Ku hlawuriwa ka ti protein eka ndlela ya secretory swina nkoka eka ku hlayisa cell compartmentalization na homeostasis. Ku engetela eka shell-mediated sorting, ntirho wa ti lipids eka kinesin sorting eka maendlelo ya secretory transport i xivutiso xa xisekelo lexi nga na nkarhi wo leha lexi nga si hlamuriwa. Laha, hi endla 3D simultaneous multicolor high-resolution real-time imaging ku kombisa in vivo leswaku ti protein letintshwa leti endliweke hi glycosylphosphatidylinositol-immobilized leti nga na swiphemu swo leha swinene swa ceramide lipid ti hlengeletiwile no hlayiwa eka ti endoplasms to hlawuleka Net exit site, leyi hambaneke na leyi tirhisiwaka hi ti protein ta transmembrane. Ku engetela kwalano, hi kombisa leswaku ku leha ka chain ya ceramide eka endoplasmic reticulum membrane i swa nkoka swinene eka ku hlawula loku ka ku hlawula. Dyondzo ya hina yi nyika vumbhoni byo sungula byo kongoma bya in vivo ku hlawula ndzhwalo wa tiphrotheyini lowu simekiweke eka ku leha ka lipid chain eka tindhawu to hlawula to rhumela ematikweni mambe eka ndlela ya xihundla.
Eka tisele ta eukaryotic, tiphrotheyini leti endliweke eka endoplasmic reticulum (ER) ti tlhela ti hleriwe hi nkarhi wa ku fambisiwa hi ndlela ya xihundla leswaku ti yisiwa eka ndhawu ya tona leyi faneleke ya tisele (1). Ku engetela eka ku hlawula loku kongomisiwaka hi jazi, ku tshame ku ringanyetiwa leswaku ti- lipid to karhi ti nga tlhela ti tirha tanihi tindhawu to huma leti hlawulaka hi ku ti hlengeleta eka tidomeni to karhi ta membrane leti tiphrotheyini to karhi (2-5). Hambiswiritano, ka ha ri na ku pfumaleka ka vumbhoni byo kongoma bya in vivo ku tiyisisa endlelo leri leri nga kotekaka leri sekeriweke eka lipid. Ku kota ku tlhantlha xiphiqo lexi xa xisekelo, hi dyondze eka comela ndlela leyi ti glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) ti rhumeriwaka hi ndlela yo hambana kusuka eka ER. GPI-APs i ku hambana ka tiphrotheyini ta le henhla ka sele leti hlanganisiweke na lipid ( 6, 7). GPI-AP i protein leyi humesiweke leyi khomanisiweke na swiphepherhele swale handle swa plasma membrane hiku tirhisa glycolipid moiety (GPI anchor). Va amukela ti-anchor ta GPI tanihi ku cinca loku hlayisaka endzhaku ka vuhundzuluxi eka ER lumen (8). Endzhaku ko namarhela, GPI-AP yi hundza eka xitirhisiwa xa Golgi (5, 9) kusuka eka ER kuya eka plasma membrane. Vukona bya ti anchor ta GPI byi endla leswaku GPI-AP yi fambisiwa hi ku hambana na ti protein leti humesiwaka hi transmembrane (ku katsa na ti protein tin’wana ta plasma membrane) hi ndlela ya secretory ( 5 , 9 , 10 ). Eka tisele ta comela, ti GPI-APs ti hambanisiwa na ti protein tin’wana leti humesiweke eka endoplasmic reticulum, kutani ti pakiwa eka ti vesicles to hlawuleka leti phutseriweke hi coat protein complex II (COPII) ( 6 , 7 ). Swihlawulekisi swa endlelo leri ra ku hlawula eka endlelo ro rhumela ehandle ka ER a swi le rivaleni, kambe ku ringanyetiwa leswaku endlelo leri ri nga ha lava ti-lipid, ngopfu-ngopfu ku pfuxetiwa ka xivumbeko xa xiphemu xa lipid xa anchor ya GPI (5, 8). Eka comela, ku pfuxetiwa ka lipid ya GPI ku sungula hi ku hatlisa endzhaku ka loko GPI yi namarherile, naswona eka swiyimo swo tala, swi endla leswaku ceramide yi boha eka 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide i ceramide leyikulu leyi humelerisiwaka hi ti cells ta comela kufikela sweswi. Yi endliwa eka ER naswona vunyingi bya yona yi rhumeriwa eka xitirhisiwa xa Golgi hiku tirhisa ti vesicles ta COPII (13). Ku rhumeriwa ka ER ka GPI-AP hi ku kongoma swi lava ku endliwa ka ceramide loku yaka emahlweni ( 14 , 15 ), naswona hi ku tlhelela, ku hundzuriwa ka ceramide ku va inositol phosphate ceramide (IPC) eka xitirhisiwa xa Golgi swi titshege hi ku endliwa ka anchor ya GPI ( 16 ). Tidyondzo ta biophysical na ti artificial membranes ti kombisile leswaku ti acyl chain ceramides to leha swinene tinga hlangana ku vumba ti ordered domains letingana swivumbeko swo hlawuleka swa physical ( 17 , 18 ). Data leyi yi yisa eka xiringanyeto xa leswaku C26 ceramide na GPI-AP na C26 ceramide ti tirhisa swivumbeko swa tona swa nyama ku hlangana eka tindzhawu leti hlelekeke kumbe swifundzha eka ndzhawu ya lipid ya ER membrane leyi nga hlelekangiki ngopfu. Ngopfu ngopfu yi vumbiwa hi ti glycerolipids to koma na leti nga tatiwangiki (C16:1 na C18:1) (19, 20). Swifundzha leswi swi ta kongomisiwa hi ku hlawula eka tindhawu to karhi to huma ta ER (ERES), laha ceramide na ceramide-based GPI-AP swi nga rhumeriwaka swin’we eka Golgi eka vesicle yin’we leyi tinyiketeleke ya COPII (5).
Eka dyondzo leyi, hi ringetile hi ku kongoma endlelo leri leri simekiweke eka lipid hi ku tirhisa super-resolution confocal real-time imaging microscopy (SCLIM), leyi nga thekiniki ya microscopy ya xiyimo xa le henhla leyi nga langutaka hi nkarhi wun’we tiphrotheyini leti leteriweke hi fluorescent Swifaniso swa mihlovo yinharhu na swiphemu swinharhu (3D) swi na xiboho xa le henhla swinene na rivilo eka tisele leti hanyaka (21, 22).
Hi sungule hi tirhisa thekinoloji ya SCLIM ku ya emahlweni hi hlamusela hilaha GPI-AP ya ntolovelo leyi nga na ntlawa wa C26 ceramide yi kamberiweke hakona ku suka eka tiphrotheyini leti humesiweke hi transmembrane endzhaku ko huma eka ER eka S. cerevisiae. Leswaku hi kambela ku hlawuriwa ka ER, hi tirhise fambiselo ra xitekela leri nga vonaka hi ku kongoma ndzhwalo lowuntshwa lowu endliweke lowu nghenaka eka ERES eka vivo ( 7 , 23 ). Tanihi ndzhwalo, hi hlawurile C26 ceramide-based GPI-AP Gas1 leyi leteriweke hi rihlaza ra fluorescent protein (GFP) na transmembrane secreted protein Mid2 leyi leteriweke hi near-infrared fluorescent protein (iRFP), leswi havumbirhi bya swona swi kongomisaka eka plasma membrane ( 24–26 ). Eka sec31-1 temperature-sensitive mutant, mindzhwalo leyi yimbirhi yi kombisiwa ehansi ka galactose-inducible promoter na constitutive ERES marker. Eka mahiselo yo tlula mpimo (37°C), hikuva ku cinca ka sec31-1 ku khumba ntirho wa xiphemu xa jazi ra COPII Sec31 ku sivela ku mila ka COPII na ku rhumeriwa ka ER, ndzhwalo lowuntshwa lowu endliweke wu hlengeletana eka ER (23). Endzhaku ko hola kuya eka mahiselo yale hansi (24°C), ti cells ta sec31-1 mutant ti vuyisiwile kusuka eka ndzhawu ya secretory, naswona ndzhwalo wuntshwa wa synthetic lowu hlengeletiweke wusungule ku rhumeriwa kusuka eka ER. Ku vonakala ka CLIM ku kombisile leswaku vunyingi bya Gas1-GFP na Mid2-iRFP leyintshwa leyi endliweke ya ha hlengeletana eka ER ya tisele ta sec31-1 mutant endzhaku ko pfaleriwa eka 37°C ivi ti humesiwa eka 24°C ku ringana 5 wa timinete (Xifaniso 1). Tanihi leswi Mid2-iRFP yi hangalasiweke eka ER membrane hinkwayo, naswona Gas1-GFP yi hlanganisiwile no hlengeletiwa eka ndzhawu ya ER membrane leyi nga yimangiki, ku hangalasiwa ka yona ku hambanile hi ku helela (Xifaniso 1, A ku ya eka C na Movie S1). Ku engetela kwalaho, tanihilaha swi kombisiweke hakona eka Xifaniso xa 1D, xitluletsongo xa Gas1-GFP a xi na Mid2-iRFP. Mimbuyelo leyi yi kombisa leswaku ti protein ta GPI-AP na transmembrane ti hambanisiwile eka tindzhawu to hambana ta ER membrane ka ha ri na nkarhi. Ntlawa wa Gas1-GFP wu le kusuhi na ERES yo karhi leyi leteriweke hi mCherry’s COPII coat protein Sec13 (Xifaniso 1, E na F, na filimi ya S1) (23).
ti cells ta sec31-1 ti kombisa swihumesi leswi hlohleteriwaka hi galactose, long acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, rihlaza) na transmembrane protein Mid2-iRFP (TMP, blue) naswona leswi Constructive ERES labeling Sec13-mCherry (ERES, magenta) yi pfaleriwile eka 37°C kuringana 30 wa timinete, yi rhurhiseriwe eka 24°C, naswona yi tekiwile swifaniso hi SCLIM endzhaku ka timinete ta 5. (A ku ya eka C) yi kombisa xifaniso lexi yimelaka lexi hlanganisiweke kumbe xin’we xa 2D xa xihahampfhuka (A), xifaniso xa 2D xa projection xa 10 z-sections (B) kumbe xifaniso xa 3D cell hemisphere xa ndzhwalo na swimaki swa ERES (C). Bar ya xikalo 1μm (A na B). Yuniti ya xikalo i 0.551μm (C). Gas1-GFP yi kumiwile eka swifundzha swa ER leswi hambaneke kumbe swirhabyani, kasi Mid2-iRFP yi kumiwile no hangalasiwa eka ER membrane hinkwayo (C). (D) Girafamu yi kombisa matimba ya fluorescence ya xiringaniso ya Gas1-GFP na Mid2-iRFP eka xirhundzu xa Gas1-GFP etlhelo ka layini ya miseve yo basa (eximatsini). AU, yuniti ya ku tihlawulela. (E na F) yi yimela xifaniso xa 3D lexi hlanganisaka nhundzu na mfungho wa ERES. Swirhabyani swa Gas1-GFP swi kumiwile ekusuhi na ERES yo karhi. Yuniti ya xikalo i 0.551μm. (F) Museve wo basa wo tiya wu fungha xirhundzu xa Gas1-GFP lexi fambelanaka na ERES. Tiphaneli ta le xikarhi na ta xinene ti kombisa xifaniso xa 3D lexi kurisiweke lexi hlanganisiweke na xivono lexi rhendzelekaka xa xitluletsongo xa Gas1-GFP lexi hlawuriweke.
Vuxaka bya ndhawu ya le kusuhi exikarhi ka xitluletsongo xa Gas1-GFP na ERES yo karhi byi kombisa leswaku Gas1-GFP yi nga nghena eka ERES yo hlawula, leyi hambaneke na ku hlawula loku tirhisiwaka hi Mid2-iRFP ku huma eka ER. Ku lulamisa ku koteka loku, hi pimile mpimo wa ERES eka nhundzu yin’we kumbe yimbirhi ntsena (Xifaniso 2, A ku ya eka C). Hi kumile leswaku ERES yo tala (70%) yi na muxaka wun’we ntsena wa ndzhwalo. Xifaniso xa le hansi xa Xifaniso xa 2C xi kombisa swikombiso swimbirhi leswi tolovelekeke swa ERES leyi nga na Gas1-GFP ntsena (Xifaniso 1) kumbe Mid2-iRFP ntsena (Xifaniso 2). Kuhambana na sweswo, kwalomu ka 20% wa ERES yina mindzhwalo yimbirhi leyi hlanganisaka eka ndzhawu yin’we. Ku kumiwile leswaku ERES yin’wana (10%) a yi ri na tinxaka timbirhi ta ndzhwalo, kambe a ti hambanisiwile eka tindhawu leti hambaneke swinene. Hikwalaho, nxopaxopo lowu wa tinhlayo wu kombisa leswaku endzhaku ka loko ER yi rhumeriwile ehandle, GPI-AP Gas1-GFP na ndzhwalo wa transmembrane Mid2-iRFP swi avanyisiwile hi ERES yo hambana (Xifaniso 2D). Vutshila lebyi byo hlawula byi fambisana swinene na nxopaxopo wa khale wa tikhemikhali ta vutomi (6) na ku kumiwa ka xivumbeko (7). Hi nga tlhela hi languta mahanyelo ya ndzhwalo lowu nga le ku hlayisiweni lowu nghenaka eERES (Xifaniso 2E na Muvi wa S2). Xifaniso xa 2E xi kombisa leswaku i xiphemu lexitsongo ntsena xa Gas1-GFP (phanele 3) kumbe Mid2-iRFP (phanele 4) lexi nghenaka eka ERES ku suka eka tlhelo rin’we naswona xi pfaleriwile eka ndhawu leyi hambaneke. Panele ya 5 ya Xifaniso xa 2E yi kombisa leswaku Gas1-GFP na Mid2-iRFP minkarhi yin’wana ti kumeka eka ERES yin’we, kambe ti nghena kusuka eka matlhelo yohambana naswona ti hlengeletiwile eka tindzhawu to hambana letinga yimelaka ti vesicles to hambana ta COPII. Hi tlhele hi tiyisisa leswaku ku hambanisiwa loku xiyiweke na ku hlawuriwa ka C26 ceramide-based GPI-AP Gas1 tanihi ERES yo hlawula swi kongomile hikuva ndzhwalo wun’wana wa transmembrane secretion, GFP-tagged plasma membrane protein Axl2 (27 ), leyi kombisaka mahanyelo yo fana na Mid2-iRFP. (Xifaniso xa S1 na Filimi ya S3). Axl2-GFP leyintshwa leyi endliweke yi hangalasiwa hi ER membrane kufana na Mid2-iRFP (Xifaniso S1, A na B), naswona yi co-localized na Mid2-iRFP eka ERES yotala (Xifaniso S1, B kuya eka D). Tiphaneli ta 1 na 2 ta Xifaniso 1. S1C yi kombisa swikombiso swimbirhi leswi tolovelekeke swa ERES laha mindzhwalo yimbirhi ya transmembrane yi hlanganisaka. Eka swiyimo leswi, nhundzu leyimbirhi yi nghena eka ERES swin’we (Xifaniso xa S1E, Phanele 3 na Muvi S3).
Ti cells ta sec31-1 leti kombisaka galactose inducible secretions, Gas1-GFP (GPI-AP, rihlaza) na Mid2-iRFP (TMP, blue) na constitutive ERES labeling Sec13-mCherry (ERES, magenta) ti vekiwile eka 37 Endzhaku ko pfaleriwa kuringana 30 wa timinete eka °C, ti yisa eka 24 °C ku humesa secretion block, na xifaniso hi SCLIM endzhaku 20 wa timinete. (A ku ya eka C) Swifaniso leswi yimelaka 2D projection (A; scale bar, 1μm) kumbe swifaniso swa 3D cell hemisphere (B na C; scale unit, 0.456μm) swa ndzhwalo na 10 wa swiphemu swa z leswi funghiweke hi ERES. Phanele ya le hansi eka (B) na phanele eka (C) yi kombisa swifaniso leswi phurosesiweke ku kombisa ntsena nhundzu leyi nga kona eka ERES (magenta) [Gas1-GFP (gray) na Mid2-iRFP (light blue)]. (C) Museve lowu pfulekeke: ERES yi rhwala ntsena xiphemu xin’we xa ndzhwalo (1 ku ya eka 4). Museve wa grey: ERES yi na ndzhwalo lowu hambanisiweke (5). Museve wo basa wo tiya: ERES leyi nga na ndzhwalo lowu nga eka ndhawu yin’we. Laha hansi: ERES yin’we leyi hlawuriweke yi na Gas1-GFP (1) kumbe Mid2-iRFP (2) ntsena. Bar ya xikalo, 100 nm. (D) Ku pima nhlayo ya photomicrograph leyi hlamuseriweke eka (C). Phesente ya xikarhi ya ERES leyi nga na ndzhwalo wun’we ntsena (Gas1-GFP kumbe Mid2-iRFP), ndzhwalo lowu hambanisiweke na ndzhwalo lowu hlanganisaka. Eka swikambelo swinharhu leswi tiyimeleke, n=432 eka 54 wa tisele. Bar ya swihoxo = SD. Xikambelo xa t lexi nga hlanganisiwangiki xa misisi yimbirhi. *** P = 0.0002. (E) Xifaniso xa 3D xa ERES leyi hlawuriweke ya ndzhwalo lowu vekiweke etlhelo lowu funghiweke hi (C). Gas1-GFP (rihlaza) (3) kumbe Mid2-iRFP (ya wasi) (4) yi nghena eka ERES (magenta) kusuka eka tlhelo rin’we naswona yi siveriwa eka ndzhawu leyintsongo endzeni ka ERES. Minkarhi yin’wana, tinxaka leti hatimbirhi ta ndzhwalo ti nghena eka ERES yin’we (5) ku suka eka tlhelo rin’we naswona ti pfaleriwa endhawini leyi nga yoxe endzeni ka ERES. Bar ya xikalo, 100 nm.
Endzhaku ka sweswo, hi ringetile xiringanyeto xa leswaku long acyl chain ceramide (C26) leyi nga kona eka ER membrane yi fambisa ku hlengeletiwa ko karhi na ku hlawula ka Gas1 eka ERES yo hlawula. Ku fikelela leswi, hi tirhise muxaka wa comela lowu cinciweke GhLag1, laha ti-endogenous ceramide synthases timbirhi Lag1 na Lac1 ti siviwe hi GhLag1 (the Lag1 homolog of cotton), leswi endleke leswaku ku va na muxaka wa comela lowu nga na muxaka wa cell membrane Ceramide wo koma ku tlula muxaka wa nhova (Xifaniso 3A) (28). Nxopaxopo wa mass spectrometry (MS) wu kombisile leswaku eka tinxaka ta muxaka wa nhova, 95% wa ceramide hinkwayo yi lehe swinene (C26) chain ceramide, kasi eka GhLag1, 85% wa ceramide yi lehe swinene (C18 na C16). ), ntsena 2% wa ceramide i ceramide ya chain yo leha swinene (C26). Hambi leswi C18 na C16 ceramides kunga ti ceramides letikulu leti kumiweke eka GhLag1 membrane kufikela sweswi, nxopaxopo wa MS wutlhele wu tiyisisa leswaku GPI anchor ya Gas1-GFP leyi kombisiweke eka muxaka wa GhLag1 yina C26 ceramide, leyi ringanisiwaka na ti wild-type lipids. Khwalithi ya kona ya fana (Fig. 3A) (26). Hikokwalaho, leswi swivula leswaku enzyme ya ceramide remodeling Cwh43 yi hlawula swinene eka C26 ceramide, tani hileswi swikombisiweke eka Xifaniso xa 26, yitsakela ku katsa GPI anchor kusuka eka nhlayo yintsongo ya C26 ceramide eka muxaka wa GhLag1. S2 (29) (29). Hambi swi ri tano, membrane ya sele ya GhLag1 hi xisekelo yi na C18-C16 ceramide ntsena, kasi Gas1-GFP ya ha ri na C26 ceramide. Mhaka leyi yi endla leswaku muxaka lowu wu va xitirhisiwa lexinene xo tlhantlha hi ku kongoma xiphiqo xa ku leha ka acyl chain ya membrane ceramide eka ER. Ntirho wa xiringanyeto wa tlilasi na ku hlela. Kutani, hi sungule hi dyondza vuswikoti bya C26 Gas1-GFP ku hlengeletana hi swirhabyani eka GhLag1 hi temperature-sensitive mutant allele ya sec31-1 hi ku tirhisa microscopy ya ntolovelo ya fluorescence, laha ku nga na chain yo leha (C18-C16) ntsena eka ER membrane Ceramide (Fig. 3). Hi xiye leswaku eka sec31-1, vunyingi bya Gas1-GFP byi hlanganisiwile hi swirhabyani, kasi Gas1-GFP eka sec31-1 GhLag1 leyi nga na long (C18-C16) long ceramide ER membrane ngopfu-ngopfu a yi nga hlengeletiwanga no hangalasiwa eka Eka ER membrane hinkwayo. Ku vula ntiyiso, hikuva C26 ceramide-based clustering yi fambelana swinene na ERES yo karhi (Xifaniso 1), hi landzerile hi lavisisile loko endlelo leri ri nga ha tlhela ri katsa ntirho wa ER export protein mechanism. GPI-AP yi tirhisa sisiteme yo hlawuleka ya COPII ya ER export, leyi lawuriwaka hi xihatla hi Ted1′s structural remodeling ya xiphemu xa glycan xa GPI anchor ( 30 , 31 ). GPI-glycan leyi hlanganisiweke nakambe yi tlhela yi tiviwa hi transmembrane cargo receptor p24 complex, leyi hi ku famba ka nkarhi yi hlawulaka ku thola Lst1, leyi nga isoform yo karhi ya subunit leyikulu yo boha ndzhwalo wa COPII Sec24, ku vumba COPII leyi fuweke hi GPI-AP Vesicles ya laveka (31-33). Hikwalaho, hi ake mutant yimbirhi leyi hlanganiseke ku susiwa ka tiphrotheyini leti tin’we (xiphemu xa p24 complex Emp24, GPI-glycan remodeling enzyme Ted1 na COPII subunit yo karhi Lst1) na muxaka wa sec31-1 mutant, naswona hi swi dyondze Xana swa koteka ku vumba Gas1-cluster GFP (Xifaniso 3). Hi xiye leswaku eka sec31-1emp24Δ na sec31-1ted1Δ, Gas1-GFP ngopfu-ngopfu a yi hlengeletiwanga naswona yi hangalasiwa eka ER membrane hinkwayo, tanihilaha swi voniweke hakona khale eka sec31-1 GhLag1, kasi eka sec31-1lst1Δ, Gas1-GFP Ku fana na sec31-1. Mimbuyelo leyi yi kombisa leswaku ku engetela eka vukona bya C26 ceramide eka ER membrane, ku hlengeletiwa ka Gas1-GFP na kona ku lava ku boha eka p24 complex, naswona a swi lavi ku thoriwa ko karhi ka Lst1. Kutani, hi lavisisile ku koteka ka leswaku ku leha ka ketane ya ceramide eka ER membrane ku nga lawula ku bohiwa ka Gas1-GFP eka p24. Hambiswiritano, hi kumile leswaku vukona bya C18-C16 ceramide eka membrane a byi khumbi ti GPI-glycans leti pfuxetiweke hi p24 complex (Swifaniso S3 na S4, A na B) kumbe ku boha eka GPI-AP na ku rhumela ehandle GPI-AP. vuswikoti. Ku thola muxaka wa le hansi wa COPII Lst1 (Xifaniso xa S4C). Hikokwalaho, ku hlengeletiwa loku titshegeke hi C26 ceramide a ku lavi ku tirhisana ka tiphrotheyini na tindlela to hambana ta tiphrotheyini to rhumela ehandle ka ER, kambe ku seketela endlelo rin’wana ro hlawula leri fambiwaka hi ku leha ka lipid. Kutani, hi kambisisile loko ku leha ka ceramide acyl chain eka ER membrane ku ri ka nkoka eka ku hlawuriwa loku humelelaka ka Gas1-GFP tanihi ERES yo hlawula. Tanihi leswi Gas1 eka muxaka wa GhLag1 lowu nga na ceramide ya ketane yo koma yi huma eka ER yi nghena eka membrane ya plasma (Xifaniso S5), hi tshemba leswaku loko ku hlawula ku fambisiwa hi ku leha ka ketane ya ceramide acyl, Gas1 eka muxaka wa GhLag1 yi nga kongomisiwa nakambe no tsemakanya. Nhundzu ya ERES leyi nga na membrane yin’we.
(A) Xirhendzevutani xa tisele ta GhLag1 ngopfu-ngopfu xi na ti-ceramide to koma ta C18-C16, kasi anchor ya GPI ya Gas1-GFP ya ha ri na C26 IPC leyi fanaka na tisele ta muxaka wa nhova. Laha henhla: nxopaxopo wa ku leha ka acyl chain ya ceramide eka cell membrane ya tinxaka ta muxaka wa nhova (Wt) na GhLag1p hi mass spectrometry (MS). Data yi yimela tiphesente ta ceramide hinkwayo. Xiringaniso xa swikambelo swinharhu leswi tiyimeleke. Bar ya swihoxo = SD. Xikambelo xa t lexi nga hlanganisiwangiki xa misisi yimbirhi. **** P <0.0001. Phaneli yale hansi: Nxopaxopo wa MS wa ku leha ka acyl chain ya IPC leyi nga kona eka Gas1-GFP (GPI-IPC) GPI anchor leyi kombisiweke eka tinxaka ta muxaka wa nhova na GhLag1p. Data yi yimela tiphesente ta xigingi xa IPC hinkwaxo. Avhareji ya swikambelo swa ntlhanu leswi tiyimeleke. Bar ya swihoxo = SD. Xikambelo xa t lexi nga hlanganisiwangiki xa misisi yimbirhi. ns, a hi swa nkoka. P = 0.9134. (B) Ti micrographs ta fluorescence ta sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ na sec31-1lst1Δ ti cells leti kombisaka galactose-induced Gas1-GFP ti pfaleriwile eka 37°C kuringana 30 wa timinete naswona ti hundziseriwe ehansi kuya eka Perform routine fluorescence microscopy endzhaku 24°C. Museve wo basa: Xitluletsongo xa ER Gas1-GFP. Museve lowu pfulekeke: Unclustered Gas1-GFP yi hangalasiwa eka ER membrane hinkwayo, leswi kombisaka ER characteristic nuclear ring staining. Bar ya xikalo, 5μm. (C) Ku pima nhlayo ya photomicrograph leyi hlamuseriweke eka (B). Tiphesente ta xikarhi ta tisele leti nga na xivumbeko xa punctate Gas1-GFP. Eka swikambelo swinharhu leswi tiyimeleke, n≥300 wa tisele. Bar ya swihoxo = SD. Xikambelo xa t lexi nga hlanganisiwangiki xa misisi yimbirhi. **** P <0.0001.
Ku tlhantlha xiphiqo lexi hi ku kongoma, hi endle SCLIM visualization ya Gas1-GFP na Mid2-iRFP eka GhLag1 na sec31-1 temperature-sensitive mutant allele (Xifaniso 4 na Movie S4). Endzhaku kaloko ER yi hlayisiwile eka 37°C naswona endzhaku yi humesiwile eka 24°C, vunyingi bya Gas1-GFP leyintshwa leyi endliweke a yi hlengeletiwanga no hangalasiwa eka membrane hinkwayo ya ER, tani hileswi swi voniweke hi ti microscopes ta ntolovelo (Xifaniso 4, A na B ). Ku engetela kwalaho, phesente leyikulu ya ERES (67%) yi katsa tinxaka timbirhi ta ndzhwalo lowu kumekaka swin’we eka yona (Xifaniso xa 4D). Tiphaneli ta 1 na 2 ta Xifaniso xa 4C ti kombisa swikombiso swimbirhi leswi tolovelekeke swa ERES leswi nga na Gas1-GFP na Mid2-GFP leswi hlanganisaka. Ku engetela kwalaho, nhundzu leyimbirhi yi thoriwile eka ERES yin’we (Xifaniso 4E, phanele 3 na filimi ya S4). Hikokwalaho, mbuyelo wa hina wu kombisa leswaku ku leha ka ceramide acyl chain eka ER membrane i xihlawulekisi xa nkoka xa nhlengeleto wa ti protein ta ER na ku hlawuriwa.
Ti cells ta Sec31-1 GhLag1 leti kombisaka swihumesi leswi hlohleteriwaka hi galactose, Gas1-GFP (GPI-AP, rihlaza) na Mid2-iRFP (TMP, blue) na constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Ti pfaleriwa eka 37°C. Yana emahlweni kuringana 30 wa timinete, ehansi kuya eka 24°C kuva u humesa secretions, kutani u teka xifaniso hi SCLIM endzhaku ka 20 wa timinete. (A ku ya eka C) Swifaniso leswi yimelaka 2D projection (A; scale bar, 1μm) kumbe swifaniso swa 3D cell hemisphere (B na C; scale unit, 0.45μm) swa 10 wa swiyenge swa z leswi funghiweke hi cargo na ERES. Phanele ya le hansi eka (B) na phanele eka (C) yi kombisa swifaniso leswi phurosesiweke ku kombisa ntsena nhundzu leyi nga kona eka ERES (magenta) [Gas1-GFP (gray) na Mid2-iRFP (light blue)]. (C) Museve lowu taleke wo basa: ERES, nhundzu yi hlangana. Museve lowu pfulekeke: ERES yi na nchumu wun’we ntsena. Phaneli ya le hansi: ERES leyi hlawuriweke yi na nhundzu leyi hlanganisaka (1 na 2) leyi funghiweke eka (C). Bar ya xikalo, 100 nm. (D) Ku pima nhlayo ya photomicrograph leyi hlamuseriweke eka (C). Eka tiyuniti ta sec31-1 na sec31-1 GhLag1, ku katsiwile ntsena ndzhwalo wun’we (Gas1-GFP kumbe Mid2-iRFP), na phesente ya xikarhi ya ERES ya ndzhwalo lowu nga yoxe na ndzhwalo lowu hlanganisaka. Eka swikambelo swinharhu leswi tiyimeleke, n = 432 eka 54 wa tisele (sec31-1) na n = 430 eka 47 wa tisele (sec31-1 GhLag1). Bar ya swihoxo = SD. Xikambelo xa t lexi nga hlanganisiwangiki xa misisi yimbirhi. *** P = 0.0002 (xiyenge xa ​​31-1) na ** P = 0.0031 (xiyenge xa ​​31-1 GhLag1). (E) Xifaniso xa 3D xa ERES leyi hlawuriweke leyi nga na ndzhwalo lowu hlanganisaka (3) lowu funghiweke eka (C). Gas1-GFP (rihlaza) na Mid2-iRFP (ya wasi) swi tshinelela ERES (magenta) kusuka eka tlhelo rin’we naswona switshama eka ndzhawu yin’we leyi siveleriweke hi ERES. Bar ya xikalo, 100 nm.
Dyondzo leyi yinyika vumbhoni byo kongoma bya in vivo leswaku ti lipid-based protein cargoes ti hlawuriwa eka tindzhawu leti hlawulaka to rhumela ematikweni mambe eka ndlela ya secretory, naswona yi paluxa nkoka wa ku leha ka acyl chain eka ku hlawula ka ku hlawuriwa. Hi ku tirhisa endlelo ra matimba na ra xiyimo xa le henhla ra microscopy leri vuriwaka SCLIM, hi kombisile Gas1-GFP leyintshwa leyi endliweke (a major plasma membrane GPI-AP with a very long acyl chain (C26) ceramide lipid portion) in yeast ) Swifundzha leswi hlengeletiweke eka ti discrete ERs swi fambelanisiwa na ERES yo karhi, kasi tiphrotheyini leti humesiweke hi transmembrane ti hangalasiwa eka membrane hinkwayo ya ER (Xifaniso xa 1). Ku engetela kwalaho, tinxaka leti timbirhi ta nhundzu ti nghena eka ERES yo hambana hi ku hlawula (Xifaniso 2). Ku leha ka acyl chain ya cellular ceramide eka membrane swihungutiwile kusuka eka C26 kuya eka C18-C16, Gas1-GFP cluster yi kavanyetiwile kuya eka discrete ER region, naswona Gas1-GFP yi tlheriseriwa ndlela ku suka eka ER na transmembrane protein hiku tirhisa ERES leyi fanaka (Figure 3 na Figure 3). 4).
Hambi leswi GPI-AP yi tirhisaka endlelo ro hlawuleka ra tiphrotheyini ku huma eka ER, hi kumile leswaku ku hambanisiwa loku titshegeke hi C26 ceramide a ku titsheganga hi ku tirhisana ka tiphrotheyini to hambana loku nga yisaka eka ku hlawuleka ka ERES (Swifaniso S4 na S5). Ematshan’wini ya sweswo, swikumiwa swa hina swi seketela endlelo rin’wana ro hlawula leri fambiwaka hi ku hlengeletiwa ka tiphrotheyini leti simekiweke eka lipid na ku hlongoriwa loku landzelaka ka ndzhwalo wun’wana. Swibumabumelo swa hina swi kombisa leswaku xifundzha xa Gas1-GFP kumbe xitluletsongo lexi fambelanaka na ERES yo karhi xi pfumala transmembrane secreted protein Mid2-iRFP, leswi kombisaka leswaku C26 ceramide-dependent GPI-AP cluster yi ta olovisa ku nghena ka yona eka ERES leyi faneleke, naswona hi nkarhi lowu fanaka, yi susa transmembrane Swihumesi swi nghena eka ERES leyi yo karhi (Swifaniso 1 na 2). Kuhambana na sweswo, vukona bya C18-C16 ceramides eka ER membrane aswi endli leswaku GPI-AP yi endla swifundzha kumbe swirhabyani, hikokwalaho aswi hlongoli kumbe ku siva ti protein leti humesiwaka hi transmembrane eka ERES leyi fanaka (Swifaniso 3 na 4). . Hikwalaho, hi ringanyeta leswaku C26 ceramide yi fambisa ku hambanisiwa na ku hlawuriwa hi ku olovisa ku hlengeletiwa ka tiphrotheyini leti hlanganisiweke na ERES yo karhi.
Xana ku fikeleriwa njhani ku hlengeletiwa loku ku titshegeke hi C26 ceramide eka ndhawu yo karhi ya ER? Ku tala ka membrane ceramide ku hambanisiwa hi tlhelo swi nga endla leswaku GPI-AP na C26 ceramide swi vumba ti lipids letintsongo na leti odaweke hi xihatla eka ndhawu ya lipid leyi nga tshamisekangiki ngopfu ya ER membrane leyi nga na ti glycerolipids to koma na leti nga tatiwangiki. Swiyenge swa khwalithi (17, 18). Swirhabyani leswi leswintsongo swa nkarhinyana swi nga ha tlhela swi hlanganisiwa swi va swirhabyani leswikulu, leswi tiyeke endzhaku ko boha eka p24 complex ( 34 ). Hi ku fambisana na leswi, hi kombisile leswaku C26 Gas1-GFP yi lava ku tirhisana na p24 complex ku vumba swirhabyani leswikulu leswi vonakaka (Xifaniso 3). P24 complex i heterozygous oligomer leyi vumbiweke hi mune wa tiphrotheyini to hambana ta p24 transmembrane eka comela ( 35 ), leswi nyikaka ku boha ka multivalent, leswi nga endlaka leswaku ku va na cross-linking ya swirhabyani leswintsongo swa GPI-AP, hi ndlela yoleyo ku humesa xitluletsongo xa Stable ( 34 ). Ku tirhisana exikarhi ka ti protein ectodomains ta GPI-APs ku nga ha tlhela ku hoxa xandla eka ku hlengeletiwa ka tona, tanihilaha swi kombisiweke hakona hi nkarhi wa ku fambisiwa ka tona ka Golgi eka tisele ta epithelial ta mammalian polarized ( 36 ). Hambiswiritano, loko C18-C16 ceramide yi ri kona eka ER membrane, loko p24 complex yi boha eka Gas1-GFP, swirhabyani leswikulu leswi hambaneke a swi nge vumbiwi. Endlelo ra le hansi ri nga ha titshega hi swivumbeko swo karhi swa nyama na tikhemikhali ta long acyl chain ceramide. Tidyondzo ta biophysical ta ti artificial membranes ti kombisa leswaku hambi leswi ti ceramides to leha (C24) na to koma (C18-C16) ta acyl chain tinga vangaka ku hambanisiwa ka phase, i ti long acyl chain ceramides (C24) ntsena letinga hlohletelaka Curvature yale henhla xikan’we naku gombonyoka ka filimi kuva yi vumba filimi hi vuntshwa. Hi ku tirhisa ku kongomisana (17, 37, 38). Ku kombisiwile leswaku transmembrane helix ya TMED2, homologue ya munhu ya Emp24, hi ku hlawula yi tirhisana na C18 ceramide-based sphingomyelin eka ti cytoplasmic lobules ( 39 ). Hi ku tirhisa ti molecular dynamics (MD) simulations, hi kumile leswaku havumbirhi bya C18 na C26 ceramides ti hlengeletana ku rhendzela ti cytoplasmic lobules ta Emp24 transmembrane helix, naswona ti na ku tsakela loku fanaka (Xifaniso S6). Swifanele ku xiya leswaku leswi swikomba leswaku transmembrane helix ya Emp24 yinga endla leswaku kuva na asymmetric distribution ya lipids eka membrane. Lebyi i vuyelo bya sweswinyana lebyi sekeriweke eka tisele ta swihadyana leswi mamisaka. Swifaniso leswi fanaka swa MD swi tlhela swi kombisa vukona bya ti- ether lipids (40) . Hikokwalaho, hi ringanyeta leswaku C26 ceramide eka ti lobules timbirhi ta ER26 yi fuwile laha kaya. Loko GPI-AP eka ti luminal lobules yi boha hi ku kongoma eka multivalent p24 na ku hlengeletiwa ka C26 ceramide ku rhendzela p24 eka ti cytoplasmic lobules, yi nga tlakusa ku hlengeletiwa ka Protein loku fambisanaka na ku gombonyoka ka membrane swi endliwa hi tintiho ( 41 ), leswi endlaka leswaku GPI-AP yi hambanisiwa yi va tindhawu leti hambaneke leti nga ekusuhi na ERES, leswi tlhelaka swi tsakela leswi govekeke swinene swifundzha swa ER membrane (42). Swiviko swa khale swi seketele endlelo leri ringanyetiweke (43, 44). Ku bohiwa ka multivalent ka oligolectins, ti pathogens kumbe ti antibodies eka ceramide-based glycosphingolipids (GSL) eka plasma membrane swi hlohlotela nhlengeleto lowukulu wa GSL, swi ndlandlamuxa ku hambanisiwa ka swiphemu naswona swi vanga ku onhaka ka membrane na ku nghenisiwa endzeni ( 44 ). Iwabuchi etc. (43) Ku kumiwile leswaku eka vukona bya ti acyl chains to leha (C24) kambe kungari to koma (C16), ligand ya multivalent leyi bohiweke eka GSL lactosylceramide yi hlohlotele ku vumbiwa ka swirhabyani leswikulu na membrane invagination, naswona cytoplasm Lyn-mediated signal transduction eka swiphepherhele yi hlanganisiwa hi ti acyl chains leti hlanganisiweke ti- neutrophil.
Eka tisele ta epithelial leti mammalian polarized, ku hlangana ka anti-Golgi network (TGN) ku ya eka mpimo wa apical plasma membrane swi lawula ku hambanisiwa na ku hlela ka GPI-AP ( 10 , 45 ). Ku hlengeletiwa loku ku fambisiwa hi GPI-AP oligomerization ( 36 ), kambe swi nga ha tlhela swi titshega hi ku leha ka ceramide chain leyi hi yi kumaka eka comela. Hambi leswi mammalian GPI-AP yi nga na ether lipid-based anchor, naswona xivumbeko xa yona xa tikhemikhali xi hambanile swinene na acyl chain ceramide yo leha swinene, nkambisiso wa sweswinyana wu kumile leswaku ti lipids leti hatimbirhi ti na evolutionarily similar physical and chemical properties And function ( 40 ). Hikokwalaho, xiphemu xa ether lipid eka ti cells ta swihadyani leswi mamisaka xinga fana na C26 ceramide eka comela, naswona ntirho wa yona iku tihlanganisa na long-chain ceramide eka membrane ku hlohletela nhlengeleto wa GPI-AP xikan’we naku hlawuriwa. Hambi leswi ku koteka loku ka ha lavaka ku kamberiwa hi ku kongoma, swikumiwa swa khale swi seketela leswaku ku fambisiwa ka long acyl chain ceramide kuya eka miri wa Golgi aswi endliwi hi ti cytoplasmic transfer proteins, kambe swiya hiku endliwa ka ti anchors ta GPI tofana na yeast. Hikwalaho, endlelo ra evolutionary conservative ri vonaka ri kota ku hlawula ku fambisa swin’we ceramide yo leha swinene ya acyl chain na GPI-AP (13, 16, 20, 46, 47) eka vesicle yin’we yo fambisa.
Eka yeast na mammalian polarized epithelial cell systems, ku hlengeletiwa ka GPI-AP na ku hambanisiwa na ti protein tin’wana ta plasma membrane hinkwaswo swihumelela loko swinga se fika ehenhla ka cell. Paladino na van’wana. (48) va kumile leswaku eka TGN ya tisele ta epithelial leti mammalian polarized, GPI-AP clustering a yi laveki ntsena eka ku hlawuriwa loku hlawulaka ka GPI-APs eka apical plasma membrane, kambe yi tlhela yi lawula nhlengeletano ya clustering ya GPI-APs na ntirho wa yona wa biological. Vuandlalo bya sele. Eka comela, dyondzo leyi yi kombisile leswaku C26 ceramide-dependent GPI-AP cluster eka ER yi nga lawula nhlengeletano ya cluster na ntirho wa ntirho wa GPI-AP eka plasma membrane ( 24 , 49 ). Hi ku fambisana na modele lowu, tisele ta GhLag1 ti na allergy eka ti inhibitors ta GPI kumbe mirhi leyi khumbaka vutshembeki bya rirhangu ra tisele ( 28 ), naswona xilaveko xa swirhabyani swa Gas1-GFP leswi tirhaka ( 49 ) swa tip ceramide leyi languteriweke eka ku hlangana ka tisele ta comela swi kombisa G Vuyelo bya physiological lebyi nga kotekaka bya tisele ta hLag1. Xihoxo xa GPI-AP. Hambiswiritano, ku ya emahlweni ku kambela loko nhlengeletano ya ntirho ya vuandlalo bya sele yi phurogiremiwile ku suka eka ER hi ndlela yo hlawula leyi sekeriweke eka ku leha ka lipid ku ta va mhaka ya ndzavisiso wa hina wa nkarhi lowu taka.
Tinxaka ta Saccharomyces cerevisiae leti tirhisiweke eka ntirho lowu ti xaxametiwile eka Tafula ra S1. Tinxaka ta MMY1583 na MMY1635 ta SCLIM eka swifaniso swa tisele leti hanyaka ti akiwile endzhaku ka W303. Tinxaka leti ti kombisaka Sec13-mCherry na fluorescent protein tag ti akiwile hiku tirhisa ndlela leyi simekiweke eka polymerase chain reaction (PCR) na pFA6a plasmid tani hi template ( 23 ). Muxaka lowu kombisaka Mid2-iRFP lowu lebuliweke hi fluorescent protein ehansi ka vulawuri bya GAL1 promoter wu akiwile hi ndlela leyi landzelaka. Ku kurisiwa ka PCR ka ntlhandlamano wa iRFP-KanMx ku suka eka vector ya pKTiRFP-KAN (nyiko ya E. O’Shea, nomboro ya plasmid ya Addgene 64687; http://n2t.net/addgene: 64687; xihlawulekisi xa switirhisiwa swa ndzavisiso (RRID): Addgene_64687) Naswona yi nghenisiwile eka C-terminus ya endogenous Mid2. Endzhaku ka loko ntlhandlamano wa genome wa Mid2-iRFP wu kurisiwile no endliwa cloned eka GAL1 promoter, wu hlanganisiwile eka ndzhawu ya Not I-Sac I ya integration plasmid pRS306. Plasmid leyi humeleleke pRGS7 yi linearized na Pst I ku hlanganisiwa eka URA3 locus.
Jini ya Gas1-GFP fusion yi kombisiwa ehansi ka vulawuri bya GAL1 promoter eka centromere (CEN) plasmid, leyi akiweke hi ndlela leyi landzelaka. Ntlhandlamano wa Gas1-GFP wu antswisiwile hi PCR ku suka eka plasmid ya pRS416-GAS1-GFP (24) (nyiko ya L. Popolo) naswona yi cloned eka ndhawu ya Xma I–Xho I ya plasmid ya CEN pBEVY-GL LEU2 (nyiko ya C) . Miller; Addgene nomboro ya plasmid ya 51225; Nkanelo wa ririmi ra Xitsonga: 51225; RRID: Xihlamusela-marito_51225). Plasmid leyi humeleleke yi thyiwile vito ra pRGS6. Jini ya Axl2-GFP fusion yi tlhela yi kombisiwa ehansi ka vulawuri bya GAL1 promoter ya pBEVY-GL LEU2 vector, naswona ku akiwa ka yona hi ndlela leyi landzelaka. Ntlhandlamano wa Axl2-GFP wu antswisiwile kusuka eka pRS304-p2HSE-Axl2-GFP plasmid (23) hi PCR, naswona wu cloned eka ndzhawu ya Bam HI-Pst I ya pBEVY-GL LEU2 vector. Plasmid leyi humeleleke yi thyiwile vito ra pRGS12. Ntlhandlamano wa ti oligonucleotides leti tirhisiweke eka ndzavisiso lowu wu xaxametiwile eka Tafula ra S2.
Muxaka lowu wu engeteriwile hi 0.2% wa adenine na 2% wa glucose [YP-dextrose (YPD)], 2% wa raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract na 2% protein ept). (YPR)] kumbe 2% galactose [YP-galactose (YPG)] tani hi xihlovo xa khaboni, kumbe eka xitirhisiwa xa le hansi xa synthetic (0.15% yeast nitrogen base na 0.5% ammonium sulfate) ku engetela ti amino acids leti faneleke na ti bases leti lavekaka eka swakudya , Naswona leyingana 2% glucose (synthetic glucose minimal medium) kumbe 2% galactose (synthetic galactose minimal medium) tani hi xihlovo xa khaboni.
Eka swifaniso swa nkarhi wa xiviri, ti cells ta sec31-1 mutant leti twisisaka mahiselo leti kombisaka construct ehansi ka GAL1 promoter ti byariwile eka YPR medium eka 24°C vusiku hinkwabyo kuya eka mid-log phase. Endzhaku ka induction eka YPG eka 24°C kuringana 1 wa awara, ti cells ti pfaleriwile eka SG eka 37°C kuringana 30 wa timinete, kutani ti hundziseriwa eka 24°C kuva ti humesiwa kusuka eka secretion block. Concanavalin A yitirhisiwile ku lulamisa ti cells eka glass slide naswona yi tekiwile swifaniso hi SCLIM. SCLIM i nkatsakanyo wa Olympus IX-71 inverted fluorescence microscope na UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), xikeni xa rivilo lerikulu na xa xiyimo xa le henhla xa xikombiso na mpfumawulo lexi rhendzelekaka disc confocal scanner (Yokogawa Electric), spectrometer ya ntolovelo, na ku kufumeta hi ku landza mukhuva Xitiyisisi xa swifaniso xa sisiteme (Hamamatsu Photonics) yi nga nyika sisiteme ya lens leyi kurisaka leyi nga na ku kurisiwa ko hetelela ka ×266.7 na khamera ya xitirhisiwa lexi hlanganisiweke na charge leyi andzisaka tielektroni (Hamamatsu Photonics) (21). Ku kumiwa ka swifaniso ku endliwa hi software ya ntolovelo (Yokogawa Electric). Eka swifaniso swa 3D, hi tirhise xitirhisiwa xa piezoelectric lexi endliweke hi ku landza mukhuva ku rhurhumela lens ya xikongomelo hi ku kongoma, naswona hi hlengeletile swiphemu swa mahlo hi 100 nm hi ku hambana eka xirhendzevutani. Xifaniso xa Z-stack xi hundzuriwa eka 3D voxel data, naswona ntirho wa theoretical point spread lowu tirhisiwaka eka rotating disc confocal microscope wu tirhisiwa eka deconvolution processing hi Volocity software (PerkinElmer). Hi ku tirhisa sofwere ya Volocity ku tisungulela threshold ya nxopaxopo wa ndhawu yin’we, ERES ku katsa na ndzhwalo yi pimiwe. Nxopaxopo wa xikeni xa layini wu endliwile hi ku tirhisa software ya MetaMorph (Molecular Devices).
Tirhisa software ya GraphPad Prism ku kumisisa nkoka wa tinhlayo. Eka xikambelo xa Student xa t-test xa misisi yimbirhi na xikambelo xa ntolovelo xa ndlela yin’we ya ku hambana (ANOVA), ku hambana exikarhi ka mintlawa ku tekiwa ku ri na nkucetelo lowukulu eka P <0.05 (*).
Eka microscopy ya fluorescence ya Gas1-GFP, ti cells ta log phase ti kurisiwile vusiku hinkwabyo eka YPD naswona ti hlengeletiwile hi centrifugation, ti hlantswiwa kambirhi hi phosphate buffered saline, naswona ti pfaleriwa eka ayisi kuringana kwalomu ka 15 wa timinete, kutani ti ya emahlweni ehansi ka microscope tani hileswi swinga hlamuseriwa khale Check (24). Microscope ya Leica DMi8 (HCX PL APO 1003/1.40 oil PH3 CS) leyi hlomisiweke hi lens ya xikongomelo, sefo ya L5 (GFP), khamera ya Hamamatsu na software ya Application Suite X (LAS X) yi tirhisiwile ku kuma. .
Tisampulu ti denatured hi SDS sample buffer eka 65°C kuringana 10 wa timinete, kutani ti hambanisiwa hi SDS-polyacrylamide gel electrophoresis (PAGE). Eka nxopaxopo wa immunoblotting, 10 μl ya xikombiso yi layichiwile hi lane. Antibody yosungula: Tirhisa rabbit polyclonal anti-Gas1 eka dilution ya 1:3000, rabbit polyclonal anti-Emp24 eka dilution ya 1:500, na rabbit polyclonal anti-GFP (nyiko kusuka eka H. Riezman) eka dilution ya 1:3000. Mouse monoclonal anti-Pgk1 antibody yitirhisiwile eka dilution ya 1:5000 (nyiko kusuka eka J. de la Cruz). Antibody ya vumbirhi: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) leyi tirhisiwaka eka dilution ya 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG yitirhisiwile eka dilution ya 1:3000 (Pierce). Ndzhawu ya nhlamulo ya masocha ya miri yi voniwe hi ndlela ya chemiluminescence ya SuperSignal West Pico reagent (Thermo Fisher Scientific).
Hilaha swi hlamuseriweke hakona eka (31), ku endliwile xikambelo xa ntumbuluko xa immunoprecipitation eka xiphemu xa ER lexi fuweke. Hiku komisa, hlamba ti cells ta comela hi TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride na nkatsakanyo wa protease inhibitor) eka 600 nm ( OD600) eka 100 optical density kambirhi. Yi tshoviwile hi vuhlalu bya nghilazi, ivi endzhaku ka sweswo thyaka ra tisele ni vuhlalu bya nghilazi swi susiwile hi ku tirhisa centrifugation. Kutani supernatant yi centrifuged eka 17,000 g kuringana 15 wa timinete eka 4°C. Pellet yitlhele yi suspended eka TNE naswona digitalis saponin yi cheriwile kuya eka nhlayo yohetelela ya 1%. Suspension yi pfaleriwile kuringana 1 wa awara hiku rhendzeleka eka 4°C, kutani swiphemu leswinga khulukiki swi susiwile hiku centrifugation eka 13,000 g eka 4°C kuringana 60 wa timinete. Eka Gas1-GFP immunoprecipitation, rhanga hi pre-incubate xikombiso na empty agarose beads (ChromoTek) eka 4°C kuringana 1 wa awara, kutani u pfaleriwa na GFP-Trap_A (ChromoTek) eka 4°C kuringana 3 wa tiawara. Ti beads letinga immunoprecipitated ti hlantswiwile ka ntlhanu hi TNE leyinga na 0.2% digoxigenin, ti hlantswiwa hi SDS sample buffer, ti hambanisiwile eka SDS-PAGE, naswona ti kamberiwile hi immunoblotting.
Hilaha swi hlamuseriweke hakona eka (31), ku tiyimisela ka cross-linking ku endliwile eka xiphemu xa ER lexi fuweke. Hiku komisa, xiphemu xa ER lexi fuweke xi pfaleriwile na 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Crosslinking reaction yi timiwile hiku cheriwa glycine (50 mM final concentration, 5 wa timinete, 20°C).
Tani hilaha swi hlamuseriweke hakona khale (50), nxopaxopo wa MS wa ceramide eka tinxaka ta muxaka wa nhova na GhLag1 wu endliwile. Hiku komisa, ti cells ti kurisiwile kuya eka exponential phase (3 kuya eka 4 OD600 units/ml) eka YPD eka 30°C, naswona 25×107 wa ti cells ti tshoveriwile. Metabolism ya tona yi timiwa hi trichloroacetic acid. Tirhisa xitofu xo humesa [ethanol, mati, ether, pyridine na 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] na 1.2 nmol ya khwalithi ya le ndzeni ya standard C17 ceramide (860517, Avanti polar lipid)). Tirhisa monomethylamine reagent [methanol, mati, n-butanol na methylamine solution (4:3:1:5 v/v)] ku endla mild alkaline hydrolysis ya extract, kutani u tirhisa n-butanol leyi taleke hi mati ku susa munyu. Eku heteleleni, extract yitlhele yi suspended eka positive mode solvent [chloroform/methanol/mati (2:7:1) + 5 mM ammonium acetate] kutani yi cheriwa eka mass spectrometer. Ku langutisisa ka multi-reaction (MRM) ku endliwile ku kuma na ku pima nhlayo ya ti molecules ta sphingolipid. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) yi hlomisiwile hi xihlovo xa robotic nanoflow ion Nanomate HD (Advion Biosciences, Ithaca, NY) ku endlela nxopaxopo wa lipid. Enerji ya ku tlumbana yi antswisiwile eka xiyenge xin’wana na xin’wana xa ceramide. Data ya MS yi kumiwile hi ndlela ya kahle. Eka ku tlheriseriwa kun’wana na kun’wana ka biological, xikombiso xa lipid i xikarhi xa mimpimo yinharhu leyi tiyimeleke.
Hilaha swi hlamuseriweke hakona eka (31), tisele (800×107) leti kombisaka Gas1-GFP ti endliwile immunoprecipitation ya ntumbuluko. Gas1-GFP leyi basisiweke yi hambanisiwile hi SDS-PAGE naswona yi hundziseriwe eka polyvinylidene fluoride (PVDF) membrane. Protein leyi yi voniwe hi mahlo hi ku penda PVDF hi amide black. Band ya Gas1-GFP yi tsemiwile kusuka eka PVDF kutani yi hlantswiwa ka 5 hi methanol na kan’we hi mati ya liquid chromatography-MS (LC-MS) grade. Hiku pfalela membrane strip na 500μl 0.3 M NaOAc (pH 4.0), buffer na 500μl leyi ha ku khulukaka 1 M sodium nitrite nkatsakanyo eka 37°C kuringana 3 wa tiawara, xiphemu xa lipid xi humesiwa kusuka eka Gas1-GFP na lysed Ku humesiwa ka inosine phosphate ceramide exikarhi ka glucosamine na inositol (51). Endzhaku ka sweswo, membrane strip yi hlantswiwile ka mune hi mati ya LC-MS grade, yi omisiwa eka mahiselo ya kamara, naswona yi hlayisiwile eka xibakabaka xa nitrogen eka -80°C kufikela ku kamberiwa. Tanihi vulawuri, xikombiso lexi nga riki na nchumu xa PVDF membrane xi tirhisiwile eka xikambelo xin’wana na xin’wana. Lipid leyi humesiweke eka Gas1-GFP yi tlhele yi kamberiwa hi MS tanihilaha swi hlamuseriweke hakona ( 50 ). Hiku komisa, swiphemu swa PVDF leswingana GPI-lipid switlhele swi suspended eka 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] naswona swi hundze electrospray ionization (ESI)-MRM/MS Analysis ya tinxaka ta sphingolipid (TSQ Vantage). Eka xiyimo lexi, datha ya MS yi kumiwile eka movha wa ion yo biha.
Hilaha swi boxiweke hakona eku sunguleni, xiphemu xa lipid xa GPI anchor xi hambanisiwile na [3H]-inositol-labeled GPI-AP ( 16 ). Ti lipids ti hambanisiwile hi thin-layer chromatography hiku tirhisa solvent system (55:45:10 chloroform-methanol-0.25% KCl) naswona ti voniwe hi mahlo hiku tirhisa FLA-7000 (Fujifilm).
Ti cells leti kombisaka Gas1-GFP (600×107) ti hlantswiwile kambirhi hi TNE buffer hi TNE buffer, naswona ti tshovekile hi vuhlalu bya nghilazi, kutani ti centrifuged ku susa swilo leswinga sala swa ti cell na vuhlalu bya nghilazi. Kutani supernatant yi centrifuged eka 17,000 g kuringana 1 wa awara eka 4°C. Pellet yi hlantswiwile eka TNE kutani yi pfaleriwa na 1 U PI-PLC (Invitrogen) eka TNE leyingana 0.2% digitalis saponin kuringana 1 wa awara eka 37°C. Endzhaku ka vutshunguri bya enzyme, membrane yi susiwile hi centrifugation eka 17,000 g eka 4°C kuringana 1 wa awara. Ku immunoprecipitate Gas1-GFP, supernatant yi pfaleriwile na GFP-Trap_A (ChromoTek) eka 4°C vusiku hinkwabyo. Gas1-GFP leyi basisiweke leyi hambanisiweke hi SDS-PAGE yi pfanganisiwe hi Coomassie brilliant blue. Band ya Gas1-GFP staining yi tsemiwile kusuka eka grey leyi rhendzeleke aqueduct, kutani endzhaku ka alkylation hi iodoacetamide naku hungutiwa hi dithiothreitol, in-gel digestion hi trypsin yi endliwile. Humesa naku omisa ti tryptic peptides na ti peptides letingana GPI-glycans. Peptide leyi omisiweke yi pfanganisiwile na 20 μl ya mati. Chela xiphemu (8μl) eka LC. Kholomo ya octadecylsilane (ODS) (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) yitirhisiwile ku hambanyisa ti peptides ehansi ka swiyimo swokarhi swa gradient. Xiphemu lexi fambafambaka i solvent A (0.08% formic acid) na solvent B (0.15% formic acid eka 80% acetonitrile). Sisiteme ya Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) yitirhisiwile ku hlantswa kholomo hi solvent A endzhaku ka 55 wa timinete eka flow rate ya 50 μl min-1 kuringana 5 wa timinete, kutani nhlayo ya solvent B yi engeteriwile kuya eka 40%. , eUnited States). Eluate yi tshama yi ri karhi yi nghenisiwa eka xihlovo xa ion xa ESI, naswona ti tryptic peptides na ti peptides leti nga na GPI-glycans ti kamberiwile hi LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Eka setup ya MS, voltage ya xihlovo xa capillary yi vekiwile eka 4.5 kV, naswona mahiselo ya transfer capillary ya hlayisiwile eka 300°C. Voltage ya capillary na voltage ya lens ya tube swi vekiwile eka 15 V na 50 V, hiku landzelelana. Data ya MS yi kumiwile hi ndlela ya ion ya kahle (resolution ya 60,000; mass accuracy ya 10 parts per million) eka mass range ya 300/m/z mass/charge ratio (m/z) 3000. Data ya MS/MS yi kumiwile hi ku tirhisa ion trap eka LTQ Orbitrap XL [tidijithali to sungula ta 3 leti data yi titshegeke ha tona, collision induced dissociation (CID)].
Swifaniso swa MD swi endliwile hi ku tirhisa software ya GROMACS (52) na nsimu ya matimba ya MARTINI 2 (53-55). Kutani ku tirhisiwile CHARMM GUI Membrane Builder (56, 57) ku aka bilayer leyi nga na dioleoylphosphatidylcholine (DOPC) na Cer C18 kumbe DOPC na Cer C26. Topology na ti coordinates ta Cer C26 swikumiwa kusuka eka DXCE hiku susa vuhlalu byo engetela kusuka eka ncila wa sphingosine. Tirhisa endlelo leri hlamuseriweke laha hansi ku ringanisela leyara yimbirhi no yi fambisa, kutani u tirhisa tikhodixini to hetelela ta sisiteme ku aka sisiteme leyi nga na Emp24. Domain ya transmembrane ya comela Emp24 (masalela 173 ku ya eka 193) yi akiwile tanihi α-helix hi ku tirhisa xivumbeko xa molekhuli ya xitirhisiwa xa visual MD (VMD) ( 58 ). Kutani, endzhaku ko susa ti lipids leti hlanganisiweke, protein yi endliwe coarsely granulated kutani yi nghenisiwa eka bilayer hiku tirhisa CHARMM GUI. Sisiteme yo hetelela yi na 1202 DOPC na 302 Cer C26 kumbe 1197 DOPC na 295 Cer C18 na Emp24. Ionize sisiteme kuya eka nhlayo ya 0.150M. Ti replicates ta mune leti tiyimeleke ti endliwile eka swivumbeko swimbirhi swa bilayer.
Lipid bilayer yi ringanisiwa hiku tirhisa endlelo ra CHARMM GUI, leri katsaka ku hunguta kutani ku ringanisiwa 405,000 wa magoza, laha swipimelo swa xiyimo swihungutiwa hakatsongotsongo naku herisiwa, naswona goza ra nkarhi ri engeteriwa kusuka eka 0.005 ps kuya eka 0.02 ps. Endzhaku ka equilibration, yi humesa 6 μs na goza ra nkarhi ra 0.02 ps. Endzhaku ko nghenisa Emp24, tirhisa endlelo leri fanaka ra CHARMM GUI ku hunguta naku ringanisela sisiteme, kutani u tsutsuma kuringana 8 s eka vuhumelerisi.
Eka tisisiteme hinkwato, hi nkarhi wa endlelo ro ringanisela, ntshikelelo wu lawuriwa hi barostat ya Berendsen (59), naswona hi nkarhi wa endlelo ra vuhumelerisi, ntshikelelo wu lawuriwa hi barostat ya Parrinello-Rahman (60). Eka swiyimo hinkwaswo, ntshikelelo wa xikarhi i 1 bar naswona ku tirhisiwa xikimi xa semi-isotropic pressure coupling. Eka maendlelo ya balance na vuhumelerisi, thermostat (61) leyingana speed recalibration yitirhisiwa ku hlanganisa mahiselo ya ti protein, lipid na solvent particles hiku landzelelana. Hi nkarhi hinkwawo wa ntirho, mahiselo lawa ya kongomisiweke i 310K. Ntirhisano wa non-bonding wu hlayeriwa hiku tumbuluxa nxaxamelo wa pairing hiku tirhisa xikimi xa Verlet lexingana 0.005 buffer tolerance. Theme ra Coulomb ri hlayeriwa hiku tirhisa nsimu ya reaction na cut-off distance ya 1.1 nm. Rito ra Vander Waals ri tirhisa xikimi xo tsema lexi nga na mpfhuka wo tsema wa 1.1 nm, naswona xikimi xo tsema xa Verlet xi tirhisiwa eka potential drift (62).
Hiku tirhisa VMD, cutoff wavelength exikarhi ka DOPC phosphate beads kumbe ceramide AM1 beads na protein i 0.7 nm, naswona nhlayo ya ti lipids leti tirhisanaka na protein ya hlayeriwa. Hi ku ya hi fomula leyi landzelaka, hlayela depletion-enrichment (DE) factor tanihi le ka (63): DE factor = (nhlayo ya total lipids eka protein 0.7) eka protein 0.7 (nhlayo ya Cer eka total lipids) .
Nhlayo leyi vikiweke yi kumiwa tanihi xiringaniso, naswona tibara ta swihoxo i tikopi ta mune leti tiyimeleke ta SE. Nkoka wa tinhlayo wa DE factor wu hlayeriwa hi t test [(averageDE-factor-1)/SE]. Hlela ntikelo wa P ku suka eka ku hangalasiwa ka ncila wun’we.
Xitirhisiwa xa GROMACS xi tirhisiwile ku hlayela mepe wa 2D wa lateral density wa sisiteme leyi nga na Emp24 endzeni ka 250 ns yo hetelela ya trace. Leswaku ku kumiwa mepe wa enrichment/depletion wa ceramide, mepe wa density wa Cer wu avanyisiwa hi nhlayo ya mepe wa Cer na DOPC, kutani wu avanyisiwa hi nhlayo ya Cer emirini. Ku tirhisiwa xikalo xa mepe wa mihlovo leyi fanaka.
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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, Manuel Muniz
3D high-resolution real-time imaging yi paluxa nkoka wa ku leha ka ceramide chain eka ku hlawula ti protein eka tindzhawu leti hlawulaka ta vuhumelerisi.
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 high-resolution real-time imaging yi paluxa nkoka wa ku leha ka ceramide chain eka ku hlawula ti protein eka tindzhawu leti hlawulaka ta vuhumelerisi.
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