Kusankha mapuloteni mu njira yotulutsira ma cell ndikofunikira kwambiri kuti maselo azikhala m'malo obisika komanso kuti maselo azikhala bwino. Kuwonjezera pa kusankha ma shell-mediated sorting, ntchito ya lipids mu kukonza kinesin mu njira yotumizira ma cell ndi funso lofunika kwambiri lomwe silinayankhidwebe. Pano, tikuchita kujambula kwa 3D nthawi imodzi kwa mitundu yosiyanasiyana kuti titsimikizire mu vivo kuti mapuloteni atsopano a glycosylphosphatidylinositol omwe ali ndi ceramide lipid moieties yayitali kwambiri amagawidwa m'magulu ndikugawidwa m'magawo apadera a endoplasms. Net exit site, yomwe ndi yosiyana ndi yomwe imagwiritsidwa ntchito ndi mapuloteni a transmembrane. Kuphatikiza apo, tikuwonetsa kuti kutalika kwa unyolo wa ceramide mu endoplasmic reticulum membrane ndikofunikira kwambiri pakusankha kosankha kumeneku. Kafukufuku wathu akupereka umboni woyamba mwachindunji mu vivo wogawa ma protein cargoes kutengera kutalika kwa unyolo wa lipid m'malo osankhidwa otumizira kunja mu njira yotulutsira ma cell.
Mu maselo a eukaryotic, mapuloteni omwe amapangidwa mu endoplasmic reticulum (ER) amasankhidwa panthawi yoyenda kudzera mu njira yotulutsira kuti afike kumalo oyenera a maselo (1). Kuwonjezera pa kusankha kopangidwa ndi coat, kwakhala kuganiziridwa kwa nthawi yayitali kuti lipids zina zimathanso kukhala malo osankha otulukira mwa kuziphatikiza m'magawo enaake a membrane kuposa mapuloteni enaake (2-5). Komabe, pakadalibe umboni wowongoka mu vivo wotsimikizira njira iyi yotheka yochokera ku lipid. Pofuna kuthetsa vutoli, tinaphunzira mu yisiti momwe mapuloteni a glycosylphosphatidylinositol (GPI) anchored (GPI-APs) amatumizidwa mosiyana kuchokera ku ER. GPI-APs ndi mapuloteni osiyanasiyana olumikizidwa ndi lipid pamwamba pa maselo (6, 7). GPI-AP ndi mapuloteni otulutsidwa omwe amamangiriridwa ku mapepala akunja a plasma membrane kudzera mu glycolipid moiety (GPI anchor). Amavomereza ma GPI anchors ngati kusintha kosinthika pambuyo pa kumasulira mu ER lumen (8). Pambuyo polumikizana, GPI-AP imadutsa mu chipangizo cha Golgi (5, 9) kuchokera ku ER kupita ku plasma membrane. Kupezeka kwa ma GPI anchors kumapangitsa kuti GPI-AP inyamulidwe mosiyana ndi mapuloteni otulutsidwa ndi transmembrane (kuphatikiza mapuloteni ena a plasma membrane) kudzera mu njira yotulutsira (5, 9, 10). Mu maselo a yisiti, ma GPI-AP amalekanitsidwa ndi mapuloteni ena otulutsidwa mu endoplasmic reticulum, kenako amaikidwa mu ma vesicles apadera okulungidwa ndi coat protein complex II (COPII) (6, 7). Zomwe zimayambitsa njira yogawa iyi mu njira yotumizira kunja kwa ER sizikudziwika bwino, koma zikuganiziridwa kuti njira iyi ingafunike lipids, makamaka kukonzanso kapangidwe ka gawo la lipid la GPI anchor (5, 8). Mu yisiti, kusintha kwa lipids za GPI kumayamba nthawi yomweyo GPI ikamamatira, ndipo nthawi zambiri, zimapangitsa kuti ceramide igwirizane ndi 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide ndiye ceramide yayikulu yopangidwa ndi maselo a yisiti mpaka pano. Imapangidwa mu ER ndipo yambiri imatumizidwa ku chipangizo cha Golgi kudzera mu COPII vesicles (13). Kutumiza GPI-AP ku ER kumafuna makamaka kupanga ceramide kosalekeza (14, 15), ndipo kusintha kwa ceramide kukhala inositol phosphate ceramide (IPC) mu chipangizo cha Golgi kumadalira kupanga GPI anchor (16). Kafukufuku wa biophysical wokhala ndi nembanemba zopanga wasonyeza kuti ma acyl chain ceramide ataliatali amatha kusonkhana kuti apange madera okonzedwa okhala ndi mawonekedwe apadera (17, 18). Deta iyi imabweretsa lingaliro lakuti C26 ceramide ndi GPI-AP yokhala ndi C26 ceramide amagwiritsa ntchito mawonekedwe awo enieni kuti agwirizane m'madera okonzedwa bwino kapena madera omwe ali ndi ER membrane lipids. Amapangidwa makamaka ndi glycerolipids zazifupi komanso zosakhuta (C16:1 ndi C18:1) (19, 20). Madera awa adzayang'ana kwambiri malo enaake otulukira a ER (ERES), komwe ceramide ndi ceramide-based GPI-AP zitha kunyamulidwa limodzi kupita ku Golgi mu COPII vesicle yomweyo (5).
Mu kafukufukuyu, tayesa mwachindunji njira imeneyi yochokera ku lipid pogwiritsa ntchito super-resolution confocal real-time imaging microscopy (SCLIM), yomwe ndi njira yamakono yowonera ma microscopy yomwe imatha kuwona mapuloteni olembedwa ndi fluorescent nthawi imodzi. Zithunzi zamitundu itatu ndi zitatu (3D) zimakhala ndi resolution yapamwamba kwambiri komanso liwiro m'maselo amoyo (21, 22).
Choyamba tinagwiritsa ntchito ukadaulo wa SCLIM kuti tifotokoze bwino momwe GPI-AP yachibadwa yokhala ndi gulu la C26 ceramide imafufuzidwira kuchokera ku mapuloteni otulutsidwa ndi transmembrane atatuluka mu ER mu S. cerevisiae. Pofuna kuwona kugawidwa kwa ER, tinagwiritsa ntchito njira ya majini yomwe imatha kuwona mwachindunji katundu watsopano wopangidwa kulowa mu ERES mu vivo (7, 23). Monga katundu, tinasankha C26 ceramide-based GPI-AP Gas1 yolembedwa ndi green fluorescent protein (GFP) ndi transmembrane secreted protein Mid2 yolembedwa ndi near-infrared fluorescent protein (iRFP), zonse zomwe zimayang'ana pa plasma membrane (24-26). Mu sec31-1 temperature-sensitivity mutant, katundu awiriwa amawonetsedwa pansi pa galactose-inducible promoter ndi constitutive ERES marker. Pa kutentha kwakukulu (37°C), chifukwa kusintha kwa sec31-1 kumakhudza ntchito ya COPII coat component Sec31 kuti ilepheretse COPII kumera ndi kutumiza kunja kwa ER, katundu watsopano wopangidwa umasonkhana pa ER (23). Pambuyo pozizira mpaka kutentha kochepa (24°C), maselo osinthika a sec31-1 adachira kuchokera ku malo obisika, ndipo katundu watsopano wopangidwa unayamba kutumizidwa kuchokera ku ER. Kuwona kwa CLIM kunawonetsa kuti ambiri mwa mapuloteni atsopano a Gas1-GFP ndi Mid2-iRFP omwe adapangidwanso adasonkhanabe mu ER ya maselo osinthika a sec31-1 atangoyamba kusungidwa pa 37°C kenako nkutulutsidwa pa 24°C kwa mphindi 5 (Chithunzi 1). Popeza Mid2-iRFP imagawidwa pa nembanemba yonse ya ER, ndipo Gas1-GFP imayikidwa ndikusonkhanitsidwa m'dera la nembanemba ya ER yosakhazikika, kufalikira kwawo ndi kosiyana kwambiri (Chithunzi 1, A mpaka C ndi Movie S1). Kuphatikiza apo, monga momwe zasonyezedwera pa Chithunzi 1D, gulu la Gas1-GFP lilibe Mid2-iRFP. Zotsatirazi zikusonyeza kuti mapuloteni a GPI-AP ndi transmembrane adalekanitsidwa m'magawo osiyanasiyana a nembanemba ya ER koyambirira. Gulu la Gas1-GFP lili pafupi ndi ERES yeniyeni yolembedwa ndi mCherry's COPII coat protein Sec13 (Chithunzi 1, E ndi F, ndi filimu S1) (23).
Maselo a sec31-1 amasonyeza kutulutsa kwa galactose komwe kumachitika chifukwa cha galactose, ceramide yayitali ya acyl (C26) GPI-AP Gas1-GFP (GPI-AP, yobiriwira) ndi puloteni ya transmembrane Mid2-iRFP (TMP, buluu) ndipo chizindikiro ichi cha Constructive ERES cholembedwa Sec13-mCherry (ERES, magenta) chinayikidwa pa 37°C kwa mphindi 30, chinasunthidwa kupita ku 24°C, ndikujambulidwa ndi SCLIM patatha mphindi 5. (A mpaka C) chikuwonetsa chithunzi choyimira choyimira chimodzi kapena chimodzi cha 2D cha ndege (A), chithunzi cha 2D chowonetsera cha 10 z-sections (B) kapena chithunzi cha 3D cell hemisphere cha cargo ndi ERES markers (C). Scale bar 1μm (A ndi B). Scale unit ndi 0.551μm (C). Gas1-GFP idapezeka m'madera kapena magulu a ER osiyana, pomwe Mid2-iRFP idapezeka ndikugawidwa mu ER membrane yonse (C). (D) Chithunzichi chikuwonetsa mphamvu ya kuwala kwa Gas1-GFP ndi Mid2-iRFP mu gulu la Gas1-GFP motsatira mzere woyera wa muvi (kumanzere). AU, gawo losasinthika. (E ndi F) akuyimira chithunzi cha 3D chomwe chimaphatikiza katundu ndi chizindikiro cha ERES. Magulu a Gas1-GFP adapezeka pafupi ndi ERES yeniyeni. Gawo la sikelo ndi 0.551μm. (F) Muvi woyera wolimba umawonetsa gulu la Gas1-GFP logwirizana ndi ERES. Mapanelo apakati ndi akumanja akuwonetsa chithunzi chokulirapo cha 3D chophatikizidwa komanso mawonekedwe ozungulira a gulu losankhidwa la Gas1-GFP.
Ubale wapafupi pakati pa gulu la Gas1-GFP ndi ERES yeniyeni ukusonyeza kuti Gas1-GFP ikhoza kulowa mu ERES yosankhidwa, yomwe ndi yosiyana ndi kusankha komwe Mid2-iRFP imagwiritsa ntchito kuti ituluke mu ER. Pofuna kuthana ndi izi, tinayesa chiŵerengero cha ERES cha katundu mmodzi kapena awiri okha (Chithunzi 2, A mpaka C). Tapeza kuti ERES zambiri (70%) zili ndi mtundu umodzi wokha wa katundu. Chithunzi cha pansi pa Chithunzi 2C chikuwonetsa zitsanzo ziwiri za ERES zokhala ndi Gas1-GFP yokha (Chithunzi 1) kapena Mid2-iRFP yokha (Chithunzi 2). Mosiyana ndi zimenezi, pafupifupi 20% ya ERES ili ndi katundu awiri omwe amalumikizana m'dera lomwelo. Zinapezeka kuti ERES zina (10%) zinali ndi mitundu iwiri ya katundu, koma zinalekanitsidwa m'madera osiyana kwambiri. Chifukwa chake, kusanthula kwa ziwerengerozi kukuwonetsa kuti ER ikatumizidwa kunja, GPI-AP Gas1-GFP ndi katundu wa transmembrane Mid2-iRFP zimagawidwa m'ma ERES osiyanasiyana (Chithunzi 2D). Kuchita bwino kosankha kumeneku kumagwirizana kwambiri ndi kusanthula kwakale kwa biochemical (6) ndi kutsimikiza kwa mawonekedwe (7). Titha kuwonanso momwe katundu wotsekedwa amagwirira ntchito akulowera ku ERES (Chithunzi 2E ndi Movie S2). Chithunzi 2E chikuwonetsa kuti gawo laling'ono lokha la Gas1-GFP (gawo 3) kapena Mid2-iRFP (gawo 4) limalowa mu ERES kuchokera mbali imodzi ndipo limatsekedwa m'dera losiyana. Gulu 5 la Chithunzi 2E likuwonetsa kuti Gas1-GFP ndi Mid2-iRFP nthawi zina zimapezeka mu ERES yomweyo, koma zimalowa kuchokera mbali zosiyanasiyana ndipo zimakhala m'madera osiyana omwe angaimire ma vesicles osiyanasiyana a COPII. Tatsimikiziranso kuti kulekanitsidwa ndi kugawidwa kwa C26 ceramide-based GPI-AP Gas1 ngati ERES yosankha ndi yeniyeni chifukwa katundu wina wa transmembrane secretion, GFP-taged plasma membrane protein Axl2 (27), kusonyeza khalidwe lofanana ndi Mid2-iRFP. (Chithunzi S1 ndi Movie S3). Axl2-GFP yopangidwa kumene imagawidwa kudzera mu nembanemba ya ER monga Mid2-iRFP (Chithunzi S1, A ndi B), ndipo imagwirizana ndi Mid2-iRFP mu ERES zambiri (Chithunzi S1, B mpaka D). Mapanelo 1 ndi 2 a Chithunzi 1. S1C ikuwonetsa zitsanzo ziwiri za ERES pomwe katundu awiri a transmembrane amalumikizana. Pazochitika izi, katundu onse awiri amalowa pamodzi mu ERES (Chithunzi S1E, Panel 3 ndi Movie S3).
Maselo a sec31-1 omwe amawonetsa kutulutsa kwa galactose, Gas1-GFP (GPI-AP, wobiriwira) ndi Mid2-iRFP (TMP, buluu) ndi zilembo za ERES zomwe zili ndi Sec13-mCherry (ERES, magenta) adayikidwa pa 37. Pambuyo poyika mu incubating kwa mphindi 30 pa °C, sinthani ku 24 °C kuti mutulutse block ya secretion, ndi chithunzi ndi SCLIM patatha mphindi 20. (A mpaka C) Zithunzi zoyimira 2D projection (A; scale bar, 1μm) kapena zithunzi za 3D cell hemisphere (B ndi C; scale unit, 0.456μm) za katundu ndi magawo 10 a z olembedwa ndi ERES. Gulu la pansi mu (B) ndi gulu mu (C) zimawonetsa zithunzi zokonzedwa kuti ziwonetse katundu yemwe ali mu ERES (magenta) [Gas1-GFP (imvi) ndi Mid2-iRFP (buluu wopepuka)]. (C) Muvi wotseguka: ERES imanyamula katundu mmodzi yekha (1 mpaka 4). Muvi wa imvi: ERES ili ndi katundu wolekanitsidwa (5). Muvi woyera wolimba: ERES ili ndi katundu wokhala pamalo amodzi. Pansipa: ERES imodzi yosankhidwa ili ndi Gas1-GFP (1) kapena Mid2-iRFP (2) yokha. Muvi wopingasa, 100 nm. (D) Kuyeza kwa chithunzi cha zithunzi chomwe chafotokozedwa mu (C). Chiwerengero chapakati cha ERES chomwe chili ndi katundu mmodzi yekha (Gas1-GFP kapena Mid2-iRFP), katundu wolekanitsidwa ndi katundu wolumikizana. Mu zoyeserera zitatu zodziyimira pawokha, n=432 m'maselo 54. Muvi wolakwika = SD. Mayeso a t osaphatikizidwa awiri. *** P = 0.0002. (E) Chithunzi cha 3D cha ERES yosankhidwa ya katundu wokhazikika wolembedwa ndi (C). Gas1-GFP (wobiriwira) (3) kapena Mid2-iRFP (buluu) (4) imalowa mu ERES (magenta) kuchokera mbali imodzi ndipo imaloledwa kufika pamalo ang'onoang'ono mkati mwa ERES. Nthawi zina, mitundu yonse iwiri ya katundu imalowa mu ERES (5) yomweyi kuchokera mbali imodzi ndipo imayikidwa pamalo enaake mkati mwa ERES. Scale bar, 100 nm.
Kenako, tinayesa lingaliro lakuti long acyl chain ceramide (C26) yomwe ili mu ER membrane imayendetsa magulu ndi kusandutsa kwa Gas1 kukhala ERES yosankha. Pachifukwa ichi, tinagwiritsa ntchito mtundu wa yisiti wosinthidwa GhLag1, momwe ma endogenous ceramide synthases awiri a Lag1 ndi Lac1 adalowedwa m'malo ndi GhLag1 (homolog ya Lag1 ya thonje), zomwe zinapangitsa kuti mtundu wa yisiti ukhale ndi nembanemba ya cell strain ya Ceramide yochepa kuposa mtundu wa wild (Chithunzi 3A) (28). Kusanthula kwa Mass spectrometry (MS) kunawonetsa kuti m'mitundu ya wild-type, 95% ya ceramide yonse ndi yayitali kwambiri (C26) chain ceramide, pomwe mu GhLag1, 85% ya ceramide ndi yayitali kwambiri (C18 ndi C16). ), 2% yokha ya ceramide ndi yayitali kwambiri (C26) chain ceramide. Ngakhale kuti ma ceramides a C18 ndi C16 ndi omwe amapezeka mu nembanemba ya GhLag1 mpaka pano, kusanthula kwa MS kunatsimikiziranso kuti nangula wa GPI wa Gas1-GFP womwe wafotokozedwa mu mtundu wa GhLag1 uli ndi C26 ceramide, womwe ndi wofanana ndi mafuta amtundu wa wild-type. Ubwino wake ndi womwewo (Chithunzi 3A) (26). Chifukwa chake, izi zikutanthauza kuti enzyme yokonzanso ya ceramide Cwh43 imasankha kwambiri C26 ceramide, monga momwe zasonyezedwera pa Chithunzi 26, imagwiritsa ntchito nangula wa GPI kuchokera ku C26 ceramide yochepa mu mtundu wa GhLag1. S2 (29). Komabe, nembanemba ya cell ya GhLag1 imakhala ndi C18-C16 ceramide yokha, pomwe Gas1-GFP ikadali ndi C26 ceramide. Izi zimapangitsa kuti mtundu uwu ukhale chida chabwino kwambiri chothetsera vuto la kutalika kwa unyolo wa acyl wa nembanemba ya ceramide mu ER. Udindo wongoganizira wa kalasi ndi kusanja. Kenako, tinayamba taphunzira za kuthekera kwa C26 Gas1-GFP kusonkhana m'magulu mu GhLag1 ndi allele yosinthika kutentha ya sec31-1 kudzera mu microscopy ya fluorescence, komwe unyolo wautali (C18-C16) wokha ulipo mu ER membrane Ceramide (Chithunzi 3). Tinaona kuti mu sec31-1, Gas1-GFP yambiri inali yodzaza m'magulu, pomwe Gas1-GFP mu sec31-1 GhLag1 yokhala ndi ceramide ER membrane yayitali (C18-C16) sinali yolumikizidwa ndikugawidwa mu ER membrane yonse. Kunena zoona, chifukwa C26 ceramide-based clustering ikugwirizana kwambiri ndi ERES yeniyeni (Chithunzi 1), kenako tinafufuza ngati njirayi ingakhudzenso ntchito ya ER export protein mechanism. GPI-AP imagwiritsa ntchito njira yapadera ya COPII yotumizira kunja kwa ER, yomwe imayendetsedwa mwachangu ndi Ted1's structural reformation ya gawo la glycan la GPI nangula (30, 31). Kenako GPI-glycan yobwezeretsanso imadziwika ndi transmembrane cargo receptor p24 complex, yomwe imagwiritsa ntchito Lst1 mosankha, yomwe ndi isoform yeniyeni ya COPII cargo binding subunit Sec24 yayikulu, ndikupanga GPI-AP-rich COPII Vesicles ndizofunikira (31-33). Chifukwa chake, tidapanga mutant iwiri yomwe idaphatikiza kuchotsedwa kwa mapuloteni amodzi awa (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 ndi COPII subunit Lst1 yeniyeni) ndi sec31-1 mutant strain, ndipo tidawaphunzira Kodi n'zotheka kupanga Gas1-cluster GFP (Chithunzi 3). Tawona kuti mu sec31-1emp24Δ ndi sec31-1ted1Δ, Gas1-GFP nthawi zambiri imakhala yopanda magulu ndipo imagawidwa mu ER membrane, monga momwe tawonera kale mu sec31-1 GhLag1, pomwe mu sec31-1lst1Δ, Gas1-GFP Monga sec31-1. Zotsatirazi zikusonyeza kuti kuwonjezera pa kukhalapo kwa C26 ceramide mu nembanemba ya ER, kusonkhana kwa Gas1-GFP kuyeneranso kumangirira ku p24 complex, ndipo sikufuna kusankhidwa kwapadera kwa Lst1. Kenako, tinafufuza kuthekera kwa kutalika kwa unyolo wa ceramide mu nembanemba ya ER kungathe kuwongolera kumangirira kwa Gas1-GFP ku p24. Komabe, tinapeza kuti kukhalapo kwa C18-C16 ceramide mu nembanemba sikukhudza GPI-glycans yomangidwanso ndi p24 complex (Zithunzi S3 ndi S4, A ndi B) kapena kumangirira ku GPI-AP ndi kutumiza GPI-AP. Kulembetsa COPII subtype Lst1 (Chithunzi S4C). Chifukwa chake, C26 ceramide-dependent clustering sikufuna kuyanjana kwa mapuloteni ndi njira zosiyanasiyana zotumizira mapuloteni a ER, koma imathandizira njira ina yosankhira yoyendetsedwa ndi kutalika kwa lipid. Kenako, tinafufuza ngati kutalika kwa unyolo wa ceramide acyl mu nembanemba ya ER ndikofunikira kuti Gas1-GFP ikhale yosankhidwa bwino. Popeza Gas1 mu mtundu wa GhLag1 wokhala ndi ceramide yofupikitsidwa imachoka mu ER ndikulowa mu plasma membrane (Chithunzi S5), tikukhulupirira kuti ngati kusankhidwa kumayendetsedwa ndi kutalika kwa unyolo wa ceramide acyl, Gas1 mu mtundu wa GhLag1 ikhoza kusinthidwa ndikuwoloka. ERES katundu wokhala ndi nembanemba yomweyo.
(A) Nembanemba ya selo ya GhLag1 makamaka imakhala ndi ma ceramides afupi a C18-C16, pomwe nangula wa GPI wa Gas1-GFP akadali ndi C26 IPC yofanana ndi maselo amtundu wa wild-type. Pamwambapa: kusanthula kutalika kwa acyl chain ya ceramide mu nembanemba ya selo ya wild-type (Wt) ndi GhLag1p strains pogwiritsa ntchito mass spectrometry (MS). Deta ikuyimira kuchuluka kwa ceramide yonse. Avereji ya mayesero atatu odziyimira pawokha. Error bar = SD. T-tailed unpaired t test. **** P ＜0.0001. Pansi pa gulu: Kusanthula kwa MS kwa kutalika kwa acyl chain ya IPC yomwe ilipo mu Gas1-GFP (GPI-IPC) GPI anchor yomwe imawonetsedwa mu wild-type ndi GhLag1p strains. Deta ikuyimira kuchuluka kwa chizindikiro chonse cha IPC. Avereji ya mayesero asanu odziyimira pawokha. Error bar = SD. T-tailed unpaired t test. ns, sizofunikira. P = 0.9134. (B) Ma micrograph a kuwala kwa sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ndi sec31-1lst1Δ omwe akuwonetsa Gas1-GFP yoyambitsidwa ndi galactose adayikidwa pa 37°C kwa mphindi 30 ndipo adaperekedwa kuti achite microscopy ya fluorescence yachizolowezi pambuyo pa 24°C. Muvi woyera: ER Gas1-GFP cluster. Muvi wotseguka: Gas1-GFP yosaphatikizidwa imagawidwa pa nembanemba yonse ya ER, kuwonetsa utoto wa ER wa mphete ya nyukiliya. Scale bar, 5μm. (C) Kuyeza kwa photomicrograph komwe kwafotokozedwa mu (B). Avereji yapakati ya maselo omwe ali ndi kapangidwe ka Gas1-GFP. Mu zoyeserera zitatu zodziyimira pawokha, maselo a n≥300. Error bar = SD. T-tailed unpaired test. **** P ＜0.0001.
Kuti tithetse vutoli mwachindunji, tinachita SCLIM visualization ya Gas1-GFP ndi Mid2-iRFP mu GhLag1 ndi sec31-1 temperature-sensitive mutant allele (Chithunzi 4 ndi Movie S4). Pambuyo poti ER yasungidwa pa 37°C kenako nkutulutsidwa pa 24°C, Gas1-GFP yambiri yopangidwa kumene sinaphatikizidwe ndikugawidwa mu ER membrane, monga momwe zawonedwera ndi ma microscopes wamba (Chithunzi 4, A ndi B). Kuphatikiza apo, kuchuluka kwakukulu kwa ERES (67%) kumaphatikizapo mitundu iwiri ya katundu wophatikizidwa mkati mwake (Chithunzi 4D). Mapanelo 1 ndi 2 a Chithunzi 4C akuwonetsa zitsanzo ziwiri za ERES zomwe zili ndi Gas1-GFP ndi Mid2-GFP yolumikizana. Kuphatikiza apo, katundu onsewa adasonkhanitsidwa mu ERES yomweyo (Chithunzi 4E, gulu 3 ndi filimu S4). Chifukwa chake, zotsatira zathu zikuwonetsa kuti kutalika kwa unyolo wa ceramide acyl mu ER membrane ndikofunikira kwambiri pakusonkhanitsa ndi kugawa mapuloteni a ER.
Maselo a Sec31-1 GhLag1 omwe amatulutsa kutulutsa kwa galactose, Gas1-GFP (GPI-AP, wobiriwira) ndi Mid2-iRFP (TMP, buluu) ndi Sec13-mCherry (ERES, magenta) yokhala ndi zilembo za ERES (ERES, magenta) amaikidwa pa kutentha kwa 37°C. Pitirizani kwa mphindi 30, tsitsani mpaka 24°C kuti mutulutse kutulutsa, ndipo chithunzicho chikhale ndi SCLIM patatha mphindi 20. (A mpaka C) Zithunzi zoyimira 2D projection (A; scale bar, 1μm) kapena zithunzi za 3D cell hemisphere (B ndi C; scale unit, 0.45μm) za magawo 10 a z omwe alembedwa ndi cargo ndi ERES. Gulu la pansi mu (B) ndi gulu mu (C) zimawonetsa zithunzi zomwe zakonzedwa kuti ziwonetse katundu yemwe alipo mu ERES (magenta) [Gas1-GFP (imvi) ndi Mid2-iRFP (buluu wopepuka)]. (C) Muvi woyera wodzazidwa: ERES, katundu amalumikizana. Muvi wotseguka: ERES ili ndi chinthu chimodzi chokha. Pansi: ERES yosankhidwa ili ndi katundu wolumikizana (1 ndi 2) wolembedwa mu (C). Muyeso wa sikelo, 100 nm. (D) Kuyeza kwa chithunzi cha micrograph chomwe chafotokozedwa mu (C). Mu sec31-1 ndi sec31-1 GhLag1 units, katundu m'modzi yekha (Gas1-GFP kapena Mid2-iRFP) ndi omwe akuphatikizidwa, ndipo peresenti yapakati ya ERES ya katundu wosiyana ndi katundu wolumikizana. Mu zoyeserera zitatu zodziyimira pawokha, n = 432 mu maselo 54 (sec31-1) ndi n = 430 mu maselo 47 (sec31-1 GhLag1). Muyeso wa cholakwika = SD. Mayeso a t osaphatikizidwa awiri. *** P = 0.0002 (sec31-1) ndi ** P = 0.0031 (sec31-1 GhLag1). (E) Chithunzi cha 3D cha ERES yosankhidwa ndi katundu wolumikizana (3) wolembedwa mu (C). Gas1-GFP (yobiriwira) ndi Mid2-iRFP (yabuluu) zimayandikira ERES (magenta) kuchokera mbali imodzi ndipo zimakhala m'dera lomwelo la ERES loletsedwa. Scale bar, 100 nm.
Kafukufukuyu akupereka umboni wowonekera bwino mu vivo kuti katundu wa mapuloteni okhala ndi lipid amagawidwa m'malo osankhidwa otumizira kunja mu njira yotulutsira, ndipo akuwonetsa kufunika kwa kutalika kwa unyolo wa acyl pakusankha magulu. Pogwiritsa ntchito njira yamphamvu komanso yapamwamba yotchedwa SCLIM, tawonetsa Gas1-GFP yatsopano yopangidwa (membrane yayikulu ya plasma GPI-AP yokhala ndi gawo lalitali kwambiri la acyl chain (C26) ceramide lipid) mu yisiti) Madera omwe ali m'magulu a ER osiyana amagwirizanitsidwa ndi ERES yeniyeni, pomwe mapuloteni otulutsidwa ndi transmembrane amagawidwa mu ER membrane yonse (Chithunzi 1). Kuphatikiza apo, mitundu iwiriyi ya zinthu imalowa mu ERES zosiyanasiyana mosankha (Chithunzi 2). Kutalika kwa unyolo wa acyl wa ceramide yamaselo mu nembanemba kumachepetsedwa kuchokera ku C26 kupita ku C18-C16, gulu la Gas1-GFP limasokonezedwa kupita ku dera la ER losiyana, ndipo Gas1-GFP imasinthidwa kuti ituluke mu ER ndi protein ya transmembrane kudzera mu ERES yomweyo (Chithunzi 3 ndi Chithunzi 3). 4).
Ngakhale GPI-AP imagwiritsa ntchito njira yapadera ya mapuloteni kuti ituluke mu ER, tapeza kuti kulekanitsidwa kwa C26 ceramide-dependent sikudalira kuyanjana kwa mapuloteni osiyanasiyana komwe kungayambitse ERES specialization (Zithunzi S4 ndi S5). M'malo mwake, zomwe tapeza zikuthandizira njira ina yogawa yomwe imayendetsedwa ndi lipid-based protein clustering ndi kuchotsa katundu wina pambuyo pake. Zomwe tawona zikusonyeza kuti dera la Gas1-GFP kapena gulu logwirizana ndi ERES linalake lilibe protein yotulutsidwa ndi transmembrane Mid2-iRFP, zomwe zikusonyeza kuti gulu la C26 ceramide-dependent GPI-AP lidzathandiza kulowa kwawo mu ERES yoyenera, ndipo nthawi yomweyo, kuchotsa transmembrane. Kutulutsa kumalowa mu ERES iyi (Zithunzi 1 ndi 2). Mosiyana ndi zimenezi, kukhalapo kwa C18-C16 ceramide mu ER membrane sikupangitsa GPI-AP kupanga madera kapena magulu, kotero samachotsa kapena kusintha mapuloteni otulutsidwa ndi transmembrane mu ERES yomweyo (Zithunzi 3 ndi 4). Chifukwa chake, tikupereka lingaliro lakuti C26 ceramide imayambitsa kulekanitsa ndi kugawa magulu mwa kuthandizira kusonkhanitsa mapuloteni olumikizidwa ndi ERES yeniyeni.
Kodi mungakwaniritse bwanji kusonkhana kwa C26 ceramide m'dera linalake la ER? Kusinthasintha kwa membrane ceramide kungapangitse GPI-AP ndi C26 ceramide kupanga lipids zazing'ono komanso zoyendetsedwa nthawi yomweyo m'malo osakhazikika a lipid a membrane ya ER yokhala ndi glycerolipids zazifupi komanso zosakhuta. Magulu abwino (17, 18). Magulu ang'onoang'ono akanthawi awa amatha kuphatikizidwanso kukhala magulu akuluakulu, okhazikika pambuyo polumikizana ndi p24 complex (34). Mogwirizana ndi izi, tawonetsa kuti C26 Gas1-GFP iyenera kuyanjana ndi p24 complex kuti ipange magulu akuluakulu owoneka (Chithunzi 3). P24 complex ndi heterozygous oligomer yopangidwa ndi mapuloteni anayi osiyanasiyana a p24 transmembrane mu yisiti (35), yomwe imapereka kulumikizana kwamitundu yambiri, komwe kungayambitse kulumikizana kwa magulu ang'onoang'ono a GPI-AP, motero kupanga gulu lalikulu la Stable (34). Kuyanjana pakati pa ma ectodomain a mapuloteni a GPI-APs kungathandizenso kuti agwirizane, monga momwe zasonyezedwera panthawi yoyenda kwawo ku Golgi m'maselo a epithelial omwe ali ndi polarized mammaly (36). Komabe, pamene C18-C16 ceramide ilipo mu ER membrane, pamene p24 complex ikugwirizana ndi Gas1-GFP, magulu akuluakulu osiyana sadzapangidwa. Njira yoyambira ingadalire mawonekedwe enieni a thupi ndi mankhwala a long acyl chain ceramide. Kafukufuku wa biophysical wa ma acyl chain membranes akuwonetsa kuti ngakhale kuti ma acyl chain ceramide aatali (C24) ndi aafupi (C18-C16) atali amatha kuyambitsa kulekana kwa gawo, ma acyl chain ceramide aatali okha (C24) ndi omwe angalimbikitse kupindika kwakukulu ndi kupindika kwa filimu kuti isinthe mawonekedwe a filimuyo. Kudzera mu referensi yogwirizana (17, 37, 38). Zawonetsedwa kuti transmembrane helix ya TMED2, yomwe ndi homologue ya anthu ya Emp24, imalumikizana mosankha ndi C18 ceramide-based sphingomyelin mu cytoplasmic lobules (39). Pogwiritsa ntchito ma simulation a molecular dynamics (MD), tapeza kuti ma ceramides onse a C18 ndi C26 amasonkhana mozungulira ma cytoplasmic lobules a Emp24 transmembrane helix, ndipo ali ndi zokonda zofanana (Chithunzi S6). Ndikofunikira kudziwa kuti izi zikusonyeza kuti transmembrane helix ya Emp24 ikhoza kutsogolera kugawa kwa lipids mu nembanemba. Izi ndi zotsatira zaposachedwa zochokera ku maselo a nyama zoyamwitsa. Ma simulation ofanana a MD akuwonetsanso kukhalapo kwa ether lipids (40). Chifukwa chake, tikuganiza kuti C26 ceramide mu ma lobules awiri a ER26 ndi olemera m'deralo. Pamene GPI-AP mu ma lobules a luminal imagwirizana mwachindunji ndi multivalent p24 ndipo kusonkhanitsa kwa C26 ceramide mozungulira p24 mu cytoplasmic lobules, kumatha kulimbikitsa kusonkhana kwa Mapuloteni ndi kupindika kwa membrane kumapangidwa kudzera m'zala (41), zomwe zimapangitsa kuti GPI-AP igawikane m'madera osiyana pafupi ndi ERES, zomwe zimathandizanso madera opindika kwambiri a ER membrane (42). Malipoti am'mbuyomu adathandizira njira yomwe idaperekedwa (43, 44). Kumangirira kwa multivalent kwa oligolectins, tizilombo toyambitsa matenda kapena ma antibodies ku ceramide-based glycosphingolipids (GSL) pa plasma membrane kumayambitsa kusonkhana kwakukulu kwa GSL, kumawonjezera kulekana kwa gawo ndikuyambitsa kusintha kwa membrane ndi internalization (44). Iwabuchi etc. (43) Zinapezeka kuti pakakhala ma acyl chains ataliatali (C24) koma osati afupiafupi (C16), multivalent ligand yolumikizidwa ku GSL lactosylceramide inayambitsa kupangika kwa magulu akuluakulu ndi membrane invagination, ndipo cytoplasm Lyn-mediated signal transduction pamapepalawa imagwirizanitsidwa ndi ma acyl chains mu ma neutrophils ogwirizana.
Mu maselo a epithelial opangidwa ndi ma mammalian, kuchuluka kwa ma anti-Golgi network (TGN) kufika pamlingo wa apical plasma membrane kumalamulira kulekanitsidwa ndi kusanja kwa GPI-AP (10, 45). Kusonkhanitsidwa kumeneku kumayendetsedwa ndi GPI-AP oligomerization (36), koma kungadalirenso kutalika kwa unyolo wa ceramide womwe timapeza mu yisiti. Ngakhale kuti GPI-AP ya mammalian ili ndi ether lipid-based anchor, ndipo kapangidwe kake ka mankhwala ndi kosiyana kwambiri ndi acyl chain ceramide yayitali kwambiri, kafukufuku waposachedwa adapeza kuti ma lipid onsewa ali ndi mphamvu zofanana zakuthupi ndi zamakemikolo ndi ntchito (40). Chifukwa chake, gawo la ether lipid m'maselo a mammalian likhoza kukhala lofanana ndi C26 ceramide mu yisiti, ndipo ntchito yake ndikulumikizana ndi ceramide yayitali mu nembanemba kuti ilimbikitse kusonkhanitsa ndi kusanja kwa GPI-AP. Ngakhale kuti kuthekera kumeneku kukufunikabe kuyesedwa mwachindunji, zomwe zapezeka kale zimatsimikizira kuti kunyamula kwa ceramide yayitali ya acyl chain kupita ku thupi la Golgi sikumachitika ndi mapuloteni osamutsa a cytoplasmic, koma kumadalira kupanga kwa ma GPI anchors monga yisiti. Chifukwa chake, njira yosinthira yosinthika ikuwoneka kuti imatha kunyamula mosankha acyl chain ceramide yayitali kwambiri ndi GPI-AP (13, 16, 20, 46, 47) mu vesicle yomweyo yoyendera.
Mu maselo a epithelial a yisiti ndi ma mammalian polarized epithelial systems, kusonkhana kwa GPI-AP ndi kulekanitsidwa ndi mapuloteni ena a plasma membrane kumachitika asanafike pamwamba pa selo. Paladino et al. (48) adapeza kuti pa TGN ya maselo a epithelial a mammalian polarized, GPI-AP clustering sikofunikira kokha kuti GPI-APs isankhidwe bwino ku apical plasma membrane, komanso imayang'anira gulu la GPI-APs ndi ntchito yake yachilengedwe. Pamwamba pa selo. Mu yisiti, kafukufukuyu adawonetsa kuti gulu la C26 ceramide-dependent GPI-AP pa ER lingathe kuwongolera gulu la cluster ndi ntchito ya GPI-AP pa plasma membrane (24, 49). Mogwirizana ndi chitsanzo ichi, maselo a GhLag1 ali ndi vuto la GPI inhibitors kapena mankhwala omwe amakhudza umphumphu wa makoma a selo (28), ndipo kufunikira kwa magulu ogwira ntchito a Gas1-GFP (49) a tip ceramide omwe akuwonetsedwa pakulumikizana kwa maselo a yisiti kumasonyeza G​​ Zotsatira zomwe zingatheke za thupi la maselo a hLag1. Cholakwika cha GPI-AP. Komabe, kuyesa kwina ngati dongosolo logwira ntchito la pamwamba pa selo lakonzedwa kuchokera ku ER pogwiritsa ntchito njira yosankhira kutengera kutalika kwa lipid kudzakhala mutu wa kafukufuku wathu wamtsogolo.
Mitundu ya Saccharomyces cerevisiae yomwe yagwiritsidwa ntchito mu ntchitoyi yalembedwa mu Table S1. Mitundu ya MMY1583 ndi MMY1635 ya SCLIM yojambulira maselo amoyo idapangidwa kumbuyo kwa W303. Mitundu iyi yowonetsa Sec13-mCherry yokhala ndi chizindikiro cha mapuloteni opepuka idapangidwa pogwiritsa ntchito njira yochokera ku polymerase chain reaction (PCR) yokhala ndi pFA6a plasmid ngati template (23). Mtundu wowonetsa Mid2-iRFP wolembedwa ndi mapuloteni opepuka pansi pa ulamuliro wa GAL1 promoter udapangidwa motere. Kukulitsa kwa PCR kwa iRFP-KanMx sequence kuchokera ku pKTiRFP-KAN vector (mphatso ya E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Ndipo idayikidwa mu C-terminus ya endogenous Mid2. Pambuyo poti njira ya majini ya Mid2-iRFP yakulitsidwa ndikuyikidwa mu cholimbikitsira cha GAL1, idaphatikizidwa mu malo a Not I-Sac I a integration plasmid pRS306. Plasmid pRGS7 yomwe idatuluka idasinthidwa ndi Pst I kuti iphatikizidwe mu malo a URA3.
Jini ya Gas1-GFP fusion imawonetsedwa motsogozedwa ndi cholimbikitsira cha GAL1 mu plasmid ya centromere (CEN), yomwe imapangidwa motere. Mndandanda wa Gas1-GFP unakulitsidwa ndi PCR kuchokera ku plasmid ya pRS416-GAS1-GFP (24) (mphatso ya L. Popolo) ndipo unayikidwa mu malo a Xma I–Xho I a CEN plasmid pBEVY-GL LEU2 (mphatso ya C). Miller; Addgene plasmid nambala 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Plasmid yomwe idatuluka idatchedwa pRGS6. Jini ya Axl2-GFP fusion imawonetsedwanso motsogozedwa ndi cholimbikitsira cha GAL1 cha vector ya pBEVY-GL LEU2, ndipo kapangidwe kake ndi motere. Ndondomeko ya Axl2-GFP inakulitsidwa kuchokera ku pRS304-p2HSE-Axl2-GFP plasmid (23) ndi PCR, ndipo inalowetsedwa mu Bam HI-Pst I site ya pBEVY-GL LEU2 vector. Plasmid yomwe inatuluka inatchedwa pRGS12. Ndondomeko ya oligonucleotides yomwe yagwiritsidwa ntchito mu kafukufukuyu yalembedwa mu Table S2.
Mtunduwu unawonjezeredwa ndi 0.2% adenine ndi 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1% Yeast extract ndi 2% protein ept). (YPR)] kapena 2% galactose [YP-galactose (YPG)] ngati gwero la kaboni, kapena mu synthetic minimal medium (0.15% yeast nitrogen base ndi 0.5% ammonium sulfate) kuti iwonjezere ma amino acid ndi maziko oyenera ofunikira pazakudya, Ndipo yokhala ndi 2% glucose (synthetic glucose minimal medium) kapena 2% galactose (synthetic galactose minimal medium) ngati gwero la kaboni.
Pa kujambula zenizeni, maselo osinthika a sec31-1 omwe amawonetsa kutentha omwe akuwonetsa kapangidwe kake pansi pa GAL1 promoter adakulitsidwa mu YPR medium pa 24°C usiku wonse mpaka pakati pa log phase. Pambuyo poyambitsa YPG pa 24°C kwa ola limodzi, maselowo adayikidwa mu SG pa 37°C kwa mphindi 30, kenako adasamutsidwira ku 24°C kuti amasulidwe kuchokera ku block ya secretion. Concanavalin A idagwiritsidwa ntchito kukonza maselowo pa slide yagalasi ndikujambulidwa ndi SCLIM. SCLIM ndi kuphatikiza kwa maikulosikopu ya Olympus IX-71 inverted fluorescence ndi lenzi ya mafuta ya UPlanSApo 100×1.4 numeral aperture (Olympus), high-speed and high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, ndi custom cooling. Chowonjezera chithunzi cha dongosololi (Hamamatsu Photonics) chingapereke dongosolo la magnifying lens ndi magnification omaliza a ×266.7 ndi kamera ya chipangizo cholumikizidwa ndi charge yomwe imachulukitsa ma electron (Hamamatsu Photonics) (21). Kupeza chithunzi kumachitika ndi pulogalamu yapadera (Yokogawa Electric). Pazithunzi za 3D, tidagwiritsa ntchito piezoelectric actuator yopangidwa mwamakonda kuti tigwedeze lenzi yolunjika molunjika, ndikusonkhanitsa magawo owoneka bwino 100 nm motalikirana mu stack. Chithunzi cha Z-stack chimasinthidwa kukhala data ya voxel ya 3D, ndipo ntchito yofalitsa mfundo yogwiritsidwa ntchito pa maikulosikopu yozungulira ya disc confocal imagwiritsidwa ntchito pochotsa convolution ndi pulogalamu ya Volocity (PerkinElmer). Pogwiritsa ntchito pulogalamu ya Volocity kuti ipange malire okhazikika kuti iwunikenso malo ogwirizana, ERES kuphatikiza katundu idayesedwa. Kusanthula kwa mzere kunachitika pogwiritsa ntchito pulogalamu ya MetaMorph (Molecular Devices).
Gwiritsani ntchito pulogalamu ya GraphPad Prism kuti mudziwe kufunika kwa ziwerengero. Pa mayeso a t-test a Student okhala ndi michira iwiri ndi mayeso wamba a kusanthula kwa kusiyana kwa njira imodzi (ANOVA), kusiyana pakati pa magulu kumaonedwa kuti kumakhudza kwambiri P <0.05 (*).
Pa microscopy ya fluorescence ya Gas1-GFP, maselo a log phase adakulitsidwa usiku wonse mu YPD ndikusonkhanitsidwa pogwiritsa ntchito centrifugation, kutsukidwa kawiri ndi phosphate buffered saline, ndikuyikidwa pa ayezi kwa mphindi zosachepera 15, kenako kupitilira pansi pa maikulosikopu monga momwe tafotokozera kale Onani (24). Maikulosikopu ya Leica DMi8 (HCX PL APO 1003/1.40 oil PH3 CS) yokhala ndi lens yolunjika, fyuluta ya L5 (GFP), kamera ya Hamamatsu ndi pulogalamu ya Application Suite X (LAS X) idagwiritsidwa ntchito kuti ipezeke.
Zitsanzozo zinachotsedwa ndi SDS sample buffer pa 65°C kwa mphindi 10, kenako zinalekanitsidwa ndi SDS-polyacrylamide gel electrophoresis (PAGE). Pa kusanthula kwa immunoblotting, 10 μl ya chitsanzo inayikidwa pa msewu uliwonse. Antibody yoyamba: Gwiritsani ntchito rabbit polyclonal anti-Gas1 pa dilution ya 1:3000, rabbit polyclonal anti-Emp24 pa dilution ya 1:500, ndi rabbit polyclonal anti-GFP (mphatso yochokera kwa H. Riezman) pa dilution ya 1:3000. Antibody ya mbewa ya monoclonal anti-Pgk1 inagwiritsidwa ntchito pa dilution ya 1:5000 (mphatso yochokera kwa J. de la Cruz). Antibody yachiwiri: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) yogwiritsidwa ntchito pa dilution ya 1:3000 (Pierce). Mankhwala a HRP-conjugated goat anti-mbewa IgG adagwiritsidwa ntchito pochepetsa 1:3000 (Pierce). Malo oteteza chitetezo cha mthupi adawonedwa pogwiritsa ntchito njira ya chemiluminescence ya SuperSignal West Pico reagent (Thermo Fisher Scientific).
Monga tafotokozera mu (31), kuyesa kwachilengedwe kwa immunoprecipitation kunachitika pa gawo la ER lolemera. Mwachidule, sambitsani maselo a yisiti ndi TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ndi protease inhibitor mixture) pa 600 nm (OD600) pa 100 optical density kawiri. Inasweka ndi mikanda yagalasi, kenako zinyalala za maselo ndi mikanda yagalasi zinachotsedwa ndi centrifugation. Supernatant kenako inayikidwa centrifuge pa 17,000 g kwa mphindi 15 pa 4°C. Pellet inabwezeretsedwa mu TNE ndipo digitalis saponin inawonjezedwa ku kuchuluka komaliza kwa 1%. Kuyimitsidwako kunayikidwa mu incubation kwa ola limodzi ndi kuzungulira pa 4°C, kenako zigawo zosasungunuka zinachotsedwa ndi centrifugation pa 13,000 g pa 4°C kwa mphindi 60. Pa Gas1-GFP immunoprecipitation, choyamba ikani chitsanzocho musanayambe ndi mikanda yopanda kanthu ya agarose (ChromoTek) pa 4°C kwa ola limodzi, kenako ikani mu GFP-Trap_A (ChromoTek) pa 4°C kwa maola atatu. Mikanda yopangidwa ndi immunoprecipitation inatsukidwa kasanu ndi TNE yokhala ndi 0.2% digoxigenin, yochotsedwa ndi SDS sample buffer, yolekanitsidwa pa SDS-PAGE, ndikuwunikidwa ndi immunoblotting.
Monga tafotokozera mu (31), kusanthula kwa cross-linking kunachitika pa chigawo cha ER cholemera. Mwachidule, chigawo cha ER cholemeracho chinayikidwa ndi 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, mphindi 20). Kusanthula kwa crosslinking kunathetsedwa powonjezera glycine (50 mM final concentration, mphindi 5, 20°C).
Monga tafotokozera kale (50), kusanthula kwa MS kwa ceramide mu wild-type ndi GhLag1 strains kunachitika. Mwachidule, maselo adakula kufika pa exponential phase (3 mpaka 4 OD600 units/ml) mu YPD pa 30°C, ndipo maselo 25×107 adasonkhanitsidwa. Kagayidwe kawo ka thupi kamathetsedwa ndi trichloroacetic acid. Gwiritsani ntchito solvent yochotsera [ethanol, madzi, ether, pyridine ndi 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] ndi 1.2 nmol ya C17 ceramide yamkati (860517, Avanti polar lipid) quality). Gwiritsani ntchito monomethylamine reagent [methanol, madzi, n-butanol ndi methylamine solution (4:3:1:5 v/v)] kuti mugwiritse ntchito alkaline hydrolysis yofatsa ya chotsitsacho, kenako gwiritsani ntchito n-butanol yodzazidwa ndi madzi kuti muchotse mchere. Pomaliza, chotsitsacho chinabwezeretsedwanso mu positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] ndipo chinalowetsedwa mu mass spectrometer. Kuyang'anira kwa Multi-reaction (MRM) kunachitika kuti kuzindikire ndi kuwerengera mamolekyu a sphingolipid. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ili ndi robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) yowunikira mafuta. Mphamvu ya collision imakonzedwa bwino pa gulu lililonse la ceramide. Deta ya MS idapezeka mu positive mode. Pa biological replica iliyonse, chizindikiro cha lipid ndi apakatikati a miyeso itatu yodziyimira payokha.
Monga tafotokozera mu (31), maselo (800×107) omwe amawonetsa Gas1-GFP adayikidwa mu immunoprecipitation yachilengedwe. Gas1-GFP yoyeretsedwayo idalekanitsidwa ndi SDS-PAGE ndikusamutsidwira ku nembanemba ya polyvinylidene fluoride (PVDF). Puloteniyo idawonetsedwa popaka utoto wa PVDF ndi amide wakuda. Gulu la Gas1-GFP linadulidwa kuchokera ku PVDF ndikutsukidwa kasanu ndi kamodzi ndi methanol ndipo kamodzi ndi madzi amadzimadzi a chromatography-MS (LC-MS). Mwa kuyika mzere wa nembanemba ndi 500μl 0.3 M NaOAc (pH 4.0), buffer ndi 500μl wosakaniza wa sodium nitrite wa 1 M wosungunuka kumene pa 37°C kwa maola atatu, gawo la lipid limatulutsidwa kuchokera ku Gas1-GFP ndikusinthidwa. Kutulutsidwa kwa inosine phosphate ceramide pakati pa glucosamine ndi inositol (51). Pambuyo pake, mzere wa nembanemba unatsukidwa kanayi ndi madzi a LC-MS, unaumitsidwa kutentha kwa chipinda, ndikusungidwa mumlengalenga wa nayitrogeni pa -80°C mpaka kusanthula. Monga chowongolera, chitsanzo chopanda kanthu cha nembanemba ya PVDF chinagwiritsidwa ntchito pa kuyesera kulikonse. Mafuta otengedwa kuchokera ku Gas1-GFP adawunikidwa ndi MS monga tafotokozera (50). Mwachidule, mizere ya PVDF yokhala ndi GPI-lipid idabwezeretsedwanso mu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ndikudutsa electrospray ionization (ESI)-MRM/MS Kusanthula kwa mitundu ya sphingolipid (TSQ Vantage). Pankhaniyi, deta ya MS idapezeka mu negative ion mode.
Monga tanenera kale, gawo la lipid la GPI anchor linalekanitsidwa ndi [3H]-inositol-labeled GPI-AP (16). Ma lipids analekanitsidwa ndi thin-layer chromatography pogwiritsa ntchito solvent system (55:45:10 chloroform-methanol-0.25% KCl) ndipo anawonetsedwa pogwiritsa ntchito FLA-7000 (Fujifilm).
Maselo omwe ali ndi Gas1-GFP (600×107) adatsukidwa kawiri ndi TNE buffer yokhala ndi TNE buffer, ndikusweka ndi mikanda yagalasi, kenako adayikidwa pakati kuti achotse zinyalala za maselo ndi mikanda yagalasi. Supernatant kenako idayikidwa pakati pa 17,000 g kwa ola limodzi pa 4°C. Pellet idatsukidwa mu TNE ndikuyikidwa mkati ndi 1 U PI-PLC (Invitrogen) mu TNE yokhala ndi 0.2% digitalis saponin kwa ola limodzi pa 37°C. Pambuyo pa chithandizo cha enzyme, nembanemba idachotsedwa poyimitsa pa 17,000 g pa 4°C kwa ola limodzi. Kuti Gas1-GFP iteteze chitetezo cha mthupi, supernatant idayikidwa mkati ndi GFP-Trap_A (ChromoTek) pa 4°C usiku wonse. Gas1-GFP yoyeretsedwa yolekanitsidwa ndi SDS-PAGE idapakidwa utoto wabuluu wa Coomassie. Mzere wopaka utoto wa Gas1-GFP unadulidwa kuchokera ku imvi yozungulira ngalande, kenako pambuyo pa alkylation ndi iodoacetamide ndi kuchepetsa ndi dithiothreitol, kugaya mu gel ndi trypsin kunachitika. Kuchotsa ndi kuumitsa ma peptide a tryptic ndi ma peptide ndi GPI-glycans. Peptide youmayo inasungunuka mu 20 μl ya madzi. Ikani gawo (8μl) mu LC. Mzere wa octadecylsilane (ODS) (Develosil 300ODS-HG-5; m'mimba mwake wamkati 150 mm × 1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) unagwiritsidwa ntchito kulekanitsa ma peptide pansi pa mikhalidwe inayake ya gradient. Gawo loyenda ndi solvent A (0.08% formic acid) ndi solvent B (0.15% formic acid mu 80% acetonitrile). Dongosolo la Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) linagwiritsidwa ntchito kuchotsa column ndi solvent A mkati mwa mphindi 55 pa liwiro la 50 μl min-1 kwa mphindi 5, kenako kuchuluka kwa solvent B kunawonjezeka kufika pa 40%., United States). Eluate inalowetsedwa mosalekeza mu gwero la ESI ion, ndipo ma peptide a tryptic ndi ma peptide okhala ndi GPI-glycans anawunikidwa ndi LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Mu MS setup, voltage ya gwero la capillary inayikidwa pa 4.5 kV, ndipo kutentha kwa transfer capillary kunasungidwa pa 300°C. Voltage ya capillary ndi tube lens voltage zinayikidwa pa 15 V ndi 50 V, motsatana. Deta ya MS idapezeka mu positive ion mode (resolution ya 60,000; mass accuracy of 10 pa milioni) mu mass range ya 300/m/z mass/charge ratio (m/z) 3000. Deta ya MS/MS imapezeka kudzera mu ion trap mu LTQ Orbitrap XL [manambala atatu oyamba omwe deta imadalira, collision induced dissociation (CID)].
Kuyeserera kwa MD kunachitika pogwiritsa ntchito pulogalamu ya GROMACS (52) ndi MARTINI 2 force field (53-55). Kenako CHARMM GUI Membrane Builder (56, 57) idagwiritsidwa ntchito popanga bilayer yokhala ndi dioleoylphosphatidylcholine (DOPC) ndi Cer C18 kapena DOPC ndi Cer C26. Topology ndi coordinates za Cer C26 zimachokera ku DXCE pochotsa mikanda yowonjezera kuchokera kumchira wa sphingosine. Gwiritsani ntchito njira yomwe yafotokozedwa pansipa kuti mulinganize wosanjikiza wawiri ndikuyendetsa, kenako gwiritsani ntchito coordinates yomaliza ya dongosololi kuti mupange dongosolo lomwe lili ndi Emp24. Malo osinthira a yisiti Emp24 (zotsalira 173 mpaka 193) adapangidwa ngati α-helix pogwiritsa ntchito kapangidwe ka molekyulu ya chida cha visual MD (VMD) (58). Kenako, atachotsa lipids zolumikizana, puloteniyo idakulungidwa pang'ono ndikuyikidwa mu bilayer pogwiritsa ntchito CHARMM GUI. Dongosolo lomaliza lili ndi 1202 DOPC ndi 302 Cer C26 kapena 1197 DOPC ndi 295 Cer C18 ndi Emp24. Ikani ioni pa dongosololi kufika pa 0.150M. Ma kopi anayi odziyimira pawokha adapangidwa pakupanga zinthu ziwiri za bilayer.
Chigawo cha lipid chimayendetsedwa bwino pogwiritsa ntchito njira ya CHARMM GUI, yomwe imaphatikizapo kuchepetsa kenako kulinganiza masitepe 405,000, pomwe zoletsa za malo zimachepetsedwa pang'onopang'ono ndikuchotsedwa, ndipo sitepe ya nthawi imawonjezeka kuchoka pa 0.005 ps kufika pa 0.02 ps. Pambuyo polinganiza, imapanga 6 µs ndi sitepe ya nthawi ya 0.02 ps. Mukayika Emp24, gwiritsani ntchito njira yomweyo ya CHARMM GUI kuti muchepetse ndikulinganiza dongosolo, kenako muthamange kwa masekondi 8 mukupanga.
Pa machitidwe onse, panthawi yolinganiza, kupanikizika kumayendetsedwa ndi Berendsen barostat (59), ndipo panthawi yopangira, kupanikizika kumayendetsedwa ndi Parrinello-Rahman barostat (60). Nthawi zonse, kupanikizika kwapakati ndi 1 bar ndipo njira yolumikizira kupanikizika kwa semi-isotropic imagwiritsidwa ntchito. Mu njira yolinganiza ndi kupanga, thermostat (61) yokhala ndi liwiro lokonzanso imagwiritsidwa ntchito kuphatikiza kutentha kwa mapuloteni, mafuta ndi tinthu ta solvent motsatana. Pa ntchito yonse, kutentha komwe kumayang'aniridwa ndi 310K. Kuyanjana kosalumikizana kumawerengedwa popanga mndandanda wolumikizirana pogwiritsa ntchito Verlet scheme yokhala ndi 0.005 buffer tolerance. Coulomb term imawerengedwa pogwiritsa ntchito reaction field ndi cut-off distance ya 1.1 nm. Vander Waals term imagwiritsa ntchito cut-off scheme yokhala ndi cut-off distance ya 1.1 nm, ndipo Verlet cut-off scheme imagwiritsidwa ntchito poyendetsa (62).
Pogwiritsa ntchito VMD, kutalika kwa malekezero pakati pa mikanda ya phosphate ya DOPC kapena mikanda ya ceramide AM1 ndi mapuloteni ndi 0.7 nm, ndipo chiwerengero cha mafuta omwe amalumikizana ndi mapuloteni chimawerengedwa. Malinga ndi fomula yotsatirayi, werengani chinthu chochepetsa kuchuluka kwa mafuta (DE) monga momwe zilili mu (63): DE factor = (kuchuluka kwa mafuta onse mu mapuloteni 0.7) mu mapuloteni 0.7 (kuchuluka kwa Cer mu mafuta onse)
Mtengo wonenedwawo umapezeka ngati avareji, ndipo mipiringidzo ya zolakwika ndi makope anayi odziyimira pawokha a SE. Kufunika kwa ziwerengero za DE factor kumawerengedwa ndi mayeso a t [(averageDE-factor-1)/SE]. Werengani mtengo wa P kuchokera ku kugawa kwa mchira umodzi.
Chida cha GROMACS chinagwiritsidwa ntchito kuwerengera mapu a 2D lateral density a dongosolo lomwe lili ndi Emp24 mkati mwa 250 ns yomaliza ya trace. Kuti mupeze mapu owonjezera/kuchepa kwa ceramide, mapu a density a Cer amagawidwa ndi kuchuluka kwa mapu a Cer ndi DOPC, kenako amagawidwa ndi kuchuluka kwa Cer m'thupi. Muyeso womwewo wa mapu umagwiritsidwa ntchito.
Kuti mudziwe zambiri zowonjezera pa nkhaniyi, chonde onani http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Iyi ndi nkhani yotseguka yogawidwa motsatira malamulo a Creative Commons Attribution-Non-Commercial License, yomwe imalola kugwiritsa ntchito, kufalitsa ndi kubwerezanso mu njira iliyonse, bola ngati kugwiritsidwa ntchito komaliza sikuli kopindulitsa pamalonda ndipo mfundo ndi yakuti ntchito yoyambirira ndi yolondola.
Dziwani: Tikukupemphani kuti mupereke imelo yanu kuti munthu amene mwamulimbikitsa patsamba lino adziwe kuti mukufuna kuti aone imeloyo ndipo si sipamu. Sitidzalemba ma adilesi aliwonse a imelo.
Funso ili limagwiritsidwa ntchito poyesa ngati ndinu mlendo ndikuletsa kutumiza sipamu yokha.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perezgier Lopero Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Kujambula zithunzi zenizeni zenizeni za 3D kukuwonetsa kufunika kwa kutalika kwa unyolo wa ceramide pakusankha mapuloteni m'malo osankhidwa.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perezgier Lopero Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Kujambula zithunzi zenizeni zenizeni za 3D kukuwonetsa kufunika kwa kutalika kwa unyolo wa ceramide pakusankha mapuloteni m'malo osankhidwa.
©2020 American Association for the Advancement of Science. maumwini onse ndi otetezedwa. AAAS ndi mnzake wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ndi COUNTER. ScienceAdvances ISSN 2375-2548.