He mea nui te whakarōpūtanga pūmua i roto i te ara huna hei pupuri i te wehewehenga pūtau me te homeostasis. Haunga te whakarōpūtanga e whakahaerehia ana e te anga, ko te mahi a ngā lipids i roto i te whakarōpūtanga kinesin i roto i te tukanga kawe huna he pātai taketake roa kāore anō kia whakautua. I konei, ka whakahaerehia e mātou he whakaahua wā-tūturu taumira teitei maha-tae 3D hei whakaatu i roto i te tinana ora kua whakarōpūhia, ā, kua whakarōpūhia ngā pūmua glycosylphosphatidylinositol-whakaū hou me ngā wāhanga lipid ceramide roa rawa atu ki roto i ngā endoplasms motuhake. He rerekē tēnei i te mea e whakamahia ana e ngā pūmua transmembrane. Hei tāpiri, ka whakaatuhia e mātou he mea nui te roa o te mekameka ceramide i roto i te membrane reticulum endoplasmic mō tēnei whiriwhiringa whakarōpū. Ko tā mātou rangahau te taunakitanga tika tuatahi i roto i te tinana ora hei whakarōpū i ngā utanga pūmua i runga i te roa o te mekameka lipid ki roto i ngā wāhi kaweake whiriwhiri i roto i te ara huna.
I roto i ngā pūtau eukaryotic, ka whakarōpūhia ngā pūmua i hangaia i roto i te reticulum endoplasmic (ER) i te wā e kawehia ana mā te ara huna hei tuku ki tō rātou ūnga pūtau tika (1). Haunga te whakarōpūtanga-whakawhitinga-uwhi, kua roa te whakapae ka taea hoki e ētahi ngako te mahi hei wāhi putanga whiriwhiri mā te whakarōpū i a rātou ki roto i ngā rohe membrane motuhake e pā ana ki ngā pūmua motuhake (2-5). Heoi, kei te kore tonu he taunakitanga tika i roto i te tinana hei whakamatau i tēnei tikanga e hangai ana ki te ngako. Hei whakaoti i tēnei raruraru taketake, i ako mātou i roto i te rewena me pēhea te kawe rerekētanga o ngā pūmua taunga glycosylphosphatidylinositol (GPI) (GPI-AP) mai i te ER. Ko ngā GPI-AP he momo pūmua mata pūtau e hono ana ki te ngako (6, 7). Ko te GPI-AP he pūmua huna e piri ana ki ngā rau o waho o te membrane plasma mā roto i te wāhanga glycolipid (taunga GPI). Ka whakaaetia e rātou ngā taunga GPI hei whakarerekētanga muri-whakamāoritanga i roto i te lumen ER (8). I muri i te whakapiringa, ka haere a GPI-AP mā roto i te taputapu Golgi (5, 9) mai i te ER ki te kiriuhi plasma. Nā te noho mai o ngā punga GPI ka kawe motuhaketia a GPI-AP mai i ngā pūmua huna transmembrane (tae atu ki ētahi atu pūmua kiriuhi plasma) i te ara huna (5, 9, 10). I roto i ngā pūtau rewena, ka wehea ngā GPI-AP mai i ētahi atu pūmua huna i roto i te reticulum endoplasmic, kātahi ka tākaihia ki roto i ngā vesicle ahurei kua takaihia e te matatini pūmua uhi II (COPII) (6, 7). Kāore i te mārama ngā mea e whakatau ana i tēnei tukanga whakarōpūtanga i roto i te tukanga kaweake ER, engari e whakaarohia ana me whai lipid tēnei tikanga, inā koa ko te whakahou hanganga o te wāhanga lipid o te punga GPI (5, 8). I roto i te rewena, ka tīmata tonu te whakahou lipid GPI i muri i te whakapiringa o te GPI, ā, i roto i te nuinga o ngā wā, ka meinga e ia te ceramide kia herea ki te waikawa ngako mekameka roa 26-waro (C26:0) (11, 12). Ko te ceramide C26 te ceramide matua i hangaia e ngā pūtau rewena tae noa ki tēnei wā. Ka hangaia i roto i te ER, ā, ko te nuinga ka tukuna ki te taputapu Golgi mā roto i ngā vesicle COPII (13). Ko te kaweake ER o te GPI-AP me whai tonu te hanganga ceramide (14, 15), ā, ko te huringa o te ceramide ki te inositol phosphate ceramide (IPC) i roto i te taputapu Golgi e whakawhirinaki ana ki te hanganga punga GPI (16). Kua whakaatuhia e ngā rangahau koiora-tinana me ngā kiriuhi horihori ka taea e ngā ceramide mekameka acyl roa rawa te whakakotahi hei hanga i ngā rohe kua whakaritea me ngā āhuatanga ā-tinana ahurei (17, 18). Ka arahina ēnei raraunga ki te whakapae ka whakamahia e te ceramide C26 me te GPI-AP me te ceramide C26 ō rātou āhuatanga ā-tinana hei whakakotahi hei rohe, hei rohe rānei kua whakaritea i roto i te taiao lipid membrane ER pōrearea. Ko te nuinga o te mea he glycerolipids poto me te kore-mākū (C16:1 me C18:1) (19, 20). Ka arotahi motuhaketia ēnei rohe ki ngā wāhi putanga ER motuhake (ERES), ka taea te kawe tahi i te ceramide me te GPI-AP e hangai ana ki te ceramide ki te Golgi i roto i te vesicle COPII motuhake (5).
I roto i tēnei rangahau, kua whakamātau tika mātou i tēnei tikanga e ahu mai ana i te ngako mā te whakamahi i te karu whakaahua wā-tūturu confocal super-resolution (SCLIM), he tikanga karu whakaahua matatau e taea ai te tirotiro i ngā pūmua kua tohua ki te fluorescence i te wā kotahi. He tino teitei te taumira me te tere o ngā whakaahua tae-toru me te toru-ahu (3D) i roto i ngā pūtau ora (21, 22).
I te tuatahi i whakamahia e mātou te hangarau SCLIM hei whakamārama anō i te āhua o te tirotiro i te GPI-AP noa me te rōpū ceramide C26 mai i ngā pūmua i hunaia e te membrane i muri i te wehenga atu i te ER i roto i te S. cerevisiae. Hei tirotiro i te whakarōpūtanga o te ER, i whakamahia e mātou he pūnaha ira ka taea te tiro tika i ngā utanga hou kua hangaia e uru ana ki te ERES in vivo (7, 23). Hei utanga, i whiriwhiria e mātou te GPI-AP Gas1 e ahu mai ana i te ceramide C26 kua tohua ki te pūmua fluorescent matomato (GFP) me te pūmua i hunaia e te membrane Mid2 kua tohua ki te pūmua fluorescent tata-infrared (iRFP), e rua e aro ana ki te membrane plasma (24–26). I roto i te mutant pāmahana-tairongo sec31-1, ka whakaatuhia ēnei utanga e rua i raro i te whakatairanga galactose-inducible me te tohu ERES pumau. I te pāmahana tino nui (37°C), nā te mea ka pā te whakarerekētanga sec31-1 ki te mahi a te wāhanga uhi COPII Sec31 ki te aukati i te whakatipu COPII me te kaweake ER, ka kohia ngā utanga hou kua hangaia ki te ER (23). I muri i te whakamatao ki te pāmahana iti (24°C), ka ora ake ngā pūtau whakarerekētanga sec31-1 mai i te rohe huna, ā, ka tīmata te kaweake i ngā utanga hangai hou kua kohia mai i te ER. I whakaatuhia e te tirohanga CLIM ko te nuinga o ngā Gas1-GFP me Mid2-iRFP hou kua kohia kei te kohia tonu i roto i te ER o ngā pūtau whakarerekētanga sec31-1 i muri i te whakatipu i te 37°C, kātahi ka tukuna i te 24°C mō te 5 meneti (Pikitia 1). Nā te mea kua tohatohahia te Mid2-iRFP ki runga i te kiriuhi ER katoa, ā, kua kukū te Gas1-GFP, ā, kua kohia ki roto i te rohe kiriuhi ER motumotu, he tino rerekē tō rātou tohatoha (Pikitia 1, A ki C me te Kiriata S1). Hei tāpiri, e ai ki te whakaaturanga i te Pikitia 1D, kāore he Mid2-iRFP i roto i te tautau Gas1-GFP. E whakaatu ana ēnei hua i wehea wawetia ngā pūmua GPI-AP me ngā pūmua transmembrane ki ngā rohe kiriuhi ER rerekē. Kei te taha tonu o te tautau Gas1-GFP tētahi ERES motuhake kua tohua ki te pūmua uhi COPII a mCherry, arā, ko Sec13 (Pikitia 1, E me F, me te kiriata S1) (23).
Ka whakaatuhia e ngā pūtau sec31-1 ngā hinu i puta mai i te galactose, he mekameka acyl roa (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, kakariki) me te pūmua transmembrane Mid2-iRFP (TMP, kikorangi) ā, i whakatōngia tēnei tapanga ERES Hanga Sec13-mCherry (ERES, magenta) i te 37°C mō te 30 meneti, ka nekehia ki te 24°C, ā, ka whakaahuahia e SCLIM i muri i te 5 meneti. E whakaatu ana (A ki C) i tētahi whakaahua 2D whakakotahi, kotahi rānei o tētahi papa (A), he whakaahua whakaaturanga 2D o ngā wāhanga-z 10 (B) he whakaahua pūtau 3D rānei o ngā tohu utanga me te ERES (C). Pae tauine 1μm (A me B). Ko te wae tauine he 0.551μm (C). I kitea a Gas1-GFP i roto i ngā rohe ER motuhake, i ngā tautau rānei, i te mea i kitea a Mid2-iRFP, ā, i tohatohahia puta noa i te kiriuhi ER (C). (D) E whakaatu ana te kauwhata i te kaha o te hihi o te Gas1-GFP me te Mid2-iRFP i roto i te tautau Gas1-GFP i te rārangi pere mā (maui). Ko te AU, te waeine ā-wā. Ko te (E me te F) e tohu ana i te ahua 3D e whakakotahi ana i te tohu taonga me te ERES. I kitea ngā tautau Gas1-GFP tata ki te ERES motuhake. Ko te waeine tauine ko te 0.551μm. (F) E tohu ana te pere mā totoka i te tautau Gas1-GFP e hono ana ki te ERES. E whakaatu ana ngā panera waenga me te taha matau i te ahua 3D kua whakanuia kua hanumi me te tirohanga hurihuri o te tautau Gas1-GFP kua tīpakohia.
Ko te whanaungatanga tata i waenga i te rōpū Gas1-GFP me tētahi ERES motuhake e tohu ana ka taea e Gas1-GFP te tomo ki roto i te ERES whiriwhiri, he rerekē tēnei i te whiriwhiringa e whakamahia ana e Mid2-iRFP hei wehe atu i te ER. Hei whakatika i tēnei tūponotanga, i inehia e mātou te ōwehenga ERES mō te kotahi, e rua rānei ngā taonga (Pikitia 2, A ki C). I kitea e mātou ko te nuinga o ngā ERES (70%) he kotahi noa te momo utanga. Ko te ahua o raro o te Pikitia 2C e whakaatu ana i ngā tauira e rua o ngā ERES me te Gas1-GFP anake (Pikitia 1) me te Mid2-iRFP anake rānei (Pikitia 2). He rerekē, tata ki te 20% o ngā ERES kei roto ngā utanga e rua e taupatupatu ana i te rohe kotahi. I kitea ko ētahi ERES (10%) he rua ngā momo utanga, engari i wehea i roto i ngā rohe rerekē. Nō reira, e whakaatu ana tēnei tātaritanga tatauranga i muri i te kaweake o te ER, ka wehea te GPI-AP Gas1-GFP me te utanga whakawhiti-membrane Mid2-iRFP ki ngā ERES rerekē (Pikitia 2D). He tino ōrite tēnei whai huatanga o te whakarōpūtanga ki te tātari matū koiora o mua (6) me te whakatau āhua (7). Ka taea hoki e tātou te tirotiro i te whanonga o te utanga kua wehea e tomo ana ki te ERES (Pikitia 2E me te Kiriata S2). E whakaatu ana te Pikitia 2E he iti noa te wāhanga o Gas1-GFP (rōpū 3) Mid2-iRFP rānei (rōpū 4) e tomo ana ki te ERES mai i te taha kotahi, ā, e herea ana ki tētahi wāhi motuhake. E whakaatu ana te Rōpū 5 o te Pikitia 2E ka kitea ētahi wā ko Gas1-GFP me Mid2-iRFP i roto i te ERES kotahi, engari ka tomo mai i ngā taha rerekē, ā, ka kukū ki ngā rohe motuhake e tohu ana i ngā vesicle COPII rerekē. I whakaūhia hoki e mātou he motuhake te wehenga me te whakarōpūtanga i kitea o te C26 ceramide-based GPI-AP Gas1 hei ERES whiriwhiri nā te mea he utanga huna transmembrane anō tētahi atu, ko te pūmua membrane plasma kua tohua ki te GFP, Axl2 (27), e whakaatu ana i te whanonga rite ki te Mid2-iRFP. (Pikitia S1 me te Kiriata S3). Ka tohatohahia te Axl2-GFP hou i hangaia mā roto i te kiriuhi ER pērā i te Mid2-iRFP (Pikitia S1, A me te B), ā, ka honoa tahitia ki te Mid2-iRFP i roto i te nuinga o ngā ERES (Pikitia S1, B ki te D). E whakaatu ana ngā Paewhiri 1 me te 2 o te Pikitia 1. E whakaatu ana te Paewhiri S1C i ngā tauira e rua o te ERES e taupatupatu ana ngā utanga whakawhiti-kiriuhi e rua. I ēnei wā, ka uru ngātahi ngā taonga e rua ki te ERES (Pikitia S1E, Paewhiri 3 me te Kiriata S3).
Ko ngā pūtau sec31-1 e whakaatu ana i ngā hunanga ka taea te whakaputa galactose, Gas1-GFP (GPI-AP, kakariki) me Mid2-iRFP (TMP, kikorangi) me te tapanga ERES pumau Sec13-mCherry (ERES, magenta) i whakanohoia ki te 37. I muri i te whakatō mō te 30 meneti i te °C, nekehia ki te 24 °C hei tuku i te poraka huna, ā, whakaahuahia ki te SCLIM i muri i te 20 meneti. (A ki te C) Ngā whakaahua whakaata 2D māngai (A; pae tauine, 1μm) ngā whakaahua taha pūtau 3D rānei (B me C; wae tauine, 0.456μm) o te utanga me ngā wāhanga-z 10 kua tohua e ERES. Ko te panera o raro i (B) me te panera i (C) e whakaatu ana i ngā whakaahua tukatuka hei whakaatu i ngā taonga anake kei roto i te ERES (magenta) [Gas1-GFP (hina) me Mid2-iRFP (kikorangi mārama)]. (C) Pere tuwhera: Kotahi noa te wahi utanga e mau ana a ERES (1 ki te 4). Pere hina: Kei roto i te ERES ngā utanga wehe (5). Pere mā totoka: Kei roto i te ERES ngā utanga kei te taha kotahi. Kei raro: Kei roto i te ERES kotahi kua tīpakohia ko Gas1-GFP anake (1) Mid2-iRFP rānei (2). Pae tauine, 100 nm. (D) Te ine i te whakaahua iti kua whakaahuatia i roto i te (C). Ko te ōrau toharite o te ERES kei roto kotahi anake te utanga (Gas1-GFP Mid2-iRFP rānei), ngā utanga wehe me ngā utanga taupatupatu. I roto i ngā whakamātautau motuhake e toru, n=432 i roto i te 54 pūtau. Pae hapa = SD. Whakamātautau t kore-tauira e rua-waewae. *** P = 0.0002. (E) Whakaahua 3D o te ERES kua tīpakohia o ngā utanga kua wehea kua tohua ki te (C). Ka uru a Gas1-GFP (kakariki) (3) Mid2-iRFP rānei (kikorangi) (4) ki te ERES (magenta) mai i te taha kotahi, ā, ka herea ki tētahi wāhi iti i roto i te ERES. I ētahi wā, ka uru ngā momo utanga e rua ki te ERES kotahi (5) mai i te taha kotahi, ā, ka herea ki tētahi wāhi motuhake i roto i te ERES. Pae tauine, 100 nm.
Muri iho, i whakamātauhia e mātou tētahi whakapae ko te ceramide mekameka acyl roa (C26) kei roto i te membrane ER te mea e akiaki ana i te whakarōpūtanga me te whakarōpūtanga motuhake o Gas1 ki roto i te ERES whiriwhiri. Hei whakatutuki i tēnei, i whakamahia e mātou he momo rewena kua whakarerekētia a GhLag1, i reira i whakakapia ai ngā synthases ceramide taketake e rua, arā, a Lag1 me Lac1, e GhLag1 (te homolog Lag1 o te miro), ka hua ake he momo rewena me te kiriuhi pūtau Ceramide he momo poto ake i te momo mohoao (Pikitia 3A) (28). I whakaatuhia e te tātaritanga papatipu (MS) i roto i ngā momo mohoao, 95% o te ceramide katoa he mekameka ceramide tino roa (C26), i roto i a GhLag1, 85% o te ceramide he tino roa (C18 me C16). ), 2% anake o te ceramide he mekameka ceramide tino roa (C26). Ahakoa ko ngā ceramide C18 me C16 ngā ceramide matua i kitea i roto i te membrane GhLag1 tae noa mai ki tēnei wā, i whakaūhia hoki e te tātaritanga MS kei roto i te punga GPI o Gas1-GFP i whakaatuhia i roto i te momo GhLag1 te ceramide C26, he rite ki ngā lipid momo-mohoao. He rite tonu te kounga (Pikitia 3A) (26). Nō reira, ko te tikanga he tino whiriwhiri te whākōkī whakahou ceramide Cwh43 mō te ceramide C26, e whakaaturia ana i te Pikitia 26, e pai ake ana te whakauru i te punga GPI mai i te iti o te ceramide C26 i roto i te momo GhLag1. S2 (29). Heoi anō, ko te membrane pūtau o GhLag1 kei roto noa i te ceramide C18-C16, ko Gas1-GFP kei roto tonu te ceramide C26. Nā tēnei āhuatanga ka waiho tēnei momo hei taputapu pai hei whakaoti rapanga i te raruraru o te roa o te mekameka acyl o te ceramide membrane i roto i te ER. Ko te tūranga whakapae o te akomanga me te whakarōpūtanga. Kātahi, i ako tuatahi mātou i te kaha o te C26 Gas1-GFP ki te kohikohi i roto i ngā tautau i roto i te GhLag1 me te iraira whakarerekētanga pāmahana-tairongo o sec31-1 mā te karuārai fluorescence tikanga, kei reira anake te mekameka roa (C18-C16) kei roto i te membrane ER Ceramide (Pikitia 3). I kite mātou i roto i te sec31-1, ko te nuinga o te Gas1-GFP i kukū i roto i ngā tautau, ko te Gas1-GFP i roto i te sec31-1 GhLag1 me te membrane ceramide ER roa (C18-C16) kāore i te nuinga o te wā i te kohikohi me te tohatoha i roto i te membrane ER katoa. Hei tika ake, nā te mea he hononga tata te kohikohinga i runga i te ceramide C26 ki te ERES motuhake (Pikitia 1), i tirotirohia e mātou i muri mai mēnā ka uru pea tēnei tukanga ki te mahi a te tikanga pūmua kaweake ER. Ka whakamahia e te GPI-AP he pūnaha COPII motuhake mō te kaweake ER, e whakahaeretia ana e te whakahoutanga hanganga a Ted1 o te wāhanga glycan o te punga GPI (30, 31). Kātahi ka kitea te GPI-glycan recombinant e te matatini p24 o te kaiwhakawhiwhi utanga whakawhiti-membrane, ka tīpakohia e ia a Lst1, he isoform motuhake o te waeine here utanga COPII matua Sec24, ka hanga he COPII whai-AP-whai kiko (31-33). Nō reira, i hangaia e mātou he mutant takirua i whakakotahi i te mukunga o ēnei pūmua takitahi (te waeine matatini p24 Emp24, te whākōkī whakahou GPI-glycan Ted1 me te waeine COPII motuhake Lst1) me te momo mutant sec31-1, ā, i akohia e mātou. Ka taea te hanga i te Gas1-cluster GFP (Pikitia 3). I kite mātou i roto i te sec31-1emp24Δ me te sec31-1ted1Δ, ko te Gas1-GFP te nuinga o te wā kāore i te nohoia, ā, kua tohatohahia puta noa i te membrane ER, e ai ki te kitenga o mua i roto i te sec31-1 GhLag1, i roto i te sec31-1lst1Δ, ko Gas1-GFP Pērā i a sec31-1. E whakaatu ana ēnei hua, haunga te noho mai o te ceramide C26 i roto i te kiriuhi ER, me herea hoki te whakarōpūtanga o Gas1-GFP ki te matatini p24, ā, kāore e hiahiatia he whakaurunga Lst1 motuhake. Kātahi ka tūhuratia e mātou te āheinga ka taea e te roa o te mekameka o te ceramide i roto i te kiriuhi ER te whakahaere i te herenga o Gas1-GFP ki te p24. Heoi, i kitea e mātou kāore te noho mai o te ceramide C18-C16 i roto i te kiriuhi e pā ki ngā GPI-glycans i hangaia anō e te matatini p24 (Pikitia S3 me S4, A me B) te herenga rānei ki te GPI-AP me te kaweake GPI-AP. Te kimi i te momo COPII Lst1 (Pikitia S4C). Nō reira, kāore te whakarōpūtanga e whakawhirinaki ana ki te ceramide C26 e hiahia ana ki ngā taunekeneke pūmua me ngā tikanga kaweake pūmua ER rerekē, engari e tautoko ana i tētahi tikanga whakarōpū rerekē e peia ana e te roa o te ngako. Kātahi ka tātarihia e mātou mēnā he mea nui te roa o te mekameka acyl ceramide i roto i te kiriuhi ER mō te whakarōpūtanga whai hua o Gas1-GFP hei ERES whiriwhiri. I te mea ka wehe atu a Gas1 i roto i te momo GhLag1 me te ceramide mekameka-poto i te ER ka tomo ki te kiriuhi plasma (Pikitia S5), e whakapono ana mātou mēnā ka peia te whakarōpūtanga e te roa o te mekameka ceramide acyl, ka taea te huri me te whakawhiti i te Gas1 i roto i te momo GhLag1. Ka tukuna ngā taonga ERES me te kiriuhi kotahi.
(A) Kei roto i te kiriuhi pūtau o GhLag1 ngā ceramide C18-C16 poto ake, engari kei te punga GPI o Gas1-GFP tonu te C26 IPC rite tonu ki ngā pūtau momo-mohoao. I runga ake nei: te tātaritanga roa o te mekameka acyl o te ceramide i roto i te kiriuhi pūtau o ngā momo-mohoao (Wt) me ngā momo GhLag1p mā te ine papatipu (MS). Ko ngā raraunga e tohu ana i te ōrau o te ceramide katoa. Ko te toharite o ngā whakamātautau motuhake e toru. Pae hapa = SD. Whakamātautau t kore-takirua e rua-waewae. **** P <0.0001. Paewhiri raro: Te tātaritanga MS o te roa o te mekameka acyl o te IPC kei roto i te punga GPI Gas1-GFP (GPI-IPC) i whakaatuhia i roto i ngā momo-mohoao me ngā momo GhLag1p. Ko ngā raraunga e tohu ana i te ōrau o te tohu IPC katoa. Te toharite o ngā whakamātautau motuhake e rima. Pae hapa = SD. Whakamātautau t kore-takirua e rua-waewae. ns, kāore i te nui. P = 0.9134. (B) I whakatōngia ngā pūtau irahiko o sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ me sec31-1lst1Δ e whakaatu ana i te Gas1-GFP i puta mai i te galactose i te 37°C mō te 30 meneti, ā, i tukuna iho ki te Mahi i te irahiko irahiko auau i muri i te 24°C. Pere mā: Taupū ER Gas1-GFP. Pere tuwhera: Kua tohatohahia te Gas1-GFP kāore i te taupū ki runga i te kiriuhi ER katoa, e whakaatu ana i te tae mowhiti karihi āhuatanga ER. Pae tauine, 5μm. (C) Te ine i te irahiko whakaahua kua whakaahuatia i roto i te (B). Te ōrau toharite o ngā pūtau me te hanganga Gas1-GFP punctate. I roto i ngā whakamātautau motuhake e toru, n≥300 ngā pūtau. Pae hapa = SD. Whakamātautau-t kore-tauira e rua-waewae. **** P <0.0001.
Hei whakaoti tika i tēnei raruraru, i whakahaerehia e mātou te whakaaturanga SCLIM o Gas1-GFP me Mid2-iRFP i roto i te GhLag1 me te ira whakarerekētanga pāmahana-tairongo sec31-1 (Pikitia 4 me te Kiriata S4). I muri i te pupuri i te ER i te 37°C, ā, i muri mai ka tukuna i te 24°C, ko te nuinga o te Gas1-GFP hou i hangaia kāore i te whakarōpūhia, i tohatohahia puta noa i te membrane ER, e ai ki te kitenga o ngā karuārai noa (Pikitia 4, A me B). Hei tāpiri, he ōrau nui o ERES (67%) kei roto ko ngā momo utanga e rua kei roto (Pikitia 4D). Ko ngā panera 1 me te 2 o te Pikitia 4C e whakaatu ana i ngā tauira e rua o ERES me te Gas1-GFP me te Mid2-GFP e taupatupatu ana. Hei tāpiri, i whakaurua ngā taonga e rua ki roto i te ERES kotahi (Pikitia 4E, panera 3 me te kiriata S4). Nō reira, e whakaatu ana ā mātou hua ko te roa o te mekameka ceramide acyl i roto i te membrane ER he mea nui hei whakatau i te whakahiato me te whakarōpūtanga o te pūmua ER.
Ngā pūtau Sec31-1 GhLag1 e whakaatu ana i ngā hunanga i puta mai i te galactose, Gas1-GFP (GPI-AP, kakariki) me Mid2-iRFP (TMP, kikorangi) me te ERES-tohu Sec13-mCherry (ERES, magenta) Whakatōngia ki te 37°C. Haere tonu mō te 30 meneti, whakaheke ki te 24°C hei tuku i ngā hunanga, ā, whakaahuahia ki te SCLIM i muri i te 20 meneti. (A ki te C) Ngā whakaahua whakaata 2D māngai (A; pae tauine, 1μm) ngā whakaahua taha pūtau 3D rānei (B me C; wae tauine, 0.45μm) o ngā wāhanga-z 10 kua tohua e te utanga me te ERES. Ko te panera o raro i (B) me te panera i (C) e whakaatu ana i ngā whakaahua tukatuka hei whakaatu i ngā taonga anake kei roto i te ERES (magenta) [Gas1-GFP (hina) me Mid2-iRFP (kikorangi mārama)]. (C) Pere mā kua whakakiia: ERES, ngā taonga e taupatupatu ana. Pere tuwhera: Kotahi anake te tūemi kei roto i te ERES. Paewhiri o raro: Kei te ERES kua tīpakohia ngā taonga taupatupatu (1 me te 2) kua tohua ki (C). Pae tauine, 100 nm. (D) Te ine i te whakaahua iti kua whakaahuatia ki (C). I roto i ngā waeine sec31-1 me te sec31-1 GhLag1, kotahi anake te utanga (Gas1-GFP, Mid2-iRFP rānei) kua whakaurua, me te ōrau toharite o te ERES mō ngā utanga takitahi me ngā utanga taupatupatu. I roto i ngā whakamātautau motuhake e toru, n = 432 i roto i te 54 pūtau (sec31-1) me te n = 430 i roto i te 47 pūtau (sec31-1 GhLag1). Pae hapa = SD. Whakamātautau t kore-taurite e rua-waewae. *** P = 0.0002 (sec31-1) me te ** P = 0.0031 (sec31-1 GhLag1). (E) Whakaahua 3D o te ERES kua tīpakohia me ngā utanga taupatupatu (3) kua tohua ki (C). Ka whakatata atu a Gas1-GFP (kakariki) me Mid2-iRFP (kikorangi) ki a ERES (magenta) mai i te taha kotahi, ā, ka noho tonu ki te rohe ERES kua aukatia. Pae tauine, 100 nm.
E whakarato ana tēnei rangahau i ngā taunakitanga tika i roto i te tinana ora e whakarōpūtia ana ngā utanga pūmua e ahu mai ana i te ngako ki ngā wāhi kaweake whiriwhiri i roto i te ara huna, ā, e whakaatu ana i te hiranga o te roa o te mekameka acyl mō te whiriwhiringa whakarōpū. Mā te whakamahi i tētahi tikanga karuārai kaha me te matatau e kiia nei ko te SCLIM, i whakaaturia e mātou te Gas1-GFP hou i hangaia (he kiriuhi plasma nui GPI-AP me te wāhanga ngako ceramide mekameka acyl roa rawa (C26)) i roto i te rewena ) Ko ngā rohe e huihui ana i roto i ngā ER motuhake e hono ana ki ngā ERES motuhake, i te mea ka tohatohahia ngā pūmua huna transmembrane puta noa i te kiriuhi ER (Pikitia 1). Hei tāpiri, ka uru ēnei momo taonga e rua ki ngā ERES rerekē me te whiriwhiri (Pikitia 2). Ka whakaitihia te roa o te mekameka acyl o te ceramide pūtau i roto i te kiriuhi mai i te C26 ki te C18-C16, ka pakaru te tautau Gas1-GFP ki roto i te rohe ER motuhake, ā, ka hurihia te Gas1-GFP ki te wehe atu i te ER me te pūmua transmembrane mā te ERES kotahi (Pikitia 3 me te Pikitia 3). 4).
Ahakoa e whakamahi ana a GPI-AP i tētahi tikanga pūmua motuhake hei puta atu i te ER, i kitea e mātou kāore te wehenga e whakawhirinaki ana ki te ceramide C26 e whakawhirinaki ana ki ngā taunekeneke pūmua rerekē ka arahi pea ki te motuhaketanga ERES (Ngā Pikitia S4 me te S5). Engari, e tautoko ana ā mātou kitenga i tētahi tikanga whakarōpū rerekē e whakahaerehia ana e te whakarōpūtanga pūmua e hangai ana ki te lipid me te tango i ētahi atu utanga i muri mai. E whakaatu ana ā mātou kitenga kāore i te rohe, i te rōpū rānei o Gas1-GFP e hono ana ki tētahi ERES motuhake te pūmua huna transmembrane Mid2-iRFP, e tohu ana ko te rōpū GPI-AP e whakawhirinaki ana ki te ceramide C26 ka āwhina i tā rātou urunga ki roto i te ERES e tika ana, ā, i te wā kotahi, ka tango i ngā huna transmembrane. Ka tomo ngā huna ki roto i tēnei ERES motuhake (Ngā Pikitia 1 me te 2). He rerekē, ko te noho o ngā ceramide C18-C16 i roto i te membrane ER kāore e meinga te GPI-AP kia hanga rohe, rōpū rānei, nō reira kāore rātou e tango, e whakakapi rānei i ngā pūmua huna transmembrane ki roto i te ERES kotahi (Ngā Pikitia 3 me te 4). Nō reira, e whakaaro ana mātou mā te ceramide C26 e ārahi te wehenga me te whakarōpūtanga mā te whakahaere i te whakarōpūtanga o ngā pūmua e hono ana ki ngā ERES motuhake.
Me pēhea te whakatutuki i tēnei whakarōpūtanga e whakawhirinaki ana ki te ceramide C26 ki roto i tētahi wāhi ER motuhake? Ko te āhua o te membrane ceramide ki te wehe taha ka meinga pea a GPI-AP me C26 ceramide ki te hanga i ngā lipid iti me te raupapa tere i roto i te taiao lipid koretake o te membrane ER kei roto ko ngā glycerolipids poto ake me te kore-mākū. Ngā tautau kounga (17, 18). Ka taea te whakakotahi i ēnei tautau rangitahi iti ki roto i ngā tautau nui ake, pumau ake i muri i te herea ki te matatini p24 (34). E rite ana ki tēnei, i whakaaturia e mātou me taunekeneke a C26 Gas1-GFP ki te matatini p24 kia hangaia ai ngā tautau nui ake e kitea ana (Pikitia 3). Ko te matatini p24 he oligomer heterozygous i titoa mai i ngā pūmua transmembrane p24 rerekē e whā i roto i te rewena (35), e whakarato ana i te herenga maha-valent, ka taea ai te hono-whiti o ngā tautau GPI-AP iti, ā, ka puta he tautau pumau nui ake (34). Tērā pea ka whai wāhi atu te taunekeneke i waenga i ngā ectodomains pūmua o ngā GPI-AP ki tō rātou whakahiatotanga, e whakaaturia ana i te wā o tā rātou kawe Golgi i roto i ngā pūtau epithelial polarized mammalian (36). Heoi, ina kei roto i te membrane ER te ceramide C18-C16, ina herea te matatini p24 ki te Gas1-GFP, kāore e hangaia he tautau nui motuhake. Ko te tikanga matua ka whakawhirinaki pea ki ngā āhuatanga ā-tinana me te matū motuhake o te ceramide mekameka acyl roa. E whakaatu ana ngā rangahau koiora ā-tinana o ngā membrane horihori ahakoa ka taea e ngā ceramide mekameka acyl roa (C24) me te poto (C18-C16) te wehewehe i te wāhanga, ko ngā ceramide mekameka acyl roa anake (C24) ka taea te whakatairanga i te Curvature teitei me te piko kiriata hei hanga anō i te kiriata. Mā te tohutoro tahi (17, 37, 38). Kua whakaaturia ko te helix transmembrane o TMED2, te homologue tangata o Emp24, ka taunekeneke whiriwhiri ki te sphingomyelin C18 ceramide-based i roto i ngā lobules cytoplasmic (39). Mā te whakamahi i ngā whakatauira irahiko ngota (MD), i kitea e mātou ka kohikohi ngā ceramide C18 me C26 huri noa i ngā lobules cytoplasmic o te Emp24 transmembrane helix, ā, he rite tonu ō rātou hiahia (Pikitia S6). He mea tika kia mōhio ko tēnei e tohu ana ka taea e te transmembrane helix o Emp24 te arahi ki te tohatoha koretake o ngā lipids i roto i te membrane. He hua hou tēnei i runga i ngā pūtau mammal. E whakaatu ana hoki ngā whakatauira MD rite i te aroaro o ngā lipids ether (40). Nō reira, e whakapae ana mātou ka whai rawa te C26 ceramide i roto i ngā lobules e rua o ER26. Ina herea tika te GPI-AP i roto i ngā lobules luminal ki te p24 maha-valent me te kohikohinga o te C26 ceramide huri noa i te p24 i roto i ngā lobules cytoplasmic, ka taea e ia te whakatairanga i te whakakotahitanga pūmua me te piko o te membrane e puta mai ana i roto i ngā maihao (41), ka wehea te GPI-AP ki ngā rohe motuhake e tata ana ki te ERES, e pai ake ana hoki ki ngā rohe tino piko o te membrane ER (42). I tautokohia e ngā pūrongo o mua te tikanga kua whakaarohia (43, 44). Ko te herenga maha-valent o ngā oligolectins, ngā pathogens, ngā antibodies rānei ki ngā glycosphingolipids (GSL) e hangai ana ki te ceramide i runga i te membrane plasma ka whakaoho i te nui o te whakakotahitanga GSL, ka whakarei ake i te wehenga wāhanga, ā, ka puta te whakarerekētanga me te whakaurunga o te membrane (44). Iwabuchi me ētahi atu (43) I kitea i te aroaro o ngā mekameka acyl roa (C24) engari kāore i te poto (C16), ko te ligand maha-valent e herea ana ki te GSL lactosylceramide i whakaoho i te hanganga o ngā tautau nui me te invagination membrane, ā, ko te cytoplasm Lyn-mediated signal transduction i runga i ngā pepa ka interdigitated e ngā mekameka acyl i roto i ngā neutrophils kua honoa.
I roto i ngā pūtau epithelial polarized o ngā mammal, ko te kukū o te whatunga anti-Golgi (TGN) ki te taumata o te membrane plasma apical e whakahaere ana i te wehenga me te whakarōpūtanga o te GPI-AP (10, 45). Ko tēnei whakakotahitanga e akiakihia ana e te oligomerization GPI-AP (36), engari tera pea ka whakawhirinaki ki te roa o te mekameka ceramide e kitea ana e tātou i roto i te rewena. Ahakoa he punga lipid-ether a te mammal GPI-AP, ā, he tino rerekē tōna hanganga matū mai i te ceramide mekameka acyl roa rawa, i kitea e tētahi rangahau tata nei he rite tonu ngā āhuatanga ā-tinana me te matū o ngā lipid e rua me te mahi (40). Nō reira, ko te wāhanga lipid ether i roto i ngā pūtau mammal he rite pea ki te ceramide C26 i roto i te rewena, ā, ko tana mahi he hono atu ki te ceramide mekameka-roa i roto i te membrane hei whakatairanga i te whakakotahitanga me te whakarōpūtanga o te GPI-AP. Ahakoa me whakamātautau tonu tēnei pea, e tautoko ana ngā kitenga o mua ko te kawe o te ceramide mekameka acyl roa ki te tinana Golgi kāore e mahia e ngā pūmua whakawhiti cytoplasmic, engari e whakawhirinaki ana ki te hanganga o ngā punga GPI pērā i te rewena. Nō reira, te āhua nei ka taea e te tikanga tiaki whanaketanga te kawe tahi i te ceramide mekameka acyl roa rawa me te GPI-AP (13, 16, 20, 46, 47) i roto i te pūwero kawe kotahi.
I roto i ngā pūnaha pūtau epithelial polarized rewena me ngā mammalian, ka puta te whakakotahitanga me te wehenga o GPI-AP mai i ētahi atu pūmua membrane plasma i mua i te taenga ki te mata o te pūtau. I kitea e Paladino et al. (48) i runga i te TGN o ngā pūtau epithelial polarized mammalian, ehara i te mea he mea tika te whakarōpūtanga GPI-AP mō te whakarōpūtanga whiriwhiri o GPI-AP ki te membrane plasma apical, engari e whakahaere ana hoki i te whakaritenga whakarōpū o GPI-AP me tana mahi koiora. Mata pūtau. I roto i te rewena, i whakaatuhia e tēnei rangahau ka taea e te rōpū GPI-AP e whakawhirinaki ana ki te ceramide C26 i runga i te ER te whakahaere i te whakaritenga rōpū me te mahi mahi a GPI-AP i runga i te membrane plasma (24, 49). E rite ana ki tēnei tauira, he mate pāwera ngā pūtau GhLag1 ki ngā aukati GPI, ki ngā rongoā rānei e pā ana ki te pono o te pakitara pūtau (28), ā, ko te hiahia mō ngā rōpū Gas1-GFP mahi (49) o te ceramide pito i tukuna i roto i te hononga o ngā pūtau rewena e tohu ana i ngā hua ā-tinana pea o ngā pūtau hLag1. Hapa GPI-AP. Heoi, ko te kaupapa o ā mātou rangahau ā muri ake nei ko te whakamātautau anō mēnā kua whakaritea te whakaritenga mahi o te mata o te pūtau mai i te ER mā te tikanga whakarōpū i runga i te roa o te ngako.
Kua whakarārangihia ngā momo Saccharomyces cerevisiae i whakamahia i roto i tēnei mahi ki te Ripanga S1. Ko ngā momo MMY1583 me MMY1635 o SCLIM mō te whakaahua pūtau ora i hangaia i te papamuri o W303. Ko ēnei momo e whakaatu ana i te Sec13-mCherry me te tohu pūmua fluorescent i hangaia mā te whakamahi i te tikanga polymerase chain reaction (PCR) me te plasmid pFA6a hei tauira (23). Ko te momo e whakaatu ana i te Mid2-iRFP i tohua ki te pūmua fluorescent i raro i te mana o te kaiwhakatairanga GAL1 i hangaia penei. Ko te whakanui PCR o te raupapa iRFP-KanMx mai i te vector pKTiRFP-KAN (koha a E. O'Shea, Addgene plasmid nama 64687; http://n2t.net/addgene: 64687; tohu rauemi rangahau (RRID): Addgene_64687) Ā, i whakaurua ki roto i te C-terminus o te Mid2 taketake. I muri i te whakanui ake i te raupapatanga ira Mid2-iRFP me te tārua ki te kaiwhakatairanga GAL1, ka whakauruhia ki te pae Not I-Sac I o te plasmid whakauru pRS306. Ko te plasmid hua pRGS7 i whakarārangihia ki te Pst I hei whakauru ki te wāhi URA3.
Ko te ira whakakotahitanga Gas1-GFP ka whakaatuhia i raro i te mana o te kaiwhakatairanga GAL1 i roto i te plasmid centromere (CEN), e hangaia ana penei. I whakanuia te raupapatanga Gas1-GFP e te PCR mai i te plasmid pRS416-GAS1-GFP (24) (koha a L. Popolo) ā, i tāruatia ki roto i te pae Xma I–Xho I o te plasmid CEN pBEVY-GL LEU2 (koha a C). Miller; Tau plasmid Addgene 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Ko te plasmid hua i tapaina ko pRGS6. Ko te ira whakakotahitanga Axl2-GFP ka whakaatuhia hoki i raro i te mana o te kaiwhakatairanga GAL1 o te weka pBEVY-GL LEU2, ā, ko tōna hanganga penei. I whakanuia te raupapatanga Axl2-GFP mai i te plasmid pRS304-p2HSE-Axl2-GFP (23) mā te PCR, ā, i tāruatia ki te pae Bam HI-Pst I o te vector pBEVY-GL LEU2. Ko te plasmid i puta ko te ingoa pRGS12. Kei te Ripanga S2 te raupapatanga o ngā oligonucleotides i whakamahia i roto i tēnei rangahau.
I tāpirihia te momo ki te 0.2% adenine me te 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1% Yeast extract and 2% protein ept). (YPR)] 2% galactose [YP-galactose (YPG)] rānei hei pūtake waro, i roto rānei i te reo iti hangai (0.15% yeast nitrogen base and 0.5% ammonium sulfate) hei tāpiri i ngā waikawa amino me ngā turanga e tika ana mō te kai, me te whai 2% glucose (synthetic glucose medium) 2% galactose rānei (synthetic galactose minimal medium) hei pūtake waro.
Mō te whakaahua wā-tūturu, i whakatipuria ngā pūtau whakarerekētanga sec31-1 tairongo-mahana e whakaatu ana i te hanganga i raro i te kaiwhakatairanga GAL1 i roto i te reo YPR i te 24°C i te pō tae noa ki te wāhanga waenga-log. Whai muri i te whakaurunga ki te YPG i te 24°C mō te 1 hāora, i whaowhia ngā pūtau ki te SG i te 37°C mō te 30 meneti, kātahi ka whakawhitia ki te 24°C kia tukuna mai i te aukati huna. I whakamahia te Concanavalin A hei whakapiri i ngā pūtau ki runga i te kiriata karāhe, ā, i whakaahuahia e SCLIM. He huinga te SCLIM o te karuārai whakahurihuri Olympus IX-71 me te arotahi hinu āputa tau UPlanSApo 100×1.4 (Olympus), te matawai kōpae hurihuri ōwehenga tere-teitei me te ōwehenga tohu-ki-haruru teitei (Yokogawa Electric), te ine āwhata ritenga, me te whakamatao ritenga. Ka taea e te whakanui ahua o te pūnaha (Hamamatsu Photonics) te whakarato i tētahi pūnaha arotahi whakanui me te whakanui whakamutunga o ×266.7 me tētahi kāmera taputapu hono-utu e whakarea ana i ngā irahiko (Hamamatsu Photonics) (21). Ka mahia te hopu ahua mā te pūmanawa ritenga (Yokogawa Electric). Mō ngā whakaahua 3D, i whakamahia e mātou he kaiwhakaoho piezoelectric ritenga hei wiri i te arotahi whāinga poutū, ā, ka kohia ngā wāhanga whatu 100 nm te wehe i roto i tētahi puranga. Ka hurihia te ahua Z-puranga ki ngā raraunga voxel 3D, ā, ko te mahi horapa pūwāhi ariā e whakamahia ana mō te karuārai kōpae hurihuri e whakamahia ana mō te tukatuka wetewete e te pūmanawa Volocity (PerkinElmer). Mā te whakamahi i te pūmanawa Volocity hei whakawhāiti aunoa i te tātaritanga tauwāhi-tahi, i inehia te ERES tae atu ki te utanga. I whakahaerehia te tātaritanga matawai rārangi mā te whakamahi i te pūmanawa MetaMorph (Molecular Devices).
Whakamahia te pūmanawa GraphPad Prism hei whakatau i te hiranga tatauranga. Mō te whakamātautau-t a te Tauira e rua-waewae me te whakamātautau tātaritanga rerekētanga kotahi-ara noa (ANOVA), e kiia ana he pānga nui ngā rerekētanga i waenga i ngā rōpū ki te P <0.05 (*).
Mō te karuārai fluorescence o Gas1-GFP, i whakatipuria ngā pūtau wāhanga log i roto i te YPD i te pō, ā, i kohia mā te centrifugation, i horoia kia rua ngā wā ki te tote phosphate buffered, ā, i whaowhia ki runga i te hukapapa mō te 15 meneti pea, kātahi ka haere tonu i raro i te karuārai e ai ki te whakaahuatanga o mua Tirohia (24). I whakamahia te karuārai Leica DMi8 (HCX PL APO 1003/1.40 hinu PH3 CS) me te arotahi whāinga, te tātari L5 (GFP), te kāmera Hamamatsu me te pūmanawa Application Suite X (LAS X) mō te hopu.
I whakakorengia ngā tauira ki te SDS sample buffer i te 65°C mō te 10 meneti, kātahi ka wehea mā te SDS-polyacrylamide gel electrophoresis (PAGE). Mō te tātari immunoblotting, i utaina te 10 μl o te tauira mō ia ara. Paturopi tuatahi: Whakamahia te rāpeti polyclonal anti-Gas1 i te waimehatanga o te 1:3000, te rāpeti polyclonal anti-Emp24 i te waimehatanga o te 1:500, me te rāpeti polyclonal anti-GFP (he koha nā H. Riezman) i te waimehatanga o te 1:3000. I whakamahia te paturopi kiore monoclonal anti-Pgk1 i te waimehatanga o te 1:5000 (he koha nā J. de la Cruz). Paturopi tuarua: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) i whakamahia i te waimehatanga o te 1:3000 (Pierce). I whakamahia te HRP-conjugated koati anti-kiore IgG i te waimehatanga o te 1:3000 (Pierce). I tirohia te rohe urupare ārai mate mā te tikanga chemiluminescence o te SuperSignal West Pico reagent (Thermo Fisher Scientific).
E ai ki te whakaahuatanga i roto i te (31), i whakahaerehia he whakamātautau ā-pūmua ā-taiao i runga i te wāhanga ER kua whakareihia. I roto i te poto, horoia ngā pūtau rewena ki te pūmau TNE [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride me te ranunga aukati protease) i te 600 nm (OD600) i te 100 kiato whatu kia rua ngā wā. I pakaruhia ki ngā pirepire karāhe, kātahi ka tangohia ngā para pūtau me ngā pirepire karāhe mā te centrifugation. Kātahi ka centrifugatehia te supernatant i te 17,000 karamu mō te 15 meneti i te 4°C. I whakatārewatia anō te pirepire ki roto i te TNE, ā, i tāpirihia te saponin digitalis ki te kukū whakamutunga o te 1%. I whaowhia te whakatārewatanga mō te 1 hāora me te hurihuri i te 4°C, kātahi ka tangohia ngā wāhanga kore-rewa mā te centrifugation i te 13,000 karamu i te 4°C mō te 60 meneti. Mō te whakapūmau ārai mate o Gas1-GFP, tuatahi, whakatōkia te tauira ki ngā pītiti agarose kau (ChromoTek) i te 4°C mō te 1 hāora, kātahi ka whakatōkia ki te GFP-Trap_A (ChromoTek) i te 4°C mō te 3 hāora. E rima ngā wā i horoia ai ngā pītiti immunoprecipitated ki te TNE kei roto ko te 0.2% digoxigenin, i tangohia ki te SDS sample buffer, i wehea ki te SDS-PAGE, ā, i tātarihia mā te immunoblotting.
E ai ki te whakaahuatanga i roto i (31), i mahia te whakatau hononga-whakawhiti ki runga i te wāhanga ER whai rawa. I roto i te poto, i whakatōkia te wāhanga ER whai rawa ki te 0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). I tineia te tauhohenga hononga-whakawhiti mā te tāpiri i te glycine (50 mM te kukū whakamutunga, 5 meneti, 20°C).
E ai ki te whakaahuatanga o mua (50), i whakahaerehia te tātaritanga MS o te ceramide i roto i ngā momo mohoao me ngā momo GhLag1. I roto i te poto, i whakatipuhia ngā pūtau ki te wāhanga taupū (3 ki te 4 OD600 wae/ml) i roto i te YPD i te 30°C, ā, i hauhakehia he 25×107 pūtau. Ka tineia ō rātou pākia ki te waikawa trichloroacetic. Whakamahia te whakarewa tango [ethanol, wai, ether, pyridine me te 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] me te 1.2 nmol o te paerewa ā-roto C17 ceramide (860517, Avanti polar lipid) kounga). Whakamahia te monomethylamine reagent [methanol, wai, n-butanol me te otinga methylamine (4:3:1:5 v/v)] hei mahi i te hydrolysis alkaline ngawari o te tango, kātahi ka whakamahia te n-butanol kua whakakiia ki te wai hei tango i te tote. Hei whakamutunga, i whakatārewatia anō te tangohanga ki roto i tētahi whakarewa aratau pai [chloroform/methanol/wai (2:7:1) + 5 mM ammonium acetate] ā, i werohia ki roto i te ine papatipu. I whakahaerehia te aroturuki tauhohenga maha (MRM) hei tautuhi me te ine i ngā ngota sphingolipid. Ko te ine papatipu quadrupole tuatoru TSQ Vantage (Thermo Fisher Scientific) he mea whakarite ki tētahi pūtake katote nanoflow robotic Nanomate HD (Advion Biosciences, Ithaca, NY) mō te tātari ngako. Ka arotauhia te kaha tukinga mō ia kāwai ceramide. I whiwhihia ngā raraunga MS i roto i te aratau pai. Mō ia tāruarua koiora, ko te tohu ngako te waenga o ngā inenga motuhake e toru.
E ai ki te whakaahuatanga i roto i te (31), ko ngā pūtau (800×107) e whakaatu ana i te Gas1-GFP i tukuna ki te immunoprecipitation tūturu. I wehea te Gas1-GFP kua purea e te SDS-PAGE, ā, i whakawhitia ki te kiriuhi polyvinylidene fluoride (PVDF). I tirohia te pūmua mā te tae i te PVDF ki te pango amide. I tapahia te roopu Gas1-GFP mai i te PVDF, ā, i horoia kia 5 ngā wā ki te methanol, ā, kotahi ki te wai chromatography-MS wai (LC-MS). Mā te whakatō i te rīpene kiriuhi ki te 500μl 0.3 M NaOAc (pH 4.0), te buffer me te 500μl ranunga konutai nitrite 1 M hou i te 37°C mō ngā hāora e 3, ka tukuna te wāhanga lipid mai i te Gas1-GFP, ā, ka wehea. Tukunga o te inosine phosphate ceramide i waenga i te glucosamine me te inositol (51). I muri i tēnā, e whā ngā wā i horoia ai te rīpene kiriuhi ki te wai LC-MS, i whakamaroketia i te pāmahana rūma, ā, i rongoatia ki te hauota i te -80°C tae noa ki te tātaritanga. Hei mana whakahaere, i whakamahia he tauira kau o te kiriuhi PVDF mō ia whakamātautau. Kātahi ka tātarihia te ngako i tangohia mai i te Gas1-GFP e te MS e ai ki te whakaahuatanga (50). I roto i te poto, i whakatārewatia anō ngā rīpene PVDF kei roto ko te GPI-lipid ki roto i te 75μl whakarewa pokepoke kino [chloroform/methanol (1:2) + 5 mM ammonium acetate] ā, i paahitia te tātaritanga electrospray ionization (ESI)-MRM/MS o ngā momo sphingolipid (TSQ Vantage). I tēnei wā, i whiwhihia ngā raraunga MS i roto i te aratau katote kino.
E ai ki te kōrero i mua ake nei, i wehea te wāhanga ngako o te punga GPI mai i te GPI-AP kua tohua ki te [3H]-inositol (16). I wehea ngā ngako mā te chromatography paparanga-angiangi mā te whakamahi i te pūnaha whakarewa (55:45:10 chloroform-methanol-0.25% KCl) ā, i whakaaturia mā te whakamahi i te FLA-7000 (Fujifilm).
Ko ngā pūtau e whakaatu ana i te Gas1-GFP (600×107) i horoia kia rua ngā wā ki te pūpare TNE me te pūpare TNE, ā, i pakaruhia ki ngā pire karāhe, kātahi ka whakapūtāhia kia tangohia ngā para pūtau me ngā pire karāhe. Kātahi ka whakapūtāhia te wai i te 17,000 karamu mō te 1 hāora i te 4°C. I horoia te pire ki te TNE, ā, i whakatōkia ki te 1 U PI-PLC (Invitrogen) ki roto i te TNE kei roto ko te 0.2% digitalis saponin mō te 1 hāora i te 37°C. I muri i te maimoatanga whākōkī, i tangohia te kiriuhi mā te whakapūtāhia i te 17,000 karamu i te 4°C mō te 1 hāora. Hei whakapūtā i te Gas1-GFP, i whakapūtāhia te wai i te GFP-Trap_A (ChromoTek) i te 4°C i te pō. Ko te Gas1-GFP kua purea i wehea e te SDS-PAGE i pania ki te kikorangi kanapa Coomassie. I tapahia te whitiki tae Gas1-GFP mai i te hina e karapoti ana i te awaawa, ā, i muri i te alkylation ki te iodoacetamide me te whakaiti ki te dithiothreitol, ka mahia te nakunaku in-gel ki te trypsin. Tangohia me te whakamaroke i ngā peptide tryptic me ngā peptide ki te GPI-glycans. I whakarewaina te peptide maroke ki te 20 μl o te wai. Werohia he wāhanga (8μl) ki roto i te LC. I whakamahia he pou octadecylsilane (ODS) (Develosil 300ODS-HG-5; diameter o roto 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) hei wehewehe i ngā peptide i raro i ngā āhuatanga gradient motuhake. Ko te wāhanga neke ko te whakarewa A (0.08% waikawa formic) me te whakarewa B (0.15% waikawa formic i roto i te 80% acetonitrile). I whakamahia he pūnaha Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) hei whakakore i te pou me te whakarewa A i roto i te 55 meneti i te tere rere o te 50 μl min-1 mō te 5 meneti, kātahi ka whakanuia te kukū o te whakarewa B ki te 40%. (Te Hononga o Amerika). I whakaurua tonutia te eluate ki roto i te pūtake katote ESI, ā, i tātarihia ngā peptide tryptic me ngā peptide me ngā GPI-glycans e te LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). I roto i te whakatakotoranga MS, i whakatakotoria te ngaohiko o te pūtake capillary ki te 4.5 kV, ā, i puritia te pāmahana o te capillary whakawhiti ki te 300°C. I whakatakotoria te ngaohiko capillary me te ngaohiko arotahi ngongo ki te 15 V me te 50 V, ia. I whiwhihia ngā raraunga MS i te aratau katote pai (taumira o te 60,000; tika o te papatipu o te 10 wāhanga ia miriona) i roto i te awhe papatipu o te 300/m/z ōwehenga papatipu/utu (m/z) 3000. Ka whiwhihia ngā raraunga MS/MS mā te mahanga katote i roto i te LTQ Orbitrap XL [ngā mati tuatahi e 3 e whakawhirinaki ana ngā raraunga ki runga, te wehenga i puta mai i te tukinga (CID)].
I whakahaerehia ngā whakatauira MD mā te whakamahi i te pūmanawa GROMACS (52) me te mara kaha MARTINI 2 (53-55). I whakamahia te CHARMM GUI Membrane Builder (56, 57) hei hanga i tētahi paparanga rua kei roto ko te dioleoylphosphatidylcholine (DOPC) me te Cer C18, DOPC rānei me te Cer C26. Ko te āhua me ngā taunga o Cer C26 i ahu mai i te DXCE mā te tango i ngā pīni tāpiri mai i te hiku sphingosine. Whakamahia te tukanga kua whakaahuatia i raro nei hei taurite i te paparanga rua, ā, whakahaerea, kātahi ka whakamahia ngā taunga whakamutunga o te pūnaha hei hanga i tētahi pūnaha kei roto ko te Emp24. I hangaia te rohe transmembrane o te rewena Emp24 (ngā toenga 173 ki te 193) hei α-helix mā te whakamahi i te hanganga ngota ngota MD (VMD) tirohanga (58). Kātahi, i muri i te tango i ngā lipid e taupatupatu ana, ka whakapūkarahia te pūmua, ā, ka whakaurua ki roto i te paparanga rua mā te whakamahi i te CHARMM GUI. Kei roto i te pūnaha whakamutunga he 1202 DOPC me te 302 Cer C26, he 1197 DOPC rānei me te 295 Cer C18 me te Emp24. Whakakotahitia te pūnaha ki te kukū o te 0.150M. E whā ngā tāruarua motuhake i hangaia mō ngā hanganga paparanga rua e rua.
Ka whakatauritea te paparanga ngako-rua mā te whakamahi i te tukanga CHARMM GUI, e uru ana ki te whakaiti me te whakataurite i ngā taahiraa 405,000, ka whakaitihia haeretia ngā here tūranga me te whakakore, ā, ka whakanuia te taahiraa wā mai i te 0.005 ps ki te 0.02 ps. I muri i te whakataurite, ka puta he 6 µs me te taahiraa wā o te 0.02 ps. I muri i te whakauru i te Emp24, whakamahia te tukanga CHARMM GUI kotahi hei whakaiti me te whakataurite i te pūnaha, kātahi ka whakahaere mō te 8 hēkona i roto i te whakaputanga.
Mō ngā pūnaha katoa, i te wā o te tukanga taurite, ka whakahaerehia te pēhanga e te barostat Berendsen (59), ā, i te wā o te tukanga whakaputa, ka whakahaerehia te pēhanga e te barostat Parrinello-Rahman (60). I ngā wā katoa, ko te pēhanga toharite he 1 pae, ā, ka whakamahia he kaupapa hononga pēhanga haurua-isotropic. I roto i te tukanga taurite me te tukanga whakaputa, ka whakamahia he thermostat (61) me te whakatikatika tere hei hono i te pāmahana o te pūmua, te ngako me ngā matūriki whakarewa. I te wā katoa o te mahi, ko te pāmahana ūnga he 310K. Ka tatauhia te taunekeneke kore-hononga mā te whakaputa i tētahi rārangi takirua mā te whakamahi i te kaupapa Verlet me te manawanui parepare 0.005. Ka tatauhia te kupu Coulomb mā te whakamahi i te mara tauhohenga me te tawhiti tapahi o te 1.1 nm. Ka whakamahia e te kupu Vander Waals he kaupapa tapahi me te tawhiti tapahi o te 1.1 nm, ā, ka whakamahia te kaupapa tapahi Verlet mō te nekeneke pea (62).
Mā te whakamahi i te VMD, ko te roangaru tapahi i waenga i ngā pīrere phosphate DOPC, ngā pīrere ceramide AM1 rānei me te pūmua he 0.7 nm, ā, ka tatauhia te maha o ngā ngako e taunekeneke ana ki te pūmua. E ai ki te tātai e whai ake nei, tatauhia te tauwehenga whakawhāiti-whakarei (DE) e ai ki te (63): tauwehenga DE = (te nui o ngā ngako katoa i roto i te pūmua 0.7) i roto i te pūmua 0.7 (te nui o Cer i roto i ngā ngako katoa)
Ka riro mai te uara kua pūrongohia hei toharite, ā, ko ngā pae hapa he tārua motuhake e whā o SE. Ka tatauhia te hiranga tatauranga o te tauwehe DE mā te whakamātautau-t [(tauwehe-DE-1)/SE]. Tātaihia te uara P mai i te tohatoha kotahi-hiku.
I whakamahia te taputapu GROMACS hei tatau i te mahere mātotoru taha 2D o te pūnaha kei roto ko Emp24 i roto i ngā 250 ns whakamutunga o te ara. Hei whiwhi i te mahere whakarei/whakaiti o te ceramide, ka wehea te mahere mātotoru o Cer e te tapeke o te mahere o Cer me DOPC, kātahi ka wehea e te kukū o Cer i roto i te tinana. Ka whakamahia te tauine mahere tae kotahi.
Mō ngā rauemi tāpiri mō tēnei tuhinga, tirohia http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
He tuhinga urunga-tuwhera tēnei e tohatohahia ana i raro i ngā tikanga o te Raihana Creative Commons Attribution-Non-Commercial, e āhei ai te whakamahinga, te tohatoha me te tārua i roto i tētahi momo pāpāho, mena kāore te whakamahinga whakamutunga mō te whai hua arumoni, ā, ko te whakaaro matua he tika te mahi taketake. Tohutoro.
Kia mōhio: Ko tā mātou anake he tuku mai i tō wāhitau īmēra kia mōhio ai te tangata e taunaki ana koe ki te whārangi e hiahia ana koe kia kite rātou i te īmēra, ā, ehara i te pāme. Kāore mātou e hopu i tētahi wāhitau īmēra.
Ka whakamahia tēnei pātai hei whakamātautau mēnā he manuhiri koe, hei ārai hoki i te tuku pāme aunoa.
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 Wagez -Linero (Sergio Lopez), Misaho Lopez (Sergio Lopez) Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
E whakaatu ana te whakaahua wā-tūturu taumira-teitei 3D i te hiranga o te roa o te mekameka ceramide mō te whakarōpūtanga pūmua i ngā pae putanga whiriwhiri.
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 Wagez -Linero (Sergio Lopez), Misaho Lopez (Sergio Lopez) Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
E whakaatu ana te whakaahua wā-tūturu taumira-teitei 3D i te hiranga o te roa o te mekameka ceramide mō te whakarōpūtanga pūmua i ngā pae putanga whiriwhiri.
©2020 American Association for the Advancement of Science. mana katoa. Ko AAAS te hoa o HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef me COUNTER. ScienceAdvances ISSN 2375-2548.