Protéines (protéines) tilatilali gundola sira la, o nafa ka bon walasa ka seliluw tilatilali ni homeostasis sabati. Ka fara fɛnw sɛgɛsɛgɛli kan minnu bɛ kɛ ni jirisunw ye, tulumafɛnw jɔyɔrɔ kinesin sɛgɛsɛgɛli la gundolabɔli taabolo la, o ye ɲininkali jɔnjɔn ye min bɛ senna kabini tuma jan, n’a ma jaabi fɔlɔ. Yan, an bɛ 3D simultaneous multicolor high-resolution real-time imaging kɛ walasa k’a jira in vivo ko glycosylphosphatidylinositol-immobilised proteines kura minnu dilannen don ni ceramide lipid yɔrɔ janba ye, olu bɛ kuluw kɔnɔ ka tila ka kɛ endoplasmes kɛrɛnkɛrɛnnenw ye Net exit site, o ni transmembrane proteines bɛ baara kɛ ni min ye, o tɛ kelen ye. Ka fara o kan, an b’a jira ko seramidi cakɛda janya min bɛ endoplasmic reticulum membrane kɔnɔ, o nafa ka bon kosɛbɛ nin sorting selectivity in na. An ka kalan in bɛ dalilu tilennen fɔlɔ di in vivo ma walasa ka farikolojɔlifɛnw doni tila ka da lipid cakɛda janya kan ka kɛ jagokɛyɔrɔ sugandilenw ye gundo sira la.
Eukaryote selilɛriw kɔnɔ, farikolojɔlifɛn minnu bɛ sɔrɔ endoplasmique reticulum (ER) kɔnɔ, olu bɛ tila ka tila ka tila ka taa ni u ye gundo sira fɛ walasa ka taa u selilu yɔrɔ bɛnnen na (1). Ka fara coat-mediated sorting kan, a tun bɛ fɔ kabini tuma jan ko lipid dɔw fana bɛ se ka kɛ bɔyɔrɔ sugandilenw ye n’u y’u dalajɛ ka kɛ membrane domaines kɛrɛnkɛrɛnnenw ye minnu ye proteines kɛrɛnkɛrɛnnenw ye (2-5). Nka, dalilu tilennenw tɛ yen hali bi minnu bɛ se ka kɛ sababu ye ka nin fɛɛrɛ in jira min bɛ se ka kɛ ni tulumafɛnw ye. Walasa ka nin gɛlɛya jɔnjɔn in ɲɛnabɔ, an ye sɛgɛsɛgɛli kɛ yezu kɔnɔ cogo min na glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) bɛ bɔ ER la cogo min na danfara la. GPI-APw ye farikolojɔlifɛn suguya caman ye minnu ni tulumafɛnw bɛ ɲɔgɔn kan ( 6, 7 ). GPI-AP ye farikolojɔlifɛn ye min bɛ bɔ kɛnɛ kan, min bɛ nɔrɔ joli-sira-funu kɛnɛma furabuluw la glycolipid (GPI anchor) fɛ. U bɛ sɔn GPI ankɔriw ma i n’a fɔ bamanankan bamanankan-falen kɔfɛ fɛn caman sɛmɛntiyalenw ER lumen kɔnɔ (8). A nɔrɔlen kɔfɛ, GPI-AP bɛ tɛmɛ Golgi minɛn fɛ (5, 9) ka bɔ ER la ka taa joli-sira-funu na. GPI ankɔriw sɔrɔli bɛ kɛ sababu ye ka GPI-AP ta ka bɔ ɲɔgɔn na ka bɔ farikolojɔlifɛnw na minnu bɛ bɔ farikolo la (o la, joli-sira-funu-poroteyini wɛrɛw bɛ olu la) gundola sira fɛ (5, 9, 10). Yezu seliluw la, GPI-APw bɛ faran ka bɔ farikolojɔlifɛn wɛrɛw la minnu bɛ bɔ endoplasmique reticulum kɔnɔ, o kɔfɛ, u bɛ pake ka kɛ bɔgɔlan kɛrɛnkɛrɛnnenw ye minnu sirilen don ni coat protein complex II (COPII) ye (6, 7). Nin danfara taabolo in dantigɛlanw ER bɔli taabolo la, olu ma jɛya, nka a bɛ fɔ ko nin fɛɛrɛ in bɛ se ka kɛ sababu ye ka tulumafɛnw de wajibiya, kɛrɛnkɛrɛnnenya la, GPI ankɔri tulumafɛnw yɔrɔ jɔcogo kura (5, 8). Leveur la, GPI lipid remodeling bɛ daminɛ o yɔrɔnin bɛɛ GPI nɔrɔlen kɔfɛ, wa a ka ca a la, a bɛ kɛ sababu ye ka seramidi siri asidi tuluma cakɛda jan 26-carbon saturated saturated (C26:0) la (11, 12). C26 seramidi ye seramidiba ye min bɛ bɔ funteni selilɛriw fɛ fo ka na se bi ma. A bɛ dilan ER kɔnɔ wa a fanba bɛ taa ni a ye Golgi minɛn na COPII bɔgɔlanw fɛ (13). GPI-AP ka ER bɔli kɛrɛnkɛrɛnnenya la, o bɛ seramidi dilanni taabolo de wajibiya (14, 15), wa o kɔfɛ, seramidi jiginni ka kɛ inositɔli fosifate seramidi (IPC) ye Golgi minɛn kɔnɔ, o bɛ bɔ GPI ankɔri dilanni na (16). Biophysique kalan minnu kɛra ni membranes artificielles ye, olu y’a jira ko seramidi cakɛda asili janyalenba bɛ se ka fara ɲɔgɔn kan ka kɛ domaines orderés ye minnu farikolo cogoya kɛrɛnkɛrɛnnenw bɛ u la (17, 18). O kunnafoniw bɛ na ni hakilina ye ko C26 seramidi ni GPI-AP ni C26 seramidi bɛ baara kɛ n’u farikolo cogoyaw ye walasa ka fara ɲɔgɔn kan ka kɛ yɔrɔw walima maraw ye minnu labɛnna ER membrane lipid sigida la min bɛ ɲagami kosɛbɛ. A bɛ kɛ kɛrɛnkɛrɛnnenya la glycérolipide surunw ni glycérolipides saturated (C16:1 ani C18:1) ye (19, 20). O mara ninnu bɛna sinsin sugandili la ER bɔyɔrɔ kɛrɛnkɛrɛnnenw kan (ERES), yɔrɔ minnu na seramidi ni seramidi basigilen GPI-AP bɛ se ka taa ni u ye ɲɔgɔn fɛ ka taa Golgi la COPII bɔgɔlan kɛrɛnkɛrɛnnen kelen kɔnɔ (5).
Nin kalan in kɔnɔ, an ye nin fɛɛrɛ in kɔrɔbɔ k’a ɲɛsin lipidi ma, an kɛtɔ ka baara kɛ ni super-resolution confocal real-time imaging microscopy (SCLIM) ye, o ye mikroskɔpi fɛɛrɛ tigɛlen ye min bɛ se ka farikolojɔlifɛnw kɔlɔsi waati kelen na minnu bɛ taamasiyɛn kɛ ni fluorescent ye Jaw kulɛri saba ni saba (3D) ja ninnu ka ɲɛfɔli ni teliya ka bon kosɛbɛ selilu ɲɛnamaw kɔnɔ (21, 22).
An ye SCLIM fɛɛrɛ fɔlɔ kɛ walasa ka ɲɛfɔli wɛrɛ kɛ cogo min na GPI-AP nɔgɔman ni C26 seramidi kulu bɛ sɛgɛsɛgɛ ka bɔ farikolojɔlifɛnw na minnu bɛ bɔ transmembrane kɔnɔ, a bɔlen kɔ ER la S. cerevisiae kɔnɔ. Walasa ka ER danfara lajɛ, an ye baara kɛ ni jamu siratigɛ ye min bɛ se ka doni kura dilannenw ye minnu bɛ don ERES kɔnɔ in vivo (7, 23). I n’a fɔ doni, an ye C26 seramidi-based GPI-AP Gas1 sugandi min sɛbɛnnen don ni green fluorescent protein (GFP) ye ani transmembrane secreted protein Mid2 min taamasiyɛn ye near-infrared fluorescent protein (iRFP) ye, olu fila bɛɛ bɛ joli-sira-funu laɲini (24–26). sec31-1 funteni-sɛgɛsɛgɛli mutant kɔnɔ, o doni fila ninnu bɛ jira galactose-inducible promoter (galactose-inducible promoteur) ni ERES marker constitutif (ERES taamasiyɛn) kɔrɔ. Ni funteni ka bon kosɛbɛ (37°C), bawo sec31-1 fɛnɲɛnamafagalan bɛ nɔ bila COPII coat component Sec31 ka baara la walasa ka COPII falenni ni ER bɔli bali, doni kura dilannenw bɛ dalajɛ ER la (23). Nɛnɛ kɔfɛ fo ka se funteni dɔgɔman ma (24°C), sec31-1 fɛnɲɛnama falenw seginna ka bɔ gundo yɔrɔ la, wa doni kura dalajɛlenw daminɛna ka bɔ ER la. CLIM yecogo y’a jira ko Gas1-GFP ni Mid2-iRFP kura minnu dilannen don, olu fanba bɛ dalajɛ hali bi sec31-1 fɛnɲɛnama falenw ka ER la, u bilalen kɔ 37°C la, ka sɔrɔ ka bɔ 24°C la miniti 5 kɔnɔ (Jaa 1). Ikomi Mid2-iRFP bɛ jɛnsɛn ER membrane bɛɛ kan, ani Gas1-GFP bɛ lajɛ ka lajɛ ER membrane yɔrɔ la min tɛ taa ɲɛ, u tilatilali tɛ kelen ye pewu (Ja 1, A ka taa C la ani Movie S1). Ka fara o kan, i n’a fɔ a jiralen bɛ cogo min na ja 1D kɔnɔ, Gas1-GFP kulu tɛ Mid2-iRFP sɔrɔ. Nin jaabi ninnu b’a jira ko GPI-AP ni transmembrane proteines farala ɲɔgɔn kan ka kɛ ER membrane yɔrɔw danfaralenw ye joona. Gas1-GFP kulu bɛ ERES kɛrɛnkɛrɛnnen dɔ kɛrɛfɛ min taamasiyɛn ye mCherry ka COPII coat proteine ​​Sec13 ye (Ja 1, E ani F, ani filimu S1) (23).
sec31-1 seliluw bɛ galaktose-induite secretions jira, asili cakɛda jan (C26) seramidi GPI-AP Gas1-GFP (GPI-AP, vert) ani transmembrane proteine ​​Mid2-iRFP (TMP, bleu) ani nin Constructive ERES labeling Sec13-mCherry (ERES, magenta) in bilala 37°C la miniti 30 kɔnɔ, ka taa 24°C, ani ja min bɛ kɛ SCLIM fɛ miniti 5 o kɔfɛ. . Skala bara 1μm (A ni B). Skala hakɛ ye 0,551μm (C) ye. Gas1-GFP sɔrɔla ER yɔrɔw la walima kuluw kɔnɔ minnu tɛ kelen ye, ka sɔrɔ Mid2-iRFP sɔrɔla ka tilatila ER membrane (C) bɛɛ kɔnɔ. (D) Jaa in bɛ Gas1-GFP ni Mid2-iRFP ka fluorescence fanga danfaralen jira Gas1-GFP kulu kɔnɔ, ka kɛɲɛ ni jiribolo finman ye (numan fɛ). AU, unité arbitraire. (E ni F) bɛ ja 3D jira min bɛ fɛnw ni ERES taamasiyɛn fara ɲɔgɔn kan. Gas1-GFP kuluw sɔrɔla ERES kɛrɛnkɛrɛnnen kɛrɛfɛ. Skala hakɛ ye 0,551μm ye. (F) Tile solide finman bɛ Gas1-GFP kulu taamasiyɛn min bɛ tali kɛ ERES la. Cɛmancɛ ni kininbolo pannew bɛ 3D ja belebeleba faralen ɲɔgɔn kan ani Gas1-GFP kulu sugandilen yecogo wulilen jira.
Yɔrɔko jɛɲɔgɔnya gɛlɛn min bɛ Gas1-GFP kulu ni ERES kɛrɛnkɛrɛnnen dɔ cɛ, o b’a jira ko Gas1-GFP bɛ se ka don ERES sugandilen kɔnɔ, o min ni Mid2-iRFP bɛ baara kɛ ni sugandili ye walasa ka bɔ ER kɔnɔ, o tɛ kelen ye. Walasa ka o seko in ɲɛnabɔ, an ye ERES hakɛ jateminɛ fɛn kelen walima fila dɔrɔn de la (Jaa 2, A ka se C ma). An y’a ye ko ERES fanba (70%) kɔnɔ, doni suguya kelen dɔrɔn de bɛ yen. Jaa 2C duguma ja bɛ ERES misali danma fila jira ni Gas1-GFP dɔrɔn ye (Ja 1) walima Mid2-iRFP dɔrɔn (Ja 2). O ni ERES 20% ɲɔgɔn ta tɛ kelen ye, doni fila bɛ yen minnu bɛ ɲɔgɔn dafa yɔrɔ kelen na. A jirala ko doni suguya fila tun bɛ ERES dɔw la (10%), nka u tun bɛ danfara la yɔrɔw la minnu tɛ kelen ye ka jɛya. O la, nin jatebɔ in b’a jira ko ER bilalen kɔ jamana kɔkan, GPI-AP Gas1-GFP ni transmembrane cargo Mid2-iRFP bɛ tila ERES danfaralenw na (Ja 2D). O sɛgɛsɛgɛli kɛcogo ɲuman in bɛ bɛn kosɛbɛ ni biyokimiki sɛgɛsɛgɛli tɛmɛnenw ye (6) ani cogoya dantigɛli (7). An bɛ se fana ka doni minnu bilala dankan na, olu ka kɛwalew kɔlɔsi minnu bɛ don ERES kɔnɔ (Jaa 2E ani Sinema S2). Jaa 2E b’a jira ko Gas1-GFP (panɛli 3) walima Mid2-iRFP (panɛli 4) yɔrɔ fitinin dɔrɔn de bɛ don ERES kɔnɔ ka bɔ fan kelen fɛ, wa a bɛ datugu yɔrɔ la min tɛ kelen ye. Jaa 2E ka panɛli 5 b’a jira ko Gas1-GFP ni Mid2-iRFP bɛ sɔrɔ tuma dɔw la ERES kelen na, nka u bɛ don ka bɔ fan wɛrɛw la, wa u bɛ lajɛ yɔrɔ danfaralenw na minnu bɛ se ka COPII bɔgɔlan suguya wɛrɛw jira. An y’a jira fana ko C26 seramidi basigilen GPI-AP Gas1 danfara n’a danfara kɔlɔsilen i n’a fɔ ERES sugandilen, o ye kɛrɛnkɛrɛnnen ye bawo transmembrane secretion cargo wɛrɛ, n’o ye GFP-tagged plasma membrane protein Axl2 (27 ), o bɛ kɛwale kelen jira ni Mid2-iRFP ye. (Ja S1 ani Filimu S3). Axl2-GFP kura min dilannen don, o bɛ jɛnsɛn ER membrane fɛ i n’a fɔ Mid2-iRFP (Ja S1, A ani B), wa a ni Mid2-iRFP bɛ sigi ɲɔgɔn fɛ ERES fanba la (Ja S1, B ka se D ma). Jaa 1 ka panɛli 1 ni 2. S1C bɛ ERES misali danma fila jira yɔrɔ minnu na doni fila minnu bɛ tɛmɛ membrane fɛ, olu bɛ ɲɔgɔn dafa. O cogo la, fɛn fila bɛɛ bɛ don ERES kɔnɔ ɲɔgɔn fɛ (Ja S1E, Panɛli 3 ani Sinema S3).
sec31-1 selilu minnu bɛ galaktose inducible secretions jira, Gas1-GFP (GPI-AP, green) ani Mid2-iRFP (TMP, blue) ani constitutive ERES labeling Sec13-mCherry (ERES, magenta) olu bilala 37 la U bilalen kɔ miniti 30 kɔnɔ °C la, ka taa 24 °C la walasa ka bɔli bloki bɔ, ka ja ta ni SCLIM ye o kɔfɛ Miniti 20 kɔnɔ. . Panɛli min bɛ duguma (B) kɔnɔ ani pankurun min bɛ (C) kɔnɔ, olu bɛ ja baaralenw jira walasa ka fɛnw dɔrɔn jira minnu bɛ ERES (magenta) kɔnɔ [Gas1-GFP (gris) ani Mid2-iRFP (bulu jɛman) (C) Tile da wulilen : ERES bɛ doni kelen dɔrɔn de ta (1 fo 4). Tile gris: ERES kɔnɔ, doni danfaralenw bɛ sɔrɔ (5). Tile solide blanc: ERES min kɔnɔ doni bɛ sɔrɔ ɲɔgɔn fɛ. Jɛ la: ERES kelen sugandilen kɔnɔ, Gas1-GFP (1) walima Mid2-iRFP (2) dɔrɔn de bɛ sɔrɔ. Skala bara, 100 nm. (D) Fotomikrografu hakɛ jateminɛ min ɲɛfɔlen don (C) kɔnɔ. ERES kɛmɛsarada la, doni kelen dɔrɔn bɛ min kɔnɔ (Gas1-GFP walima Mid2-iRFP), doni danfaralenw ani doni minnu bɛ ɲɔgɔn kan. Kɔrɔbɔli yɛrɛmahɔrɔnyalen saba la, n=432 ye seli 54 kɔnɔ. Misali barika = SD. T test min tɛ fila-fila ye. *** P = 0,0002 ye. (E) 3D ja min bɛ ERES sugandilenw kan doni minnu bɛ dankan na, minnu taamasiyɛn ye (C) ye. Gas1-GFP (bulu) (3) walima Mid2-iRFP (bulu) (4) bɛ don ERES (magenta) kɔnɔ ka bɔ fan kelen fɛ, wa a bɛ dan yɔrɔ fitinin dɔ ma ERES kɔnɔ. Tuma dɔw la, doni suguya fila bɛɛ bɛ don ERES kelen na (5) ka bɔ fan kelen na, wa u bɛ dan yɔrɔ la min yɔrɔ ka jan ERES kɔnɔ. Skala bara, 100 nm.
O kɔfɛ, an ye hakilina dɔ kɔrɔbɔ ko seramidi asili cakɛda jan (C26) min bɛ ER membrane kɔnɔ, o bɛ Gas1 kulu kɛrɛnkɛrɛnnenw ni a tilacogo bila ka kɛ ERES sugandilenw ye. O kama, an ye yezu suguya sɛmɛntiyalen dɔ kɛ GhLag1, min kɔnɔ, seramidi sintazi fila minnu bɛ kɔnɔna na, Lag1 ni Lac1, olu bilala GhLag1 nɔ na (Lag1 homologue of cotton), o kɛra sababu ye ka yezu suguya dɔ sɔrɔ min ka selilɛri membrane Ceramide suguya ka surun ka tɛmɛ kungo suguya kan (Ja 3A) (28). Mass spectrometry (MS) sɛgɛsɛgɛli y’a jira ko kungo kɔnɔ suguyaw la, seramidi bɛɛ lajɛlen 95% ye cakɛda seramidi ye min janya ka bon kosɛbɛ (C26), k’a sɔrɔ GhLag1 la, seramidi 85% janya ka bon kosɛbɛ (C18 ani C16). ), seramidi 2% dɔrɔn de ye seramidi cakɛda janba (C26) ye. Hali ni C18 ni C16 seramidiw ye seramidi kunbabaw ye minnu sɔrɔla GhLag1 membrane kɔnɔ fo ka na se bi ma, MS sɛgɛsɛgɛli fana y’a jira ko Gas1-GFP ka GPI ankɔri min jiralen bɛ GhLag1 suguya la, C26 seramidi bɛ o kɔnɔ, o min bɛ se ka suma ni kungo kɔnɔ fɛnɲɛnamafagalanw ye. A jogo ye kelen ye (Ja 3A) (26). O la, o kɔrɔ ye ko seramidi remodeling enzyme Cwh43 bɛ sugandi kosɛbɛ C26 seramidi la, i n’a fɔ a jiralen bɛ cogo min na ja 26 na, a ka fisa a ka GPI ankɔri don a la ka bɔ C26 seramidi hakɛ fitinin na GhLag1 suguya la. S2 (29) ye. O bɛɛ n’a ta, GhLag1 ka selilɛri kɔnɔ, C18-C16 seramidi dɔrɔn de bɛ sɔrɔ a jɔyɔrɔba la, k’a sɔrɔ C26 seramidi bɛ Gas1-GFP la hali bi. O ko in b’a to nin suguya in bɛ kɛ baarakɛminɛn ɲuman ye walasa ka kɛrɛnkɛrɛnnenya la ka gɛlɛya ɲɛnabɔ kɛrɛnkɛrɛnnenya la, min ye membrane ceramide ka asili cakɛda janya ye ER kɔnɔ. Kalanso ni tila-tila jɔyɔrɔ hakilinatalen. O kɔfɛ, an ye fɔlɔ ka C26 Gas1-GFP seko sɛgɛsɛgɛ ka dalajɛ kuluw kɔnɔ GhLag1 kɔnɔ ni sec31-1 alelɛ fɛnɲɛnamafagalan ye min bɛ funteni jateminɛ ni fluorescence microscopie conventionnelle ye, yɔrɔ min na cakɛda jan (C18-C16) dɔrɔn de bɛ ER membrane Ceramide kɔnɔ (Ja 3). An y’a kɔlɔsi ko sec31-1 kɔnɔ, Gas1-GFP fanba tun bɛ kɛ kuluw kɔnɔ, k’a sɔrɔ Gas1-GFP min bɛ sec31-1 GhLag1 kɔnɔ ni seramidi ER membrane jan jan (C18-C16) ye, o tun tɛ kuluba ye ani a tun bɛ jɛnsɛn In the entire ER membrane kɔnɔ. Walasa ka tiɲɛ fɔ, bawo C26 seramidi basigilen kulusi bɛ tali kɛ kosɛbɛ ERES kɛrɛnkɛrɛnnenw na (Ja 1), o kɔfɛ, an ye sɛgɛsɛgɛli kɛ n’a y’a sɔrɔ nin wale in bɛ se ka kɛ ER bɔli poroteyini fɛɛrɛ fana ka baara ye. GPI-AP bɛ baara kɛ ni COPII sigida kɛrɛnkɛrɛnnen ye ER bɔli kama, min bɛ labɛn kosɛbɛ Ted1 ka GPI ankɔri ka glycan yɔrɔ labɛnni kura fɛ (30, 31). O kɔfɛ, GPI-glycan min bɛ ɲɔgɔn sɔrɔ kokura, o bɛ dɔn ni transmembrane cargo receptor p24 complex ye, o min bɛ Lst1 ta ka sugandi, o min ye COPII doni sirili yɔrɔba Sec24 ka isoform kɛrɛnkɛrɛnnen ye, ka GPI-AP-rich COPII Vesicles are necessary (31-33). O la, an ye fɛn falen fila dɔ dilan min ye nin farikolojɔlifɛn kelen ninnu bɔli fara ɲɔgɔn kan (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 ani COPII subunit kɛrɛnkɛrɛnnen Lst1) ni sec31-1 fɛn falen suguya ye, ka u sɛgɛsɛgɛ Yala a bɛ se ka kɛ ka Gas1-cluster GFP dilan (Jaa 3). An y’a kɔlɔsi ko sec31-1emp24Δ ni sec31-1ted1Δ kɔnɔ, Gas1-GFP tɛ kuluba la, wa a bɛ jɛnsɛn ER membrane bɛɛ kɔnɔ, i n’a fɔ a tun bɛ ye cogo min na ka tɛmɛ sec31-1 GhLag1 kɔnɔ, k’a sɔrɔ sec31-1lst1Δ la, Gas1-GFP I n’a fɔ sec31-1. Nin jaabi ninnu b’a jira ko ka fara C26 seramidi sɔrɔli kan ER membrane kɔnɔ, Gas1-GFP kulusi fana ka kan ka siri p24 complexe la, wa a tɛ Lst1 kɛrɛnkɛrɛnnen sɔrɔli wajibiya. O kɔfɛ, an ye sɛgɛsɛgɛli kɛ ko seramidi cakɛda janya min bɛ ER membrane kɔnɔ, o bɛ se ka Gas1-GFP sirili labɛn p24 la. Nka, an y’a ye ko C18-C16 seramidi sɔrɔli membrane kɔnɔ, o tɛ nɔ bila GPI-glycans minnu jɔra kokura p24 complexe fɛ (Ja S3 ani S4, A ani B) walima ka siri GPI-AP la ani ka GPI-AP bila jamana kɔkan. se ko. Aw bɛ COPII suguya fitinin Lst1 ta (Jaa S4C). O la, C26 seramidi-dependant clustering tɛ poroteyini jɛɲɔgɔnyaw wajibiya ni ER export proteine ​​kɛcogo wɛrɛw ye, nka a bɛ dɛmɛ don sorting fɛɛrɛ wɛrɛ la min bɛ ɲɛminɛ lipid janya fɛ. O kɔfɛ, an ye sɛgɛsɛgɛli kɛ ni seramidi asili cakɛda janya min bɛ ER membrane kɔnɔ, o nafa ka bon Gas1-GFP danfara ɲuman na i n’a fɔ ERES sugandilen. Ikomi Gas1 min bɛ GhLag1 suguya la ni seramidi cakɛda surun ye, o bɛ bɔ ER la ka don joli-sira-funu kɔnɔ (Ja S5), an dalen b’a la ko ni danfara bɛ boli seramidi asili cakɛda janya fɛ, Gas1 min bɛ GhLag1 suguya la, o bɛ se ka sira wɛrɛ bila ka tɛmɛ. ERES fɛnw ni membrane kelen ye.
(A) GhLag1 ka selilɛri kɔnɔ, C18-C16 seramidi surunw bɛ sɔrɔ kosɛbɛ, k’a sɔrɔ Gas1-GFP ka GPI ankɔri bɛ ni C26 IPC kelen ye hali bi ni kungo kɔnɔ selilu ye. Sanfɛ : seramidi asili cakɛda janya sɛgɛsɛgɛli kungo suguya (Wt) ni GhLag1p suguyaw ka selilɛri kɔnɔ ni mass spectrometry (MS) ye. Donanw bɛ seramidi bɛɛ lajɛlen kɛmɛsarada la jira. Kɔrɔbɔli yɛrɛmahɔrɔnyalen saba cɛmancɛ la. Misali barika = SD. T test min tɛ fila-fila ye. **** P <0,0001 ye. Panɛli duguma : MS sɛgɛsɛgɛli IPC ka asili cakɛda janya kan min bɛ Gas1-GFP (GPI-IPC) GPI ankɔri kɔnɔ min jiralen don kungo suguya ni GhLag1p suguyaw la. Donanw bɛ IPC taamasiyɛn bɛɛ lajɛlen kɛmɛsarada la jira. Kɔrɔbɔli yɛrɛmahɔrɔnyalen duuru cɛmancɛ la. Misali barika = SD. T test min tɛ fila-fila ye. ns, nafa tɛ. P = 0,9134 ye. (B) sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ani sec31-1lst1Δ selilɛriw minnu bɛ galaktose-induite Gas1-GFP jira, olu ka fluorescence micrographes (fluorescence micrographes) bilala 37°C la miniti 30 kɔnɔ, ka tɛmɛ ka taa Perform routine fluorescence microscopy o kɔfɛ 24°C ye. Tile finman: ER Gas1-GFP kulu. Tile da wulilen : Gas1-GFP min ma kulu, o bɛ tilatila ER membrane bɛɛ kan, o bɛ ER taamasiɲɛ nucléaire ring staining jira. Skala bara, 5μm. (C) Fotomikrografu hakɛ jateminɛ min ɲɛfɔlen don (B) kɔnɔ. Kɛmɛsarada la, selilu minnu bɛ ni Gas1-GFP jɔcogo ye min bɛ kɛ ni punctate ye. Kɔrɔbɔli yɛrɛmahɔrɔnyalen saba kɔnɔ, n≥300 selilɛriw. Misali barika = SD. T test min tɛ fila-fila ye. **** P <0,0001 ye.
Walasa ka nin gɛlɛya in ɲɛnabɔ k’a ɲɛsin a yɛrɛ ma, an ye Gas1-GFP ni Mid2-iRFP yecogo SCLIM kɛ GhLag1 kɔnɔ ni sec31-1 temperature-sensitive mutant allele ye (Ja 4 ani Movie S4). ER maralen kɔfɛ 37°C la, o kɔfɛ a bɔra 24°C la, Gas1-GFP kura dilannen fanba ma fara ɲɔgɔn kan ka tilatila ER membrane bɛɛ kɔnɔ, i n’a fɔ a kɔlɔsira cogo min na ni mikroskɔpu kɔrɔlenw ye (Jaa 4, A ani B ). Ka fara o kan, ERES kɛmɛsarada la, doni suguya fila bɛ yen minnu bɛ ɲɔgɔn sɔrɔ a kɔnɔ (Jaa 4D). Jaa 4C panɛli 1 ni 2 bɛ ERES misali danma fila jira ni Gas1-GFP ni Mid2-GFP bɛ ɲɔgɔn kan. Ka fara o kan, o fɛn fila bɛɛ tun bɛ don ERES kelen kɔnɔ (Ja 4E, panɛli 3 ani filimu S4). O la, an ka jaabiw b’a jira ko seramidi asili cakɛda janya min bɛ ER membrane kɔnɔ, o ye ER poroteyini dalajɛli ni a danfara dantigɛlan nafama ye.
Sec31-1 GhLag1 selilu minnu bɛ galakɔtɔsi ka fɛnɲɛnamafagalanw jira, Gas1-GFP (GPI-AP, vert) ani Mid2-iRFP (TMP, bleu) ani constitutif ERES-labeled Sec13-mCherry (ERES, magenta) U bɛ bila 37°C la. Aw bɛ tɛmɛ ni a ye miniti 30 kɔnɔ, ka a jigin fo 24°C walasa ka fɛnɲɛnamafagalanw bɔ, ka ja ta ni SCLIM ye miniti 20 kɔfɛ. (A ka taa C la) 2D projection ja jiralenw (A; scale bar, 1μm) walima 3D cellule hemisphere ja (B ani C; scale unit, 0,45μm) z-yɔrɔ 10 minnu taamasiyɛn ye doni ni ERES ye. Panɛli min bɛ duguma (B) kɔnɔ ani pankurun min bɛ (C) kɔnɔ, olu bɛ ja baaralenw jira walasa ka fɛnw dɔrɔn jira minnu bɛ ERES (magenta) kɔnɔ [Gas1-GFP (gris) ani Mid2-iRFP (bulu jɛman) (C) Tile min falen don finman na: ERES, fɛnw bɛ ɲɔgɔn dafa. Tile da wulilen: ERES kɔnɔ, fɛn kelen dɔrɔn de bɛ yen. Panɛli duguma: ERES sugandilen bɛ ni fɛnw ye minnu bɛ ɲɔgɔn kan (1 ani 2) minnu taamasiyɛn bɛ (C) kɔnɔ. Skala bara, 100 nm. (D) Fotomikrografu hakɛ jateminɛ min ɲɛfɔlen don (C) kɔnɔ. sec31-1 ni sec31-1 GhLag1 unitw kɔnɔ, doni kelen dɔrɔn de bɛ don a kɔnɔ (Gas1-GFP walima Mid2-iRFP), ani ERES kɛmɛsarada la, doni danfaralenw ni doni minnu bɛ ɲɔgɔn kan. Kɔrɔbɔli yɛrɛmahɔrɔnyalen saba la, n = 432 ye seli 54 kɔnɔ (sec31-1) ani n = 430 seli 47 kɔnɔ (sec31-1 GhLag1). Misali barika = SD. T test min tɛ fila-fila ye. *** P = 0,0002 (sec31-1) ani ** P = 0,0031 (sec31-1 GhLag1). (E) ERES sugandilenw ja 3D ni doni minnu bɛ ɲɔgɔn kan (3) minnu taamasiyɛn bɛ (C) kɔnɔ. Gazi1-GFP (bilen) ni Mid2-iRFP (bulu) bɛ gɛrɛ ERES (magenta) la ka bɔ fan kelen na ka to ERES dantigɛlen yɔrɔ kelen na. Skala bara, 100 nm.
Nin kalan in bɛ dalilu tilennenw di in vivo ma ko farikolojɔli dumuniw doni minnu sinsinnen bɛ tulumafɛnw kan, olu bɛ tila ka kɛ fɛnw bilayɔrɔ sugandilenw ye gundo sira fɛ, wa a bɛ asili cakɛda janya nafa jira danfara sugandili la. An kɛlen ka baara kɛ ni mikroskɔpi fɛɛrɛ barikama ye min bɛ se ka tigɛ, n’o bɛ wele ko SCLIM, an ye Gas1-GFP (plasma membrane GPI-AP belebeleba dɔ jira min dilannen don ni asili cakɛda janba ye (C26) ceramide lipid portion) yezu kɔnɔ ) Yɔrɔ minnu lajɛlen don ER danfaralenw kɔnɔ, olu bɛ tali kɛ ERES kɛrɛnkɛrɛnnenw na, k’a sɔrɔ transmembrane secreted proteins bɛ tilatila ER membrane bɛɛ kɔnɔ (Jaa 1nan). Ka fara o kan, nin fɛn suguya fila ninnu bɛ don ERES danfaralenw na sugandili la (Jaa 2). Selilu seramidi min bɛ membrane kɔnɔ, o ka asili cakɛda janya bɛ dɔgɔya ka bɔ C26 la ka se C18-C16 ma, Gas1-GFP kulu bɛ tiɲɛ ka don ER yɔrɔ danfaralen na, ani Gas1-GFP bɛ sira wɛrɛ bila ka bɔ ER la ni transmembrane proteine ​​ye ERES kelen fɛ (Jaa 3 ani Jaa 3). 4).
Hali ni GPI-AP bɛ baara kɛ ni farikolojɔli fɛɛrɛ kɛrɛnkɛrɛnnen ye walasa ka bɔ ER la, an y’a ye ko C26 seramidi-dafalen danfara tɛ tali kɛ farikolojɔlifɛnw ka jɛɲɔgɔnya danfaralenw na minnu bɛ se ka kɛ sababu ye ka ERES kɛrɛnkɛrɛnnenya la (Ja S4 ani S5). O nɔ na, ​​an ka sɔrɔw bɛ danfara kɛcogo wɛrɛ dɛmɛ min bɛ ɲɛminɛ ni farikolojɔli dumuniw kuluw ye minnu sinsinnen bɛ tulumafɛnw kan ani doni wɛrɛw bɔli kɔfɛ. An ka kɔlɔsiliw b’a jira ko Gas1-GFP yɔrɔ walima kulu min bɛ tali kɛ ERES kɛrɛnkɛrɛnnen dɔ la, o tɛ ni transmembrane secreted protein Mid2-iRFP ye, o b’a jira ko C26 ceramide-dependent GPI-AP cluster bɛna u don nɔgɔya ERES kɔnɔ min bɛ tali kɛ o la, wa o waati kelen na, ka transmembrane bɔ a la The secretions enter this particular ERES (Ja 1 ani 2). O ni C18-C16 seramidiw sɔrɔli ER membrane kɔnɔ, o tɛ kɛ sababu ye ka GPI-AP kɛ yɔrɔw walima kuluw ye, o la u tɛ transmembrane secreted proteins bɔ kɛnɛ kan walima ka u bila ERES kelen na (Jaa 3 ni 4). . O la, an b’a jira ko C26 seramidi bɛ faranfasi ni danfara bila senkan ni farikolojɔlifɛnw dalajɛli nɔgɔyali ye minnu ni ERES kɛrɛnkɛrɛnnenw bɛ tali kɛ ɲɔgɔn na.
Nin C26 seramidi-dependant clustering in bɛ se ka sɔrɔ cogo di ka don ER yɔrɔ kɛrɛnkɛrɛnnen dɔ la? Ni fɛnɲɛnamafagalan seramidi ka teli ka faranfasi kɛrɛfɛ, o bɛ se ka kɛ sababu ye ka GPI-AP ni C26 seramidi kɛ lipidi misɛnninw ye ani minnu bɛ sigi sen kan yɔrɔnin kelen, ER membrane lipid sigida la min tɛ sabati kosɛbɛ, glycérolipidi surunw ni saturabaliw bɛ minnu na. Jogoɲumanya kuluw (17, 18). O waatilama kulu misɛnnin ninnu bɛ se ka fara ɲɔgɔn kan ka taa a fɛ ka kɛ kulu belebelebaw ye, minnu sabatilen don kosɛbɛ, u sirilen kɔ p24 complexe (34) la. Ka bɛn o ma, an y’a jira ko C26 Gas1-GFP ka kan ka jɛɲɔgɔnya kɛ ni p24 complexe ye walasa ka kuluba yelenw dilan (Jaa 3). p24 complexe ye oligomère hétérozigote ye min bɛ kɛ ni p24 transmembrane proteines naani ye minnu tɛ kelen ye yezu kɔnɔ (35), o bɛ multivalent sirili di, o bɛ se ka kɛ sababu ye ka GPI-AP kulu misɛnninw ka cross-linking kɛ, o la, Stable cluster belebeleba bɛ sɔrɔ (34). GPI-APw ka poroteyini ectodomains ni ɲɔgɔn cɛ jɛɲɔgɔnya fana bɛ se ka kɛ sababu ye ka u fara ɲɔgɔn kan, i n’a fɔ a jiralen bɛ cogo min na u ka Golgi tali waati la baganw ka polarize epithelial cellules kɔnɔ (36). Nka ni C18-C16 seramidi bɛ ER membrane kɔnɔ, ni p24 complexe sirilen don Gas1-GFP la, kuluba danfaralenw tɛna kɛ. Fɛɛrɛ min bɛ a jukɔrɔ, o bɛ se ka bɔ asili cakɛda jan seramidi farikolo ni a kemikaliya kɛrɛnkɛrɛnnenw na. Biophysique kalan minnu kɛra membrane artificielle kan, olu y’a jira ko hali ni asili cakɛda seramidi jan (C24) ni surun (C18-C16) fila bɛɛ bɛ se ka kɛ sababu ye ka fasa faranfasi, asili cakɛda jan seramidi (C24) dɔrɔn de bɛ se ka Curvature caman ni filimu kurulen sabati walasa ka filimu cogoya sɛmɛntiya. Fɛnɲɛnɛmafagalanw fɛ (17, 37, 38). A jirala ko TMED2 ka transmembrane helix, n’o ye Emp24 hadamaden ka homologue ye, o bɛ ɲɔgɔn sugandi ni C18 seramidi basigilen sphingomyelin ye sitoplasma lobules kɔnɔ (39). Ni an ye baara kɛ ni molekiyɔmu dinamiki (MD) simulasiw ye, an y’a ye ko C18 ni C26 seramidi fila bɛɛ bɛ dalajɛ Emp24 transmembrane helix ka sitoplasma lobules lamini na, wa u ka fɛɛrɛw bɛ ɲɔgɔn ta (Ja S6). A ka kan ka kɔlɔsi ko o b’a jira ko Emp24 ka transmembrane helix bɛ se ka kɛ sababu ye ka tulumafɛnw tilatilali asimɛtiri la membrane kɔnɔ. Nin ye kɔsa in na jaabi ye min sinsinnen bɛ baganw ka selilɛriw kan. O ɲɔgɔnna MD simulations fana bɛ etɛri lipidiw sɔrɔli jira (40) . O la, an b’a miiri ko C26 seramidi min bɛ ER26 lobule fila la, o bɛ nafa sɔrɔ sigida la. Ni GPI-AP min bɛ luminal lobules kɔnɔ, o sirilen don k’a ɲɛsin p24 multivalent ma ani C26 ceramide dalajɛli p24 lamini na sitoplasma lobules kɔnɔ, o bɛ se ka Protéines aggregation ni membrane curvature min bɛ taa n’a ye, o bɛ sɔrɔ bolokɔniw fɛ (41), o bɛ kɛ sababu ye GPI-AP ka faranfasi ka kɛ yɔrɔw ye minnu bɛ ERES kɛrɛfɛ, o fana bɛ kɛ sababu ye ka kurulenba sɔrɔ maraw minnu bɛ ER membrane (42) la. Raporo tɛmɛnenw ye fɛɛrɛ sugandilen in dɛmɛ (43, 44). Oligolɛkiniw, banakisɛfagalanw walima farikolo tangalanw sirili ni seramidi-based glycosphingolipids (GSL) ye joli-sira-funu kan, o bɛ GSL caman dalajɛli daminɛ, ka fasa faranfasi bonya ani ka kɛ sababu ye ka ɲɛgɛnɛsiraw bɔgɔbɔ ani ka u don a kɔnɔ (44). Iwabuchi wdfl (43) A jirala ko ni asili cakɛda janw (C24) nka u man surun (C16) bɛ yen, ligandi multivalent min sirilen bɛ GSL lactosylceramide la, o ye kulubaw ni membrane invagination (membrane invagination) dilan, ani sitoplasma Lyn-mediated signal transduction on the stemmes, o bɛ ɲɔgɔn cɛ ni asili cakɛdaw ye minnu faralen don ɲɔgɔn kan neutrophils (banakisɛfagalanw).
Baganw ka polarize epithelial cellules kɔnɔ, anti-Golgi network (TGN) hakɛ fo ka se apical plasma membrane hakɛ ma, o bɛ GPI-AP farali ni a tilacogo kɔlɔsi (10, 45). O farali in bɛ kɛ ni GPI-AP oligomɛrizi ye ( 36 ), nka a bɛ se ka kɛ fana ka bɔ seramidi cakɛda janya la an bɛ min sɔrɔ yezu la. Hali ni baganw ka GPI-AP bɛ ni etɛri lipid basigilen ankɔri ye, wa a kemikaliya cogoya ni asili cakɛda seramidi janyalenba tɛ kelen ye kosɛbɛ, kɔsa in na, sɛgɛsɛgɛli dɔ y’a jira ko lipidi fila bɛɛ farikolo ni kemikaliya cogoyaw ni baarakɛcogo bɛ ɲɔgɔn ta jiginni siratigɛ la ani u ka baara (40). O la, etɛri lipidi yɔrɔ min bɛ baganw ka selilɛriw kɔnɔ, o bɛ se ka kɛ i n’a fɔ C26 seramidi min bɛ yezu kɔnɔ, wa a jɔyɔrɔ ye ka jɛɲɔgɔnya kɛ ni seramidi cakɛda jan ye min bɛ membrane kɔnɔ walasa ka GPI-AP dalajɛli ni a tilacogo sabati. Hali n’a y’a sɔrɔ ko nin seko in ka kan ka sɛgɛsɛgɛ k’a ɲɛsin a yɛrɛ ma hali bi, sɔrɔ tɛmɛnenw b’a jira ko seramidi cakɛda jan asililen tali ka taa Golgi farikolo la, o tɛ kɛ ni sitoplasma jiginni poroteyini ye, nka a bɛ bɔ GPI ankɔriw dilanni na i n’a fɔ funteni. O la, a bɛ iko fɛnɲɛnɛmako kɔrɔsigi fɛɛrɛ bɛ se ka seramidi asili cakɛda janba ni GPI-AP (13, 16, 20, 46, 47) ta ɲɔgɔn fɛ ni sugandili ye taasibila fɛnɲɛnamafagalan kelen kɔnɔ.
Yezu ni baganw ka polarize epithelial cell systems kɔnɔ, GPI-AP dalajɛli ni a farali ka bɔ plasma membrane proteine ​​wɛrɛw la, o bɛɛ bɛ kɛ sani a ka se cellule surface ma. Paladino ni a ɲɔgɔnnaw. . Kɔnɔnatumuw sanfɛla. Yezu la, nin ɲinini in y’a jira ko C26 seramidi-dependant GPI-AP kulu min bɛ ER kan, o bɛ se ka GPI-AP kulu labɛncogo n’a baarakɛcogo ɲɛnabɔ joli-sira-funu kan (24, 49). Ka bɛn ni nin misali in ye, GhLag1 seliluw fari tɛ sɔn GPI balilanw ma walima fura minnu bɛ nɔ bila selilu kogo dafalen na ( 28 ), wa mago bɛ Gas1-GFP kuluw baarakɛtaw la ( 49 ) minnu bɛ kɛ ni tip ceramide ye min projected in the mating of yeast cells, o b’a jira ko G Possible physiological consequences of hLag1 cells. GPI-AP fili ye. Nka, ka sɛgɛsɛgɛli wɛrɛ kɛ ni selilu sanfɛla baarakɛcogo labɛnni porogaramura ka bɔ ER la ni ɲɔgɔndan fɛɛrɛ ye min sinsinnen bɛ tulumafɛnw janya kan, o bɛna kɛ an ka ɲinini nataw kun ye.
Saccharomyces cerevisiae suguya minnu bɛ kɛ nin baara in na, olu bɛ sɔrɔ jatebɔsen S1 kɔnɔ. SCLIM suguya MMY1583 ni MMY1635 minnu bɛ kɛ ka selilu ɲɛnamaw ja lajɛ, olu jɔra W303 kɔfɛ. Nin suguya minnu bɛ Sec13-mCherry jira ni poroteyini taamasiyɛn ye min bɛ yeelen bɔ, olu dilannen don ni polimerazi cakɛda (PCR) fɛɛrɛ ye ni pFA6a plasmide ye i n’a fɔ misali (23). Mid2-iRFP jiracogo suguya min taamasiyɛn kɛra ni poroteyini fluorescent ye GAL1 promotɛri ka fanga kɔrɔ, o dilanna nin cogo la. iRFP-KanMx sinsinni PCR fɛ ka bɔ pKTiRFP-KAN fɛnɲɛnamafagalan na (E. O’Shea ka nilifɛn, Addgene plasmide nimɔrɔ 64687; http://n2t.net/addgene: 64687; ɲinini nafolo jiralan (RRID): Addgene_64687) Wa a donna endogène Mid2 ka C-terminus kɔnɔ. Mid2-iRFP jamu sinsinni kɛlen kɔ ka bonya ka kɛ GAL1 promotɛri kɔnɔ, a donna Not I-Sac I yɔrɔ la, min bɛ sɔrɔ fɛnɲɛnamafagalan pRS306 la. Plasmide pRGS7 min sɔrɔla, o kɛra lineari ye ni Pst I ye walasa ka don URA3 yɔrɔ la.
Gas1-GFP fusion gène bɛ jira GAL1 promoteur ka fanga kɔrɔ centromere (CEN) plasmide kɔnɔ, o min bɛ dilan nin cogo la. Gas1-GFP sinsinni fanga bonyana ni PCR ye ka bɔ pRS416-GAS1-GFP plasmide (24) la (L. Popolo ka nilifɛn) ani ka clone kɛ CEN plasmide pBEVY-GL LEU2 (C ka nilifɛn) ka Xma I–Xho I yɔrɔ la Miller; A farala plasmide nimɔrɔ 51225 kan; Ɲɛjirali dɔw: 51225; RRID: Fàrafinna_51225). Plasmide min sɔrɔla, o tɔgɔ dara ko pRGS6. Axl2-GFP fusili jamu fana bɛ jira pBEVY-GL LEU2 fɛnɲɛnamafagalan ka GAL1 promotɛri ka fanga kɔrɔ, wa a jɔcogo bɛ nin cogo la. Axl2-GFP sinsinni cayara ka bɔ pRS304-p2HSE-Axl2-GFP plasmide (23) la ni PCR ye, ka clone kɛ pBEVY-GL LEU2 fɛnɲɛnamafagalan Bam HI-Pst I yɔrɔ la. Plasmide min sɔrɔla, o tɔgɔ dara ko pRGS12. Oligonuklɛtidi minnu kɛra nin kalan in na, olu sigiyɔrɔmali bɛ sɔrɔ jatebɔsen S2 kɔnɔ.
Nin suguya in farala adenine 0,2% ni gulukose 2% [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract ani 2% protein ept). (YPR)] or 2% galactose [YP-galactose (YPG)] as a carbon source, or in a synthetic minimal medium (0.15% yeast nitrogen base and 0.5% ammonium sulfate) to supplement appropriate amino acids and bases required for nutrition , And containing 2% glucose (synthetic glucose minimal medium) or 2% galactose (galactose minimal medium synthétique) i n’a fɔ karɔbɔli sɔrɔyɔrɔ.
Walasa ka ja lajɛ waati yɛrɛ la, sec31-1 fɛnɲɛnama falen minnu bɛ funteni jateminɛ, n’olu bɛ fɛn dilannen jira GAL1 promotɛri kɔrɔ, olu falenna YPR kɔnɔ 24°C la su fɛ fo ka se log fasa cɛmancɛ ma. YPG in bilalen kɔfɛ 24°C la sanga 1 kɔnɔ, aw bɛ seliluw bila SG la 37°C la miniti 30 kɔnɔ, ka sɔrɔ ka u bila 24°C la walasa ka bɔ bɔli bloki la. Konkanavalin A kɛra ka seliluw siri galasi slide kan ani ka ja ta SCLIM fɛ. SCLIM ye Olympus IX-71 inverted fluorescence microscope ni UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), teliyaba ni signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, ani custom cooling faralen ɲɔgɔn kan The system’s image intensifier (Hamamatsu Fotoniki) bɛ se ka lensɛri bonyalan dɔ di ni bonya laban ye ×266,7 ye ani minɛn kamera min bɛ charge-couplé min bɛ ɛlektrɔnikiw caya (Hamamatsu Photonics) (21). Jaw sɔrɔli bɛ kɛ ni porogaramuw ye minnu bɛ kɛ ka kɛɲɛ ni mɔgɔw sago ye (Yokogawa Electric). 3D jaw kama, an ye baara kɛ ni piezoelectric actuator ye min dilannen don ka kɛɲɛ ni mɔgɔw sago ye walasa ka objective lens (laɲini) lamaga jɔlen na, ka optique yɔrɔw lajɛ minnu ni ɲɔgɔn cɛ ye 100 nm ye stack kɔnɔ. Z-stack ja bɛ wuli ka kɛ 3D voxel data ye, ani teori point spread baarakɛcogo min bɛ kɛ disiki konfokali mikroskɔpu wulilen na, o bɛ kɛ ka deconvolution baarakɛcogo kɛ Volocity porogaramu fɛ (PerkinElmer). Ni baara kɛra ni Volocity porogaramu ye walasa ka dantigɛli kɛ otomatiki la ka ɲɛsin sigiyɔrɔma sɛgɛsɛgɛli ma, ERES doni sen bɛ o la, o suman. Layini sɛgɛsɛgɛli kɛra ni porogaramu MetaMorph ye (Molecular Devices).
Baara kɛ ni GraphPad Prism porogaramu ye walasa ka jateminɛ nafa dɔn. Kalanden ka t-test (Student’s t-test) min bɛ kɛ ni wulu fila ye ani sɛgɛsɛgɛli gansan min bɛ kɛ ni fɛn kelen-kelen bɛɛ ye (ANOVA), danfara minnu bɛ kuluw ni ɲɔgɔn cɛ, olu jatera ko olu bɛ nɔba bila P <0,05 (*) la.
Gas1-GFP ka fluorescence microscopie kama, log phase seliluw falenna su kelen kɔnɔ YPD kɔnɔ ka u lajɛ ni centrifugation ye, ka u ko siɲɛ fila ni phosphate buffered saline ye, ka u bila jisuma kan miniti 15 ni kɔ, ka sɔrɔ ka taa ɲɛ microscope kɔnɔ i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ Check (24). Leica DMi8 mikroskɔpu (HCX PL APO 1003/1.40 tulu PH3 CS) min bɛ ni objective lens, L5 (GFP) filɛri, Hamamatsu kamera ani Application Suite X (LAS X) porogaramu ye, o kɛra sababu ye ka sɔrɔ. .
A sɛgɛsɛgɛli kɛra ni SDS sɛgɛsɛgɛli tampon ye 65°C la miniti 10 kɔnɔ, o kɔfɛ, u farala ɲɔgɔn kan ni SDS-polyacrylamide gel electrophoresis (PAGE) ye. Walasa ka immunoblotting sɛgɛsɛgɛli kɛ, 10 μl de bɛ doni ta lane kelen kɔnɔ. Banakisɛfagalan fɔlɔ : Aw bɛ laɲɔ poliklɔni anti-Gas1 kɛ ni a dilancogo ye 1:3000 ye, laɲɔ poliklɔni anti-Emp24 ni a dilancogo ye 1:500 ye, ani laɲɔ poliklɔni anti-GFP (H. Riezman ka nilifɛn) ni a dilancogo ye 1:3000 ye. Sogo monoklɔni anti-Pgk1 banakisɛfagalan kɛra ni a dilancogo ye 1:5000 (J. de la Cruz ka nilifɛn). Banakisɛfagalan filanan : Horseradish peroxidase (HRP) conjugué bakɔrɔn anti-lapin immunoglobuline G (IgG) min bɛ kɛ ni a dilancogo ye 1:3000 (Pierce). HRP-conjugué bakɔrɔn anti-mouse IgG kɛra ni a dilancogo ye 1:3000 (Pierce). Banakisɛfagalanw ka jaabi yɔrɔ kɔlɔsira ni SuperSignal West Pico reagent (Thermo Fisher Scientific) ka kemiluminesɛnsi fɛɛrɛ ye.
I n’a fɔ a ɲɛfɔlen don cogo min na (31) kɔnɔ, farikolo tangacogo nafama dɔ kɛra ER tilayɔrɔba nafama kan. Kuma surun na, aw bɛ yeelenbɔlanw ko ni TNE tampon ye [50 mM tris-HCl (pH 7,5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ani protease inhibitor ɲagaminen) 600 nm ( OD600) la ni ɲɛkisɛ 100 ye siɲɛ fila. A tun karilen don ni galasi biɲɛw ye, o kɔfɛ, u tun bɛ selilɛri nɔgɔw ni galasi biɲɛw bɔ ni santirifugare ye. O kɔfɛ, ji min bɛ sanfɛ, o bɛ kɛ santirifugu la garamu 17 000 la miniti 15 kɔnɔ 4°C la. Aw bɛ furakisɛ in wuli kokura TNE la ka digiditali saponin fara a kan fo ka se a hakɛ laban ma min ye 1% ye. Aw bɛ suspension in tobi lɛri 1 kɔnɔ ni a jiginni ye 4°C la, o kɔfɛ, a yɔrɔ minnu tɛ se ka wuli, olu bɛ bɔ ni centrifugation ye garamu 13.000 la 4°C la miniti 60 kɔnɔ. Walasa ka Gas1-GFP immunoprecipitation sɔrɔ, aw bɛ fɔlɔ ka a lajɛ ni agarose biɲɛ lankolonw ye (ChromoTek) 4°C la lɛri 1 kɔnɔ, o kɔ aw bɛ a labɔ ni GFP-Trap_A (ChromoTek) ye 4°C la fo lɛri 3. Immunoprecipitated biɲɛw ko siɲɛ duuru ni TNE ye min kɔnɔ digoxigenin 0,2% bɛ sɔrɔ, ka u ko ni SDS sample buffer ye, ka u faranfasi SDS-PAGE kan, ka u sɛgɛsɛgɛ ni immunoblotting ye.
I n’a fɔ a ɲɛfɔlen don cogo min na (31) kɔnɔ, ɲɔgɔndan dantigɛli kɛra ER tilayɔrɔba nafama kan. Kuma surun na, ER tilayɔrɔba min falen don, o bɛ tobi ni ditiyobisi(succinimidyl propionate) 0,5 mM ye (Pierce, Thermo Fisher Scientific, Rockford, IL, Etazuni; 20°C, miniti 20). Kɛrɛnkɛrɛnnenya la, o kɛra sababu ye ka glycine (50 mM laban hakɛ, miniti 5, 20°C) fara a kan.
I n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ (50), seramidi sɛgɛsɛgɛli MS kɛra kungo suguya ni GhLag1 suguyaw la. Kuma surun na, seliluw falenna fo ka se fasa exponentiel ma (3 fo 4 OD600 units/ml) YPD kɔnɔ 30°C la, ani selilu 25×107 lajɛra. U farikolojɔli bɛ faga ni asidi trikuloroasetiki ye. Aw bɛ furaji bɔlen kɛ [etanɔli, ji, ɛtiri, piridini ani amoniyɔmu idɔrɔkisidi 4,2 N (15:15:5:1:0,018 v/v)] ani kɔnɔna nafama C17 seramidi (860517, Avanti polar lipid) jogo 1,2 nmol). Aw bɛ monometilamini fɛnɲɛnamafagalan [mɛtanɔli, ji, n-butanɔli ani mɛtilamini jilama (4:3:1:5 v/v)] kɛ ka a bɔlen in jilamali nɔgɔman kɛ, o kɔ aw bɛ n-butanɔli jilama kɛ ka kɔgɔ bɔ a la. A laban na, a bɔlen bɛ segin ka kɛ furaji ye min bɛ kɛ cogo ɲuman na [kulorofɔrimu/mɛtanɔli/ji (2:7:1) + asetati amoniyɔmu 5 mM] ka sɔrɔ ka a pikiri kɛ masala jateminɛnan na. Multi-reaction monitoring (MRM) kɛra walasa ka sphingolipid molecules dɔnni n’u hakɛ jateminɛ. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) bɛ ni robo nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) ye walasa ka lipid sɛgɛsɛgɛli kɛ. Kɔnɔnafili fanga bɛ ɲɛnabɔ ka ɲɛ seramidi suguya kelen-kelen bɛɛ la. MS kunnafoniw sɔrɔla cogo ɲuman na. Biologique replicate kelen-kelen bɛɛ la, lipid signal ye sumanikɛlan yɛrɛmahɔrɔnyalen saba cɛmancɛ ye.
I n’a fɔ a ɲɛfɔlen don cogo min na (31) kɔnɔ, Gas1-GFP jirali seliluw (800×107) kɛra immunoprecipitation naturelle ye. Gas1-GFP saniyalen farala ɲɔgɔn kan ni SDS-PAGE ye, ka a bila polivinilideni fluoride (PVDF) membrane kɔnɔ. Protéine in jirala ni PVDF nɔgɔli ye ni amide black ye. Gas1-GFP bandi tigɛra ka bɔ PVDF la ka ko siɲɛ 5 ni mɛtanɔli ye ani siɲɛ kelen ni ji jilama ye min bɛ wele ko liquid chromatography-MS (LC-MS) grade. Ni aw ye membrane strip tobi ni 500μl 0,3 M NaOAc (pH 4,0), tampon ani 500μl 1 M nitiriti sodiyɔmu ɲagaminen kura ye 37°C la sanga 3 kɔnɔ, tulumafɛn tilayɔrɔba bɛ bɔ Gas1-GFP la ka lyse Release of inosine phosphate ceramide between glucosamine and inositol (51). O kɔfɛ, membrane strip ko siɲɛ naani ni LC-MS grade ji ye, ka ja so funteni na, ka mara azote fiɲɛ na -80°C fo ka se a sɛgɛsɛgɛli ma. K’a kɛ kɔrɔsili ye, PVDF membrane (minɛn) gansan dɔ kɛra ka kɛɲɛ ni kɔrɔbɔli kelen-kelen bɛɛ ye. O kɔfɛ, tulumafɛn min bɔra Gas1-GFP la, o sɛgɛsɛgɛra ni MS ye i n’a fɔ a ɲɛfɔlen don cogo min na (50). Kuma surun na, PVDF sɛrɛkili minnu kɔnɔ GPI-lipid bɛ sɔrɔ, olu bilala kokura 75μl fɛnɲɛnamafagalan na [chloroform/methanol (1:2) + 5 mM amoniyɔmu asetati] ani ka tɛmɛ electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage) kan. O cogo la, MS kunnafoniw sɔrɔla ion negative cogo la.
I n’a fɔ a fɔra cogo min na ka tɛmɛ, GPI ankɔri lipidi yɔrɔ farala ka bɔ GPI-AP la min tɔgɔ ye ko [3H]-inositol (16). Lipidiw farala ɲɔgɔn kan ni chromatography finman ye ni solvent system (55:45:10 chloroform-methanol-0,25% KCl) ye, ka u jira ni FLA-7000 (Fujifilm) ye.
Gas1-GFP (600×107) jirali selilu minnu kolen don siɲɛ fila ni TNE tampon ye ni TNE tampon ye, ka u kari ni gilasi biɲɛw ye, ka sɔrɔ ka u kɛ santirifugare la walasa ka selilu nɔgɔw ni gilasi biɲɛw bɔ. O kɔfɛ, ji min bɛ sanfɛ, o bɛ kɛ santirifugu la garamu 17 000 la lɛri 1 kɔnɔ 4°C la. Aw bɛ furakisɛ ko TNE la ka a bila ni 1 U PI-PLC (Invitrogen) ye TNE kɔnɔ min kɔnɔ digitalis saponin 0,2% bɛ sɔrɔ lɛri 1 kɔnɔ 37°C la. Enzyme furakɛli kɔfɛ, a bɛ membrane bɔ ni centrifugation ye garamu 17.000 la 4°C la sanga 1 kɔnɔ. Walasa ka Gas1-GFP farikolo tanga, a sanfɛla bɛ tobi ni GFP-Trap_A (ChromoTek) ye 4°C la su fɛ. Gas1-GFP saniyalen min farala ɲɔgɔn kan ni SDS-PAGE ye, o nɔgɔlen don ni Coomassie brilliant blue ye. Gas1-GFP nɔgɔbɔlan tigɛra ka bɔ jibolisira lamini na, o kɔfɛ, alkilili kɔfɛ ni iyodoasetamidi ye ani ka dɔgɔyali kɛ ni ditiyotiriyitɔli ye, jeli kɔnɔ dumuni kɛli ni tripsini ye. Aw bɛ triptiki pepitiriw ni pepitiriw bɔ ka u ja ni GPI-glycans ye. Pepitiri jalen in bɛ wuli ji 20 μl la. Aw bɛ a yɔrɔ dɔ (8μl) pikiri kɛ LC kɔnɔ. Oktadesilisilani (ODS) koloma (Develosil 300ODS-HG-5; kɔnɔna cɛmancɛ ye milimɛtɛrɛ 150×1,0 ye; Nomura Chemical, Aichi mara la, Japon) kɛra ka pepitiriw fara ɲɔgɔn kan ni jiginni kɛrɛnkɛrɛnnenw ye. Fasa mobili ye furaji A (asidi formiki 0,08%) ani furaji B (asidi foromiki 0,15% asetonitirili 80% la). Accela HPLC sitɛmu (Thermo Fisher Scientific, Boston, Massachusetts) kɛra ka kolotuguda ko ni furaji A ye miniti 55 kɔnɔ ni jibɔ hakɛ ye min ye 50 μl ye miniti 1 kɔnɔ, o kɔfɛ, furaji B hakɛ cayara ka se 40% ma. , Etazuni jamana na). Eluate tun bɛ to ka don ESI ion source kɔnɔ, ani triptic peptides ni peptides minnu bɛ ni GPI-glycans ye, olu sɛgɛsɛgɛra ni LTQ Orbitrap XL ye (hybride linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). MS sigicogo la, kapilɛri sɔrɔyɔrɔ ka wulicogo tun bɛ kɛ 4,5 kV ye, ani kapilɛri jiginni funteni tun bɛ to 300°C la. Kapilɛri wulicogo ni tubabukan lensɛri wulicogo tun sigilen don 15 V ani 50 V kan. MS kunnafoniw sɔrɔla ion pozitif cogo la (resolution 60.000; mass accuracy of 10 parts per million) la mass range of 300/m/z mass/charge ratio (m/z) 3000. MS/MS kunnafoniw sɔrɔla ion trap fɛ LTQ Orbitrap XL kɔnɔ [daɲɛ 3 fɔlɔ minnu kan kunnafoniw bɛ tali kɛ, collision induced dissociation (CID)) ye.
MD simulations kɛra ni GROMACS (52) porogaramu ye ani MARTINI 2 fanga foro (53-55). O kɔfɛ, CHARMM GUI Membrane Builder (56, 57) kɛra ka bilayer dɔ dilan min kɔnɔ dioleoylphosphatidylcholine (DOPC) ni Cer C18 walima DOPC ni Cer C26 bɛ sɔrɔ. Cer C26 ka topidiya ni a ka koordinatɛriw bɛ bɔ DXCE la ni biɲɛ wɛrɛw bɔli ye sphingosine kɔ la. Baara kɛ ni taabolo ye min ɲɛfɔlen don jukɔrɔ walasa ka layini fila balansi ani k’a boli, o kɔfɛ, baara kɛ ni sistɛmu ka koordinatɛri labanw ye walasa ka sitɛmu dɔ jɔ min kɔnɔ Emp24 bɛ sɔrɔ. Yezu Emp24 ka transmembrane domaine (residu 173 fo 193) dilannen don i n’a fɔ α-helix ni visual MD (VMD) baarakɛminɛn molekiyɔmu jɔcogo ye (58). O kɔfɛ, tulumafɛnw bɔlen kɔ minnu bɛ ɲɔgɔn kan, farikolojɔlifɛn in kɛra fɛnmugumugu ye ka a don bilayɔrɔ la ni CHARMM GUI ye. Sisitɛmu laban kɔnɔ, DOPC 1202 ani Cer C26 302 walima DOPC 1197 ani Cer C18 ani Emp24 295 bɛ sɔrɔ. Aw bɛ ionize kɛ sistɛmu la ka se a hakɛ ma min ye 0,150M ye. Replikati yɛrɛmahɔrɔnyalen naani kɛra bilayer compositions fila kama.
Lipid bilayer bɛ balansi kɛ ni CHARMM GUI fɛɛrɛ ye, o min bɛ kɛ ni sen 405.000 dɔgɔyali ye, ka sɔrɔ ka balansi kɛ, yɔrɔ min na jɔyɔrɔko gɛlɛyaw bɛ dɔgɔya dɔɔni dɔɔni ka ban, ani waati taabolo bɛ bonya ka bɔ 0,005 ps na ka se 0,02 ps ma. Balannako kɔfɛ, a bɛ 6 μs Bɔ ni waati taamaʃyɛn ye 0,02 ps. Emp24 donni kɔfɛ, aw bɛ CHARMM GUI kɛcogo kelen in kɛ walasa ka sistɛmu dɔgɔya ani ka balansi don a la, ka sɔrɔ ka boli s 8 kɔnɔ a dilanni na.
Sitimɛw bɛɛ la, balansi kɛcogo la, degun bɛ kɔlɔsi Berendsen barostat fɛ (59), ani sɛnɛ kɛcogo la, degun bɛ kɔlɔsi Parrinello-Rahman barostat fɛ (60). O bɛɛ la, tansiyɔn cɛmancɛ ye 1 bar ye ani tansiyɔn semi-isotrope coupling scheme bɛ Kɛ. Balanna ni sɛnɛ kɛcogo la, thermostat (61) ni teliya seginni bɛ kɛ ka farikolojɔli dumuniw, tulumafɛnw ani furabuluw funteni fara ɲɔgɔn kan. O baara bɛɛ kɔnɔna na, funteni laɲinilen ye 310K ye. Jɛɲɔgɔnya min tɛ jɛɲɔgɔnya ye, o jate bɛ kɛ ni ɲɔgɔndan lisi dɔ labɛnni ye ni Verlet ka fɛɛrɛ ye ni 0,005 buffer tolerance ye. Kulomb ka waati jate bɛ Kɛ ni waleyali-yɔrɔ ye ani tigɛ-yɔrɔ ye min ye 1,1 nm ye. Vander Waals daɲɛ bɛ baara kɛ ni tigɛcogo ye min tigɛcogo ye 1,1 nm ye, ani Verlet tigɛcogo bɛ kɛ ka ɲɛsin seko ni dɔnko jiginni ma (62).
Ni baara kɛra ni VMD ye, DOPC fositɛri biɲɛw walima seramidi AM1 biɲɛw ni farikolojɔli dumuniw cɛ tigɛcogo janya ye 0,7 nm ye, wa tulumafɛnw hakɛ min bɛ ɲɔgɔn sɔrɔ ni farikolojɔlifɛn ye, o bɛ jate. Ka kɛɲɛ ni nin formula in ye, aw bɛ depletion-enrichment (DE) factor jate i n’a fɔ a bɛ cogo min na (63) kɔnɔ: DE factor = (tumafɛn bɛɛ lajɛlen hakɛ min bɛ farikolojɔli dumuni na 0,7) farikolojɔlan 0,7 kɔnɔ (Cer hakɛ min bɛ tulumafɛnw bɛɛ lajɛlen na)
Nafa min fɔra, o bɛ sɔrɔ i n’a fɔ cɛmancɛ, wa filiw ye SE ka kopi yɛrɛmahɔrɔnyalen naani ye. DE factor nafa jateminɛlen bɛ kɛ ni t test ye [(averageDE-factor-1)/SE]. P hakɛ jatebɔ ka bɔ kelen-kelen jatebɔ la.
GROMACS baarakɛminɛn kɛra ka 2D kɛrɛfɛ density karti jatebɔ kɛ sistɛmu kɔnɔ min kɔnɔ Emp24 bɛ sɔrɔ 250 ns labanw kɔnɔ trace kɔnɔ. Walasa ka seramidi nafa/dɛsɛ karti sɔrɔ, Cer ka density karti bɛ tila ni Cer ni DOPC ka karti faralen ye ɲɔgɔn kan, o kɔfɛ, a bɛ tila ni Cer hakɛ ye farikolo la. O kulɛri karti sɛgɛsɛgɛli kelen de bɛ kɛ.
Walasa ka kunnafoni wɛrɛw sɔrɔ nin barokun in kama, aw bɛ se ka http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1 lajɛ
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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, Manuɛl Muniz
3D ja min bɛ se ka kɛ waati yɛrɛ la, o bɛ seramidi cakɛda janya nafa jira farikolojɔlifɛnw tilatilali la bɔli yɔrɔ sugandilenw na.
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, Manuɛl Muniz
3D ja min bɛ se ka kɛ waati yɛrɛ la, o bɛ seramidi cakɛda janya nafa jira farikolojɔlifɛnw tilatilali la bɔli yɔrɔ sugandilenw na.
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