Ukuhlungwa kwamaphrotheyini endleleni yokukhipha kuqakathekile ekugcineni ukuhlukaniswa kwamaseli nokuhlala kuhle. Ngaphezu kokuhlunga okulawulwa zigobolondo, indima yamafutha ekuhlungeni kwe-kinesin emsebenzini wokuthuthwa kwe-secretory ngumbuzo oyisisekelo wesikhathi eside ongakaphendulwa. Lapha, senza i-3D simultaneous multicolor high-resolution real-time imaging ukufakazela ku-vivo bona amaphrotheyini asanda kwenziwa we-glycosylphosphatidylinositol-immobilized anama-ceramide lipid moieties amade khulu ahlanganiswa begodu ahlukaniswe abe ma-endoplasm akhethekileko we-Net exit site, ehlukileko kunaleyo esetjenziswa maphrotheyini we-transmembrane. Ukungezelela, sitjengisa bona ubude beketango le-ceramide ku-endoplasmic reticulum membrane buqakathekile ekukhetheni lokhu. Irhubhululo lethu linikela ngobufakazi bokuthoma obuqondileko be-vivo ukuhlukanisa imithwalo yamaphrotheyini ngokuya ngobude beketango lamafutha ibe ziindawo ezikhethiweko zokuthumela ngaphandle endleleni yokukhipha.
Emaseli we-eukaryotic, amaphrotheyini enziwe ku-endoplasmic reticulum (ER) abese ahlukaniswa ngesikhathi sokuthuthwa ngendlela yokukhiqiza ukuze athunyelwe endaweni yawo efaneleko yamaseli (1). Ngaphezu kokuhlunga okulawulwa yi-coat, bekusolo kucatshangelwa bona amafutha athileko angasebenza njengeendawo zokuphuma ezikhethiweko ngokuwahlanganisa abe ziindawo ezithileko ze-membrane ezithize amaphrotheyini athileko (2-5). Nanyana kunjalo, kusese nokutlhogeka kobufakazi obuqondileko be-vivo ukufakazela indlela le esekelwe emafutheni. Ukurarulula umraro lo, sifunde ngemvubelo bona amaphrotheyini ahlangeneko we-glycosylphosphatidylinositol (GPI) (ama-GPI-AP) athunyelwa njani ngokuhlukileko avela e-ER. Ama-GPI-APs ziinhlobo ezihlukahlukeneko zamaphrotheyini weseli ahlanganiswe namafutha ( 6, 7). I-GPI-AP yiprotheyini ekhiqizwako enamathele emaphepheni wangaphandle we-plasma membrane nge-glycolipid moiety (i-GPI anchor). Bamukela ama-anchor we-GPI njengeentjhuguluko ezilondolozako ngemva kokuhumusha ku-ER lumen (8). Ngemva kokunamathelana, i-GPI-AP idlula nge-Golgi apparatus (5, 9) ukusuka ku-ER ukuya e-plasma membrane. Ukuba khona kwama-anchor we-GPI kwenza bona i-GPI-AP ithuthwe ngokwehlukana namaphrotheyini akhiqizwe nge-transmembrane (okufaka hlangana amanye amaphrotheyini we-plasma membrane) endleleni yokukhipha (5, 9, 10). Emaseli wemvubelo, ama-GPI-AP ahlukaniswa kwamanye amaphrotheyini akhiqizwako ku-endoplasmic reticulum, bese afakwa abe mavesikili ahlukileko ahlanganiswe yi-coat protein complex II (COPII) (6, 7). Iinqunto zenqubo yokuhlukanisa enqubweni yokuthunyelwa kwe-ER azicaci, kodwana kuqagelwa bona indlela le ingatlhoga amafutha, khulukhulu ukulungiswa kabutjha kwesakhiwo sengcenye yamafutha we-GPI anchor (5, 8). Ku-yeast, ukuvuselelwa kwamafutha we-GPI kuthoma msinyana ngemva kobana i-GPI inamathele, begodu esikhathini esinengi, kubangela bona i-ceramide ibophelele ku-26-carbon long-chain saturated fatty acid (C26:0) (11, 12). I-C26 ceramide yi-ceramide ekulu ekhiqizwa maseli wemvubelo bekube nje. Yenziwa ku-ER begodu inengi layo lithunyelwa e-Golgi apparatus ngokusebenzisa ama-COPII vesicles (13). Ukuthunyelwa ngaphandle kwe-ER ye-GPI-AP kutlhoga ukwenziwa kwe-ceramide (14, 15), begodu, ukutjhugululwa kwe-ceramide ibe yi-inositol phosphate ceramide (IPC) ku-Golgi apparatus kuya ngokwenziwa kwe-GPI anchor (16). Iimfundo ze-biophysical nge-membrane yokwenziwa zitjengise bona ama-acyl chain ceramides amade khulu angahlangana enze ama-domain ahlelekileko anezinto ezihlukileko (17, 18). Idatha le irholela emcabangweni wokobana i-C26 ceramide kanye ne-GPI-AP ene-C26 ceramide isebenzisa izakhiwo zayo ezibonakalako ukuhlangana zibe ziindawo ezihlelekileko nofana iindawo ezisebujameni obuhlelekileko be-ER membrane lipid. Ibunjwa khulu ngama-glycerolipids amafitjhani nangakagcwaliswa (C16:1 no-C18:1) (19, 20). Iindawo lezi zizokukhethwa ngokukhethekileko eendaweni ezithileko zokuphuma kwe-ER (i-ERES), lapho i-ceramide ne-ceramide-based GPI-AP ingathuthwa khona ndawonye ukuya e-Golgi nge-COPII vesicle efanako (5).
Esifundweni lesi, sihlole ngqo indlela le esekelwe emafutheni ngokusebenzisa i-super-resolution confocal real-time imaging microscopy (SCLIM), okuyindlela yokuhlola ye-microscopy ephezulu engabona ngasikhathi sinye amaphrotheyini anelebula ngokukhanya.
Siqale ngokusebenzisa ithekhnoloji ye-SCLIM ukuragela phambili nokuhlathulula bona i-GPI-AP ejayelekileko enesiqhema se-C26 ceramide yahlolwa njani emaphrotheyini akhiqizwe nge-transmembrane ngemva kokutjhiya i-ER ku-S. cerevisiae. Ukuze sihlole ukuhlukaniswa kwe-ER, sisebenzise ihlelo lofuzo elingakghona ukubona ngokunqophileko imithwalo esanda kwenziwa ingena ku-ERES ku-vivo (7, 23). Njengomthwalo, sakhetha i-C26 ceramide-based GPI-AP Gas1 ebhalwe nge-green fluorescent protein (GFP) kanye ne-transmembrane secreted protein Mid2 ebhalwe nge-near-infrared fluorescent protein (iRFP), zombili ezihlose i-plasma membrane (24–26). Ku-sec31-1 eguquguqukako ezwela amazinga womtjhiso, imithwalo emibili le ivezwa ngaphasi komkhuthazi we-galactose kanye nomaka we-ERES. Emazingeni womtjhiso amakhulu (37°C), ngombana ukutjhuguluka kwe-sec31-1 kuthinta umsebenzi we-COPII coat component Sec31 ukuvimbela ukumila kwe-COPII nokuthunyelwa kwe-ER, umthwalo osanda kwenziwa ubuthelelana ku-ER (23). Ngemva kokupholisa ukuya emazingeni womtjhiso aphasi (24°C), amaseli aguqukileko we-sec31-1 alulama endaweni yokukhiqiza, begodu umthwalo omutjha wokwenziwa oqongelelweko wathoma ukuthunyelwa ngaphandle ukusuka e-ER. Ukubonwa kwe-CLIM kutjengise bona inengi le-Gas1-GFP esanda kwenziwa kanye ne-Mid2-iRFP isabuthelela ku-ER yamaseli we-sec31-1 ngemva kokufukamela ku-37°C bese ikhutjhwa ku-24°C imizuzu emi-5 (Umfanekiso 1). Njengombana i-Mid2-iRFP isabalaliswe kiyo yoke i-ER membrane, begodu i-Gas1-GFP ihlanganiswe begodu ibuthelelwe endaweni ye-ER membrane engaqhubekiko, ukusabalala kwayo kuhluke khulu (Umfanekiso 1, A ukuya ku-C kanye ne-Movie S1). Ukungezelela, njengombana kutjengiswe kuMfanekiso 1D, iqoqo le-Gas1-GFP alinayo i-Mid2-iRFP. Imiphumela le itjengisa bona i-GPI-AP namaphrotheyini we-transmembrane ahlukaniswe abe ziindawo ezihlukileko ze-ER membrane ekuthomeni. Iqoqo le-Gas1-GFP liseduze ne-ERES ethileko ebhalwe ngeprotheyini ye-mCherry's COPII coat Sec13 (Umfanekiso 1, E no-F, kanye nefilimu S1) (23).
amaseli we-sec31-1 aveza ukukhipha okubangelwa yi-galactose, iketango elide le-acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, oluhlaza) kanye nephrotheyini ye-transmembrane Mid2-iRFP (TMP, eluhlaza okwesibhakabhaka) begodu le Constructive ERES ukulebula i-Sec13-mCherry (E) yafakwa ku-CRES 30 imizuzu, yathuthelwa ku-24°C, begodu yathathwa yi-SCLIM ngemva kwemizuzu emi-5. (A ukuya ku-C) kutjengisa isithombe esihlanganisiweko namkha esisodwa se-2D sendiza (A), isithombe se-2D se-z-sections ezili-10 (B) namkha isithombe se-3D cell hemisphere ye-cargo nama-ERES markers (C). Ibha yesikali 1μm (A no-B). Iyunithi yesikali yi-0.551μm (C). I-Gas1-GFP yatholakala eendaweni ezihlukileko ze-ER nofana amaqoqo, ngesikhathi i-Mid2-iRFP yatholakala begodu yasabalaliswa kiyo yoke i-ER membrane (C). (D) Igrafu itjengisa ukukhanya okuhlobeneko kwe-Gas1-GFP ne-Mid2-iRFP esiqhemeni se-Gas1-GFP emgqeni womcibisholo omhlophe (kwesokunxele). I-AU, iyunithi engakhethiko. (E no-F) zijamele isithombe se-3D esihlanganisa ipahla netshwayo le-ERES. Amaqoqo we-Gas1-GFP atholakala eduze ne-ERES ethileko. Iyunithi yesikali ngu-0.551μm. (F) Umcibisholo omhlophe oqinileko uphawula iqoqo le-Gas1-GFP elihlobene ne-ERES. Amaphaneli aphakathi nakwesokudla atjengisa isithombe esikhulisiweko se-3D esihlanganisiweko kanye nombono ojikeleziweko weqoqo le-Gas1-GFP elikhethiweko.
Ubudlelwano obuseduze phakathi kweqoqo le-Gas1-GFP kanye ne-ERES ethileko butjengisa bona i-Gas1-GFP ingangena ku-ERES ekhethiweko, okuhlukileko ekukhetheni okusetjenziswa yi-Mid2-iRFP ukutjhiya i-ER. Ukutjheja lokhu okungenzeka, salinganisa isilinganiso se-ERES sempahla eyodwa nofana ezimbili kwaphela (Umfanekiso 2, A ukuya ku-C). Sithole bona ama-ERES amanengi (ama-70%) anomhlobo owodwa wemithwalo. Isithombe esingezansi somfanekiso 2C sitjengisa iimbonelo ezimbili ezijayelekileko ze-ERES ene-Gas1-GFP kwaphela (umfanekiso 1) nofana i-Mid2-iRFP kwaphela (umfanekiso 2). Ngokuphikisako, pheze ama-20% we-ERES anemithwalo emibili edlula endaweni yinye. Kwatholakala bona amanye ama-ERES (10%) anemihlobo emibili yemithwalo, kodwana bekahlukaniswe eendaweni ezihlukileko. Ngalokho-ke, ukuhlaziywa kwesibalo kutjengisa bona ngemva kokuthi i-ER ithunyelwe ngaphandle, i-GPI-AP Gas1-GFP kanye ne-transmembrane cargo Mid2-iRFP ihlukaniswe yaba ma-ERES ahlukileko (Umfanekiso 2D). Ukusebenza kuhle kokuhlunga kuhambisana khulu nokuhlaziywa kwamakhemikhali wezinto eziphilako kwangaphambilini (6) nokuzimisela kwesakhiwo (7). Singaphinde sibone ukuziphatha kwemithwalo evalelweko engena e-ERES (Umfanekiso 2E kanye ne-Movie S2). Umfanekiso 2E utjengisa bona yingcenye encani ye-Gas1-GFP (iphaneli 3) nofana i-Mid2-iRFP (iphaneli 4) engena ku-ERES ukusuka ngehlangothini linye begodu ivalelwe endaweni ehlukileko. Iphaneli 5 yomfanekiso 2E itjengisa bona i-Gas1-GFP ne-Mid2-iRFP kesinye isikhathi zitholakala ku-ERES efanako, kodwana zingena zivela emahlangothini ahlukileko begodu zigxile eendaweni ezihlukileko ezingajamela amavesikili ahlukileko we-COPII. Siphinde saqinisekisa bona ukuhlukaniswa nokuhlukaniswa kwe-C26 ceramide-based GPI-AP Gas1 njenge-ERES ekhethiweko kukhethekileko ngombana enye imithwalo yokukhipha i-transmembrane, i-GFP-tagged plasma membrane protein Axl2 (27 ), etjengisa ukuziphatha okufanako ne-Mid2-iRFP. (Isithombe S1 kanye nefilimu S3). I-Axl2-GFP esanda kwenziwa isabalaliswa nge-ER membrane njenge-Mid2-iRFP (Umfanekiso S1, A no-B), begodu ihlala ndawonye ne-Mid2-iRFP ku-ERES eminengi (Umfanekiso S1, B ukuya ku-D). Amaphaneli 1 no-2 womfanekiso 1. I-S1C itjengisa iimbonelo ezimbili ezijayelekileko ze-ERES lapho imithwalo emibili ye-transmembrane ihlangana khona. Kilokhu, zombili iimpahla zingena e-ERES ndawonye (Umfanekiso S1E, Iphaneli 3 kanye ne-Movie S3).
Amaseli we-sec31-1 aveza i-galactose inducible secretions, i-Gas1-GFP (GPI-AP, eluhlaza) kanye ne-Mid2-iRFP (TMP, eluhlaza) kanye ne-ERES ehlanganisa i-Sec13-mCherry (ERES, i-magenta) abekwa ku-37 Ngemva kokufukamela imizuzu ema-30 ku-°C 24, ukukhululwa ukuya ekuvimbeni isithombe, i-ion nge-SCLIM ngemva kwemizuzu ema-20. (A ukuya ku-C) Iinthombe ezijameleko ze-2D (A; ibha yesikali, 1μm) nofana iinthombe ze-3D ye-hemisphere yamaseli (B no-C; iyunithi yesikali, 0.456μm) yemithwalo kanye nama-z-sections ali-10 atshwaywe yi-ERES. Iphaneli engezansi ku-(B) nephaneli ku-(C) itjengisa iinthombe ezisetjenzisiweko ukutjengisa iimpahla ezikhona ku-ERES (i-magenta) [Gas1-GFP (grey) kanye ne-Mid2-iRFP (okukhanyako okuhlaza satjani)]. (C) Umcibisholo ovulekileko: I-ERES ithwala umthwalo munye kwaphela (1 ukuya ku-4). Umcibisholo ompunga: I-ERES imumethe imithwalo ehlukanisiweko (5). Umcibisholo oqinileko omhlophe: i-ERES equkethe imithwalo ebekwe ndawonye. Ngezansi: I-ERES eyodwa ekhethiweko ine-Gas1-GFP (1) nofana i-Mid2-iRFP (2) kwaphela. Ibha yesikali, 100 nm. (D) Ukulinganisa kwe-photomicrograph echazwe ku-(C). Iphesenti eliphakathi le-ERES equkethe umthwalo munye kwaphela (i-Gas1-GFP nofana i-Mid2-iRFP), umthwalo ohlukanisiweko kanye nomthwalo ohlanganako. Eemhlolweni ezintathu ezizijameleko, n=432 kumaseli ama-54. Ibha yephutha = SD. Ukuhlolwa kwe-t okungabhangqwanga okunemisila emibili. *** P = 0.0002. (E) Isithombe se-3D se-ERES ekhethiweko yemithwalo ehlukanisiweko ephawulwe ngo-(C). I-Gas1-GFP (eluhlaza satjani) (3) nofana i-Mid2-iRFP (eluhlaza satjani) (4) ingena e-ERES (e-magenta) ukusuka ngehlangothini linye begodu ikhawulelwe endaweni encani ngaphakathi kwe-ERES. Kwesinye isikhathi, yomibili imihlobo yemithwalo ingena ku-ERES efanako (5) ukusuka ngehlangothini elifanako begodu ivalelwe endaweni ehlukileko ngaphakathi kwe-ERES. Ibha yesikali, 100 nm.
Okulandelako, sihlole umbono wokobana i-acyl chain ceramide (C26) ekhona ku-ER membrane iqhuba ukuhlanganiswa nokuhlukaniswa kwe-Gas1 ibe yi-ERES ekhethiweko. Ukuze sifikelele lokhu, sisebenzise umhlobo wemvubelo otjhugululiweko i-GhLag1, lapho ama-endogenous ceramide synthases amabili i-Lag1 ne-Lac1 athathelwa indawo yi-GhLag1 (i-Lag1 homolog yekoteni), okurholele ekutheni kube nomhlobo wemvubelo onomhlobo we-Ceramide ye-cell membrane emfitjhani kune-wild type (Umfanekiso 3A) (28). Ukuhlaziywa kwe-mass spectrometry (MS) kutjengise bona emihlobeni ye-wild-type, i-95% ye-ceramide epheleleko yinde khulu (C26) i-ceramide, kuthi ku-GhLag1, i-85% ye-ceramide yinde khulu (C18 ne-C16). ), yi-2% kwaphela ye-ceramide ede khulu (C26) iketanga le-ceramide. Nanyana i-C18 ne-C16 ceramides zii-ceramide ezikulu ezitholwe ku-GhLag1 membrane bekube nje, ukuhlaziywa kwe-MS kuphinde kwaqinisekisa bona i-GPI anchor ye-Gas1-GFP evezwe emhlobeni we-GhLag1 ine-C26 ceramide, efana nama-lipids we-wild-type. Ikhwalithi iyafana (Umfanekiso 3A) (26). Ngalokho-ke, lokhu kutjho bona i-enzyme yokuvuselela i-ceramide i-Cwh43 ikhetha khulu i-C26 ceramide, njengombana kutjengiswe kuMfanekiso 26, ikhetha ukufaka isikhonkwane se-GPI esivela enanini elincani le-C26 ceramide ku-GhLag1 strain. S2 (29). Nanyana kunjalo, isikhumba seseli se-GhLag1 siqukethe i-C18-C16 ceramide kwaphela, lokha i-Gas1-GFP isene-C26 ceramide. Iqiniso leli lenza umhlobo lo ube lithulusi elihle lokurarulula umraro wobude beketango le-acyl le-membrane ceramide ku-ER. Indima ecatshangelwayo yeklasi nokuhlunga. Bese, sathoma ngokufunda ikghono le-C26 Gas1-GFP lokubuthelela ngamaqoqo ku-GhLag1 nge-allele ye-sec31-1 eguquguqukako ezwelako nge-conventional fluorescence microscopy, lapho kukhona iketango elide (i-C18-C16) ku-ER membrane Ceramide (Umfanekiso 3). Sitjheje bona ku-sec31-1, inengi le-Gas1-GFP belihlanganiswe ngamaqoqo, kuthi i-Gas1-GFP ku-sec31-1 GhLag1 ene-ceramide ER membrane ende (C18-C16) beyingakahlanganiswa begodu isabalaliswe kiyo yoke i-ER membrane. Ukutjho ngokunembileko, ngombana ukuhlanganiswa okusekelwe ku-C26 ceramide kuhlobana khulu ne-ERES ethileko (Umfanekiso 1), ngokulandelako saphenya bona ikambiso le ingafaka hlangana na nomsebenzi wendlela yokuthumela amaphrotheyini we-ER. I-GPI-AP isebenzisa ihlelo elikhethekileko le-COPII lokuthunyelwa kwe-ER, elilawulwa ngokusebenzako yi-Ted1′s structural remodeling ye-glycan portion ye-GPI anchor (30, 31). I-GPI-glycan ehlangeneko ibe seyibonwa yi-transmembrane cargo receptor p24 complex, eyokuthi ikhethe i-Lst1, okuyi-isoform ethileko ye-COPII cargo binding subunit ekulu i-Sec24, eyakha i-GPI-AP-rich COPII Vesicles ziyatlhogeka (31-33). Ngalokho-ke, sakha i-mutant ephindwe kabili ehlanganisa ukususwa kwamaphrotheyini la (i-p24 complex component Emp24, i-GPI-glycan remodeling enzyme Ted1 kanye ne-COPII subunit ekhethekileko Lst1) nge-sec31-1 mutant strain, begodu sazifunda Ingabe kungenzeka ukwakha i-Gas1-Gluster GFP (Umfanekiso 3). Sitjheje bona ku-sec31-1emp24Δ kanye ne-sec31-1ted1Δ, i-Gas1-GFP ayikahlanganiswa begodu isabalaliswe kiyo yoke i-ER membrane, njengombana bekubonwe ngaphambilini ku-sec31-1 GhLag1, kuthi ku-sec31-1lst1Δ, i-Gas1-GFP Ifana ne-sec31-1-1. Imiphumela le itjengisa bona ngaphezu kokuba khona kwe-C26 ceramide ku-ER membrane, ukuhlanganiswa kwe-Gas1-GFP nakho kutlhoga ukubophelela ku-p24 complex, begodu akutlhogi ukuqatjhwa kwe-Lst1. Bese, sihlolisisa ukuthi kungenzeka bona ubude beketango le-ceramide ku-ER membrane bungalawula ukubophelela kwe-Gas1-GFP ku-p24. Kodwana, sithole bona ukuba khona kwe-C18-C16 ceramide ngaphakathi kwe-membrane akuthinti i-GPI-glycans eyakhiwe kabutjha yi-p24 complex (Imifanekiso S3 no-S4, A no-B) nofana ukubophelela ku-GPI-AP nokuthumela i-GPI-AP. ikghono. Qasha i-COPII subtype Lst1 (Umfanekiso S4C). Ngalokho-ke, ukuhlanganiswa okuthembele ku-C26 ceramide akutlhogi ukusebenzisana kwamaphrotheyini neendlela ezihlukileko zokuthunyelwa kwamaphrotheyini we-ER, kodwana kusekela enye indlela yokuhlunga eqhutshwa bude be-lipid. Bese, sihlaziya bona ubude beketango le-ceramide acyl ku-ER membrane buqakathekile na ekuhlukaniseni okuphumelelako kwe-Gas1-GFP njenge-ERES ekhethiweko. Njengombana i-Gas1 ku-GhLag1 strain ene-short-chain ceramide itjhiya i-ER bese ingena ku-plasma membrane (Umfanekiso S5), sikholelwa bona nangabe ukuhlunga kuqhutshwa bude be-ceramide acyl chain, i-Gas1 ku-GhLag1 strain ingaqondiswa kabutjha begodu iwele. Iimpahla ze-ERES ezine-membrane efanako.
(A) I-cell membrane ye-GhLag1 ikakhulukazi iqukethe ama-C18-C16 ceramides amafitjhani, lokha i-GPI anchor ye-Gas1-GFP isene-C26 IPC efanako njengamaseli we-wild-type. Ngehla: ukuhlaziywa kobude beketango le-acyl le-ceramide ku-cell membrane ye-wild-type (Wt) kanye ne-GhLag1p strains nge-mass spectrometry (MS). Idatha imele iphesenteji ye-ceramide epheleleko. Isilinganiso sokuhlolwa okuzijameleko okuthathu. Ibha yephutha = SD. Ukuhlolwa kwe-t okungabhangqwanga okunemisila emibili. **** P ＜0.0001. Iphaneli engezansi: Ukuhlaziywa kwe-MS kobude be-acyl chain ye-IPC ekhona ku-Gas1-GFP (GPI-IPC) i-GPI anchor evezwe ku-wild-type kanye ne-GhLag1p strains. Idatha imele iphesenteji yesiginali ye-IPC epheleleko. Isilinganiso sokuhlolwa okuzijameleko okuhlanu. Ibha yephutha = SD. Ukuhlolwa kwe-t okungabhangqwanga okunemisila emibili. ns, akukaqakatheki. P = 0.9134. (B) Ama-micrographs we-fluorescence we-sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ kanye namaseli we-sec31-1lst1Δ aveza i-galactose-induced Gas1-GFP afukanywa ku-37°C imizuzu ema-30 bese enza bona kube ne-fluoresce isibonakhulu ngemva kwama-24°C. Umcibisholo omhlophe: iqoqo le-ER Gas1-GFP. Umcibisholo ovulekileko: I-Gas1-GFP engahlanganisiweko isabalaliswe kiyo yoke i-ER membrane, etjengisa ukugcotshwa kwendandatho yenyukliya ye-ER. Ibha yesikali, 5μm. (C) Ukulinganisa kwe-photomicrograph echazwe ku-(B). Iphesenteji ephakathi yamaseli anesakhiwo se-Gas1-GFP. Eemhlolweni ezintathu ezizijameleko, n≥300 amaseli. Ibha yephutha = SD. Ukuhlolwa kwe-t okungabhangqwanga okunemisila emibili. **** P ＜0.0001.
Ukurarulula umraro lo, senze ukubonwa kwe-SCLIM kwe-Gas1-GFP ne-Mid2-iRFP ku-GhLag1 nge-allele ye-sec31-1 etjhugulukako (Umfanekiso 4 kanye ne-Movie S4). Ngemva kokuthi i-ER igcinwe ku-37°C begodu ngemva kwalokho ikhutjhwe ku-24°C, inengi le-Gas1-GFP esanda kwenziwa azange ihlanganiswe begodu isabalaliswe kiyo yoke i-ER membrane, njengombana kubonwe ziinthombe ezijayelekileko (Umfanekiso 4, A no-B ). Ukungezelela, iphesenteji ekulu ye-ERES (67%) ifaka hlangana imihlobo emibili yemithwalo ebekwe ndawonye kiyo (Umfanekiso 4D). Amaphaneli 1 no-2 womfanekiso 4C atjengisa iimbonelo ezimbili ezijayelekileko ze-ERES ezine-Gas1-GFP ne-Mid2-GFP ezidlulako. Ukungezelela, zombili iimpahla zafakwa ku-ERES efanako (Umfanekiso 4E, iphaneli 3 kanye nefilimu S4). Ngalokho-ke, imiphumela yethu itjengisa bona ubude be-ceramide acyl chain ku-ER membrane buyinto eqakathekileko equnta ukuhlanganiswa kwamaphrotheyini we-ER nokuhlukaniswa.
Amaseli we-Sec31-1 GhLag1 aveza i-galactose-induced secretions, i-Gas1-GFP (GPI-AP, eluhlaza) kanye ne-Mid2-iRFP (TMP, eluhlaza) kanye ne-ERES-labeled Sec13-mCherry (ERES, magenta) Fukamela ku-37°C. Ragela phambili imizuzu ema-30, wehlela ku-24°C ukukhupha ukukhipha, bese uthatha isithombe nge-SCLIM ngemva kwemizuzu ema-20. (A ukuya ku-C) Iinthombe ezijameleko ze-2D (A; ibha yesikali, 1μm) nofana iinthombe ze-3D ye-hemisphere yamaseli (B no-C; iyunithi yesikali, 0.45μm) yeengaba ezili-10 ze-z ezitshwaywe nge-cargo ne-ERES. Iphaneli engezansi ku-(B) nephaneli ku-(C) itjengisa iinthombe ezisetjenzisiweko ukutjengisa iimpahla ezikhona ku-ERES (i-magenta) [Gas1-GFP (grey) kanye ne-Mid2-iRFP (okukhanyako okuhlaza satjani)]. (C) Umcibisholo omhlophe ogcwele: ERES, ukuhlangana kwempahla. Vula umcibisholo: I-ERES inento yinye kwaphela. Iphaneli engezansi: I-ERES ekhethiweko inempahla ezidlulako (1 no-2) ezitshwaywe ku-(C). Ibha yesikali, 100 nm. (D) Ukulinganisa kwe-photomicrograph echazwe ku-(C). Emayunithini we-sec31-1 kanye ne-sec31-1 GhLag1, kufakiwe umthwalo munye kwaphela (i-Gas1-GFP nofana i-Mid2-iRFP), kanye nephesenteji ephakathi ye-ERES yemithwalo ehlukileko nemithwalo edlulako. Eemhlolweni ezintathu ezizijameleko, n = 432 kumaseli ama-54 (sec31-1) kanye n = 430 kumaseli ama-47 (sec31-1 GhLag1). Ibha yephutha = SD. Ukuhlolwa kwe-t okungabhangqwanga okunemisila emibili. *** P = 0.0002 (sec31-1) kanye ** P = 0.0031 (sec31-1 GhLag1). (E) Isithombe se-3D se-ERES ekhethiweko enemithwalo edlulako (3) etshwaywe ku-(C). I-Gas1-GFP (eluhlaza satjani) kanye ne-Mid2-iRFP (eluhlaza satjani) itjhidela ku-ERES (e-magenta) ukusuka ngehlangothini elifanako begodu ihlale endaweni efanako ye-ERES evinjelwe. Ibha yesikali, 100 nm.
Irhubhululo leli linikela ubufakazi obuqondileko be-vivo bokuthi imithwalo yamaphrotheyini asekelwe emafutheni ihlukaniswe yaba ziindawo ezikhethiweko zokuthunyelwa ngaphandle endleleni yokukhipha, begodu iveza ukuqakatheka kobude beketango le-acyl ekukhetheni ukuhlukaniswa. Ngokusebenzisa indlela enamandla nesezingeni le-microscopy ebizwa nge-SCLIM, sitjengise i-Gas1-GFP esanda kwenziwa (i-plasma membrane ekulu ye-GPI-AP ene-acyl chain ende khulu (C26) ceramide lipid portion) ku-yeast) Iindawo ezihlanganiswe ngama-ER ahlukileko zihlotjaniswa ne-ERRES ethileko, lokha amaphrotheyini we-ERRES asabalaliswe nge-membrane (Umfanekiso 1). Ukungezelela, imihlobo emibili yepahla le ingena ku-ERES ehlukileko ngokukhetha (Umfanekiso 2). Ubude beketango le-acyl le-ceramide yamaseli ku-membrane buyehliswa ukusuka ku-C26 ukuya ku-C18-C16, iqoqo le-Gas1-GFP liphazanyiswa liye esifundeni se-ER esihlukileko, begodu i-Gas1-GFP itjhugululwa ukutjhiya i-ER nephrotheyini ye-transmembrane nge-ERES efanako (Umfanekiso 3 noMfanekiso 3). 4).
Nanyana i-GPI-AP isebenzisa indlela ekhethekileko yamaphrotheyini ukuphuma ku-ER, sithole bona ukuhlukaniswa okuthembele ku-C26 ceramide akuthembeli ekusebenzisaneni kwamaphrotheyini ahlukileko okungaholela ekukhetheni i-ERES (Imifanekiso S4 no-S5). Esikhundleni salokho, imiphumela yethu isekela enye indlela yokuhlukanisa eqhutshwa yi-lipid-based protein clustering kanye nokukhishwa kweminye imithwalo. Okutjhejiweko kwethu kutjengisa bona isifunda se-Gas1-GFP nofana isiqhema esihlobene ne-ERES ethileko asinawo amaphrotheyini akhiqizwa yi-transmembrane i-Mid2-iRFP, okutjengisa bona isiqhema se-GPI-AP esithembele ku-C26 ceramide sizokwenza lula ukungena kwabo ku-ERES efaneleko, begodu ngesikhathi esifanako, singafaki i-transmembrane. Ngokuphikisako, ukuba khona kwama-C18-C16 ceramides ku-ER membrane akubanga bona i-GPI-AP yenze iindawo nofana amaqoqo, njeke ayikhiphi nofana ithathe indawo yamaphrotheyini akhiqizwe yi-transmembrane ku-ERES efanako (Imifanekiso 3 no-4). . Ngalokho-ke, siphakamisa bona i-C26 ceramide iqhuba ukuhlukaniswa nokuhlukaniswa ngokusiza ukuhlanganiswa kwamaphrotheyini ahlotjaniswa ne-ERES ethileko.
Ungakwenza njani ukuhlanganiswa okuthembele ku-C26 endaweni ethileko ye-ER? Ukuthambekela kwe-membrane ceramide ukuhlukana ngapha nangapha kungabanga bona i-GPI-AP ne-C26 ceramide yenze ama-lipids amancani ahlelekileko msinyana endaweni engakajayeleki yamafutha we-ER membrane enama-glycerolipids amafitjhani begodu angagcwali. Amaqoqo wekhwalithi (17, 18). Amaqoqo amancani wesikhatjhana angahlanganiswa abe maqoqo amakhulu, azinzileko ngemva kokubophelela ku-p24 (34). Ngokuvumelana nalokhu, sitjengise bona i-C26 Gas1-GFP idinga ukusebenzisana ne-p24 ukwakha amaqoqo amakhulu abonakalako (Umfanekiso 3). I-p24 complex yi-heterozygous oligomer eyakhiwe ngamaphrotheyini amane ahlukileko we-p24 transmembrane ku-yeast (35), okunikela ukubopha okuhlukahlukeneko, okungaholela ekuhlanganisweni kwamaqoqo amancani we-GPI-AP, ngaleyo ndlela kukhiqize iqoqo elikhulu elizinzileko (34). Ukusebenzisana hlangana namaphrotheyini we-ectodomains we-GPI-APs nakho kungaba nomthelela ekuhlanganisweni kwawo, njengombana kutjengisiwe ngesikhathi sokuthuthwa kwawo kwe-Golgi kumaseli we-epithelial ahlukanisiweko weenlwana ezimunyisako (36). Kodwana, lokha i-C18-C16 ceramide nayikhona ku-ER membrane, lokha i-p24 complex nayibophelela ku-Gas1-GFP, amaqoqo amakhulu ahlukileko angeze akhiwa. Indlela yokwenza izinto ingathembela eemfanelweni ezithileko ze-acyl chain ceramide. Iimfundo ze-biophysical zama-membrane wokufakelwa zitjengisa bona nanyana zombili ama-acyl chain ceramides amade (C24) namafitjhani (C18-C16) angabangela ukuhlukaniswa kwesigaba, ama-acyl chain ceramides amade (C24) kwaphela angakhuthaza ukugoba okuphezulu nokugoba kwefilimu ukubumba kabutjha ifilimu. Ngokusebenzisana (17, 37, 38). Kutjengiswe bona i-transmembrane helix ye-TMED2, i-homologue yabantu ye-Emp24, isebenzisana ngokukhethekileko ne-C18 ceramide-based sphingomyelin kuma-cytoplasmic lobules (39). Ngokusebenzisa ukulingiswa kwe-molecular dynamics (MD), sithole bona zombili i-C18 ne-C26 ceramides zibuthelelana eduze kwama-cytoplasmic lobules we-Emp24 transmembrane helix, begodu zinokukhetha okufanako (Umfanekiso S6). Kuqakathekile ukutjheja bona lokhu kutjengisa bona i-transmembrane helix ye-Emp24 ingadosela ekusabalalisweni okungalinganiko kwamafutha ngaphakathi kwe-membrane. Lo ngumphumela wakamuva osekelwe kumaseli weenlwana ezimunyisako. Ukulingiswa okufanako kwe-MD nakho kutjengisa ubukhona bamafutha we-ether (40) . Ngalokho-ke, siqagela bona i-C26 ceramide ema-lobules amabili we-ER26 inothiswe endaweni. Lokha i-GPI-AP ema-lobules we-luminal ibophelela ngqo ku-multivalent p24 kanye nokubuthelela kwe-C26 ceramide eduze ne-p24 ema-lobules we-cytoplasmic, kungakhuthaza ukuhlanganiswa kwamaphrotheyini okukhambisanako nokugoba kwe-membrane kwenziwa ngeminwe (41), okwenza bona i-GPI-AP ihlukane ibe ziindawo ezihlukileko eziseduze ne-ERES, nazo zithanda iindawo eziphezulu ze-RES ulwelwesi lwe-ER (42). Imibiko yangaphambilini isekele indlela ehlongozwayo (43, 44). Ukubophelela okuhlukahlukeneko kwama-oligolectins, ama-pathogens nofana ama-antibodies kuma-glycosphingolipids (GSL) asekelwe ku-ceramide ku-plasma membrane kubangela ukuhlanganiswa okukhulu kwe-GSL, kuthuthukisa ukuhlukaniswa kwesigaba begodu kubangela ukuguquguquka kwe-membrane nokufaka ngaphakathi (44). Iwabuchi njll. (43) Kwatholakala bona ebukhoneni bama-acyl chains amade (C24) kodwana angasi amafitjhani (C16), i-multivalent ligand ebotjhelelwe ku-GSL lactosylceramide yenza ukwakheka kwamaqoqo amakhulu nokungena kwe-membrane, begodu ukudluliswa kwesiginali ye-cytoplasm Lyn-mediated emaphepheni kwenziwa ngama-cytoplasm neutrophils ngama-couple.
Emaseli we-epithelial ahlukanisiweko weenlwana ezimunyisako, ukugxila kwe-anti-Golgi network (TGN) ukuya ezingeni le-apical plasma membrane kulawula ukuhlukaniswa nokuhlunga kwe-GPI-AP (10, 45). Ukuhlanganiswa lokhu kuqhutshwa yi-GPI-AP oligomerization (36), kodwana kungabuye kuya ngobude beketango le-ceramide esilifumana ku-yeast. Nanyana i-GPI-AP yeenlwana ezimunyisako ine-ether lipid-based anchor, begodu isakhiwo sayo samakhemikhali sihluke khulu ku-acyl chain ceramide ede khulu, irhubhululo lakamuva lithole bona womabili amafutha anezinto ezifanako zomzimba namakhemikhali nomsebenzi (40). Ngalokho-ke, ingcenye ye-ether lipid kumaseli weenlwana ezimunyisako ingafana ne-C26 ceramide ku-yeast, begodu indima yayo kukuhlobana ne-long-chain ceramide ku-membrane ukukhuthaza ukuhlanganiswa kwe-GPI-AP nokuhlunga. Nanyana lokhu kungenzeka kusatlhogeka bona kuhlolwe ngqo, okutholwe ngaphambilini kusekela bona ukuthuthwa kwe-acyl chain ceramide ede ukuya emzimbeni we-Golgi akukwenziwa ngamaphrotheyini wokudlulisa we-cytoplasmic, kodwana kuya ngokuhlanganiswa kwama-anchor we-GPI afana nemvubelo. Ngalokho-ke, indlela yokuziphendukela kwemvelo ibonakala ikghona ukuthutha ngokukhetha i-acyl chain ceramide ede khulu ne-GPI-AP (13, 16, 20, 46, 47) nge-vesicle efanako yokuthutha.
Eenhlelweni zamaseli we-yeast nezilwane ezimunyisako, ukuhlanganiswa kwe-GPI-AP nokuhlukaniswa kwamanye amaphrotheyini we-plasma membrane koke kwenzeka ngaphambi kokufikelela ebusweni beseli. U-Paladino nabanye. (48) wathola bona ku-TGN yamaseli we-epithelial ahlanganisiweko weenlwana ezimunyisako, ukuhlanganiswa kwe-GPI-AP akutlhogeki kwaphela ekuhlukaniseni ama-GPI-AP ukuya ku-apical plasma membrane, kodwana kulawula nokuhlelwa kwe-GPI-APs nomsebenzi wayo wezinto eziphilako. Ubuso beseli. Kumvubelo, irhubhululo leli litjengise bona i-C26 ceramide-dependent GPI-AP cluster ku-ER ingalawula ihlelo le-cluster nomsebenzi osebenzako we-GPI-AP ku-plasma membrane (24, 49). Ngokuvumelana nale modeli, amaseli we-GhLag1 ayagula kuma-GPI inhibitors nofana imithi ethinta ubuqotho bodonga lwamaseli (28), begodu isidingo samaqoqo we-Gas1-GFP asebenzako (49) we-tip ceramide ekhonjiswa ekuhlanganeni kwamaseli we-yeast kutjengisa i-G Imiphumela yomzimba engaba khona yamaseli we-hLag1. Iphutha le-GPI-AP. Kodwana, ukuhlola okungeziweko bona ukuhleleka okusebenzako kwephasi leseli kuhlelwe ukusuka ku-ER ngendlela yokuhlunga ngokuya ngobude bamafutha kuzokuba yindaba yokurhubhulula kwethu kwesikhathi esizako.
Iinhlobo ze-Saccharomyces cerevisiae ezisetjenziswe emsebenzini lo zifakwe kuThebuli S1. I-MMY1583 kanye ne-MMY1635 imihlobo ye-SCLIM yokuthatha iinthombe zamaseli aphilako yakhiwe ngemva kwe-W303. Iinhlobo eziveza i-Sec13-mCherry enethegi yamaphrotheyini akhanyako zakhiwe kusetjenziswa indlela esekelwe ku-polymerase chain reaction (PCR) ene-pFA6a plasmid njengethempulethi (23). Umhlobo oveza i-Mid2-iRFP ebhalwe ngephrotheyini ekhanyako ngaphasi kokulawula komkhuthazi we-GAL1 wakhiwe ngalendlela elandelako. Ukukhuliswa kwe-PCR kwe-iRFP-KanMx evela ku-pKTiRFP-KAN vector (isipho saka-E. O'Shea, inomboro yeplasmidi ye-Addgene 64687; http://n2t.net/addgene: 64687; isihlonzi semithombo yokurhubhulula (RRID): Addgene_64687) Begodu ifakwe ngaphakathi kwe-Cend end-endous ye-Mid. Ngemva kokuthi ukulandelana kwe-genome ye-Mid2-iRFP kwakhuliswa begodu kwafakwa ku-GAL1 promoter, yahlanganiswa endaweni ye-Not I-Sac I ye-plasmid yokuhlanganiswa pRS306. I-plasmid pRGS7 ephumelako yahlanganiswa ne-Pst I ukuhlanganiswa ne-URA3 locus.
I-Gas1-GFP fusion gene ivezwa ngaphasi kokulawula kwe-GAL1 promoter ku-centromere (CEN) plasmid, eyakhiwe ngalendlela elandelako. Ukulandelana kwe-Gas1-GFP kwakhuliswa nge-PCR ukusuka ku-pRS416-GAS1-GFP plasmid (24) (isipho saka-L. Popolo) begodu yakhiwe endaweni ye-Xma I–Xho I ye-CEN plasmid pBEVY-GL LEU2 (isipho saka-C). U-Miller; Inomboro ye-addgene plasmid 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Iplasmidi ephumelako yabizwa ngokuthi yi-pRGS6. I-Axl2-GFP fusion gene nayo ivezwa ngaphasi kokulawula kwe-GAL1 promoter ye-pBEVY-GL LEU2 vector, begodu ukwakhiwa kwayo kunje. Ukulandelana kwe-Axl2-GFP kwakhuliswa ukusuka ku-pRS304-p2HSE-Axl2-GFP plasmid (23) nge-PCR, begodu yakhiwe endaweni ye-Bam HI-Pst I ye-pBEVY-GL LEU2 vector. Iplasmidi ephumelako yabizwa ngokuthi yi-pRGS12. Ukulandelana kwama-oligonucleotide asetjenziswe esifundweni lesi kufakwe kuThebuli S2.
Umhlobo lo wangezelelwa nge-0.2% ye-adenine kanye ne-2% ye-glucose [YP-dextrose (YPD)], i-2% ye-raffinose [YP-raffinose] ecebileko ye-yeast extract protein p (YP) medium (1 % ye-yeast extract kanye ne-2% ye-protein ept). (YPR)] nofana i-2% galactose [YP-galactose (YPG)] njengomthombo wekhabhoni, nofana nge-synthetic minimal medium (0.15% ye-yeast nitrogen base kanye ne-0.5% ammonium sulfate) ukungezelela ama-amino acid afaneleko kanye nama-base atlhogekako ekudleni, begodu ine-2% ye-glucose (2% glucose glucose) nofana i-minimum ye-glucose i-galactose (i-galactose encani yokwenziwa) njengomthombo wekhabhoni.
Ukuthatha iinthombe ngesikhathi sangempela, amaseli we-sec31-1 atjhugulukako aveza ukwakhiwa ngaphasi komkhuthazi we-GAL1 akhuliswa ngaphakathi kwe-YPR ku-24°C ubusuku boke ukuya esigabeni se-log esiphakathi. Ngemva kokufakwa ku-YPG ku-24°C i-awa elilodwa, amaseli afukanywa ku-SG ku-37°C imizuzu ema-30, bese adluliselwa ku-24°C ukukhululwa ebhlokini lokukhipha. I-Concanavalin A yasetjenziselwa ukulungisa amaseli esilayidini seglasi begodu yathathwa nge-SCLIM. I-SCLIM yihlanganisela ye-Olympus IX-71 inverted fluorescence microscope kanye ne-UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), isikena se-disc confocal esijikelezako esinejubane elikhulu nesine-signal-to-noise ratio ephezulu (Yokogawa Electric), i-spectrometer eyenziwe ngokwezifiso, i-thetomiter ye-intensive systems (Hamamatsu Photonics) inganikela ngendlela yokukhulisa ilensi ngokukhulisa kokugcina okungu-×266.7 kanye nekhamera yedivayisi ehlanganisiwe ephindaphinda ama-elekthronikhi (Hamamatsu Photonics) (21). Ukutholwa kweenthombe kwenziwa ngesofthiwe eyenziwe ngokwezifiso (i-Yokogawa Electric). Ngeenthombe ze-3D, sisebenzise i-piezoelectric actuator eyenziwe ngokwezifiso ukudlidliza ilensi yomnqopho ngokujame phezulu, begodu sabuthelela iingcenye ezibonakalako ziqhelelene nge-100 nm esitokisini. Isithombe se-Z-stack sitjhugululwa sibe yidatha ye-3D voxel, begodu umsebenzi wokusakazeka kwephuzu elisetjenziselwa i-rotating disc confocal microscope usetjenziselwa ukucubungula i-deconvolution ngesofthiwe ye-Volocity (PerkinElmer). Ngokusebenzisa isofthiwe ye-Volocity ukubeka umkhawulo wokuhlaziywa kwendawo, i-ERES efaka hlangana imithwalo yalinganiswa. Ukuhlaziywa kwelayini kwenziwa kusetjenziswa i-MetaMorph software (Molecular Devices).
Sebenzisa isofthiwe ye-GraphPad Prism ukuthola ukuqakatheka kwezibalo. Nge-t-test ye-Student enemisila emibili kanye ne-ordinary one-way analysis of variance (ANOVA) test, umehluko phakathi kwamaqembu uthathwa njengomthelela omkhulu ku-P <0.05 (*).
Ukuze uthole i-fluorescence microscopy ye-Gas1-GFP, amaseli we-log phase akhuliswa ubusuku boke ku-YPD begodu aqoqwa nge-centrifugation, ahlanzwa kabili nge-phosphate buffered saline, begodu afakwa eqhweni okungenani imizuzu eli-15, bese aragela phambili ngaphasi kwe-microscope njengombana kuchaziwe ngaphambilini Hlola (24). I-Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ene-lens ye-objective, isihlungi se-L5 (GFP), ikhamera ye-Hamamatsu kanye nesofthiwe ye-Application Suite X (LAS X) yasetjenziselwa ukuthola. .
Amasampula atjhugululwa nge-SDS isampula bufa ku-65°C imizuzu eli-10, bese ahlukaniswa nge-SDS-polyacrylamide gel electrophoresis (PAGE). Ukuhlaziywa kwe-immunoblotting, i-10 μl yesampula yafakwa emzileni ngamunye. I-antibody yokuthoma: Sebenzisa i-rabbit polyclonal anti-Gas1 nge-dilution ye-1:3000, i-rabbit polyclonal anti-Emp24 nge-dilution ye-1:500, kanye ne-rabbit polyclonal anti-GFP (isipho esivela ku-H. Riezman) nge-dilution ye-1:3000. I-mouse monoclonal anti-Pgk1 antibody yasetjenziswa nge-dilution ye-1:5000 (isipho esivela ku-J. de la Cruz). I-antibody yesibili: I-horseradish peroxidase (HRP) ehlanganiswe nembuzi elwa nomvundla i-immunoglobulin G (IgG) esetjenziswe ngokuhlanjululwa okungu-1:3000 (u-Pierce). I-HRP-conjugated goat anti-mouse IgG yasetjenziswa ngokunciphisa okungu-1:3000 (u-Pierce). Indawo yokuphendula kwamasotja womzimba yabonwa ngendlela ye-chemiluminescence ye-SuperSignal West Pico reagent (Thermo Fisher Scientific).
Njengombana kuchaziwe ku-(31), ukuhlolwa kwe-immunoprecipitation kwemvelo kwenziwa engcenyeni ye-ER enothileko. Ngamafuphi, hlamba amaseli wemvubelo nge-TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kanye nomxube we-protease inhibitor) ku-600 nm ( OD600) ku-100 optical density kabili. Yaphulwa ngobuhlalu beglasi, bese kuthi iimvuthuluka zamaseli kanye nobuhlalu beglasi kwasuswa ngokusebenzisa i-centrifugation. I-pernatant yase i-centrifuged ku-17,000 g imizuzu eli-15 ku-4°C. I-pellet yabuyiselwa ku-TNE begodu i-digitalis saponin yengezwa yaba sezingeni lokugcina le-1%. Ukumiswa kwafukamela i-awa elilodwa ngokujikeleziswa ku-4°C, bese kuthi iingcenye ezingancibilikiko zisuswe ngokutjhinga ku-13,000 g ku-4°C imizuzu ema-60. Ukwenza i-Gas1-GFP immunoprecipitation, qala ngaphambilini ukufukamela isampula ngama-agarose beads angenalitho (ChromoTek) ku-4°C i-awa eli-1, bese ufukamela nge-GFP-Trap_A (ChromoTek) ku-4°C ama-awa ama-3. Ama-immunoprecipitated beads ahlanjwa amahlandla amahlanu nge-TNE ene-0.2% ye-digoxigenin, ahlanjululwe nge-SDS sample buffer, ahlukaniswe ku-SDS-PAGE, begodu ahlaziywa nge-immunoblotting.
Njengombana kuchaziwe ku-(31), ukuzimisela kokuhlanganisa kwenziwa engcenyeni ye-ER enothileko. Kafitjhani, ingcenye ye-ER enothileko yafukanywa nge-0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Ukusabela kokuhlanganisa kwacinywa ngokungezelela i-glycine (50 mM ukugxila kokugcina, imizuzu emi-5, 20°C).
Njengombana kuchaziwe ngaphambilini (50), ukuhlaziywa kwe-MS kwe-ceramide emhlobeni we-wild-type kanye ne-GhLag1 kwenziwa. Ngamafuphi, amaseli akhuliswa abe sesigabeni esikhulu (3 ukuya ku-4 OD600 amayunithi/ml) ku-YPD ku-30°C, begodu amaseli ama-25×107 avunwa. Ukugaya ukudla kwazo kucinywa nge-trichloroacetic acid. Sebenzisa isisombululo sokukhipha [i-ethanol, amanzi, i-ether, i-pyridine kanye ne-4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] kanye ne-1.2 nmol yekhwalithi yangaphakathi ye-C17 ceramide (860517, Avanti polar lipid)). Sebenzisa i-monomethylamine reagent [methanol, amanzi, n-butanol kanye nesisombululo se-methylamine (4:3:1:5 v/v)] ukwenza i-mild alkaline hydrolysis ye-extract, bese usebenzisa i-n-butanol egcwele amanzi ukususa itswayi. Ekugcineni, i-extract yaphinde yamiswa ku-positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] bese yafakwa ngaphakathi kwe-mass spectrometer. Ukutjheja ukusabela okunengi (i-MRM) kwenziwa ukukhomba nokulinganisa amamolekhuli we-sphingolipid. I-TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ifakwe irobhothi ye-nanoflow ion source i-Nanomate HD (Advion Biosciences, Ithaca, NY) ukuhlaziywa kwamafutha. Amandla wokutjhayisana alungiselelwe isigaba ngasinye se-ceramide. Idatha ye-MS itholwe ngendlela ehle. Ekuphindaphindweni ngalinye kwezinto eziphilako, itshwayo le-lipid liphakathi kweenlinganiso ezintathu ezizijameleko.
Njengombana kuchaziwe ku-(31), amaseli (800×107) aveza i-Gas1-GFP abekwa ngaphasi kwe-immunoprecipitation yemvelo. I-Gas1-GFP ehlanjululweko yahlukaniswa nge-SDS-PAGE begodu yadluliselwa ku-polyvinylidene fluoride (PVDF) membrane. Iphrotheyini yabonwa ngokufakwa i-PVDF nge-amide emnyama. Ibhendi ye-Gas1-GFP yasikwa ku-PVDF begodu yahlanzwa amahlandla amahlanu nge-methanol begodu kanye ngamanzi wezinga le-liquid chromatography-MS (LC-MS). Ngokufukamela umtjhini we-membrane nge-500μl 0.3 M NaOAc (pH 4.0), i-buffer kanye ne-500μl ye-1 M ye-sodium nitrite ehlanganisiweko ku-37°C ama-awa ama-3, ingcenye ye-lipid ikhutjhwa ku-Gas1-GFP begodu ikhutjhwe i-lysed Release of in-in-inphosophamiophosemide ku-ceraamine-gluecode (51). Ngemva kwalokho, umtjhini we-membrane wahlanzwa kane ngamanzi wezinga le-LC-MS, womiswe emazingeni womtjhiso wegumbi, begodu wagcinwa emmoyeni we-nitrogen ku--80°C bekwaba kulapho kuhlaziywa khona. Njengokulawula, isampula enganalitho ye-PVDF membrane yasetjenziselwa ukuhlolwa ngalinye. I-lipid ekhutjhwe ku-Gas1-GFP yahlaziywa yi-MS njengombana kuchaziwe (50). Ngamafuphi, imitjhini ye-PVDF ene-GPI-lipid yabuyiselwa ku-75μl ye-negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] begodu yadlula i-electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Kulokhu, idatha ye-MS itholwe ngendlela ye-ion embi.
Njengombana kutjhiwo ngaphambilini, ingcenye ye-lipid ye-GPI anchor yahlukaniswa ne-[3H]-inositol-labeled GPI-AP (16). Amafutha ahlukaniswe nge-chromatography ye-thin-layer kusetjenziswa isistimu ye-solvent (55:45:10 chloroform-methanol-0.25% KCl) begodu abonwa kusetjenziswa i-FLA-7000 (Fujifilm).
Amaseli aveza i-Gas1-GFP (600×107) ahlanzwa kabili nge-TNE buffer ene-TNE buffer, begodu aphulwa ngama-glass beads, bese atjhugululwa ukususa iimvuthuluka zamaseli nama-glass beads. I-pernatant yase i-centrifuged ku-17,000 g nge-awa elilodwa ku-4°C. I-pellet yahlanzwa ku-TNE begodu yafukanywa nge-1 U PI-PLC (Invitrogen) ku-TNE ene-0.2% ye-digitalis saponin i-awa yi-1 ku-37°C. Ngemva kokwelatjhwa nge-enzayimu, ulwelwesi lwasuswa ngokutjhinga nge-17,000 g ku-4°C nge-awa elilodwa. Ukuvikela i-Gas1-GFP, i-supernatant yafukanywa nge-GFP-Trap_A (ChromoTek) ku-4°C ubusuku boke. I-Gas1-GFP ehlanjululweko ehlukaniswe yi-SDS-PAGE yafakwa ibala nge-Coomassie eluhlaza okwesibhakabhaka. I-Gas1-GFP staining band yanqunywa ku-grey ezungeze umsele wamanzi, bese kuthi ngemva koku-alkylation nge-iodoacetamide nokunciphisa nge-dithiothreitol, ukugaya ngaphakathi kwe-gel nge-trypsin kwenziwa. Khipha begodu womise ama-tryptic peptides nama-peptides nge-GPI-glycans. I-peptide eyomileko yancibilikiswa ngaphakathi kwama-20 μl wamanzi. Hlaba ingcenye (8μl) ngaphakathi kwe-LC. Ikholomu ye-octadecylsilane (ODS) (Develosil 300ODS-HG-5; ububanzi bangaphakathi 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) yasetjenziselwa ukuhlukanisa ama-peptide ngaphasi kweemeko ezithileko ze-gradient. Isigaba esihambako yi-solvent A (0.08% ye-formic acid) kanye ne-solvent B (0.15% ye-formic acid ku-80% ye-acetonitrile). Ihlelo le-Accela HPLC (i-Thermo Fisher Scientific, Boston, Massachusetts) lasetjenziselwa ukuhlanza ikholomu nge-solvent A kungakapheli imizuzu ema-55 ngezinga lokugeleza elingu-50 μl min-1 imizuzu emi-5, bese kuthi ukugxila kwe-solvent B kwanyuswa kwaba ngu-40%. , I-United States). I-eluate beyifakwa ngokuragela phambili emthonjeni we-ESI ion, begodu ama-tryptic peptides nama-peptides ane-GPI-glycans ahlaziywa yi-LTQ Orbitrap XL (i-hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Ekuhlelweni kwe-MS, ivoltheji yomthombo we-capillary yabekwa ku-4.5 kV, begodu amazinga womtjhiso we-capillary yokudlulisa agcinwa ku-300°C. I-capillary voltage kanye ne-tube lens voltage zabekwa ku-15 V no-50 V, ngokulandelana. Idatha ye-MS itholwe ngendlela ye-ayoni ehle (i-resolution ye-60,000; ukunemba kwesisindo seengcenye ezili-10 ngesigidi) ngebanga le-mass le-300/m/z mass/charge ratio (m/z) 3000. Idatha ye-MS/MS itholwa nge-ion trap ku-LTQ Orbit [XL, idatha yokuthoma ukutjhayisana okubangelwa kuhlukana (CID)].
Ukulingiswa kwe-MD kwenziwa kusetjenziswa isofthiwe ye-GROMACS (52) kanye ne-MARTINI 2 force field (53-55). I-CHARMM GUI Membrane Builder (56, 57) yasetjenziswa ukwakha i-bilayer ene-dioleoylphosphatidylcholine (DOPC) kanye ne-Cer C18 nofana i-DOPC ne-Cer C26. I-topology kanye nama-coordinates we-Cer C26 atholakala ku-DXCE ngokususa ubuhlalu obungezelelweko emsileni we-sphingosine. Sebenzisa ikambiso echazwe ngenzasi ukulinganisa isendlalelo esiphindwe kabili bese uyisebenzisa, bese usebenzisa amakhodi wokugcina wesistimu ukwakha isistimu ene-Emp24. I-transmembrane domain ye-yeast Emp24 (iinsalela 173 ukuya ku-193) yakhiwa njenge-α-helix kusetjenziswa i-visual MD (VMD) tool molecular structure (58). Bese, ngemva kokususa amafutha ahlangeneko, iphrotheyini yaba yi-coarse granulated bese ifakwa ngaphakathi kwe-bilayer kusetjenziswa i-CHARMM GUI. Irherho lokugcina liqukethe i-1202 DOPC kanye ne-302 Cer C26 nofana i-1197 DOPC kanye ne-295 Cer C18 ne-Emp24. Yenza i-ayoni ihlelo libe sezingeni elingu-0.150M. Ukuphindaphinda okune okuzijameleko kwenziwa ngeenhlanganisela ezimbili.
I-lipid bilayer ilinganiswa kusetjenziswa ikambiso ye-CHARMM GUI, ebandakanya ukunciphisa bese kulinganiselwa amagadango ama-405,000, lapho imikhawulo yendawo yehliswa kancanikancani begodu iqedwe, begodu igadango lesikhathi likhuphuka ukusuka ku-0.005 ps ukuya ku-0.02 ps. Ngemva kokulinganisa, ikhiqiza i-6 μs ngesinyathelo sesikhathi se-0.02 ps. Ngemva kokufaka i-Emp24, sebenzisa ikambiso efanako ye-CHARMM GUI ukunciphisa nokulinganisa ihlelo, bese ugijima ama-8 s ekukhiqizeni.
Kiwo woke amahlelo, ngesikhathi senqubo yokulinganisa, umfutho ulawulwa yi-Berendsen barostat (59), begodu ngesikhathi senqubo yokukhiqiza, umfutho ulawulwa yi-Parrinello-Rahman barostat (60). Kizo zoke iimeko, umtjhini ophakathi nendawo ngu-1 bar begodu kusetjenziswa isikimu sokuhlanganisa umtjhini ongakajayeleki. Ehlelweni lokulinganisa nokukhiqiza, i-thermostat (61) ene-speed recalibration isetjenziselwa ukuhlanganisa izinga lokutjhisa lamaprotheyini, amafutha kanye nama-solvent particles ngokulandelana. Ngesikhathi sokusebenza koke, izinga lokutjhisa eliqothelweko lingu-310K. Ukusebenzisana okungabophiko kubalwa ngokwenza irhelo lokubhangqa ngokusebenzisa isikimu se-Verlet esine-0.005 yokubekezelela ibhafa. Ithemu ye-Coulomb ibalwa kusetjenziswa indawo yokusabela kanye nebanga lokusika elingu-1.1 nm. Ithemu le-Vander Waals lisebenzisa isikimu sokusika esinebanga lokusika eliyi-1.1 nm, begodu isikimu sokusika se-Verlet sisetjenziselwa ukutjhinga okungenzeka (62).
Ngokusebenzisa i-VMD, i-cutoff wavelength hlangana nama-DOPC phosphate beads nofana i-ceramide AM1 beads kanye nephrotheyini ngu-0.7 nm, begodu inani lamafutha asebenzisana nephrotheyini liyabalwa. Ngokuya ngefomula elandelako, bala i-depletion-enrichment (DE) njengaku-(63): i-DE = (inani lamafutha apheleleko kuphrotheyini 0.7) kuphrotheyini 0.7 (inani le-Cer emafutheni woke)
Inani elibikiweko litholwa njengesilinganiso, begodu imigoqo yamaphutha amakhophi amane azijameleko we-SE. Ukuqakatheka kwe-DE factor kubalwa nge-t test [(averageDE-factor-1)/SE]. Bala inani le-P ukusuka ekusabalalisweni okunomsila owodwa.
Ithulusi le-GROMACS lasetjenziselwa ukubala imephu ye-2D ye-lateral density yesistimu ene-Emp24 ngaphakathi kwama-250 ns wokugcina womkhondo. Ukuze kutholakale imephu yokunothisa/yokuphela kwe-ceramide, imephu yokuminyana ye-Cer ihlukaniswe ngesamba semephu ye-Cer ne-DOPC, bese ihlukaniswa ngokugxila kwe-Cer emzimbeni. Isikali semephu yombala ofanako sisetjenziswa.
Ukuthola izinto ezingezelelweko ze-atikili le, sibawa ubone i-http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Le yi-atikili evulekileko esabalaliswa ngaphasi kwemibandela ye-Creative Commons Attribution-Non-Commercial License, evumela ukusetjenziswa, ukusabalalisa nokukhiqizwa kabutjha nganoma ngiyiphi indlela, inqobo nje nangabe ukusetjenziswa kokugcina kungasi kwezokurhweba begodu umqondo kukuthi umsebenzi wokuthoma ulungile. Ireferensi.
Tjheja: Sikubawa bona unikele ngekheli lakho le-imeyili ukwenzela bona umuntu omncomako ekhasini azi bona ufuna bona abone i-imeyili begodu akusiyo i-spam. Angeze sathatha amakheli we-imeyili.
Umbuzo lo usetjenziselwa ukuhlola bona usivakatjhi begodu uvimbele ukuthunyelwa kwe-spam okuzenzakalelayo.
USofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), U-Auxiliadora Aguilera-Romero, Ana Maria Perez -Lingio Sererope), Wazerhope (Serhopez) (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ukuthathwa kwemifanekiso ye-3D ephezulu yesikhathi sangempela kuveza ukuqakatheka kobude beketango le-ceramide ekuhlungeni amaphrotheyini eendaweni ezikhethiweko zokuphuma.
USofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), U-Auxiliadora Aguilera-Romero, Ana Maria Perez -Lingio Sererope), Wazerhope (Serhopez) (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ukuthathwa kwemifanekiso ye-3D ephezulu yesikhathi sangempela kuveza ukuqakatheka kobude beketango le-ceramide ekuhlungeni amaphrotheyini eendaweni ezikhethiweko zokuphuma.
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