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Kuvumbura no gukoresha neza ibicuruzwa bisanzwe birashobora gufasha kuzamura imibereho yumuntu.Imiti ikura yibihingwa ikoreshwa cyane nkibyatsi byo kurwanya nyakatsi.Bitewe no gukenera gukoresha ubwoko butandukanye bwimiti yica ibyatsi, hakenewe kumenya ibice hamwe nuburyo bushya bwibikorwa.Muri ubu bushakashatsi, twavumbuye igitabo N -alkoxypyrrole compound, coumamonamide, kiva muri Streptomyces werraensis MK493-CF1 maze dushyiraho inzira yuzuye ya synthesis.Binyuze mubikorwa byibinyabuzima, twabonye ko aside ya urs-monoamic ari intangiriro ya urs-monoamide kandi ishoboraimikurire yikimera.Twongeyeho, twateje imbere ibikomoka kuri acide ya urbenonic, harimo ibikomoka kuri urbenyloxy (UDA), bifite ibikorwa byinshi by’ibyatsi bitagize ingaruka mbi ku mikurire y’uturemangingo twa HeLa.Twasanze kandi ibikomoka kuri acide ya urmotonic bihagarika microtubules yibihingwa;hiyongereyeho, KAND igira ingaruka kuri filime ya actin kandi itera urupfu;Izi ngaruka zinyuranye ziratandukanye nizizwi na microtubule inhibitor kandi zitanga uburyo bushya bwibikorwa bya acide ursonic, ibyo bikaba byerekana inyungu zingenzi mugutezimbere ibyatsi bishya.
Kuvumbura no gushyira mubikorwa ibikorwa byiza byingirakamaro nibibukomokaho nuburyo bwo kuzamura imibereho yumuntu.Secondary metabolite ikorwa na mikorobe, ibimera nudukoko byatumye iterambere ryinshi mubuvuzi nubuhinzi.Antibiyotike nyinshi hamwe nibiyobyabwenge birwanya leukemia byakozwe mubicuruzwa bisanzwe.Byongeyeho, ubwoko butandukanye bwaimiti yica udukoko, fungiside na herbiside bivanwa muri ibyo bicuruzwa bisanzwe kugirango bikoreshwe mu buhinzi.By'umwihariko, kurwanya ibyatsi bibi ni ibikoresho byingenzi byo kongera umusaruro w’ibihingwa mu buhinzi bugezweho, kandi ubwoko butandukanye bw’ibimera bumaze gukoreshwa mu bucuruzi.Inzira nyinshi zingirabuzimafatizo mu bimera, nka fotosintezeza, metabolisme ya aside amine, synthesis y'urukuta rw'utugingo ngengabuzima, kugenzura mitito, ibimenyetso bya phytohormone, cyangwa sintezamubiri ya poroteyine, bifatwa nk'ibisanzwe byibasira ibyatsi.Imvange zibuza imikorere ya microtubule nicyiciro rusange cyibyatsi bigira ingaruka kumikurire yibimera bigira ingaruka kumikorere ya mitoto2.
Microtubules ni igice cya cytoskeleton kandi kibitswe cyane muri selile eukaryotic.Tubuline heterodimer igizwe na α-tubuline na β-tubuline ikora umurongo wa microtubule protofilaments, hamwe na protofilament 13 igizwe na silindrike.Microtubules igira uruhare runini mu ngirabuzimafatizo, harimo kumenya imiterere y'utugingo, kugabana ingirabuzimafatizo, hamwe no gutwara imitsi3,4.Utugingo ngengabuzima turimo microtubules munsi ya intera ya plasma membrane, kandi ibyo bita microtubules bita cortical microtubules bibwira ko bigenzura imitunganyirize ya microfibrile ya selile binyuze mumabwiriza ya selile ya synthase.Cortical microtubules ya selile epidermal selile, igaragara muri zone yo kwaguka byihuse kumutwe wumuzi, iherereye kuruhande, kandi microfibers ya selile ikurikiza izo microtubules ikagabanya icyerekezo cyo kwaguka kwakagari, bityo bigatera kwaguka kwa selile anisotropique.Kubwibyo, imikorere ya microtubule ifitanye isano rya hafi na morphologie yibimera.Gusimbuza aside amine muri gen zigizwe na tubuline zitera skew ya microtubule ya cortical cortical hamwe no gukura ibumoso cyangwa iburyo muri Arabidopsis 6,7.Mu buryo nk'ubwo, ihinduka rya poroteyine zijyanye na microtubule zigenga imbaraga za microtubule nazo zishobora gutuma imizi igoramye 8,9,10,11,12,13.Byongeye kandi, kuvura hamwe na microtubule-ihungabanya imiti yica ibyatsi nka disopyramide, izwi kandi nka pretilachlor, nayo itera imizi y’ibumoso ikura imizi14.Aya makuru yerekana ko kugenzura neza imikorere ya microtubule ari ngombwa mu kumenya icyerekezo cyo gukura kw'ibimera.
Haravumbuwe ubwoko butandukanye bwa microtubule inhibitor, kandi iyi miti yagize uruhare runini mubushakashatsi bwa cytoskeletal, ndetse no mubuhinzi nubuvuzi2.By'umwihariko, oryzalin, dinitroaniline ivanze, disopyramide, ibimera bifitanye isano na benzamide, hamwe na byo birashobora kubuza imikorere ya microtubule bityo bikabuza gukura kw'ibimera.Kubwibyo, zikoreshwa cyane nkibimera.Nyamara, kubera ko microtubules ari ikintu cyingenzi kigize ibimera n’inyamanswa, ibyangiza byinshi bya microtubule ni cytotoxic kubwoko bwombi.Kubwibyo, nubwo bizwi ko bifite akamaro nkibimera, umubare muto wimiti igabanya ubukana ikoreshwa mubikorwa bifatika.
Streptomyces ni ubwoko bwumuryango Streptomyces, ikubiyemo bacteri zo mu kirere, gram-nziza, za bagiteri kandi izwi cyane kubera ubushobozi bwo gukora metabolite ya kabiri.Kubwibyo, ifatwa nkimwe mumasoko yingenzi yibinyabuzima bishya bikora biologiya.Mu bushakashatsi burimo, twavumbuye ikigo gishya cyitwa coumamonamide, cyatandukanijwe na Streptomyces werraensis MK493-CF1 na S. werraensis ISP 5486. Twifashishije isesengura ryerekanwa hamwe nisesengura ryuzuye, imiterere ya coumamonamide yaranzwe kandi skeleton idasanzwe ya N-alkoxypyrrole. yari yiyemeje.synthesis.Acide ya Ursmonic, ikomatanya hagati ya ursmonoamide n'ibiyikomokaho, wasangaga ibuza gukura no kumera kw'igihingwa kizwi cyane cyitwa Arabidopsis thaliana.Mu bushakashatsi bwakozwe ku miterere-yibikorwa, twasanze ibivanze na C9 byahinduwe kuri acide ya ursonic, bita nonyloxy derivative ya acide ursonic (KAND), byongera cyane ingaruka zibuza gukura no kumera.Ikigaragara ni uko ibimera bishya byavumbuwe bikura nabyo byagize ingaruka ku mikurire y’itabi n’umwijima kandi ntabwo byari cytotoxic kuri bagiteri cyangwa selile HeLa.Byongeye kandi, bimwe mu bikomoka kuri acide ya urmotonic itera imizi ya fenotipike igoramye, bivuze ko ibiyikomokaho bigira ingaruka kuri microtubules mu buryo butaziguye cyangwa butaziguye.Dufatiye kuri iki gitekerezo, ibyo twabonye kuri microtubules yanditseho immunohistochemic cyangwa proteine za fluorescent byerekana ko kuvura KAND depolymerize microtubules.Mubyongeyeho, kuvura hamwe na aside kumamotonic ikomoka kuri acrof microfilaments.Kubwibyo, twavumbuye uburyo bushya bwo gukura bwibimera bifite uburyo bwihariye bwibikorwa birimo kurimbura cytoskeleton.
Strain MK493-CF1 yatandukanijwe nubutaka bwa Shinagawa-ku, Tokiyo.Strain MK493-CF1 yakoze amashami meza ya mycelium.Urukurikirane rw'ibice bya 16S ribosomal RNA gene (1422 bp) byagaragaye.Iyi mitekerereze isa cyane na S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: imiterere isanzwe, 99,93%).Hashingiwe kuri iki gisubizo, hemejwe ko iyi ntera yari ifitanye isano rya hafi nubwoko bwa S. werraensis.Kubwibyo, twise by'agateganyo iyi ntera S. werraensis MK493-CF1.S. werraensis ISP 5486T nayo itanga ibinyabuzima bimwe.Kubera ko habaye ubushakashatsi buke hakiri kare mu kubona ibicuruzwa kamere muri iyi mikorobe, hakozwe ubundi bushakashatsi bw’imiti.Nyuma yo guhinga S. werraensis MK493-CF1 kumurima wa sayiri ukoresheje fermentation ikomeye kuri 30 ° C muminsi 14, imiti yakuweho 50% EtOH.Ml 60 yicyitegererezo yarumye kugirango ibone 59.5 mg yumusemburo.Ibicuruzwa biva mu mahanga byakorewe icyiciro cya HPLC cyo guha N-mikorerexy-1H-pyrrole-2-carboxamide (1, yitwa coumamonamide, 36.0 mg).Umubare wuzuye wa 1 ni hafi 60% yikuramo.Kubwibyo, twahisemo kwiga birambuye imiterere ya kumamotoamide 1.
Coumamonamide 1 ni ifu yera ya amorphous yera kandi nini cyane ya spekrometrike (HRESIMS) yemeza C6H8N2O2 (Ishusho 1).Igice cya C2 cyasimbuwe pyrrole yuru ruganda kirangwa na δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH muburyo bwa 1H NMR: 4.5 Hz , H-5) na δH 6.78 (1H, d, J = 2,5 Hz, H-6), hamwe na 13C NMR yerekana ko hariho atome enye za karuboni sp2.Kuba hari itsinda rya amide kumwanya wa C2 ryasuzumwe isano ya HMBC kuva kuri C-3 proton kugeza kuri karubone ya amide kuri δC 161.1.Mubyongeyeho, impinga ya 1 H na 13 C NMR kuri δH 4.10 (3H, S) na δC 68.3 byerekana ko hariho amatsinda ya N-mikorerexy muri molekile.Nubwo umwanya wukuri witsinda rya methoxy wari utaramenyekana ukoresheje isesengura rya spekitroscopique nko kongera itandukaniro rya spekitroscopi hamwe n’incamake ya Overhauser (NOEDF), N-mikorerexy-1H-pyrrole-2-carboxamide yabaye umukandida wa mbere w’abakandida.
Kugirango umenye imiterere nyayo ya 1, hakozwe synthesis yose (Ishusho 2a).Kuvura ubucuruzi buboneka 2-aminopyridine 2 hamwe na m-CPBA byatumye N-oxyde 3 ihwanye numusaruro mwinshi.Nyuma ya 2-aminoazidation ya 2, reaction ya cyclocondensation yasobanuwe na Abramovich yakorewe muri benzene kuri 90 ° C kugirango ibone 1-hydroxy-1H-pyrrole-2-karubone 5 muri garama.Umuvuduko 60% (ibyiciro bibiri).15.16.Methylation na hydrolysis ya 4 noneho byatanze 1-mikorerexy-1H-pyrrole-2-acide karubasi (bita "cumotonic aside", 6) mumusaruro mwiza (70%, intambwe ebyiri).Hanyuma, amidation ikoresheje aside chloride hagati ya 6 ukoresheje ammonia y'amazi yahaye Kumamoto amide 1 kumusaruro wa 98%.Amakuru yose yerekanwe ya synthesize 1 yasaga na 1 yihariye, kuburyo imiterere ya 1 yagenwe;
Rusange rusange hamwe nisesengura ryibinyabuzima bya urbenamide na aside urbenic.(a) Synthesis yuzuye ya Kumamoto amide..Umwanya munini = cm 1.
Ubwa mbere, twasuzumye ibikorwa byibinyabuzima bya urbenamide nabahuza bayo kubushobozi bwabo bwo guhindura imikurire.Twongeyeho ubunini butandukanye bwa ursmonamide 1 cyangwa aside ya ursmonic 6 kuri MS agar iciriritse kandi gifite umuco wa Arabidopsis thaliana ingemwe kuri ubu buryo.Ubu bushakashatsi bwerekanye ko kwibanda cyane (500 μM) ya 6 yabujije imizi gukura (Ishusho 2b).Ibikurikira, twabyaye inkomoko zitandukanye dusimbuza umwanya wa N1 wa 6 hanyuma dukora imiterere - ubushakashatsi bwibikorwa byerekeranye nabo (inzira ya analogue synthesis isobanurwa mumakuru yo gushyigikira (SI)).Ingemwe za Arabidopsis zahinzwe ku buryo burimo 50 μM zikomoka kuri acide ya ursonic, hanyuma hapimwa uburebure bw'imizi.nkuko bigaragara ku ishusho.Nkuko bigaragara ku gishushanyo cya 3a, b, na S1, acide coumamo ifite uburebure butandukanye bwiminyururu ya alkoxy (9, 10, 11, 12, na 13) cyangwa iminyururu minini ya alkoxy (15, 16, na 17) kumwanya wa N1.Ibikomokaho byagaragaje kubuza gukura kwimizi.Twongeyeho, twasanze gukoresha 200 μM 10, 11, cyangwa 17 byabujije kumera (Ishusho 3c na S2).
Kwiga imiterere-ibikorwa isano ya Kumamoto amide hamwe nibindi bifitanye isano.(a) Imiterere na synthesis gahunda yo kugereranya..Asterisks yerekana itandukaniro rikomeye hamwe no kuvura sham (t ikizamini, p<0.05).n>18. Amakuru yerekanwe asobanura ± SD.nt bisobanura “kutageragezwa” kuko imbuto zirenga 50% ntizimera..Asterisks yerekana itandukaniro rinini hamwe no kuvura sham (ikizamini cya chi-kare).n = 96.
Igishimishije, kongeramo iminyururu ya alkyl kurenza C9 byagabanije ibikorwa byo kubuza, byerekana ko ibibyimba bifitanye isano na aside kumamotoic bisaba iminyururu yuruhande runaka kugirango yerekane ibikorwa byibinyabuzima.
Kuberako isesengura ryimikorere-ibikorwa byerekanaga ko C9 yahinduwe acide ya ursonic kandi nonyloxy ikomoka kuri acide ya ursonic (nyuma yiswe KAND 11) niyo yari ingirakamaro cyane yo gukura kw'ibimera, twakoze ibisobanuro birambuye biranga KANDI 11. Kuvura Arabidopsis hamwe na 50 μM KANDI 11 hafi ya yose yabujije kumera, mu gihe intumbero yo hasi (40, 30, 20, cyangwa 10 μM) ya KAND 11 yabujije imizi gukura mu buryo bushingiye ku gipimo (Ishusho 4a, b).Kugirango tumenye niba KAND 11 igira ingaruka kumuzi ya meristem, twasuzumye meristem yumuzi yandujwe na iyode ya propidium (PI) hanyuma dupima ubunini bwa meristem.Ubunini bwa meristem yingemwe zahinzwe muburyo burimo 25 μM KAND-11 bwari 151.1 ± 32.5 mm, mugihe ubunini bwa meristem yingemwe zahinzwe muburyo bugenzura burimo DMSO bwari 264.7 ± 30.8 mm (Ishusho 4c, d) , byerekana ko KAND-11 igarura ibikorwa bya selile.gukwirakwira.Imizi meristem.Bihuye nibi, ubuvuzi bwa KAND 11 bwagabanije umubare wikimenyetso cyo kugabana selile CDKB2; 1p :: CDKB2; ikimenyetso cya 1-GUS mumuzi meristem (Ishusho 4e) 17.Ibisubizo byerekana ko KANDI 11 ibuza imizi kugabanya ibikorwa byo gukwirakwiza selile.
Isesengura ryingaruka zibuza aside irbenonic (ibikomoka kuri urbenyloxy) kumikurire..(b) Umubare w'uburebure bw'imizi.Inzandiko zerekana itandukaniro rikomeye (Ikizamini cya Tukey HSD, p<0.05).n>16. Amakuru yerekanwa asobanura ± SD..Umwanya munini = 100 µm.(d) Umubare wubunini bwa meristem (n = 10 kugeza 11).Itandukaniro rishingiye ku mibare ryagenwe hakoreshejwe t-test (p<0.05).Utubari twerekana impuzandengo ya meristem.(e) Itandukaniro ritandukanye ryo gutandukanya (DIC) microscopi yumuzi meristem irimo CDKB2 yubaka;1pro: CDKB2;1-GUS yandujwe kandi yandujwe ku mbuto zimaze iminsi 5 zihingwa ku masahani ya MS hamwe na 25 µM KAND assay.
Fytotoxicity ya KAND 11 yongeye kugeragezwa hifashishijwe ikindi gihingwa cya dicotyledonous, itabi (Nicotiana tabacum), hamwe n’ibinyabuzima by’icyitegererezo cy’ubutaka, umwijima (Marchantia polymorpha).Nkuko byagenze kuri Arabidopsis, itabi SR-1 ingemwe zihingwa hagati zirimo 25 μM KAND 11 zabyaye imizi migufi (Ishusho 5a).Byongeye kandi, imbuto 40 kuri 48 zimaze kumera ku isahani irimo 200 μM KANDI 11, mu gihe imbuto zose uko ari 48 zimaze kumera ku bitangazamakuru bivura urw'agashinyaguro, byerekana ko ubunini bwa KAND bwagize akamaro (p<0.05;chi test -square) yabujije kumera kwitabi.(Isanamu 5b).Byongeye kandi, kwibumbira hamwe kwa KAND 11 kwabujije imikurire ya bagiteri mu mwijima wasaga n’ubushakashatsi bwiza muri Arabidopsis (Ishusho 5c).Ibisubizo byerekana ko KANDI 11 ishobora kubuza imikurire yibihingwa bitandukanye.Twahise dukora ubushakashatsi kuri cytotoxicitike y’imyororokere ifitanye isano n’idubu monoamide mu bindi binyabuzima, aribyo selile ya HeLa y’abantu na Escherichia coli strain DH5α, nk’abahagarariye ingirabuzimafatizo zo mu bwoko bwa bagiteri na bagiteri.Mu ruhererekane rw'ikwirakwizwa ry'utugingo ngengabuzima, twabonye ko coumamonamide 1, aside coumamonamidic 6, na KAND 11 itagize ingaruka ku mikurire y'uturemangingo twa HeLa cyangwa E. coli ku gipimo cya 100 μM (Ishusho 5d, e).
Gukura kubuza KAND 11 mubinyabuzima bitari Arabidopsis.. Isahani ya MS irimo 200 μM KAND 11. igihe.(d) Ikwirakwizwa ry'utugingo ngengabuzima twa HeLa.Umubare w'ingirabuzimafatizo zishobora gupimwa mugihe cyagenwe ukoresheje ibikoresho byo kubara selile 8 (Dojindo).Nkigenzura, selile HeLa zavuwe hamwe na 5 μg / ml actinomycine D (Act D), ibuza kwanduza RNA polymerase kandi igatera urupfu.Isesengura ryakozwe muri bitatu.(e) E. coli ikwirakwizwa ry'utugari.Imikurire ya E. coli yasesenguwe no gupima OD600.Nkigenzura, selile zavuwe hamwe na 50 μg / ml ampisilline (Amp), ibuza urukuta rwa bagiteri.Isesengura ryakozwe muri bitatu.
Kugirango dusobanure uburyo bwibikorwa bya cytotoxicity iterwa nuruvange rufitanye isano na uramide, twongeye gusesengura inkomoko ya acide ya urbenic hamwe ningaruka zo kubuza.nkuko bigaragara ku ishusho.Nkuko bigaragara ku gishushanyo cya 2b, 6a, ingemwe zahinzwe ku isahani ya agar irimo intungamubiri nyinshi (200 μM) ya aside urmotonic 6 itanga imizi migufi kandi igoramye ibumoso (θ = - 23.7 ± 6.1), mu gihe ingemwe zikura ku buryo bwo kugenzura, ingemwe zitanga imizi igororotse (θ = - 3.8 ± 7.1).Uku gukura kuranga bizwi ko guturuka kumikorere mibi ya cortical microtubules14,18.Dukurikije ubu bushakashatsi, imiti ya microtubule-ihungabanya umutekano disopyramide na oryzalin yatumye imizi imera nk'iyikura mu miterere yacu yo gukura (Ishusho 2b, 6a).Muri icyo gihe, twagerageje inkomoko ya acide ya urmotonic hanyuma duhitamo byinshi muri byo, ku bitekerezo bimwe na bimwe, byatumye imizi ikura.Ibicuruzwa 8, 9, na 15 byahinduye icyerekezo cyo gukura kwimizi kuri 75 μM, 50 μM, na 40 μM, byerekana ko ibyo bivanga bishobora guhungabanya microtubule neza (Ishusho 2b, 6a).Twagerageje kandi inkomoko ya acide ya ursolike ikomeye, KAND 11, ku gipimo cyo hasi (15 µM) dusanga ko gukoresha KAND 11 byabujije imizi kandi ko icyerekezo cyo gukura kw'imizi kitaringaniye, nubwo bakunze kunyerera ibumoso ( Igicapo C3)..Kubera ko ibiyobyabwenge byinshi bya microtubule-bihungabanya umutekano rimwe na rimwe bibuza imikurire y’ibihingwa aho gutera imizi, nyuma twasuzumye bishoboka ko KAND 11 igira ingaruka kuri microtubules tureba microtubules cortical selile selile epidermal selile.Immunohistochemie ikoresheje antibodiyide anti-tub-tubuline mu ngirabuzimafatizo ya epidermal yimizi yatewe hamwe na 25 μM KAND 11 yerekanye ibura rya microtubule hafi ya cortical hafi ya selile epidermal muri zone ndende (Ishusho 6b).Ibisubizo byerekana ko aside kumamotonic n'ibiyikomokaho bikora mu buryo butaziguye cyangwa butaziguye kuri microtubules kugira ngo bibahungabanye kandi ko ibyo bivanga ari microtubule inhibitor.
Acide Ursonic nibiyikomokaho bihindura microtubules ya cortical muri Arabidopsis thaliana..Hasesenguwe kandi ingaruka zibintu bibiri bizwiho kubuza microtubules: disopyramide na oryzalin.Inet yerekana igipimo gikoreshwa mugupima imizi yo gukura.Asterisks yerekana itandukaniro rikomeye hamwe no kuvura sham (t ikizamini, p<0.05).n>19. Umwanya munini = cm 1.(b) Microtubules ya Cortical muri selile epidermal selile muri zone ndende.Microtubules yo mu bwoko bwa Arabidopsis yo mu gasozi Imizi ya Col ikura ku isahani ya MS ifite cyangwa idafite 25 μM KAND 11 yagaragajwe no kwanduza immunohistochemic ukoresheje antibodiya yibanze ya tub-tubuline na antibodiya ya kabiri ya Alexa Fluor-conjugated.Umwanya munini = 10 µm.(c) Imiterere ya mitoto ya microtubules mumuzi meristem.Microtubules yagaragaye hakoreshejwe irangi rya immunohistochemic.Imiterere ya Mitotic, harimo zone ya prophase, spindles, na fragmoplasts, byabaruwe uhereye kumashusho asebanya.Imyambi yerekana mitoto ya microtubule.Asterisks yerekana itandukaniro rikomeye hamwe no kuvura sham (t ikizamini, p<0.05).n>9. Umwanya muto = 50 µm.
Nubwo Ursa ifite ubushobozi bwo guhagarika imikorere ya microtubule, imikorere yayo iteganijwe kuba itandukanye na microtubule depolymerizing agent.Kurugero, kwibanda cyane kuri microtubule depolymerizing nka disopyramide na oryzalin bitera kwaguka kwa anisotropique kwaguka epidermal, mugihe KAND 11 itabikora.Byongeye kandi, gufatanya gukoresha KAND 11 na disopyramide byatumye habaho gukura kwa disopyramide iterwa no gukura kwimizi kandi KAND 11 yabujijwe gukura gukura (Ishusho S4).Twasesenguye kandi igisubizo cya hypersensitive disopyramide 1-1 (phs1-1) mutant kuri KAND 11. phs1-1 ifite ihinduka rya mutuline ya tubulin kinase ihinduka kandi itanga imizi migufi iyo ivuwe na disopyramide9,20.phs1-1 ingemwe za mutant zikura kumurima wa agar urimo KAND 11 zifite imizi migufi isa niyakuze kuri disopyramide (igishusho S5).
Twongeyeho, twabonye mikorobe ya microtubule, nka zone ya prophase, spindles, na phragmoplasts, mumuzi meristem y ingemwe zavuwe na KAND 11. Dukurikije ibyo twabonye kuri CDKB2; 1p :: CDKB2; 1-GUS, igabanuka rikabije rya umubare wa microtubules ya mitotic yagaragaye (Ishusho .6c).
Kugirango tugaragaze cytotoxicity ya KAND 11 kumurongo wa subcellular, twakoresheje itabi BY-2 ihagarika hamwe na KAND 11 hanyuma tureba ibisubizo byabo.Twabanje kongeramo KAND 11 kuri selile BY-2 yerekana TagRFP-TUA6, yanditseho microtubules, kugirango tumenye ingaruka za KAND 11 kuri microtubules cortical.Ubucucike bwa microtubule bwa Cortical bwasuzumwe hifashishijwe isesengura ryamashusho, ryagereranije ijanisha rya pigiseli ya cytoskeletike hagati ya pigiseli ya cytoplasme.Ibisubizo by'ubushakashatsi byagaragaje ko nyuma yo kuvurwa hamwe na 50 μM cyangwa 100 μM KAND 11 mu gihe cy'isaha 1, ubucucike bwaragabanutse cyane kugera kuri 0,94 ± 0,74% cyangwa 0.23 ± 0.28%, mu gihe ubucucike bw'utugari twavuwe na DMSO, bungana na 1.61 ± 0.34 % (Ishusho 7a).Ibisubizo birahuye nubushakashatsi bwakozwe muri Arabidopsis ko kuvura KAND 11 bitera depolymerisation ya microtubules ya cortical (Ishusho 6b).Twasuzumye kandi umurongo wa BY-2 hamwe na GFP-ABD yanditseho filime ya actin nyuma yo kuvurwa hamwe na KAND 11 hamwe kandi twabonye ko kuvura KAND 11 byahungabanije filime ya actin.Umuti hamwe na 50 μM cyangwa 100 μM KAND 11 kuri 1 h wagabanije cyane ubucucike bwa filime ya actin kugera kuri 1,20 ± 0,62% cyangwa 0,61 ± 0.26%, mu gihe ubucucike mu tugari twavuwe na DMSO bwari 1.69 ± 0.51% (Ishusho 2).7b).Ibisubizo bitandukanye ningaruka za propyzamide, itagira ingaruka kuri firimu ya actin, na latrunculin B, actin depolymerizer idafite ingaruka kuri microtubules (SI Igicapo S6).Byongeye kandi, kuvura hamwe na coumamonamide 1, aside coumamonamide 6, cyangwa KAND 11 ntabwo byagize ingaruka kuri microtubules mu tugari twa HeLa (SI Igicapo S7).Rero, uburyo bwibikorwa bya KAND 11 bemeza ko butandukanye nubwa cytoskeleton izwi.Byongeye kandi, ubushakashatsi bwakozwe na microscopique kuri selile BY-2 zavuwe na KAND 11 bwerekanye ko hapfuye urupfu rw’akagari mu gihe cyo kuvura KAND 11 kandi byerekana ko umubare w’ingirabuzimafatizo za Evans zifite ubururu utigeze wiyongera cyane nyuma yiminota 30 yo kwivuza KAND 11, mu gihe nyuma yiminota 90 yo kuvurwa hamwe na 50 μM cyangwa 100 μM KAND, umubare w'ingirabuzimafatizo zapfuye wiyongereye kugera kuri 43.7% cyangwa 80.1%, (Ishusho 7c).Ufatiye hamwe, aya makuru yerekana ko igitabo gishya cya ursolique ikomoka kuri KAND 11 ari igihingwa cyihariye cya cytoskeletal inhibitor hamwe nuburyo butamenyekana bwibikorwa.
KAND yibasira microtubules ya cortical, filime ya actin, hamwe nubuzima bwitabi BY-2.(a) Kwerekana amashusho ya microtubules ya cortical muri selile BY-2 imbere ya TagRFP-TUA6.BY-2 selile zavuwe na KAND 11 (50 μM cyangwa 100 μM) cyangwa DMSO zasuzumwe na microscopi conocal.Ubwinshi bwa microtubule ubwinshi bwabazwe uhereye kuri micrografi ya selile 25 yigenga.Inzandiko zerekana itandukaniro rikomeye (Ikizamini cya Tukey HSD, p<0.05).Umwanya munini = 10 µm.(b) Cortical actin filaments muri selile BY-2 igaragara imbere ya GFP-ABD2.BY-2 selile zavuwe na KAND 11 (50 μM cyangwa 100 μM) cyangwa DMSO zasuzumwe na microscopi conocal.Ubucucike bwa cortical actin filaments yabazwe uhereye kuri micrografi ya selile 25 yigenga.Inzandiko zerekana itandukaniro rikomeye (Ikizamini cya Tukey HSD, p<0.05).Umwanya munini = 10 µm.(c) Kwitegereza selile zapfuye BY-2 na Evans irangi ry'ubururu.BY-2 selile zavuwe na KAND 11 (50 μM cyangwa 100 μM) cyangwa DMSO zasuzumwe na microscopi yumurima.n = 3.Umwanya munini = 100 µm.
Kuvumbura no gukoresha ibicuruzwa bishya byatumye habaho iterambere ryinshi mubice bitandukanye byubuzima bwabantu, harimo ubuvuzi nubuhinzi.Ubushakashatsi bwamateka bwakozwe kugirango haboneke ibintu byingirakamaro biva mumitungo kamere.By'umwihariko, actinomycetes izwiho kuba ingirakamaro nka antibiyotike ya antiparasitike ya nematode bitewe n'ubushobozi bwabo bwo gukora metabolite itandukanye ya kabiri nka avermectin, ikomatanya rya ivermectin na bleomycine n'ibiyikomokaho, bikoreshwa mu buvuzi nka anticancer agent21,22.Mu buryo nk'ubwo, ibimera bitandukanye biva mu bimera byavumbuwe muri actinomycetes, bimwe muri byo bikaba bimaze gukoreshwa mu bucuruzi1,23.Kubwibyo, isesengura rya metabolite ya actinomycete yo gutandukanya ibicuruzwa karemano nibikorwa byibinyabuzima bifatwa bifatwa nkingamba zifatika.Muri ubu bushakashatsi, twavumbuye uruganda rushya, coumamonamide, ruva muri S. werraensis hanyuma turagereranya neza.Acide Ursonic ni intangiriro ya urbenamide n'ibiyikomokaho.Irashobora gutera imizi iranga gutondeka, kwerekana ibikorwa biciriritse kandi bikomeye, kandi byangiza bitaziguye cyangwa bitaziguye microtubules.Nyamara, uburyo bwibikorwa bya acide ya urmotonic irashobora gutandukana nubwa microtubule inhibitor zihari, kubera ko KAND 11 nayo ihagarika filime ya actin kandi igatera urupfu rwingirabuzimafatizo, byerekana uburyo bwo kugenzura aside urmotonic nibiyikomokaho bigira uruhare runini muburyo bwa cytoskeletal..
Ibindi bisobanuro biranga aside urbenonic bizafasha gusobanukirwa neza nuburyo bwibikorwa bya acide ya urbenonic.By'umwihariko, intego ikurikira ni ugusuzuma ubushobozi bwa aside ya ursonic guhuza na microtubules yagabanutse kugirango hamenyekane niba aside ya ursonic n'ibiyikomokaho bikora kuri microtubules no kubitandukanya, cyangwa niba ibikorwa byabo bivamo ihungabana rya microtubule.Byongeye kandi, mugihe microtubules itari intego itaziguye, kumenya aho ibikorwa byakorewe hamwe na molekuline yibasiwe na aside ya ursonic kuri selile yibimera bizafasha kurushaho gusobanukirwa nimiterere yibintu bifitanye isano nuburyo bushoboka bwo kunoza ibikorwa byibyatsi.Ubushakashatsi bwakozwe na bioactivite bwerekanye ubushobozi budasanzwe bwa cytotoxique ya aside ya ursonic ku mikurire y’ibimera nka Arabidopsis thaliana, itabi n’umwijima, mu gihe E. coli cyangwa HeLa selile zagize ingaruka.Uburozi buke cyangwa butagira ingirabuzimafatizo zinyamanswa ninyungu zikomoka kuri acide ya ursonic niba zakozwe nka herbiside zo gukoresha mumirima yubuhinzi ifunguye.Mubyukuri, kubera ko microtubules ari ibintu bisanzwe muri eukaryote, kubuza guhitamo ibimera nikintu cyingenzi gisabwa kubimera.Kurugero, propyzamide, microtubule depolymerizing agent ihuza na tubuline kandi ikabuza polymerisiyonike, ikoreshwa nka herbicide kubera uburozi buke bwingirabuzimafatizo 24.Bitandukanye na disopyramide, benzamide ifitanye isano ifite intego zitandukanye.Usibye ibimera bya microtubules, RH-4032 cyangwa benzoxamide inabuza microtubules yingirangingo zinyamaswa cyangwa oomycetes, kandi zalilamide ikoreshwa nka fungiside kubera phytotoxicite nkeya 25,26,27.Idubu rimaze kuvumburwa n'ibiyikomokaho byerekana cytotoxicitike ihitamo ibimera, ariko birakwiye ko tumenya ko ibindi byahinduwe bishobora guhindura umwihariko wabyo, bikaba bishobora gutanga ibikomoka ku bindi byo kurwanya ibihumyo cyangwa oomycetes.
Imiterere yihariye ya acide ya urbenonic nibiyikomokaho ni ingirakamaro mu iterambere ryabo nka herbiside kandi ikoreshwa nkibikoresho byubushakashatsi.Akamaro ka cytoskeleton mugucunga imiterere yibimera bizwi cyane.Ubushakashatsi bwakozwe mbere bwerekanye ko ibimera byahinduye uburyo bukomeye bwimikorere ya cortical microtubule mugucunga microtubule kugirango igenzure neza morfogenezi.Umubare munini wa molekile ushinzwe kugenzura ibikorwa bya microtubule wamenyekanye, kandi ubushakashatsi bujyanye buracyakomeza3,4,28.Kugeza ubu gusobanukirwa kwimikorere ya microtubule muri selile yibihingwa ntabwo isobanura neza uburyo bwimikorere ya cortical microtubule.Kurugero, nubwo disopyramide na oryzalin byombi bishobora kugabanya microtubules, disopyramide itera kugoreka imizi mugihe oryzalin igira ingaruka zoroheje.Byongeye kandi, ihinduka rya tubuline, rihindura microtubules, naryo ritera dextrorotation mu mizi, mu gihe paclitaxel, nayo ihindura imbaraga za microtubule, ntabwo.Kubwibyo, kwiga no kumenya intego ya molekile ya acide ya ursolique igomba gutanga ubumenyi bushya mugutunganya microtubules yibimera.Mu buryo nk'ubwo, kugereranya ejo hazaza hifashishijwe imiti igira uruhare runini mu kuzamura imikurire igoramye, nka disopyramide, hamwe n’imiti idakora neza nka oryzalin cyangwa aside kumamotoric, bizatanga ibimenyetso byerekana uko imikurire igoramye iba.
Ku rundi ruhande, uburyo bwo kwirinda bwerekeranye na cytoskeletal bundi buryo bushoboka bwo gusobanura cytotoxicity ya aside ya ursonic.Kwanduza virusi cyangwa kwinjiza elicitor mu ngirabuzimafatizo rimwe na rimwe bitera kurimbuka kwa cytoskeleton ndetse no gupfa kwakagari29.Kurugero, oomycete ikomoka kuri cryptoxanthin yavuzwe ko ihungabanya microtubules na actin filaments mbere yurupfu rwa selile y itabi, bisa nibibaho no kuvura KAND 30,31.Isano riri hagati y ibisubizo byokwirwanaho hamwe nibisubizo bya selile byatewe na aside ya ursonic byatumye dushidikanya ko bitera inzira zisanzwe, nubwo ingaruka yihuse kandi ikomeye ya acide ursonic kuruta cryptoxanthin igaragara.Nyamara, ubushakashatsi bwerekanye ko guhagarika filime ya actin itera urupfu rwimikorere ya selile, itajyana buri gihe na microtubule ihungabana29.Byongeye kandi, hasigaye kurebwa niba virusi cyangwa elicitor itera imizi igoramye, nkuko inkomoko ya aside ya ursonic ibikora.Rero, ubumenyi bwa molekile ihuza ibisubizo byokwirwanaho na cytoskeleton nikibazo gishimishije gukemurwa.Mugukoresha ahari uburemere buke bwa molekuline ijyanye na acide ya ursonic, hamwe nibikomokaho bifite imbaraga zitandukanye, barashobora gutanga amahirwe yo kwibasira imikorere ya selile itazwi.
Ufatiye hamwe, kuvumbura no gushyira mubikorwa ibice bishya bigabanya imbaraga za microtubule bizatanga uburyo bukomeye bwo gukemura uburyo bukomeye bwimikorere ya molekile ishingiye kumiterere yimiterere yimiterere yibimera.Ni muri urwo rwego, aside irmotonic iherutse guterwa, ifata microtubules na actin filaments kandi igatera urupfu rwa selile, irashobora gutanga amahirwe yo gusobanura isano iri hagati yo kugenzura microtubule nubundi buryo.Rero, isesengura ryibinyabuzima na biologiya ukoresheje aside urbenonic bizadufasha gusobanukirwa nuburyo bwimikorere ya molekuline igenzura ibihingwa cytoskeleton.
Shyira S. werraensis MK493-CF1 mumashanyarazi ya mL 500 ya Erlenmeyer irimo urujijo rurimo mL 110 yimbuto igizwe na galaktose ya 2% (w / v), 2% (w / v) Paste ya Essence, 1% (w / v) Ibigize Bacto .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) ibigori bivamo ibigori (KOGOSTCH Co., Ltd., Ubuyapani), 0.2% (w / v) (NH4) 2SO4 na 0.2% CaCO3 mumazi yimana.(pH 7.4 mbere yo kuboneza urubyaro).Imico yimbuto yashizwemo kumashanyarazi (180 rpm) kuri 27 ° C muminsi 2.Guhinga umusaruro ukoresheje fermentation ya leta ikomeye.Umuco w'imbuto (7 ml) wimuriwe muri ml 500 ya K-1 irimo 40 g yumusaruro ugizwe na g 15 ya sayiri ikanda (MUSO Co., Ltd., Ubuyapani) na g 25 zamazi ya deioni (pH ntabwo yahinduwe mbere yo kuboneza urubyaro).).Gusembura byakozwe kuri 30 ° C mu mwijima iminsi 14.Ibikoresho bya fermentation byakuwe hamwe na ml 40 / icupa EtOH hamwe na centrifuged (1500 g, 4 ° C, min 10).Umuco ndengakamere (ml 60) wakuwe hamwe nuruvange rwa 10% MeOH / EtOAc.Urwego kama rwashizwemo umwuka mubi wagabanutse kugirango haboneke ibisigisigi (59.5 mg), byakorewe HPLC hamwe no gukuraho buhoro buhoro (iminota 0-10: 90%) kumurongo winyuma (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × uburebure bwa mm 250) H2O / CH3CN, iminota 10-35: 90% H2O / CH3CN kugeza 70% H2O / CH3CN (gradient), iminota 35-45: 90% H2O / EtOH, iminota 45-155: 90% H2O / EtOH kugeza kuri 100% EtOH (gradient)
Kumamotoamide (1);1H-NMR (500 MHz, CDCl3) δ 6.93 (t, J = 2.5 Hz, 1H), 6.76 (dd, J = 4.3, 1.8 Hz 1H), 6.05 (t, J = 3.8 Hz, 1H).), 4.08 (s, 3H);13C-NMR (125 MHz, CDCl3) δ 161.1, 121.0, 119.9, 112.2, 105.0, 68.3;ESI-HRMS [M + H] +: [C6H9N22
Imbuto za Columbia (Col-0) zabonetse muri Arabidopsis Biological Resource Centre (ABRC) uruhushya rwo gukoresha ubushakashatsi.Imbuto ya Col-0 yarakwirakwijwe kandi ikomeza kubungabungwa muri laboratoire kandi ikoreshwa nkibimera byo mu bwoko bwa Arabidopsis.Imbuto za Arabidopsis zashyizwe hejuru kandi zihingwa mu gice cya kabiri cyimbaraga za Murashige na Skoog zirimo 2% sucrose (Fujifilm Wako Pure Chemical), 0,05% (w / v) 2- (4-morpholino) aside Ethanesulfonike (MES) (Fujifilm Wako Pure Chemical) ).) na 1.5% agar (Fujifilm Wako Chemical Pure), pH 5.7, kuri 23 ° C n'umucyo uhoraho.Imbuto za phs1-1 mutant zatanzwe na T. Hashimoto (Nara Institute of Science and Technology).
Imbuto zo mu bwoko bwa SR-1 zatanzwe na T. Hashimoto (Nara Institute of Science and Technology) kandi zikoreshwa nk'ibihingwa by'itabi byo mu gasozi.Imbuto z'itabi zashizwe hejuru hanyuma zishirwa mumazi meza mugihe cyamajoro atatu kugirango ziteze kumera, hanyuma zishyirwa mubisubizo byimbaraga zirimo igice cya 2% sucrose, 0,05% (w / v) MES, na 0.8% gum glan (Fujifilm Wako Pure Chemical) Murashige.na Skoog medium) hamwe na pH 5.7 kandi yashyizwe kuri 23 ° C munsi yumucyo uhoraho.
Strain Tak-1 yatanzwe na T. Kohchi (Kaminuza ya Kyoto) kandi yakoreshejwe nkigice gisanzwe cyubushakashatsi kubushakashatsi bwumwijima.Gemma yakuwe mu bimera by’umuco hanyuma bigashyirwa ku gikoresho cya Gamborg B5 (Fujifilm Wako Pure Chemical) kirimo 1% sucrose na geli ya 0.3% kandi byashyizwe kuri 23 ° C munsi yumucyo uhoraho.
Itabi BY-2 selile (Nicotiana tabacum L. cv. Bright Umuhondo 2) ryatanzwe na S. Hasezawa (Kaminuza ya Tokiyo).Ingirabuzimafatizo za BY-2 zavomwe inshuro 95 muburyo bwa Linsmeier na Skoog zahinduwe kandi zongerwaho buri cyumweru hamwe na acide 2,4-dichlorophenoxyacetic 32.Guhagarika ingirabuzimafatizo byavanze kuri shitingi izunguruka saa 130 rpm kuri 27 ° C mu mwijima.Koza selile hamwe ninshuro 10 zingana nuburyo bushya hanyuma uhagarike muburyo bumwe.BY-2 imirongo ya selile transgenic yerekana neza ikimenyetso cya microtubule TagRFP-TUA6 cyangwa ikimenyetso cya actin filament GFP-ABD2 munsi ya virusi ya mosaic virusi 35S yamamaza nkuko byasobanuwe 33,34,35.Iyi mirongo ya selile irashobora kubungabungwa no guhuzwa hakoreshejwe inzira isa niyakoreshejwe kumurongo wambere wa BY-2.
Utugingo ngengabuzima twa HeLa twakuze mu buryo bwa Dulbecco bwahinduwe na Eagle (DMEM) (Life Technologies) hiyongeraho 10% ya serumu y'inda y'inda, 1.2 U / ml penisiline, na 1.2 μg / ml streptomycine muri 37 ° C hamwe na 5% CO2.
Ubushakashatsi bwose bwasobanuwe muri iyi nyandiko yandikishijwe intoki bwakozwe hakurikijwe amabwiriza n’ubuyobozi bw’Ubuyapani.
Ibicuruzwa byashongeshejwe muri dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) nkibisubizo byimigabane hanyuma bivangwa muri MS medium ya Arabidopsis n itabi cyangwa Gamborg B5 ikoresheje umwijima.Kugirango imizi ikure neza, imbuto zirenga 10 kuri buri sahani zabibwe kuri agar medium irimo ibice byerekanwe cyangwa DMSO.Imbuto zashyizwe mu cyumba cyo gukura iminsi 7.Ingemwe zafotowe kandi hapimwa uburebure bwimizi.Kubyerekeranye no kumera kwa Arabidopsis, imbuto 48 kuri buri sahani zabibwe kuri agar medium irimo 200 μM compound cyangwa DMSO.Imbuto za Arabidopsis zahinzwe mu cyumba cyo gukura kandi umubare w’ingemwe zimaze kubarwa nyuma yiminsi 7 zimaze kumera (dag).Kubijyanye no kumera kw'itabi, imbuto 24 kuri buri sahani zabibwe kuri agar medium irimo 200 μM KAND cyangwa DMSO.Imbuto z'itabi zahinzwe mu cyumba cyo gukura kandi umubare w'ingemwe zimaze kubarwa nyuma y'iminsi 14.Kubijyanye no gukura kwumwijima, insoro 9 kuri buri sahani zashyizwe ku cyuma cya agar kirimo ubunini bwa KAND cyangwa DMSO kandi byashyizwe mu cyumba cyo gukura iminsi 14.
Koresha ingemwe zandujwe na mg / ml 5 ya propidium iyode (PI) kugirango ubone ishusho ya meristem.Ibimenyetso bya PI byagaragaye na microscopi ya fluorescence ikoresheje TCS SPE conocal laser scanning microscope (Leica Microsystems).
Gusiga amateka ya gistochemiki hamwe na β-glucuronidase (GUS) byakozwe hakurikijwe protocole yasobanuwe na Malami na Benfey36.Imbuto zashyizwe muri 90% acetone ijoro ryose, zisiga 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acide muri buffer ya GUS kumasaha 1 hanyuma igashyirwa mumuti wa chloraldehyde..
Inguni zumuzi zapimwe ku mbuto zimaze iminsi 7 zikura ku masahani ashyizwe.Gupima inguni yumuzi uhereye ku cyerekezo cya rukuruzi ya rukuruzi nkuko byasobanuwe mu ntambwe ya 6.
Gahunda ya microtubules ya cortical yagaragaye nkuko byasobanuwe, hamwe na bike byahinduwe kuri protocole 37.Antibody anti-β-tubuline (KMX-1, Merk Millipore: MAB3408) na Alexa Fluor 488-conjugated anti-imbeba IgG (Thermo Fisher Scientific: A32723) yakoreshejwe nka antibodi yibanze nayisumbuye kuri 1: 1000 na 1: 100, bikurikiranye.Amashusho ya Fluorescence yabonetse hakoreshejwe TCS SPE ya conocal laser scanning microscope (Leica Microsystems).Shaka Z-stack amashusho hanyuma ukore ubukana ntarengwa ukurikije amabwiriza yabakozwe.
Ikwirakwizwa rya selile ya HeLa ryakozwe hifashishijwe ibikoresho byo kubara selile 8 (Dojindo) ukurikije amabwiriza yabakozwe.
Iterambere rya E. coli DH5α ryasesenguwe no gupima ubwinshi bwakagari mu muco ukoresheje spekitifotometero kuri 600 nm (OD600).
Ishirahamwe rya Cytoskeletal muri selile ya BY-2 ryagaragaye hakoreshejwe microscope ya fluorescence ifite ibikoresho byo gusikana CSU-X1 (Yokogawa) hamwe na kamera ya sCMOS (Zyla, Ikoranabuhanga rya Andor).Ubucucike bwa Cytoskeletal bwasuzumwe nisesengura ryamashusho, ryagereranije ijanisha rya pigiseli ya cytoskeletal hagati ya pigiseli ya cytoplasmeque mumashusho asebanya ukoresheje software ya ImageJ nkuko byasobanuwe 38,39.
Kugirango umenye urupfu rw'utugingo ngengabuzima BY-2, alikot yo guhagarika ingirabuzimafatizo yashyizwemo 0,05% Evans ubururu mu minota 10 ku bushyuhe bw'icyumba.Guhitamo Evans ibara ry'ubururu bwa selile zapfuye biterwa no gusohora irangi muri selile zifatika na plasma membrane40.Ingirabuzimafatizo zanduye zagaragaye hakoreshejwe microscope yumucyo (BX53, Olympus).
Ingirabuzimafatizo za HeLa zahinzwe muri DMEM zongerwaho 10% FBS muri incubator itose kuri 37 ° C na 5% CO2.Ingirabuzimafatizo zavuwe hamwe na 100 μM KAND 11, aside kumamonamic 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), cyangwa 100 ng / ml Nocodmaze (Sigma) kuri 6 h kuri 37 ° C.Ingirabuzimafatizo zashyizwe hamwe na MetOH kuminota 10 hanyuma hamwe na acetate kuminota 5 mubushyuhe bwicyumba.Ingirabuzimafatizo zihamye zashyizwemo antibody y'ibanze ya tub-tubuline (1D4A4, Proteintech: 66240-1) yavanze muri 0.5% BSA / PBS mu masaha 2, yoza inshuro 3 hamwe na TBST, hanyuma yinjizwamo antibody y'ihene ya Alexa Fluor.488 Isaha 1.- Imbeba IgG (Thermo Fisher Scientific: A11001) na 15 ng / ml 4 ′, 6-diamidino-2-fenylindole (DAPI) ivanze muri 0.5% BSA / PBS.Nyuma yo gukaraba hamwe na TBST inshuro eshatu, selile zanduye zagaragaye kuri microscope ya Nikon Eclipse Ti-E.Amashusho yafashwe hamwe na kamera ikonje ya Hamamatsu ORCA-R2 CCD ikoresheje software ya MetaMorph (Ibikoresho bya Molecular).
Igihe cyo kohereza: Jun-17-2024