"Lne4 257en Mebt_Tot Biouoru Ccnsr Homt PrnriUSA Lysophosphatidylcholine Reversibly Arrests Exocytosis and Viral Membrane Merger* Fusion at a Stage between Triggering and (Recejved for publication; June 22, 1993,and revised frm, August 20, 1993) Steven 8. Vogel, Eugenia 4. Leikina, and Leonid V Chernomordikt From the Laboratory %f Thearelicol and Physical Biology: Natianal Institute of Child Health and Huncn Wrue Dnman of Health, Bethesda, Maryland 20892 Natanarntutes lize them We recently found that the addition of the events occurzing between exoCenous Little Tem KnoWn lysolipids pubaequent fuaion steps: To bet #een contacting membranes inhibited fusicn in brane fuslon triggering and regardless of their particu- reversible inhibitor of disparate biologicul procegges applied dissect this process we fusion,  lysophosphatidylcholine; PH, GTP-S, GTP) (12), This inhibition atTeat trigger calcium membrane virus-mediated syncytla formation Next requisite fusion exocytosia related sclubilization 'cuot respective fusion triggera) wus adminis- membrane lysig (12}. The Ca" or H: (the nents, irreversible denaturation, Thcn inhibitor was withdrawn the phenomenon suggested tered and later removed apparently uniyersa character enqued demonstrating that triggering for all fusion that lysolipids inhibit an Jnterm cdato commor activated state, which luter cuuses the formation fa8 ard 8.0w. Fusior reachon? wbile different into the fused gtate Imerefor develope To determine which step biological fusion twrgeted by utilize different triggera; the pivotal fuaion "alow "fast" fusion processey lysolipids test eramples involvlng membrane merger is trigger-independent atep studied contrasting share common mechanistc stepa; and lipid-sensitive: triggered biolopical fusion: Low pH-triggered syn examples cytia formation in baculovins-infected celle (13, 14) is an ex protuin of the en ample of slow fusion mlediated by Lnot The gecond system, the erocytotic fusion Of 8ea cells involves menbrane dock- Vnis Triggered membrane fubion veloped ing &nd subacquent mcmbrare merger; Recent developments reaction that occura urchin egg cortica) granules, suggest conservation of docking mechanisms disparate fu- including humans (15} This most aimals , egg? 2). In contrast, the regulatory components of which can eke place ample of fast , ~triggered fusion_ Tenctioni Cc which respond to different triggers limit of 10-35 mg alter the introduction o ATe Incsi Junton membrane within an uopcr docking i the mechanism membrane likely calcium (16). Because triggering occura ILOWDAa subSequunt Wqul Calcium-trig- these systema, they &re well suited for atudying the final in different fuision reactions merger conseryc pH-triggered viral fusion gered exocytcsis Quretse fubion membrane anodlox steps exampleg o cell biological processes that still share common EXPERIMENTAL PROCEDURES addition to uging different fusion (3-5} In Aeerpe membrane purcheged fuuing they also differ in which leaflets of the Material:_All lipidg dyus memn- membrant trigger8 _ exoplasmic" Avunli Pclar Lipida (Himinzhem AL) excep: for Q tadegyl rhc lunine braney make initial contact (-endoplasmic Vereus Stock RIBL which was purchaecd #om Moleculur Probea (Eugere; OR} 7) Perhaps the most striking difiterence apeed; fusion) (6, 0.5% (wtw} 4quecus ilutnni Ayealpldg Were Icahun prcpArCo calcium-triggered exocytotic fusion, such Eynantc Seen Stock eution MRI8 (100 uK)nnd n-Iaamne Inodamng dispersion be 1000 tires faster than viral fusion (5} Based Bulfonyl) diacylphosphabidylethanolamine Prepared frcm eg8 (Rhod- tarminale propoged that while thesc chebe dhtterencey Fr- Tere mndc Elianol guperticially similar; they may have rere a tacked Corticnl Exocytosis Lytechinus pictus egg8 Grarull brane rearrangemente jet of isptonic difterent mechanism; (5}. In particular; the - polylysine-coated Flag3 Bldae teun Iycd fundamentally coldiun-free buffer 08 previcusly described (17} except PKCME bufler (50 triggered exocytosis Wa, hypothesized to reflect & Eitu- pidity EGTA, Fipcs; pH 6.7, 425 mv KCL; 10 MaCl, ation where the introduction of calcium enabled @inal single- Cortoa uxed insteed cortex bentamidine) Eiium step reaction exaindles 0r membrane fusion (5). In contrast; perfused +ith GP chamber mourted microgcopc ncrtusion only triggering and mem- "slycire; 50 mu Fipe%, pH 6.7, Intii Lurion JreKnoxm Involye InM banzamdno contnin? ouft scep (8) CaClz and#5nt brane merger but also 12 my DM-nitrophan ( caged caldjum_ interna-diale commitment Amhochem the calcium Indicator Rhod-2 (10 p Molecular und the insertion of & hydrophobic viral peptide into the adulc amerimente Lut Probed) wns included the bulicr - mealure caped celclum Teltaed. membrane (9-11) accomplished by The Teleasn Clcuo DOlLO DM-nicronhe Ultimately; comparison of viral and exocytotic fusion mecha- application of a brief pulae of UV light (380 nm) thruzh the microacope the fusion intermediates requires the identification elcctric shutter: With this design ouly the Mats epifluorescence port with between triggering and membrane merger:  Progress this caldium, Alowing witbin tne Gield ctjostd COmce of the ghort-lived nature of the slide_ Fusion TarMonitord WALDO direction has heen :lox bechue multinle eroenment? NamE (LKC-90O, LK stabi- digital videc dtoceyeor pbsence cf inhibicora cptic? ennGEL intermediate strictures and Lhc modihed Zeiss Rewrarch Triangle Park, NC} Haxke Etlc Auonitense Wan Taanirt uxnt cont (181- Caalum indicntor publication ol thia article Fcre defraycd in part by the the microacope ocular with a 690-nm long pass fiter: The costs Lodiodc ntucheo payment of pag" charges. This article must therefore be hereby marked ITC-[6 cmputor Itorluze (Instrutech Tlke signals rcrc digitizedby 18 USC 1734 soley procesied by commercial sftwurc (Igor; Wave - Sicblon Eltecnt, AYY) and anueriemerf accordance Com andlta duik fuct whom cpmespordence shauld be addrexxed: Laborator o The 1The abbreviations ufed are: GTP-S,quaratice 5'3-0-(thiokriphos: 6C-101, NCHHD , NIH, and Pkyaical Biology. Bldg: 10, Tejcn phate; Pipos, 1,4-piperazinediethanesulonic acid; DIC , differentinl in- Betheada,MD 20592. Tel : 301-496-0740; Fax 301402-0263 Electtonic rArerencr contragt_ mail: Ichem Bhelix nih FoY; 25764
"Lne4 257en PrnriUSA Mebt_Tot Homt Biouoru Ccnsr Lysophosphatidylcholine Reversibly Arrests Exocytosis and Viral Fusion at a Stage between Triggering and Membrane Merger* (Recejved for publication; June 22, 1993,and revised frm, August 20, 1993) Steven 8. Vogel, Eugenia 4. Leikina, and Leonid V Chernomordikt From the Laboratory %f Thearelicol and Physical Biology: Natianal Institute of Child Health and Huncn Wrue Dnman Natanarntutes of Health, Bethesda, Maryland 20892 Little KnoWn of the events occurzing between Tem lize them We recently found that the addition exoCenous and pubaequent fuaion steps: To lysolipids bet #een contacting membranes inhibited fusicn in brane fuslon triggering dissect this process we applied reversible inhibitor of disparate biologicul procegges regardless of their particu- membrane fusion,  lysophosphatidylcholine; atTeat trigger calcium PH, GTP-S, GTP) (12), This inhibition exocytosia virus-mediated syncytla formation Next related sclubilization requisite fusion 'cuot Ca" or H: (the respective fusion triggera) wus adminis- nents, irreversible denaturation, membrane lysig (12}. The tered and later removed Thcn inhibitor was withdrawn apparently uniyersa character the phenomenon suggested fusion enqued demonstrating that triggering that lysolipids inhibit an Jnterm cdato commor for all cuuses the formation activated state, which luter Fusior reachon? fa8 ard 8.0w. develope into the fused gtate Imerefor wbile different To determine which step biological fusion twrgeted by fuaion processey utilize different triggera; the pivotal lysolipids test eramples "alow "fast" fusion atep involvlng membrane merger is trigger-independent share common mechanistc stepa; studied contrasting and lipid-sensitive: examples triggered biolopical fusion: Low pH-triggered syn cytia formation in baculovins-infected celle (13, 14) is an ex ample of slow fusion mlediated by Lnot protuin of the en Triggered membrane fubion cells involves menbrane dock- veloped Vnis The gecond system, the erocytotic fusion Of 8ea ing &nd subacquent mcmbrare merger; Recent developments urchin egg cortica) granules, reaction that occura suggest conservation of docking mechanisms disparate fu- egg? most aimals , including humans (15} This Tenctioni 2). In contrast, the regulatory components of ample of fast , Cc ~triggered fusion_ which can eke place membrane Junton which respond to different triggers ATe Incsi within an uopcr limit of 10-35 mg alter the introduction o likely Wqul ILOWDAa i the mechanism membrane calcium (16). Because triggering occura subSequunt docking merger conseryc in different fuision reactions Calcium-trig- these systema, they &re well suited for atudying the final gered exocytcsis anodlox pH-triggered viral fusion Quretse steps membrane fubion exampleg o cell biological processes that still share common Aeerpe membrane fusion (3-5} In addition to uging different EXPERIMENTAL PROCEDURES they also differ in which leaflets of the fuuing memn- Material:_All lipidg membrant dyus purcheged trigger8 _ braney make initial contact (-endoplasmic Vereus exoplasmic" Avunli Pclar Lipida (Himinzhem AL) excep: for Q tadegyl rhc lunine apeed; RIBL which was purchaecd #om Moleculur Probea (Eugere; OR} Stock fusion) (6, 7) Perhaps the most striking difiterence ilutnni Ayealpldg Were Icahun prcpArCo 0.5% (wtw} 4quecus calcium-triggered exocytotic fusion, such Seen Eynantc dispersion Stock eution MRI8 (100 uK)nnd n-Iaamne Inodamng tarminale be 1000 tires faster than viral fusion (5} Based Bulfonyl) diacylphosphabidylethanolamine Prepared frcm eg8 (Rhod- chebe dhtterencey propoged that while thesc  Fr- Tere mndc Elianol brane rearrangemente guperticially similar; they may have Corticnl Grarull Exocytosis Lytechinus pictus egg8 rere a tacked fundamentally difterent mechanism; (5}. In particular; the - polylysine-coated Flag3 Bldae teun Iycd jet of isptonic coldiun-free buffer 08 previcusly described (17} except PKCME bufler (50 pidity triggered exocytosis Wa, hypothesized to reflect & Eitu- Fipcs; pH 6.7, 425 mv KCL; 10 MaCl, EGTA, ation where the introduction of calcium enabled @inal single- bentamidine) uxed insteed cortex Eiium  Cortoa step reaction membrane fusion (5). In contrast; exaindles 0r mourted microgcopc ncrtusion chamber perfused +ith GP Intii Lurion JreKnoxm Involye only triggering and mem- ouft "slycire; 50 mu Fipe%, pH 6.7, InM banzamdno contnin? interna-diale commitment scep (8) 12 my DM-nitrophan ( caged caldjum_ Amhochem and#5nt brane merger but also CaClz und the insertion of & hydrophobic viral peptide into the Lut adulc amerimente the calcium Indicator Rhod-2 (10 p Molecular Probed) wns included the bulicr - mealure caped celclum Teltaed. membrane (9-11) The Teleasn Clcuo DOlLO DM-nicronhe accomplished by Ultimately; comparison of viral and exocytotic fusion mecha- application of a brief pulae of UV light (380 nm) thruzh the microacope Mats requires the identification the fusion intermediates epifluorescence port with elcctric shutter: With this design ouly the between triggering and membrane merger:  Progress this COmce witbin tne Gield ctjostd caldium, Alowing direction has heen :lox bechue of the ghort-lived nature of the multinle eroenment? NamE slide_ Fusion TarMonitord WALDO pbsence cf inhibicora stabi- cptic? ennGEL digital videc dtoceyeor (LKC-90O, LK intermediate strictures and Lhc Haxke Rewrarch Triangle Park, NC} modihed Zeiss Etlc cont (181- Caalum indicntor Auonitense Wan Taanirt uxnt The costs publication ol thia article Fcre defraycd in part by the Lodiodc ntucheo the microacope ocular with a 690-nm long pass fiter: payment of pag" charges. This article must therefore be hereby marked Tlke signals rcrc digitizedby ITC-[6 cmputor Itorluze (Instrutech anueriemerf accordance 18 USC Sicblon 1734 soley Com Eltecnt, AYY) and procesied by commercial sftwurc (Igor; Wave - andlta duik fuct whom cpmespordence shauld be addrexxed: Laborator o The Tejcn and Pkyaical Biology. Bldg: 10, 6C-101, NCHHD , NIH, 1The abbreviations ufed are: GTP-S,quaratice 5'3-0-(thiokriphos: Betheada,MD 20592. Tel : 301-496-0740; Fax 301402-0263 Electtonic phate; Pipos, 1,4-piperazinediethanesulonic acid; DIC , differentinl in- rArerencr contragt_ mail: Ichem Bhelix nih FoY; 25764