Protein ƒe hatsotsowo me toto le adzamemɔa me le vevie ŋutɔ be woalé lãmenugbagbeviwo ƒe mama ɖe akpa vovovowo me kple woƒe homeostasis me ɖe asi. Tsɔ kpe ɖe lãmiwo ƒe vovototodedeameme to gowo dzi ŋu la, akpa si amiwo wɔna le kinesin ƒe vovototodedeameme me le adzameʋuʋu ƒe ɖoɖoa me nye nyabiase vevi aɖe si li ɣeyiɣi didi aɖee nye sia si ŋu womekpɔ ŋuɖoɖo na haɖe o. Le afisia la, míewɔa 3D le ɣeyiɣi ɖeka me amadede vovovowo ƒe nɔnɔmetatawo ɖeɖe le ɣeyiɣi ŋutɔŋutɔ me tsɔ ɖoa ​​kpe edzi le vivo be protein yeye siwo wowɔ kple glycosylphosphatidylinositol-immobilized protein siwo me ceramide lipid ƒe akpa didi ŋutɔ le la ƒoa ƒu eye woda wo ɖe hatsotso tɔxɛwo me ɖe endoplasms tɔxɛwo me Net exit site, si to vovo na esi transmembrane proteinwo zãna. Tsɔ kpe ɖe eŋu la, míeɖee fia be kɔsɔkɔsɔ ƒe didime si le ceramide me le endoplasmic reticulum membrane me le vevie ŋutɔ na vovototodedeameme ƒe tiatia sia. Míaƒe numekukua na kpeɖodzi gbãtɔ si le agbe me tẽ be woatsɔ ade protein ƒe agba siwo wotu ɖe lipid kɔsɔkɔsɔ ƒe didime dzi ɖe teƒe tiatia siwo woɖona ɖe duta le adzamemɔa me.
Le eukaryotic lãmenugbagbeviwo me la, emegbe woɖea protein siwo wowɔ le endoplasmic reticulum (ER) me la ɖe hatsotsowo me le wo tsɔtsɔ yi teƒe bubu to adzamemɔa dzi be woatsɔ wo ayi woƒe lãmenugbagbeviwo ƒe teƒe si sɔ (1). Tsɔ kpe ɖe coat-mediated sorting ŋu la, wosusui ɣeyiɣi didi aɖe be lipid aɖewo hã ate ŋu anye dodoƒe tiatia to wo ƒoƒo ƒu ɖe membrane domains tɔxɛ siwo me protein tɔxɛwo le me (2-5). Gake kpeɖodzi aɖeke megali tẽ le agbe me si ɖo kpe mɔnu sia si ate ŋu anɔ te ɖe ami dzi dzi o. Be míakpɔ kuxi vevi sia gbɔ la, míesrɔ̃ nu tso alesi wotsɔa glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) yia duta le mɔ vovovowo nu tso ER me ŋu le amɔwɔʋãnu me. GPI-APs nye lãmenugbagbeviwo ƒe anyigbadzi protein vovovo siwo do ƒome kple ami ( 6, 7 ). GPI-AP nye protein si woɖena ɖe go si léna ɖe plasma membrane ƒe gotagome aŋgbawo ŋu to glycolipid ƒe akpa (GPI anchor) dzi. Woxɔa GPI sekewo be wonye tɔtrɔ siwo wowɔna le gbegɔmeɖeɖe megbe le ER lumen me (8). Ne wotsɔe kpe ɖe eŋu vɔ la, GPI-AP toa Golgi-mɔ̃a me (5, 9) tso ER la me yia ʋumenugbagbeviɣia me. GPI ƒe sekewo ƒe anyinɔnɔ nana be wotsɔa GPI-AP ɖe vovo tso protein siwo woɖena tsoa lãkusi me (siwo dome protein siwo le ʋumenugbagbevi dzĩ me bubuwo hã le) gbɔ to ʋuʋudedimɔa dzi ( 5 , 9 , 10 ). Le amɔwɔʋãnu lãmenugbagbeviwo me la, woɖea GPI-APwo ɖa tso protein bubu siwo woɖena ɖe go le endoplasmic reticulum me, eye emegbe woblaa wo ɖe vesicles tɔxɛ siwo wotsɔ coat protein complex II (COPII) xatsa me (6, 7). Nusiwo dea dzesi vovototodedeameme ƒe ɖoɖo sia le ER ƒe dɔdɔ ƒe ɖoɖoa me me mekɔ o, gake wosusui be mɔnu sia ate ŋu abia be woatsɔ ami, vevietɔ ami ƒe akpa si le GPI sekea ƒe wɔwɔme gbugbɔgawɔ (5, 8). Le amɔwɔʋãnu me la, GPI ƒe ami ƒe tɔtrɔ dzea egɔme enumake ne GPI la kpe ɖe eŋu, eye le go geɖe me la, enaa ceramide blana ɖe 26-carbon long-chain saturated fatty acid (C26:0) ŋu (11, 12). C26 ceramide ye nye ceramide vevitɔ si amɔwɔʋãnu lãmenugbagbeviwo wɔ vaseɖe fifia. Wowɔa esia le ER me eye wotsɔa eƒe akpa gãtɔ yia Golgi mɔ̃a me to COPII vesicles (13) dzi. GPI-AP ƒe ER ɖoɖo ɖe duta bia koŋ be woawɔ ceramide ƒe wɔwɔme si ayi edzi ( 14 , 15 ), eye le esia ta la, ceramide ƒe tɔtrɔ wòazu inositol phosphate ceramide (IPC) le Golgi dɔwɔnua me nɔ te ɖe GPI anchor synthesis dzi ( 16 ). Nu gbagbewo ŋuti numekuku siwo wowɔ kple lãkusi siwo wowɔ kple asi ɖee fia be acyl kɔsɔkɔsɔ ƒe ceramide didi ŋutɔwo ate ŋu aƒo ƒu awɔ domenyinyi siwo le ɖoɖo nu siwo si dzɔdzɔmeŋusẽ ƒe nɔnɔme tɔxɛwo le ( 17 , 18 ). Nyatakaka siawo na wosusui be C26 ceramide kple GPI-AP kple C26 ceramide zãa woƒe dzɔdzɔmenɔnɔmewo tsɔ ƒoa ƒu zua nuto alo nuto siwo le ɖoɖo nu le ER membrane lipid nɔnɔme si me tɔtɔ vie la me. Glycerolipid kpuiwo kple esiwo me mekɔ o (C16:1 kple C18:1) koŋue le eme (19, 20). Woatia nuto siawo ɖe ER ƒe dodoƒe tɔxɛwo (ERES), afisi woate ŋu atsɔ ceramide kple ceramide-si wotu ɖe GPI-AP dzi ɖekae ayi Golgi le COPII vesicle ɖeka ma ke si woɖo ɖi me (5).
Le numekuku sia me la, míedo mɔ̃ sia si wotu ɖe ami dzi la kpɔ tẽ to super-resolution confocal real-time imaging microscopy (SCLIM) zazã me, si nye nusuekpɔmɔ̃ deŋgɔ aɖe si ate ŋu alé ŋku ɖe protein siwo wotsɔ fluorescent de dzesii ŋu le ɣeyiɣi ɖeka me Amadede etɔ̃ kple akpa etɔ̃ (3D) nɔnɔmetatawo ƒe nɔnɔmetatawo ƒe vovototo kple duƒuƒu lolo ŋutɔ le lãmenugbagbevi gbagbewo me (21, 22).
Míezã SCLIM mɔ̃ɖaŋununya gbã tsɔ ɖe alesi wodo GPI-AP si sɔ kple C26 ceramide ƒuƒoƒo kpɔ tso protein siwo woɖe tso lãmenugbagbeviwo me le dzodzo le ER me le S. cerevisiae me la me yi ŋgɔe. Be míate ŋu alé ŋku ɖe ER ƒe hatsotsowo ŋu la, míezã domenyiŋusẽfianu si ate ŋu akpɔ agba yeye siwo wowɔ la le susu me tẽ be woage ɖe ERES me le agbe me ( 7 , 23 ). Abe agba ene la, míetia C26 ceramide-si wotu ɖe GPI-AP Gas1 si wotsɔ green fluorescent protein (GFP) de dzesii kple transmembrane secreted protein Mid2 si wotsɔ near-infrared fluorescent protein (iRFP) de dzesii, siwo katã woɖo taɖodzinu na plasma membrane (24-26). Le sec31-1 dzoxɔxɔ-sensitive mutant me la, woɖea agba eve siawo fiana le galactose-inducible promoter kple constitutive ERES marker te. Le dzoxɔxɔ si gbɔ eme (37°C), esi sec31-1 ƒe tɔtrɔ kpɔa ŋusẽ ɖe COPII ƒe awudziwui ƒe akpa si nye Sec31 ƒe dɔwɔwɔ dzi be wòaxe mɔ ɖe COPII ƒe miemiã kple ER ƒe dɔdɔ nu ta la, agba yeye siwo wowɔ la ƒoa ƒu ɖe ER (23). Esi wofae va ɖo dzoxɔxɔ si bɔbɔ (24°C) vɔ la, sec31-1 ƒe lãmenugbagbevi siwo trɔ la gahaya tso teƒe si woɖea nu le, eye wodze agba yeye siwo wowɔ siwo woƒo ƒu la ɖoɖo ɖe duta tso ER la me. CLIM ƒe nukpɔkpɔ ɖee fia be Gas1-GFP kple Mid2-iRFP yeye siwo wowɔ yeyee ƒe akpa gãtɔ gakpɔtɔ ƒoa ƒu ɖe sec31-1 lãmenugbagbevi siwo trɔ la ƒe ER me le woƒe ƒoƒo le 37°C megbe eye emegbe woɖe asi le wo ŋu le 24°C hena miniti 5 (Nɔnɔmetata 1). Esi wònye be Mid2-iRFP kaka ɖe ER ƒe lãkusi bliboa dzi, eye Gas1-GFP ƒoa ƒu heƒoa ƒu ɖe ER lãkusi ƒe nuto si me mekɔ o me ta la, woƒe mama to vovo kura (Nɔnɔmetata 1, A vaseɖe C kple Sinima S1). Gakpe ɖe eŋu la, abe alesi woɖee fia le nɔnɔmetata 1D me ene la, Mid2-iRFP mele Gas1-GFP ƒuƒoƒoa me o. Nusiwo do tso eme siawo ɖee fia be woma GPI-AP kple transmembrane proteinwo ɖe ER membrane nuto vovovowo me kaba. Gas1-GFP ƒuƒoƒoa te ɖe ERES tɔxɛ aɖe si wotsɔ mCherry ƒe COPII coat protein Sec13 de dzesii ŋu (Nɔnɔmetata 1, E kple F, kple sinima S1) (23).
sec31-1 lãmenugbagbeviwo ɖea galactose-si woɖena fiana, acyl kɔsɔkɔsɔ didi aɖe (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, amadede dzẽ) kple transmembrane protein Mid2-iRFP (TMP, blɔ) eye wotsɔ Constructive ERES labeling Sec13-mCherry (ERES, magenta) sia de 37°C hena miniti 30, ʋu yi 24°C, eye woɖe fotoe to SCLIM dzi le miniti 5 megbe. (A vaseɖe C) fia yameʋu (A) ƒe 2D nɔnɔmetata si wotsɔ ƒo ƒu alo ɖeka si le tsitre ɖi na, 2D ƒe nɔnɔmetata si woɖe ɖe go si ƒe z-afã 10 (B) alo agba kple ERES dzesiwo ƒe 3D lãmenugbagbeviwo ƒe afã ƒe nɔnɔmetata (C). Nudzidzenu ƒe ʋuƒo 1μm (A kple B). Nudzidzenu ƒe xexlẽme nye 0.551μm (C). Wokpɔ Gas1-GFP le ER nuto alo ƒuƒoƒo siwo le vovo me, gake wokpɔ Mid2-iRFP eye womae ɖe ER ƒe lãkusi (C) katã me. (D) Nɔnɔmetata la fia Gas1-GFP kple Mid2-iRFP ƒe keklẽŋusẽ ƒe sesẽme le Gas1-GFP ƒe ƒuƒoƒoa me le aŋutrɔ ɣia ƒe fli dzi (miame). AU, unit si wowɔna le wo ɖokui si. (E kple F) tsi tre ɖi na 3D nɔnɔmetata si ƒo adzɔnuwo kple ERES dzesi nu ƒu. Wokpɔ Gas1-GFP ƒe ƒuƒoƒo siwo te ɖe ERES tɔxɛa ŋu. Nudzidzenu ƒe xexlẽme nye 0.551μm. (F) Aŋutrɔ sesẽ ɣia de dzesi Gas1-GFP ƒe ƒuƒoƒo si do ƒome kple ERES. Titina kple ɖusime ƒe akpaawo ɖe 3D nɔnɔmetata si wodzi ɖe edzi si wotsɔ ƒo ƒui kple Gas1-GFP ƒuƒoƒo si wotia ƒe nukpɔkpɔ si wogbugbɔ trɔ la fia.
Teƒe ƒe ƒomedodo kplikplikpli si le Gas1-GFP ƒuƒoƒoa kple ERES tɔxɛ aɖe dome ɖee fia be Gas1-GFP ateŋu age ɖe ERES tiatia me, si to vovo na tiatiawɔwɔ si Mid2-iRFP zãna tsɔ doa le ER me. Be míakpɔ nusi ate ŋu adzɔ sia gbɔ la, míebu ERES ƒe xexlẽme ƒe agbɔsɔsɔme na adzɔnu ɖeka alo eve ko (Nɔnɔmetata 2, A vaseɖe C). Míekpɔe be agba ƒomevi ɖeka koe le ERES akpa gãtɔ (70%) me. Nɔnɔmetata 2C ƒe nɔnɔmetata si le ete la ɖe ERES ƒe kpɔɖeŋu eve siwo bɔ siwo me Gas1-GFP koe le (Nɔnɔmetata 1) alo Mid2-iRFP koe le (Nɔnɔmetata 2) fia. To vovo na esia la, agba eve siwo ƒoa xlã wo nɔewo le nuto ɖeka me le ERES ƒe 20% lɔƒo. Wokpɔe be agba ƒomevi eve le ERES aɖewo (10%) me, gake woɖe wo ɖe aga le teƒe siwo to vovo kɔte. Eyata akɔntabubu sia ɖee fia be le ER la ɖoɖo ɖe duta megbe la, woma GPI-AP Gas1-GFP kple transmembrane cargo Mid2-iRFP ɖe ERES vovovowo me (Nɔnɔmetata 2D). Nuwo ƒe vovototodedeameme ƒe ŋutete sia sɔ kple nugbagbeŋutinunya me dzodzro si wowɔ va yi (6) kple nɔnɔme ŋuti nyametsotso (7) ŋutɔ. Míate ŋu alé ŋku ɖe agba siwo woɖe ɖe vovo siwo ge ɖe ERES me ƒe nuwɔna hã ŋu (Nɔnɔmetata 2E kple Sinima S2). Nutata 2E ɖee fia be Gas1-GFP (panel 3) alo Mid2-iRFP (panel 4) ƒe akpa sue aɖe koe gena ɖe ERES me tso akpa ɖeka eye wotu ɖe teƒe si woɖe ɖe vovo. Nɔnɔmetata 2E ƒe akpa 5 lia ɖee fia be Gas1-GFP kple Mid2-iRFP nɔa ERES ɖeka me ɣeaɖewoɣi, gake wogena ɖe eme tso axa vovovowo eye woƒoa ƒu ɖe nuto vovovowo me siwo ate ŋu atsi tre ɖi na COPII ƒe ʋuʋudedi vovovowo. Míeɖo kpe edzi hã be C26 ceramide-si wotu ɖe GPI-AP Gas1 ƒe mama kple hatsotso si wokpɔ be enye tiatia ERES la le etɔxɛ elabena transmembrane secretion cargo bubu, si nye GFP-tagged plasma membrane protein Axl2 (27 ), si ɖe nuwɔna si sɔ kple Mid2-iRFP fia. (Nɔnɔmetata S1 kple Sinima S3). Axl2-GFP yeye si wowɔ la ma to ER lãkusi me abe Mid2-iRFP ene (Nɔnɔmetata S1, A kple B), eye wòle teƒe ɖeka kple Mid2-iRFP le ERES akpa gãtɔ me (Nɔnɔmetata S1, B vaseɖe D). Panel 1 kple 2 le Figure 1. S1C fia ERES ƒe kpɔɖeŋu eve siwo bɔ afisi agba eve siwo toa lãkusi me ƒoa ƒu ɖo. Le nɔnɔme siawo me la, adzɔnu eveawo siaa gena ɖe ERES me ɖekae (Nɔnɔmetata S1E, Panel 3 kple Movie S3).
Wotsɔ sec31-1 lãmenugbagbevi siwo ɖea galactose inducible secretions fiana, Gas1-GFP (GPI-AP, green) kple Mid2-iRFP (TMP, blue) kple constitutive ERES labeling Sec13-mCherry (ERES, magenta) da ɖe 37 Esi wotsɔ wo de lãme na wo aɖabaƒoƒo 30 le °C megbe la, woʋu yi 24 °C be woaɖe asi le secretion block la ŋu, eye woatsɔ SCLIM awɔ nɔnɔmetata le ema megbe Aɖabaƒoƒo 20 sɔŋ. (A vaseɖe C) Teƒenɔla 2D projection nɔnɔmetatawo (A; scale bar, 1μm) alo 3D cell hemisphere nɔnɔmetatawo (B kple C; scale unit, 0.456μm) le agbaa kple z-afã 10 siwo wode dzesii kple ERES. Akpa si le ete le (B) me kple akpa si le (C) me ɖea nɔnɔmetata siwo ŋu wotrɔ asi le fiana be woaɖe adzɔnu siwo le ERES (magenta) [Gas1-GFP (gray) kple Mid2-iRFP (blɔ keklẽ)] me ko afia. (C) Aŋutrɔ si le ʋuʋu ɖi: Agba ɖeka koe ERES tsɔna (1 vaseɖe 4). Aŋutsrɔe ɣi: Agba siwo woɖe ɖe vovo (5) le ERES me. Aŋutrɔ sesẽ ɣi: ERES si me agba siwo le teƒe ɖeka le. Le ete: Gas1-GFP (1) alo Mid2-iRFP (2) koe le ERES ɖeka si wotia la me. Nudzidzenu ƒe ʋuƒo, 100 nm. (D) Fotoɖemɔ̃ si ŋu woƒo nu tsoe le (C) me ƒe agbɔsɔsɔme. ERES ƒe alafa memamã le mama dedie nu si me agba ɖeka koe le (Gas1-GFP alo Mid2-iRFP), agba siwo woɖe ɖe vovo kple agba siwo ƒo xlã wo nɔewo. Le dodokpɔ etɔ̃ siwo wowɔ le wo ɖokui si me la, n=432 le lãmenugbagbevi 54 me. Vodada ƒe dzesi = SD. T dodokpɔ si me asike eve mele o. *** P = 0.0002 ƒe xexlẽme. (E) Agba siwo woɖe ɖe vovo ƒe ERES tiatia siwo wode dzesii kple (C) ƒe 3D nɔnɔmetata. Gas1-GFP (amadede dzẽ) (3) alo Mid2-iRFP (blɔ) (4) gena ɖe ERES (magenta) me tso akpa ɖeka eye woɖoe ɖe teƒe sue aɖe le ERES me. Ɣeaɖewoɣi la, agba ƒomevi eveawo siaa gena ɖe ERES ɖeka me (5) tso akpa ɖeka eye wotua wo ɖe teƒe aɖe si sa ɖe aga le ERES la me. Nudzidzenu ƒe ʋuƒo, 100 nm.
Eyome, míedo nukpɔsusu aɖe kpɔ be acyl chain ceramide didi (C26) si le ER membrane me la ʋãa Gas1 ƒe ƒuƒoƒo tɔxɛ kple eƒe hatsotsowo me toto wòzua ERES tiatia. Be míawɔ esia la, míezã amɔwɔʋãnu ƒomevi si ŋu wotrɔ asi le si nye GhLag1, si me wotsɔ GhLag1 (si nye Lag1 homolog of cotton) ɖɔ li endogenous ceramide synthases Lag1 kple Lac1, si na be yeast ƒomevi aɖe si ƒe lãmenugbagbeviwo ƒe lãkusi Ceramide ƒomevi le kpuie wu gbemelã ƒomevi (Nɔnɔmetata 3A) (28). Mass spectrometry (MS) numekuku ɖee fia be le gbeme ƒomeviwo me la, ceramide bliboa ƒe 95% nye kɔsɔkɔsɔ ƒe ceramide didi ŋutɔ (C26), gake le GhLag1 me la, ceramide ƒe 85% didi ŋutɔ (C18 kple C16). ), ceramide ƒe 2% koe nye ceramide si ƒe kɔsɔkɔsɔ didi ŋutɔ (C26). Togbɔ be C18 kple C16 ceramides ye nye ceramide vevi siwo wokpɔ le GhLag1 ƒe lãkusi me vaseɖe fifia hã la, MS numekuku hã ɖo kpe edzi be C26 ceramide le Gas1-GFP ƒe GPI seke si woɖe fia le GhLag1 ƒomevi me, si sɔ kple gbemelã ƒomeviwo ƒe amiwo. Nɔnɔmea sɔ (Fig. 3A) (26). Eyata esia fia be ceramide remodeling enzyme Cwh43 tiaa C26 ceramide ŋutɔ, abe alesi woɖee fia le nɔnɔmetata 26 me ene la, elɔ̃na be yeade GPI seke si tso C26 ceramide sue aɖe me le GhLag1 ƒomevi me. S2 (29) ƒe agbalẽa. Ke hã, C18-C16 ceramide koe le GhLag1 ƒe lãmenugbagbeviwo ƒe lãkusi me koŋ, evɔ C26 ceramide gakpɔtɔ le Gas1-GFP me. Nyateƒenya sia na be ƒomevi sia nye dɔwɔnu nyui aɖe si woatsɔ akpɔ kuxi si nye acyl kɔsɔkɔsɔ ƒe didime si le membrane ceramide me le ER me gbɔ koŋ. Akpa si klass kple vovototodedeameme wɔna le nususugblɔ me. Emegbe, míesrɔ̃ nu tso ŋutete si le C26 Gas1-GFP ŋu be wòaƒo ƒu ɖe hatsotsowo me le GhLag1 me kple dzoxɔxɔ-sensitive mutant allele si nye sec31-1 to fluorescence microscopy si bɔ me, afisi kɔsɔkɔsɔ didi (C18-C16) koe le ER membrane Ceramide me (Fig. 3). Míede dzesii be le sec31-1 me la, Gas1-GFP ƒe akpa gãtɔ ƒo ƒu ɖe hatsotsowo me, esime Gas1-GFP le sec31-1 GhLag1 si me didi (C18-C16) didi ceramide ER lãkusi me koŋ meƒo ƒu o eye woma ɖe Le ER lãkusi bliboa me. Ne míagblɔe tututu la, esi wònye be C26 ceramide-based clustering do ƒome kplikplikpli kple ERES tɔxɛwo (Nɔnɔmetata 1) ta la, míeku nu me emegbe nenye be ɖoɖo sia ate ŋu alɔ ER ƒe protein ƒe dɔwɔwɔ ɖe duta ƒe mɔnu hã ɖe eme hã. GPI-AP zãa COPII ɖoɖo tɔxɛ aɖe na ER dɔdɔ, si dzi Ted1 ƒe xɔtuɖoɖo gbugbɔgawɔ le GPI seke ƒe akpa si nye glycan ( 30 , 31 ). Emegbe wokpɔa GPI-glycan si wogbugbɔ ƒo ƒu la dzea sii to transmembrane cargo receptor p24 complex dzi, si hã tiaa Lst1, si nye isoform tɔxɛ aɖe na COPII agbatsɔtsɔ ƒe akpa gãtɔ Sec24, si wɔa GPI-AP-rich COPII Vesicles hiã (31-33). Eyata míewɔ nu eve ƒe tɔtrɔ si ƒo protein ɖeka siawo (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 kple COPII subunit tɔxɛ Lst1) ƒe ɖeɖeɖa nu ƒu kple sec31-1 mutant ƒomevi, eye míesrɔ̃ nu tso wo ŋu Ðe wòanya wɔ be woawɔ Gas1-cluster GFP (Nɔnɔmetata 3). Míede dzesii be le sec31-1emp24Δ kple sec31-1ted1Δ me la, Gas1-GFP koŋ nye esi womeƒo ƒu ɖe hatsotsowo me o eye wòma ɖe ER lãkusi bliboa me, abe alesi wokpɔe va yi le sec31-1 GhLag1 me ene, esime le sec31-1lst1Δ me la, Gas1-GFP Abe sec31-1 ene. Nusiwo do tso eme siawo ɖee fia be tsɔ kpe ɖe C26 ceramide ƒe anyinɔnɔ le ER lãkusi me ŋu la, ehiã be Gas1-GFP ƒe ƒuƒoƒo hã nabla ɖe p24 ƒe ƒuƒoƒoa ŋu, eye mehiã be woaxɔ Lst1 koŋ o. Emegbe, míedzro alesi wòate ŋu adzɔe be ceramide ƒe kɔsɔkɔsɔ ƒe didime le ER lãkusi me ate ŋu aɖɔ Gas1-GFP ƒe kadodo ɖe p24 ŋu ɖo. Ke hã, míekpɔe be C18-C16 ceramide ƒe anyinɔnɔ le lãkusia me mekpɔa ŋusẽ ɖe GPI-glycans siwo p24 complex (Nɔnɔmetata S3 kple S4, A kple B) gbugbɔ tu alo bla ɖe GPI-AP ŋu eye woɖoa GPI-AP ɖe duta dzi o. ŋutete. Xɔ COPII ƒe hatsotso sue Lst1 (Nɔnɔmetata S4C). Eyata C26 ceramide-dzikpɔkpɔ ƒe ƒuƒoƒo mehiã protein ƒe kadodo kple ER dɔdɔ ƒe protein ƒe mɔnu vovovowo o, ke boŋ edoa alɔ vovototodedeameme ƒe mɔnu bubu si lipid ƒe didime ʋãna. Emegbe, míedzro eme be ceramide acyl kɔsɔkɔsɔ ƒe didime le ER lãkusi me le vevie na Gas1-GFP ƒe hatsotso nyuitɔ abe ERES tiatia ene hã. Esi wònye be Gas1 si le GhLag1 ƒomevi si me kɔsɔkɔsɔ kpui ƒe ceramide le me dona le ER me eye wògena ɖe plasma membrane me (Nɔnɔmetata S5) ta la, míexɔe se be ne ceramide acyl kɔsɔkɔsɔa ƒe didimee ʋãa vovototodedeameme la, woate ŋu atrɔ Gas1 si le GhLag1 ƒomevi me eye woatsoe. ERES adzɔnuwo kple lãkusi ɖeka ma ke.
(A) C18-C16 ceramide kpuiwo koŋue le GhLag1 ƒe lãmenugbagbeviwo ƒe lãkusi me, gake C26 IPC si le gbemelãwo me la gakpɔtɔ le Gas1-GFP ƒe GPI seke me. Le etame: acyl kɔsɔkɔsɔ ƒe didime me dzodzro le ceramide ŋu le lãmenugbagbevi ƒe lãkusi me le gbemelã ƒomevi (Wt) kple GhLag1p ƒomeviwo me to mass spectrometry (MS) me. Nyatakakaawo tsi tre ɖi na ceramide bliboa ƒe alafa memamã. Dodokpɔ etɔ̃ siwo wowɔ le wo ɖokui si ƒe mamã dedie. Vodada ƒe dzesi = SD. T dodokpɔ si me asike eve mele o. **** P <0.0001 ƒe ɣeyiɣia. Anyigba ƒe akpa si le ete: MS numekuku le IPC si le Gas1-GFP (GPI-IPC) GPI seke si woɖe fia le gbemelã ƒomevi kple GhLag1p ƒomeviwo me ƒe acyl kɔsɔkɔsɔ ƒe didime ŋu. Nyatakakaawo tsi tre ɖi na IPC ƒe dzesi bliboa ƒe alafa memamã. Dodokpɔ atɔ̃ siwo wowɔ le wo ɖokui si le mamã dedie nu. Vodada ƒe dzesi = SD. T dodokpɔ si me asike eve mele o. ns, mele vevie o. P = 0.9134 ƒe xexlẽme. (B) Fluorescence micrographs of sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ and sec31-1lst1Δ cells expressing galactose-induced Gas1-GFP were incubated at 37°C for 30 minutes and passed down to Perform routine fluorescence microscopy after 24°C ƒe dzoxɔxɔ. Aŋutrɔ ɣi: ER Gas1-GFP ƒuƒoƒo. Aŋutrɔ si le ʋuʋu ɖi: Woma Gas1-GFP si womeƒo ƒu o ɖe ER ƒe lãkusi bliboa dzi, si ɖe ER ƒe dzesi nuklia ƒe asigɛ ƒe amadede fia. Nudzidzenu ƒe ʋuƒo, 5μm. (C) Fotoɖemɔ̃ si ŋu woƒo nu tsoe le (B) me ƒe agbɔsɔsɔme. Lãmenugbagbevi siwo si punctate Gas1-GFP wɔwɔme le ƒe alafa memamã le mama dedie nu. Le dodokpɔ etɔ̃ siwo wowɔ le wo ɖokui si me la, lãmenugbagbevi n≥300. Vodada ƒe dzesi = SD. T dodokpɔ si me asike eve mele o. **** P <0.0001 ƒe ɣeyiɣia.
Be míakpɔ kuxi sia gbɔ tẽ la, míewɔ SCLIM-nukpɔkpɔ le Gas1-GFP kple Mid2-iRFP me le GhLag1 me kple sec31-1 dzoxɔxɔ-sensitive mutant allele (Nɔnɔmetata 4 kple Movie S4). Esi wolé ER la ɖe 37°C eye emegbe woɖe asi le eŋu le 24°C vɔ la, womeƒo Gas1-GFP yeye si wowɔ la ƒe akpa gãtɔ nu ƒu hema ɖe ER ƒe lãkusi bliboa me o, abe alesi wokpɔe to nusuekpɔmɔ̃ siwo wozãna ɖaa me ene (Nɔnɔmetata 4, A kple B ). Gakpe ɖe eŋu la, ERES ƒe alafa memamã gã aɖe (67%) lɔ agba ƒomevi eve siwo le eme ɖekae (Nɔnɔmetata 4D). Nɔnɔmetata 4C ƒe akpa 1 kple 2 ɖe ERES ƒe kpɔɖeŋu eve siwo bɔ kple Gas1-GFP kple Mid2-GFP siwo tsi tre ɖe wo nɔewo ŋu fia. Tsɔ kpe ɖe eŋu la, woxɔ adzɔnu eveawo siaa ɖe ERES ɖeka me (Nɔnɔmetata 4E, akpa 3 kple sinima S4). Eyata míaƒe emetsonuwo ɖee fia be ceramide acyl kɔsɔkɔsɔ ƒe didime le ER ƒe lãkusi me nye nu vevi aɖe si dea dzesi ER protein ƒe ƒuƒoƒo kple eƒe hatsotsowo.
Sec31-1 GhLag1 lãmenugbagbevi siwo ɖea galactose-siwo hena vɛ, Gas1-GFP (GPI-AP, amadede dzẽ) kple Mid2-iRFP (TMP, blɔ) kple constitutive ERES-labeled Sec13-mCherry (ERES, magenta) fiana Wodea dzoe le 37°C. Yi edzi aɖabaƒoƒo 30, ɖiɖi va ɖo 24°C be wòaɖe tsi siwo dona le eme la ɖa, eye nàtsɔ SCLIM awɔ nɔnɔmetatae le miniti 20 megbe. (A vaseɖe C) Teƒenɔla 2D projection nɔnɔmetatawo (A; scale bar, 1μm) alo 3D cell hemisphere nɔnɔmetatawo (B kple C; scale unit, 0.45μm) le z-afã 10 siwo wotsɔ agba kple ERES de dzesii la me. Akpa si le ete le (B) me kple akpa si le (C) me ɖea nɔnɔmetata siwo ŋu wotrɔ asi le fiana be woaɖe adzɔnu siwo le ERES (magenta) [Gas1-GFP (gray) kple Mid2-iRFP (blɔ keklẽ)] me ko afia. (C) Aŋutrɔvi si yɔ fũu ɣi: ERES, adzɔnuwo ƒo xlã wo nɔewo. Aŋutrɔ si ʋu: Nu ɖeka koe le ERES me. Akpa si le ete: ERES si wotia la ƒe adzɔnuwo (1 kple 2) siwo wotsɔ de dzesii le (C) me la le wo nɔewo dzi. Nudzidzenu ƒe ʋuƒo, 100 nm. (D) Fotoɖemɔ̃ si ŋu woƒo nu tsoe le (C) me ƒe agbɔsɔsɔme. Le sec31-1 kple sec31-1 GhLag1 units me la, agba ɖeka koe (Gas1-GFP alo Mid2-iRFP) de eme, eye ERES ƒe alafa memamã le mama dedie nu na agba siwo woɖe ɖe aga kple agba siwo tsi tre ɖe wo nɔewo ŋu. Le dodokpɔ etɔ̃ siwo wowɔ le wo ɖokui si me la, n = 432 le lãmenugbagbevi 54 me (sec31-1) eye n = 430 le lãmenugbagbevi 47 me (sec31-1 GhLag1). Vodada ƒe dzesi = SD. T dodokpɔ si me asike eve mele o. *** P = 0.0002 (sec31-1) kple ** P = 0.0031 (sec31-1 GhLag1). (E) ERES tiatia aɖe ƒe 3D nɔnɔmetata si me agba siwo ƒo xlã wo nɔewo (3) si wode dzesii le (C) me. Gas1-GFP (amadede dzẽ) kple Mid2-iRFP (blɔ) tena ɖe ERES (magenta) ŋu tso akpa ɖeka eye wonɔa teƒe ɖeka si woxe mɔ ɖe ERES nu. Nudzidzenu ƒe ʋuƒo, 100 nm.
Numekuku sia na kpeɖodzi tẽ le agbe me be woda protein ƒe agba siwo wotu ɖe ami dzi ɖe hatsotsowo me ɖe teƒe tiatia siwo wotsɔ ɖoa ​​duta le adzamemɔa me, eye wòɖe acyl kɔsɔkɔsɔ ƒe didime ƒe vevienyenye fia na hatsotsowo me tiatia. Míezã nusuekpɔmɔ̃ sẽŋu aɖe si de ŋgɔ wu si woyɔna be SCLIM tsɔ ɖe Gas1-GFP (si nye plasma membrane GPI-AP gã aɖe si me acyl kɔsɔkɔsɔ didi ŋutɔ (C26) ceramide lipid ƒe akpa aɖe le) si wowɔ yeyee le amɔwɔʋãnu me fia ) Nuto siwo ƒo ƒu ɖe ER siwo le vovo me la do ƒome kple ERES tɔxɛwo, esime protein siwo woɖe ɖe go to lãkusi me la ma to ER lãkusi bliboa me (Nɔnɔmetata 1). Gakpe ɖe eŋu la, adzɔnu ƒomevi eve siawo gena ɖe ERES vovovowo me tiatiatɔe (Nɔnɔmetata 2). Woɖea lãmenugbagbeviwo ƒe ceramide si le lãkusia me ƒe acyl kɔsɔkɔsɔ ƒe didime dzi kpɔtɔna tso C26 va ɖo C18-C16, wogblẽa Gas1-GFP ƒuƒoƒoa me yia ER nuto si le vovo me, eye wogbugbɔa mɔ na Gas1-GFP be wòadzo le ER la me kple transmembrane protein la to ERES ma ke dzi (Nɔnɔmetata 3 kple Nɔnɔmetata 3). 4).
Togbɔ be GPI-AP zãa protein ƒe mɔnu tɔxɛ aɖe tsɔ doa go le ER hã la, míekpɔe be C26 ceramide-dzikpɔkpɔ ƒe mama meɖoa ŋu ɖe protein ƒe kadodo vovovo siwo ate ŋu ahe ERES ƒe etɔxɛnyenye vɛ ŋu o (Nɔnɔmetata S4 kple S5). Ke boŋ nusiwo ŋu míeke ɖo ɖo kpe vovototodedeameme ƒe mɔnu bubu aɖe si protein ƒe ƒuƒoƒo si wotu ɖe ami dzi kple agba bubuwo ɖeɖeɖa emegbe ʋãna dzi. Míaƒe ŋkuléleɖenuŋu ɖee fia be Gas1-GFP nuto alo ƒuƒoƒo si do ƒome kple ERES tɔxɛ aɖe la mekpɔ protein si woɖena tso lãkusi me si nye Mid2-iRFP o, si ɖee fia be GPI-AP ƒuƒoƒo si nɔa te ɖe C26 ceramide dzi la ana woƒe gege ɖe ERES si sɔ me nanɔ bɔbɔe, eye le ɣeyiɣi ma ke me la, woaɖe transmembrane ɖa The secretions enter this particular ERES (Nɔnɔmetata 1 kple 2). To vovo na ema la, C18-C16 ceramides ƒe anyinɔnɔ le ER ƒe lãkusi me menaa GPI-AP wɔa nuto alo ƒuƒoƒo o, eyata womeɖea protein siwo dona tso lãkusi me la ɖa alo ɖɔlia wo ɖe ERES ɖeka ma ke me o (Nɔnɔmetata 3 kple 4). . Eyata míedo susua ɖa be C26 ceramide ʋãa mama kple hatsotsowo to protein siwo do ƒome kple ERES tɔxɛwo ƒe ƒuƒoƒo me.
Aleke woawɔ aɖo C26 ceramide-dzikpɔkpɔ ƒe ƒuƒoƒo sia gbɔ ɖe ER nuto aɖe koŋ me? Alesi membrane ceramide dina be yeaklã le axadzi ate ŋu ana GPI-AP kple C26 ceramide nawɔ lipid sue siwo woɖo ɖe ɖoɖo nu enumake le lipid nɔnɔme si mesɔ o wu le ER membrane si me glycerolipids kpuiwo kple esiwo meyɔ fũ o me. Dzɔdzɔmeŋusẽ ƒe hatsotsowo (17, 18). Woate ŋu aƒo ɣeyiɣi kpui aɖe ƒe ƒuƒoƒo sue siawo nu ƒu ɖe ƒuƒoƒo gã siwo li ke wu me ne wobla ɖe p24 ƒe ƒuƒoƒoa ŋu vɔ ( 34 ). Le ɖekawɔwɔ me kple esia la, míeɖee fia be ehiã be C26 Gas1-GFP nawɔ nu kple p24 complex la be wòawɔ ƒuƒoƒo gã siwo woate ŋu akpɔ (Nɔnɔmetata 3). p24 complex nye heterozygous oligomer si me p24 transmembrane protein vovovo ene le le amɔwɔʋãnu me ( 35 ), si naa multivalent binding, si ate ŋu ana cross-linking na GPI-AP clusters suewo, si ana Stable cluster si lolo wu nado ( 34 ). GPI-APs ƒe protein ectodomains dome kadodo hã ate ŋu awɔ akpa aɖe le woƒe ƒuƒoƒo me, abe alesi woɖee fia le woƒe Golgi mɔzɔzɔ me le no me lãmenugbagbeviwo ƒe polarized epithelial cells me ene ( 36 ). Gake ne C18-C16 ceramide le ER ƒe lãkusi me, ne p24 ƒe ƒuƒoƒoa bla ɖe Gas1-GFP ŋu la, ƒuƒoƒo gã siwo to vovo madzɔ o. Mɔnu si le ete ate ŋu anɔ te ɖe acyl kɔsɔkɔsɔ didi si nye ceramide ƒe dzɔdzɔmeŋusẽ kple atike ƒe nɔnɔme tɔxɛwo dzi. Nu gbagbewo ŋuti numekuku siwo wowɔ le lãkusi siwo wowɔ ŋu ɖee fia be togbɔ be acyl kɔsɔkɔsɔ ƒe ceramide didi (C24) kple kpui (C18-C16) siaa ate ŋu ana phase ƒe mama nava hã la, acyl kɔsɔkɔsɔ didi ƒe ceramide (C24) koe ate ŋu ado Curvature gã kple film ƒe bɔbɔ ɖe ŋgɔ be wòatrɔ asi le film la ŋu. To wo nɔewo ƒe nuƒoƒo tso wo nɔewo ŋu me (17, 37, 38). Woɖee fia be TMED2 ƒe transmembrane helix, si nye amegbetɔ ƒe Emp24 ƒe homologue, tiaa nu kple C18 ceramide-based sphingomyelin le cytoplasmic lobules me ( 39 ). Míezã molecule dynamics (MD) simulations tsɔ kpɔe be C18 kple C26 ceramides siaa ƒoa ƒu ɖe Emp24 transmembrane helix ƒe cytoplasmic lobules ŋu, eye woƒe didiwo sɔ (Nɔnɔmetata S6). Enyo be míade dzesii be esia ɖee fia be Emp24 ƒe transmembrane helix ateŋu ahe lipids ƒe mama si mesɔ o vɛ le membrane la me. Esia nye nusi do tso eme nyitsɔ laa si wotu ɖe no me lãmenugbagbeviwo dzi. MD ƒe nɔnɔmetata mawo tɔgbe ɖe ether lipids ƒe anyinɔnɔ hã fia (40) . Eyata míesusu be C26 ceramide si le ER26 ƒe lobule eveawo me la sɔ gbɔ ɖe teƒea. Ne GPI-AP si le luminal lobules me bla ɖe multivalent p24 ŋu tẽ eye C26 ceramide ƒe ƒuƒoƒo ƒo xlã p24 le cytoplasmic lobules me la, ate ŋu ado Protein ƒe ƒuƒoƒo si kpe ɖe eŋu ɖe ŋgɔ eye membrane curvature dona to asibidɛwo dzi ( 41 ), si ana GPI-AP ma ɖe vovo ɖe nuto vovovo siwo te ɖe ERES ŋu me, si hã doa dzidzɔ na nuto siwo ƒo xlãe ŋutɔ nuto siwo le ER lãkusia me ( 42 ). Nyatakaka siwo woxɔ va yi da asi ɖe mɔnu si wodo ɖa dzi ( 43 , 44 ). Oligolectins, dɔlékuiwo alo dɔlékuiwutikewo ƒe kadodo geɖe kple ceramide-based glycosphingolipids (GSL) le plasma membrane dzi hea GSL ƒe ƒuƒoƒo gã aɖe vɛ, doa phase ƒe mama ɖe ŋgɔ eye wòhea membrane ƒe tɔtrɔ kple ememe nɔnɔ vɛ ( 44 ). Iwabuchi etc. (43) Wokpɔe be le acyl kɔsɔkɔsɔ didi (C24) gake menye kpui (C16) ƒe anyinɔnɔ me la, multivalent ligand si bla ɖe GSL lactosylceramide ŋu na ƒuƒoƒo gãwo kple membrane invagination dzɔ, eye cytoplasm Lyn-mediated signal transduction le agbalẽviawo dzi la, acyl kɔsɔkɔsɔ siwo wotsɔ ƒo ƒui la tsɔa wo nɔewo ƒoa ƒui neutrophils ƒe lãmenugbagbevi siwo woyɔna be neutrophils.
Le no me lãmenugbagbeviwo ƒe polarized epithelial cells me la, anti-Golgi network (TGN) ƒe agbɔsɔsɔ va ɖo apical plasma membrane ƒe seƒe kpɔa GPI-AP ƒe mama kple eƒe hatsotsowo me toto dzi ( 10 , 45 ). GPI-AP oligomerization ( 36 ) ye ʋãa nuƒoƒoƒu sia, gake ate ŋu anɔ te ɖe ceramide kɔsɔkɔsɔ ƒe didime si míekpɔna le amɔwɔʋãnu me hã dzi. Togbɔ be no me lãwo ƒe GPI-AP ƒe seke si wotu ɖe ether lipid dzi le esi, eye eƒe atike ƒe wɔwɔme to vovo kura tso acyl kɔsɔkɔsɔ didi ŋutɔ ceramide gbɔ hã la, numekuku aɖe si wowɔ nyitsɔ laa ɖee fia be lãmi eveawo siaa ƒe dzɔdzɔmeŋusẽ kple atike ƒe nɔnɔmewo sɔ le nɔnɔmetɔtrɔ me Eye woƒe dɔwɔwɔ sɔ (40). Eyata ether lipid ƒe akpa si le no me lãmenugbagbeviwo me ate ŋu asɔ kple C26 ceramide si le amɔwɔʋãnu me, eye eƒe dɔe nye be wòade ha kple ceramide si ƒe kɔsɔkɔsɔ didi le lãkusia me be wòado GPI-AP ƒe ƒuƒoƒo kple vovototodedeameme ɖe ŋgɔ. Togbɔ be egahiã kokoko be woado nusi ate ŋu adzɔ sia kpɔ tẽ hã la, nusiwo ŋu woke ɖo va yi ɖo kpe edzi be menye cytoplasmic transfer proteins ye wɔa acyl kɔsɔkɔsɔ didi la ƒe tsɔtsɔ yi Golgi ŋutilã me o, ke boŋ enɔ te ɖe GPI sekewo abe amɔwɔʋãnu ene ƒe wɔwɔme dzi. Eyata edze abe nɔnɔmetɔtrɔ ƒe mɔnu si léa nuteƒewɔwɔ me ɖe asi la te ŋu tiaa acyl kɔsɔkɔsɔ didi ŋutɔ ceramide kple GPI-AP (13, 16, 20, 46, 47) ɖekae tsɔ yia ʋuɖoɖo ƒe ʋuka ɖeka me ene.
Le amɔwɔʋãnu kple no me lãmenugbagbeviwo ƒe polarized epithelial cell systems me la, GPI-AP ƒe ƒuƒoƒo kple eƒe mama tso plasma membrane protein bubuwo gbɔ katã dzɔna hafi ɖoa lãmenugbagbeviwo ƒe anyime. Paladino kple exɔlɔ̃wo. (48) kpɔe be le no me lãmenugbagbevi siwo woɖe ɖe vovo ƒe TGN dzi la, menye ɖeko GPI-AP ƒe ƒuƒoƒo hiã na GPI-APwo ƒe hatsotso tiatia ɖe apical plasma membrane me ko o, ke eɖɔa GPI-APs ƒe ƒuƒoƒo ƒe ɖoɖowɔwɔ kple Eƒe nugbagbeŋutinunya hã ɖo. Lãmenugbagbeviwo ƒe anyigba. Le amɔwɔʋãnu me la, numekuku sia ɖee fia be C26 ceramide-dzikpɔkpɔ GPI-AP ƒuƒoƒo si le ER ate ŋu aɖɔ ƒuƒoƒo ƒe ɖoɖowɔwɔ kple GPI-AP ƒe dɔwɔwɔ ƒe dɔwɔwɔ le plasma membrane dzi ( 24 , 49 ). Le ɖekawɔwɔ me kple kpɔɖeŋu sia la, GhLag1 lãmenugbagbeviwo ƒe nulɔdɔ le GPI mɔxenuwo alo atike siwo kpɔa ŋusẽ ɖe lãmenugbagbeviwo ƒe gli ƒe blibonyenye dzi ( 28 ), eye alesi wòhiã be Gas1-GFP ƒuƒoƒo siwo wɔa dɔ ( 49 ) le tip ceramide si wobu le yeast cells ƒe ƒoƒo me ɖee fia be G Possible physiological consequences of hLag1 cells. GPI-AP ƒe vodada. Gake dodokpɔ bubu nenye be wowɔ ɖoɖo ɖe lãmenugbagbeviwo ƒe anyigba ƒe dɔwɔwɔ ƒe ɖoɖowɔwɔ ŋu tso ER to vovototodedeameme mɔnu si wotu ɖe ami ƒe didime dzi dzi anye míaƒe numekuku si míava wɔ le etsɔme.
Woŋlɔ Saccharomyces cerevisiae ƒomevi siwo wozã le dɔ sia me ɖe Tabla S1 me. Wowɔ SCLIM ƒe ƒomevi siwo nye MMY1583 kple MMY1635 hena lãmenugbagbevi gbagbewo ƒe nɔnɔmetatawo ɖeɖe le W303 ƒe megbe. Wowɔ ƒomevi siawo siwo ɖea Sec13-mCherry fiana kple fluorescent protein tag to mɔnu si wotu ɖe polymerase chain reaction (PCR) dzi kple pFA6a plasmid abe nɔnɔmetata ene ( 23 ). Wowɔ ƒome si ɖea Mid2-iRFP si wotsɔ fluorescent protein de dzesii le GAL1 promoter ƒe dzikpɔkpɔ te la fiana ale. PCR ƒe iRFP-KanMx ƒe dzidziɖedzi tso pKTiRFP-KAN vector (nunana si E. O'Shea, Addgene plasmid xexlẽdzesi 64687; http://n2t.net/addgene: 64687; numekuku dɔwɔnu ƒe dzesidenu (RRID): Addgene_64687) Eye wotsɔe de C-nuwuƒe si le endogenous Mid2. Esi wodo Mid2-iRFP domenyiŋusẽfianu ƒe ɖoɖoa ɖe ŋgɔ eye wowɔ eƒe nɔnɔmetata ɖe GAL1 ƒe ŋgɔdola me vɔ la, wotsɔe de integration plasmid pRS306 ƒe Not I-Sac I teƒea. Wotsɔ Pst I wɔ plasmid pRGS7 si do tso eme la ɖe fli me be wòawɔ ɖeka kple URA3 ƒe teƒea.
Woɖea Gas1-GFP fusion domenyiŋusẽfianu la fiana le GAL1 ƒe ŋgɔdola ƒe dzikpɔkpɔ te le centromere (CEN) plasmid me, si wowɔ abe alesi gbɔna ene. Wodo ŋusẽ Gas1-GFP ƒe ɖoɖoa to PCR me tso pRS416-GAS1-GFP plasmid (24) (L. Popolo ƒe nunana) me eye wowɔ eƒe nɔnɔmetata ɖe CEN plasmid pBEVY-GL LEU2 (C nunana) ƒe Xma I–Xho I teƒe . Miller; Addgene plasmid xexlẽdzesi 51225; http://n2t.net/addgene: 51225, Ƒe 1999 ƒe ɣleti gbãtɔ me. RRID: Nusiwo wotsɔ kpe ɖe eŋu_51225). Wotsɔ ŋkɔ na plasmid si do tso eme la be pRGS6. Axl2-GFP fusion domenyiŋusẽfianu hã dzena le pBEVY-GL LEU2 dɔlékuiwutike ƒe GAL1 promoter ƒe dzikpɔkpɔ te, eye eƒe wɔwɔme le ale. Wodo ŋusẽ Axl2-GFP ƒe ɖoɖoa tso pRS304-p2HSE-Axl2-GFP plasmid (23) me to PCR me, eye wowɔ eƒe nɔnɔmetata ɖe Bam HI-Pst I teƒe si le pBEVY-GL LEU2 dɔlékuiwutike me. Wotsɔ ŋkɔ na plasmid si do tso eme la be pRGS12. Woŋlɔ oligonucleotide siwo wozã le numekuku sia me ƒe ɖoɖo ɖe wo nɔewo yome ɖe Tabla S2 me.
Wotsɔ adenine 0.2% kple sukli 2% [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract kple 2% protein ept) kpe ɖe ƒomea ŋu. (YPR)] alo 2% galactose [YP-galactose (YPG)] abe carbon dzɔtsoƒe ene, alo le nu suetɔ kekeake si wowɔ me (0.15% yeast nitrogen base kple 0.5% ammonium sulfate) be woatsɔ akpe ɖe amino acid kple base siwo sɔ siwo hiã na nunyiame ŋu , Eye glucose 2% (synthetic glucose minimal medium) alo galactose 2% le eme (galactose minimal medium si wowɔ) abe carbon dzɔtsoƒe ene.
Le ɣeyiɣi ŋutɔŋutɔ me nɔnɔmetatawo ɖeɖe gome la, wodo sec31-1 ƒe lãmenugbagbevi siwo trɔna siwo sea dzoxɔxɔ nu siwo ɖea nusi wowɔ le GAL1 ƒe ŋgɔdola te fiana le YPR me le 24°C me zã bliboa va ɖo log ƒe titina. Esi wotsɔ lãmenugbagbeviawo de YPG me le 24°C me gaƒoƒo 1 vɔ la, wotsɔ lãmenugbagbeviawo de SG me le 37°C me aɖabaƒoƒo 30, eye emegbe wotsɔ wo yi 24°C me be woaɖe asi le wo ŋu tso ʋuʋudedi ƒe mɔxenu la me. Wozã Concanavalin A tsɔ ɖɔ lãmenugbagbeviawo ɖo ɖe glass slide dzi eye wotsɔ SCLIM wɔ nɔnɔmetata. SCLIM nye Olympus IX-71 inverted fluorescence microscope kple UPlanSApo 100×1.4 xexlẽdzesiwo ƒe aperture oil lens (Olympus), duƒuƒu sesẽ kple dzesi-to-toɣliɖeɖe ƒe sɔsɔme gã rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, kple custom cooling ƒe ƒuƒoƒo The system’s image intensifier (Hamamatsu Photonics) ate ŋu ana lens si lolona ƒe ɖoɖo si ƒe lolome mamlɛtɔ nye ×266.7 kple charge-coupled device camera si dzia elektrɔnikwo ɖe edzi (Hamamatsu Photonics) (21). Kɔmpiutadziɖoɖo si wowɔ ɖe ɖoɖo nu (Yokogawa Electric) ye wɔa nɔnɔmetatawo xɔxɔ. Le 3D nɔnɔmetatawo gome la, míezã piezoelectric actuator si wowɔ ɖe ɖoɖo nu tsɔ ʋuʋu taɖodzinu ƒe lens la le tsitrenu, eye míeƒo nukpɔkpɔ ƒe akpa siwo dome didi 100 nm nu ƒu ɖe ƒuƒoƒo me. Wotrɔa Z-stack nɔnɔmetata la wòzua 3D voxel data, eye wozãa nukpɔsusu ƒe teƒe kaka ƒe dɔwɔwɔ si wozãna na rotating disc confocal microscope hena deconvolution dɔwɔwɔ to Volocity software (PerkinElmer) dzi. To Volocity kɔmpiuta dɔwɔɖoɖo zazã be woatsɔ aɖo dzidzenu le eɖokui si na teƒe ɖeka me dzodzro me la, wodzidze ERES si me agbawo hã le. Wozã MetaMorph kɔmpiuta dɔwɔɖoɖo (Molecular Devices) tsɔ wɔ fliwo me dzodzro.
Zã GraphPad Prism kɔmpiuta dɔwɔɖoɖo nàtsɔ anya akɔntabubu ƒe vevienyenye. Le Student ƒe t-dodokpɔ si me asike eve le kple mɔ ɖeka dzi numekuku dzro (ANOVA) dodokpɔ si bɔ gome la, wobu vovototo siwo le ƒuƒoƒoawo dome be wokpɔ ŋusẽ gã aɖe ɖe P <0.05 (*) dzi.
Le Gas1-GFP ƒe fluorescence microscopy gome la, wodo log phase cells la zã bliboa le YPD me eye woƒo wo nu ƒu to centrifugation me, woklɔ wo zi eve kple phosphate buffered saline, eye wotsɔ wo de tsikpe dzi aɖabaƒoƒo 15 ya teti, eye emegbe woyi edzi le microscope te abe alesi wogblɔe va yi ene Check (24). Wozã Leica DMi8 nusuekpɔmɔ̃ (HCX PL APO 1003/1.40 ami PH3 CS) si me wotsɔ taɖodzinu ƒe ŋkumekpɔnu, L5 (GFP) filter, Hamamatsu fotoɖemɔ̃ kple Application Suite X (LAS X) kɔmpiuta dɔwɔɖoɖo le hena exɔ. .
Wotsɔ SDS sample buffer trɔ asi le kpɔɖeŋuawo ŋu le 65°C hena miniti 10, eye emegbe wotsɔ SDS-polyacrylamide gel electrophoresis (PAGE) ma wo dome. Le immunoblotting numekuku me la, wotsɔ kpɔɖeŋu 10 μl de mɔ ɖesiaɖe dzi. Dɔlékuiwutike gbãtɔ: Zã avugbɔ̃e ƒe polyclonal anti-Gas1 le tsi si me wode 1:3000 me, avugbɔe ƒe polyclonal anti-Emp24 le tsi me 1:500, kple avugbɔe ƒe polyclonal anti-GFP (nunana tso H. Riezman gbɔ) le tsi 1:3000 me. Wozã sisiblisi ƒe monoclonal anti-Pgk1 dɔlékuiwutike le tsi si me wode 1:5000 (nunana tso J. de la Cruz gbɔ) me. Dɔlékuiwutike evelia: Horseradish peroxidase (HRP) si wotsɔ ƒo ƒu wɔ gbɔ̃ ƒe anti-rabbit immunoglobulin G (IgG) si wozãna le tsi si me wode 1:3000 me (Pierce). Wozã gbɔ̃ ƒe IgG si wotsɔ HRP ƒo ƒui tsɔ tsi tre ɖe sisiblisi ŋu le tsi si me wode 1:3000 me (Pierce). Wotsɔ SuperSignal West Pico reagent (Thermo Fisher Scientific) ƒe chemiluminescence mɔnu kpɔ dɔlélenutsiŋutete ƒe ŋuɖoɖo ƒe akpaa.
Abe alesi woɖe eme le (31) me ene la, wowɔ dzɔdzɔme dɔlélenutsiŋutete ƒe dodokpɔ le ER ƒe akpa si wodo ŋusẽe la ŋu. Kpuie ko la, klɔ amɔwɔʋãnu lãmenugbagbeviwo kple TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kple protease inhibitor tsakatsaka) le 600 nm ( OD600) me le 100 optical density me zi eve. Wotsɔa ahuhɔ̃e-kuwo gbãnɛ, eye emegbe wotsɔa centrifugation ɖea lãmenugbagbeviwo ƒe gbeɖuɖɔ kple ahuhɔ̃e-kuwo ɖa. Emegbe wotsɔa tsi si le edzi la ƒua gbe le centrifug me le g 17,000 me hena miniti 15 le dzoxɔxɔ 4°C me. Wogatsɔ pellet la de TNE me eye wotsɔ digitalis saponin de eme vaseɖe esime eƒe agbɔsɔsɔme mamlɛtɔ nye 1%. Wotsɔa tsi si wotsɔ ƒoe la dea lãme gaƒoƒo 1 eye wotrɔa wo le dzoxɔxɔ 4°C me, eye emegbe woɖea akpa siwo me mekɔ o la ɖa to centrifugation me le 13,000 g le 4°C me hena miniti 60. Le Gas1-GFP dɔlélenutsiŋutete ƒe ʋuʋu gome la, do ŋgɔ tsɔ agarose-ku ƒuƒlu (ChromoTek) do ŋgɔ na kpɔɖeŋua le 4°C me gaƒoƒo 1, eye emegbe nàtsɔ GFP-Trap_A (ChromoTek) ade 4°C me gaƒoƒo 3. Wotsɔ TNE si me digoxigenin 0.2% le klɔ beads siwo ŋu dɔlélenutsiŋutete le la zi atɔ̃, wotsɔ SDS sample buffer kɔ wo me, woɖe wo ɖe vovo le SDS-PAGE dzi, eye woda wo kpɔ to immunoblotting me.
Abe alesi woɖe eme le (31) me ene la, wowɔ cross-linking determination le ER ƒe akpa si wodo ŋusẽe la dzi. Kpuie ko la, wotsɔ ER ƒe akpa si wodo ŋusẽe la de 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, U.S.A.; 20°C, miniti 20). Wotsi crosslinking reaction la to glycine (50 mM ƒe agbɔsɔsɔ mamlɛtɔ, miniti 5, 20°C) dede eme me.
Abe alesi míegblɔe va yi ene (50) la, wowɔ MS numekuku le ceramide ŋu le gbemelã ƒomeviwo kple GhLag1 ƒomeviwo me. Kpuie ko la, wodzi lãmenugbagbeviwo va ɖo exponential phase (3 va ɖo 4 OD600 units/ml) le YPD me le 30°C me, eye woŋe lãmenugbagbevi 25×107. Wotsɔa trichloroacetic acid tsia woƒe lãmenugbagbeviwo ƒe dɔwɔwɔ nu. Zã atike si woɖena tsoa eme [ethanol, tsi, ether, pyridine kple 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] kple 1.2 nmol si nye ememe dzidzenu C17 ceramide (860517, Avanti polar lipid) ƒe nyonyome). Zã monomethylamine reagent [methanol, tsi, n-butanol kple methylamine solution (4:3:1:5 v/v)] nàtsɔ awɔ alkaline hydrolysis fafɛ le nusi woɖe tso eme la me, eye emegbe nàzã n-butanol si me tsi yɔ fũu atsɔ aɖe dze ɖa. Mlɔeba la, wogatsɔ nusi woɖe tso eme la de atike si nye positive mode solvent [chloroform/methanol/tsi (2:7:1) + 5 mM ammonium acetate] me eye wodoe ɖe mass spectrometer la me. Wowɔ multi-reaction monitoring (MRM) hena sphingolipid molecules ƒe dzesidede kple woƒe agbɔsɔsɔme. Wotsɔ robot nanoflow ion dzɔtsoƒe Nanomate HD (Advion Biosciences, Ithaca, NY) de TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) me hena ami me dzodzro. Wotrɔ asi le ƒoƒo ƒe ŋusẽ ŋu wònyo wu na ceramide ƒe hatsotso ɖesiaɖe. Woxɔ MS nyatakakawo le nɔnɔme nyui me. Le nugbagbewo ƒe gbugbɔgawɔ ɖesiaɖe gome la, ami ƒe dzesi nyea dzidzenu etɔ̃ siwo wowɔ le wo ɖokui si ƒe titina.
Abe alesi woɖe eme le (31) me ene la, wotsɔ lãmenugbagbevi (800×107) siwo ɖea Gas1-GFP fiana la de dzɔdzɔme dɔlélenutsiŋutete ƒe ʋuʋu me. Wotsɔ SDS-PAGE ɖe Gas1-GFP si ŋu wokɔ la ɖe vovo eye wotsɔe de polyvinylidene fluoride (PVDF) membrane me. Wokpɔ protein la le susu me to PVDF si wotsɔ amide black de amae me. Wotso Gas1-GFP ƒe ƒuƒoƒoa tso PVDF me eye woklɔe zi 5 kple methanol eye zi ɖeka kple tsi si nye liquid chromatography-MS (LC-MS) grade tsi. To membrane strip la tsɔtsɔ de 500μl 0.3 M NaOAc (pH 4.0), buffer kple 500μl 1 M sodium nitrite tsakatsaka si wofa yeyee le 37°C me gaƒoƒo 3 me la, woɖea lipid ƒe akpa si le Gas1-GFP me eye woɖea asi le eŋu Release of inosine phosphate ceramide between glucosamine and inositol (51). Le ema megbe la, woklɔa membrane strip la zi ene kple LC-MS grade tsi, wotsɔe ƒu gbe le xɔme ƒe dzoxɔxɔ me, eye wodzrae ɖo ɖe nitrogen yame le -80°C vaseɖe esime wodae kpɔ. Abe alesi woawɔ akpɔ egbɔ ene la, wozã PVDF membrane ƒe kpɔɖeŋu ƒuƒlu aɖe na dodokpɔ ɖesiaɖe. Emegbe wotsɔ MS da ami si woɖe tso Gas1-GFP me kpɔ abe alesi wogblɔe ene ( 50 ). Kpuie ko la, wogbugbɔ tsɔ PVDF-ka siwo me GPI-lipid le la de 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] me eye woto electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage) me. Le go sia me la, woxɔ MS nyatakakawo le ion manyomanyo ƒe nɔnɔme me.
Abe alesi míegblɔe va yi ene la, woɖe GPI sekea ƒe ami ƒe akpaa ɖa tso GPI-AP si wotsɔ [3H]-inositol de dzesii gbɔ ( 16 ). Woɖe amiawo ɖe vovo to thin-layer chromatography me to solvent system (55:45:10 chloroform-methanol-0.25% KCl) zazã me eye wotsɔ FLA-7000 (Fujifilm) kpɔ wo le susu me.
Woklɔ lãmenugbagbevi siwo ɖea Gas1-GFP (600×107) fiana la zi eve kple TNE buffer si me TNE buffer le, eye wotsɔ glass beads gbã wo, eye emegbe wotsɔ wo centrifuged tsɔ ɖe lãmenugbagbeviwo ƒe gbeɖuɖɔ kple glass beads ɖa. Emegbe wotsɔa tsi si le edzi la ƒua gbe le centrifux me le 17,000 g me gaƒoƒo 1 le 4°C. Woklɔ pellet la le TNE me eye wotsɔ 1 U PI-PLC (Invitrogen) de TNE si me 0.2% digitalis saponin le me gaƒoƒo 1 le 37°C me. Le enzyme ƒe dɔwɔwɔ vɔ megbe la, woɖe lãkusia ɖa to centrifugation me le 17,000 g me le 4°C me hena gaƒoƒo 1. Be dɔlélenutsiŋutete nawɔ Gas1-GFP la, wotsɔ GFP-Trap_A (ChromoTek) de tsi si le edzi la me le 4°C me zã bliboa. Wotsɔ Coomassie brilliant blue de ama Gas1-GFP si ŋu wokɔ si woɖe ɖe vovo to SDS-PAGE dzi. Wotso Gas1-GFP ƒe amadede ƒe ka la ɖa le amadede ɣi si ƒo xlã tsimɔa ŋu, eye emegbe le alkylation kple iodoacetamide kple edziɖeɖe kple dithiothreitol megbe la, wowɔ in-gel digestion kple trypsin. Woɖe tryptic peptides kple peptides siwo me GPI-glycans le ɖa eye nàƒu wo. Wotsɔ peptide ƒuƒu la ƒo ɖe tsi 20 μl me. Do akpa aɖe (8μl) ɖe LC la me. Wozã octadecylsilane (ODS) sɔti (Develosil 300ODS-HG-5; ememe didime 150 mm×1.0 mm; Nomura Chemical, Aichi Nutome, Japan) tsɔ ma peptidewo ɖe vovo le nɔnɔme tɔxɛ siwo me wole zɔzɔm le me. Akpa si zɔnae nye atike A (0.08% formic acid) kple atike B (0.15% formic acid le 80% acetonitrile me). Wozã Accela HPLC ɖoɖo (Thermo Fisher Scientific, Boston, Massachusetts) tsɔ kɔ sɔti la me kple atike A le miniti 55 me le sisi ƒe agbɔsɔsɔ si nye 50 μl min-1 hena miniti 5, eye emegbe wodzi atike B ƒe agbɔsɔsɔ ɖe edzi va ɖo 40%. , United States). Wotsɔa eluate la dea ESI ion dzɔtsoƒea me ɣesiaɣi, eye wotsɔ LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific) da tryptic peptide kple peptide siwo me GPI-glycans le la kpɔ. Le MS ɖoɖoa me la, woɖo ʋuka si me ʋu sina tona ƒe dzoxɔxɔ ɖe 4.5 kV, eye wotsɔ ʋuka si me ʋu sina tona ƒe dzoxɔxɔ nanɔ 300°C. Woɖo ʋuka me ʋu ƒe ŋusẽ kple tube ƒe ŋkume ƒe ŋusẽ ɖe 15 V kple 50 V dzi. Woxɔ MS nyatakakawo le ion ƒe nɔnɔme nyui me (si ƒe vovototo nye 60,000; agbɔsɔsɔme ƒe nyateƒetoto nye akpa 10 le miliɔn ɖeka me) le kpekpeme ƒe didime si nye 300/m/z mass/charge ratio (m/z) 3000. Woxɔ MS/MS nyatakakawo to ion mɔ̃ si le LTQ Orbitrap XL [xexlẽdzesi 3 gbãtɔ siwo dzi nyatakakaawo nɔ te ɖo, collision induced dissociation (CID)].
Wowɔ MD ƒe nɔnɔmetatawo to GROMACS (52) kɔmpiuta dɔwɔɖoɖo kple MARTINI 2 ŋusẽ ƒe teƒe (53-55) zazã me. Emegbe wozã CHARMM GUI Membrane Builder (56, 57) tsɔ wɔ ƒuƒoƒo eve si me dioleoylphosphatidylcholine (DOPC) kple Cer C18 alo DOPC kple Cer C26 le. Wokpɔa Cer C26 ƒe topology kple coordinates tso DXCE me to beads siwo sɔ gbɔ ɖe sphingosine ƒe asike me ɖeɖeɖa me. Zã ɖoɖo si woɖe fia le ete nàtsɔ ada asɔ le ƒuƒoƒo eveawo me eye nàwɔe, emegbe nàzã ɖoɖoa ƒe kɔordinate mamlɛawo nàtsɔ atu ɖoɖo si me Emp24 le. Wowɔ amɔwɔʋãnu Emp24 ƒe transmembrane domain (nu susɔe 173 va ɖo 193) abe α-helix ene to visual MD (VMD) dɔwɔnu ƒe molecule structure (58) zazã me. Emegbe esi woɖe ami siwo tsi tre ɖe wo nɔewo ŋu ɖa vɔ la, woɖe protein la ɖa le mɔ ƒuƒuie nu eye wotsɔ CHARMM GUI de bilayer la me. Nuɖoanyi mamlɛtɔa me 1202 DOPC kple 302 Cer C26 alo 1197 DOPC kple 295 Cer C18 kple Emp24 le. Ionize ɖoɖoa va ɖo 0.150M ƒe agbɔsɔsɔme. Wowɔ ene siwo wogbugbɔ wɔ le wo ɖokui si na bilayer compositions eve.
Woda asɔ le lipid bilayer la me to CHARMM GUI ƒe mɔnu zazã me, si bia be woaɖe afɔɖeɖe 405,000 dzi akpɔtɔ eye emegbe woada asɔ, afisi woɖea teƒe ƒe mɔxenuwo dzi kpɔtɔna vivivi eye woɖea wo ɖa le, eye wodzia ɣeyiɣi ƒe afɔɖeɖea ɖe edzi tso 0.005 ps va ɖo 0.02 ps. Le dadasɔ megbe la, ewɔa 6 μs kple ɣeyiɣi ƒe afɔɖeɖe si nye 0.02 ps. Ne ède Emp24 eme vɔ la, zã CHARMM GUI ƒe ɖoɖo ma ke nàtsɔ aɖe ɖoɖoa dzi akpɔtɔ eye nàda asɔ, eye emegbe nàƒu du hena s 8 le ewɔwɔ me.
Le ɖoɖowo katã gome la, le dadasɔ ƒe ɖoɖoa me la, Berendsen barostat (59) ye kpɔa nyaƒoɖeamenua dzi, eye le ewɔwɔ me la, Parrinello-Rahman barostat (60) ye kpɔa nyaƒoɖeamenua dzi. Le nɔnɔmeawo katã me la, nyaƒoɖeamenu le mama dedie nu nye 1 bar eye wozãa semi-isotropic nyaƒoɖeamenu ƒe kadodo ƒe ɖoɖo. Le dadasɔ kple ewɔwɔ ƒe ɖoɖoa me la, wozãa dzoxɔxɔdzidzenu (61) si me wogbugbɔ dzidzea duƒuƒu le tsɔ ƒoa protein, lipid kple solvent ƒe akpa suewo ƒe dzoxɔxɔ nu ƒu ɖekae. Le dɔwɔwɔ bliboa me la, dzoxɔxɔ si woɖo taɖodzinu na nye 310K. Wobu akɔnta le kadodo si me kadodo mele o ŋu to evewɔwɔ ƒe xexlẽdzesi wɔwɔ me to Verlet ɖoɖo si me 0.005 buffer tolerance le zazã me. Wobua Coulomb ƒe nya la to reaction field kple cut-off distance si nye 1.1 nm zazã me. Vander Waals ƒe nya la zãa cut-off scheme si ƒe cut-off didime nye 1.1 nm, eye wozãa Verlet cut-off scheme na potential drift (62).
Ne wozã VMD la, ƒutsotsoe ƒe didime si wotso le DOPC phosphate beads alo ceramide AM1 beads kple protein la dome nye 0.7 nm, eye wobua lipids siwo wɔa dɔ kple protein la ƒe xexlẽme. Le mɔfiame si gbɔna nu la, bu akɔnta le depletion-enrichment (DE) factor ŋu abe alesi wòle le (63) me ene: DE factor = (ami siwo katã le protein la me ƒe agbɔsɔsɔ 0.7) le protein la me 0.7 (Cer ƒe agbɔsɔsɔ le lipids bliboa me) .
Woxɔa asixɔxɔ si ŋu woka nya ta le abe mamã dedie ene, eye vodada ƒe dzesiwo nye SE ƒe kɔpi ene siwo le wo ɖokui si. Wobu DE factor ƒe akɔntabubu ƒe vevienyenye to t dodokpɔ [(averageDE-factor-1)/SE] me. Bu P ƒe asixɔxɔ tso asike ɖeka ƒe mama me.
Wozã GROMACS dɔwɔnu tsɔ bu akɔnta le 2D lateral density map si le ɖoɖo si me Emp24 le le 250 ns mamlɛawo me le trace la me. Be woateŋu akpɔ ceramide ƒe enrichment/depletion map la, womaa Cer ƒe density map ɖe Cer kple DOPC ƒe map ƒe ƒuƒoƒo me, eye emegbe womaae ɖe Cer ƒe agbɔsɔsɔ le ŋutilã me nu. Wozãa amadede ƒe anyigbatata ƒe dzidzenu ma ke.
Ne èdi nyatakaka kpeɖeŋutɔ na nyati sia la, taflatse kpɔ http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Esia nye nyati si woateŋu akpɔ le mɔ gbadza nu si woma le Creative Commons Attribution-Non-Commercial License ƒe sewo nu, si ɖe mɔ ɖe eŋudɔwɔwɔ, mama kple egbugbɔgawɔ le mɔnu ɖesiaɖe me, nenye be zazã mamlɛtɔ menye asitsatsa ƒe viɖe ta o eye gɔmeɖoanyia nye be dɔ gbãtɔa sɔ. Ɖeɖe fiã.
De dzesii: Ðeko míebia tso asiwò be nàtsɔ wò e-mail adrɛs ana ale be amesi nèkafu na axaa nanya be yedi be yeakpɔ e-mail la eye be menye nyatakaka gbegblẽe wònye o. Mímalé e-mail adrɛs aɖeke ɖe mɔ̃ dzi o.
Wozãa biabia sia tsɔ doa amedzro kpɔ nenye be amedzroe nènye eye woxea mɔ na nyatakaka gbegblẽwo ɖoɖo ɖe amewo le wo ɖokui si.
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 Perez -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D ɣeyiɣi ŋutɔŋutɔ me nɔnɔmetata siwo ƒe lolome deŋgɔ ɖe ceramide kɔsɔkɔsɔ ƒe didime ƒe vevienyenye fia na protein ƒe vovototodedeameme le teƒe tiatia siwo woɖea nu le.
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 Perez -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D ɣeyiɣi ŋutɔŋutɔ me nɔnɔmetata siwo ƒe lolome deŋgɔ ɖe ceramide kɔsɔkɔsɔ ƒe didime ƒe vevienyenye fia na protein ƒe vovototodedeameme le teƒe tiatia siwo woɖea nu le.
©2020 Amerikatɔwo ƒe Dzɔdzɔmeŋutinunya ƒe Ŋgɔyiyi Habɔbɔ. gomenɔamesiwo katã le esi. AAAS nye HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef kple COUNTER ƒe hadɔwɔla. Dzɔdzɔmeŋutinunya ƒe Ŋgɔyiyi ISSN 2375-2548.