Poko pa protein i yo ma wok kud i iye piretek pi gwoko cell compartmentalization man homeostasis. Mi medo ikum poko piny ma jutimo ku cing, tic pa lipids i poko kin kinesin i yore mi tero piny ma wok kud i iye ubedo penji ma pire tek ma podi judwoko ngo. Kae, watimo 3D simultaneous multicolor high-resolution real-time imaging ni nyutho in vivo nia glycosylphosphatidylinositol-immobilized proteins ma nyen ku ceramide lipid moieties ma bor jupoko man jupoko gi i endoplasms Net exit site, ma tung tung ku ma transmembrane proteins tiyo kudu. Mi medo, wanyutho nia bor pa ceramide in endoplasmic reticulum membrane piretek pi poko piny eni. Somu mwa umiyu lanyuth mir acel ma nenre in vivo pi poko piny ma jucwalo ku protein malubiri ku bor pa lipid chain i kabedo ma juyero i yore mi secretory.
I cell pa eukaryotic, proteins ma juyubu i endoplasmic reticulum (ER) jupoko gi ikare ma jucwalo kud i yore mi secretory pi tero gi kama mitire (1). Mi medo ikum poko piny, jubedo ka paru nia lipids moko copo bedu ni kabedo mi wok woko ni kadhu kud i membrane domains ma tung tung (2-5). Ento, podi lembe moko mbe ma nyutho in vivo ma copo nyutho nia lipid-based mechanism copo bedu. Pi ni daru peko eni, waponjo i yeast kite ma glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) juwodho ko kud i ER. GPI-APs utiye kit piny matung tung ma mako ikum cell ( 6, 7). GPI-AP ubedo protein ma juwodho ma ucungo iwi lum ma woko pa plasma membrane nikadhu kud i glycolipid moiety (GPI anchor). Giyio GPI anchors calu aloka loka ma jutimo i ER lumen (8). I ngei mako, GPI-AP ukadhu kud i Golgi apparatus (5, 9) kud i ER nitundu i plasma membrane. Bedo tiye pa GPI anchors miyo GPI-AP ucidhi tung tung ku transmembrane secreted proteins (ma uketho i iye plasma membrane proteins mange) i yore mi secretory (5, 9, 10). I yeast cells, GPI-APs jupoko ku proteins mukende ma juwodho kud i endoplasmic reticulum, man jupoko gi i vesicles ma tung tung ma jugwoko ku coat protein complex II (COPII) (6, 7). Lembe ma nyayu poko eni i ER export process ungiyu ngo, ento juparu niya yore eni copo kwayu lipids, asaga structural remodeling pa lipid portion pa GPI anchor (5, 8). I yeast, GPI lipid remodeling ucaku ndhundhu ingeye ma GPI ucungo, man pol kare, eketho ceramide ucungo ikum 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide ubedo ceramide madit ma juyubu ku yeast cells nitundu kawoni. Juyike i ER man pol pare jucwalo woko i Golgi apparatus nikadhu kud i COPII vesicles (13). ER export pa GPI-AP mito ceramide synthesis (14, 15), man i ngeye, loko ceramide ni inositol phosphate ceramide (IPC) i Golgi apparatus ucungo iwi GPI anchor synthesis (16). Somo mi biophysical ku membrane ma dhanu timo unyutho nia acyl chain ceramides ma bor copo rii karacelu mi yubu domains ma rwom pare ukoc (17, 18). Lembe eni ucwalu paru niya C26 ceramide man GPI-AP ku C26 ceramide gitiyo ku kite migi mi nen ni bedo i kabedo ma ber nyo kabedo i ER membrane lipid environment ma nya wange nyai. Ebedo ku glycerolipids ma nok man ma unsaturated (C16:1 man C18:1) (19, 20). Kabedo eni jubi ketho wigi ikum kabedo moko ma wok kud i ER (ERES), kama ceramide ku ceramide-based GPI-AP copo bedu i Golgi i COPII vesicle acel (5).
I lembe maeni, wa temo yore maeni mi lipid ku tic ku super-resolution confocal real-time imaging microscopy (SCLIM), ma en e yore mi microscopy ma nyen ma copo neno fluorescently labeled proteins i saa acel.
Wacaku tiyo ku diro mi SCLIM ni medo nyutho kite ma GPI-AP maber ku C26 ceramide group jupoko kudu kud i transmembrane secreted proteins ingey weko ER in S. cerevisiae. Pi ni neno poko pa ER, watiyo ku yore mi gene ma copo neno piny ma nyen ma juyubu ma mondo i ERES in vivo (7, 23). Calu piny, wayero C26 ceramide-based GPI-AP Gas1 ma jugoro ku green fluorescent protein (GFP) man transmembrane secreted protein Mid2 ma jugoro ku near-infrared fluorescent protein (iRFP), ma gin ario zoo gicungo ikum plasma membrane (24–26). I sec31-1 temperature-sensitive mutant, piny aryo eni nyuthiri i the galactose-inducible promoter man constitutive ERES marker. I lyethu ma tek (37°C), pi en sec31-1 mutation nyayu tic pa COPII coat component Sec31 mi juku COPII germination man ER export, piny manyen ma juyubu cokiri i ER (23). I ngei lyel i lyethu ma piny (24°C), sec31-1 mutant cells udok kud i kabedu mi secretory, man piny manyen ma jucoko ucaku wodhu kud i ER. Nen pa CLIM unyutho nia pol pa Gas1-GFP man Mid2-iRFP ma nyen ma juyubu podi ucokiri i ER pa sec31-1 mutant cells ingey bedo i 37°C man juwodhe i 24°C pi dakika 5 (lanyut 1). Calu Mid2-iRFP jupoko iwi ER membrane zo, man Gas1-GFP jupoko man jucokiri i kabedo mi ER membrane ma rwinyo ngo, poko migi ukoc (lanyut 1, A ku C man filim S1). Mi medo, calu ma ju nyuthu i cal mir 1D, dul pa Gas1-GFP epe ku Mid2-iRFP. Adwogi eni nyutho nia GPI-AP man transmembrane proteins jupoko gi i ER membrane regions matung tung con. Gas1-GFP cluster utiye inget ERES moko ma jugoro ku mCherry's COPII coat protein Sec13 (lanyut 1, E ku F, man filim S1) (23).
sec31-1 cells express galactose-induced secretions, a long acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, green) and the transmembrane protein Mid2-iRFP (TMP, blue) and this Constructive ERES labeling Sec13-mCherry (E) were magcuted for RES13°C, 37 dakika, judwoko i 24°C, man jufoto ku SCLIM dakika abic i ngeye. (A ku C) nyutho cal ma rwate nyo acel pa 2D pa ndege (A), cal pa 2D pa 10 z-sections (B) nyo cal pa 3D pa cell hemisphere pa cargo man ERES markers (C). Scale bar 1μm (A ku B). Piny ma jupimo kudu ubedo 0.551μm (C). Gas1-GFP junwango i ER regions ma tung tung nyo dul, ma Mid2-iRFP ju nwango man jupoko i ER membrane (C). (D) Graph nyuthu rwom pa ler pa Gas1-GFP man Mid2-iRFP i Gas1-GFP cluster i thenge ma rangi pa thwol (kor cing). AU, piny ma rwinyo. (E ku F) nyutho cal 3D ma rwate ku piny man lanyuth pa ERES. Gas1-GFP clusters junwango ceng ku ERES. Piny ma jupimo kudu ubedo 0.551μm. (F) Lanyuth ma tar ma tek nyuthu dul pa Gas1-GFP ma mako ERES. Kabedo ma ikin man ma cinge yor acam nyuthu cal ma dit ma 3D man nen pa Gas1-GFP cluster ma juyeru.
Bede ma ceng ikum Gas1-GFP cluster ku ERES moko nyutho nia Gas1-GFP copo mondo i ERES ma juyeru, ma tung tung ku selectivity ma Mid2-iRFP tiyo kudu pi weko ER. Mi cobo peko eni, wapimo ERES ratio pi piny acel nyo aryo kende (lanyut 2, A ku C). Wanwango ni pol pa ERES (70%) utiye ku kit jeni acel kende. Cal ma piny pa cal 2C nyutho lanyuth ario pa ERES ku Gas1-GFP kende (cal 1) nyo Mid2-iRFP kende (cal 2). I ayi pa tung tung, 20% pa ERES utiye ku piny aryo ma ucungo iwi kabedu acel. Junwangu niya ERES moko (10%) utiye ku kit jeni ario, ento jupoko gi kama tung tung. Pieno, kwan maeni nyutho nia ingey ER juwodho woko, GPI-AP Gas1-GFP man transmembrane cargo Mid2-iRFP jupoko gi i ERES matung tung (lanyut 2D). Kit mi poko piny eni rwate ku lembe mukadho mi biochemical analysis (6) man morphological determination (7). Wacopo neno bende kura pa piny ma juketho i quarantine ma mondo i ERES (lanyut 2E man filim S2). Cal 2E nyutho nia dul ma nok kende pa Gas1-GFP (panel 3) nyo Mid2-iRFP (panel 4) re ma mondo i ERES kud i thenge acel man juketho i kabedo ma tung tung. Panel 5 mi cal 2E nyutho nia Gas1-GFP ku Mid2-iRFP kare moko nwangire i ERES acel, ento gimondo kud i thenge matung tung man gibedo i kabedo matung tung ma copo nyutho COPII vesicles matung tung. Wamoko bende nia poko man poko pa C26 ceramide-based GPI-AP Gas1 calu selective ERES utiye lembe ma pire tek pilembe transmembrane secretion cargo mange, GFP-tagged plasma membrane protein Axl2 (27 ), nyutho kura marom ku Mid2-iRFP. (Cal S1 man filim S3). Axl2-GFP ma nyen ma juyubu jupoko nikadhu kud i ER membrane calu Mid2-iRFP (lanyut S1, A ku B), man jupoko ku Mid2-iRFP i ERES mapol (lanyut S1, B ku D). Panel 1 ku 2 mi cal 1. S1C nyutho lanyuth ario pa ERES kama jeni ario ma wok kud i iye gicokiri. I lembe maeni, piny ario zoo gimondo i ERES karacelu (lanyut S1E, Panel 3 man filim S3).
Sec31-1 cells ma nyuthu galactose inducible secretions, Gas1-GFP (GPI-AP, green) man Mid2-iRFP (TMP, blue) man constitutive ERES labeling Sec13-mCherry (ERES, magenta) ju ketho gi i 37 I ngei bedo pi dakika 30 i °C, juwodho ion, man juwodh ion ku SCLIM ingey dakika 20. (A ku C) Cal ma nyutho 2D (A; scale bar, 1μm) nyo 3D cell hemisphere (B ku C; scale unit, 0.456μm) pa piny ma jucwalo man 10 z-sections ma juketho i ERES. Kabedo ma piny in (B) man kabedo in (C) nyuthu cal ma juyubu pi nyuthu piny ma nwangre i ERES (magenta) [Gas1-GFP (gray) man Mid2-iRFP (light blue)]. (C) Open arrow: ERES tingo piny acel kende (1 ku 4). Arrow macol: ERES utiye ku piny ma jupoko (5). Arrow ma rangine tar: ERES ma tye ku piny ma jucwalo. Piny: ERES acel ma juyeru utiye ku Gas1-GFP (1) nyo Mid2-iRFP (2) kende. Scale bar, 100 nm. (D) Kwantif pa photomicrograph ma jukoro i (C). Wel pa ERES ma tingo jeni acel kende (Gas1-GFP nyo Mid2-iRFP), jeni ma jupoko man jeni ma jupoko. I lembe adek ma jutimo, n=432 i 54 cells. Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) Cal 3D pa ERES ma juyeru pa piny ma juketho i quarantine ma jugoro ku (C). Gas1-GFP (macol) (3) nyo Mid2-iRFP (macol) (4) mondo i ERES (magenta) kud i thenge acel man juketho i kabedo moko manok i ERES. Pol kare, kit jeni ario zoo gimondo i ERES acel (5) kud i thenge acel man gibedo i kabedu moko kende i ERES. Scale bar, 100 nm.
I ngeye, wa temo paru moko ma nia long acyl chain ceramide (C26) ma nwangre i ER membrane telo wi poko man poko pa Gas1 i ERES ma juyeru. Pi timo eni, watiyo ku kit thobi ma juloko GhLag1, ma i iye ceramide synthases aryo Lag1 ku Lac1 juloko ku GhLag1 (Lag1 homolog pa pamba), ma ukelo kit thobi ku cell membrane Ceramide strain ma nok uloyu wild type (Figure 3A) (28). Mass spectrometry (MS) analysis unyutho nia i wild-type strains, 95% pa ceramide ceke ubedo bor (C26) chain ceramide, man i GhLag1, 85% pa ceramide ubedo mabor akeca (C18 ku C16). ), 2% kende pa ceramide utiye mabor (C26) ceramide. Kadi bende C18 ku C16 ceramides gibedo ceramides madongo ma junwango i GhLag1 membrane nitundu kawoni, MS analysis de unyutho nia GPI anchor pa Gas1-GFP ma juwodho i GhLag1 utiye ku C26 ceramide, ma juporo ku wild-type lipids. Ber pa piny utiye marom (cal 3A) (26). Pieno, eni nyutho nya ceramide remodeling enzyme Cwh43 utiye maber akeca pi C26 ceramide, calu ma ju nyuthu i cal mir 26, eketho GPI anchor kud i C26 ceramide manok in GhLag1 strain. S2 (29). Ento, cell membrane pa GhLag1 utiye ku C18-C16 ceramide kende, ma Gas1-GFP podi utiye ku C26 ceramide. Lembe eni weko kit eni ubedo piny maber mi cobo peko pa acyl chain length pa membrane ceramide i ER. Tic ma juparu pa dul man poko. I ngeye, wacaku ponjo kero pa C26 Gas1-GFP mi cokiri i dul i GhLag1 ku allele ma loko loko pa sec31-1 nikadhu kud i fluorescence microscopy, kama dul ma bor (C18-C16) kende re ma nwangre i ER membrane Ceramide (cal 3). Waneno ni i sec31-1, pol pa Gas1-GFP ubedo i dul, ma Gas1-GFP i sec31-1 GhLag1 ku long (C18-C16) long ceramide ER membrane ubedo ngo dul man jupoko i ER membrane zo. Mi bedu maleng, pi C26 ceramide-based clustering utiye kago ku ERES moko (lanyut 1), wabe lubo tok lembe eni copo bedu ku tic pa ER export protein mechanism. GPI-AP tiyo ku COPII system moko ma pire tek pi ER export, ma juyike ku Ted1′s structural remodeling pa glycan portion pa GPI anchor (30, 31). Recombinant GPI-glycan dong ungeyere ku transmembrane cargo receptor p24 complex, ma en e dwoko Lst1, ma en e isoform moko pa COPII cargo binding subunit Sec24, ma ketho GPI-AP-rich COPII Vesicles are necessary (31-33). Pieno, wacwio mutant ario ma uketho rwom pa proteins acel acel (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 man COPII subunit Lst1) ku sec31-1 mutant strain, man waponjo gi. Ka nyo ecopu bedu Gas1-cluster Gluster (Figure 3FP). Waneno ni i sec31-1emp24Δ man sec31-1ted1Δ, Gas1-GFP utiye ma ungiyo ngo i ER membrane, calu ma juneno con i sec31-1 GhLag1, man i sec31-1lst1Δ, Gas1-GFP calu sec31-1-1. Adwogi eni nyutho nia ikum bedo tiye pa C26 ceramide in ER membrane, dul pa Gas1-GFP de mitu ni mako p24 complex, man epe ku yeny pa Lst1. I ngeye, wa ngiyo ka nyo bor pa ceramide in ER membrane copo doro mako pa Gas1-GFP ku p24. Ento, wanwango ni bedo pa C18-C16 ceramide i membrane nyayu ngo adwogi ikum GPI-glycans ma juyubu kendu ku p24 complex ( cal S3 ku S4, A ku B) nyo mako GPI-AP man cwalo GPI-AP woko. tego. Nwangu COPII subtype Lst1 (lanyut S4C). Pieno, C26 ceramide-dependent clustering umitu ngo timo tic ku protein ku ER export protein mechanisms matung tung, ento ekonyo yore mukende mi poko piny ma lubo bor pa lipid. I ngeye, wa ngiyo ka ceramide acyl chain length in ER membrane piretek pi poko pa Gas1-GFP calu selective ERES. Calu Gas1 i GhLag1 strain ku short-chain ceramide weko ER man emondo i plasma membrane (Figure S5), wayiyu niya ka poko ubedo ku bor pa ceramide acyl chain, Gas1 i GhLag1 strain copo dwoko man kadhu. ERES piny ku membrane marom.
(A) Cell membrane pa GhLag1 utiye ku C18-C16 ceramides ma nok, man GPI anchor pa Gas1-GFP podi utiye ku C26 IPC marom ku wild-type cells. Malu: acyl chain length analysis pa ceramide in cell membrane pa wild-type (Wt) man GhLag1p strains ku mass spectrometry (MS). Lembe ne nyuthu wel pa ceramide. Ikind lembe adek ma jutimo giri. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001. Kabedo ma piny: MS analysis pa acyl chain length pa IPC ma nwangre i Gas1-GFP (GPI-IPC) GPI anchor ma jukoro i wild-type man GhLag1p strains. Lembe nyuthu wel pa IPC signal. Ikind lembe abic ma jutimo giri. Error bar = SD. Two-tailed unpaired t test. ns, piretek ungo. P = 0.9134. (B) Fluorescence micrographs pa sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ man sec31-1lst1Δ cells ma nyuthu galactose-induced Gas1-GFP juyike i 37°C pi dakika 30 man jutimo fluorescence microscopy ingey 24°C. Lanyuth matar: ER Gas1-GFP cluster. Open arrow: Unclustered Gas1-GFP jupoko iwi ER membrane zo, ma nyutho ER characteristic nuclear ring staining. Scale bar, 5μm. (C) Kwantif pa photomicrograph ma jukoro i (B). Wel ma romo pa cell ma ku punctate Gas1-GFP structure. I lembe adek ma jutimo, n≥300 cells. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001.
Mi daru peko eni, watimo SCLIM nen pa Gas1-GFP man Mid2-iRFP in GhLag1 ku sec31-1 temperature-sensitive mutant allele (lanyut 4 man filim S4). I ngei ma ER ubedo i 37°C man i ngeye juwodho i 24°C, pol pa Gas1-GFP ma nyen ma juyubu ubedo ngo ma jucokiri man jupoko ngo i ER membrane, calu ma juneno ku microscopes macon (lanyut 4, A man B). Mi medo, wel madit pa ERES (67%) uketho i iye kit jeni ario ma juketho i iye (lanyut 4D). Kabedo 1 ku 2 mi cal 4C nyutho lanyuth ario pa ERES ku Gas1-GFP man Mid2-GFP. Mi medo, piny ario zoo jukwanu gi i ERES acel (lanyut 4E, panel 3 man filim S4). Pieno, adwogi mwa nyutho nia bor pa ceramide acyl chain in ER membrane ubedo piny mapire tek ma copo ketho ER protein aggregation man poko.
Sec31-1 GhLag1 cells expressing galactose-induced secretions, Gas1-GFP (GPI-AP, green) and Mid2-iRFP (TMP, blue) and constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Incubate at 37°C. Med pi dakika 30, jwigo piny ni 24°C mi wodhu piny ma wok kud i iye, man cal ku SCLIM ingey dakika 20. (A ku C) Cal ma nyutho 2D (A; scale bar, 1μm) nyo 3D cell hemisphere (B ku C; scale unit, 0.45μm) pa 10 z-sections ma jugoro ku cargo man ERES. Kabedo ma piny in (B) man kabedo in (C) nyuthu cal ma juyubu pi nyuthu piny ma nwangre i ERES (magenta) [Gas1-GFP (gray) man Mid2-iRFP (light blue)]. (C) Arrow ma upong ku rangi matar: ERES, piny ma jucwalo ucungo. Open arrow: ERES utiye ku piny acel kende. Kabedo ma piny: ERES ma juyeru utiye ku piny ma rwate (1 ku 2) ma jugoro i (C). Scale bar, 100 nm. (D) Kwantif pa photomicrograph ma jukoro i (C). I kind sec31-1 man sec31-1 GhLag1, jeni acel kende (Gas1-GFP nyo Mid2-iRFP) re ma juketho i iye, man wel pa ERES pi jeni ma jupoko man jeni ma rwinyo. I lembe adek ma jutimo, n = 432 i 54 cells (sec31-1) man n = 430 i 47 cells (sec31-1 GhLag1). Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) man ** P = 0.0031 (sec31-1 GhLag1). (E) Cal 3D pa ERES ma juyeru ku piny ma jucwalo (3) ma jugoro i (C). Gas1-GFP (macol) man Mid2-iRFP (macol) bino i ERES (magenta) kud i thenge acel man gibedo i kabedo acel ma ERES juketho i iye. Scale bar, 100 nm.
Somo eni mio lanyuth ma nen kamaleng ikum lipid-based protein cargoes jupoko gi i kabedo ma juyero i yore mi secretory, man enyutho beru pa acyl chain length pi poko lembe. Ni tiyo ku diru mi microscopy ma tek man ma nyen ma julwongo ni SCLIM, wa nyutho Gas1-GFP (GPI-AP madit mi plasma membrane ku acyl chain mabor (C26) ceramide lipid portion) i yeast) Kabedo ma jucokiri i ERs ma tung tung gi rwate ku ERES ma tung tung, ma transmembrane proteins gi poko i membrane ERRES (lanyut 1). Mi medo, kit piny ario eni gimondo i ERES matung tung (lanyut 2). Bor pa acyl chain pa cellular ceramide i membrane judwoko piny kud i C26 ni C18-C16, Gas1-GFP cluster jupoko i ER region ma poko, man Gas1-GFP judwoko ni weko ER ku transmembrane protein nikadhu kud i ERES marom (lanyut 3 ku cal 3). 4).
Kadi bende GPI-AP tiyo ku yore moko ma pire tek mi wok kud i ER, wanwango ni C26 ceramide-dependent separation ucungo ngo iwi tung tung pa protein ma copo telu ERES specialization (Figures S4 ku S5). Kakare, lembe ma wanwango cwaku yore mukende mi poko piny ma wok kud i lipid-based protein clustering man weko piny mukende. Lembe ma waneno nyutho nia Gas1-GFP region nyo cluster ma mako ERES moko epe ku transmembrane secreted protein Mid2-iRFP, ma nyutho nia C26 ceramide-dependent GPI-AP cluster bikonyo gi ni mondo i ERES ma rwate, man i kare marom, ebiwodho transmembrane. I ayi pa tung tung, bedo tiye pa C18-C16 ceramides in ER membrane uketho GPI-AP timo ngo kabedo nyo clusters, ci giwodho ngo nyo giloko ngo transmembrane secreted proteins i ERES marom (lanyut 3 ku 4). . Pieno, waparu niya C26 ceramide telo wi poko man poko piny ni konyo poko pa proteins ma mako ERES moko.
Kite mi nwangu C26 ceramide-dependent clustering i kabedo moko mi ER? Kite ma membrane ceramide copo pokere kudu copo ketho GPI-AP ku C26 ceramide ni bedo lipids ma nok man ma rwinyore ndhundhu i kabedo ma lipid ma rwinyo ngo pa ER membrane ma tingo glycerolipids ma nok man ma rwinyo ngo. Dul maber (17, 18). Dul ma nok ma rii pi kare manok ecopu medere ni bedo dul madongo, ma ucungo maber ingey mako p24 complex (34). Malube ku lembe eni, wanyutho nia C26 Gas1-GFP umitu etim tic ku p24 complex mi yubu dul madongo ma nen (lanyut 3). P24 complex ubedo heterozygous oligomer ma juketho i iye p24 transmembrane proteins angwen matung tung i yeast (35), ma mio multivalent binding, ma copo telo wi cross-linking pa GPI-AP clusters ma nok, ma copo kelo Stable cluster madit (34). Wec ikind protein ectodomains pa GPI-APs copo bedu bende ni ketho gi karacelu, calu ma ju nyuthu i kare ma gi tero Golgi i mammalian polarized epithelial cells (36). Ento, ka C18-C16 ceramide utiye i ER membrane, ka p24 complex mako Gas1-GFP, dul madongo ma tung tung bibedo ngo. Lembe ma nyayu timo copo bedu nimakere ku kit ma nen ku wang man kemikol pa long acyl chain ceramide. Somo mi biophysical pa artificial membranes nyutho nia kadi nangu acyl chain ceramides ma bor (C24) man ma nok (C18-C16) copo nyayu apoka poka, acyl chain ceramides ma bor (C24) kende re ma copo kelo Curvature ma malu man film bending mi loko kit pa filim. Ni kadhu kud i lembe ma juporo (17, 37, 38). Junyutho nia transmembrane helix pa TMED2, ma en e human homologue pa Emp24, timo tic ku C18 ceramide-based sphingomyelin in cytoplasmic lobules (39). Ni tiyo ku molecular dynamics (MD) simulations, wanwango ni C18 ku C26 ceramides gicokiri i cytoplasmic lobules pa Emp24 transmembrane helix, man gitiye ku paru marom (Figure S6). Ber ni ngeyo ni eni nyutho nia transmembrane helix pa Emp24 copo telu poko pa lipids i membrane. Eni ubedo adwogi manyen ma wok kud i kum lei makwiny. MD simulations marom bende nyutho bedo pa ether lipids (40) . Pieno, waparu niya C26 ceramide in lobules aryo pa ER26 utiye maber. Ka GPI-AP i luminal lobules ucungo iwi multivalent p24 man cokiri pa C26 ceramide i p24 i cytoplasmic lobules, ecopu ketho Protein aggregation man membrane curvature juyubu nikadhu kud i cing (41), ma weko GPI-AP pokore i kabedo ma tung tung ma ceng ku ERES, ma bende konyo kabedo ma malu pa RES ER membrane (42). Lapor mukadho ucwaku yore ma juyero (43, 44). Dikiri pa oligolectins, pathogens nyo antibodies ni ceramide-based glycosphingolipids (GSL) iwi plasma membrane nyayu GSL madwong, medo apoka poka pa dul man nyayu aloka loka pa membrane man internalization (44). Iwabuchi etc. (43) Junwangu niya i bedo pa acyl chains ma bor (C24) ento ma nok ungo (C16), multivalent ligand ma mako GSL lactosylceramide uketho yik pa clusters madongo man membrane invagination, man cytoplasm Lyn-mediated signal transduction iwi leaflets ubedo interitatated by acyl chains in couples neutrophils.
I kind ler ma dongo dongo, rwom pa anti-Golgi network (TGN) ni rwom pa apical plasma membrane doro poko man poko pa GPI-AP (10, 45). Dikiri eni ucungo iwi GPI-AP oligomerization (36), ento ecopu bedu bende iwi bor pa ceramide ma wanwango i yeast. Kadi bende GPI-AP pa lei makwiny utiye ku ether lipid-based anchor, man kit pa kemikol pare opokiri ku acyl chain ceramide ma bor, somu manyen unwangu niya lipid ario eni gitiye ku kit man tic marom (40). Pieno, dul pa ether lipid in cell pa lei makwiny copo bedu marom ku C26 ceramide in yeast, man tic pare utiye ni ribri ku long-chain ceramide in membrane pi konyo GPI-AP aggregation man sorting. Kadi bende lembe eni podi mitire ni temo, lembe ma junwango con nyutho nia tero pa long acyl chain ceramide ni kum pa Golgi timo ngo ku cytoplasmic transfer proteins, ento ucungo iwi yubu pa GPI anchors calu yeast. Pieno, yore mi gwoko piny nen ni ecopu tingo acyl chain ceramide man GPI-AP (13, 16, 20, 46, 47) i vesicle acel.
I yeast man mammalian polarized epithelial cell systems, GPI-AP aggregation man separation ku plasma membrane proteins mukende timere ma podi etuc ungo i cell surface. Paladino et al. (48) unwango ni iwi TGN pa ler ma julwongo ni mammalian polarized epithelial cells, GPI-AP clustering epe kende pi poko GPI-APs ni apical plasma membrane, ento de eketho yub pa GPI-APs man tic migi. Kabedo pa cell. I yeast, somo eni unyutho nia C26 ceramide-dependent GPI-AP cluster iwi ER copo doro yub pa cluster man tic pa GPI-AP iwi plasma membrane (24, 49). Malube ku labol eni, GhLag1 cells gitiye ku allergic ni GPI inhibitors nyo yath ma nyayu peko i cell wall integrity (28), man mitu pa Gas1-GFP clusters (49) mi tip ceramide ma juporo i mating pa yeast cells nyutho G� Possible physiological consequences pa hLag1 cells. GPI-AP error. Ento, medo temo ka nyo yub pa cell surface juyike kud i ER ku yore mi poko piny malubiri ku bor pa lipid bibedo the lembe ma wabeponjo i anyim.
Kit pa Saccharomyces cerevisiae ma jutiyo kudu i tic eni jukiewo gi i Table S1. MMY1583 ku MMY1635 pa SCLIM pi neno cell ma kwo juyike i ngeye pa W303. Kit ma Sec13-mCherry ku fluorescent protein tag juyike ku tic ku polymerase chain reaction (PCR)-based method ku pFA6a plasmid calu template (23). Kit ma nyuthu Mid2-iRFP ma jugoro ku fluorescent protein ithe loc pa GAL1 promoter juyike calu ma e. PCR amplification pa iRFP-KanMx sequence kud i pKTiRFP-KAN vector (mic pa E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Man juketho i C-terminus pa Midgeneus. I ngei ma Mid2-iRFP genome sequence ju medo man ju clone i GAL1 promoter, ju ketho i Not I-Sac I site pa integration plasmid pRS306. Plasmid pRGS7 ma wok eca juyike ku Pst I ni ketho i URA3 locus.
Gas1-GFP fusion gene nyutre ithe telowic pa GAL1 promoter in centromere (CEN) plasmid, ma juyike nimakere ku e. Gas1-GFP sequence jumedo ku PCR kud i pRS416-GAS1-GFP plasmid (24) (mic pa L. Popolo) man juyike i Xma I-Xho I site pa CEN plasmid pBEVY-GL LEU2 (mic pa C). Miller; Addgene plasmid number 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Plasmid ma wok eca julwongo ni pRGS6. Axl2-GFP fusion gene de nyutre i the loc pa GAL1 promoter pa pBEVY-GL LEU2 vector, man yiku pare utiye ma e. Axl2-GFP sequence ju medo kud i pRS304-p2HSE-Axl2-GFP plasmid (23) ku PCR, man ju clone i Bam HI-Pst I site pa pBEVY-GL LEU2 vector. Plasmid ma wok eca julwongo ni pRGS12. Lembe ma oligonucleotides ma jutiyo kudu i lembe maeni jukiewo i Table S2.
Kit ne ju medo ku 0.2% adenine man 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract and 2% protein ept). (YPR)] nyo 2% galactose [YP-galactose (YPG)] calu piny ma nyayu carbon, nyo i synthetic minimal medium (0.15% yeast nitrogen base man 0.5% ammonium sulfate) pi medo amino acids man bases ma mitere pi cam, man ma tingo 2% glucose (2% glucose glucose) nyo minimum medium galactose (synthetic galactose minimal medium) calu piny ma kelo carbon.
Pi neno cal i kare mandha, sec31-1 mutant cells ma winjo lyethu ma nyutho construct ithe GAL1 promoter jupidu gi i YPR medium i 24°C dyewor nitundu mid-log phase. I ngei ketho i YPG i 24°C pi sawa acel, juyike cells i SG i 37°C pi dakika 30, man judwokogi i 24°C mi wodhu kud i secretion block. Concanavalin A jutiyo kudu pi ketho dul kom iwi gilasi man jufoto ku SCLIM. SCLIM ubedo dul pa Olympus IX-71 inverted fluorescence microscope man UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed man high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, intensive systems and the systems of images (Hamamatsu Photonics) ecopu mio lens ma dit ku rwom ma lageng pa ×266.7 man camera ma tiyo ku mac ma medo electrons (Hamamatsu Photonics) (21). Nwangu cal jutimo ku jam tic moko (Yokogawa Electric). Pi cal 3D, watiyo ku piezoelectric actuator ma juyike pi loko lens ma nen ku wang, man wacoko piny ma nen ku wang 100 nm i dul acel. Cal pa Z-stack juloko ni 3D voxel data, man theoretical point spread function ma jutiyo kudu pi rotating disc confocal microscope jutiyo kudu pi deconvolution processing ku Volocity software (PerkinElmer). Ni tiyo ku Volocity software mi ketho threshold pi co-location analysis, ERES ma uketho i iye cargo jupimu. Line scan analysis utimere ku kony pa MetaMorph software (Molecular Devices).
Ti ku GraphPad Prism software ni nyang ikum wel ma pire tek. Pi two-tailed Student's t-test man ordinary one-way analysis of variance (ANOVA) test, tung tung ikind ju nen ni bedu ku adwogi madit ikum P <0.05 (*).
Pi fluorescence microscopy pa Gas1-GFP, log phase cells jupidho dyewor i YPD man jucoko ku centrifugation, ju lwoko wang aryo ku phosphate buffered saline, man ju gwoko iwi ice pi dakika 15, man ju medo gi i the microscope calu ma jukoro con Check (24). Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ma juketho i iye objective lens, L5 (GFP) filter, Hamamatsu camera man Application Suite X (LAS X) software jutiyo kudu pi nwangu piny. .
Jam lanyuth ne juyike ku SDS sample buffer i 65°C pi dakika 10, man jupoko ku SDS-polyacrylamide gel electrophoresis (PAGE). Pi immunoblotting analysis, 10 μl pa lanyuth jubolo i lane acel acel. Antibody ma pire tek: Ti ku rabbit polyclonal anti-Gas1 i dilution pa 1:3000, rabbit polyclonal anti-Emp24 i dilution pa 1:500, man rabbit polyclonal anti-GFP (mic pa H. Riezman) i dilution pa 1:3000. Mouse monoclonal anti-Pgk1 antibody jutiyo kudu i dilution pa 1:5000 (mic pa J. de la Cruz). Antibody mir aryo: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) ma jutiyo kudu i dilution pa 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG jutiyo kudu i dilution pa 1:3000 (Pierce). Kabedo mi gwoko kum dhanu nen ku yore mi chemiluminescence pa SuperSignal West Pico reagent (Thermo Fisher Scientific).
Calu ma jukoro i (31), jutimo sayusac mi immunoprecipitation iwi ER fraction ma juyiko. I lembe ma nok, lwok yeast cells ku TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride man protease inhibitor mixture) i 600 nm (OD600) i 100 optical density wang aryo. Juturo ku cing gilasi, man ingeye juwodhu lum man cing gilasi ku centrifugation. Piny ma ucungo iwi pii eca juyike i 17,000 g pi dakika 15 i 4°C. Pellet ne ju dwoko i TNE man digitalis saponin ju medo i rwom ma lac pa 1%. Juyiko piny ma juciko pi sawa acel ku loko i 4°C, man i ngeye juwodho piny ma nyai ungo ku centrifugation i 13,000 g i 4°C pi dakika 60. Pi Gas1-GFP immunoprecipitation, mir acel iket lanyuth ku agarose beads (ChromoTek) i 4°C pi sawa acel, man i ngeye iket ku GFP-Trap_A (ChromoTek) i 4°C pi sawa 3. Jam piny ma juyiko ku cing ju lwoko wang abic ku TNE ma tye ku 0.2% digoxigenin, juwodho ku SDS sample buffer, jupoko iwi SDS-PAGE, man juporo ku immunoblotting.
Calu ma jukoro i (31), timo sayusac ikum dul pa ER. I lembe ma nok, dul pa ER ma juyiko ku 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Adwogi pa crosslinking ne jwigo ku medu glycine (50 mM final concentration, 5 minutes, 20°C).
Calu ma jukoro con (50), MS analysis pa ceramide in wild-type man GhLag1 jutimo. I lembe ma nok, ju dongo ikum dul kom (3 ku 4 OD600 units/ml) i YPD i 30°C, man ju kwalo dul kom 25×107. Kit ma gi tiyo kudu ju jwigo ku trichloroacetic acid. Ti ku piny ma nyayu pii [ethanol, pii, ether, pyridine man 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] man 1.2 nmol pa internal standard C17 ceramide (860517, Avanti polar lipid) quality). Ti ku monomethylamine reagent [methanol, pii, n-butanol man methylamine solution (4:3:1:5 v/v)] mi timo mild alkaline hydrolysis pa extract, man i ngeye ti ku water-saturated n-butanol mi kwanyo kadu. I ajiki, juwodho piny ne i pii ma ber [chloroform/methanol/pi (2:7:1) + 5 mM ammonium acetate] man jubolo i mass spectrometer. Multi-reaction monitoring (MRM) jutimo pi ngeyo man pimo wel pa sphingolipid molecules. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) utiye ku robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) pi nyang ikum lipid. Teko mi turo piny juyike pi dul pa ceramide acel acel. MS data ju nwango i yore maber. Pi piny acel acel ma judwoko wange, lanyuth pa lipid ubedo ikind piny adek ma jupimo giri.
Calu ma jukoro i (31), dul kom (800×107) ma nyutho Gas1-GFP jutimo igi immunoprecipitation. Gas1-GFP ma juyike jupoko ku SDS-PAGE man judwoko i polyvinylidene fluoride (PVDF) membrane. Protein ne nenre ku rangi pa PVDF ku amide macol. Gas1-GFP band jupoko kud i PVDF man ju lwoko wang abic ku methanol man wang acel ku pii ma rwom pare lapiny. Ku ketho membrane strip ku 500μl 0.3 M NaOAc (pH 4.0), buffer man 500μl freshly dissolved 1 M sodium nitrite mixture i 37°C pi sawa 3, lipid fraction juwodho kud i Gas1-GFP man lysed Release of ininphoosphaminephos in gluefamiol i kind ceramiode (51). I ngeye, juyiko membrane strip wang angwen ku pii ma LC-MS, juyike i rwom ma piny, man jugwoko i nitrogen atmosphere i -80°C nitundu jutimo sayusac. Calu lanyuth, lanyuth ma oling pa PVDF membrane jutiyo kudu pi temo acel acel. Piny ma juwodho kud i Gas1-GFP jutimo sayusac iwie ku MS calu ma juloko (50). I lembe ma nok, PVDF strips ma tye ku GPI-lipid ju dwoko gi i 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] man ukadhu electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). I lembe maeni, MS data ju nwango i negative ion mode.
Calu ma juweco pire acaka, dul pa lipid pa GPI anchor jupoko kud i [3H]-inositol-labeled GPI-AP (16). Lipids jupoko ku thin-layer chromatography ma jutiyo kudu ku solvent system (55:45:10 chloroform-methanol-0.25% KCl) man juporo ku FLA-7000 (Fujifilm).
Cells ma nyutho Gas1-GFP (600×107) ju lwoko wang aryo ku TNE buffer ku TNE buffer, man ju turo ku glass beads, man ju centrifuge pi kwanyu cell debris man glass beads. Piny ma ucungo iwi pii eca juyike i 17,000 g pi sawa acel i 4°C. Pellet ne juyike i TNE man juyike ku 1 U PI-PLC (Invitrogen) i TNE ma tye ku 0.2% digitalis saponin pi sawa acel i 37°C. I ngei tic ku enzyme, juwodho membrane ku centrifugation i 17,000 g i 4°C pi sawa acel. Mi immunoprecipitate Gas1-GFP, juyike ku GFP-Trap_A (ChromoTek) i 4°C dyewor. Gas1-GFP ma juyiko maber ma jupoko ku SDS-PAGE juyike ku Coomassie macol ma ler. Gas1-GFP staining band jupoko kud i gray ma ucungu aqueduct, man ingey alkylation ku iodoacetamide man reduction ku dithiothreitol, in-gel digestion ku trypsin jutimo. Kwany man jwigo tryptic peptides man peptides ku GPI-glycans. Peptide ma muthwo juyike i 20 μl pa pii. Med dul (8μl) i LC. Octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) jutiyo kudu pi poko peptides i thenge moko. The mobile phase is solvent A (0.08% formic acid) and solvent B (0.15% formic acid in 80% acetonitrile). Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) jutiyo kudu pi jwigo column ku solvent A ikind dakika 55 i flow rate pa 50 μl min-1 pi dakika 5, man i ngeye jumedo rwom pa solvent B ni 40%. , Amerika). Eluate ne jumedo i ESI ion source, man tryptic peptides man peptides ku GPI-glycans juporo ku LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). I MS setup, voltage pa capillary source ubedo 4.5 kV, man temperature pa transfer capillary ubedo 300°C. Voltage pa capillary man voltage pa tube lens juyike ni 15 V man 50 V, kubang wadi. MS data ju nwango i positive ion mode (resolution pa 60,000; mass accuracy pa 10 parts per million) i mass range pa 300/m/z mass/charge ratio (m/z) 3000. MS/MS data ju nwango nikadhu kud i ion trap in LTQ Orbit [XL XL data ma kwong, ma ucungo iwi namba ma kwong collision induced dissociation (CID)].
MD simulations jutimo ku GROMACS (52) software man MARTINI 2 force field (53-55). CHARMM GUI Membrane Builder (56, 57) jutiyo kudu pi yiku bilayer ma tye ku dioleoylphosphatidylcholine (DOPC) man Cer C18 nyo DOPC man Cer C26. Topology man coordinates pa Cer C26 juwodho kud i DXCE ku kwanyu beads mapol kud i sphingosine tail. Ti ku yore ma jukoro piny eno ni kethu rwom pa dul ario man timo kudu, man ti ku coordinates mir ajiki pa yub ni gieru yub ma tye ku Emp24. Transmembrane domain pa yeast Emp24 (residues 173 to 193) juyike calu α-helix ku tic ku visual MD (VMD) tool molecular structure (58). I ngeye, ingey wodhu lipids ma ucungo iwi gi, protein ne jupoko man jubolo i bilayer ku tic pa CHARMM GUI. Lembe ma tok cen otie ku 1202 DOPC man 302 Cer C26 nyo 1197 DOPC man 295 Cer C18 man Emp24. Ionize piny ni rwom pa 0.150M. Replicates angwen ma tung tung jutimo pi bilayer aryo.
Lipid bilayer juyike ku tic pa CHARMM GUI, ma uketho i iye dwoko piny man dwoko piny 405,000 steps, kama position constraints judwoko piny man juwodho, man time step jumedo nicaku 0.005 ps ni 0.02 ps. I ngei rwom, ewodho 6 μs ku rwom pa 0.02 ps. I ngei ketho Emp24, ti ku CHARMM GUI marom ni dwoko piny man ni dwoko piny, man idok i tic pi 8 s i tic.
Pi lembe ceke, i saa mi ketho piny i rwom acel, tego ne juyike ku Berendsen barostat (59), man i kare mi yubu piny, tego ne juyike ku Parrinello-Rahman barostat (60). I lembe ceke, rwom pa rwom ubedo 1 bar man rwom pa rwom pa rwom ma lageng jutiyo kudu. I lembe mi rwom man mi yubu piny, thermostat (61) ku speed recalibration jutiyu kudu pi rwom pa lyethu pa protein, lipid man solvent particles. I kind tic eca zo, lyethu ma mitire ubedo 310K. Wec ma epe ku cing ju kwanu ku yubu dul ma rwate ku Verlet scheme ku 0.005 buffer tolerance. Coulomb term ju kwanu ku tic ku reaction field man cut-off distance pa 1.1 nm. Lembe pa Vander Waals tiyo ku yore mi poko piny ku bor pa 1.1 nm, man yore mi poko piny pa Verlet tiyu ku yore mi poko piny (62).
Ni tiyo ku VMD, cutoff wavelength ikind DOPC phosphate beads nyo ceramide AM1 beads ku protein utiye 0.7 nm, man wel pa lipids ma timo tic ku protein ju kwanu. Nimakere ku formula ma e, kwan depletion-enrichment (DE) factor calu in (63): DE factor = (wel pa lipids i protein 0.7) i protein 0.7 (wel pa Cer in total lipids)
Wel ma jukoro eca ju nwango calu wel ma romo, man error bars utiye kopi angwen ma tung tung pa SE. Lembe ma pire tek pa DE factor ju kwanu ku t test [(averageDE-factor-1)/SE]. Kwan P value kud i acel-tailed distribution.
Jam tic pa GROMACS jutiyo kudu pi kwanu 2D lateral density map pa piny ma tingo Emp24 ikind 250 ns ma tok cen pa trace. Pi ni nwangu map mi dongo/nyoth pa ceramide, map mi pek pa Cer jupoko ku wel pa map pa Cer ku DOPC, man jupoko ku dwong pa Cer i kum. Map scale marom jutiyo kudu.
Pi lembe mukende ma medo ikum lembe maeni, nen http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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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 -Lingio Serero, Lopez (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging nyutho beru pa ceramide chain length pi poko protein i kabedo ma juyeru.
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 -Lingio Serero, Lopez (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging nyutho beru pa ceramide chain length pi poko protein i kabedo ma juyeru.
©2020 Kanica mi Amerika pi dongo ngec mi ngec. twero ceke jugwoko. AAAS ubedo wadi pa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef man COUNTER. ScienceAdvances ISSN 2375-2548.