Gutoranya poroteyine mu nzira y’ugusohora ni ngombwa kugira ngo utugingo ngengabuzima tugume dufise utugingo ngengabuzima kandi tugume dukora neza. Uretse ugutoranya bifashijwe n’ibikoko, uruhara rw’amavuta mu gutoranya kinesine mu nzira yo gutwara amabanga ni ikibazo c’ishimikiro kimaze igihe kirekire kitararonka inyishu. Aha, dukora amashusho y’igihe nyaco y’amabara menshi y’igihe kimwe kugira ngo twemeze mu buzima ko poroteyine ziherutse gukora glycosylphosphatidylinositol-immobilized zifise ibice vy’amavuta maremare cane vy’amavuta y’umubiri zikoranirizwa hamwe kandi zigashirwa mu bice vy’umubiri bitandukanye biva ku nzira y’ugusohoka kw’umubiri, ari vyo. Ikindi, twerekana ko uburebure bw’uruzitiro rwa ceramide mu ruhu rw’uruzitiro rw’imbere mu mubiri ari ngirakamaro cane muri iyo nzira yo gutoranya. Ico cigwa cacu kiratanga ikimenyamenya ca mbere gitomoye co gushikiriza imitwaro y’amaproteine ​​ishingiye ku burebure bw’uruzitiro rw’amavuta mu bibanza vy’ugushiramwo ibintu hanze mu nzira y’amazi.
Mu ngingo ngenderwako z’ibinyabuzima bita eukaryote, izo poroteyine zikozwe mu nzira y’amaraso (ER) zica zitorwa mu gihe zitwarwa biciye mu nzira y’amabanga kugira ngo zishikirizwe aho zibereye mu ngingo ngenderwako (1). Uretse ugutoranya bifashijwe n’impuzu, vyari vyaramaze igihe kirekire bivugwa ko amavuta amwamwe ashobora kandi gukora nk’aho asohokera mu kuyashira mu bice vy’umubiri vyihariye vy’amaproteine ​​yihariye (2-5). Ariko rero, haracariho ibimenyamenya bitaziguye vyerekana ko ubwo buryo bushoboka bushingiye ku mavuta. Kugira ngo dutorere umuti ico kibazo c’ishimikiro, twarize mu musemburo ingene poroteyine zishingiye ku glycosylphosphatidylinositol (GPI) zirungikwa hanze mu buryo butandukanye zivuye mu ER. GPI-APs ni ubwoko butandukanye bw’amaproteine ​​yo ku ruhande rw’ingingo zifatanye n’amaraso ( 6, 7). GPI-AP ni poroteyine isohoka ifatanye n’ibipande vyo hanze vy’uruzitiro rwa plasma biciye ku gice c’amaraso citwa glycolipide (GPI anchor). Bemera amananga ya GPI nk’ihinduka ry’inyuma y’uguhindura mu ER lumen (8). Inyuma y’aho GPI-AP ifatanye, ica mu gikoresho ca Golgi (5, 9) iva muri ER ikaja mu ruhu rw’amaraso. Kuba hariho amananga ya GPI bituma GPI-AP itwarwa itandukanye n’amaproteine ​​asohoka mu nzira y’amaraso (harimwo n’izindi poroteyine zisohoka mu nzira y’amaraso) mu nzira y’amaraso (5, 9, 10). Mu ngingo z’umusemburo, GPI-APs zitandukanywa n’izindi poroteyine zisohoka mu nzira y’amaraso, hanyuma zigashirwa mu bihimba vy’umubiri bidasanzwe bizingiwe n’igice c’umubiri citwa coat protein complex II (COPII) (6, 7). Ivyo bigena iyo nzira yo gushikiriza mu nzira yo gushora ER hanze ntibitomoye, ariko bivugwa ko iyo nzira ishobora gusaba amavuta, cane cane uguhindura imiterere y’igice c’amavuta c’inanga ya GPI (5, 8). Mu musemburo, uguhindura amavuta ya GPI gutangura ubwo nyene GPI imaze kwifatanya, kandi kenshi, bituma ceramide ifatanya n’aside y’ibinure y’uruzitiro rurerure y’imitsi 26 (C26:0) (11, 12). C26 ceramide ni ceramide nyamukuru ivugwa n’ingingo z’umusemburo gushika ubu. Ikoranywa muri ER kandi nyinshi muri zo zirungikwa mu gikoresho ca Golgi biciye mu bihimba vya COPII (13). Ivyoherezwa hanze vya ER vya GPI-AP bisaba canecane ugukorana kw’ivyuma vy’ivyuma (14, 15), kandi na vyo nyene, uguhindura ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma (IPC) mu gikoresho ca Golgi bivana n’ivyuma vy’ivyuma vy’ivyuma (16). Ivyigwa vy’ubuhinga bwa biophysique bikoresheje utugingo ngengabuzima tw’abantu vyerekanye ko ceramides zifise uruzitiro rurerure cane rwitwa acyl zishobora gufatanya kugira ngo zibe ibice vy’umubiri bifise imiterere yihariye (17, 18). Ivyo bimenyetso bishikana ku ciyumviro c’uko C26 ceramide na GPI-AP biri kumwe na C26 ceramide bikoresha imiterere yavyo kugira ngo bifatanye mu turere canke mu turere dufise urutonde mu bidukikije vy’amavuta y’umubiri biteye akaga. Ahanini igizwe n’ibihimba vy’umubiri bigufi kandi bitagira amazi (C16:1 na C18:1) (19, 20). Izo ntara zizokwibanda ku bibanza vyihariye vy’ugusohoka kwa ER (ERES), aho ceramide na ceramide ishingiye kuri GPI-AP bishobora gutwarwa hamwe bikaja muri Golgi mu gice kimwe c’umubiri cihariye ca COPII (5).
Muri iki cigwa, twagerageje ata guca ku ruhande ubwo buryo bushingiye ku mavuta dukoresheje ubuhinga bwo gupima amashusho mu gihe nyaco (SCLIM), ari bwo buhinga bushasha bwo gupima amavuta bushobora kwihweza icarimwe poroteyine zifise amabara y’umuco. (21, 22).
Twabanje gukoresha ubuhinga bwa SCLIM kugira ngo dusobanure neza ingene GPI-AP isanzwe n’umugwi wa C26 ceramide yasuzumwe iva ku ma proteine ​​asohoka mu nzira y’amaraso amaze kuva muri ER muri S. cerevisiae. Kugira ngo tugenzure ukuntu ER ishirwa mu migwi, twakoresheje ubuhinga bw’ivy’ivyaduka bushobora kubona ata guca ku ruhande imizigo mishasha yakozwe yinjira muri ERES in vivo (7, 23). Nk’umuzigo, twahisemwo C26 ceramide-based GPI-AP Gas1 ifise ikimenyetso c’ibara ry’icatsi kibisi (GFP) n’ibara ry’ibara ry’icatsi kibisi ry’ibara ry’agahama Mid2 ifise ibara ry’ibara ry’icatsi kibisi ry’ibara ry’agahama (iRFP), ivyo vyose bikaba vyerekeye urukoba rw’ibara ry’agahama (24–26). Mu muvuduko w’ubushuhe witwa sec31-1, izo nzira zibiri zigaragazwa munsi y’umusemburo utera galactose be n’ikimenyetso ca ERES. Ku bushuhe bukaze cane (37°C), kubera ko ihinduka rya sec31-1 rigira ico rihinduye ku gikorwa c’igice c’impuzu ya COPII Sec31 kugira ngo kibuze COPII gukura no gutanga ER hanze, imizigo mishasha ikozwe irundanywa kuri ER (23). Inyuma yo gukonja ku bushuhe buke (24°C), izo nsoro zihinduka sec31-1 zarasubiye mu karere k’ibanga, maze imizigo mishasha y’ubuhinga bwa none yari yirundanijwe itangura gusohoka hanze ivuye muri ER. Ivyerekanwa vya CLIM vyerekanye ko vyinshi mu bihimba bishasha vya Gas1-GFP na Mid2-iRFP vyari bikiri mu ER y’ingingo z’umubiri zihinduka sec31-1 inyuma yo guterwa ku 37°C hanyuma zikarekurwa ku 24°C mu minota 5 (Ishusho 1). Kubera ko Mid2-iRFP ikwiragizwa ku ruhu rwose rwa ER, Gas1-GFP na yo ikaba yibanda kandi ikoranirizwa mu karere k’uruzitiro rwa ER rudakomeza, ukuntu ikwiragizwa biratandukanye rwose (Ishusho 1, A gushika kuri C na Movie S1). Ikindi, nk’uko vyerekanwa ku gishushanyo 1D, igice ca Gas1-GFP nta Mid2-iRFP gifise. Ivyo bimenyetso vyerekana ko GPI-AP n’amaproteine ​​y’imbere mu mubiri vyatandukanijwe mu turere dutandukanye tw’umubiri wa ER kare. Ico kigwi ca Gas1-GFP kiri hafi y’iyi ERES yihariye yanditsweko poroteyine y’impuzu ya mCherry COPII Sec13 (Ishusho 1, E na F, na filime S1) (23).
sec31-1 utugingo ngengabuzima tugaragaza ivyimburwa biva ku galactose, uruzitiro rurerure rw’ivyatsi (C26) rwitwa ceramide GPI-AP Gas1-GFP (GPI-AP, icatsi kibisi) be n’ivyo bihimba vy’umubiri vyitwa Mid2-iRFP (TMP, ubururu) kandi ivyo bihimba vy’umubiri vyubatswe na Sec13-mEScu mu 2012. iminota 5, bijanwa ku 24°C, hanyuma bifatwa amashusho na SCLIM haciye iminota 5. (A gushika kuri C) yerekana ishusho y’indege (A), ishusho y’indege y’ibice 10 vy’umubiri (B) canke ishusho y’ibice 10 vy’umubiri (B) canke ishusho y’ibice vy’umubiri vy’imizigo n’ibimenyetso vya ERES (C). Igiti c’urugero 1μm (A na B). Igipimo c’urugero ni 0,551μm (C). Gas1-GFP yabonetse mu turere canke mu bice vy’umubiri bitandukanye, mu gihe Mid2-iRFP yabonetse kandi ikwiragizwa mu ruhu rwose rwa ER (C). (D) Igishushanyo kirerekana uburemere bw’umuco w’izuba wa Gas1-GFP na Mid2-iRFP mu kigwi ca Gas1-GFP ku murongo w’umwampi werurutse (ibubamfu). AU, igice c’ububisha. (E na F) zigereranya ishusho y’uburebure bwa 3D ihuriza hamwe ibintu n’ikimenyetso ca ERES. Ivyiyumviro vya Gas1-GFP vyabonetse hafi y’aho ERES yihariye. Igipimo c’urugero ni 0.551μm. (F) Umwampi ukomeye w’umweru ugaragaza igice ca Gas1-GFP gifitaniye isano na ERES. Ivyumba vyo hagati n’ivyo mu buryo vyerekana ishusho ya 3D yagutse ihuriweko be n’ishusho izunguruka y’ikigwi ca Gas1-GFP catowe.
Isano ry’ahantu hagati y’ikigwi ca Gas1-GFP n’ERES yihariye ryerekana ko Gas1-GFP ishobora kwinjira muri ERES itoranya, ivyo bikaba bitandukanye n’ugutoranya gukoreshwa na Mid2-iRFP kugira ngo ive muri ER. Kugira ngo ivyo bishoboke, twagereranyije igipimo ca ERES ku kintu kimwe canke bibiri gusa (Ishusho 2, A gushika kuri C). Twasanze ama ERES menshi (70%) arimwo ubwoko bumwe gusa bw’imitwaro. Ishusho iri musi y’igishushanyo 2C yerekana ingero zibiri zisanzwe z’ERES zifise Gas1-GFP gusa (igishushanyo 1) canke Mid2-iRFP gusa (igishushanyo 2). Mu buryo bunyuranye, hafi 20% vya ERES birimwo imizigo ibiri ihuriranye mu karere kamwe. Vyabonetse ko ama ERES amwe amwe (10%) arimwo ubwoko bubiri bw’imitwaro, ariko yari yitandukanije mu bibanza bitandukanye bigaragara. Rero, iri sesangura ry’imibare ryerekana ko ER imaze gusohoka hanze, GPI-AP Gas1-GFP n’umuzigo w’imbere mu mubiri Mid2-iRFP bigabanywamwo ERES zitandukanye (Ishusho 2D). Ukwo gutoranya neza guhuye cane n’isesengura ry’ibinyabuzima ry’imbere (6) n’ugupima imiterere y’ibinyabuzima (7). Turashobora kandi kwihweza inyifato y’imizigo yashizwe mu gace yinjira muri ERES (Ishusho 2E na Filime S2). Igishushanyo 2E kirerekana ko igice gitoyi gusa ca Gas1-GFP (igice ca 3) canke Mid2-iRFP (igice ca 4) cinjira muri ERES kivuye ku ruhande rumwe kandi kikaba gifunzwe mu kibanza kidasanzwe. Igipande ca 5 c’igishushanyo ca 2E kirerekana ko Gas1-GFP na Mid2-iRFP rimwe na rimwe ziboneka muri ERES imwe, ariko zija zivuye ku mpande zitandukanye kandi zikaba ziri mu turere dutandukanye dushobora guserukira utugingo ngengabuzima dutandukanye twa COPII. Twemeje kandi ko ugutandukanya n’ugushirwa mu migwi kwa C26 ceramide-based GPI-AP Gas1 nk’ERES itoranya ari ukudasanzwe kubera ko uwundi muzigo w’ugusohoka kw’ibihimba vy’umubiri, proteine ​​y’ibihimba vy’umubiri vy’umubiri Axl2 (27 ), yerekana inyifato isa n’iya Mid2-iRFP. (Ishusho S1 na Filime S3). Axl2-GFP nshasha yakozwe ikwiragizwa biciye mu nzira ya ER nka Mid2-iRFP (Ishusho S1, A na B), kandi ihuriye hamwe na Mid2-iRFP muri ERES nyinshi (Ishusho S1, B gushika kuri D). Igipande ca 1 n’ica 2 c’igishushanyo ca 1. S1C yerekana ingero zibiri zisanzwe za ERES aho imizigo ibiri y’ibihimba vy’umubiri ihurirana. Muri ivyo bihe, ivyo bicuruzwa vyose bica muri ERES hamwe (Ishusho S1E, Igipande ca 3 na Filime S3).
Ivyo bihimba vy’umubiri vyerekana ivyimburwa bishobora guterwa n’ivyo bita galactose, Gas1-GFP (GPI-AP, icatsi kibisi) na Mid2-iRFP (TMP, ubururu) be n’ivyo bihimba vy’umubiri bifise ikimenyetso ca ERES Sec13-mCherry (ERES, magenta) vyashizwe ku 37 Inyuma yo guterwa ku °C iminota 30. block, n’ishusho na SCLIM inyuma y’iminota 20. (A gushika kuri C) Amashusho aserukira 2D (A; umurongo w’urugero, 1μm) canke amashusho y’igice c’umubiri c’ingingo 3D (B na C; urugero, 0.456μm) y’imizigo n’ibice 10 vy’urugero rwa z vyerekanwa na ERES. Igipande co hasi kiri muri (B) n’igipande kiri muri (C) vyerekana amashusho yatunganijwe kugira ngo yerekane gusa ibintu biri muri ERES (ibara ry’agahama) [Gas1-GFP (ibara ry’umuhondo) na Mid2-iRFP (ibara ry’ubururu ry’agahama)]. (C) Umwampi wuguruye: ERES itwara gusa umuzigo umwe (1 gushika kuri 4). Umwampi w’umweru: ERES irimwo imizigo itandukanye (5). Umwampi ukomeye w’umweru: ERES irimwo imizigo iri hamwe. Mu nsi: ERES imwe yatowe irimwo Gas1-GFP (1) canke Mid2-iRFP (2) gusa. Igiti c’urugero, 100 nm. (D) Ugupima igitigiri c’ivyo bishushanyo vy’amafoto bivugwa muri (C). Ijanisha ry’ivya ERES birimwo umuzigo umwe gusa (Gas1-GFP canke Mid2-iRFP), umuzigo utandukanye n’umuzigo ushize hamwe. Mu ngeragezwa zitatu zigenga, n=432 mu nsoro 54. Ikosa ry'umurongo = SD. Igerageza ry’imirizo ibiri ritagira ababiri. *** P = 0,0002. (E) Ishusho ya 3D ya ERES yatowe y’imizigo yashizwe mu gacerere ifise ikimenyetso (C). Gas1-GFP (icatsi kibisi) (3) canke Mid2-iRFP (ubururu) (4) yinjira muri ERES (ibara ry’agahama) ivuye ku ruhande rumwe kandi igarukira ahantu hatoyi muri ERES. Rimwe na rimwe, ubwo bwoko bwompi bw’imitwaro bwinjira muri ERES imwe (5) buvuye ku ruhande rumwe, bugaca buja mu kibanza kiri ukwaco kiri muri iyo ERES. Igiti c’urugero, 100 nm.
Inyuma y’aho, twagerageje iciyumviro c’uko uruzitiro rurerure rwa acyl ceramide (C26) ruri mu ruhu rwa ER rutuma habaho ugukorana n’ugutoranya Gas1 mu ERES itoranywa. Kugira ngo ivyo bishoboke, twakoresheje ubwoko bw’umusemburo wahinduwe GhLag1, aho izo ceramide synthases zibiri z’imbere mu mubiri Lag1 na Lac1 zasubirijwe na GhLag1 (umusemburo Lag1 homolog y’ipampa), bikaba vyatumye haba ubwoko bw’umwambiro ufise uruzitiro rw’ingingo ubwoko bwa Ceramide bugufi kuruta ubwoko bw’ishamba (Ishusho 3A) (2). Isesengurwa ry’uburemere bw’ibintu (MS) ryerekanye ko mu mice y’ubwoko bw’ishamba, 95% vy’ivyuma vyose vy’umubiri ari ivyuma vy’uruzitiro rurerure cane (C26), mu gihe muri GhLag1, 85% vy’ivyuma vy’umubiri ari ivy’uruzitiro rurerure cane (C18 na C16). ), 2% gusa vy’iseramide ni iseramide y’uruzitiro rurerure cane (C26). Naho C18 na C16 ceramides ari zo ceramides nyamukuru zabonetse mu ruhu rwa GhLag1 gushika ubu, isesengura rya MS na ryo nyene ryemeje ko umugozi wa GPI wa Gas1-GFP ugaragazwa mu bwoko bwa GhLag1 urimwo C26 ceramide, igereranywa n’amavuta y’ubwoko bw’ishamba. Uburyo ni bumwe (Ishusho 3A) (26). Ivyo rero bisigura ko enzyme Cwh43 ihindura ceramide ihitamwo cane ceramide C26, nk’uko vyerekanwa ku gishushanyo ca 26, ihitamwo gushiramwo nanga ya GPI iva ku ceramide ntoyi ya C26 iri mu bwoko bwa GhLag1. S2 (29). Naho ari ukwo, uruzitiro rw’ingingo rwa GhLag1 mu bisanzwe rurimwo gusa C18-C16, mu gihe Gas1-GFP iguma ifise C26. Ivyo bituma uwo mugera uba igikoresho ciza co gutorera umuti ingorane y’uburebure bw’uruzitiro rwa acyl rw’ivyuma vyitwa membrane ceramide mu ER. Uruhara rw’ivyiyumviro rw’umugwi n’ugutoranya. Hanyuma, twabanje kwiga ubushobozi bwa C26 Gas1-GFP bwo kwirundanira mu bice muri GhLag1 n’umugera uhinduka w’ubushuhe wa sec31-1 biciye ku buhinga busanzwe bwo gukoresha imikorosikopi y’umuco, aho uruzitiro rurerure (C18-C16) ruriho gusa mu ruhu rwa ER Ceramide (Ishusho 3). Twabonye ko muri sec31-1, igice kinini ca Gas1-GFP cari cihariye mu bice, mu gihe Gas1-GFP muri sec31-1 GhLag1 ifise uruzitiro rurerure (C18-C16) rw’uruzitiro rurerure rwa ceramide ER ahanini ntiyari yihariye kandi igakwiragizwa mu Mu ruzitiro rwose rwa ER. Kugira ngo tuvuge ukuri, kubera ko C26 ceramide-based clustering ifitaniye isano cane n’ERES yihariye (Ishusho 1), twaciye dukora itohoza nimba iyo nzira ishobora kandi kuba irimwo igikorwa c’uburyo bwo gutanga poroteyine hanze ya ER. GPI-AP ikoresha uburyo budasanzwe bwa COPII bwo gutuma ER irungikwa hanze, bugenzurwa n’uguhindura imiterere ya Ted1 y’igice ca glycan c’inanga ya GPI (30, 31). Ivyo bihimba vy’umubiri bifatanya n’ibindi bihimba vy’umubiri bita p24, ivyo bikaba bica bitora Lst1, ari vyo bihimba vy’umubiri bifatanya n’ibindi bihimba vy’umubiri bifatanya n’ibindi bihimba vy’umubiri Sec24, bikaba bikora igice kinini c’umubiri gifatanya imizigo ya COPII Sec24, bikaba ari ngombwa (31). Ku bw’ivyo, twakoze umugera ugira kabiri wahuje ugukuraho izo poroteyine zimwe zimwe (igice ca p24 gikomeye Emp24, enzyme ihindura GPI-glycan Ted1 n’igice c’umugera wa COPII Lst1) n’umugera wa sec31-1, maze tuvyiga Mbega birashoboka ko umuntu ashobora guhingura Gas1-clu3. Twabonye ko mu gice ca 31-1emp24Δ no mu gice ca 31-1ted1Δ, Gas1-GFP ahanini itagira amatsinda kandi igakwiragizwa mu ruhu rwose rwa ER, nk’uko vyabonetse mbere mu gice ca 31-1 GhLag1, mu gihe mu gice ca 31-1lst1Δ, Gas1-GFP Nk’igice ca 3. Ivyo bimenyetso vyerekana ko uretse kuba hariho C26 ceramide mu ruhu rwa ER, ugukorana kwa Gas1-GFP na kwo nyene gukeneye kwifatanya n’umugwi wa p24, kandi ntibisaba gutora Lst1 yihariye. Hanyuma, twasuzumye ko uburebure bw’uruzitiro rwa ceramide mu ruhu rwa ER bushobora gutunganya ukuntu Gas1-GFP ifatanya na p24. Ariko rero, twabonye ko kuba hariho ceramide ya C18-C16 mu ruhu rw’umubiri bitagira ico bikoze ku GPI-glycans zasubiwemwo n’umugwi wa p24 (Ibishushanyo S3 na S4, A na B) canke ku kwifatanya na GPI-AP no gutanga GPI-AP hanze. ubushobozi. Gutora COPII ubwoko buto Lst1 (Ishusho S4C). Ku bw’ivyo, C26 ceramide-dependent clustering ntibisaba ko poroteyine zikorana n’uburyo butandukanye bwo gutuma poroteyine zisohoka hanze, ariko zishigikira uburyo bundi bwo gutoranya burongowe n’uburebure bw’amavuta. Hanyuma, twasuzumye nimba uburebure bw’uruzitiro rwa ceramide acyl mu ruhu rwa ER ari ngirakamaro ku bijanye no gushikiriza neza Gas1-GFP nk’ERES itoranya. Kubera ko Gas1 mu bwoko bwa GhLag1 bufise ceramide y’uruzitiro rugufi iva muri ER ikaja mu ruhu rwa plasma (Ishusho S5), twizera ko iyo ugutoranya birongowe n’uburebure bw’uruzitiro rwa ceramide acyl, Gas1 yo mu bwoko bwa GhLag1 ishobora guhindura inzira no kujabuka. Ibintu vya ERES bifise uruzitiro rumwe.
(A) Uruzitiro rw’ingingo rwa GhLag1 ahanini rurimwo ama ceramides C18-C16 magufi, mu gihe nanga ya GPI ya Gas1-GFP igifise C26 IPC imwe n’ingingo z’ubwoko bw’ishamba. Hejuru: isesengura ry’uburebure bw’uruzitiro rwa acyl rwa ceramide mu ruhu rw’ingingo z’ubwoko bw’ibikoko (Wt) n’ubwoko bwa GhLag1p hakoreshejwe ubuhinga bwo gupima uburemere bw’ibintu (MS). Ivyo bimenyetso bigereranya ijana kw’ijana ry’ivyo bihimba vyose vy’umubiri. Igitigiri c’ivyageragejwe bitatu vyigenga. Ikosa ry'umurongo = SD. Igerageza ry’imirizo ibiri ritagira ababiri. **** P <0,0001. Igipande co hasi: Isesengura rya MS ry’uburebure bw’uruzitiro rwa acyl rwa IPC ruri mu nanga ya Gas1-GFP (GPI-IPC) GPI yerekanwa mu bwoko bw’ishamba n’imiryango ya GhLag1p. Amakuru agereranya ijana kw’ijana ry’ikimenyetso cose ca IPC. Igitigiri c’ivyageragejwe bitanu vyigenga. Ikosa ry'umurongo = SD. Igerageza ry’imirizo ibiri ritagira ababiri. ns, si ngombwa. P = 0,9134. (B) Ivyerekanwa vy’imirongo y’izuba vy’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo 1Δ yerekana Gas1-GFP iterwa na galactose vyatewe ku bushuhe bwa 37°C ku minota 3000. inyuma ya 24°C. Umwampi werurutse: Itsinda rya ER Gas1-GFP. Umwampi wuguruye: Gas1-GFP itagira amatsinda ikwiragizwa ku ruhu rwose rwa ER, yerekana ibara ry’uruzitiro rw’imitsi y’umubiri ruranga ER. Igiti c’urugero, 5μm. (C) Ugupima igitigiri c’ivyo bishushanyo vy’amafoto bivugwa muri (B). Ijanisha ry’ingingo ngenderwako zifise imiterere ya Gas1-GFP. Mu ngeragezwa zitatu zigenga, n≥300 z’ingingo. Ikosa ry'umurongo = SD. Igerageza ry’imirizo ibiri ritagira ababiri. **** P <0,0001.
Kugira ngo dutorere umuti ico kibazo, twakoze ivyerekanwa vya SCLIM vya Gas1-GFP na Mid2-iRFP muri GhLag1 n’umugera wa sec31-1 ushobora guhindura ubushuhe (Ishusho 4 na Filime S4). Inyuma y’aho ER igumye ku bushuhe bwa 37°C hanyuma ikarekurwa ku bushuhe bwa 24°C, igice kinini c’ivyo bihimba bishasha vya Gas1-GFP ntivyari vyifashe hamwe kandi ntivyakwiragijwe mu ruhu rwose rwa ER, nk’uko bigaragara n’ibikoresho bisanzwe bikoreshwa mu kubona ibintu (Ishusho 4, A na B ). Ikindi, igice kinini c’i ERES (67%) kirimwo ubwoko bubiri bw’imizigo iri kumwe muri yo (Ishusho 4D). Ivyumba 1 na 2 vy’igishushanyo 4C vyerekana ingero zibiri zisanzwe z’ERES zifise Gas1-GFP na Mid2-GFP zifatanye. Ikindi, ivyo bicuruzwa vyose vyari vyaronswe muri ERES imwe (Ishusho 4E, urupapuro rwa 3 na filime S4). Ku bw’ivyo, ivyo twabonye vyerekana ko uburebure bw’uruzitiro rwa ceramide acyl mu ruhu rwa ER ari ikintu gihambaye kigaragaza ugukorana n’ugushikiriza poroteyine za ER.
Sec31-1 Utugingo ngengabuzima twa GhLag1 tugaragaza ivyimburwa biterwa n’ivyatsi bita galactose, Gas1-GFP (GPI-AP, icatsi kibisi) na Mid2-iRFP (TMP, ubururu) be n’ivyatsi bifise ikimenyetso ca ERES (ERES, icatsi kibisi) Biterwa ku bushuhe bwa 37°C. Bandanya iminota 30, ushire ku 24°C kugira ngo urekure amazi, hanyuma ukoreshe ishusho na SCLIM inyuma y’iminota 20. (A gushika kuri C) Amashusho aserukira 2D yerekana (A; umurongo w’urugero, 1μm) canke amashusho y’igice c’umubiri c’ingingo 3D (B na C; urugero, 0.45μm) y’ibice 10 z vyerekanwa n’imitwaro na ERES. Igipande co hasi kiri muri (B) n’igipande kiri muri (C) vyerekana amashusho yatunganijwe kugira ngo yerekane gusa ibintu biri muri ERES (ibara ry’agahama) [Gas1-GFP (ibara ry’umuhondo) na Mid2-iRFP (ibara ry’ubururu ry’agahama)]. (C) Umwampi wuzuye umweru: ERES, ibintu birasangira. Umwampi wuguruye: ERES irimwo ikintu kimwe gusa. Igipande co hasi: ERES yatowe ifise ibintu bifatanye (1 na 2) vyerekanwa muri (C). Igiti c’urugero, 100 nm. (D) Ugupima igitigiri c’ivyo bishushanyo vy’amafoto bivugwa muri (C). Mu bice vya sec31-1 na sec31-1 GhLag1, umuzigo umwe gusa (Gas1-GFP canke Mid2-iRFP) ni wo ushizwemwo, n’ijanisha ry’ijanisha rya ERES ku mizigo iri ukwayo n’imizigo ihurikiye hamwe. Mu ngeragezwa zitatu zigenga, n = 432 mu ngingo 54 (sec31-1) na n = 430 mu ngingo 47 (sec31-1 GhLag1). Ikosa ry'umurongo = SD. Igerageza ry’imirizo ibiri ritagira ababiri. *** P = 0,0002 (igice ca 31-1) na ** P = 0,0031 (igice ca 31-1 GhLag1). (E) Ishusho ya 3D y’i ERES yatowe ifise imizigo ihuriyeko (3) yerekanwa muri (C). Gas1-GFP (icatsi kibisi) na Mid2-iRFP (ubururu) zegera ERES (ibara ry’agahama) zivuye ku ruhande rumwe zigaguma mu karere kamwe ERES ibujijwe. Igiti c’urugero, 100 nm.
Iyi nyigisho itanga ikimenyamenya gitomoye c’uko imizigo y’amaproteine ​​ishingiye ku mavuta ishirwa mu bibanza vy’ugushora hanze mu nzira y’amabanga, kandi igaragaza akamaro k’uburebure bw’uruzitiro rwa acyl ku guhitamwo gushirwa mu migwi. Dukoresheje ubuhinga bukomeye kandi bugezweho bwo gupima ibihimba vy’umubiri vyitwa SCLIM, twarerekanye ko Gas1-GFP (igihimba kinini c’umubiri GPI-AP gifise uruzitiro rurerure cane rw’amavuta y’umubiri (C26) ceramide) mu musemburo ) Uturere dukoranijwe mu bihimba vy’umubiri vy’ibanga bitandukanye biciye ku bihimba vy’umubiri vy’umubiri bitandukanye, n’uruzitiro rwa ER (Ishusho 1). Ikindi, ubwo bwoko bubiri bw’ibicuruzwa bwinjira muri ERES zitandukanye mu buryo butoranijwe (Ishusho 2). Uburebure bw’uruzitiro rwa acyl rwa ceramide y’ingingo mu ruhu buragabanywa kuva kuri C26 gushika kuri C18-C16, igice ca Gas1-GFP kigahungabana kikaja mu karere ka ER katandukanye, maze Gas1-GFP igahindurwa inzira kugira ngo ive muri ER ifise poroteyine y’uruzitiro biciye muri iyo ERES nyene (Ishusho 3 n’Ishusho3). 4).
Naho GPI-AP ikoresha uburyo bwihariye bw’amaproteine ​​kugira ngo isohoke muri ER, twabonye ko ugutandukanya gushingiye kuri C26 ceramide kutashingiye ku migenderanire itandukanye y’amaproteine ​​ishobora gutuma ERES igira ubuhinga bwihariye (Ibishushanyo S4 na S5). Ahubwo, ivyo twabonye birashigikira uburyo bundi bwo gushikiriza ibintu mu migwi bushingiye ku gukoranya poroteyine zishingiye ku mavuta y’umubiri be n’ugukuraho ibindi bintu. Ivyo twihweje vyerekana ko akarere ka Gas1-GFP canke igice gifitaniye isano n’ERES yihariye kitagira poroteyine Mid2-iRFP isohoka mu ruhu, ivyo bikaba vyerekana ko igice ca GPI-AP gishingiye kuri C26 ceramide kizotuma binjira muri ERES ibereye, kandi muri ico gihe nyene, kigakuraho iyo transmembrane yihariye The ESER 2). Mu buryo bunyuranye, kuba hariho ama ceramides C18-C16 mu ruhu rwa ER ntibituma GPI-AP igira uturere canke amatsinda, rero ntizikuraho canke ngo zisubirize poroteyine zisohoka mu ruhu rwa ER zibe muri iyo ERES nyene (Ibishushanyo 3 na 4). . Ku bw’ivyo, dusaba ko C26 ceramide ituma habaho ugutandukanya n’ugushira mu migwi mu gufasha gukoranya poroteyine zifitaniye isano n’ibindi bihimba vy’umubiri.
Ni gute twoshika kuri ivyo bihimba vy’umubiri bishingiye ku ceramide C26 mu karere ka ER kanaka? Ivyo bishobora gutuma ceramide ya GPI-AP na C26 igira amavuta mato kandi atunganijwe mu kanya nk’ako gukubita mu bidukikije vy’amavuta bidasanzwe vy’uruzitiro rwa ER birimwo amavuta magufi kandi atagiramwo amavuta. Ivyiyumviro vy’uburyo (17, 18). Ivyo bice bitobito vy’igihe gito birashobora kurushiriza gufatanya bikaba ibice bininibinini kandi bikomeye cane bimaze gufatanya n’igice ca p24 (34). Mu guhuza n’ivyo, twarerekanye ko C26 Gas1-GFP ikeneye gukorana n’umugwi wa p24 kugira ngo ikore amatsinda manini aboneka (Ishusho 3). Igitera p24 ni oligomere heterozygous igizwe n’amaproteine ​​ane atandukanye ya p24 transmembrane mu musemburo (35), itanga ubufatanye bw’ibintu vyinshi, bishobora gutuma habaho uguhuza kw’ibice bitobito vya GPI-AP, gutyo bikazana igice kinini ca Stable (34). Ugukorana hagati y’ibice vy’inyuma vy’amaproteine ​​vya GPI-APs na vyo nyene birashobora gutuma bikorana, nk’uko vyerekanwa mu gihe c’ugutwara kwavyo kwa Golgi mu ngingo z’umubiri z’ibikoko vy’ibinyamabere (36). Ariko rero, iyo C18-C16 ceramide iri mu ruhu rwa ER, iyo p24 complex ifatanye na Gas1-GFP, ntihazoba amatsinda manini manini atandukanye. Uburyo bushingiyeko bushobora kuva ku mico yihariye y’umubiri n’imiterere y’uruzitiro rurerure rw’ivyo bihimba vy’umubiri vyitwa ceramide. Ivyigwa vyakozwe ku bihimba vy’umubiri vyerekana ko naho ama ceramide maremare (C24) n’ama ceramide maremare (C18-C16) ashobora gutuma habaho ugutandukana kw’ibice, ama ceramide maremare y’uruzitiro (C24) ni yo yonyene ashobora gutuma habaho ugukora cane no gupfukama kw’ifilime kugira ngo ihindure ibara ry’iyi filime. Biciye mu gufatanya (17, 37, 38). Vyerekanwe ko igihimba c’umubiri citwa TMED2, ari co gihimba c’umubiri gihuye n’umubiri wa Emp24, gikorana n’igihimba c’umubiri citwa sphingomyeline gishingiye kuri C18 ceramide mu bihimba vy’umubiri (39). Dukoresheje ibigereranyo vy’inguvu z’ibihimba (MD), twabonye ko ceramides zompi C18 na C26 zirundanira hirya no hino y’ibihimba vy’umubiri vy’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri w’umubiri (Ishusho S6). Ni vyiza kumenya ko ivyo vyerekana ko umurongo w’uruzitiro rwa Emp24 ushobora gutuma amavuta y’amaraso mu ruzitiro agabanywa mu buryo budahuye. Ivyo ni ibiherutse gusohoka bishingiye ku ngirabuzima fatizo z’ibikoko vy’ibinyamabere. Ivyo bigereranyo bisa n'ivyo vyerekana kandi ko hariho amavuta y'umubiri (40) . Ku bw’ivyo, turakeka ko C26 ceramide iri mu bihimba bibiri vya ER26 itunganywa n’aho hantu. Igihe GPI-AP mu bihimba vy’umubiri bifatanye ataco bimaze na p24 n’ukwirundanira kwa C26 ceramide hirya no hino ya p24 mu bihimba vy’umubiri, birashobora gutuma habaho uguteranira hamwe kw’amaproteine ​​n’ugukorana kw’ibihimba vy’umubiri biva ku ntoke (41), bikaba bituma GPI-AP na yo nyene itandukana mu karere gakomeye. uturere tw’uruzitiro rwa ER (42). Raporo za kera zashigikiye uburyo bwari bwateguwe (43, 44). Ivyo bihimba vy’umubiri bifatanya n’ibihimba vy’umubiri bita oligolectins, ibitera indwara canke ibihimba vy’umubiri birwanya indwara bita glycosphingolipids (GSL) bishingiye kuri ceramide ku rubavu rwa plasma bituma habaho uguteranira hamwe kwinshi kwa GSL, bituma habaho ugutandukana kw’ibice kandi bituma urubavu ruhinduka kandi rugaca ruja imbere (44). Iwabuchi etc. (43) Vyabonetse ko iyo hariho iminyororo miremire (C24) ariko itari migufi (C16), ligand y’imirongo myinshi ifatanye na GSL lactosylceramide yatumye habaho amatsinda manini manini n’ugutera kw’imitsi, kandi uguhindura ikimenyetso c’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi y’imitsi.
Mu ngirabuzimafatizo z’ibikoko vy’ibinyamabere, ukwibanda kw’uruzitiro rwa anti-Golgi (TGN) ku rugero rw’uruzitiro rw’amaraso rw’imbere ni rwo rugenzura ugutandukanya n’ugutoranya GPI-AP (10, 45). Ivyo bihimba vy’umubiri biterwa n’uguhinduka kw’ibihimba vy’umubiri (36), ariko kandi bishobora kuvana n’uburebure bw’uruzitiro rwa ceramide dusanga mu musemburo. Naho GPI-AP y’ibikoko vy’ibinyamabere ifise nanga ishingiye ku mavuta ya ether, kandi imiterere yayo y’imiti itandukanye cane n’iyi ceramide y’uruzitiro rurerure cane rwitwa acyl, inyigisho iherutse gukorwa yerekanye ko ayo mavuta yompi afise imiterere y’umubiri n’imiterere y’imiti isa n’iy’ubwihindurize (40). Ku bw’ivyo, igice c’amavuta ya etere kiri mu ngingo z’ibikoko vy’ibinyamabere gishobora gusa n’ica C26 ceramide kiri mu musemburo, kandi uruhara rwaco ni ugufatanya n’igice c’uruzitiro rurerure c’i ceramide kiri mu ruhu kugira ngo gitere imbere mu gukoranya no gutoranya GPI-AP. Naho ivyo bishoboka bikiri ngombwa ko bigeragezwa ata guca ku ruhande, ivyo vyabonetse imbere y’aho birashigikira ko gutwara ceramide y’uruzitiro rurerure rw’acyl mu mubiri wa Golgi bitakorwa n’amaproteine ​​y’uguhindura cytoplasmic, ariko bivana n’ugukorana kw’ibifatanya GPI nk’umwambiro. Ku bw’ivyo, uburyo bwo kubungabunga ivy’ubwihindurize busa n’ubushobora guhitamwo gutwara hamwe uruzitiro rurerure cane rwa acyl ceramide na GPI-AP (13, 16, 20, 46, 47) mu nzira imwe yo gutwara.
Mu nzira y’ingingo z’umubiri zifise umusemburo n’iz’ibikoko vy’ibinyamabere, ugukorana kwa GPI-AP n’ugutandukana n’izindi poroteyine z’umubiri zikorana n’amaraso, vyose bishika imbere y’uko bishika ku ruhande rw’ingingo. Paladino n’abandi. (48) basanze ku TGN y’ingingo z’umubiri z’ibikoko vy’ibinyamabere, gukoranya GPI-AP atari ngombwa gusa kugira ngo GPI-APs zishire mu migwi mu buryo bubereye, ariko kandi zitunganya imiterere y’ugukorana kw’ingingo za GPI-APs n’ibikorwa vyazo vy’ibinyabuzima. Uburebure bw'agace. Mu musemburo, iki cigwa cerekanye ko igice ca GPI-AP gishingiye kuri C26 ceramide ku ER gishobora gutunganya imitunganirize y’igice n’ibikorwa vy’ibikorwa vya GPI-AP ku ruhu rwa plasma (24, 49). Mu buryo buhuye n’iki kigereranyo, utugingo ngengabuzima twa GhLag1 turagira allergie ku bibuza GPI canke imiti igira ingaruka ku buzima bw’uruhome rw’utugingo ngengabuzima (28), kandi ivy’ugukenera amatsinda ya Gas1-GFP akora (49) y’isonga ry’umusemburo ryerekanywe mu gukorana n’utugingo ngengabuzima tw’umusemburo vyerekana G Inkurikizi zishobora guterwa n’utugingo ngengabuzima twa hLag1. Ikosa rya GPI-AP. Ariko rero, ukugerageza nimba imitunganirize y’ibikorwa vy’uruhande rw’ingingo rwateguwe kuva kuri ER hakoreshejwe uburyo bwo gutoranya bushingiye ku burebure bw’amaraso, ni vyo tuzokora ubushakashatsi muri kazoza.
Ubwoko bwa Saccharomyces cerevisiae bwakoreshejwe muri iki gikorwa buri mu ndome S1. Ubwoko bwa MMY1583 na MMY1635 bwa SCLIM bwo gufata amashusho y’ingingo z’umubiri bwakozwe inyuma ya W303. Izo nzira zigaragaza Sec13-mCherry zifise ikimenyetso c’iporoteyine zica ibibatsi zakozwe hakoreshejwe uburyo bushingiye ku nzira y’uruhererekane rwa polymerase (PCR) hakoreshejwe plasmide ya pFA6a nk’akarorero (23). Ubwoko bugaragaza Mid2-iRFP bufise ikimenyetso c’iporoteyine y’umuco igenzurwa n’umurongozi wa GAL1 bwakozwe gutya. Igikoresho co gukuraho urutonde rwa iRFP-KanMx kivuye ku mugera wa pKTiRFP-KAN (ingabire ya E. O’Shea, inomero ya plasmide ya Addgene 64687; http://n2t.net/addgene: 64687; Inyuma y’aho urutonde rw’ibihimba vy’umubiri vy’umubiri wa Mid2-iRFP rwagwijwe maze rugahindurwa mu murongo wa GAL1, rwashizwe mu kibanza ca Not I-Sac I c’umubiri w’umubiri pRS306. Ico gice ca plasmide pRGS7 carashizwe ku murongo na Pst I kugira ngo kije mu gice ca URA3.
Igitera c’ugukorana kwa Gas1-GFP kigaragazwa n’umugera wa GAL1 mu plasmide ya centromere (CEN), iyo plasmide yubatswe gutya. Urutonde rwa Gas1-GFP rwakozwe na PCR ruvuye kuri plasmide ya pRS416-GAS1-GFP (24) (ingabire ya L. Popolo) maze ruhindurwa mu kibanza ca Xma I–Xho I c'umugera wa CEN pBEVY-GL LEU2 (ingabire ya C) . Umusuku; Igitigiri c’ivyatsi vy’umubiri 51225; 51225; RRID: Inyongera_51225). Ico gikoko cavuyemwo citwa pRGS6. Igitera Axl2-GFP fusion gene na co nyene kigaragazwa munsi y’ubugenzuzi bw’umurongozi wa GAL1 w’umugera wa pBEVY-GL LEU2, kandi ubuhinga bwaco ni ubu bukurikira. Urutonde rwa Axl2-GFP rwakomejwe ruva ku plasmide ya pRS304-p2HSE-Axl2-GFP (23) hakoreshejwe ubuhinga bwa PCR, hanyuma ruca ruhindurwa mu kibanza ca Bam HI-Pst I c’umugera wa pBEVY-GL LEU2. Ico gikoko cavuyemwo citwa pRGS12. Urutonde rw’ibihimba vy’umubiri vyakoreshejwe muri iki cigwa ruri mu ndome S2.
Ubwo bwoko bwari bwongereweko 0,2% vy’adenine n’2% vy’isukari [YP-dextrose (YPD)], 2% vy’umusemburo w’umusemburo p (YP) (1 % vy’umusemburo n’2% vy’umusemburo ept). (YPR)] canke 2% galactose [YP-galactose (YPG)] nk’inkomoko y’umuyagankuba, canke mu gikoresho gikoreshwa n’abantu (0,15% y’umusemburo w’azote n’0,5% y’umusemburo w’amoniyumu) ​​kugira ngo wongereko aside amino n’ibindi bibereye bikenewe kugira ngo umuntu aronke ingaburo , Kandi kirimwo 2% y’isukari canke glucose2 ( (agalactose y’ubuhinga bwa none) nk’inkomoko y’umuyagankuba.
Kugira ngo umuntu abone amashusho mu gihe nyaco, utugingo ngengabuzima duhinduka dufise ubushuhe tugaragaza ivyubatswe munsi y’umurongozi wa GAL1 twakuriye mu gitereko ca YPR ku 24°C ijoro ryose gushika mu gihe c’imbere. Inyuma yo guterwa muri YPG ku 24°C mu kiringo c’isaha 1, izo nsoro zari zitewe muri SG ku 37°C mu kiringo c’iminota 30, hanyuma zijanwa mu 24°C kugira ngo zive mu gice c’umubiri gisohoka. Concanavalin A yakoreshejwe mu gushiramwo izo nsoro ku gicapo c’ibirahuri maze zifatwa n’amashusho na SCLIM. SCLIM ni ihuriro ry’imikorosikope y’umuco w’izuba Olympus IX-71 n’ibara ry’amavuta ry’imibare UPlanSApo 100×1.4 (Olympus), umuvuduko munini kandi w’umuvuduko munini w’ikimenyetso n’ijwi ryinshi ry’urugero rw’ikimenyetso n’urusaku ruzunguruka (Yokogawa Electric n’urugero rw’umuco w’ibara ry’umuco (Yokogawa Electric) (Hamamatsu Photonics) irashobora gutanga ubuhinga bwo gukura amatara bufise ubushobozi bwo gukura bwa nyuma bugera kuri ×266,7 be n’igikoresho gifata amasanamu gifise ubushobozi bwo gukura ama elegitoroni (Hamamatsu Photonics) (21). Ugutora amashusho bikorwa n'ubuhinga busanzwe (Yokogawa Electric). Ku mashusho y’uburebure bwa 3D, twakoresheje igikoresho c’umuyagankuba cakozwe ku buryo bubereye kugira ngo kinyiganyize urushinge rw’intumbero mu buryo buhagaze, maze dukorakoranya ibihimba vy’amaso bitandukanye 100 nm mu kirundo. Ishusho y’ikirundo ca Z ihindurwa mu makuru y’amajwi 3D, kandi igikorwa co gukwiragiza amabara y’ivyiyumviro gikoreshwa ku gikoresho co gupima igipimo c’amabara gikoreshwa mu gukora ibikorwa vy’ugukuraho ibintu bikoresheje porogarama ya Volocity (PerkinElmer). Mu gukoresha porogarama ya Volocity kugira ngo igere ku nzira y’ugusesangura aho umuntu ari, ERES harimwo n’imizigo yaragerejwe. Isesengurwa ry’umurongo ryakozwe hakoreshejwe porogarama ya MetaMorph (Ibikoresho vy’ibihimba).
Koresha porogaramu ya GraphPad Prism kugira ngo umenye akamaro k’imibare. Ku kigeragezo ca t c’umunyeshure c’imirongo ibiri n’ikigeragezo gisanzwe c’isesengura ry’uburyo bumwe (ANOVA), itandukaniro hagati y’imigwi rifatwa ko rigira ingaruka zikomeye kuri P <0.05 (*).
Ku bijanye n’ugupima umuco w’izuba wa Gas1-GFP, utugingo ngengabuzima tw’inzira y’ibiti twakuze ijoro ryose muri YPD maze dukwegeranywa hakoreshejwe ubuhinga bwa centrifugation, twozwa incuro zibiri n’umunyu ufise phosphate buffered, hanyuma duterwa ku barafu nibura iminota 15, hanyuma duca dukorwa munsi y’umuco w’izuba nk’uko vyavuzwe imbere y’aho (Check24). Ivyo bikoresho vyakoreshejwe mu kuronka iyo mikorosikope yitwa Leica DMi8 (HCX PL APO 1003/1.40 oil PH3 CS) ifise urushinge rwo gucapura, akayunguruzo ka L5 (GFP), kamera ya Hamamatsu n’ubuhinga bwa Application Suite X (LAS X). .
Ivyo bipimo vyahinduwe n’ivyo bipimo vya SDS ku bushuhe bwa 65°C mu minota 10, hanyuma bitandukanywa hakoreshejwe ubuhinga bwa SDS-polyacrylamide gel electrophoresis (PAGE). Ku bijanye n’isesengura ry’immunoblotting, 10 μl y’ivyafashwe vyashizwe ku murongo wose. Igitera umubiri: Koresha umuti w’inkwavu urwanya Gas1 ku rugero rwa 1:3000, umuti w’inkwavu urwanya Emp24 ku rugero rwa 1:500, n’umuti w’inkwavu urwanya GFP (ingabire ya H. Riezman) ku rugero rwa 1:3000. Imbeba irwanya Pgk1 yakoreshejwe mu gupima 1:5000 (ingabirano ya J. de la Cruz). Igitera indwara y’ibicurane: Igitera indwara y’ibicurane (HRP) gikoreshwa mu gukingira indwara y’ibicurane G (IgG) gikoreshwa mu gupima 1:3000 (Pierce). IgG y’impene irwanya imbeba ifatanye na HRP yakoreshejwe ku rugero rwa 1:3000 (Pierce). Ico kibanza c’ubudahangarwa bw’umubiri carabonetse hakoreshejwe uburyo bwo gukoresha ubuhinga bwa chemiluminescence bukoresheje ubuhinga bwa SuperSignal West Pico (Thermo Fisher Scientific).
Nk’uko vyavuzwe muri (31), igerageza ry’ubudahangarwa bw’umubiri ryakozwe ku gice ca ER catunganijwe. Muri make, woze ingingo z’umusemburo n’umuti wo gukingira TNE [50 mM tris-HCl (pH 7,5), 150 mM NaCl, 5 mM EDTA, 1 mM umuvango w’umusemburo w’umusemburo n’umusemburo w’umusemburo) ku 600 nm ( OD600) ku 100 mM. Yaravunitse n’amabuye y’ibirahuri, hanyuma ibisigazwa vy’ingingo n’amabuye y’ibirahuri bikakurwamwo biciye ku nzira y’ugucapura. Ivyo bihimba vy’umubiri vyari hejuru yavyo vyaciye bishirwa mu gitereko c’amazi ku 17.000 g mu minota 15 ku 4°C. Iryo pellet ryasubiwemwo muri TNE hanyuma digitalis saponin yongerwako gushika ku rugero rwa nyuma rwa 1%. Ivyo bihimba vy’umubiri vyaciwe mu mfuruka y’isaha 1 bihindurwa ku bushuhe bwa 4°C, hanyuma ivyo bihimba bidashobora gusenyuka bikurwamwo biciye mu gucapura ku 13.000 g ku bushuhe bwa 4°C mu minota 60. Ku bijanye n’ugukingira umubiri Gas1-GFP, banza ushiremwo amabuye y’agarose ataco amaze (ChromoTek) ku 4°C mu kiringo c’isaha 1, hanyuma ushiremwo GFP-Trap_A (ChromoTek) ku 4°C mu kiringo c’amasaha 3. Ivyo bihimba vy’umubiri vyari vyarasukuwe incuro zitanu na TNE irimwo 0,2% ya digoxigenin, bica bivanwa n’umuti wo gukingira umubiri wa SDS, bitandukanywa kuri SDS-PAGE, hanyuma bisuzumwa hakoreshejwe ubuhinga bwa immunoblotting.
Nk’uko vyavuzwe muri (31), ugupima uguhuza kwakozwe ku gice ca ER catunganijwe. Mu magambo make, igice ca ER catunganijwe caratewe n’umuti witwa dithiobis (succinimidyl propionate) 0,5 mM (Pierce, Thermo Fisher Scientific, Rockford, IL, Leta Zunze Ubumwe za Amerika; 20°C, iminota 20). Ivyo bihimba vy’umubiri vyazimye mu kwongerako glycine (50 mM y’iherezo, iminota 5, 20°C).
Nk’uko vyavuzwe mbere (50), hakozwe isesengura rya MS ry’i ceramide mu bwoko bw’ibikoko n’ubwoko bwa GhLag1. Muri make, utugingo ngengabuzima twakuze gushika ku rwego rw’uburemere (3 gushika kuri 4 OD600 units/ml) muri YPD ku 30°C, maze utugingo ngengabuzima 25×107 turasarurwa. Ivyo bihimba vy’umubiri bizimwa n’aside trichloroacétique. Koresha umuti wo gukura [etanoli, amazi, etere, piridine n’amazi y’amazi y’amazi y’amazi 4,2 N (15:15:5:1:0,018 v/v)] n’amazi y’amazi y’amazi y’amazi y’amazi (860517, Avanti polar lipid) 1,2 nmol y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi y’amazi. Koresha igikoresho co gupima amazi [methanol, amazi, n-butanol n’umunyu wa methylamine (4:3:1:5 v/v)] kugira ngo ukore igikorwa co guhindura amazi y’ivyo bihimba, hanyuma ukoreshe n-butanol yuzuye amazi kugira ngo ukure umunyu. Ubwa nyuma, ivyo bihimba vyasubiwemwo mu buryo bwiza [kloroforme/methanol/amazi (2:7:1) + 5 mM acetate d’amonium] hanyuma biterwa mu gikoresho co gupima uburemere bw’ibintu. Ivyiyumviro vy’ugukurikirana ivyiyumviro vyinshi (MRM) vyakozwe kugira ngo hamenyekane no gupima igitigiri c’ibihimba vy’umubiri bita sphingolipid. Igikoresho co gupima uburemere bw’ibintu bifise amabara ane (Thermo Fisher Scientific) gifise ubuhinga bwo gupima uburemere bw’ibintu bifise amavuta y’umubiri (Nanomate HD) kugira ngo hasuzumwe amavuta y’umubiri. Inguvu z’ugutombora ziratunganijwe neza ku rwego rwose rw’ivyuma vy’ivyuma. Amakuru ya MS yaronswe mu buryo bwiza. Ku bijanye n’ihinduka ryose ry’ibinyabuzima, ikimenyetso c’amaraso ni co gica hagati y’ingero zitatu zigenga.
Nk’uko vyavuzwe muri (31), utugingo ngengabuzima (800×107) tugaragaza Gas1-GFP twakoreshejwe ubuhinga bwo gukingira umubiri. Gas1-GFP yasukuwe yatandukanijwe na SDS-PAGE hanyuma ijanwa mu gipfukisho c’ivyuma vy’ivyuma (PVDF). Iryo poroteyine ryabonetse mu gutera ibara PVDF n’umukara w’amide. Igipande ca Gas1-GFP caraciwe muri PVDF maze gisukurwa incuro 5 n’amazi y’ibara ry’agahama (methanol) rimwe na rimwe n’amazi y’urugero rwa chromatography-MS (LC-MS). Mu gutera umurongo w’uruzitiro n’imiti 500 μl ya NaOAc 0,3 M (pH 4,0), umuti wo gukingira n’imiti 500 μl y’umunyu w’amazi 1 M uherutse gusenyuka ku bushuhe bwa 37°C mu kiringo c’amasaha 3, igice c’amaraso kirarekurwa muri Gas1-GFP na inophamine y’inophamine hagati y’ivyo bihimba vy’umubiri. (51). Inyuma y’ivyo, iyo nzira y’uruzitiro yarayozwe incuro zine n’amazi y’agaciro ka LC-MS, yumishwa mu bushuhe bwo mu cumba, hanyuma ibikwa mu kirere c’azote ku -80°C gushika isuzumwe. Nk’ikigereranyo, hakoreshejwe igipimo c’ubusa c’uruzitiro rwa PVDF ku ngeragezwa yose. Ivyo bihimba vy’umubiri vyakuwe muri Gas1-GFP vyaciye bisuzumwa na MS nk’uko vyavuzwe (50). Muri make, imirongo ya PVDF irimwo GPI-lipid yasubiwemwo mu 75μl y’umunyu w’ububi [kloroforme/methanol (1:2) + 5 mM y’acetate y’amoniyumu] hanyuma irengana n’ugusesangura kw’ubwoko bw’amavuta y’umubiri (TSQ Vantage). Muri ivyo, amakuru ya MS yaronswe mu buryo bw’iyon mbi.
Nk’uko twabibonye, ​​igice c’amaraso c’inanga ya GPI caratandukanye n’igice ca GPI-AP canditsweko [3H]-inositol (16). Ivyo bihimba vy’umubiri vyatandukanywe hakoreshejwe ubuhinga bwo gupima ibara ry’umubiri (55:45:10 kloroforme-methanol-0,25% KCl) hanyuma bigaragazwa hakoreshejwe ubuhinga bwa FLA-7000 (Fujifilm).
Utugingo ngengabuzima tugaragaza Gas1-GFP (600×107) twayozwe incuro zibiri n’umuti wa TNE n’umuti wa TNE, hanyuma turamenagurwa n’amabuye y’ibirahuri, hanyuma turajanwa mu gitereko c’ibisigazwa vy’utugingo ngengabuzima n’amabuye y’ibirahuri. Ivyo bihimba vy’umubiri vyari hejuru y’ivyo bihimba vyaciye bishirwa mu gitereko c’amazi ku 17.000 g mu kiringo c’isaha 1 ku 4°C. Ico gice c’umubiri carayozwe muri TNE hanyuma giterwa n’amazi 1 U PI-PLC (Invitrogen) muri TNE irimwo 0,2% ya saponine ya digitalis mu kiringo c’isaha 1 ku bushuhe bwa 37°C. Inyuma y’ugutera enzyme, iyo membrane yarakuweho biciye mu gucapura ku 17.000 g ku 4°C mu kiringo c’isaha 1. Kugira ngo Gas1-GFP ishobore gukingira umubiri, ivyo bihimba vyari hejuru yavyo vyashizwe mu gitereko ca GFP-Trap_A (ChromoTek) ku bushuhe bwa 4°C ijoro ryose. Gas1-GFP yasukuwe itandukanijwe na SDS-PAGE yari yashizweko ibara ry’ubururu bukeye bwa Coomassie. Igipande c’ibara rya Gas1-GFP caraciwe ku rangi y’umweru ikikuje umugende w’amazi, hanyuma inyuma y’uguhindura ibara ry’umubiri n’iyodoacetamide no kugabanya n’ibara ry’umubiri ryitwa dithiothreitol, hakorwa ugufungura mu gel hakoreshejwe trypsine. Gukura no kwumisha amapeptide y’ivyatsi n’amapeptide afise GPI-glycans. Ico gice c’umubiri cyumye cari gishizwe mu mazi 20 μl. Nimushire igice (8μl) muri LC. Inkingi ya octadecylsilane (ODS) (Develosil 300ODS-HG-5; umurongo w’imbere 150 mm×1,0 mm; Nomura Chemical, mu ntara ya Aichi, mu Buyapani) yarakoreshejwe mu gutandukanya amapeptide mu bihe vy’ibara ry’umubiri. Igice gigendagenda ni umunyuro A (0,08% aside formique) n’umunyuro B (0,15% aside formique muri 80% acetonitrile). Hakoreshejwe ubuhinga bwa Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) kugira ngo iyo nkingi isukurwe n’umuti A mu minota 55 ku rugero rw’amazi 50 μl min-1 mu minota 5, hanyuma umubare w’umuti B wongerwa kugera kuri 40%. , muri Leta Zunze Ubumwe za Amerika). Ivyo bihimba vy’umubiri vyaguma vyinjizwa mu nzira y’iyon za ESI, kandi ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma vy’ivyuma; Thermo Fisher Scientific). Mu gutegura MS, umuriro w’isoko y’amazi y’amaraso wari ushizwe kuri 4,5 kV, kandi ubushuhe bw’amazi y’amazi y’uguhindura bwagumye kuri 300°C. Inguvu z’umuriro w’amazi n’iz’amatara y’amazi vyari vyashizwe kuri 15 V na 50 V, uko bikurikirana. Amakuru ya MS yaronswe mu buryo bw’iyon nziza (ubushobozi bwo gutorera umuti 60.000; uburemere bw’ibice 10 ku miliyoni) mu rutonde rw’uburemere rwa 300/m/z uburemere/uburemere (m/z) 3000. Amakuru ya MS/MS aboneka biciye ku mutego w’iyon mu XLthe Ors3 ku mbere n’ugusenyuka kwatewe n’ugutombora (CID)].
Ivyiyumviro vya MD vyakozwe hakoreshejwe porogarama ya GROMACS (52) n’ibarabara ry’inguvu rya MARTINI 2 (53-55). Igikoresho co kwubaka uruzitiro rwa CHARMM GUI (56, 57) carakoreshejwe mu kwubaka uruzitiro rubiri rurimwo DOPC na Cer C18 canke DOPC na Cer C26. Ivyerekeye topologie n’imirongo ya Cer C26 biva kuri DXCE mu gukuraho amabuye y’inyongera ku murizo wa sphingosine. Koresha uburyo buvugwa aha hepfo kugira ngo ugereranije igice ca kabiri maze ugikoreshe, hanyuma ukoreshe ama coordinates ya nyuma ya sisitemu kugira ngo wubake sisitemu irimwo Emp24. Igihugu c’umusemburo Emp24 (ibisigazwa 173 gushika ku 193) cubatswe nk’igice c’umubiri citwa α-helix hakoreshejwe igikoresho c’amaso MD (VMD) c’imiterere y’ibihimba vy’umubiri (58). Hanyuma, bamaze gukuraho amavuta yari apfutse, iyo poroteyine yarahinduwe utugingo ngengabuzima dutoduto maze yinjizwa mu gice c’umubiri hakoreshejwe ubuhinga bwa CHARMM GUI. Uburyo bwa nyuma burimwo 1202 DOPC na 302 Cer C26 canke 1197 DOPC na 295 Cer C18 na Emp24. Ionize iyo system ku rugero rwa 0.150M. Ivyiyumviro bine vyigenga vyakozwe ku bihimba bibiri vy’imirongo ibiri.
Ivyo bihimba bibiri vy’amaraso biraringanizwa hakoreshejwe uburyo bwa CHARMM GUI, bujanye no kugabanya hanyuma bagaringaniza intambwe 405.000, aho inzitizi z’aho umuntu ari zigabanywa buhoro buhoro zigakurwaho, intambwe y’igihe na yo ikaduga kuva kuri 0.005 ps gushika kuri 0.02 ps. Inyuma y’uguringaniza, itanga 6 μs n’intambwe y’igihe ya 0,02 ps. Amaze kwinjiza Emp24, ukoreshe iyo nzira nyene ya CHARMM GUI kugira ngo ugabanye kandi ushire uburinganire muri sisitemu, hanyuma ukore amasegonda 8 mu gukora.
Ku mice yose, mu gihe c’uguringaniza, umuvuduko ugenzurwa n’igipimo ca Berendsen (59), mu gihe c’uguhingura, umuvuduko ugenzurwa n’igipimo ca Parrinello-Rahman (60). Mu bihe vyose, umuvuduko w’amazi ni 1 bar kandi hakoreshwa umugambi wo gufatanya umuvuduko w’amazi w’igice c’isotropic. Mu nzira yo gupima no gukora, hakoreshwa igikoresho co gupima ubushuhe (61) gifise umuvuduko wo gusubira gupima kugira ngo gifatanye ubushuhe bw’ibice vy’amaproteine, amavuta n’ibinyabuzima. Mu gihe cose c’igikorwa, ubushuhe bugenewe ni 310K. Ivyo bihuza bitari vyo biharurwa mu gutanga urutonde rw’ivyo bihuza hakoreshejwe umugambi wa Verlet ufise 0.005 buffer tolerance. Ijambo rya Coulomb riharurwa hakoreshejwe ikibanza c’inyishu n’urugendo rw’uguca rwa 1,1 nm. Ijambo rya Vander Waals rikoresha umugambi wo gucapura ufise urugendo rwo gucapura rwa 1,1 nm, kandi umugambi wo gucagura wa Verlet ukoreshwa ku bijanye n’uguhungabana gushobora kubaho (62).
Hakoreshejwe VMD, uburebure bw’umuvuduko wo guca hagati y’amabuye ya DOPC phosphate canke amabuye ya ceramide AM1 na poroteyine ni 0,7 nm, hanyuma umubare w’amavuta akorana na poroteyine uraharurwa. Dushingiye kuri iyi formile, harura igitigiri c’ugutakaza-gutunga (DE) nk’uko biri muri (63): igitigiri c’amavuta yose = (umubare w’amavuta yose ari muri poroteyine 0,7) muri poroteyine 0,7 (umubare w’amavuta yose ari muri Cer) .
Agaciro kavuzwe karonswa nk’agaciro, kandi imirongo y’amakosa ni kopi zine zigenga za SE. Igiharuro c'agaciro k'umubare wa DE giharurwa n'ikigeragezo ca t [(averageDE-factor-1)/SE]. Harura agaciro ka P uhereye ku gusangira kw’umurizo umwe.
Igikoresho ca GROMACS carakoreshejwe mu kubara ikarita y’uburemere bw’ibarabara 2D y’urutonde rurimwo Emp24 mu 250 ns za nyuma z’urugendo. Kugira ngo umuntu aronke ikarita y’ugutunganywa/ugutakaza ceramide, ikarita y’uburemere bwa Cer igabanywa n’umubare w’ikarata ya Cer na DOPC, hanyuma igabanywa n’uburemere bwa Cer mu mubiri. Urugero rw’ikarata y’ibara nyene ni rwo rukoreshwa.
Ku bijanye n’ibikoresho vy’inyongera vy’iyi ngingo, raba kuri http://advances.sciencemag.org/cgi/ibirimwo/vyuzuye/6/50/eaba8237/DC1.
Iyi ni ingingo ishobora gukoreshwa n’abantu bose igakwiragizwa hakurikijwe amategeko y’uruhusha rwo gukoresha, gukwiragiza no gusubiramwo mu buryo ubwo ari bwo bwose, igihe cose ikoreshwa rya nyuma ritari ryo gushaka inyungu z’ubudandaji kandi ikintu nyamukuru ari uko igikorwa c’intango ari co. Urutonde.
Iciyumviro: Turagusaba gusa gutanga aderesi yawe ya email kugira ngo uwo usaba kuri iyo paji amenye ko ushaka ko abona iyo email kandi ko atari spam. Nta ma email yose tuzofata.
Iki kibazo gikoreshwa mu kugerageza nimba uri umushitsi no kubuza gutanga ubutumwa butari bwo.
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 Lorgihope -Linero), Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D-resolution nini igihe nyaco amashusho yerekana akamaro k’uburebure bw’uruzitiro rwa ceramide mu gutoranya poroteyine mu bibanza vy’ibisohoka vyatoranijwe.
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 Lorgihope -Linero), Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D-resolution nini igihe nyaco amashusho yerekana akamaro k’uburebure bw’uruzitiro rwa ceramide mu gutoranya poroteyine mu bibanza vy’ibisohoka vyatoranijwe.
©2020 Ishirahamwe ry’Abanyamerika ry’Iterambere ry’Ubuhinga. uburenganzira bwose burarindwa. AAAS ni umufatanyabikorwa wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef na COUNTER. Iterambere ry'ubuhinga ISSN 2375-2548.