5 and 25 s, and mean photon counts were between 105 and 106 cps. Images were recorded 256 × 256 pixels from a 1024 × 1024-image scan with 256 time bins over a 12-ns period. FLIM data were acquired for between 5 and 60 s for a 1024 × 1024-image frame. FLIM Data Analysis and FRET Calculations—Off-line FLIM data analysis used pixel-based fitting software (SPCImage, Becker & Hickl). The fluorescence was assumed to follow a multiexponential decay. In addition, an adaptive offset correction was performed. A constant offset takes into consideration the time-independent baseline due to dark noise of the detector and the background caused by room light, calculated from the average number of photons per channel in front of the rising part of the fluorescence trace. To fit the parameters of the multiexponential decay to the fluorescence decay trace measured by the system, a convolution with the instrumental response function was carried out. The optimization of the fit parameters was performed by using the Levenberg-Marquardt algorithm, minimizing the weighted chi-square quantity. The approach can be used to separate the interacting from the non-interacting donor fraction in our FRET systems. The slow (long lifetime) component τ2 was determined by control assays with mCerulean alone or expressed with (non-interacting) Venus in the way described above. This value is finally used as a fixed τ2 lifetime for all other experiments. Fluorescence lifetimes here are expressed as the weighted mean of two fitted exponential functions in each pixel. As controls for nonspecific FRET or FRET between green fluorescent proteins that may form dimers spontaneously when overexpressed in cells, we determined the fluorescence lifetimes of mCerulean-SSOI alone, mCerulean alone, or mCerulean-SSOI co-expressed with Venus (not shown). No FRET was detected in any of these experiments. Likewise, experiments using mCeruleanA206K-fused SSOI revealed no self-dimerization between fluorescent proteins (not shown). Itraconazole Treatment—For testing the effect of itraconazole on growth and protein secretion, two pMV63/67 transformants expressing Venus·SNCI and mCer·SSOI fusion proteins were grown in a minimal medium (31Penttilaö M. Nevalainen H. Rattoö M. Salminen E. Knowles J. Gene (Amst.). 1987; 61: 155-164Crossref PubMed Scopus (507) Google Scholar) with 2% lactose in shake flasks for 3 days. Each culture was divided into four aliquots, and itraconazole (10, 50, and 100 μm in Me2SO) was added to three aliquots. Samples from the culture supernatants were taken once a day for 5 days, and endoglucanase activities were analyzed as described in Ref. 35Bailey M.J. Nevalainen K.M.H. Enzyme Microb. Technol. 1981; 3: 153-157Crossref Scopus (250) Google Scholar. For imaging, the T. reesei strains were grown on plates as described above. After 5 days, 10 or 50 μm itraconazole in Me2SO was added to an agar block cut out from the cultures, and the samples were incubated at 28 °C for 16 h. Before imaging, the samples were washed with 1× phosphate-buffered saline buffer. Protein Sequence Alignment—Protein sequences were aligned with ClustalW (36Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). Phylogenetic trees were constructed with Phylip. 3J. Felsestein, personal communication. Its programs seqboot, protpars, and consense were used to make unrooted 100 times bootstrapped parsimony trees. Trees were drawn with Treeview (38Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). Comparative modeling of T. reesei SNCI and SSO proteins was done using the Swiss-Model server (39Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4376) Google Scholar). The models were viewed using the DeepView-Swiss-PdbViewer (40Gribskov M. McLachlan A.D. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4355-4358Crossref PubMed Scopus (1108) Google Scholar, 41Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). Detection of mRNA Levels—The analysis of mRNA levels of snc1 and sso1 genes was carried out using recently developed transcriptional analysis with aid of affinity capture (TRAC) method (42Rautio J.J. Kataja K. Satokari R. Penttilaö M. Soöderlund H. Saloheimo M. J. Microbiol. Methods. 2006; 65: 404-416Crossref PubMed Scopus (40) Google Scholar) using samples from the same cultures from which the endoglucanese activities were measured. Data Analysis—All data are presented as mean ± S.E. Cloning and Sequence Analysis of the T. reesei SNAREs—Complementation of a S. cerevisiae SSOI disruptant strain that had a temperature-sensitive" @default.
• W2078584735 created "2016-06-24" @default.
• W2078584735 creator A5002905598 @default.
• W2078584735 creator A5007171299 @default.
• W2078584735 creator A5009032164 @default.
• W2078584735 creator A5034695858 @default.
• W2078584735 creator A5036922921 @default.
• W2078584735 creator A5043243404 @default.
• W2078584735 date "2007-08-01" @default.
• W2078584735 modified "2023-10-15" @default.
• W2078584735 title "Spatially Segregated SNARE Protein Interactions in Living Fungal Cells" @default.
• W2078584735 cites W119116669 @default.
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• W2078584735 doi "https://doi.org/10.1074/jbc.m700916200" @default.
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