Rarraba furotin a cikin hanyar ɓoyewa yana da mahimmanci don kiyaye rarrabuwar ƙwayoyin halitta da kuma homeostasis. Baya ga rarrabawa ta hanyar harsashi, rawar da lipids ke takawa a cikin rarraba kinesin a cikin tsarin jigilar sirri wata tambaya ce ta daɗe tana wanzuwa wacce ba a amsa ta ba tukuna. A nan, muna yin hoton 3D mai launuka da yawa a lokaci guda mai ƙuduri na gaske don tabbatar da cewa sabbin sunadaran da aka haɗa da glycosylphosphatidylinositol tare da dogayen sassan lipid na ceramide an haɗa su kuma an rarraba su zuwa cikin endoplasms na musamman. Wurin fita daga Net, wanda ya bambanta da wanda furotin transmembrane ke amfani da shi. Bugu da ƙari, mun nuna cewa tsawon sarkar ceramide a cikin membrane na endoplasmic reticulum yana da mahimmanci ga wannan zaɓin rarrabuwa. Bincikenmu ya ba da shaidar farko kai tsaye a cikin jiki don rarraba kayan furotin bisa ga tsawon sarkar lipid zuwa wuraren fitarwa na zaɓi a cikin hanyar ɓoyewa.
A cikin ƙwayoyin eukaryotic, ana rarraba sunadaran da aka haɗa a cikin endoplasmic reticulum (ER) yayin jigilar su ta hanyar hanyar ɓoye don isar da su zuwa inda suka dace da ƙwayoyin halitta (1). Baya ga rarrabuwar da aka yi da murfin, an daɗe ana hasashen cewa wasu lipids suma za su iya zama wuraren fita na zaɓi ta hanyar haɗa su cikin takamaiman sassan membrane waɗanda takamaiman sunadarai (2-5). Duk da haka, har yanzu akwai rashin shaidar kai tsaye a cikin jiki don tabbatar da wannan hanyar da za a iya amfani da ita ta hanyar lipid. Domin magance wannan matsalar ta asali, mun yi nazari a cikin yisti yadda ake fitar da sunadarai masu ɗaure glycosylphosphatidylinositol (GPI) (GPI-APs) daban-daban daga ER. GPI-APs nau'ikan sunadaran saman tantanin halitta ne da ke da alaƙa da lipid (6, 7). GPI-AP furotin ne da aka ɓoye wanda aka haɗe da takaddun waje na membrane na plasma ta hanyar glycolipid moiety (GPI anga). Suna karɓar anga GPI a matsayin gyare-gyaren bayan fassarar ra'ayi a cikin ER lumen (8). Bayan haɗewa, GPI-AP yana ratsa ta cikin kayan aikin Golgi (5, 9) daga ER zuwa membrane na plasma. Kasancewar angarorin GPI yana sa a jigilar GPI-AP daban daga sunadaran da aka fitar da transmembrane (gami da sauran sunadaran membrane na plasma) tare da hanyar ɓoyewa (5, 9, 10). A cikin ƙwayoyin yisti, ana raba GPI-APs da sauran sunadaran da aka fitar a cikin endoplasmic reticulum, sannan a naɗe su cikin vesicles na musamman waɗanda aka naɗe ta hanyar haɗakar furotin mai rufi II (COPI) (6, 7). Ba a fayyace abubuwan da ke ƙayyade wannan tsari na rarrabuwa a cikin tsarin fitarwa na ER ba, amma ana hasashen cewa wannan tsari na iya buƙatar lipids, musamman sake fasalin ɓangaren lipid na anga GPI (5, 8). A cikin yisti, sake fasalin lipid na GPI yana farawa nan da nan bayan GPI ya haɗa, kuma a lokuta da yawa, yana sa ceramide ya ɗaure zuwa 26-carbon mai cike da kitse mai tsayi (C26:0) (11, 12). C26 ceramide shine babban ceramide da ƙwayoyin yisti ke samarwa zuwa yanzu. An haɗa shi a cikin ER kuma yawancinsa ana fitar da shi zuwa na'urar Golgi ta hanyar vesicles na COPII (13). Fitar da ER na GPI-AP musamman yana buƙatar ci gaba da haɗa ceramide (14, 15), kuma bi da bi, canza ceramide zuwa inositol phosphate ceramide (IPC) a cikin na'urar Golgi ya dogara da haɗa GPI anga (16). Nazarin biophysical tare da membranes na wucin gadi ya nuna cewa ceramides sarkar acyl masu tsayi sosai na iya haɗuwa don samar da yankuna masu tsari tare da halayen jiki na musamman (17, 18). Waɗannan bayanan sun haifar da hasashen cewa ceramide na C26 da GPI-AP tare da ceramide na C26 suna amfani da halayensu na zahiri don haɗuwa zuwa yankuna ko yankuna masu tsari a cikin yanayin lipid na membrane na ER mai rikitarwa. Ya ƙunshi galibin glycerolipids gajeru da marasa cika (C16:1 da C18:1) (19, 20). Za a zaɓi waɗannan yankuna a kan takamaiman wuraren fita na ER (ERES), inda za a iya haɗa ceramide da GPI-AP mai tushen ceramide zuwa Golgi a cikin vesicle ɗaya na COPII (5).
A cikin wannan binciken, mun gwada wannan hanyar da aka yi amfani da ita kai tsaye ta hanyar amfani da na'urar daukar hoto ta zamani mai cikakken ƙuduri (SCLIM), wacce wata dabara ce ta zamani mai amfani da na'urar daukar hoto wadda za ta iya lura da sunadaran da aka yiwa lakabi da fluorescent a lokaci guda. Hotunan masu launi uku da masu girma uku (3D) suna da babban ƙuduri da sauri a cikin ƙwayoyin halitta masu rai (21, 22).
Da farko mun yi amfani da fasahar SCLIM don ƙara bayyana yadda aka tantance GPI-AP na yau da kullun tare da ƙungiyar ceramide ta C26 daga furotin da aka fitar bayan sun bar ER a cikin S. cerevisiae. Domin duba rarrabuwar ER, mun yi amfani da tsarin kwayoyin halitta wanda zai iya ganin sabbin kaya da aka haɗa kai tsaye suna shiga cikin ERES a cikin jiki (7, 23). A matsayin kaya, mun zaɓi C26 ceramide-based GPI-AP Gas1 mai lakabi da furotin mai haske kore (GFP) da furotin mai narkewa Mid2 mai lakabi da furotin mai haske kusa da infrared (iRFP), duka biyun suna kai hari ga membrane na plasma (24-26). A cikin maye gurbin da ke da alaƙa da zafin jiki na sec31-1, waɗannan kaya guda biyu ana bayyana su a ƙarƙashin mai haɓaka galactose-inducible da alamar ERES mai tsari. A yanayin zafi mai tsanani (37°C), saboda maye gurbin sec31-1 yana shafar aikin sashin murfin COPII Sec31 don hana germination na COPII da fitarwa na ER, sabbin kaya da aka haɗa suna taruwa a ER (23). Bayan sanyaya zuwa ƙaramin zafin jiki (24°C), ƙwayoyin halitta masu maye gurbin sec31-1 sun dawo daga yankin ɓoyewa, kuma an fara fitar da sabbin kayan roba da aka tara daga ER. Hotunan CLIM sun nuna cewa yawancin sabbin Gas1-GFP da Mid2-iRFP da aka haɗa har yanzu suna taruwa a cikin ER na ƙwayoyin halitta masu maye gurbin sec31-1 bayan sun kumbura a 37°C sannan a sake su a 24°C na tsawon mintuna 5 (Hoto na 1). Tunda Mid2-iRFP yana rarraba akan dukkan membrane na ER, kuma Gas1-GFP yana taruwa kuma an tattara shi a yankin membrane na ER mara ci gaba, rarrabawarsu ta bambanta gaba ɗaya (Hoto na 1, A zuwa C da Movie S1). Bugu da ƙari, kamar yadda aka nuna a Hoto na 1D, ƙungiyar Gas1-GFP ba ta da Mid2-iRFP. Waɗannan sakamakon sun nuna cewa an raba furotin GPI-AP da transmembrane zuwa yankuna daban-daban na membrane na ER da wuri. Ƙungiyar Gas1-GFP tana kusa da wani takamaiman ERES mai lakabi da furotin mai launi na mCherry's COPII Sec13 (Hoto na 1, E da F, da fim ɗin S1) (23).
Kwayoyin sec31-1 suna bayyana fitar da galactose daga cikin galactose, wani dogon sarkar acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, kore) da kuma furotin mai narkewa Mid2-iRFP (TMP, shuɗi) kuma an saka wannan mai suna Sec13-mCherry (ERES, magenta) a zafin jiki na 37°C na tsawon mintuna 30, an motsa shi zuwa 24°C, kuma SCLIM ta ɗauki hotonsa bayan mintuna 5. (A zuwa C) yana nuna hoton da aka haɗa ko guda ɗaya na jirgin sama (A), hoton da aka nuna na 2D na sassan z 10 (B) ko hoton hemisphere na ƙwayoyin 3D na kaya da alamun ERES (C). Ma'aunin ma'auni 1μm (A da B). Naúrar ma'aunin ita ce 0.551μm (C). An gano Gas1-GFP a yankuna ko gungu na ER daban-daban, yayin da aka gano Mid2-iRFP kuma aka rarraba shi a cikin membrane na ER (C). (D) Jadawalin yana nuna ƙarfin haske na Gas1-GFP da Mid2-iRFP a cikin ƙungiyar Gas1-GFP tare da layin kibiya fari (hagu). AU, naúrar da ba ta dace ba. (E da F) suna wakiltar hoton 3D wanda ya haɗa kayayyaki da alamar ERES. An gano tarin Gas1-GFP kusa da takamaiman ERES. Naúrar sikelin ita ce 0.551μm. (F) Farin kibiya mai ƙarfi yana nuna tarin Gas1-GFP da ke da alaƙa da ERES. Allon tsakiya da dama suna nuna hoton 3D da aka haɗa da aka juya da kuma kallon da aka zaɓa na ƙungiyar Gas1-GFP.
Dangantakar sarari tsakanin rukunin Gas1-GFP da takamaiman ERES yana nuna cewa Gas1-GFP na iya shigar da ERES masu zaɓi, wanda ya bambanta da zaɓin da Mid2-iRFP ke amfani da shi don barin ER. Don magance wannan yuwuwar, mun ƙididdige rabon ERES don kaya ɗaya ko biyu kawai (Hoto na 2, A zuwa C). Mun gano cewa yawancin ERES (70%) suna ɗauke da nau'in kaya ɗaya kawai. Hoton ƙasa na Hoto na 2C yana nuna misalai biyu na ERES tare da Gas1-GFP kawai (Hoto na 1) ko Mid2-iRFP kawai (Hoto na 2). Akasin haka, kusan 20% na ERES ya ƙunshi kaya biyu waɗanda suka yi karo a yanki ɗaya. An gano cewa wasu ERES (10%) sun ƙunshi nau'ikan kaya guda biyu, amma an ware su a wurare daban-daban a sarari. Saboda haka, wannan nazarin ƙididdiga ya nuna cewa bayan an fitar da ER, GPI-AP Gas1-GFP da kayan transmembrane Mid2-iRFP an raba su zuwa ERES daban-daban (Hoto na 2D). Wannan ingancin rarrabawa ya yi daidai da binciken sinadarai na baya (6) da kuma tantance yanayin halitta (7). Haka nan za mu iya lura da halayen kayan da aka keɓe da ke shiga ERES (Hoto na 2E da Fim S2). Hoto na 2E ya nuna cewa ƙaramin ɓangare ne kawai na Gas1-GFP (panel 3) ko Mid2-iRFP (panel 4) ke shiga ERES daga gefe ɗaya kuma an killace shi a wani yanki daban. Panel na 5 na Hoto na 2E ya nuna cewa Gas1-GFP da Mid2-iRFP wani lokacin ana samun su a cikin ERES iri ɗaya, amma suna shiga daga ɓangarori daban-daban kuma suna taruwa a yankuna daban-daban waɗanda za su iya wakiltar vesicles daban-daban na COPII. Mun kuma tabbatar da cewa rabuwa da aka lura da rarrabuwar C26 da aka yi da ceramide GPI-AP Gas1 a matsayin zaɓi na ERES takamaiman ne saboda wani kayan ɓoyewa na transmembrane, furotin plasma membrane mai alamar GFP Axl2 (27), yana nuna irin wannan hali ga Mid2-iRFP. (Hoto na S1 da Fim S3). An rarraba sabon Axl2-GFP ta cikin membrane na ER kamar Mid2-iRFP (Hoto na S1, A da B), kuma an haɗa shi da Mid2-iRFP a mafi yawan ERES (Hoto na S1, B zuwa D). Faifai na 1 da 2 na Hoto na 1. S1C yana nuna misalai guda biyu na ERES inda kayan transmembrane guda biyu suka haɗu. A cikin waɗannan yanayi, duka kayayyaki suna shiga ERES tare (Hoto na S1E, Panel na 3 da Movie S3).
An sanya ƙwayoyin sec31-1 da ke bayyana fitar da galactose mai narkewa, Gas1-GFP (GPI-AP, kore) da Mid2-iRFP (TMP, shuɗi) da kuma alamar ERES mai tsari Sec13-mCherry (ERES, magenta) a 37 Bayan an saka su na tsawon mintuna 30 a °C, a matsa zuwa 24 °C don sakin toshewar, da kuma hoton tare da SCLIM bayan mintuna 20. (A zuwa C) Hotunan hasashen 2D masu wakiltar (A; sandar sikelin, 1μm) ko hotunan hemisphere na tantanin halitta na 3D (B da C; naúrar sikelin, 0.456μm) na kayan da sassan z 10 da aka yiwa alama da ERES. Ƙananan panel a cikin (B) da panel a cikin (C) suna nuna hotuna da aka sarrafa don nuna kawai kayan da ke cikin ERES (magenta) [Gas1-GFP (launin toka) da Mid2-iRFP (shuɗi mai haske)]. (C) Kibiyar Buɗaɗɗiya: ERES tana ɗauke da kaya ɗaya kawai (1 zuwa 4). Kibiyar launin toka: ERES tana ɗauke da kaya daban-daban (5). Farin kibiyar ƙarfi: ERES tana ɗauke da kaya tare. A ƙasa: ERES guda ɗaya da aka zaɓa ya ƙunshi Gas1-GFP (1) ko Mid2-iRFP (2) kawai. Madaurin sikelin, 100 nm. (D) Ƙididdige hoton micrograph da aka bayyana a cikin (C). Matsakaicin kaso na ERES wanda ke ɗauke da kaya ɗaya kawai (Gas1-GFP ko Mid2-iRFP), kaya daban-daban da kaya masu haɗuwa. A cikin gwaje-gwaje uku masu zaman kansu, n=432 a cikin ƙwayoyin 54. Madaurin kuskure = SD. Gwajin t mara haɗin kai mai wutsiya biyu. *** P = 0.0002. (E) Hoton 3D na zaɓaɓɓun ERES na kayan da aka keɓe waɗanda aka yiwa alama da (C). Gas1-GFP (kore) (3) ko Mid2-iRFP (shuɗi) (4) yana shiga ERES (magenta) daga gefe ɗaya kuma an iyakance shi zuwa ƙaramin yanki a cikin ERES. Wani lokaci, nau'ikan kaya guda biyu suna shiga iri ɗaya na ERES (5) daga gefe ɗaya kuma ana iyakance su zuwa wani yanki da ke keɓancewa a cikin ERES. Ma'aunin sikelin, 100 nm.
Na gaba, mun gwada wani zato cewa dogon sarkar acyl ceramide (C26) da ke cikin membrane na ER yana tura takamaiman tarawa da rarraba Gas1 zuwa zaɓaɓɓun ERES. Don wannan dalili, mun yi amfani da nau'in yisti GhLag1 da aka gyara, inda aka maye gurbin Lag1 da Lac1 na ciki guda biyu na ceramide da GhLag1 (homolog na Lag1 na auduga), wanda ya haifar da nau'in yisti tare da membrane na tantanin halitta Ceramide ya fi guntu fiye da nau'in daji (Hoto na 3A) (28). Binciken Mass spectrometry (MS) ya nuna cewa a cikin nau'in daji, 95% na jimlar ceramide yana da tsayi sosai (C26) sarkar ceramide, yayin da a cikin GhLag1, 85% na ceramide yana da tsayi sosai (C18 da C16). ), 2% kawai na ceramide yana da tsayi sosai (C26) sarkar ceramide. Duk da cewa ceramides na C18 da C16 sune manyan ceramides da aka gano a cikin membrane na GhLag1 zuwa yanzu, binciken MS ya kuma tabbatar da cewa anga GPI na Gas1-GFP da aka bayyana a cikin nau'in GhLag1 yana ɗauke da ceramide na C26, wanda yayi daidai da lipids na daji. Ingancin iri ɗaya ne (Hoto na 3A) (26). Saboda haka, wannan yana nufin cewa enzyme na sake fasalin ceramide Cwh43 yana da zaɓi sosai ga ceramide na C26, kamar yadda aka nuna a Hoto na 26, ya fi dacewa ya haɗa da anga GPI daga ƙaramin adadin ceramide na C26 a cikin nau'in GhLag1. S2 (29). Duk da haka, membrane na tantanin halitta na GhLag1 ya ƙunshi ceramide na C18-C16 kawai, yayin da Gas1-GFP har yanzu yana da ceramide na C26. Wannan gaskiyar ta sa wannan nau'in ya zama kayan aiki mai kyau don magance matsalar tsawon sarkar acyl na membrane ceramide a cikin ER. Matsayin da aka ɗauka na ajin da rarrabuwa. Bayan haka, da farko mun yi nazarin ikon C26 Gas1-GFP na taruwa a cikin gungu a cikin GhLag1 tare da allele mai canza yanayin zafi na sec31-1 ta hanyar amfani da na'urar hangen nesa ta al'ada, inda sarkar dogon (C18-C16) kawai ke wanzuwa a cikin membrane na ER Ceramide (Hoto na 3). Mun lura cewa a cikin sec31-1, yawancin Gas1-GFP an tattara su a cikin gungu, yayin da Gas1-GFP a cikin sec31-1 GhLag1 tare da dogon membrane na ceramide ER mai tsawo (C18-C16) ba a haɗa shi ba kuma an rarraba shi a cikin dukkan membrane na ER. Don zama daidai, saboda tarin ceramide na C26 yana da alaƙa da takamaiman ERES (Hoto na 1), mun sake bincika ko wannan tsari na iya haɗawa da aikin tsarin furotin na fitarwa na ER. GPI-AP yana amfani da tsarin COPII na musamman don fitarwa na ER, wanda ke aiki ta hanyar gyaran tsarin Ted1 na ɓangaren glycan na anga na GPI (30, 31). Sai a gane GPI-glycan mai sake haɗawa ta hanyar haɗakar mai karɓar kaya ta hanyar transmembrane cargo receptor p24, wanda hakan zai ɗauki Lst1 da kansa, wanda shine takamaiman isoform na babban sashin ɗaukar kaya na COPII Sec24, yana samar da ƙwayoyin cuta masu wadata na GPI-AP (31-33). Saboda haka, mun gina wani nau'in maye gurbi mai kama da juna wanda ya haɗu da share waɗannan sunadaran guda ɗaya (sashi mai rikitarwa na p24 Emp24, enzyme na sake fasalin GPI-glycan Ted1 da takamaiman sashin COPII Lst1) tare da nau'in maye gurbi na sec31-1, kuma muka yi nazarin su Shin zai yiwu a samar da GFP na Gas1 (Hoto na 3). Mun lura cewa a cikin sec31-1emp24Δ da sec31-1ted1Δ, Gas1-GFP galibi ba a haɗa shi ba kuma yana yaɗuwa a cikin membrane na ER, kamar yadda aka gani a baya a cikin sec31-1 GhLag1, yayin da a cikin sec31-1lst1Δ, Gas1-GFP Kamar sec31-1. Waɗannan sakamakon sun nuna cewa ban da kasancewar ceramide na C26 a cikin membrane na ER, tarin Gas1-GFP kuma yana buƙatar ɗaurewa da hadaddun p24, kuma baya buƙatar takamaiman ɗaukar Lst1. Sannan, mun bincika yuwuwar cewa tsawon sarkar ceramide a cikin membrane na ER zai iya daidaita ɗaurewar Gas1-GFP zuwa p24. Duk da haka, mun gano cewa kasancewar ceramide na C18-C16 a cikin membrane ba ya shafar GPI-glycans da aka sake ginawa ta hanyar complex na p24 (Figures S3 da S4, A da B) ko ɗaurewa ga GPI-AP da fitarwa ikon GPI-AP. Rijista nau'in COPII Lst1 (Hoto na S4C). Saboda haka, tarin C26 da ke dogara da ceramide ba ya buƙatar hulɗar furotin tare da hanyoyin furotin na fitarwa na ER daban-daban, amma yana goyan bayan wata hanyar rarrabawa ta madadin da tsawon lipid ke jagoranta. Sannan, mun bincika ko tsawon sarkar ceramide acyl a cikin membrane na ER yana da mahimmanci don ingantaccen rarrabuwa na Gas1-GFP a matsayin zaɓi na ERES. Tunda Gas1 a cikin nau'in GhLag1 tare da ceramide mai gajeren sarka ya bar ER kuma ya shiga membrane na plasma (Hoto na S5), mun yi imanin cewa idan rarrabawar ta dogara ne da tsawon sarkar ceramide acyl, za a iya tura Gas1 a cikin nau'in GhLag1 da kuma ketare shi. Kayayyakin ERES tare da membrane iri ɗaya.
(A) Famfon tantanin halitta na GhLag1 galibi yana ɗauke da gajerun ceramides na C18-C16, yayin da maƙallin GPI na Gas1-GFP har yanzu yana da C26 IPC iri ɗaya kamar ƙwayoyin daji. A sama: nazarin tsawon sarkar acyl na ceramide a cikin membrane na ƙwayoyin daji na nau'in wild-type (Wt) da GhLag1p ta hanyar mass spectrometry (MS). Bayanan suna wakiltar kashi na jimillar ceramide. Matsakaicin gwaje-gwaje uku masu zaman kansu. Maƙallin kuskure = SD. Gwajin t mara haɗin kai biyu. **** P ＜0.0001. Allon ƙasa: Binciken MS na tsawon sarkar acyl na IPC da ke cikin maƙallin GPI na Gas1-GFP (GPI-IPC) da aka bayyana a cikin nau'in wild-type da GhLag1p. Bayanan suna wakiltar kashi na jimillar siginar IPC. Matsakaicin gwaje-gwaje biyar masu zaman kansu. Maƙallin kuskure = SD. Gwajin t mara haɗin kai biyu. ns, ba mahimmanci ba. P = 0.9134. (B) An saka ƙananan hotunan haske na sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ da sec31-1lst1Δ waɗanda ke bayyana Gas1-GFP da galactose ya haifar a zafin jiki na 37°C na tsawon mintuna 30 sannan aka tura su zuwa yin gwajin haske na yau da kullun bayan 24°C. Kibiya fari: Ƙungiyar ER Gas1-GFP. Kibiya buɗewa: Gas1-GFP mara tarin abubuwa yana yaɗuwa akan dukkan membrane na ER, yana nuna tabon zoben nukiliya na halayen ER. Madaurin sikelin, 5μm. (C) Adadin hoton micrograph da aka bayyana a cikin (B). Matsakaicin kaso na ƙwayoyin halitta tare da tsarin Gas1-GFP mai mannewa. A cikin gwaje-gwaje uku masu zaman kansu, ƙwayoyin n≥300. Madaurin kuskure = SD. Gwajin t mara haɗin kai biyu. **** P ＜0.0001.
Domin magance wannan matsalar kai tsaye, mun yi amfani da hoton SCLIM na Gas1-GFP da Mid2-iRFP a cikin GhLag1 tare da allele mai canza yanayin zafi na sec31-1 (Hoto na 4 da Movie S4). Bayan an riƙe ER a 37°C sannan aka sake shi a 24°C, yawancin sabbin Gas1-GFP da aka haɗa ba a haɗa su ba kuma an rarraba su a cikin membrane na ER, kamar yadda aka lura ta hanyar na'urorin microscope na gargajiya (Hoto na 4, A da B). Bugu da ƙari, babban kaso na ERES (67%) ya haɗa da nau'ikan kaya guda biyu waɗanda ke tare a ciki (Hoto na 4D). Faifai na 1 da 2 na Hoto na 4C sun nuna misalai biyu na ERES tare da Gas1-GFP da Mid2-GFP masu haɗuwa. Bugu da ƙari, an tattara dukkan kayayyaki biyu a cikin ERES iri ɗaya (Hoto na 4E, panel na 3 da fim ɗin S4). Saboda haka, sakamakonmu ya nuna cewa tsawon sarkar acyl na ceramide a cikin membrane na ER muhimmin abu ne na tantance tarin furotin na ER da rarrabuwa.
Kwayoyin GhLag1 na Sec31-1 da ke bayyana fitar da galactose daga galactose, Gas1-GFP (GPI-AP, kore) da Mid2-iRFP (TMP, shuɗi) da kuma ERES mai lakabin Sec13-mCherry (ERES, magenta) a cikin 37°C. Ci gaba na minti 30, sauka zuwa 24°C don fitar da ... Kibiyar Buɗewa: ERES ta ƙunshi abu ɗaya kawai. Ƙananan faifan: ERES ɗin da aka zaɓa yana da kayayyaki masu haɗuwa (1 da 2) waɗanda aka yiwa alama a cikin (C). Madaurin sikelin, 100 nm. (D) Ƙididdige hoton micrograph ɗin da aka bayyana a cikin (C). A cikin raka'a na sec31-1 da sec31-1 GhLag1, an haɗa kaya ɗaya kawai (Gas1-GFP ko Mid2-iRFP), da matsakaicin kaso na ERES don kayan da aka keɓe da kayan da aka haɗa. A cikin gwaje-gwaje uku masu zaman kansu, n = 432 a cikin ƙwayoyin 54 (sec31-1) da n = 430 a cikin ƙwayoyin 47 (sec31-1 GhLag1). Madaurin kuskure = SD. Gwajin t mai wutsiya biyu mara haɗin kai. *** P = 0.0002 (sec31-1) da ** P = 0.0031 (sec31-1 GhLag1). (E) Hoton 3D na ERES da aka zaɓa tare da kayan da aka haɗa (3) waɗanda aka yiwa alama a cikin (C). Gas1-GFP (kore) da Mid2-iRFP (shuɗi) suna kusantar ERES (magenta) daga gefe ɗaya kuma suna ci gaba da kasancewa a cikin yanki ɗaya da aka ƙayyade na ERES. Ma'aunin sikelin, 100 nm.
Wannan binciken ya bayar da shaida kai tsaye a cikin jiki cewa an rarraba kayan furotin da aka samo daga lipid zuwa wuraren fitarwa na zaɓaɓɓu a cikin hanyar ɓoyewa, kuma ya bayyana mahimmancin tsawon sarkar acyl don zaɓin rarrabuwa. Ta amfani da dabarar microscopy mai ƙarfi da zamani mai suna SCLIM, mun nuna sabon Gas1-GFP da aka haɗa (babban membrane na plasma GPI-AP tare da dogon sarkar acyl (C26) ceramide lipid rabo) a cikin yisti) Yankunan da aka haɗa a cikin ERs masu rarraba suna da alaƙa da takamaiman ERES, yayin da furotin da aka fitar da transmembrane an rarraba su a cikin membrane na ER (Hoto na 1). Bugu da ƙari, waɗannan nau'ikan kayayyaki guda biyu suna shiga ERES daban-daban zaɓi (Hoto na 2). Tsawon sarkar acyl na ceramide ta sel a cikin membrane an rage shi daga C26 zuwa C18-C16, an wargaza ƙungiyar Gas1-GFP zuwa yankin ER mai rarrabuwa, kuma an sake tura Gas1-GFP don barin ER tare da furotin mai rarrabuwa ta hanyar ERES iri ɗaya (Hoto na 3 da Hoto na 3). 4).
Duk da cewa GPI-AP yana amfani da wata hanyar furotin ta musamman don fita daga ER, mun gano cewa rabuwar da ke dogara da ceramide ta C26 ba ta dogara da bambancin hulɗar furotin wanda zai iya haifar da ƙwarewa ga ERES ba (Figures S4 da S5). Madadin haka, bincikenmu yana goyan bayan wata hanyar rarrabuwa ta daban wacce ke haifar da tarin furotin mai tushen lipid da kuma cire wasu kaya daga baya. Abubuwan da muka lura sun nuna cewa yankin Gas1-GFP ko rukuni da ke da alaƙa da takamaiman ERES ba shi da furotin da ke fitowa daga transmembrane Mid2-iRFP, wanda ke nuna cewa rukunin GPI-AP na C26 wanda ke dogara da ceramide zai sauƙaƙa shigarsu cikin ERES masu dacewa, kuma a lokaci guda, cire transmembrane. Abubuwan da ke fitowa daga transmembrane suna shiga wannan takamaiman ERES (Figures 1 da 2). Sabanin haka, kasancewar ceramides na C18-C16 a cikin membrane na ER ba ya haifar da GPI-AP ta samar da yankuna ko gungu, don haka ba sa ware ko maye gurbin furotin da aka fitar daga transmembrane zuwa cikin ERES iri ɗaya (Figures 3 da 4). Saboda haka, muna ba da shawarar cewa C26 ceramide yana haifar da rabuwa da rarrabuwa ta hanyar sauƙaƙe tarin sunadaran da ke da alaƙa da takamaiman ERES.
Ta yaya za a cimma wannan tarin da ya dogara da ceramide na C26 zuwa wani yanki na musamman na ER? Halin da membrane ceramide ke rabawa a gefe na iya sa GPI-AP da C26 ceramide su samar da ƙananan lipids da aka tsara nan take a cikin yanayin lipid mara tsari na membrane na ER wanda ke ɗauke da gajerun glycerolipids da ba su da cikakken kitse. Tarin inganci (17, 18). Waɗannan ƙananan tari na ɗan lokaci za a iya ƙara haɗa su cikin manyan tari masu ƙarfi bayan an ɗaure su da hadaddun p24 (34). Daidai da wannan, mun nuna cewa C26 Gas1-GFP yana buƙatar yin hulɗa da hadaddun p24 don samar da manyan tari masu gani (Hoto na 3). Hadadden p24 wani oligomer ne mai heterozygous wanda ya ƙunshi furotin transmembrane guda huɗu daban-daban na p24 a cikin yisti (35), wanda ke ba da haɗin multivalent, wanda zai iya haifar da haɗin ƙananan tari na GPI-AP, ta haka yana samar da babban tari mai ƙarfi (34). Hulɗar da ke tsakanin ectodomains na furotin na GPI-APs na iya taimakawa wajen haɗa su, kamar yadda aka nuna a lokacin jigilar su ta Golgi a cikin ƙwayoyin epithelial masu rarrafe (36). Duk da haka, lokacin da ceramide na C18-C16 yake a cikin membrane na ER, lokacin da hadaddun p24 ya haɗu da Gas1-GFP, ba za a samar da manyan gungu daban-daban ba. Tsarin da ke ƙasa na iya dogara ne akan takamaiman halayen zahiri da na sinadarai na ceramide na sarkar acyl mai tsawo. Nazarin biophysical na membranes na wucin gadi ya nuna cewa kodayake duka dogayen (C24) da gajerun (C18-C16) ceramides na sarkar acyl na iya haifar da rabuwar lokaci, ceramides na sarkar acyl mai tsawo (C24) kawai zasu iya haɓaka babban lanƙwasa da lanƙwasa fim don sake fasalin fim ɗin. Ta hanyar ma'amala tsakanin juna (17, 37, 38). An nuna cewa helix na transmembrane na TMED2, homologue na ɗan adam na Emp24, yana hulɗa da sphingomyelin na tushen C18 ceramide a cikin lobules na cytoplasmic (39). Ta amfani da kwaikwayon molecular dynamics (MD), mun gano cewa ceramides na C18 da C26 suna taruwa a kusa da cytoplasmic lobules na Emp24 transmembrane helix, kuma suna da irin wannan fifiko (Hoto na S6). Yana da kyau a lura cewa wannan yana nuna cewa helix na transmembrane na Emp24 na iya haifar da rarraba lipids a cikin membrane mara daidaituwa. Wannan sakamako ne na baya-bayan nan dangane da ƙwayoyin dabbobi masu shayarwa. Irin waɗannan kwaikwayon MD kuma suna nuna kasancewar lipids na ether (40). Saboda haka, muna hasashen cewa C26 ceramide a cikin lobules biyu na ER26 yana da wadata a cikin gida. Lokacin da GPI-AP a cikin ƙananan lobules ɗin ya haɗu kai tsaye da multivalent p24 kuma tarin C26 ceramide a kusa da p24 a cikin cytoplasmic lobules, yana iya haɓaka haɗakar Protein da ke tare kuma ana samar da lanƙwasa membrane ta cikin yatsu (41), wanda ke sa GPI-AP ya rabu zuwa yankuna daban-daban da ke kusa da ERES, wanda kuma yana fifita yankuna masu lanƙwasa na membrane ER (42). Rahotannin da suka gabata sun goyi bayan tsarin da aka gabatar (43, 44). Haɗa oligolectins, ƙwayoyin cuta ko ƙwayoyin rigakafi ga glycosphingolipids (GSL) na tushen ceramide akan membrane plasma yana haifar da babban tarin GSL, yana haɓaka rabuwar lokaci kuma yana haifar da nakasa membrane da ciki (44). Iwabuchi da sauransu (43) An gano cewa a gaban dogayen sarƙoƙin acyl (C24) amma ba gajeru ba (C16), ligand mai yawa da aka ɗaure zuwa lactosylceramide na GSL ya haifar da samuwar manyan gungu da kuma shigar membrane, kuma transduction na siginar da aka yi ta hanyar Lyn akan takaddun yana haɗuwa da sarƙoƙin acyl a cikin neutrophils masu haɗin gwiwa.
A cikin ƙwayoyin epithelial masu rarrafe na dabbobi masu shayarwa, yawan haɗin hanyar sadarwa ta anti-Golgi (TGN) zuwa matakin membrane na plasma na apical yana sarrafa rabuwa da rarrabawa na GPI-AP (10, 45). Wannan haɗuwa tana gudana ne ta hanyar oligomerization na GPI-AP (36), amma kuma yana iya dogara da tsawon sarkar ceramide da muka samu a cikin yisti. Kodayake GPI-AP na dabbobi masu shayarwa yana da anga mai tushen ether lipid, kuma tsarin sinadaransa ya bambanta sosai da ceramide na sarkar acyl mai tsayi, wani bincike na baya-bayan nan ya gano cewa duka lipids suna da halaye na zahiri da na sinadarai iri ɗaya a cikin juyin halitta (40). Saboda haka, ɓangaren lipid na ether a cikin ƙwayoyin dabbobi masu shayarwa na iya kama da ceramide na C26 a cikin yisti, kuma aikinsa shine haɗawa da ceramide mai tsayi a cikin membrane don haɓaka tarin GPI-AP da rarrabawa. Ko da yake har yanzu ana buƙatar gwada wannan yiwuwar kai tsaye, binciken da aka yi a baya ya tabbatar da cewa jigilar dogon sarkar acyl ceramide zuwa jikin Golgi ba ta hanyar sunadaran canja wurin cytoplasmic ba ne, amma ya dogara ne akan haɗakar abubuwan haɗin GPI kamar yisti. Saboda haka, hanyar juyin halitta mai ra'ayin mazan jiya tana da ikon zaɓar jigilar dogon sarkar acyl ceramide da GPI-AP (13, 16, 20, 46, 47) a cikin vesicle ɗin jigilar kaya iri ɗaya.
A cikin tsarin ƙwayoyin epithelial masu rarrafe da yisti da kuma na dabbobi masu shayarwa, tarin GPI-AP da rabuwa da sauran sunadaran membrane na plasma duk suna faruwa kafin su isa saman tantanin halitta. Paladino et al. (48) sun gano cewa a kan TGN na ƙwayoyin epithelial masu rarrafe da dabbobi masu shayarwa, tarin GPI-AP ba wai kawai ya zama dole ba don rarrabuwar GPI-APs zuwa membrane na plasma mai apical, har ma yana daidaita tsarin tarin GPI-APs da ayyukan halittunsa. Surface na ƙwayoyin halitta. A cikin yisti, wannan binciken ya nuna cewa ƙungiyar GPI-AP mai dogara da C26 ceramide akan ER na iya tsara tsarin rukuni da aikin GPI-AP akan membrane na plasma (24, 49). Daidai da wannan samfurin, ƙwayoyin GhLag1 suna da rashin lafiyar masu hana GPI ko magunguna waɗanda ke shafar amincin bangon tantanin halitta (28), kuma buƙatar rukunin Gas1-GFP masu aiki (49) na ceramide na tip da aka hango a cikin haɗuwar ƙwayoyin yisti yana nuna G​​​​ Sakamakon ilimin halittar ƙwayoyin hLag1. Kuskuren GPI-AP. Duk da haka, ƙarin gwaji ko an tsara tsarin aikin saman tantanin halitta daga ER ta hanyar rarrabawa bisa ga tsawon lipid zai zama batun bincikenmu na gaba.
An jera nau'ikan Saccharomyces cerevisiae da aka yi amfani da su a wannan aikin a cikin Jadawali na S1. An gina nau'ikan MMY1583 da MMY1635 na SCLIM don ɗaukar hoton tantanin halitta a bayan W303. An gina waɗannan nau'ikan da ke bayyana Sec13-mCherry tare da alamar furotin mai haske ta amfani da hanyar polymerase chain reaction (PCR) tare da pFA6a plasmid a matsayin samfuri (23). An gina nau'in da ke bayyana Mid2-iRFP mai suna tare da furotin mai haske a ƙarƙashin ikon mai haɓaka GAL1 kamar haka. Haɓaka PCR na jerin iRFP-KanMx daga vector na pKTiRFP-KAN (kyautar E. O'Shea, lambar plasmid Addgene 64687; http://n2t.net/addgene: 64687; mai gano albarkatun bincike (RRID): Addgene_64687) Kuma an saka shi cikin C-terminus na Mid2 na ciki. Bayan an ƙara yawan jerin kwayoyin halittar Mid2-iRFP kuma an haɗa shi cikin mai haɓaka GAL1, an haɗa shi cikin shafin Not I-Sac I na haɗin plasmid pRS306. An haɗa pRGS7 na plasmid da aka samu tare da Pst I don haɗawa cikin wurin URA3.
Ana bayyana kwayar halittar Gas1-GFP a ƙarƙashin ikon mai haɓaka GAL1 a cikin plasmid na centromere (CEN), wanda aka gina kamar haka. An ƙara yawan jerin Gas1-GFP ta PCR daga plasmid na pRS416-GAS1-GFP (24) (kyauta ta L. Popolo) kuma an haɗa shi cikin wurin Xma I–Xho I na plasmid na CEN pBEVY-GL LEU2 (kyauta ta C). Miller; Lambar plasmid na Adgene 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). An sanya wa plasmid ɗin da ya haifar suna pRGS6. Hakanan ana bayyana kwayar halittar Axl2-GFP a ƙarƙashin ikon mai haɓaka GAL1 na vector na pBEVY-GL LEU2, kuma gininsa kamar haka ne. An ƙara yawan jerin Axl2-GFP daga plasmid pRS304-p2HSE-Axl2-GFP (23) ta hanyar PCR, kuma an haɗa shi cikin wurin Bam HI-Pst I na vector pBEVY-GL LEU2. An sanya wa plasmid ɗin da ya haifar suna pRGS12. Jerin oligonucleotides da aka yi amfani da su a wannan binciken an jera su a cikin Tebur S2.
An ƙara wa wannan nau'in sinadarin adenine 0.2% da 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) matsakaici (1% yeast extract da 2% protein ept). (YPR)] ko 2% galactose [YP-galactose (YPG)] a matsayin tushen carbon, ko kuma a cikin ƙaramin matsakaici na roba (0.15% yeast nitrogen tushe da 0.5% ammonium sulfate) don ƙara amino acid da tushe masu dacewa da ake buƙata don abinci mai gina jiki, Kuma yana ɗauke da 2% glucose (ƙaramin matsakaici na glucose synthetic) ko 2% galactose (ƙaramin matsakaici na galactose synthetic) a matsayin tushen carbon.
Don ɗaukar hoto na ainihin lokaci, ƙwayoyin halitta masu canza yanayin zafi na sec31-1 waɗanda ke bayyana ginin a ƙarƙashin mai haɓaka GAL1 an girma su a cikin matsakaicin YPR a 24°C na dare zuwa matakin tsakiyar log. Bayan shigar da YPG a 24°C na tsawon awa 1, an saka ƙwayoyin a cikin SG a 37°C na tsawon mintuna 30, sannan aka canja su zuwa 24°C don su fita daga toshewar fitar da ruwa. An yi amfani da Concanavalin A don gyara ƙwayoyin a kan gilashin zamewa kuma SCLIM ta ɗauki hoton su. SCLIM haɗin na'urar hangen nesa ta Olympus IX-71 mai juyewa da kuma ruwan tabarau na mai lamba UPlanSApo 100×1.4 (Olympus), na'urar daukar hoto mai juyawa ta diski mai sauri da kuma babban sigina zuwa amo (Yokogawa Electric), na'urar hangen nesa ta musamman, da kuma sanyaya ta musamman. Mai ƙara girman hoton tsarin (Hamamatsu Photonics) zai iya samar da tsarin gilashin ƙara girma tare da ƙara girman ×266.7 na ƙarshe da kyamarar na'urar da aka haɗa da caji wanda ke ninka electrons (Hamamatsu Photonics) (21). Ana yin siyan hoto ta hanyar software na musamman (Yokogawa Electric). Don hotunan 3D, mun yi amfani da na'urar kunna piezoelectric da aka ƙera musamman don girgiza ruwan tabarau a tsaye, kuma muka tattara sassan gani 100 nm daban-daban a cikin tari. Ana canza hoton Z-stack zuwa bayanai na voxel na 3D, kuma ana amfani da aikin watsawar maki na ka'idar da ake amfani da shi don na'urar hangen nesa ta diski mai juyawa don sarrafa deconvolution ta hanyar software na Volocity (PerkinElmer). Ta hanyar amfani da software na Volocity don yin iyaka ta atomatik don nazarin wuri ɗaya, an auna ERES gami da kaya. An gudanar da nazarin layi ta amfani da software na MetaMorph (Na'urorin Molecular).
Yi amfani da manhajar GraphPad Prism don tantance mahimmancin ƙididdiga. Ga gwajin t-dalibi mai wutsiya biyu da kuma gwajin bambancin hanya ɗaya na yau da kullun (ANOVA), ana ɗaukar bambance-bambance tsakanin ƙungiyoyi a matsayin suna da tasiri mai mahimmanci akan P <0.05 (*).
Don yin amfani da na'urar hangen nesa ta Gas1-GFP, an girma ƙwayoyin log phase a cikin dare ɗaya a cikin YPD kuma an tattara su ta hanyar centrifugation, an wanke su sau biyu da sinadarin phosphate buffered saline, sannan aka saka su a kan kankara na tsawon akalla mintuna 15, sannan aka ci gaba da amfani da na'urar hangen nesa kamar yadda aka bayyana a baya Duba (24). An yi amfani da na'urar hangen nesa ta Leica DMi8 (HCX PL APO 1003/1.40 mai PH3 CS) wacce aka sanye da ruwan tabarau na zahiri, matattarar L5 (GFP), kyamarar Hamamatsu da software na Application Suite X (LAS X) don siyan su.
An cire samfuran daga jiki da SDS sample buffer a zafin 65°C na tsawon mintuna 10, sannan aka raba su da SDS-polyacrylamide gel electrophoresis (PAGE). Don nazarin immunoblotting, an ɗora 10 μl na samfurin a kowace layi. Babban antibody: Yi amfani da rabbit polyclonal anti-Gas1 a dilution na 1:3000, rabbit polyclonal anti-Emp24 a dilution na 1:500, da rabbit polyclonal anti-GFP (kyauta daga H. Riezman) a dilution na 1:3000. An yi amfani da beraye monoclonal anti-Pgk1 antibody a dilution na 1:5000 (kyauta daga J. de la Cruz). Secondary antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) da aka yi amfani da shi a dilution na 1:3000 (Pierce). An yi amfani da IgG na maganin hana beraye mai haɗin HRP a cikin 1:3000 (Pierce). An lura da yankin amsawar garkuwar jiki ta hanyar hanyar chemiluminescence ta SuperSignal West Pico reagent (Thermo Fisher Scientific).
Kamar yadda aka bayyana a cikin (31), an yi gwajin rigakafi na halitta akan ƙashin ER mai wadatarwa. A takaice, a wanke ƙwayoyin yisti da TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride da cakuda protease inhibitor) a 600 nm (OD600) a 100 na gani sau biyu. An karya shi da beads na gilashi, sannan aka cire tarkacen ƙwayoyin halitta da beads na gilashi ta hanyar centrifugation. Daga nan aka sanya centrifugation a 17,000 g na minti 15 a 4°C. An sake ɗaure pellet ɗin a cikin TNE kuma an ƙara saponin dijitalis zuwa taro na ƙarshe na 1%. An sanya dakatarwar na tsawon awa 1 tare da juyawa a 4°C, sannan aka cire abubuwan da ba sa narkewa ta hanyar centrifugation a 13,000 g a 4°C na minti 60. Don rigakafin Gas1-GFP, da farko a saka samfurin a cikin kwano da ƙwai mara komai na agarose (ChromoTek) a zafin jiki na 4°C na tsawon awa 1, sannan a saka a cikin kwano da GFP-Trap_A (ChromoTek) a zafin jiki na 4°C na tsawon awanni 3. An wanke ƙwai masu kariya daga garkuwar jiki sau biyar da TNE mai ɗauke da 0.2% digoxigenin, an cire shi da samfurin SDS, an raba shi a kan SDS-PAGE, sannan a yi nazari a kansa ta hanyar immunoblotting.
Kamar yadda aka bayyana a cikin (31), an yi gwajin haɗin gwiwa a kan ɓangaren ER mai wadatarwa. A taƙaice, an haɗa ɓangaren ER mai wadatarwa da dithiobis (succinimidyl propionate) 0.5 mM (Pierce, Thermo Fisher Scientific, Rockford, IL, Amurka; 20°C, minti 20). An kashe amsawar haɗin gwiwa ta hanyar ƙara glycine (ƙarshen maida hankali na 50 mM, minti 5, 20°C).
Kamar yadda aka bayyana a baya (50), an yi nazarin MS na ceramide a cikin nau'in daji da GhLag1. A takaice, an girma ƙwayoyin zuwa matakin exponential (3 zuwa 4 OD600 raka'a/ml) a cikin YPD a 30°C, kuma an girbe ƙwayoyin 25×107. Ana kashe metabolism ɗin su da trichloroacetic acid. Yi amfani da sinadarin cirewa [ethanol, ruwa, ether, pyridine da 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] da 1.2 nmol na intracent C17 ceramide (860517, Avanti polar lipid) inganci). Yi amfani da monomethylamine reagent [methanol, ruwa, n-butanol da methylamine solution (4:3:1:5 v/v)] don yin alkaline hydrolysis mai sauƙi na cirewar, sannan a yi amfani da n-butanol mai cike da ruwa don cire gishiri. A ƙarshe, an sake haɗa sinadarin a cikin wani sinadari mai kyau [chloroform/methanol/ruwa (2:7:1) + 5 mM ammonium acetate] sannan aka yi allura a cikin mass spectrometer. An yi sa ido kan yawan amsawa (MRM) don gano da kuma ƙididdige ƙwayoyin sphingolipid. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) yana da na'urar robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) don nazarin lipid. An inganta kuzarin karo ga kowane nau'in ceramide. An samo bayanan MS a cikin yanayin tabbatacce. Ga kowane kwafi na halitta, siginar lipid ita ce matsakaicin ma'auni guda uku masu zaman kansu.
Kamar yadda aka bayyana a cikin (31), ƙwayoyin (800×107) da ke bayyana Gas1-GFP an yi musu gwajin rigakafi na halitta. An raba Gas1-GFP mai tsabta ta hanyar SDS-PAGE kuma an mayar da shi cikin membrane na polyvinylidene fluoride (PVDF). An ga furotin ta hanyar yin fenti a PVDF da baki na amide. An yanke madaurin Gas1-GFP daga PVDF kuma an wanke shi sau 5 da methanol kuma sau ɗaya da ruwa mai daraja na ruwa (LC-MS). Ta hanyar haɗa madaurin membrane da cakuda 500μl 0.3 M NaOAc (pH 4.0), buffer da 500μl na cakuda sodium nitrite 1 M da aka narkar a 37°C na tsawon awanni 3, ana fitar da kaso na lipid daga Gas1-GFP kuma an yi lysed. Sakin inosine phosphate ceramide tsakanin glucosamine da inositol (51). Bayan haka, an wanke layin membrane sau huɗu da ruwan LC-MS, an busar da shi a zafin ɗaki, sannan aka adana shi a cikin yanayin nitrogen a -80°C har sai an yi nazari. A matsayin abin sarrafawa, an yi amfani da samfurin membrane na PVDF mara komai don kowane gwaji. Daga nan sai MS ta yi nazarin lipid ɗin da aka cire daga Gas1-GFP kamar yadda aka bayyana (50). A takaice, an sake dakatar da layukan PVDF da ke ɗauke da GPI-lipid a cikin 75μl na mold negative solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] kuma an wuce electrospray ionization (ESI)-MRM/MS Nazarin nau'in sphingolipid (TSQ Vantage). A wannan yanayin, an sami bayanan MS a cikin yanayin ion negative.
Kamar yadda aka ambata a baya, an raba ɓangaren lipid na anga GPI daga [3H]-inositol-labeled GPI-AP (16). An raba lipids ɗin ta hanyar chromatography mai sirara ta amfani da tsarin narkewa (55:45:10 chloroform-methanol-0.25% KCl) kuma an nuna su ta amfani da FLA-7000 (Fujifilm).
An wanke ƙwayoyin da ke ɗauke da Gas1-GFP (600×107) sau biyu da TNE buffer tare da TNE buffer, sannan aka karya su da gilashin beads, sannan aka sanya musu centrifuge don cire tarkacen ƙwayoyin halitta da beads na gilashi. Sannan aka sanya musu centrifuge a 17,000 g na tsawon awa 1 a 4°C. An wanke battle ɗin a cikin TNE kuma aka saka musu 1 U PI-PLC (Invitrogen) a cikin TNE wanda ke ɗauke da 0.2% saponin dijital na tsawon awa 1 a 37°C. Bayan maganin enzyme, an cire membrane ɗin ta hanyar centrifugation a 17,000 g a 4°C na tsawon awa 1. Don hana Gas1-GFP yin rigakafi, an saka musu GFP-Trap_A (ChromoTek) a 4°C na dare. An yi wa Gas1-GFP mai tsabta da SDS-PAGE fenti da shuɗi mai haske. An yanke madaurin tabo na Gas1-GFP daga launin toka da ke kewaye da bututun ruwa, sannan bayan alkylation da iodoacetamide da ragewa da dithiothreitol, an yi narkewar gel tare da trypsin. An cire kuma an busar da peptides da peptides na tryptic tare da GPI-glycans. An narkar da busasshen peptide a cikin 20 μl na ruwa. A yi allurar wani yanki (8μl) a cikin LC. An yi amfani da ginshiƙin octadecylsilane (ODS) (Develosil 300ODS-HG-5; diamita na ciki 150 mm × 1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) don raba peptides a ƙarƙashin takamaiman yanayi na gradient. Matakin wayar hannu shine solvent A (0.08% formic acid) da solvent B (0.15% formic acid a cikin 80% acetonitrile). An yi amfani da tsarin Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) don cire ginshiƙin da ruwan zafi A cikin mintuna 55 a cikin ƙimar kwararar 50 μl min-1 na mintuna 5, sannan aka ƙara yawan ruwan zafi B zuwa 40%. , Amurka). An ci gaba da shigar da eluate cikin tushen ion na ESI, kuma an yi nazarin peptides na tryptic da peptides tare da GPI-glycans ta hanyar LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). A cikin saitin MS, an saita ƙarfin wutar lantarki na tushen capillary zuwa 4.5 kV, kuma an kiyaye zafin capillary ɗin canja wuri a 300°C. An saita ƙarfin lantarki na capillary da ƙarfin ruwan tabarau na bututu zuwa 15 V da 50 V, bi da bi. An samo bayanan MS a cikin yanayin ion mai kyau (ƙuduri na 60,000; daidaiton taro na sassa 10 a kowace miliyan) a cikin kewayon taro na 300/m/z rabon taro/caji (m/z) 3000. Ana samun bayanan MS/MS ta hanyar tarkon ion a cikin LTQ Orbitrap XL [lambobi 3 na farko waɗanda bayanan suka dogara da su, rarrabuwar da aka haifar da karo (CID)].
An yi kwaikwayon MD ta amfani da software na GROMACS (52) da filin ƙarfin MARTINI 2 (53-55). Sannan aka yi amfani da CHARMM GUI Membrane Builder (56, 57) don gina wani layi mai siffar bilayer da ke ɗauke da dioleoylphosphatidylcholine (DOPC) da Cer C18 ko DOPC da Cer C26. Tsarin da daidaitawar Cer C26 an samo su ne daga DXCE ta hanyar cire ƙarin beads daga wutsiyar sphingosine. Yi amfani da tsarin da aka bayyana a ƙasa don daidaita Layer biyu da gudanar da shi, sannan a yi amfani da daidaitawa na ƙarshe na tsarin don gina tsarin da ke ɗauke da Emp24. An gina yankin transmembrane na yisti Emp24 (ragowar 173 zuwa 193) a matsayin α-helix ta amfani da tsarin kwayoyin halitta na kayan aikin MD (VMD) na gani (58). Sannan, bayan cire lipids masu haɗuwa, an haɗa furotin da kyau kuma an saka shi cikin layi ta amfani da CHARMM GUI. Tsarin ƙarshe ya ƙunshi DOPC 1202 da 302 Cer C26 ko 1197 DOPC da 295 Cer C18 da Emp24. Sanya tsarin a cikin ion har zuwa 0.150M. An yi kwafi huɗu masu zaman kansu don haɗakar layuka biyu.
Ana daidaita layin lipid ta amfani da tsarin CHARMM GUI, wanda ya haɗa da ragewa sannan daidaita matakai 405,000, inda ake rage ƙuntatawa a hankali da kawar da su, kuma ana ƙara matakin lokaci daga 0.005 ps zuwa 0.02 ps. Bayan daidaitawa, yana samar da 6 µs tare da matakin lokaci na 0.02 ps. Bayan saka Emp24, yi amfani da tsarin CHARMM GUI iri ɗaya don ragewa da daidaita tsarin, sannan a yi aiki na 8 seconds a samarwa.
Ga dukkan tsarin, a lokacin daidaita ma'auni, ana sarrafa matsin lamba ta Berendsen barostat (59), kuma a lokacin aiwatar da samarwa, ana sarrafa matsin lamba ta Parrinello-Rahman barostat (60). A duk lokuta, matsakaicin matsin lamba shine sandar 1 kuma ana amfani da tsarin haɗa matsin lamba na semi-isotropic. A cikin tsarin daidaitawa da samarwa, ana amfani da na'urar auna zafi (61) tare da sake daidaita saurin don haɗa zafin furotin, lipid da barbashi masu narkewa bi da bi. A duk lokacin aikin, zafin da aka nufa shine 310K. Ana ƙididdige hulɗar da ba ta haɗa ba ta hanyar samar da jerin haɗuwa ta amfani da tsarin Verlet tare da haƙurin buffer 0.005. Ana ƙididdige kalmar Coulomb ta amfani da filin amsawa da nisan yankewa na 1.1 nm. Kalmar Vander Waals tana amfani da tsarin yankewa tare da nisan yankewa na 1.1 nm, kuma ana amfani da tsarin yankewa na Verlet don yuwuwar zamewa (62).
Ta amfani da VMD, tsawon lokacin yankewa tsakanin beads na DOPC phosphate ko beads na ceramide AM1 da furotin shine 0.7 nm, kuma ana ƙididdige adadin lipids da ke hulɗa da furotin. Dangane da dabarar da ke ƙasa, ana ƙididdige factor depletion-enrichment (DE) kamar yadda yake a cikin (63): DE factor = (adadin jimillar lipids a cikin furotin 0.7) a cikin furotin 0.7 (adadin Cer a cikin jimillar lipids)
Ana samun ƙimar da aka ruwaito a matsayin matsakaici, kuma sandunan kuskuren kwafi huɗu ne masu zaman kansu na SE. Ana ƙididdige mahimmancin ƙididdiga na DE factor ta hanyar gwajin t [(averageDE-factor-1)/SE]. Lissafa ƙimar P daga rarrabawar wutsiya ɗaya.
An yi amfani da kayan aikin GROMACS don ƙididdige taswirar yawan gefe na 2D na tsarin da ke ɗauke da Emp24 a cikin 250 ns na ƙarshe na alamar. Domin samun taswirar wadatarwa/ƙarewar ceramide, ana raba taswirar yawan Cer ta jimlar taswirar Cer da DOPC, sannan a raba ta da yawan Cer a jiki. Ana amfani da sikelin taswirar launi iri ɗaya.
Don ƙarin kayan aiki don wannan labarin, da fatan za a duba 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 -Linero), Sergio Lopez (Sergio Wakoga) (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Hoton 3D mai ƙuduri mai girma a ainihin lokaci yana bayyana mahimmancin tsawon sarkar ceramide don rarrabe furotin a wuraren fitarwa na musamman.
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 Wakoga) (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Hoton 3D mai ƙuduri mai girma a ainihin lokaci yana bayyana mahimmancin tsawon sarkar ceramide don rarrabe furotin a wuraren fitarwa na musamman.
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