Ndatenda nekushanyira Nature.com.Shanduro yebrowser yauri kushandisa ine tsigiro yeCSS shoma.Kuti uwane mibairo yakanaka, tinokurudzira kuti ushandise shanduro itsva yebrowser yako (kana kudzima Compatibility Mode muInternet Explorer).Zvichakadaro, kuti tive nechokwadi cherutsigiro runoenderera, tiri kuratidza saiti isina kutaera kana JavaScript.
Kuwanikwa uye kushandiswa kunobatsira kwezvinhu zvakasikwa kunogona kubatsira kuvandudza hupenyu hwevanhu.Mishonga inodzivirira kukura kwezvirimwa inoshandiswa zvakanyanya semishonga yekurwisa sora.Nekuda kwekushandiswa kwemhando dzakasiyana dzemishonga yesora, panoda kuziva makomisheni ane nzira itsva dzekuita.Muchidzidzo ichi, takawana riini N -alkoxypyrrole compound, coumamonamide, kubva kuStreptomyces werraensis MK493-CF1 uye yakagadzira iyo yakakwana synthesis process.Kuburikidza nebiological chiitiko assays, takaona kuti urs-monoamic acid inogadzirwa yepakati yeurs-monoamide uye inogona.chirimwa kukura inhibitor.Pamusoro pezvo, takagadzira zvakasiyana siyana zveurbenonic acid, zvinosanganisira urbenyloxy derivative (UDA), iyo ine high herbicidal kuita isingakanganise kukura kwemasero eHeLa.Takaonawo kuti urmotonic acid derivatives inovhiringidza ma microtubules emiti;uyezve, KAND inokanganisa actin filaments uye inokonzera kufa kwesero;Izvi zvakasiyana-siyana zvinokonzerwa neaya anozivikanwa microtubule inhibitors uye anopa nzira itsva yekuita ursonic acid, iyo inomiririra zvakakosha mukugadzirwa kwemishonga mitsva yesora.
Kuwanikwa uye kushandiswa kunoshanda kwezvigadzirwa zvechisikigo zvinobatsira uye zvinobva kwavari inzira yekuvandudza hupenyu hwemunhu.Sekondari metabolites inogadzirwa nema microorganisms, zvidyarwa uye zvipembenene zvakakonzera kufambira mberi kukuru mukurapa nekurima.Mishonga yakawanda inorwisa mabhakitiriya uye anti-leukemia yakagadzirwa kubva kune zvigadzirwa zvepanyama.Mukuwedzera, marudzi akasiyana-siyana emishonga yezvipembenene, fungicides nemishonga yesora inotorwa kubva muzvigadzirwa zvechisikigo izvi kuti zvishandiswe mukurima.Kunyanya, mishonga yekudzivirira sora zvishandiso zvakakosha zvekuwedzera goho mukurima kwemazuva ano, uye mhando dzakasiyana dzemakomboni dzatove kushandiswa mukutengesa.Maitiro akati wandei muzvirimwa, senge photosynthesis, amino acid metabolism, cell wall synthesis, mutemo we mitosis, phytohormone siginecha, kana protein synthesis, inoonekwa seyakajairika tarisiro yemishonga yesora.Mishonga inodzivisa microtubule kushanda ikirasi yakajairika yemishonga yesora inokanganisa kukura kwechirimwa nekukanganisa mitotic regulation2.
Microtubules zvikamu zve cytoskeleton uye zvakachengetwa zvakanyanya mumasero eukaryotic.Iyo tubulin heterodimer ine α-tubulin uye β-tubulin inogadzira linear microtubule protofilaments, ine 13 protofilaments inogadzira chimiro checylindrical.Microtubules inotamba mabasa akawanda mumasero ezvirimwa, kusanganisira kuona sero chimiro, kupatsanurwa kwesero, uye intracellular transport3,4.Masero emiti ane microtubules pasi pe interphase plasma membrane, uye aya anonzi cortical microtubules anofungidzirwa kuti anodzora sangano re cellulose microfibrils kuburikidza nekugadzirisa cellulose synthase complexes4,5.Cortical microtubules yemidzi epidermal masero, aripo munzvimbo yekukurumidza kureba kwemudzi wetipi, anowanikwa nechemberi, uye cellulose microfibers anotevera mamicrotubules aya uye anodzikamisa nzira yekuwedzera kwesero, nekudaro zvinosimudzira anisotropic cell elongation.Naizvozvo, basa re microtubule rakabatana zvakanyanya nekudyara morphology.Amino acid inotsiva mumajini encoding tubulin inokonzera skew ye cortical microtubule arrays uye kuruboshwe- kana kurudyi-kurutivi kukura muArabidopsis 6,7.Saizvozvowo, kuchinja kwemapuroteni ane microtubule-akabatanidzwa anodzora microtubule dynamics anogonawo kutungamirira kune kukanganisa midzi kukura8,9,10,11,12,13.Mukuwedzera, kurapwa ne microtubule-kuvhiringidza herbicides yakadai se disopyramide, inozivikanwawo se pretilachlor, inokonzerawo kuruboshwe-sided oblique root growth14.Aya data anoratidza kuti kurongeka chaiko kweiyo microtubule basa kwakakosha pakuona mafambiro ekukura kwechirimwa.
Mhando dzakasiyana-siyana dze microtubule inhibitors dzakawanikwa, uye mishonga iyi yakaita zvakakosha pakutsvakurudza kwe cytoskeletal, pamwe nekurima nemishonga2.Kunyanya, oryzalin, dinitroaniline compounds, disopyramide, benzamide-related compounds, uye analogs avo anogona kudzivisa microtubule basa uye nokudaro kudzivisa kukura kwemiti.Nokudaro, anoshandiswa zvakanyanya semishonga yesora.Nekudaro, sezvo ma microtubules ari chinhu chakakosha chezvirimwa nemhuka maseru, mazhinji microtubule inhibitors ari cytotoxic kune ese ese masero.Nokudaro, pasinei nekushandiswa kwavo kunozivikanwa semishonga yesora, nhamba shoma ye antimicrotubule agents inoshandiswa pazvinangwa zvinoshanda.
Streptomyces iboka remhuri yeStreptomyces, iyo inosanganisira aerobic, gram-positive, filamentous bacteria uye inozivikanwa zvikuru nekukwanisa kwayo kuburitsa huwandu hwechipiri metabolites.Naizvozvo, inoonekwa seimwe yeakanyanya kukosha masosi ezvitsva biologically inoshanda zvakasikwa zvigadzirwa.Muchidzidzo chemazuva ano, takawana chikwata chitsva chinonzi coumamonamide, icho chakanga chakaparadzaniswa kubva kuStreptomyces werraensis MK493-CF1 uye S. werraensis ISP 5486. Kushandisa spectral analysis uye full spectral analysis, chimiro che coumamonamide chakaonekwa uye chakasiyana cheN-alkoxypyrrole skeleton. yakanga yakatsunga.synthesis.Ursmonic acid, inogadzirwa yepakati yeursmonoamide uye zvinobva pairi, yakawanikwa ichitadzisa kukura uye kumera kweiyo yakakurumbira modhi chirimwa Arabidopsis thaliana.Muchidzidzo chehukama-chiito chehukama, takaona kuti komboni ine C9 yakagadziridzwa kuita ursonic acid, inonzi nonyloxy derivative yeursonic acid (KAND), inosimudzira zvakanyanya inhibitory mhedzisiro pakukura nekumera.Zvikuru, iyo ichangobva kuwanikwa chirimwa yekukura inhibitor yakakanganisawo kukura kwefodya uye liverwort uye yakanga isiri cytotoxic kubhakitiriya kana masero eHeLa.Uyezve, mamwe urmotonic acid anobuda anokonzera kukanganisa mudzi phenotype, zvichireva kuti izvi zvinobva kune zvakananga kana zvisina kunanga zvinokanganisa microtubules.Zvinoenderana nepfungwa iyi, macherechedzo edu emamicrotubules akanyorwa angave immunohistochemically kana nemapuroteni efluorescent anoratidza kuti kurapwa kweKAND kunobvisa microtubules.Mukuwedzera, kurapwa nekumamotonic acid derivatives kwakavhiringidza actin microfilaments.Nekudaro, isu takawana nyowani yekukura kwechirimwa inhibitor ine yakasarudzika nzira yekuita inosanganisira kuparadzwa kwe cytoskeleton.
Strain MK493-CF1 yakaparadzaniswa nevhu muShinagawa-ku, Tokyo.Strain MK493-CF1 yakagadzira zvakanaka-bazi stromal mycelium.Kutevedzana kwechikamu che16S ribosomal RNA gene (1422 bp) kwakatemwa.Iri dambudziko rakanyanya kufanana neS. werraensis (NBRC 13404T = ISP 5486, 1421 / 1422 bp, T: strain strain, 99.93%).Zvichienderana nemhedzisiro iyi, zvakatemwa kuti rudzi urwu rwakanga rwakabatana zvakanyanya nerudzi rweS. werraensis.Naizvozvo, takapa zita rekuti rudzi urwu S. werraensis MK493-CF1.S. werraensis ISP 5486T inogadzirawo zvakafanana bioactive compounds.Sezvo pakanga paine tsvakiridzo yepakutanga yekuwana zvigadzirwa zvechisikigo kubva kune iyi microorganism, kumwe kuongorora kwemakemikari kwakaitwa.Mushure mekurima kweS. werraensis MK493-CF1 pabhari yepakati nekuvirisa kwakasimba-nyika pa30 ° C kwemazuva gumi nemana, iyo yepakati yakatorwa ne50% EtOH.60 ml yesampuli yakaomeswa kuti iwane 59.5 mg ye crude extract.Iyo isina kucheneswa yakatorwa yakaiswa reverse chikamu HPLC kupa N-methoxy-1H-pyrrole-2-carboxamide (1, inonzi coumamonamide, 36.0 mg).Huwandu hwe1 hunenge 60% yehutsvina yakabviswa.Naizvozvo, takasarudza kudzidza zvakadzama zvimiro zvekumamotoamide 1.
Coumamonamide 1 is white amorphous powder uye high resolution mass spectrometry (HRESIMS) inosimbisa C6H8N2O2 (Fig. 1).C2-inotsiva pyrrole fragment yeiyi komputa inoratidzwa ne δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH mu 1H NMR 5 Hz: 4. , H-5) uye δH 6.78 (1H, d, J = 2.5 Hz, H-6), uye 13C NMR spectrum inoratidza kuvepo kwema sp2 carbon atomu.Kuvapo kweboka reamide panzvimbo yeC2 yakaongororwa neHMBC kuwirirana kubva kuC-3 proton kusvika kune amide carbonyl carbon pa δC 161.1.Mukuwedzera, 1 H uye 13 C NMR inokwira pa δH 4.10 (3H, S) uye δC 68.3 inoratidza kuvapo kwemapoka eN-methoxy mumorekuru.Kunyangwe nzvimbo chaiyo yeboka remethoxy yanga isati yatemerwa pachishandiswa spectroscopic ongororo senge yakawedzera mutsauko spectroscopy uye nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide yakava yekutanga mumiriri musanganiswa.
Kuti uone chimiro chakarurama che1, kusanganiswa kwese kwakaitwa (Fig. 2a).Kurapa kwe2-aminopyridine 2 inotengeswa ine m-CPBA yakaguma neN-oxide 3 inoenderana muhuwandu hwegoho.Mushure me2-aminoazidation ye2, cyclocondensation reaction yakatsanangurwa naAbramovich yakaitwa mubenzene pa90 ° C kuti iwane yaidiwa 1-hydroxy-1H-pyrrole-2-carbonitrile 5 mumagiramu.Speed 60% (matanho maviri).15, 16.Methylation uye hydrolysis ye4 yakabva yapa 1-methoxy-1H-pyrrole-2-carboxylic acid (inonzi "cumotonic acid", 6) mugoho rakanaka (70%, matanho maviri).Pakupedzisira, amidation kuburikidza neacid chloride yepakati 6 uchishandisa aqueous ammonia yakapa Kumamoto amide 1 mu98% goho.Yese spectral data ye synthesized 1 yakanga yakafanana neyo yega 1, saka chimiro che1 chakatemwa;
General synthesis uye kuongororwa kweiyo biological chiitiko cheurbenamide uye urbenic acid.(a) Total synthesis yeKumamoto amide.(b) Mbeu dzemazuva manomwe dzesango dzeArabidopsis Columbia (Col) dzakarimwa paMurashige neSkoog (MS) mahwendefa ane coumamonamide 6 kana coumamonamide 1 pane zvakaratidzwa.Scale bar = 1 cm.
Chekutanga, isu takaongorora zvipenyu zviitwa zveurbenamide uye zvepakati zvekugona kwavo kuenzanisa kukura kwechirimwa.Takawedzera zvakasiyana-siyana zveursmonamide 1 kana ursmonic acid 6 kune MS agar yepakati uye yakasimwa midzi yeArabidopsis thaliana pane iyi svikiro.Iyi miedzo yakaratidza kuti kukwirira kwakanyanya (500 μM) ye6 inhibited root root (Fig. 2b).Tevere, takagadzira zvakasiyana-siyana zvakatorwa nekutsiva iyo N1 chinzvimbo che6 uye kuita chimiro-chiitwa zvidzidzo zvehukama pavari (iyo analogue synthesis process inotsanangurwa muRuzivo Rwokutsigira (SI)).Mbeu dzeArabidopsis dzakarimwa pasvikiro rine 50 μM ursonic acid derivatives, uye kureba kwemidzi kwakayerwa.sezvazvinoratidzwa pamufananidzo.Sezvinoratidzwa muFigure 3a, b, uye S1, coumamo acids ine hurefu hwakasiyana hwemutsara alkoxy cheni (9, 10, 11, 12, uye 13) kana hombe alkoxy cheni (15, 16, uye 17) panzvimbo yeN1.Zvinyorwa zvakaratidza kudziviswa kukuru kwekukura kwemidzi.Mukuwedzera, takaona kuti kushandiswa kwe200 μM 10, 11, kana 17 kunodzivisa kumera (Figs. 3c uye S2).
Kudzidza kwechimiro-chiitwa hukama hweKumamoto amide uye ane hukama makomisheni.(a) Chimiro uye synthesis chirongwa cheanalogues.(b) Huwandu hwehurefu hwemidzi yemhodzi dzemazuva manomwe dzakakura paMS medium ine kana isina 50 μM coumamonamide derivatives.Asterisks inoratidza misiyano yakakura nekurapa kwesham (t bvunzo, p<0.05).n>18. Data inoratidzwa sezvinoreva ± SD.nt zvinoreva kuti “haina kuongororwa” nekuti inodarika 50% yembeu haina kumera.(c) Huyero hwekumera kwembeu dzakabikwa dzakaiswa kwemazuva manomwe muMS medium ine kana isina 200 μM coumamonamide nemamwe makomisheni ane hukama.Asterisks inoratidza misiyano yakakura nesham kurapwa (chi-square test).n=96.
Sezvineiwo, kuwedzerwa kwealkyl side chains kureba kupfuura C9 kwakaderedza inhibitory basa, zvichiratidza kuti kumamotoic acid-inoenderana makomisheni inoda macheni epadivi ehumwe saizi kuratidza basa ravo rehupenyu.
Nokuti chimiro-chiito chehukama chekuongorora chakaratidza kuti C9 yakagadziridzwa kuva ursonic acid uye nonyloxy derivative yeursonic acid (inozonzi KAND 11) ndiyo yakanyanya kubudirira yekukura kwemiti inhibitor, takaita humwe hunhu hweKAND 11. Kurapa kweArabidopsis. ne 50 μM KAND 11 inenge yakadzivirirwa zvachose kumera, nepo kuderera kwakadzika (40, 30, 20, kana 10 μM) yeKAND 11 yakadzivisa kukura kwemidzi nenzira inoenderana nedosi (Fig. 4a, b).Kuongorora kana KAND 11 inokanganisa midzi meristem kushanda, takaongorora midzi meristem yakasvibiswa nepropidium iodide (PI) uye kuyerwa meristem nharaunda saizi.Hukuru hwemhodzi yemadzinde akarimwa pasvikiro rine 25 μM KAND-11 hwaive 151.1 ± 32.5 μm, ukuwo ukuru hwemadzinde akarimwa pachishandiswa chekudzora chine DMSO chaive 264.7 ± 30.8 μm (Fig. 4c, d) , iyo inoratidza kuti KAND-11 inodzoreredza kuita kweserura.kupararira.Root meristem.Zvinoenderana neizvi, kurapwa kweKAND 11 kwakaderedza kuwanda kwesero division marker CDKB2; 1p :: CDKB2; 1-GUS chiratidzo mumudzi meristem (Fig. 4e) 17.Mhedzisiro iyi inoratidza kuti KAND 11 inhibits kukura kwemidzi nekudzikisa basa rekuwedzera kwesero.
Kuongororwa kweiyo inhibitory mhedzisiro yeurbenonic acid derivatives (urbenyloxy derivatives) pakukura.A(b) Kuwanda kwehurefu hwemidzi.Tsamba dzinoratidza misiyano yakakura (Tukey HSD bvunzo, p<0.05).n>16. Data inoratidzwa sezvinoreva ± SD.(c) Confocal microscopy yepropidium iodide-stained wild-type Col midzi inokura paMS plates ine kana isina 25 μM KAND 11. Mabhuraketi machena anoratidza midzi meristem.Scale bar = 100 µm.(d) Quantification yemidzi meristem size (n = 10 kusvika 11).Misiyano yehuwandu yakatemwa uchishandisa t-bvunzo (p<0.05).Mabhawa anomiririra avhareji meristem size.(e) Differential interference musiyano (DIC) microscopy yemudzi meristem ine CDKB2 kuvaka;1pro: CDKB2;1-GUS yakasvibiswa uye yakasvibiswa pamadzinde ane mazuva mashanu akakura paMS plates ane kana asina 25 µM KAND assay.
Iyo phytotoxicity yeKAND 11 yakawedzera kuedzwa uchishandisa imwe dicotyledonous chirimwa, fodya (Nicotiana tabacum), uye hombe yemiti yemhando yemiti, liverwort (Marchantia polymorpha).Sezviri kuitika kuArabidopsis, mbesa dzefodya SR-1 dzakarimwa papakati dzine 25 μM KAND 11 dzakabereka midzi mipfupi (Fig. 5a).Pamusoro pezvo, makumi mana embeu makumi mana nesere dzakamera pamahwendefa ane 200 μM KAND 11, nepo mhodzi makumi mana nesere dzakamera pama media ekunyomba, zvichiratidza kuti huwandu hwepamusoro hweKAND hwaive hwakakosha (p.< 0.05;chi test -square) inodzivisa kumera kwefodya.(Mufananidzo 5b).Mukuwedzera, kusungirirwa kweKAND 11 iyo yakadzivisa kukura kwebhakitiriya muroparwort yakanga yakafanana nekugadzirisa kunobudirira muArabidopsis (Fig. 5c).Mhedzisiro iyi inoratidza kuti KAND 11 inogona kutadzisa kukura kwembesa dzakasiyana.Takazoongorora zvingaita cytotoxicity yebere monoamide-inoenderana nemakemikari mune zvimwe zvipenyu, kureva masero eHeLa evanhu uye Escherichia coli strain DH5α, sevamiririri vemasero emhuka akakwirira uye mabhakitiriya, maererano.Muchikamu chekuongorora kwekuwedzera kwemasero, takaona kuti coumamonamide 1, coumamonamidic acid 6, uye KAND 11 haina kukanganisa kukura kweHeLa kana E. coli masero pazvikamu zve 100 μM (Fig. 5d, e).
Kukura inhibition yeKAND 11 mune zvisiri-Arabidopsis zvipenyu.(a) Mbeu dzefodya dzerudzi rweSR-1 dzemavhiki maviri dzakarimwa paMS plates dzine 25 μM KAND 11. (b) Midzi yefodya yemhando yeSR-1 yemavhiki maviri yakarimwa yakachinjika. MS plates ine 200 μM KAND 11. ( c) Mavhiki maviri ekare emusango emhando yeTak-1 liverwort buds akakurira paGamborg B5 plates ane akataridza kuwanda kweKAND 11. Miseve mitsvuku inoratidza spores yakamira kukura mukati memavhiki maviri incubation. period.(d) Cell proliferation assay yeHeLa masero.Nhamba yemasero anoshanda akayerwa panguva dzakatarwa nguva uchishandisa sero kuverenga kit 8 (Dojindo).Sekutonga, masero eHeLa akabatwa ne5 μg/ml actinomycin D (Act D), iyo inodzivisa RNA polymerase transcription uye kukonzera kufa kwesero.Ongororo yakaitwa mumatatu.(e) E. coli cell proliferation assay.E. coli kukura kwakaongororwa nekuyera OD600.Sekutonga, maseru akabatwa ne50 μg/ml ampicillin (Amp), iyo inhibits bacterial cell wall synthesis.Ongororo yakaitwa mumatatu.
Kuti tinzwisise maitiro echiito che cytotoxicity inokonzerwa neuramide-inoenderana makomisheni, takaongororazve urbenic acid derivatives ine mwero inhibitory mhedzisiro.sezvazvinoratidzwa pamufananidzo.Sezvinoratidzwa muFigure 2b, 6a, mbesa dzakarimwa pamahwendefa eagar ane huwandu hwakanyanya (200 μM) yeurmotonic acid 6 yakagadzira midzi mipfupi uye yekuruboshwe-yakakombama (θ = - 23.7 ± 6.1), nepo mbesa dzakarimwa panzira yekudzora, the madzinde aibudisa midzi inenge yakatwasuka (θ = – 3.8 ± 7.1).Uhu humiro hwekukura hunozivikanwa hunobva mukusashanda kwecortical microtubules14,18.Zvinoenderana nekutsvaga uku, iyo microtubule-destabilizing zvinodhaka disopyramide uye oryzalin yakakonzera midzi yakafanana inorereka pasi pemamiriro edu ekukura (Figs. 2b, 6a).Panguva imwe cheteyo, takaedza urmotonic acid derivatives uye takasarudza akati wandei ayo, pane kumwe kudzikisira, akakonzera oblique midzi kukura.Mishonga 8, 9, uye 15 yakashandura nzira yekukura kwemidzi pa 75 μM, 50 μM, uye 40 μM, maererano, zvichiratidza kuti izvi zvinosanganiswa zvinogona kunyatsogadzirisa microtubules (Fig. 2b, 6a).Isu takaedzawo yakanyanya kusimba ursolic acid inotorwa, KAND 11, pane yakaderera (15 µM) uye takaona kuti kushandiswa kweKAND 11 kwakavharira midzi kukura uye kuti nzira yekukura kwemidzi yakanga isina kuenzana, kunyangwe ivo vaifarira kutsvedza kuruboshwe. Mufananidzo C3)..Nekuti kuwanda kwemishonga inokonzeresa microtubule dzimwe nguva inodzivirira kukura kwechirimwa kwete kukonzera kurereka kwemidzi, isu takazoongorora mukana wekuti KAND 11 inokanganisa mamicrotubules nekucherechedza cortical microtubules mumidzi epidermal cell.Immunohistochemistry inoshandisa anti-β-tubulin antibodies mumasero epidermal emidzi yemidzi inobatwa ne 25 μM KAND 11 yakaratidza kuparara kweanenge ese cortical microtubules mumasero epidermal munzvimbo ye elongation (Fig. 6b).Mhedzisiro iyi inoratidza kuti kumamotonic acid nezvinobuda pairi zvinobata zvakananga kana zvisina kunanga pamamicrotubules kuti avavhiringe uye kuti aya macompound inovel microtubule inhibitors.
Ursonic acid uye zvinobva mairi zvinoshandura cortical microtubules muArabidopsis thaliana.(a) Root inclination angle yakayerwa pamberi pezvakasiyana siyana zveurmotonic acid kubva pane zvakaratidzwa.Migumisiro yezvikamu zviviri zvinozivikanwa kuti inhibit microtubules: disopyramide uye oryzalin zvakaongororwawo.Iyo inset inoratidza mwero unoshandiswa kuyera midzi yekukura angle.Asterisks inoratidza misiyano yakakura nekurapa kwesham (t bvunzo, p<0.05).n>19. Scale bar = 1 cm.(b) Cortical microtubules mune epidermal masero munzvimbo yakareba.Microtubules musango-rudzi Arabidopsis Col midzi inokura paMS mahwendefa ane kana asina 25 μM KAND 11 akaonekwa ne immunohistochemical staining achishandisa β-tubulin primary antibodies uye Alexa Fluor-conjugated secondary antibodies.Scale bar = 10 µm.(c) Mitotic chimiro che microtubules mumudzi meristem.Microtubules akaonekwa achishandisa immunohistochemical staining.Mitotic zvimiro, kusanganisira prophase zones, spindles, uye phragmoplasts, akaverengerwa kubva confocal mifananidzo.Miseve inoratidza mitotic microtubule zvimiro.Asterisks inoratidza misiyano yakakura nekurapa kwesham (t bvunzo, p<0.05).n>9. Scale bar = 50 µm.
Kunyangwe Ursa ichigona kukanganisa basa re microtubule, maitiro ayo ekuita anotarisirwa kuve akasiyana kubva kune akajairwa microtubule depolymerizing agents.Semuenzaniso, huwandu hwepamusoro hwe microtubule depolymerizing agents dzakadai se disopyramide uye oryzalin inokonzera anisotropic kuwedzera kwemasero epidermal, asi KAND 11 haina.Mukuwedzera, kushandiswa kwekubatana kweKAND 11 uye disopyramide kwakaguma nekubatanidza disopyramide-induced root growth response uye KAND 11-induced growth inhibition yakaonekwa (Fig. S4).Takaongororawo mhinduro ye hypersensitive disopyramide 1-1 (phs1-1) mutant kuKAND 11. phs1-1 ine non-canonical tubulin kinase point mutation uye inobereka midzi mipfupi kana ichibatwa ne disopyramide9,20.phs1-1 mbesa dzemutant dzakarimwa pa agar medium ine KAND 11 dzaive nemidzi mipfupi yakafanana neinorimwa pa disopyramid (fig. S5).
Mukuwedzera, takacherechedza mitotic microtubule zvivako, zvakadai seprophase zones, spindles, uye phragmoplasts, mumidzi meristem yemiti inobatwa neKAND 11. Inopindirana nekucherechedza kweCDKB2; 1p:: CDKB2; 1-GUS, kuderera kukuru mu nhamba ye mitotic microtubules yakaonekwa (Fig. .6c).
Kuratidza cytotoxicity yeKAND 11 pakugadziriswa kwe subcellular, takabata fodya BY-2 masero ekumisikidza neKAND 11 uye takaona mhinduro yavo.Takatanga kuwedzera KAND 11 kune BY-2 masero anoratidza TagRFP-TUA6, iyo fluorescent inonyora microtubules, kuongorora mhedzisiro yeKAND 11 pane cortical microtubules.Cortical microtubule density yakaongororwa pachishandiswa ongororo yemifananidzo, iyo yakaratidza chikamu che cytoskeletal pixels pakati pe cytoplasmic pixels.Mhedzisiro yekuongorora yakaratidza kuti mushure mekurapwa ne50 μM kana 100 μM KAND 11 kweawa imwe, kuwanda kwakaderera zvakanyanya kusvika 0.94 ± 0.74% kana 0.23 ± 0.28%, ukuwo kuwanda kwemasero akabatwa neDMSO, akasvika ku10.63 ± 1. % (Mufananidzo 7a).Migumisiro iyi inopindirana nekucherechedza muArabidopsis kuti KAND 11 kurapwa inokonzera depolymerization ye cortical microtubules (Fig. 6b).Isu takaongororawo BY-2 mutsara neGFP-ABD-yakanyorwa actin filaments mushure mekurapa neiyo yakafanana yeKAND 11 uye takaona kuti KAND 11 kurapwa kwakakanganisa actin filaments.Kurapa ne 50 μM kana 100 μM KAND 11 ye1 h zvakanyanya kuderedza actin filament density kusvika 1.20 ± 0.62% kana 0.61 ± 0.26%, maererano, asi kuwanda kwemasero akabatwa neDMSO aiva 1.69 ± 0.51% (Fig 0.52).7b).Izvi zvinoguma zvinopesana nemigumisiro yepropyzamide, iyo isingabatsiri actin filaments, uye latrunculin B, an actin depolymerizer isingakanganisi microtubules (SI Mufananidzo S6).Mukuwedzera, kurapwa ne coumamonamide 1, coumamonamide acid 6, kana KAND 11 haina kukanganisa microtubules muHeLa masero (SI Mufananidzo S7).Saka, maitiro ekuita kweKAND 11 anofungidzirwa kuti akasiyana kubva kune anozivikanwa cytoskeleton disruptors.Mukuwedzera, kutarisa kwedu kuduku kweBY-2 masero akabatwa neKAND 11 kwakaratidza kutanga kwesero kufa panguva yeKAND 11 kurapwa uye yakaratidza kuti chikamu cheEvans blue-stained masero akafa haana kuwedzera zvakanyanya mushure me30 min yeKAND 11 kurapwa, nepo. mushure memaminitsi makumi mapfumbamwe ekurapwa ne50 μM kana 100 μM KAND, nhamba yemasero akafa yakawedzera kusvika 43.7% kana 80.1%, maererano (Fig. 7c).Zvakatorwa pamwechete, idzi data dzinoratidza kuti inoveli ursolic acid inotorwa KAND 11 chirimwa-chaiyo cytoskeletal inhibitor ine yaimbozivikanwa maitiro ekuita.
KAND inokanganisa cortical microtubules, actin filaments, uye kushanda kwefodya BY-2 masero.(a) Kuonekwa kwecortical microtubules muBY-2 masero pamberi peTagRFP-TUA6.BY-2 maseru akabatwa neKAND 11 (50 μM kana 100 μM) kana DMSO akaongororwa neconfocal microscopy.Cortical microtubule density yakaverengerwa kubva kumamicrographs e25 akazvimirira maseru.Tsamba dzinoratidza misiyano yakakura (Tukey HSD bvunzo, p<0.05).Scale bar = 10 µm.(b) Cortical actin filaments muBY-2 masero anoonekwa pamberi peGFP-ABD2.BY-2 maseru akabatwa neKAND 11 (50 μM kana 100 μM) kana DMSO akaongororwa neconfocal microscopy.Kuwanda kwe cortical actin filaments yakaverengerwa kubva kumamicrographs e25 akazvimirira maseru.Tsamba dzinoratidza misiyano yakakura (Tukey HSD bvunzo, p<0.05).Scale bar = 10 µm.(c) Kucherechedzwa kweakafa BY-2 masero naEvans bhuruu staining.BY-2 maseru akabatwa neKAND 11 (50 μM kana 100 μM) kana DMSO akaongororwa neinopenya-munda maikorosikopu.n=3.Scale bar = 100 µm.
Kuwanikwa uye kushandiswa kwezvigadzirwa zvitsva zvechisikigo kwakonzera kufambira mberi kwakasiyana-siyana kwehupenyu hwevanhu, kusanganisira mushonga nekurima.Ongororo yezvakaitika kare yakaitwa kuti iwane makemikari anobatsira kubva kune zvakasikwa zviwanikwa.Kunyanya, actinomycetes inozivikanwa kuti inobatsira se antiparasitic antibiotics ye nematodes nekuda kwekukwanisa kwavo kugadzira zvakasiyana-siyana zvechipiri metabolites zvakadai seavermectin, iyo inotungamira yeivermectin uye bleomycin uye madhirivatives ayo, anoshandiswa mumishonga se anticancer agent21,22.Saizvozvowo, zvakasiyana-siyana zvemishonga yesora zvakawanikwa kubva ku actinomycetes, mamwe acho atoshandiswa mukutengesa1,23.Naizvozvo, kuongororwa kwe actinomycete metabolites yekuparadzanisa zvigadzirwa zvechisikigo nezvinoda biological zviitiko zvinoonekwa senge zano rinoshanda.Muchidzidzo ichi, takawana komboni itsva, coumamonamide, kubva kuS. werraensis uye takabudirira kuigadzira.Ursonic acid inogadzirwa yepakati yeurbenamide uye inobuda nayo.Inogona kukonzera hunhu kupeta kwemidzi, kuratidza zvine mwero kusvika kune yakasimba herbicidal chiitiko, uye zvakananga kana zvisina kunanga kukuvadza chirimwa microtubules.Zvisinei, maitiro ekuita kweurmotonic acid angasiyana neaya aripo microtubule inhibitors, sezvo KAND 11 inokanganisawo actin filaments uye inokonzera kufa kwesero, ichiratidza nzira yekutonga iyo urmotonic acid uye zvigadzirwa zvayo zvinokanganisa zvakasiyana-siyana zve cytoskeletal structures..
Kumwe kutsanangurwa kwakadzama kweurbenonic acid kuchabatsira kunzwisisa zviri nani magadzirirwo ekuita kweurbenonic acid.Kunyanya, chinangwa chinotevera ndechekuongorora kugona kweursonic acid kusunga kune yakaderedzwa microtubules kuti ione kana ursonic acid uye maderivatives ayo anoshanda zvakananga pamamicrotubules uye depolymerize iwo, kana kuti kuita kwavo kunoguma nekusagadzikana kwemicrotubule.Mukuwedzera, munyaya iyo microtubules isiri iyo yakananga chinangwa, kuziva nzvimbo yekuita uye molecular targets yeursonic acid pamasero emiti ichabatsira kuwedzera kunzwisisa maitiro emakemikari ane hukama uye nzira dzinobvira dzekuvandudza herbicidal basa.Yedu bioactivity assay yakaratidza yakasarudzika cytotoxic kugona kweursonic acid pakukura kwezvirimwa zvakaita seArabidopsis thaliana, fodya uye liverwort, nepo E. coli kana masero eHeLa asina kukanganiswa.Hudiki kana husina chepfu kumasero emhuka mukana weursonic acid inobuda kana ikagadzirwa semishonga yesora kuti ishandiswe muminda yakavhurika yekurima.Zvechokwadi, sezvo ma microtubules ari maitiro akajairwa mueukaryotes, kusarudzika kwavo mumiti chinhu chakakosha chemishonga yesora.Semuenzaniso, propyzamide, microtubule depolymerizing agent inosunga zvakananga ku tubulin uye inhibits polymerization, inoshandiswa semushonga wesora nekuda kwekuderera kwayo muchetura kumasero emhuka24.Kusiyana nedisopyramide, benzamides vane hukama vane zvakasiyana-siyana zvinotarirwa.Pamusoro pekudyara microtubules, RH-4032 kana benzoxamide inodzivisawo microtubules yemasero emhuka kana oomycetes, maererano, uye zalilamide inoshandiswa sefungicide nekuda kwekuderera kwayo phytotoxicity25,26,27.Bere richangobva kuwanikwa uye zvarinobuda zvinoratidzira yakasarudzika cytotoxicity pazvidyarwa, asi zvakakosha kuziva kuti kumwe kugadziridzwa kunogona kushandura hunhu hwavo, zvichigona kupa zvimwe zvakabva pakutonga kwepathogenic fungi kana oomycetes.
Iyo yakasarudzika zvimiro zveurbenonic acid uye zvinobva mairi zvinobatsira pakukura kwavo semishonga yesora uye kushandiswa sezvishandiso zvekutsvagisa.Kukosha kweiyo cytoskeleton mukudzora chimiro chechirimwa chesero kunozivikanwa zvakanyanya.Zvidzidzo zvepakutanga zvakaratidza kuti zvidyarwa zvakashandura nzira dzakaoma dze cortical microtubule sangano nekudzora microtubule dynamics kudzora zvakanaka morphogenesis.Huwandu hukuru hwemamorekuru ane mutoro wekutonga kwe microtubule chiitiko akaonekwa, uye tsvakiridzo yakabatana ichiri kuenderera mberi3,4,28.Kunzwisisa kwedu kwazvino kweiyo microtubule dynamics mumasero ezvirimwa haitsananguri zvizere maitiro eiyo cortical microtubule sangano.Semuenzaniso, kunyangwe zvese disopyramide uye oryzalin zvinogona depolymerize microtubules, disopyramide inokonzeresa kukanganiswa kwemidzi nepo oryzalin iine hunyoro.Uyezve, kuchinja mu tubulin, iyo inogadzirisa microtubules, inokonzerawo dextrorotation mumidzi, asi paclitaxel, iyo inogadzirisawo microtubule dynamics, haidaro.Naizvozvo, kudzidza uye kuona mamorekuru anonangwa eursolic acid kunofanirwa kupa humbowo hutsva mukutonga kwechirimwa cortical microtubules.Saizvozvowo, kuenzanisa kwemangwana kwemakemikari anobudirira mukusimudzira kukura kwakakanganiswa, zvakadai se disopyramide, uye makemikari asinganyanyi kushanda, akadai se oryzalin kana kumamotoric acid, achapa mazano ekuti kukura kwakakanganiswa kunoitika sei.
Kune rimwe divi, kudzivirira-kwakabatana cytoskeletal rearrangements imwe mukana wekutsanangura cytotoxicity yeursonic acid.Kutapukira kwepathogen kana kuiswa kweelicitor mumasero ezvirimwa dzimwe nguva kunokonzeresa kuparadzwa kwecytoskeleton uye inotevera sero kufa29.Semuenzaniso, oomycete-derived cryptoxanthin yakanzi inokanganisa microtubules uye actin filaments pamberi pefodya sero kufa, zvakafanana nezvinoitika neKAND kurapwa30,31.Kufanana pakati pemhinduro dzekudzivirira uye mhinduro dzemaserura dzakakonzerwa neursonic acid dzakatitungamira kuti tifungidzire kuti dzinokonzeresa maitiro emasero, kunyangwe inokurumidza uye yakasimba mhedzisiro yeursonic acid kupfuura cryptoxanthin inoonekwa.Zvisinei, zvidzidzo zvakaratidza kuti kukanganisa kwe actin filaments kunokurudzira kufa kwesero, iyo isingawanzoperekedzwa ne microtubule disruption29.Pamusoro pezvo, zvinoramba zvichionekwa kuti pathogen kana elicitor inokonzera kukanganisa midzi kukura, sezvinoita ursonic acid derivatives.Nekudaro, ruzivo rwemamorekuru runobatanidza mhinduro dzekudzivirira uye cytoskeleton idambudziko rinokwezva rinofanira kugadziriswa.Nekushandisa kuvepo kweakaderera mamorekuru uremu makomisheni ane hukama neursonic acid, pamwe nemhando yezvinobvamo zvine akasiyana potency, vanogona kupa mikana yekunongedza zvisingazivikanwe maserura masisitimu.
Kutorwa pamwe chete, kuwanikwa uye kushandiswa kwemakomisheni matsva anogadziridza microtubule dynamics ichapa nzira dzine simba dzekugadzirisa dzakaoma mamorekuru nzira dziri pasi pechirimwa chimiro chechimiro.Muchirevo chechinyorwa chino, iyo ichangobva kugadzirwa kommoni urmotonic acid, iyo inokanganisa microtubules uye actin filaments uye induces sero kufa, inogona kupa mukana wekududzira kubatana pakati pe microtubule control uye idzi dzimwe nzira.Nekudaro, makemikari uye biological ongororo tichishandisa urbenonic acid ichatibatsira kunzwisisa mamorekuru ekudzora maitirwo anodzora chirimwa cytoskeleton.
Inoculate S. werraensis MK493-CF1 mu500 mL yakavhiringika Erlenmeyer flask ine 110 mL yembeu yepakati ine 2% (w/v) galactose, 2% (w/v) Essence paste, 1% ( w/v) Bacto composition .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) chibage chechibage (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 uye 0.2% CaCO3 mumvura yakasvibiswa.(pH 7.4 isati yaitwa sterilization).Mbeu tsika dzakaiswa pane rotary shaker (180 rpm) pa27 ° C kwemazuva maviri.Kurimwa kwekugadzira kuburikidza neiyo solid state fermentation.Mbeu tsika (7 ml) yakaendeswa mu500 ml K-1 flask ine 40 g yepakati yekugadzira ine 15 g yebhari yakatsikirirwa (MUSO Co., Ltd., Japan) uye 25 g yemvura yakasvibiswa (pH isina kugadziriswa. isati yaitwa sterilization).).Kuvirisa kwakaitwa pa30°C murima kwemazuva gumi nemana.Iyo yekuvirisa zvinhu yakabudiswa ne 40 ml / bhodhoro EtOH uye centrifuged (1500 g, 4 ° C, 10 min).Iyo tsika yepamusoro (60 ml) yakatorwa nemusanganiswa we10% MeOH/EtOAc.Iyo organic layer yakanyungudutswa pasi pekumanikidzwa kwakaderedzwa kuwana zvakasara (59.5 mg), iyo yakaiswa pasi peHPLC ine gradient elution (0-10 maminitsi: 90%) pane reverse phase column (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID. 10 mm × kureba 250 mm) H2O/CH3CN, 10–35 maminitsi: 90% H2O/CH3CN kusvika 70% H2O/CH3CN (gradient), 35–45 maminitsi: 90% H2O/EtOH, 45–155 maminitsi: 90% H2O / EtOH kusvika 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) pachiyero chekuyerera che 1.5 ml / min, coumamonamide (1, 36.0 mg) yakaparadzaniswa sechena amorphous powder.
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]+: [C6H9N2O2]+ kukosha kwakaverengwa: 141.0659, kuyerwa kukosha: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm– 1.
Mbeu dzeColumbia (Col-0) dzakatorwa kubva kuArabidopsis Biological Resource Center (ABRC) nemvumo yekushandisa patsvagiridzo.Mbeu dzeCol-0 dzakaparadzirwa uye dzakachengetwa pasi pemamiriro edu emurabhoritari uye dzakashandiswa semiti yemusango Arabidopsis.Mbeu dzeArabidopsis dzakaitwa sterilized uye dzakagadzirwa muhafu-simba Murashige uye Skoog yepakati ine 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino) ethanesulfonic acid (MES) ( Fujifilm Wako Pure Chemical ).) uye 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, pa23 °C uye chiedza chinogara chiripo.Mbeu dze phs1-1 mutant dzakapihwa naT. Hashimoto (Nara Institute of Science and Technology).
Mbeu dzeStrain SR-1 dzakapihwa naT. Hashimoto (Nara Institute of Science and Technology) uye dzakashandiswa semiti yefodya yemusango.Mbeu dzefodya dzakaitwa sterilized uye dzakanyikwa mumvura isina utachiona kwehusiku hutatu kuti dzimere, dzozoiswa mugaba rehalf-simba rine 2% sucrose, 0.05% (w/v) MES, uye 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige.uye Skoog yepakati) ine pH 5.7 uye yakachengetwa pa23°C pasi pechiedza chisingaperi.
Strain Tak-1 yakapihwa naT. Kohchi (Kyoto University) uye yakashandiswa seyakajairwa yekuyedza unit yeliverwort kudzidza.Gemma yakawanikwa kubva kumiti yakaomeswa yakasimwa ichibva yaiswa paGamborg B5 yepakati (Fujifilm Wako Pure Chemical) ine 1% sucrose uye 0.3% gellan chingamu uye yakanyudzwa pa23 ° C pasi pechiedza chisingaperi.
Fodya BY-2 masero (Nicotiana tabacum L. cv. Bright Yellow 2) akapiwa naS. Hasezawa (University of Tokyo).BY-2 masero akaderedzwa 95-fold in modified Linsmeier uye Skoog medium uye akawedzerwa vhiki nevhiki ne2,4-dichlorophenoxyacetic acid 32.Kumiswa kwesero kwakasanganiswa pane rotary shaker pa130 rpm pa27 ° C murima.Geza maseru nekagumi kuwanda kwehunyoro hwesvikiro uye womisazve mukati mepakati imwechete.BY-2 transgenic cell mitsara inonyatso kuratidza microtubule marker TagRFP-TUA6 kana actin filament marker GFP-ABD2 pasi pe cauliflower mosaic virus 35S promotor akagadzirwa sekutsanangurwa33,34,35.Iyi mitsara yesero inogona kuchengetedzwa uye kuwiriraniswa uchishandisa maitiro akafanana neaya anoshandiswa kune yekutanga BY-2 cell line.
Masero eHeLa akagadzirwa muDulbecco's modified Eagle's medium (DMEM) (Life Technologies) yakawedzerwa ne10% fetal bovine serum, 1.2 U/ml penicillin, uye 1.2 μg/ml streptomycin mu37°C incubator ine 5% CO2.
Zvese zviedzo zvinotsanangurwa muchinyorwa ichi zvakaitwa zvinoenderana neJapan biosafety mirau uye nhungamiro.
Macompounds akanyungudutswa mudimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) semhinduro dzemasheya uye yakanyungudutswa muMS yepakati yeArabidopsis nefodya kana Gamborg B5 yepakati yeliverwort.Kune midzi yekukura inhibition assay, inopfuura 10 mhodzi pandiro yakadyarwa pa agar medium ine yakaratidzwa makomisheni kana DMSO.Mbeu dzakaiswa mukamuri yekukura kwemazuva manomwe.Mbeu dzakatorwa mifananidzo uye kureba kwemidzi kwakayerwa.Pakuongorora kumera kweArabidopsis, mhodzi makumi mana nesere pandiro yakadyarwa pa agar medium ine 200 μM compound kana DMSO.Mbeu dzeArabidopsis dzakarimwa muimba yekukura uye nhamba yemadzinde akamera yakaverengwa mazuva manomwe mushure mekumera (dag).Pakuongorora kumera kwefodya, mhodzi makumi maviri neina pandiro yakadyarwa pa agar medium ine 200 μM KAND kana DMSO.Mbeu dzefodya dzairimwa muimba yekukura uye huwandu hwemadzinde amera hwakaverengwa mushure memazuva gumi nemana.Kune iyo liverwort growth inhibition assay, 9 embryos kubva mundiro yega yega yakaputirwa pa agar medium ine yakaratidzwa kutariswa kweKAND kana DMSO uye yakavharirwa mukamuri yekukura kwemazuva gumi nemana.
Shandisa madzinde ane 5 mg/ml propidium iodide (PI) kuona kurongeka kwemidzi.Zviratidzo zvePI zvakaonekwa nefluorescence microscopy vachishandisa TCS SPE confocal laser scanning microscope (Leica Microsystems).
Histochemical staining yemidzi ne β-glucuronidase (GUS) yakaitwa maererano neprotocol inotsanangurwa naMalami naBenfey36.Mbeu dzakagadziriswa mu90% acetone usiku hwose, yakasvibiswa ne 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid muGUS buffer kweawa imwe uye yakaiswa mu hydrated chloraldehyde solution.(8 g chloral hydrate, 2 ml mvura uye 1 ml glycerol) uye yakacherechedzwa nekukanganisa kusiyanisa maikorosikopu uchishandisa Axio Imager M1 maikorosikopu (Carl Zeiss).
Midzi angles yaiyerwa pamadzinde ane mazuva manomwe akakura pamahwendefa akaiswa vertically.Pima kona yemudzi kubva kudivi regiravhiti vheta sekutsanangurwa kwazvino padanho rechitanhatu.
Kurongeka kwekortical microtubules kwakaonekwa sekutsanangurwa, nekugadziriswa kuduku kune protocol 37.Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) uye Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) yakashandiswa semishonga yekutanga uye yechipiri pa1: 1000 uye 1: 100 dilutions, zvichiteerana.Mifananidzo yeFluorescence yakawanikwa uchishandisa TCS SPE confocal laser scanning microscope (Leica Microsystems).Tora Z-stack mifananidzo uye gadzira yakanyanya kusimba fungidziro zvinoenderana nemirairo yemugadziri.
HeLa cell proliferation assay yakaitwa uchishandisa Cell Counting Kit 8 (Dojindo) maererano nemirairo yemugadziri.
Kukura kweE. coli DH5α kwakaongororwa nekuyera kuwanda kwesero mutsika uchishandisa spectrophotometer pa600 nm (OD600).
Cytoskeletal organisation in transgenic BY-2 masero akaonekwa achishandisa fluorescence microscope yakagadzirirwa CSU-X1 confocal scanning device (Yokogawa) uye sCMOS kamera (Zyla, Andor Technology).Cytoskeletal density yakaongororwa nekuongorora kwemufananidzo, iyo yakaverengera chikamu che cytoskeletal pixels pakati pe cytoplasmic pixels mumifananidzo yakakomberedzwa uchishandisa ImageJ software sezvakatsanangurwa38,39.
Kuti uone kufa kwesero muBY-2 maseru, aliquot yekumisikidzwa kwesero yakaiswa ne0.05% Evans yebhuruu kwemaminetsi gumi pakupisa kwekamuri.Kusarudza Evans bhuruu kusvibiswa kwemasero akafa kunoenderana nekuwedzera kwedhayi kubva kumasero anoshanda neiyo intact plasma membrane40.Masero akasvibiswa akaonekwa achishandisa microscope inopenya-munda (BX53, Olympus).
HeLa maseru akakura muDMEM akawedzerwa ne10% FBS muhumidified incubator pa37°C uye 5% CO2.Masero akabatwa ne 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), kana 100 ng/ml Nocodmaze (Sigma) kwe6 h pa37°C.Masero akaiswa neMetOH ye10 min uye zvakare neacetate ye5 min pakushambidzika kwekamuri.Masero akagadziriswa akaiswa ne-β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) yakasvibiswa mu 0.5% BSA/PBS kwemaawa maviri, yakashambidzwa 3 nguva neTBST, uye yozoiswa neAlex Fluor antibody yembudzi.4881 awa.– Mouse IgG (Thermo Fisher Scientific: A11001) uye 15 ng/ml 4′, 6-diamidino-2-phenylindole (DAPI) diluted mu 0.5% BSA/PBS.Mushure mekugeza neTBST katatu, masero ane mavara akaonekwa paNikon Eclipse Ti-E yakadzora maikorosikopu.Mifananidzo yakatorwa nekamera yakatonhodzwa yeHamamatsu ORCA-R2 CCD ichishandisa MetaMorph software (Molecular Devices).
Nguva yekutumira: Jun-17-2024