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Kuwanikwa uye kushandiswa kwakanaka kwezvigadzirwa zvechisikigo kunogona kubatsira kuvandudza hupenyu hwevanhu. Makemikari anodzivirira kukura kwezvirimwa anoshandiswa zvakanyanya semishonga yekuuraya sora. Nekuda kwekushandiswa kwemhando dzakasiyana dzemishonga yekuuraya sora, pane chikonzero chekuona mishonga ine nzira itsva dzekuita. Muchidzidzo ichi, takawana mushonga mutsva weN-alkoxypyrrole, coumamonamide, kubva kuStreptomyces werraensis MK493-CF1 uye takasimbisa maitiro ese ekugadzira. Kuburikidza nekuyedza kwemaitiro ehupenyu, takaona kuti urs-monoamic acid inhengo yepakati ye urs-monoamide uye inogonachinodzivisa kukura kwezvirimwaPamusoro pezvo, takagadzira mishonga yakasiyana-siyana yeurbenonic acid derivatives, kusanganisira urbenyloxy derivative (UDA), ine simba guru rekuuraya zvipembenene zvisingakanganisi kukura kwemasero eHeLa. Takaonawo kuti mishonga yeurmotonic acid derivatives inokanganisa microtubules yemiti; pamusoro pezvo, KAND inokanganisa actin filaments uye inokonzera kufa kwemasero; Mhedzisiro iyi yakasiyana neiyo inozivikanwa microtubule inhibitors uye inoratidza nzira itsva yekushanda kweursonic acid, iyo inomiririra mukana wakakosha mukugadzirwa kwemishonga mitsva yekuuraya zvipembenene.
Kuwanikwa uye kushandiswa kwezvigadzirwa zvechisikigo zvinobatsira uye zvinobva mazviri inzira yekuvandudza hupenyu hwevanhu. Zvigadzirwa zvechipiri zvinogadzirwa netupukanana, zvirimwa nezvipembenene zvakatungamira mukufambira mberi kukuru mukurapa nekurima. Mishonga yakawanda inorwisa mabhakitiriya uye mishonga yekudzivirira leukemia yakagadzirwa kubva muzvigadzirwa zvechisikigo. Pamusoro pezvo, mhando dzakasiyana dzemishonga yezvipembenene, mishonga yefungicide nesora inotorwa kubva muzvigadzirwa izvi zvechisikigo kuti ishandiswe mukurima. Kunyanya, mishonga yesora inorwisa sora zvishandiso zvakakosha zvekuwedzera goho rezvirimwa mukurima kwemazuva ano, uye mhando dzakasiyana dzemishonga dzatove dzichishandiswa mukutengeserana. Maitiro akati wandei emasero muzvirimwa, akadai se photosynthesis, metabolism yeamino acid, kugadzirwa kwemadziro emasero, kudzorwa kwe mitosis, phytohormone signaling, kana kugadzirwa kweprotein, zvinoonekwa sezvinangwa zvemishonga yesora. Mishonga inodzivirira mashandiro e microtubule iboka rakajairika remishonga yesora inokanganisa kukura kwezvirimwa nekuchinja mitotic regulation2.
Microtubules zvikamu zve cytoskeleton uye zvinochengetwa zvakanyanya mumasero eukaryotic. Tubulin heterodimer ine α-tubulin ne β-tubulin zvinoumba linear microtubule protofilaments, ine 13 protofilaments dzinoumba cylindrical structure. Microtubules dzinoita mabasa akawanda mumasero emiti, kusanganisira kuona chimiro chemasero, kupatsanurwa kwemasero, uye kutakurwa kwemukati memasero3,4. Masero emiti ane microtubules pasi pe interphase plasma membrane, uye aya anonzi cortical microtubules anofungidzirwa kuti anodzora kurongeka kwe cellulose microfibrils kuburikidza nekudzora cellulose synthase complexes4,5. Cortical microtubules yemidzi epidermal cells, iripo munzvimbo yekureba nekukurumidza kwemusoro wemudzi, inowanikwa laterally, uye cellulose microfibers inotevera microtubules idzi uye inoganhurira kutungamira kwekukura kwemasero, nokudaro ichisimudzira anisotropic cell elongation. Nokudaro, basa re microtubule rakabatana zvakanyanya nechimiro chemiti. Kutsiviwa kweamino acid mumajini anoisa tubulin kunokonzera kutsveyama kwema cortical microtubule arrays uye kukura kwekuruboshwe kana kurudyi muArabidopsis 6,7. Saizvozvowo, kuchinja kwemapuroteni ane chekuita ne microtubule anodzora microtubule dynamics kunogonawo kutungamira kukura kwemidzi kwakakanganisika8,9,10,11,12,13. Pamusoro pezvo, kurapwa nemishonga inouraya herbicides inokuvadza microtubule yakaita se disopyramide, inozivikanwawo se pretilachlor, kunokonzerawo kukura kwemidzi yakachekwa kuruboshwe14. Data iri rinoratidza kuti kutonga kwakarurama kwebasa re microtubule kwakakosha pakusarudza gwara rekukura kwezvirimwa.
Mhando dzakasiyana dzemishonga inodzivirira mabhakitiriya dzakawanikwa, uye mishonga iyi yakaita basa guru mukutsvagisa kwemasero emuviri, pamwe nekurima nemishonga. 2. Kunyanya, oryzalin, makemikari edinitroaniline, disopyramide, makemikari ane chekuita nebenzamide, uye mamwe mafananidzo avo anogona kudzivirira kushanda kwemabhakitiriya uye nekudaro anodzivirira kukura kwezvirimwa. Nokudaro, anoshandiswa zvakanyanya semishonga inouraya zvipembenene. Zvisinei, sezvo mabhakitiriya ari chikamu chakakosha chemasero emiti nemhuka, mishonga yakawanda inodzivirira mabhakitiriya ine chepfu kune ese marudzi emasero. Nokudaro, pasinei nekushanda kwayo semishonga inouraya zvipembenene, huwandu hushoma hwemishonga inorwisa mabhakitiriya inoshandiswa pakuita mabasa anoshanda.
Streptomyces rudzi rwemhuri yeStreptomyces, inosanganisira mabhakitiriya ane aerobic, gram-positive, filamentous uye inozivikanwa zvikuru nekukwanisa kwayo kugadzira mhando dzakasiyana dzema metabolites echipiri. Nokudaro, inoonekwa seimwe yenzvimbo dzakakosha dzezvigadzirwa zvitsva zvechisikigo zvinoshanda muhupenyu. Muchidzidzo chazvino, takawana chinhu chitsva chinonzi coumamonamide, chakabviswa kubva kuStreptomyces werraensis MK493-CF1 naS. werraensis ISP 5486. Tichishandisa spectral analysis uye full spectral analysis, chimiro che coumamonamide chakatsanangurwa uye N-alkoxypyrrole skeleton yayo yakasiyana yakatsanangurwa. synthesis. Ursmonic acid, synthetic intermediate ye ursmonoamide nezvimwe zvinobva mairi, yakawanikwa ichidzivisa kukura nekumera kwemuti unozivikanwa we Arabidopsis thaliana. Muchidzidzo chehukama hwechimiro-basa, takaona kuti chinhu chine C9 chakagadziriswa kuita ursonic acid, chinonzi nonyloxy derivative ye ursonic acid (KAND), chinowedzera zvakanyanya kudzivirira kukura nekumera. Zvinonyanya kukosha kuti chinhu chitsva ichi chinodzivirira kukura kwezvirimwa chakakanganisawo kukura kwefodya neliverwort uye chakanga chisina chepfu kubhakitiriya kana masero eHeLa. Uyezve, mamwe ma urmotonic acid derivatives anokonzera chimiro chemidzi chakakanganisika, zvichireva kuti ma derivatives aya anokanganisa zvakananga kana zvisina kunanga ma microtubules. Zvichienderana nepfungwa iyi, kucherechedza kwedu ma microtubules akanyorwa se immunohistochemically kana nema fluorescent proteins kunoratidza kuti kurapwa kweKAND kunobvisa polymerizes ma microtubules. Pamusoro pezvo, kurapwa ne kumamotonic acid derivatives kwakakanganisa actin microfilaments. Saka, takawana chinhu chitsva chinodzivirira kukura kwezvirimwa chine mashandiro akasiyana ekuita anosanganisira kuparadzwa kwe cytoskeleton.
Kusanganiswa kweMK493-CF1 kwakabviswa muvhu muShinagawa-ku, Tokyo. Kusanganiswa kweMK493-CF1 kwakagadzira stromal mycelium ine matavi akanaka. Chikamu chechikamu che16S ribosomal RNA gene (1422 bp) chakaongororwa. Kusanganiswa uku kwakafanana zvikuru neS. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: typical strain, 99.93%). Zvichibva pamhedzisiro iyi, zvakaonekwa kuti kusanganiswa uku kwakabatana zvakanyanya nerudzi rweS. werraensis. Saka, takatumidza rudzi urwu kuti S. werraensis MK493-CF1 kwenguva pfupi. S. werraensis ISP 5486T inogadzirawo mishonga yakafanana yehupenyu. Sezvo pakanga pasina tsvakurudzo yekutanga yekuwana zvigadzirwa zvechisikigo kubva kuhutachiona uhu, kumwe kutsvagurudza kwemakemikari kwakaitwa. Mushure mekurimwa kweS. werraensis MK493-CF1 panzvimbo yebhari kuburikidza nekuvirisa solid-state pa30°C kwemazuva gumi nemana, nzvimbo yacho yakabviswa ne50% EtOH. 60 ml yesampuro yakaomeswa kuti pawanikwe 59.5 mg yechinhu chisina kuchena. Chinhu chisina kuchena chakaiswa muchikamu cheHPLC che reverse phase kuti chipe N-methoxy-1H-pyrrole-2-carboxamide (1, inonzi coumamonamide, 36.0 mg). Huwandu hwese hwe1 hunenge 60% yechinhu chisina kuchena. Saka, takasarudza kudzidza zvakadzama nezvehunhu hwekumamotoamide 1.
Coumamonamide 1 ihupfu huchena husina chimiro uye high resolution mass spectrometry (HRESIMS) hunosimbisa C6H8N2O2 (Mufananidzo 1). Chidimbu cheC2-substituted pyrrole chemushonga uyu chinoratidzwa neδH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH mu 1H NMR spectrum: 4.5 Hz, H-5) uye δH 6.78 (1H, d, J = 2.5 Hz, H-6), uye 13C NMR spectrum inoratidza kuvapo kwemaatomu mana e sp2 carbon. Kuvapo kweboka re amide panzvimbo yeC2 kwakaongororwa neHMBC correlation kubva paC-3 proton kuenda kuamide carbonyl carbon pa δC 161.1. Pamusoro pezvo, 1 H uye 13 C NMR peaks paδH 4.10 (3H, S) uye δC 68.3 zvinoratidza kuvapo kwemapoka eN-methoxy mumorekuru. Kunyangwe nzvimbo chaiyo yeboka remethoxy yakanga isati yawanikwa uchishandisa spectroscopic analysis yakadai se enhanced difference spectroscopy uye nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide yakava compound yekutanga.
Kuti vaone chimiro chakarurama che1, kuumbwa kwese kwakaitwa (Mufananidzo 2a). Kurapa kwe2-aminopyridine 2 inotengeswa ne m-CPBA kwakaita kuti N-oxide 3 ienderane neiyo muhuwandu hwehuwandu. Mushure me2-aminoazidation ye2, cyclocondensation reaction yakatsanangurwa naAbramovich yakaitwa mubenzene pa90°C kuti iwane 1-hydroxy-1H-pyrrole-2-carbonitrile 5 mu gramu yaidiwa. Speed 60% (matanho maviri). 15,16. Methylation uye hydrolysis ye4 zvakazopa 1-methoxy-1H-pyrrole-2-carboxylic acid (inonzi "cumotonic acid", 6) muhuwandu hwakanaka (70%, matanho maviri). Pakupedzisira, amidation kuburikidza ne acid chloride intermediate 6 uchishandisa aqueous ammonia yakapa Kumamoto amide 1 muhuwandu hwe98%. Data rese re spectral re synthesized 1 rakafanana neasolated 1, saka chimiro che1 chakatsanangurwa;
Kugadzirwa kwakazara uye kuongororwa kwebasa remubiki reurbenamide neurbenic acid. (a) Kugadzirwa kwese kweKumamoto amide. (b) Mbeu dzemusango dzeArabidopsis Columbia (Col) dzine mazuva manomwe dzakarimwa pamaplate eMurashige neSkoog (MS) ane coumamonamide 6 kana coumamonamide 1 pahuwandu hwakaratidzwa. Chikero chechiyero = 1 cm.
Kutanga, takaongorora mashandiro emubiological eurbenamide nezvinhu zviri pakati payo kuti tione kugona kwayo kugadzirisa kukura kwemiti. Takawedzera huwandu hwakasiyana hwe ursmonamide 1 kana ursmonic acid 6 kuMS agar medium uye mbesa dzeArabidopsis thaliana dzakakura panzvimbo iyi. Kuyedza uku kwakaratidza kuti huwandu hwakawanda (500 μM) hwe6 hwakadzivisa kukura kwemidzi (Mufananidzo 2b). Tevere, takagadzira zvinhu zvakasiyana-siyana zvinobviswa nekuisa nzvimbo yeN1 ye6 uye takaita zvidzidzo zvehukama hwechimiro pairi (maitiro ekugadzira analogue anotsanangurwa muSupporting Information (SI)). Mbesa dzeArabidopsis dzakarimwa panzvimbo ine 50 μM ursonic acid derivatives, uye kureba kwemidzi kwakayerwa. sezvakaratidzwa pamufananidzo. Sezvakaratidzwa muMifananidzo 3a, b, uye S1, ma coumamo acids ane hurefu hwakasiyana hwema linear alkoxy chains (9, 10, 11, 12, uye 13) kana ma large alkoxy chains (15, 16, uye 17) panzvimbo yeN1. Ma derivatives acho akaratidza kudzivirira kukuru kukura kwemidzi. Pamusoro pezvo, takaona kuti kushandiswa kwe 200 μM 10, 11, kana 17 kwakadzivirira kumera (Mifananidzo 3c uye S2).
Kudzidza hukama hwechimiro-basa reKumamoto amide nezvimwe zvine chekuita nazvo. (a) Chimiro uye dhizaini yekubatanidza maanalogues. (b) Kuyera kureba kwemidzi yembeu dzine mazuva manomwe dzakakura paMS medium dzine kana dzisina 50 μM coumamonamide derivatives. Nyeredzi dzinoratidza mutsauko mukuru nekurapwa kwenhema (t test, p< 0.05). n>18. Data rinoratidzwa seavhareji ± SD. nt zvinoreva kuti "hazvina kuongororwa" nekuti inopfuura 50% yembeu hadzina kumera. (c) Kuyera huwandu hwekumera kwembeu dzakarapwa dzakachengetwa kwemazuva manomwe muMS medium kana dzisina 200 μM coumamonamide nezvimwe zvine chekuita nazvo. Nyeredzi dzinoratidza mutsauko mukuru nekurapwa kwenhema (chi-square test). n=96.
Zvinofadza kuti kuwedzerwa kwealkyl side chains kwenguva yakareba kupfuura C9 kwakaderedza basa rekudzivirira, zvichiratidza kuti macompounds ane chekuita nekumamotoic acid anoda macheni eparutivi ane saizi yakati kuti aratidze basa rawo repamuviri.
Nekuti ongororo yehukama hwechimiro-basa yakaratidza kuti C9 yakagadziriswa kuita ursonic acid uye nonyloxy derivative ye ursonic acid (inozivikanwa seKAND 11) ndiyo yainyanya kubatsira pakukura kwemiti, takaita tsananguro yakadzama yeKAND 11. Kurapa Arabidopsis ne50 μM KAND 11 kwakadzivisa zvachose kumera, nepo huwandu hwakaderera (40, 30, 20, kana 10 μM) hweKAND 11 hwakadzivisa kukura kwemidzi nenzira inoenderana nedosi (Mufananidzo 4a, b). Kuti tione kana KAND 11 ichikanganisa kugara zvakanaka kwemidzi, takaongorora midzi yemeristems yakasanganiswa nepropidium iodide (PI) uye takayera saizi yenzvimbo yemeristem. Saizi yembeu dzakakura pamudzi une 25 μM KAND-11 yaive 151.1 ± 32.5 μm, nepo saizi yembeu dzakakura pamudzi une DMSO yaive 264.7 ± 30.8 μm (Mufananidzo 4c, d), zvinoratidza kuti KAND-11 inodzoreredza mashandiro emasero. Kupararira kwemidzi yembeu. Zvichienderana neizvi, kurapwa kweKAND 11 kwakaderedza huwandu hwechiratidzo chekupatsanurwa kwemasero CDKB2;1p::CDKB2;1-GUS mumudzi wembeu (Mufananidzo 4e) 17. Mhedzisiro iyi inoratidza kuti KAND 11 inodzivirira kukura kwemidzi nekuderedza kushanda kwemasero.
Kuongororwa kwemhedzisiro yekudzivirira kwe urbenonic acid derivatives (urbenyloxy derivatives) pakukura. (a) Mbeu dzemusango dzine mazuva manomwe dzakakura pamaplate eMS dzine huwandu hwakaratidzwa hweKAND 11. Scale bar = 1 cm. (b) Kuyera kureba kwemidzi. Mabhii anoratidza mutsauko wakakosha (Tukey HSD test, p.< 0.05). n>16. Data rinoratidzwa seavhareji ± SD. (c) Confocal microscopy yemidzi yemhando yeCol yepropidium iodide-stained wild-type Col yakakurirwa pamaplate eMS ane kana asina 25 μM KAND 11. Mabhureki machena anoratidza root meristem. Scale bar = 100 µm. (d) Kuyera saizi yeroot meristem (n = 10 kusvika 11). Kusiyana kwenhamba kwakatsanangurwa uchishandisa t-test (p< 0.05). Mabhawa anomiririra avhareji yehukuru hwemeristem. (e) Mikroskopi yeDifferential Interference Contrast (DIC) yemudzi wemeristem une CDKB2 construct; 1pro: CDKB2; 1-GUS yakapendwa uye yakapendwa pambeu dzine mazuva mashanu dzakakura pamaplate eMS dzine kana dzisina 25 µM KAND assay.
Kukuvadza kweKAND 11 kwakaedzwazve uchishandisa chimwe chirimwa chine dicotyledonous, fodya (Nicotiana tabacum), uye chimwe chinhu chikuru chemhando yezvirimwa zvepasi, liverwort (Marchantia polymorpha). Sezvakaitika kuArabidopsis, mbesa dzefodya dzeSR-1 dzakadyarwa panzvimbo ine 25 μM KAND 11 dzakabereka midzi mipfupi (Mufananidzo 5a). Pamusoro pezvo, 40 pambeu 48 dzakamera pamaplate ane 200 μM KAND 11, nepo mbeu dzese 48 dzakamera panzvimbo dzakagadziriswa, zvichiratidza kuti huwandu hwakawanda hweKAND hwaive hwakakura (p.< 0.05; chi test -square) yakadzivisa kumera kwefodya. (Mufananidzo 5b). Pamusoro pezvo, kuwanda kweKAND 11 kwakadzivirira kukura kwebhakitiriya mu liverwort kwakafanana nekuwanda kunoshanda muArabidopsis (Mufananidzo 5c). Mhedzisiro iyi inoratidza kuti KAND 11 inogona kudzivirira kukura kwemiti yakasiyana-siyana. Takazoongorora kuti zvinogona kukonzera chepfu here mumasero ane chekuita nebheya monoamide mune zvimwe zvipenyu, zvinoti masero eHeLa evanhu uye Escherichia coli strain DH5α, sevamiriri vemasero emhuka nebhakitiriya epamusoro, zvichiteerana. Muongororo dzakawanda dzekuwedzera kwemasero, takaona kuti coumamonamide 1, coumamonamidic acid 6, uye KAND 11 hazvina kukanganisa kukura kwemasero eHeLa kana E. coli pahuwandu hwe100 μM (Mufananidzo 5d,e).
Kudziviswa kwekukura kweKAND 11 muzvisikwa zvisiri zveArabidopsis. (a) Mbeu dzefodya dzemhando yeSR-1 dzemavhiki maviri dzakakura pamaplate eMS akamiswa akatwasuka ane 25 μM KAND 11. (b) Mbeu dzefodya dzemhando yeSR-1 dzemavhiki maviri dzakakura pamaplate eMS akamiswa akatwasuka ane 200 μM KAND 11. (c) Mabudhi emhando yeTak-1 emhando yeTak-1 emavhiki maviri akakura pamaplate eGamborg B5 ane huwandu hwakaratidzwa hweKAND 11. Miseve mitsvuku inoratidza spores dzakamira kukura mukati menguva yemavhiki maviri ekukura. (d) Kuyedzwa kwemasero eHeLa. Huwandu hwemasero anoshanda hwakayerwa panguva yakatarwa uchishandisa kit 8 yekuverenga masero (Dojindo). Senzira yekudzora, masero eHeLa akarapwa ne5 μg/ml actinomycin D (Act D), iyo inodzivirira RNA polymerase transcription uye inokonzera kufa kwemasero. Kuongorora kwakaitwa katatu. (e) Kuyedzwa kwemasero eE. coli proliferation. Kukura kweE. coli kwakaongororwa nekuyera OD600. Senzira yekudzora, masero akarapwa ne50 μg/ml ampicillin (Amp), iyo inodzivisa kugadzirwa kwemadziro emasero ebhakitiriya. Ongororo dzakaitwa katatu.
Kuti tinzwisise mashandiro anoita cytotoxicity inokonzerwa neuramide-related compounds, takaongororazve urbenic acid derivatives zvine mwero wekudzivirira. sezvakaratidzwa pamufananidzo. Sezvakaratidzwa muMifananidzo 2b, 6a, mbesa dzakakura pamaplate eagar dzine huwandu hwakawanda (200 μM) ye urmotonic acid 6 dzakaburitsa midzi mipfupi uye yakakombama kuruboshwe (θ = – 23.7 ± 6.1), nepo mbesa dzakakura panzvimbo yekudzora, mbesa dzakaburitsa midzi inenge yakatwasuka (θ = – 3.8 ± 7.1). Kukura uku kwechimiro che oblique kunozivikanwa kuti kunokonzerwa nekusashanda zvakanaka kwe cortical microtubules14,18. Zvichienderana nekuwana uku, mishonga inoparadza microtubule disopyramide ne oryzalin yakakonzera kutsveyama kwemidzi kwakafanana pasi pemamiriro edu ekukura (Mifananidzo 2b, 6a). Panguva imwe chete, takaedza urmotonic acid derivatives uye takasarudza akati wandei ayo, pane mamwe ma concentrations, akakonzera kukura kwemidzi ye oblique. Makomichi 8, 9, uye 15 akachinja mafambiro ekukura kwemidzi pa75 μM, 50 μM, uye 40 μM, zvichiteerana, zvichiratidza kuti makomichi aya anogona kukanganisa ma microtubules (Mufananidzo 2b, 6a). Takaedzawo ursol acid derivative ine simba zvikuru, KAND 11, pahuwandu hwakaderera (15 µM) uye takaona kuti kushandiswa kweKAND 11 kwakadzivisa kukura kwemidzi uye kuti mafambiro ekukura kwemidzi akanga asina kuenzana, kunyangwe zvaiwanzotsvedza kuruboshwe (Mufananidzo C3). . Nekuti huwandu hwakawanda hwemishonga inoparadza ma microtubule dzimwe nguva hunodzivirira kukura kwemiti pane kukonzera kutsvedza kwemidzi, takazoongorora mukana wekuti KAND 11 inokanganisa ma microtubules nekuona ma cortical microtubules mumasero emidzi ye epidermal. Immunohistochemistry ichishandisa ma anti-β-tubulin antibodies mumasero e epidermal emidzi yembeu yakarapwa ne25 μM KAND 11 yakaratidza kunyangarika kweanenge ma cortical microtubules ese mumasero e epidermal munzvimbo yakarebesa (Mufananidzo 6b). Mhedzisiro iyi inoratidza kuti kumamotonic acid nezvimwe zvinobva mairi zvinoshanda zvakananga kana zvisina kunanga pama microtubules kuti zvivavhiringidze uye kuti mishonga iyi inodzivirira microtubule itsva.
Ursonic acid nezvimwe zvinobva mairi zvinoshandura cortical microtubules muArabidopsis thaliana. (a) Kona yekutsvedza kwemidzi yakayerwa nekuvapo kwemarudzi akasiyana-siyana eurmotonic acid derivatives pahuwandu hwakaratidzwa. Mhedzisiro yemakemikari maviri anozivikanwa kudzivirira microtubules: disopyramide ne oryzalin zvakaongororwawo. Chinyorwa chiri mukati chinoratidza chiyero chinoshandiswa kuyera angle yekukura kwemidzi. Nyeredzi dzinoratidza mutsauko mukuru nekurapwa kwenhema (t test, p< 0.05). n>19. Chipimo chepakati = 1 cm. (b) Ma microtubules e cortical mumasero e epidermal munzvimbo yekureba. Ma microtubules mu wild-type Arabidopsis Col roots akakurira pama MS plates ane kana asina 25 μM KAND 11 akaonekwa ne immunohistochemical staining achishandisa β-tubulin primary antibodies uye Alexa Fluor-conjugated secondary antibodies. Chipimo chepakati = 10 µm. (c) Chimiro che mitotic che microtubules mu root meristem. Ma microtubules akaonekwa achishandisa immunohistochemical staining. Zvimiro zve mitotic, zvinosanganisira prophase zones, spindles, uye phragmoplasts, zvakaverengwa kubva kumifananidzo ye confocal. Miseve inoratidza mitotic microtubule structures. Asterisks inoratidza mutsauko mukuru ne sham treatment (t test, p< 0.05). n>9. Chikero chebha = 50 µm.
Kunyangwe Ursa ichikwanisa kukanganisa mashandiro e microtubule, mashandiro ayo anotarisirwa kusiyana nemaitiro akajairika e microtubule depolymerizing agents. Semuenzaniso, kuwanda kwakanyanya kwe microtubule depolymerizing agents dzakadai se disopyramide ne oryzalin kunokonzera anisotropic kuwedzera kwemasero epidermal, nepo KAND 11 isingadaro. Pamusoro pezvo, kushandiswa pamwe chete kweKAND 11 ne disopyramide kwakaita kuti pave nemhinduro yekukura kwemidzi inokonzerwa ne disopyramide uye KAND 11-induced growth inhibition yakaonekwa (Mufananidzo 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 yakarapwa ne disopyramide9,20. phs1-1 mutant seeds yakakurira pa agar medium ine KAND 11 yaive nemidzi mipfupi yakafanana neyakakurira pa disopyramid (mufananidzo S5).
Pamusoro pezvo, takaona mitotic microtubule structures, dzakadai seprophase zones, spindles, uye phragmoplasts, mumidzi yembeu dzakarapwa neKAND 11. Zvichienderana nezvakaonekwa neCDKB2;1p::CDKB2;1-GUS, kudzikira kukuru kwehuwandu hwemitotic microtubules kwakaonekwa (Mufananidzo .6c).
Kuti tinzwisise huturu hweKAND 11 pamasero emukati memasero, takarapa masero eFY-2 anomiswa nefodya neKAND 11 uye takaona mhinduro yawo. Takatanga tawedzera KAND 11 kumasero eBY-2 anoratidza TagRFP-TUA6, iyo inonyora microtubules zviri fluorescent, kuti tiongorore mhedzisiro yeKAND 11 pamasero emukati memasero. Kuwanda kwemasero emukati memasero kwakaongororwa uchishandisa ongororo yemufananidzo, iyo yakayera huwandu hwemapikiseli emumafupa pakati pemapikiseli emukati memasero. Mhedzisiro yekuongorora yakaratidza kuti mushure mekurapwa ne50 μM kana 100 μM KAND 11 kweawa imwe chete, huwandu hwacho hwakaderera zvakanyanya kusvika pa0.94 ± 0.74% kana 0.23 ± 0.28%, zvichiteerana, nepo huwandu hwemasero akarapwa neDMSO, hwakasvika pa1.61 ± 0.34% (Mufananidzo 7a). Mhedzisiro iyi inoenderana nezvakataurwa muArabidopsis kuti kurapwa kweKAND 11 kunokonzera depolymerization ye cortical microtubules (Mufananidzo 6b). Takaongororawo mutsetse weBY-2 neGFP-ABD-labeled actin filaments mushure mekurapwa ne concentration yakafanana neKAND 11 uye takaona kuti kurapwa kweKAND 11 kwakakanganisa actin filaments. Kurapa ne50 μM kana 100 μM KAND 11 kweawa imwe chete kwakaderedza zvakanyanya density ye actin filament kusvika pa1.20 ± 0.62% kana 0.61 ± 0.26%, zvichiteerana, nepo density mumasero akarapwa neDMSO yaive 1.69 ± 0.51% (Mufananidzo 2). 7b). Mhedzisiro iyi inopesana nemhedzisiro yepropyzamide, iyo isingakanganisi actin filaments, uye latrunculin B, actin depolymerizer isingakanganisi microtubules (Mufananidzo SI S6). Pamusoro pezvo, kurapwa ne coumamonamide 1, coumamonamide acid 6, kana KAND 11 hakuna kukanganisa microtubules mumasero eHeLa (SI Mufananidzo S7). Saka, mashandiro eKAND 11 anofungidzirwa kuti akasiyana neevanozivikanwa vanovhiringidza cytoskeleton. Pamusoro pezvo, kutarisa kwedu microscopic kwemasero eBY-2 akarapwa neKAND 11 kwakaratidza kutanga kwekufa kwemasero panguva yekurapwa kweKAND 11 uye kwakaratidza kuti huwandu hwemasero akafa eEvans hwakasvibiswa nebhuruu hauna kuwedzera zvakanyanya mushure memaminitsi makumi matatu ekurapwa kweKAND 11, nepo mushure memaminitsi makumi mapfumbamwe ekurapwa ne50 μM kana 100 μM KAND, huwandu hwemasero akafa hwakawedzera kusvika 43.7% kana 80.1%, zvichiteerana (Mufananidzo 7c). Kana zvabatanidzwa, data iri rinoratidza kuti ursolic acid derivative KAND 11 itsva inodzivirira cytoskeletal yemiti ine maitiro aisazivikanwa ekuita.
KAND inokanganisa ma microtubules e cortical, actin filaments, uye kugona kushanda kwemasero e BY-2 efodya. (a) Kuona ma microtubules e cortical mumasero e BY-2 pamberi peTagRFP-TUA6. Masero e BY-2 akarapwa neKAND 11 (50 μM kana 100 μM) kana DMSO akaongororwa ne confocal microscopy. Cortical microtubule density yakaverengerwa kubva kuma micrographs emasero makumi maviri nemashanu akazvimiririra. Mabhii anoratidza mutsauko wakakosha (Tukey HSD test, p.< 0.05). Scale bar = 10 µm. (b) Cortical actin filaments muBY-2 cells dzakaonekwa pamberi peGFP-ABD2. BY-2 cells dzakarapwa neKAND 11 (50 μM kana 100 μM) kana DMSO dzakaongororwa ne confocal microscopy. Kuwanda kwe cortical actin filaments kwakaverengerwa kubva kuma micrographs e25 masero akazvimiririra. Mabhii anoratidza mutsauko wakakosha (Tukey HSD test, p.< 0.05). Scale bar = 10 µm. (c) Kuonekwa kwemasero eBY-2 akafa neEvans blue staining. Masero eBY-2 akarapwa neKAND 11 (50 μM kana 100 μM) kana DMSO akaongororwa nebright-field microscopy. n=3. Scale bar = 100 µm.
Kuwanikwa nekushandiswa kwezvigadzirwa zvitsva zvechisikigo kwakatungamira mukufambira mberi kukuru muhupenyu hwevanhu, kusanganisira mushonga nekurima. Tsvagiridzo yenhoroondo yakaitwa kuti pave nemishonga inobatsira kubva muzviwanikwa zvechisikigo. Kunyanya, actinomycetes anozivikanwa semishonga inorwisa tupukanana yezvipembenene zvemanyoka nekuda kwekukwanisa kwavo kugadzira mishonga yakasiyana-siyana yakadai seavermectin, iyo inotungamira mu ivermectin ne bleomycin nezvimwe zvinobva mairi, inoshandiswa mukurapa semushonga wekenza21,22. Saizvozvowo, mishonga yakasiyana-siyana yemishonga yakawanikwa kubva ku actinomycetes, mimwe yacho yatove kushandiswa mukutengeserana1,23. Nokudaro, kuongororwa kwe actinomycete metabolites kuti tiparadzanise zvigadzirwa zvechisikigo zvine mabasa ehupenyu anodiwa kunoonekwa senzira inoshanda. Muchidzidzo ichi, takawana compound itsva, coumamonamide, kubva kuS. werraensis uye takaigadzira zvinobudirira. Ursonic acid inhengo yekugadzira ye urbenamide nezvimwe zvinobva mairi. Inogona kukonzera kukotama kwemidzi, kuratidza basa rekuuraya mishonga zvine mwero kusvika kune simba, uye kukanganisa zvakananga kana zvisina kunanga microtubules yemiti. Zvisinei, mashandiro anoita urmotonic acid anogona kusiyana neema microtubule inhibitors aripo, sezvo KAND 11 ichikanganisawo actin filaments uye ichikonzera kufa kwemasero, zvichiratidza nzira yekutonga iyo urmotonic acid nezvimwe zvinobva mairi zvinopesvedzera zvakasiyana-siyana zve cytoskeletal structures.
Kutsanangurwa kwakazara kwe urbenonic acid kuchabatsira kunzwisisa zviri nani mashandiro ekushanda kwe urbenonic acid. Kunyanya, chinangwa chinotevera ndechekuongorora kugona kwe ursonic acid kubatana ne microtubules dzakaderera kuti vaone kana ursonic acid ne derivatives dzayo zvichishanda zvakananga pa microtubules uye zvichidzibvisa polymer, kana kuti kushanda kwadzo kunoguma nekusagadzikana kwe microtubules. Pamusoro pezvo, kana microtubules isiri chinangwa chakananga, kuziva nzvimbo yekushanda uye mamorekuru e ursonic acid pamasero emiti kuchabatsira kunzwisisa hunhu hwemakemikari ane hukama uye nzira dzinogoneka dzekuvandudza mashandiro emishonga inouraya zvipembenene. Kuyedza kwedu kwe bioactivity kwakaratidza kugona kwakasiyana kwe cytotoxic kwe ursonic acid pakukura kwezvirimwa zvakaita se Arabidopsis thaliana, fodya ne liverwort, nepo masero e E. coli kana HeLa asina kukanganiswa. Chepfu shoma kana kusavapo kumasero emhuka mukana we ursonic acid derivatives kana akagadzirwa semishonga yekushandiswa muminda yakavhurika yekurima. Zvechokwadi, sezvo microtubules ari maumbirwo akajairika mu eukaryotes, kudzivirira kwavo muzvirimwa chinhu chakakosha chinodiwa pamishonga yekuuraya zvipembenene. Semuenzaniso, propyzamide, chinhu chinodzivirira utachiona kubva muzvipembenene zve microtubule chinosunga zvakananga ku tubulin uye chinodzivisa polymerization, chinoshandiswa semushonga wesora nekuda kwehupfu hwayo hushoma kumasero emhuka24. Kusiyana ne disopyramide, ma benzamides ane hukama ane hunhu hwakasiyana hwechinangwa. Pamusoro pe microtubules yemiti, RH-4032 kana benzoxamide inodzivirirawo microtubules yemasero emhuka kana oomycetes, zvichiteerana, uye zalilamide inoshandiswa semushonga wefungi nekuda kwehupfu hwayo hushoma25,26,27. Bere idzva rakawanikwa nezvinobuda mariri zvinoratidza huturu hwakasiyana pamiti, asi zvakakosha kuziva kuti zvimwe zvigadziriso zvinogona kuchinja hunhu hwazvo, zvichigona kupa zvimwe zvinobuda mariri zvekutonga fungus kana oomycetes.
Hunhu hwakasiyana hwe urbenonic acid nezvimwe zvinobva mairi zvinobatsira pakukura kwayo semishonga yekuuraya zvipembenene uye kushandiswa kwayo sezvishandiso zvekutsvagisa. Kukosha kwe cytoskeleton mukudzora chimiro chemasero emiti kunozivikanwa zvakanyanya. Zvidzidzo zvekare zvakaratidza kuti zvirimwa zvakagadzira nzira dzakaoma dzekurongeka kwe cortical microtubule nekudzora microtubule dynamics kuti zvidzore morphogenesis nemazvo. Huwandu hwakawanda hwemamorekuru anokonzera kutonga kwebasa re microtubule hwakaonekwa, uye tsvakiridzo inoenderana nahwo ichiri kuenderera mberi3,4,28. Kunzwisisa kwedu pari zvino kwe microtubule dynamics mumasero emiti hakutsananguri zvizere maitiro ekurongeka kwe cortical microtubule. Semuenzaniso, kunyangwe zvese disopyramide ne oryzalin zvichigona kubvisa polymerize microtubules, disopyramide inokonzera kukanganisa kwemidzi zvakanyanya nepo oryzalin ine mhedzisiro iri nyore. Uyezve, shanduko mu tubulin, iyo inodzikamisa microtubules, inokonzerawo dextrotation mumidzi, nepo paclitaxel, iyo inodzikamisawo microtubule dynamics, haidaro. Nokudaro, kudzidza nekuona mamorekuru e ursic acid kunofanira kupa ruzivo rutsva mukudzora plant cortical microtubules. Saizvozvowo, kuenzanisa makemikari anoshanda mukusimudzira kukura kwakakanganisika, akadai se disopyramide, uye makemikari asingashande zvakanaka, akadai se oryzalin kana kumamotoric acid, kuchapa zviratidzo zvekuti kukura kwakakanganisika kunoitika sei.
Kune rumwe rutivi, kurongwazve kwemasero etsinga ane chekuita nekudzivirira ndeimwe nzira yekutsanangura huturu hwe ursonic acid. Kutapukirwa kwehutachiona kana kuiswa kwe elicitor mumasero emiti dzimwe nguva kunokonzera kuparadzwa kwe cytoskeleton uye kufa kwemasero kunotevera29. Semuenzaniso, cryptoxanthin inobva ku oomycete yakashumwa kuti inokanganisa microtubules ne actin filaments vasati vafa nefodya, zvakafanana nezvinoitika neKAND treatment30,31. Kufanana pakati pemhinduro dzekudzivirira nemhinduro dzemasero zvinokonzerwa ne ursonic acid kwakaita kuti tifunge kuti zvinokonzeresa maitiro emasero akajairika, kunyangwe mhedzisiro inokurumidza uye yakasimba ye ursonic acid kupfuura cryptoxanthin iri pachena. Zvisinei, zvidzidzo zvakaratidza kuti kukanganiswa kwe actin filaments kunokurudzira kufa kwemasero, izvo zvisingawanzo kuperekedzwa nekukanganiswa kwe microtubule29. Pamusoro pezvo, zvichiri kuonekwa kana chirwere kana elicitor ichikonzera kukura kwemidzi kwakashata, sezvinoita ursonic acid derivatives. Saka, ruzivo rwemamorekuru runobatanidza mhinduro dzekudzivirira ne cytoskeleton idambudziko rinokwezva rinofanira kugadziriswa. Nekushandisa kuvapo kwemakomikoro ane huremu hushoma ane chekuita ne ursonic acid, pamwe chete nezvimwe zvinhu zvakasiyana-siyana zvine simba rakasiyana, zvinogona kupa mikana yekutarisa mashandiro emasero asingazivikanwe.
Kana zvabatanidzwa, kuwanikwa nekushandiswa kwemakemikari matsva anogadzirisa simba re microtubule kuchapa nzira dzakasimba dzekugadzirisa mashandiro akaomarara emamorekuru ari pasi pekuona chimiro chemasero emiti. Mumamiriro ezvinhu aya, compound urmotonic acid yakagadzirwa munguva pfupi yapfuura, iyo inokanganisa microtubules ne actin filaments uye inokonzera kufa kwemasero, inogona kupa mukana wekunzwisisa hukama huripo pakati pekudzora microtubules nedzimwe nzira idzi. Saka, ongororo yemakemikari nebiological uchishandisa urbenonic acid ichatibatsira kunzwisisa maitiro ekudzora mamorekuru anodzora cytoskeleton yemiti.
Isa mushonga weS. werraensis MK493-CF1 mubhodhoro reErlenmeyer rine 500 mL rine 110 mL yembeu ine 2% (w/v) galactose, 2% (w/v) Essence paste, 1% (w/v) Bacto composition. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) corn extract (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 uye 0.2% CaCO3 mumvura yakabviswa ion. (pH 7.4 isati yapiswa). Mbeu dzakakura dzakaiswa mu rotary shaker (180 rpm) pa27°C kwemazuva maviri. Kukura kwembeu kuburikidza nekuvirisa solid state. Kukura kwembeu (7 ml) kwakaiswa mubhodhoro re500 ml reK-1 rine 40 g yenzvimbo yekugadzira ine 15 g yebhari yakadzvanywa (MUSO Co., Ltd., Japan) uye 25 g yemvura yakabviswa ion (pH haina kugadziriswa isati yabviswa sterilization). Kuvirisa kwakaitwa pa30°C murima kwemazuva gumi nemana. Chinhu chekuvirisa chakabviswa ne40 ml/bhodhoro reEtOH ndokuiswa mukati memvura (1500 g, 4°C, 10 min). Supernatant yekuvirisa (60 ml) yakabviswa nemusanganiswa we10% MeOH/EtOAc. Rutivi rweorganic rwakabviswa mvura pasi pekumanikidzwa kwakaderera kuti pave nechinhu chakasiyiwa (59.5 mg), icho chakaiswa muHPLC ne gradient elution (0–10 maminitsi: 90%) pa 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) pa flow rate ye 1.5 ml/min, coumamonamide (1, 36.0 mg) yakaparadzaniswa seupfu huchena husina chimiro.
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 kwakaverengerwa: 141.0659, kukosha kwakayerwa: 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 mukutsvaga. Mbeu dzeCol-0 dzakaparadzirwa uye dzakachengetwa pasi pemamiriro edu emurabhoritari uye dzakashandiswa semiti yemusango yeArabidopsis. Mbeu dzeArabidopsis dzakacheneswa pamusoro uye dzakarimwa muMurashige neSkoog medium ine hafu ye 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 chisingachinji. Mbeu dze phs1-1 mutant dzakapihwa naT. Hashimoto (Nara Institute of Science and Technology).
Mbeu dzemhando yeSR-1 dzakapihwa naT. Hashimoto (Nara Institute of Science and Technology) uye dzakashandiswa sezvirimwa zvefodya zvemusango. Mbeu dzefodya dzakacheneswa pamusoro ndokunyikwa mumvura isina utachiona kwehusiku hutatu kuti dzikure, dzobva dzaiswa mumushonga une simba rehafu une 2% sucrose, 0.05% (w/v) MES, uye 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. uye Skoog medium) ine pH 5.7 uye dzakaiswa pa23°C pasi pechiedza chisingachinji.
Strain Tak-1 yakapihwa naT. Kohchi (Kyoto University) uye yakashandiswa sechikamu chekuedza chekudzidza kweliverwort. Gemma yakawanikwa kubva mumiti yakasimwa sterilize ndokuzoiswa paGamborg B5 medium (Fujifilm Wako Pure Chemical) ine 1% sucrose uye 0.3% gellan gum ndokuiswa pa23°C pasi pechiedza chinoenderera mberi.
Masero efodya eBY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) akapihwa naS. Hasezawa (Yunivhesiti yeTokyo). Masero eBY-2 akasanganiswa ka95-fold muLinsmeier yakagadziriswa uye Skoog medium uye akawedzerwa vhiki rega rega ne2,4-dichlorophenoxyacetic acid 32. Kusungirirwa kwemasero kwakasanganiswa pamuchina unotenderera pa130 rpm pa27°C murima. Geza masero ne10 ka10 huwandu hwemedium itsva uye dzokerazve mumedium imwechete. Mitsetse yemasero eBY-2 transgenic inonyatsoratidza microtubule marker TagRFP-TUA6 kana actin filament marker GFP-ABD2 pasi pe cauliflower mosaic virus 35S promoter yakagadzirwa sezvakatsanangurwa33,34,35. Mitsetse yemasero iyi inogona kuchengetedzwa uye kuwiriraniswa uchishandisa maitiro akafanana neaya akashandiswa pamutsetse wemasero eBY-2 wekutanga.
Masero eHeLa akarimwa muDulbecco's modified Eagle's medium (DMEM) (Life Technologies) yakawedzerwa 10% fetal bovine serum, 1.2 U/ml penicillin, uye 1.2 μg/ml streptomycin mu 37°C incubator ine 5% CO2.
Kuedza kwese kwakatsanangurwa muchinyorwa ichi kwakaitwa zvichienderana nemitemo nemirairo yekuchengetedzwa kwezvipenyu muJapan.
Mishonga yakanyungudutswa mu dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) semishonga yemuto uye yakasanganiswa mu MS medium ye Arabidopsis nefodya kana Gamborg B5 medium ye liverwort. Pakuyedzwa kwekudzivirira kukura kwemidzi, mbeu dzinopfuura gumi pandiro imwe neimwe dzakadyarwa pa agar medium ine mishonga yakaratidzwa kana DMSO. Mbeu dzakachengetwa mukamuri yekukura kwemazuva manomwe. Michero yakatorwa mifananidzo uye kureba kwemidzi kwakayerwa. Pakuyedzwa kwe Arabidopsis germination, mbeu makumi mana nemasere pandiro imwe neimwe dzakadyarwa pa agar medium ine 200 μM compound kana DMSO. Mbeu dze Arabidopsis dzakadyarwa mukamuri yekukura uye huwandu hwembeu dzakamera hwakaverengwa mazuva manomwe mushure mekumera (dag). Pakuyedzwa kwefodya germination, mbeu makumi maviri neina pandiro imwe chete dzakadyarwa pa agar medium ine 200 μM KAND kana DMSO. Mbeu dzefodya dzakadyarwa mukamuri yekukura uye huwandu hwembeu dzakamera hwakaverengwa mushure memazuva gumi nemana. Pakuyedzwa kwekudzivirira kukura kwe liverwort, ma embryo mapfumbamwe kubva muplate imwe neimwe akaiswa pamusoro pe agar medium ine huwandu hwakaratidzwa hweKAND kana DMSO ndokuiswa mukamuri yekukura kwemazuva gumi nemana.
Shandisai mbesa dzakapendwa ne5 mg/ml yepropidium iodide (PI) kuti muone kurongeka kwemidzi yemiti. Zviratidzo zvePI zvakaonekwa ne fluorescence microscopy uchishandisa TCS SPE confocal laser scanning microscope (Leica Microsystems).
Kuiswa kwemidzi mu histochemical ne β-glucuronidase (GUS) kwakaitwa zvichienderana nemaitiro akatsanangurwa naMalami naBenfey36. Mbeu dzakaiswa mu 90% acetone husiku hwese, dzakaiswa 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid muGUS buffer kweawa imwe chete ndokuiswa mu hydrated chloraldehyde solution. (8 g chloral hydrate, 2 ml mvura uye 1 ml glycerol) uye dzakaonekwa ne differential interference contrast microscopy uchishandisa Axio Imager M1 microscope (Carl Zeiss).
Makona emidzi akayerwa pambeu dzine mazuva manomwe dzakakura dzakamira dzakamira. Yera kona yemudzi kubva padivi regiravhiti sezvakatsanangurwa padanho rechitanhatu.
Kurongeka kwema "cortical microtubules" kwakaonekwa sezvakatsanangurwa, nekuchinja kudiki kune protocol 37. Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) uye Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) zvakashandiswa se "primary" uye "secondary antibodies" pa1:1000 uye 1:100 dilutions, zvichiteerana. Mifananidzo yeFluorescence yakawanikwa uchishandisa TCS SPE confocal laser scanning microscope (Leica Microsystems). Wana mifananidzo yeZ-stack uye gadzira maximum intensity projections zvichienderana nemirairo yemugadziri.
Kuyedzwa kwekuwedzera kwemasero eHeLa kwakaitwa uchishandisa Cell Counting Kit 8 (Dojindo) zvichienderana nemirairo yemugadziri.
Kukura kweE. coli DH5α kwakaongororwa nekuyera huwandu hwemasero mukukura kwemhuka uchishandisa spectrophotometer pa600 nm (OD600).
Kurongeka kwema cytoskeletal mumasero eBY-2 akachinjika kwakaonekwa uchishandisa maikorosikopu ye fluorescence yakashongedzerwa neCSU-X1 confocal scanning device (Yokogawa) uye kamera ye sCMOS (Zyla, Andor Technology). Kuwanda kwema cytoskeletal kwakaongororwa nekuongorora mifananidzo, iyo yakayera huwandu hwema cytoskeletal pixels pakati pema cytoplasmic pixels mumifananidzo ye confocal uchishandisa ImageJ software sezvakatsanangurwa38,39.
Kuti vaone kufa kwemasero mumasero eBY-2, chikamu chemasero chakaiswa muchimiro chebhuruu che0.05% Evans kwemaminitsi gumi patembiricha yemukamuri. Kutsvukirira kwemasero akafa kwemavara ebhuruu eEvans kunoenderana nekubuda kwedhayi kubva mumasero anoshanda neplasma membrane isingakanganise40. Masero ane mavara akaonekwa achishandisa maikorosikopu ine ruvara rwakajeka (BX53, Olympus).
Masero eHeLa akakurira muDMEM akawedzerwa 10% FBS mu incubator ine humidified pa37°C uye 5% CO2. Masero akarapwa ne100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), kana 100 ng/ml Nocodmaze (Sigma) kwemaawa matanhatu pa37°C. Masero akagadziriswa neMetOH kwemaminitsi gumi uye ipapo neacetate kwemaminitsi mashanu patembiricha yemumba. Masero akagadziriswa akaiswa neβ-tubulin primary antibody (1D4A4, Proteintech: 66240-1) yakasanganiswa mu0.5% BSA/PBS kwemaawa maviri, akagezwa katatu neTBST, uye akazoiswa neAlexa Fluor goat antibody. 488 awa imwe chete. – Mouse IgG (Thermo Fisher Scientific: A11001) uye 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) yakasanganiswa mu 0.5% BSA/PBS. Mushure mekugezwa neTBST katatu, masero ane mavara akaonekwa paNikon Eclipse Ti-E inverted microscope. Mifananidzo yakatorwa neHamamatsu ORCA-R2 CCD kamera yakatonhodzwa uchishandisa software yeMetaMorph (Molecular Devices).
Nguva yekutumira: Chikumi-17-2024