347 12.

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Abashoferi berekana ingingo eshatu kuri slide.Koresha inyuma na buto ikurikira kugirango unyuze kumurongo, cyangwa utubuto twa slide mugenzuzi kugirango urangire unyuze kuri buri slide.

347 Ibiranga Umuyoboro

347 12.7 * 1.24mm Ibyuma bidafite ingese

Hanze ya Diameter: 6.00 mm OD kugeza kuri 914.4 mm OD, Ingano igera kuri 24 ”NB iraboneka Ex-stock, OD Ingano yicyuma kiboneka Ex-stock

SS 347 Umuyoboro mwinshi: 0.3mm - mm 50, SCH 5, SCH10, SCH 40, SCH 80, SCH 80S, SCH 160, SCH XXS, SCH XS
WT: SCH5S, SCH10S, SCH40S, SCH80S, SCH160S, nibindi (0.5-12mm) Cyangwa ubunini budasanzwe kugirango buhindurwe nkuko bisabwa

Ubwoko: SS 347 Imiyoboro idafite icyerekezo |SS 347 Imiyoboro ya ERW |SS 347 Imiyoboro isudira |SS 347 Imiyoboro Yakozwe |SS 347 CDW Imiyoboro, Imiyoboro ya LSAW / Kudoda-gusudira / Kongera gushushanya

Ifishi: SS 347 Imiyoboro Yizengurutse / Imiyoboro, SS 347 Imiyoboro ya kare / Imiyoboro, SS 347 Umuyoboro urukiramende / Umuyoboro, SS 347 Igituba gikonje, SS 347 “U”, SS 347 Igiceri cya Cake, SS 347 Amazi ya Hydraulic

Uburebure: Ikintu kimwe gisanzwe, cyikubye kabiri & Uburebure busabwa Impera: Impera yikibaya, Impera ya Beveled, Yandagaye

Kurinda Impera: Ibipapuro bya plastiki |Hanze Kurangiza: 2B, No.4, No.1, No.8 Indorerwamo Kurangiza imiyoboro idafite ibyuma, Kurangiza nkuko bisabwa nabakiriya

Imiterere yo Gutanga: Bishyizwe hamwe kandi Byatoranijwe, Byogejwe, Byegeranye, Byakonje

Ubugenzuzi, Raporo y'Ibizamini: Impamyabumenyi y'Ikizamini, EN 10204 3.1, Raporo y’imiti, Raporo y’ubukanishi, Raporo y’ibizamini bya PMI, Raporo y’ubugenzuzi bw’amashusho, Raporo y’ubugenzuzi bw’abandi, Raporo ya Laboratwari yemejwe na NABL, Raporo y'ibizamini byangiza, Raporo y'ibizamini bidasenya.

Gupakira: Bipakiye mumasanduku yimbaho, imifuka ya plastiki, imirongo yicyuma ihambiriye, cyangwa nkuko abakiriya babisabye

Umwihariko: Ingano nibisobanuro bitari hejuru birashobora gukorwa kubisabwa

SS 347 Ingano yubunini Urwego: 1/2 cm NB, OD kugeza kuri 24

ASTM A312 347: Umuyoboro wa austenitike utagira ikizinga kandi ugororotse ugenewe ubushyuhe bwinshi na serivisi rusange yangiza.Kuzuza ibyuma ntabwo byemewe mugihe cyo gusudira.

ASTM A358 347: Guhuza amashanyarazi gusudira umuyoboro wa austenitis kugirango ubora kandi / cyangwa serivisi yubushyuhe bwo hejuru.Mubisanzwe umuyoboro ugera kuri santimetero 8 ukorwa kuri ibi bisobanuro.Kongera ibyuma byuzuza byemewe mugihe cyo gusudira.

ASTM A790 34.

ASTM A409 347.

ASTM A376 347: Umuyoboro wa austenitike utagira ingano kubushyuhe bwo hejuru.

ASTM A813 347: Umuyoboro umwe, umwe cyangwa kabiri-gusudira umuyoboro wa austenitike kubushyuhe bwinshi hamwe nibisanzwe byangiza.

ASTM A814 347: Gukonjesha gukonje gusudira umuyoboro wa austenitis kubushyuhe bwinshi na serivisi rusange yangiza.

347H Imiyoboro idafite ibyuma

Ibyuma bitagira umwanda 347H Umuyoboro wa mashini

Ibyuma bitagira umwanda 347H Imiyoboro Ibintu bifatika

Impamyabumenyi ihwanye na 347H Umuyoboro w'icyuma

Igiteranyo cya Amyloid alpha-synuclein (αS) ni ikimenyetso cyindwara ya Parkinson nizindi synucleinopathies.Vuba aha, poroteyine ya tau ikunze kuba ifitanye isano n'indwara ya Alzheimer ifitanye isano na αS patologie kandi ugasanga ifatanije na αS ikungahaye cyane, nubwo uburyo bwa molekile bwo guhuza poroteyine zombi butarasobanuka neza.Dutangariza hano ko icyiciro cya αS gitandukana mumazi ya kanseri binyuze mumashanyarazi ya electrostatike hamwe na polypeptide yuzuye neza nka tau.Ukurikije isano ya αS kuri polycations nigipimo cya valence igabanuka ryurusobe rwa coagulation, uturemangingo tunyuramo vuba cyangwa coescence ikurikirwa no guteranya amyloide gahoro.Muguhuza suite yubuhanga buhanitse bwa biofiziki, twashoboye kuranga gutandukanya amazi-yamazi αS / Tau icyiciro no gutandukanya ibintu byingenzi biganisha kumasemburo ya heterogeneous igizwe na proteyine zombi muri kanseri ya proteine ​​yuzuye.
Usibye ibice bya membrane, gutandukanya umwanya mungirabuzimafatizo bishobora no kugerwaho no gukora proteine ​​ikungahaye kuri poroteyine, imeze nkamazi yuzuye yiswe biomolecular condensates cyangwa ibitonyanga, binyuze mubikorwa bizwi nko gutandukanya ibice byamazi (LLPS).Ibitonyanga byakozwe nuburyo butandukanye bwigihe gito, mubisanzwe hagati ya proteyine cyangwa proteyine na RNA, kandi bigakora imirimo itandukanye mubuzima hafi ya bwose.Umubare munini wa poroteyine za LLP zishoboye kwerekana urutonde rwibintu bito bito bidahungabanye cyane muri kamere no muburyo bwa kondens ya biomolecular3,4,5.Ubushakashatsi bwinshi bwakozwe mubushakashatsi bwerekanye imiterere ihindagurika, akenshi itunganijwe, kandi itandukanye ya poroteyine zigize utwo dusimba tumeze nk'amazi, nubwo bike bizwi ku byerekeranye na molekuline yihariye igenzura imikurire n'imikurire y'izo kondegene ku buryo bukomeye-busa leta..
Amakuru mashya ashyigikira hypothesis ivuga ko aberrant proteine ​​iterwa na LLPS no guhindura ibitonyanga muburyo bukomeye bishobora kuba inzira zingirabuzimafatizo ziganisha ku gushiraho uburozi bw’ubumara butangirika akenshi buranga indwara zangirika.Intungamubiri nyinshi za LLPS zifitanye isano na poroteyine (IDP), akenshi zishishwa cyane kandi zoroshye, zimaze igihe kinini zifitanye isano na neurodegeneration binyuze mu nzira yo guteranya amyloide.By'umwihariko, IDP ya biomolecular nka FUS7 cyangwa TDP-438 cyangwa proteyine zifite domaine nini zigoye cyane nka hnRNPA19 byagaragaye ko zisaza zisa na gel cyangwa se zikomeye binyuze munzira yitwa fluidisation.ibice.Kuri fonctionnement fonctionnement (LSPT) nkigikorwa cyigihe cyangwa mugusubiza ibyahinduwe nyuma yubusobanuro cyangwa ihinduka ryimiterere ya pathologiya1,7.
Indi IDP ifitanye isano na LLPS muri vivo ni Tau, poroteyine iterwa na microtubule ifitanye isano na amyloide igira uruhare mu ndwara ya Alzheimer10 ariko ikaba iherutse no kugira uruhare mu ndwara ya Parkinson (PD) hamwe na poroteyine za kirimbuzi 11, 12, 13 zifitanye isano.Tau yerekanwe guhita yitandukanya nigisubizo / cytoplazme kubera imikoranire myiza ya electrostatike14, bikavamo gushiraho ibitonyanga bikungahaye kuri tau bizwi nka coacervates ya electrostatike.Byagaragaye kandi ko ubu bwoko bwimikoranire idasanzwe nimbaraga zitera kanseri ya biomolecular nyinshi muri kamere15.Ku bijyanye na poroteyine ya tau, igiteranyo cya electrostatike gishobora guterwa no kwegeranya byoroheje, aho uturere twinshi twashizwemo na poroteyine bitera inzira yo gutandukana, cyangwa hamwe no guteranya ibintu bitoroshye binyuze mu mikoranire na polimeri zishinja nabi nka RNA.
Vuba aha, α-synuclein (αS), IDP amyloide igira uruhare muri PD nizindi ndwara ziterwa na neurodegenerative hamwe zizwi ku izina rya synucleinopathie 17,18, yerekanwe mu ngero ngengabuzima n’inyamaswa19,20 yibanze muri poroteyine hamwe n’imyitwarire isa n’amazi.Mu bushakashatsi bwa vitro bwerekanye ko αS ihura na LLPS mu kwegeranya byoroheje binyuze mu mikoranire yiganjemo hydrophobique, nubwo iyi nzira isaba intungamubiri za poroteyine nyinshi cyane kandi igihe kirekire cyo gukuramo 19,21.Niba αS irimo kondensate zigaragara muri vivo zakozwe niyi cyangwa izindi nzira za LLPS ziracyari ikibazo cyingenzi kidakemutse.Mu buryo nk'ubwo, nubwo kwegeranya αS amyloide byagaragaye muri neuron muri PD no mu zindi synucleinopathies, uburyo nyabwo αS ikoreramo amyloide yo mu nda itagaragara neza, kuko gukabya gukabije kwa poroteyine bitagaragara ko bitera iyi nzira yonyine.Ibindi byangiritse bya selile birasabwa kenshi, byerekana ko ahantu hamwe na selile cyangwa microen ibidukikije bisabwa kugirango hahindurwe inteko zidasanzwe αS amyloide.Imiterere imwe ya selile ikunda kwibumbira hamwe ishobora kuba imbere ya proteine ​​ya kondensate 23.
Igishimishije, αS na tau byagaragaye ko bifatanya muguhuza indwara ziranga abantu barwaye indwara ya Parkinson hamwe nizindi synucleinopathies 24,25 kandi ubushakashatsi bwerekanye ko isano iri hagati ya poroteyine zombi 26.27 byerekana isano iri hagati yo guteranya αS na tau mu ndwara zifata ubwonko.uburwayi.αS na tau byagaragaye ko zikorana kandi zigateza imbere guterana kwa vitro no muri vivo 28,29 hamwe na heterogeneous agregate igizwe nizo poroteyine zombi byagaragaye mu bwonko bw’abarwayi bafite synucleinopathies 30.Nyamara, bike bizwi kubyerekeranye na molekulike ishingiro ryimikoranire hagati ya αS na tau hamwe nuburyo bwo guhuriza hamwe.αS byavuzwe ko ikorana na tau binyuze mu gukurura electrostatike hagati ya C-terminal ikabije cyane ya C-terminal ya αS hamwe n’akarere gakungahaye kuri protine rwagati ya tau, nayo ikungahaye ku bisigazwa byuzuye neza.
Muri ubu bushakashatsi, twerekana ko αS ishobora rwose kwitandukanya nigitonyanga ikoresheje kanseri ya electrostatike igoye imbere ya tau proteine, bitandukanye n’imikoranire yayo nizindi polypeptide zishishwa neza nka poly-L-lysine (pLK), kandi muriki gikorwa.αS ikora nka molekile ya scafold kumurongo wumuyoboro.Twabonye itandukaniro rigaragara mugikorwa cyo gukura kwa coacervate ya electrostatike αS, ifitanye isano no gutandukanya agaciro nimbaraga zimikoranire ya poroteyine zigira uruhare murusobe rwa coacervate.Igishimishije, twabonye uburyo bwo guhuriza hamwe poroteyine za αS na tau amyloide muri coacervate zimaze igihe kirekire kandi tumenye ibintu bimwe byingenzi biganisha ku guhuriza hamwe izo poroteyine zombi muri coacervate.Hano turasobanura muburyo burambuye iyi nzira, nuburyo bushoboka bwa molekuline bushoboka bushingiye kuri colocalisation ya proteine ​​ebyiri murwego rwihariye rwindwara.
αS ifite umurizo wa anionic C-terminal cyane kuri pH itabogamye (Ishusho 1a), kandi twavuze ko ishobora kunyura LLPS binyuze mu guhuza ibigo bya electrostatike hamwe na molekile ya polypeptide idahwitse.Twakoresheje poli-L-lysine isigaye 100 (pLK) nka molekile yintangarugero yo gutangira bitewe nuburyo bwiza bwa polymeric bwuzuye kandi butajegajega kuri pH 32. Bwa mbere, twemeje ko pLK ikorana na Ct domaine ya αS ikoresheje Solution NMR spectroscopy (Isanamu 1b) ukoresheje 13C / 15N yanditseho αS imbere yo kwiyongera αS: ibipimo bya pLK.Imikoranire ya pLK na Ct-domaine ya αS irigaragaza muguhindura ihindagurika ryimiti no kugabanuka kwimbaraga zo hejuru muri kano karere ka poroteyine.Igishimishije, iyo twavanze αS na pLK kuri αS yibanze hafi.5-25 µM imbere ya polyethylene glycol (5-15% PEG-8) (isanzwe ya LLPS: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) twahise tunyura mumurima mugari wa proteine .ibitonyanga byagaragaye hakoreshejwe fluorescence (WF) hamwe na microscopi yumucyo (BF) (Ishusho 1c).1-5 µm ibitonyanga birimo αS yibanze (wongeyeho 1 µM AlexaFluor488 yanditseho αS, AF488-αS), imitungo yabo ya electrostatike irashobora gukomoka kubirwanya 10% 1,6-hexanediol (1,6-HD) hamwe no kwiyumvamo kwiyongera kwa NaCl kwibanda (Ishusho 1c).Imiterere imeze nkamazi ya coacervate yikigo cya electrostatike αS / pLK igaragazwa nubushobozi bwabo bwo guhurira muri milisegonda (Ishusho 1d).Twifashishije turbidimetry, twagereranije imiterere yibitonyanga muri ibi bihe, twemeza imiterere ya electrostatike yimikoranire nyamukuru ijyanye no guhagarara kwayo (Ishusho 1e), tunasuzuma ingaruka ziterwa na polymer zitandukanye mubikorwa bya LLPS (Ishusho 1f).Nubwo ibitonyanga bitonyanga bigaragara murwego runini rwa polymer, inzira ni nziza cyane iyo pLK irenze αS.LLPs nayo yagaragaye ikoresheje imiti itandukanye yimura dextran-70 (70 kDa) cyangwa ikoresha uburyo butandukanye bwikitegererezo, harimo ibitonyanga byerekana ibirahure, amariba ya microplate yibikoresho bitandukanye, Eppendorf cyangwa capillaries ya quartz.
Igishushanyo cyerekana uturere dutandukanye twa poroteyine muri WT-αS na ΔCt-αS zikoreshwa muri ubu bushakashatsi.Indangantego ya amphipathic N-terminal, hydrophobic amyloide-ikora (NAC), hamwe nubutaka bwa C-terminal bwerekanwe mubururu, orange, numutuku.Ikarita ya Net yishyurwa (NCPR) ikarita ya WT-αS irerekanwa.b NMR isesengura ryimikoranire ya αS / pLK mugihe hatabayeho macromolecular.Mugihe ubunini bwa pLK bwiyongera (αS: ibipimo bya pLK ya 1: 0.5, 1: 1.5, na 1:10 byerekanwe mubyatsi bibisi, icyatsi, nicyatsi kibisi cyijimye).c Coacervate αS / pLK (igipimo cya molar 1:10) kuri 25 µM (1 µM AF488 yanditseho αS cyangwa Atto647N yanditseho pLK kumashusho ya WF) muri buffer ya LLPS (hejuru) cyangwa hiyongeraho 500 mM NaCl (hepfo ibumoso) cyangwa nyuma ya 10 % 1,6-hexanediol (1,6-HD; hepfo iburyo).Umwanya munini = 20 µm.d Amashusho ya microscopique ahagarariye ya BF ibitonyanga bya αS / pLK (igipimo cya molar 1:10) kuri concentration ya 25 μM;imyambi yerekana guhuza ibitonyanga (imyambi itukura n'umuhondo) mumatonyanga mashya (umwambi wa orange) muri 200 ms).Umwanya munini = 20 µm.e Gukwirakwiza urumuri (kuri 350 nm) αS / pLK guteranya muri buffer ya LLPS mbere na nyuma yo kongeramo 500 mM NaCl cyangwa 10% 1,6-HD kuri 25 µM αS (N = 3 icyitegererezo cyigana, bivuze no gutandukana bisanzwe nabyo byerekanwe).f Ishusho ya BF (hejuru) hamwe no gusesengura urumuri (kuri 350 nm, hepfo) ya αS / pLK guteranya kuri 25 μM αS hamwe no kwiyongera αS: igipimo cya molarike ya pLK (icyitegererezo cya N = 3 cyigana, bivuze kandi gutandukana bisanzwe nabyo byerekanwe).Umwanya munini = 10 µm.Igipimo cyibipimo ku ishusho imwe yerekana igipimo cyamashusho yose mugice kimwe.Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Dushingiye ku byo twabonye kuri αS / pLK ya elegitoroniki ya electrostatike hamwe nubushakashatsi bwibanze kuri αS nka molekile yumukiriya wa tau / RNA binyuze mu mikoranire itaziguye na tau31, twavuze ko αS na tau zishobora gufatanya n’umuti mugihe RNA idahari. umwirondoro.binyuze mumashanyarazi ya electrostatike, na αS ni proteine ​​ya scafold muri coacervates ya αS / Tau (reba ikwirakwizwa rya tau mu gishushanyo 2e).Twabonye ko iyo 10 μM αS na 10 μM Tau441 (zirimo 1 μM AF488-αS na 1 μM Atto647N-Tau,) zavanze hamwe muri buffer ya LLPS, zikora byoroshye poroteyine zirimo poroteyine zombi, nkuko bigaragara kuri microscopi ya WF.(Ishusho 2a).Gukoroniza poroteyine zombi mu bitonyanga byemejwe na microscopi ya conocal (CF) (Ishusho y'inyongera. 1a).Imyitwarire nkiyi yagaragaye mugihe dextran-70 yakoreshejwe nkigikorwa cyo guteranya (Ishusho yinyongera. 1c).Twifashishije FITC yanditseho PEG cyangwa dextran, twasanze abakozi bateraniye hamwe bagabanijwe ku buryo butandukanye, nta gutandukanya cyangwa ishyirahamwe (Ishusho yinyongera. 1d).Ahubwo, irerekana ko muri iyi sisitemu bateza imbere gutandukanya ibyiciro binyuze muri macromolecular yoguhuza abantu, kubera ko PEG ari umukozi uhuza abantu benshi, nkuko bigaragara mubindi bikoresho bya LLP 33,34.Ibitonyanga bikungahaye kuri poroteyine byumvaga NaCl (1 M) ariko ntibyari kuri 1,6-HD (10% v / v), byemeza imiterere ya electrostatike (Ishusho yinyongera. 2a, b).Imyitwarire yabo y'amazi yemejwe no kureba milisegonda ihuza ibintu bitonyanga hakoreshejwe microscopi ya BF (Ishusho 2b).
amashusho ya microscopi ya Confocal (CF) ya αS / Tau441 coacervates muri buffer ya LLPS (10 μM ya buri poroteyine, 0.5 μM ya AF488 yanditseho αS na Atto647N yanditseho Tau441).b Guhuza itandukaniro ryerekana itandukaniro (DIC) amashusho ya αS / Tau441 yibitonyanga (10 μM kuri buri proteine).c Igishushanyo cyicyiciro gishingiye ku gukwirakwiza urumuri (kuri 350 nm) ya Tau441 LLPS (0-15 µM) udahari (ibumoso) cyangwa uhari (iburyo) wa 50 µM αS.Amabara ashyushye yerekana gutatana.d Gukwirakwiza urumuri rwa αS / Tau441 LLPS ntangarugero hamwe no kwiyongera kwa αS (Tau441 kuri 5 µM, N = 2–3 byisubiramo byerekana).e Igishushanyo mbonera cyerekana poroteyine zimwe na zimwe za tau hamwe nuturere dutandukanye twa poroteyine zikoreshwa muri ubu bushakashatsi: indangarugero ya N-terminal (umutuku), akarere gakungahaye kuri protine (ubururu), indangarubuga ya microtubule (MTBD, igaragara muri orange), na amyloide-igizwe na spiral.uturere twa filament (PHF) iherereye muri MTBD (imvi).Ikarita ya Net yishyurwa (NCPR) ikarita ya Tau441 irerekanwa.f Ukoresheje 1 µM AF488 yanditseho αS na Atto647N yanditseho ΔNt-, ukoresheje 1 µM AF488 yanditseho αS cyangwa ΔCt-αS imbere ya ΔNt-Tau (hejuru, 10 µM kuri poroteyine) cyangwa K18 (hepfo, 50 µM kuri poroteyine ))) micrografi ya WF yegeranye muri LLPS cyangwa K18 buffer.Ibipimo by'ibipimo mu ishusho imwe byerekana igipimo cy'amashusho yose mu kibaho kimwe (20 µm kuri paneli a, b na f).Ibyibanze kuri paneli c na d bitangwa nkamadosiye yibanze.
Kugirango tugerageze uruhare rwa αS muriki gikorwa cya LLPS, twabanje gukora ubushakashatsi ku ngaruka za αS ku gutonyanga kwa nephelometrie dukoresheje kwiyongera kwa NaCl (Ishusho 2c).Iyo umunyu mwinshi mwinshi mubitegererezo birimo αS, niko agaciro gakwirakwiza urumuri (kuri 350 nm), byerekana uruhare ruhamye rwa αS muri sisitemu ya LLPS.Ingaruka nkiyi irashobora kugaragara mukwongera αS kwibanda (hanyuma rero αS: Tau441 igereranyo) hafi.Kwiyongera inshuro 10 ugereranije no kwibanda kwa tau (5 µM) (Ishusho 2d).Kugirango tugaragaze ko αS ari poroteyine ya scafold muri coacervates, twahisemo gukora iperereza ku myitwarire ya LLPS yahungabanye ya Tau mutant, idafite akarere ka N-terminal karimo nabi (ibisigara 1-150, reba Ishusho 2e) yitwa ΔNt-Tau.Microscopi ya WF na nephelometrie yemeje ko ΔNt-Tau ubwayo itigeze ihura na LLPS (Igishusho 2f na Supplementary Fig. yagaruwe hamwe nubucucike bwigitonyanga hafi yigitonyanga cyuzuye cyibisubizo byuzuye bya Tau na αS mubihe bisa nibitekerezo bya protein.Iyi nzira irashobora kandi kugaragara mubihe byimiterere ya macromolecular yuzuye (Ishusho yinyongera. 2c).Uruhare rwa C-terminal αS karere, ariko ntabwo aruburebure bwarwo bwose, mugikorwa cya LLPS cyerekanwe no kubuza gushiraho ibitonyanga ukoresheje C-terminal yaciwe αS variant ibura ibisigara 104–140 (Ishusho 1a) ya (ΔCt- αS) poroteyine (Igishusho 2f hamwe n’inyongera. 2d).Gukoroniza kwa αS na ΔNt-Tau byemejwe na microscopi ya fluorescence (Ishusho yinyongera. 1b).
Kugirango urusheho kugerageza uburyo bwa LLPS hagati ya Tau441 na αS, hakoreshejwe ubundi buryo bwa Tau, aribwo igice cyahujwe na helical filament core (PHF) igice cya microtubule-binding domaine (MTBD), iyo kirimo ibice bine biranga gusubiramo, mubisanzwe bizwi kandi nk'igice cya K18 (reba Ishusho 2e).Vuba aha byavuzwe ko αS ikunda guhuza na tau proteine ​​iherereye muri domine ikungahaye kuri protine mukurikirane kibanziriza microtubule-binding domaine.Nyamara, akarere ka PHF nako gakungahaye ku bisigazwa byashizwemo neza (reba Ishusho ya 2e), cyane cyane lysine (ibisigisigi 15%), byaduteye gusuzuma niba aka karere nako gatanga umusanzu mu guhuza ikigo cya αS / Tau.Twabonye ko K18 yonyine idashobora gukurura LLPS yibitekerezo bigera kuri 100 μM mubihe byageragejwe (buffer ya LLPS ifite 15% PEG cyangwa 20% dextran) (Ishusho 2f).Ariko, mugihe twongeyeho 50 µM αS kuri 50 µM K18, uburyo bwihuse bwibitonyanga bya poroteyine birimo K18 na αS byagaragaye na nephelometrie (Ishusho yinyongera. 2d) na microscopi ya WF (Ishusho 2f).Nkuko byari byitezwe, ΔCt-αS ntiyashoboye kugarura imyitwarire ya LLPS ya K18 (Ishusho 2f).Twibutse ko guteranya αS / K18 bisaba intungamubiri za poroteyine zo hejuru kugirango zitera LLPS ugereranije na αS / ΔNt-Tau cyangwa αS / Tau441, ibindi bingana.Ibi bihujwe nubufatanye bukomeye bwakarere ka αS C-terminal hamwe na Tau ikungahaye cyane kuri Tau ugereranije na microtubule-binding domain, nkuko byasobanuwe mbere 31.
Urebye ko ΔNt-Tau idashobora gukora LLPS mugihe αS idahari, twahisemo iyi variant ya Tau nkicyitegererezo cyo kuranga αS / Tau LLPS ukurikije ubworoherane bwayo muri sisitemu ya LLPS hamwe na Tau yuzuye (isotype, Tau441 / Tau441).hamwe na complexe (heterotypic, αS / Tau441) inzira yo guteranya.Twagereranije urwego rwo gukusanya αS (nkigice cya poroteyine yegeranye, fαS, c) muri sisitemu ya αS / Tau na αS / ΔNt-Tau na centrifugation hamwe no gukwirakwiza icyiciro cya SDS-PAGE (reba 2e), twabonye indangagaciro zisa cyane. Kuri poroteyine zose hamwe.By'umwihariko, twabonye fαS, c 84 ± 2% na 79 ± 7% kuri αS / Tau na αS / ΔNt-Tau, byerekana ko imikoranire itandukanye hagati ya αS na tau iruta imikoranire ya molekile ya tau.hagati.
Imikoranire hamwe na polykasi zitandukanye ningaruka zuburyo bwa kondegene kuri αS kinetics yakozwe bwa mbere nubushakashatsi bwa fluorescence nyuma yuburyo bwa Photobleaching (FRAP).Twagerageje αS / Tau441, αS / ΔNt-Tau na αS / pLK coacervates (100 μM αS hiyongereyeho 2 μM αS AF488-αS na 100 μM Tau441 cyangwa ΔNt-Tau cyangwa 1 mM pLK).Amakuru yabonetse muminota 30 yambere nyuma yo kuvanga icyitegererezo.Uhereye ku mashusho ahagarariye FRAP (Igishusho 3a, αS / Tau441)na ΔNt-Tau, byihuta cyane hamwe na pLK.Coefficient yabazwe kuri αS imbere muri coacervate ukurikije FRAP (nkuko byasobanuwe na Kang nabandi 35) ni D = 0.013 ± 0.009 µm2 / s na D = 0.026 ± 0.008 µm2 / s kuri αS / Tau441 na αS / ΔNt- kuri αS / Sisitemu.pLK, Tau, na D = 0.18 ± 0.04 µm2 / s, (Ishusho 3c).Nyamara, coefficient de diffuzione αS mugice cyatatanye ni ordre nyinshi zubunini burenze ibyiciro byose byegeranye, nkuko byagenwe na Fluorescence Correlation Spectroscopy (FCS, reba Ishusho ya 3) mubihe bimwe (buffer ya LLPS) ariko mugihe hatabayeho polykasi. (D = 8 ± 4 µm2 / s).Kubwibyo, kinetics yubusobanuro bwa αS yagabanutse cyane muri coacervate ugereranije na proteyine mugice cyatatanye bitewe ningaruka zivugwa na molekile zuzuye, nubwo coacervate zose zigumana ibintu bimeze nkamazi mugihe cyamasaha yambere nyuma yo gushingwa, bitandukanye nicyiciro cya tau.byihuse kinetics muri pLK condensate.
a - c FRAP isesengura imbaraga za αS (2% AF488 yanditseho αS) muri coacervates ya electrostatike.Amashusho ahagarariye αS / Tau441 FRAP yerekana muri bitatu yerekanwa muri (a), aho uruziga rutukura rwerekana uduce twiza.Igipimo cyibipimo ni 5 µm.b Impuzandengo ya FRAP umurongo hamwe na (c) ibarwa ikwirakwizwa rya diffuzione (D) kubitonyanga 5-6 (N) ibitonyanga bitandukanye bivuye mubushakashatsi butatu ukoresheje 100 µM αS hamwe na equimolar yibanze ya Tau441 (umutuku) cyangwa ΔNt-Tau (ubururu) cyangwa pLK (icyatsi) inshuro icumi kwibanda kuri LLPS.Gutandukana bisanzwe kumurongo wa FRAP byerekanwe mubicucu.Kugereranya, coefficient de diffuzione αS mugice cyatatanye yagenwe muri bitatu ukoresheje fluorescence ihuriro rya spekitroscopi (FCS) (reba Ishusho ya 3 nuburyo bwamakuru menshi).d Gukomeza X-band ya EPR yerekanwe ya 100 μM TEMPOL-122-αS muri buffer ya LLPS nta polycasiyo (umukara) cyangwa imbere ya 100 μM Tau441 (umutuku) cyangwa ΔNt-Tau (ubururu) cyangwa 1 mM pLK (icyatsi).Inet yerekana uburyo bunini bwo kureba umurongo ukomeye wumurongo aho impinduka zikomeye zigaragara.e Guhuza imirongo ya 50 μM TEMPOL-122-αS hamwe na polykasi zitandukanye mugihe LLPS idahari (nta PEG).Kugabanuka kwa amplitude ya bande III ugereranije nitsinda rya II (IIII / III) ryurwego rusanzwe rwa EPR rwerekanwe kongera ibipimo bya molar ya Tau441 (umutuku), ΔNt-Tau (ubururu) na pLK (icyatsi).Imirongo y'amabara yerekana ihuye namakuru ukoresheje uburyo bukomeye bwo guhuza hamwe n imbuga zisa kandi zigenga zihuza kuri buri murongo.Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Nkuzuzanya, twasesenguye imbaraga za αS muri coacervates zitandukanye dukoresheje icyerekezo cya spin label (SDSL) hamwe na electron paramagnetic resonance ikomeza (CW-EPR).Ubu buryo bwerekanye ko ari ingirakamaro cyane mu kumenyekanisha imiterere n’ingaruka za IDP hamwe n’ibisubizo bifatika bisigaye 36,37,38.Kugira ngo ibyo bishoboke, twubatse ibisigazwa bya sisitemu muri mutant imwe ya Cys kandi dukoresha 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Ibikomoka kuri Maleimide biranga.Byumwihariko, twashyizemo iperereza rya TEMPOL kumwanya wa 122 cyangwa 24 αS (TEMPOL-122-αS na TEMPOL-24-αS).Mugihe cyambere, twibanze kuri C-terminal ya proteine, igira uruhare mubikorwa na polycations.Ahubwo, umwanya wa 24 urashobora kuduha amakuru kubyerekeranye ningaruka rusange za poroteyine muri kondensate.Muri ibyo bihe byombi, ibimenyetso bya EPR byabonetse kuri poroteyine zo mu cyiciro cyatatanye bihuye na nitroxide radicals mu buryo bwihuta.Nyuma yo gutandukana kwicyiciro imbere ya tau cyangwa pLK (100 μM TEMPOL-αS, Tau441 cyangwa ΔNt-Tau ku kigereranyo cya 1: 1 cyangwa pLK ku kigereranyo cya 1:10), kwiyongera k'uburemere bugereranije byagaragaye muri EPR Ikirangantego cya αS.Umurongo wigihombo wagutse, byerekana kugabanuka kwa αS reorientation kinetics mu bitonyanga ugereranije na poroteyine mugice cya dilute (Ishusho 3d, Ishusho yinyongera. 4a).Izi mpinduka zigaragara cyane kumwanya wa 122. Mugihe kumwanya wa 24 kuba pLK itagize ingaruka kumikorere yubushakashatsi, kumwanya wa 122 imiterere yumurongo wikurikiranya yahindutse kuburyo bugaragara (Ishusho yinyongera 4a).Mugihe twagerageje kwerekana icyerekezo kuri 122 ya sisitemu ebyiri αS / polycation dukoresheje moderi ya isotropique (Ishusho yinyongera 5a) ikunze gukoreshwa mugusobanura imbaraga za spin-label IDP 38,39, ntitwashoboye kongera kubaka ibizamini byubushakashatsi..Kwigana ibintu byerekana umwanya wa 24 kuzenguruka (Ishusho yinyongera. 5a).Ibi birerekana ko hari imyanya ikunzwe mumwanya wa spin iboneza rya C-terminal akarere ka αS imbere ya polycations.Iyo usuzumye agace ka αS mugice cyegeranye mugihe cyubushakashatsi bwa EPR (84 ± 2%, 79 ± 7%, na 47 ± 4% kuri αS / Tau441, αS / ΔNt-Tau, na αS / pLK, reba - Inyongera Igishushanyo cya 2e yisesengura ryamakuru c), birashobora kugaragara ko kwaguka byagaragaye nuburyo bwa EPR byerekana cyane cyane imikoranire yakarere ka C-terminal ka αS hamwe na polykasi zitandukanye mugice cyegeranye (impinduka nyamukuru mugihe ukoresheje TEMPOL-122- αS), kandi ntabwo ari intungamubiri za poroteyine.Ubwiyongere bwa microciscosity bugaragara muri probe.Nkuko byari byitezwe, EPR ya poroteyine mubihe bitari LLPS yagaruwe rwose mugihe 1 M NaCl yongewe kumvange (Ishusho yinyongera 4b).Muri rusange, amakuru yacu yerekana ko impinduka zagaragajwe na CW-EPR zigaragaza cyane cyane imikoranire yakarere ka C-terminal ya αS hamwe na polykasi zitandukanye mugice cyegeranye, kandi iyi mikoranire isa nkaho ikomeye na pLK kuruta Tau.
Kugirango tubone amakuru menshi yuburyo bujyanye na poroteyine ziri muri coacervate, twahisemo kwiga sisitemu ya LLPS dukoresheje NMR mubisubizo.Ariko, twashoboraga gusa kumenya agace ka αS gasigaye mugice cyatatanye, gishobora guterwa no kugabanuka kwa poroteyine imbere muri coacervate hamwe nicyiciro cyimbitse munsi yumuti mubisesengura rya NMR.Mugihe twasesenguye imiterere nimbaraga za poroteyine zisigaye mugice cyatatanye cyicyitegererezo cya LLPS dukoresheje NMR (Ishusho yinyongera. 5c, d), twabonye ko poroteyine yitwaye hafi ya yose imbere ya pLK na ΔNt-Tau, byombi Byari muburyo bwa kabiri hamwe ningaruka za protein umugongo, byagaragajwe nubushakashatsi bwerekeranye no guhinduranya imiti ya kabiri no kuruhuka R1ρ.Amakuru ya NMR yerekana ko C-terminus ya αS igira igihombo kinini cyo guhuza n'imiterere mugihe igumana imiterere yayo idahwitse, nkibindi bikurikirana bya poroteyine, kubera imikoranire yayo na polycasiyo.
Kubera ko ikimenyetso cya CW-EPR cyagutse cyagaragaye muri TEMPOL-122-αS icyiciro cyegeranye kigaragaza imikoranire ya poroteyine na polycasiyo, twakoze titre ya EPR kugirango dusuzume isano ihuza αS na polykasi zitandukanye mugihe LLPS idahari (nta kwirundanya kwa Buffer LLPS), yerekana ko imikoranire ari imwe mu byiciro byoroheje kandi byibanze (ibyo bikaba byemezwa namakuru yacu, Igishushanyo cya 4a ninyongera 6).Icyari kigamijwe kwari ukureba niba coacervates zose, nubwo zisanzwe zimeze nkamazi, zigaragaza imyitwarire itandukanye ishingiye kurwego rwa molekile.Nkuko byari byitezwe, icyerekezo cya EPR cyagutse hamwe no kongera ubukana bwa polycation, byerekana kugabanuka kwimiterere ya molekile bitewe n’imikoranire ya molekuline yabafatanyabikorwa bose hafi yo kwiyuzuzamo (Ishusho 3e, Ishusho yinyongera 6).pLK yageze kuri kwiyuzuzamo ku kigereranyo cyo hasi (polycation: αS) ugereranije na ΔNt-Tau na Tau441.Mubyukuri, kugereranya amakuru hamwe nicyitegererezo cyo guhuza cyerekana ko imbuga zisa kandi zigenga zerekana ko imbuga zigaragara zihoraho za pLK (~ 5 μM) ni gahunda yubunini buri munsi ya Tau441 cyangwa ΔNt-Tau (~ 50 μM ).µM).Nubwo iyi ari igereranya, ibi byerekana ko αS ifite isano iri hejuru ya polycasi yoroshye hamwe nakarere keza keza.Urebye iri tandukaniro riri hagati ya αS na polycations zitandukanye, twavuze ko ibintu byamazi bishobora guhinduka mugihe runaka bityo bikababazwa nuburyo butandukanye bwa LSPT.
Urebye ibidukikije byuzuyemo abantu benshi muri poroteyine coacervate hamwe na amyloide ya poroteyine, twarebye imyitwarire ya coacervate mugihe kugirango tumenye inzira LSPT ishoboka.Twifashishije microscopi ya BF na CF (Isanamu 4), twabonye ko αS / Tau441 coacervates ku rugero runini mugukemura, ikora ibitonyanga binini bihuza kandi bigahindura ubuso hepfo yiziba / kunyerera nkibitonyanga byuzuye, nkuko byari byitezwe (Ishusho yinyongera . 7d);twita izi nyubako zubatswe hasi "protein rafts".Izi nyubako zagumye zitemba kuko zagumanye ubushobozi bwo guhuza (Ishusho yinyongera. 7b) kandi byashoboraga kuboneka mumasaha menshi nyuma yuko LLPS itangiye (Ishusho ya 4 nishusho yinyongera 7c).Twabonye ko uburyo bwo guhanagura butoneshwa hejuru ya hydrophilique aho kuba ibikoresho bya hydrophobi (Igicapo cyongeweho.Ikigaragara ni uko αS / ΔNt-Tau coescence hamwe na rafting byagabanutse cyane, mugihe kondensate ya αS / pLK yagabanutse cyane (Ishusho 4).Mugihe gito cya incububasi, ibitonyanga αS / pLK byashoboye guhuriza hamwe no guhanagura hejuru ya hydrophilique, ariko iki gikorwa cyahise gihagarara kandi nyuma yamasaha 5 yubushakashatsi, gusa ibyabaye bike bya coescence kandi ntamazi yabonetse.- inzibacyuho ya gel.
Uhagarariye BF (paneli ya greyscale) na CF (panne iburyo, AF488 yanditseho αS mu cyatsi) by'icyitegererezo cya coacervate irimo 100 µM αS (1% fluorescent label) muri buffer ya LLPS imbere ya 100 µM Tau441 (hejuru) fluorescence) amashusho ya microscopique ΔNt -Tau (hagati) cyangwa 1 mM pLK (hepfo) mugihe cyubushakashatsi butandukanye hamwe nuburebure bwibanze (z, intera kuva hepfo yisahani neza).Ubushakashatsi bwagiye busubirwamo inshuro 4-6 zidashingiye kuri buri kimwe hamwe nibisubizo bimwe.αS / Tau441 coacervates itose nyuma yamasaha 24, ikora ibiti binini kuruta ishusho.Igipimo cyibipimo kumashusho yose ni 20 µm.
Twahise tubaza niba ibinini binini bisa na poroteyine byakozwe muri αS / Tau441 LLPS byatera amyloide guteranya poroteyine zose zize.Twakurikiranye gukura kwa αS / Tau441 ibitonyanga mugihe hamwe na microscopi ya WF mubihe bimwe nkuko byavuzwe haruguru, ariko dukoresha 1 μM AF488 yanditseho αS na Atto647N yanditseho Tau441 (Ishusho 5a).Nkuko byari byitezwe, twabonye protein yuzuye murwego rwo gukura.Igishimishije, uhereye kuri ca.Nyuma yamasaha 5, inyubako zikomeye zidafite uruziga zagaragaye imbere muri ruhago, twise "amanota", zimwe muri zo zashyizwe hamwe na αS, izindi zikungahazwa muri Tau441 (Ishusho 5a, imyambi yera).Iyi myanya yamye igaragara mumurongo murwego runini kuri αS / ΔNt-Tau kuruta kuri αS / ΔNt-Tau.Nta mwanya wihariye wagaragaye mubitonyanga bya sisitemu ya pLK na Tau idafite ubushobozi bwo guhuza / guhanagura.Kugirango tumenye niba ibyo birangantego birimo αS na Tau441 ari igiteranyo kimeze nka amyloide, twakoze ubushakashatsi busa dukoresheje microscopi ya CF aho Tau441 yanditseho Atto647N na 12.5 μM amyloide yihariye ya thioflavin-T (ThT) yongewemo kuva yatangira.irangi.Nubwo ThT-irangi ya αS / Tau441 ibitonyanga cyangwa ibiti bitigeze bigaragara na nyuma ya 24 h ya incububasi (Igicapo 5b, umurongo wo hejuru - ibitonyanga bisigaye hejuru ya poroteyine), inyubako nziza ya ThT irimo Atto647N-Tau441 imbere muri ruhago yari ifite intege nke cyane.ibi bigana ingano, imiterere, hamwe n’ahantu hasobanuwe mbere (Igicapo 5b, umurongo wo hagati nu munsi), byerekana ko ibibara bishobora guhura na amyloide isa na agregate ikorwa muri coacervates ishaje.
WF 25. .Ubushakashatsi butandatu bwasubiwemo bwigenga hamwe nibisubizo bisa.b CF microscopique ishusho ya 25 μM αS imbere ya 25 μM Tau441 (1 μM Atto647N yanditseho Tau441) na 12.5 μM thioflavin-T (ThT).Ibitonyanga bya poroteyine bifite uburemere hamwe na poroteyine zashyizwe hamwe byerekanwe ahantu hejuru no hagati.Umurongo wo hasi werekana amashusho yigitonyanga nigitonyanga kuva 3 yigenga yigana.Imyambi yera yerekana utudomo twiza muri panne zombi.Igipimo cyibipimo kumashusho yose ni 20 µm.
Kugira ngo dusuzume mu buryo burambuye impinduka zabaye mu muyoboro wa poroteyine wa coacervate mu gihe cyo kuva mu mazi ujya mu bikomeye, twakoresheje amashusho ya fluorescence ubuzima bwose (FLIM) na Förster resonance yohereza ingufu za microscopi (FRET) (Ishusho ya 6 n’ishusho ya 8 na 9).Twakekereje ko gukura kwa coacervate kurwego rwarushijeho kuba rwinshi cyangwa se rukaba rumeze nkurwego rukomatanyije rwa poroteyine biganisha ku mikoranire ya hafi hagati ya poroteyine na probe ya fluorescent ifatanye nayo, bikaba bishobora gutanga ingaruka zo kuzimya zigaragarira mubuzima bugufi (τ) , nkuko byasobanuwe mbere40., 41, 42.Mubyongeyeho, kubirango byanditseho kabiri (AF488 na Atto647N nkumuterankunga wa FRET hamwe n amarangi yabakiriye), uku kugabanuka kwa τ kurashobora kandi guherekezwa na coacervate hamwe no kwiyongera kwa FRET (E) mugihe cya LSPT.Twakurikiranye uruzitiro hamwe nimiterere yibihe mugihe cya LLPS αS / Tau441 na αS / ΔNt-Tau (25 µM ya buri proteine ​​muri buffer ya LLPS irimo 1 µM AF488 yanditseho αS na / cyangwa Atto647N yanditseho Tau441 cyangwa ΔNt-Tau).Twabonye icyerekezo rusange muburyo ubuzima bwa fluorescence mubuzima bwa AF488 (τ488) na Atto647N (τ647N) bwagabanutseho gato uko coacervate ikuze (Ishusho 6 nigishushanyo cya 8c).Igishimishije, iri hinduka ryongerewe imbaraga cyane kumudomo uri mu rufunzo (Igishusho 6c), byerekana ko izindi poroteyine zabaye kuri utudomo.Mu rwego rwo gushyigikira ibi, nta mpinduka nini yabayeho mubuzima bwa fluorescence yagaragaye kubitonyanga bya αS / ΔNt-Tau bifite imyaka 24 h (Umugereka winyongera. muri coacervates.Twabibutsa ko utudomo dufite ubunini butandukanye nibirimo guhinduka muri αS, cyane cyane kuri sisitemu ya αS / Tau441 (Ishusho yinyongera. 8e).Kugabanuka kwubuzima bwa fluorescence mubuzima bwaherekejwe no kwiyongera kwimbaraga, cyane cyane kuri Atto647N yanditseho Tau441 (Igicapo cyinyongera. nyuma yo gukurura, poroteyine ziri mu mashanyarazi ihagaze neza.Ugereranije na αS / ΔNt-Tau, twarebye hasi τ647N hamwe na hejuru ya τ488 indangagaciro mu mwanya wa αS / Tau441, iherekejwe n’indangagaciro za FRET zo hasi kandi nyinshi.Birashoboka, ibi birashobora kuba bifitanye isano nuko muri sisitemu ya αS / Tau441, ubwinshi bwagaragaye kandi buteganijwe αS ubwinshi muri agregate ni butandukanye, akenshi usanga substoichiometric ugereranije na Tau, kubera ko Tau441 ubwayo ishobora no gukorerwa LLPS no guteranya (Ishusho yinyongera 8e) .Nyamara, urugero rwa coescence yibitonyanga, kwibumbira mu rufunzo, kandi, icy'ingenzi, kwegeranya poroteyine mu mazi asa na coacervates ni menshi iyo Tau441 na αS byombi bihari.
microscopi ya Lifetime fluorescence (FLIM) ya αS / Tau441 na αS / ΔNt-Tau kuri 25 μM ya buri poroteyine (1 μM AF488 yanditseho αS na 1 μM Atto647N yanditseho Tau441 cyangwa ΔNt-Tau) muri buffer ya LLPS.Inkingi zerekana amashusho ahagarariye ingero za LLPS mugihe cyo gukura (30 min, 5 h na 24 h).Ikadiri itukura yerekana akarere karimo ibibanza αS / Tau441.Ubuzima bumara bwerekanwa nkibara ryamabara.Umwanya muto = 20 µm kumashusho yose.b Zoomed-muri FLIM ishusho yakarere katoranijwe, yerekanwe mumasanduku itukura mumwanya a.Urwego rwubuzima rwerekanwe ukoresheje ibipimo byamabara nkibiri mu kibaho a.Umwanya munini = 5 µm.c Histogramu yerekana AF488 (yometse kuri αS) cyangwa Atto647N (ifatanye na Tau) kubwoko butandukanye bwa poroteyine (ibitonyanga-D-, raft-R- nudusimba-P) byagaragaye mumashusho ya FLIM yanditswe kuri αS-) igihe cyo kugabura igihe cya Tau441 na αS / ΔNt-Tau coacervate ntangarugero (N = 17-32 ROI kuri D, 29-44 ROI kuri R, na 21-51 ROI kumanota).Indangagaciro na median zerekanwa nkumuhondo kare hamwe numurongo wumukara imbere mubisanduku.Imipaka yo hepfo no hejuru yisanduku yerekana quartile ya mbere na gatatu, kimwe, kandi byibuze nagaciro ntarengwa murwego rwikubye inshuro 1.5 (IQR) byerekanwe nka whiskers.Abasohoka berekanwa nka diyama yumukara.Ubusobanuro bwibarurishamibare hagati yabantu babiri bagabanijwe byagenwe hakoreshejwe ibyitegererezo bibiri-t-ikizamini, ukeka ko bitandukanijwe.Imirongo ibiri-t-igeragezwa p-indangagaciro yerekanwa hamwe ninyenyeri kuri buri jambo ryagereranijwe (* p-agaciro> 0.01, ** p-agaciro> 0.001, *** p-agaciro> 0.0001, **** p-agaciro> 0.00001), ns Yerekana uburangare (p-agaciro> 0.05).Indangagaciro nyazo zitangwa mu mbonerahamwe ya 1, kandi amakuru yumwimerere atangwa nkamadosiye yibanze.
Kugirango turusheho kwerekana imiterere ya amyloide yimitsi / igiteranyo, twakoresheje ingero za coacervate zidafite isuku mugihe cyamasaha 24 hamwe na (1 M) NaCl nyinshi, bigatuma habaho gutandukanya hamwe na proteine ​​coacervate.Iyo igiteranyo cyitaruye (ni ukuvuga igisubizo gitatanye cya agregate) cyagaragaye hakoreshejwe microscopi yingufu za atome (AFM), twabonye imyifatire yiganjemo imiterere ya metero nini ifite uburebure bwa metero 15 nm, ikunda guhuza mugihe cyumunyu mwinshi, bisa imyitwarire ya fibrile isanzwe ya amyloide kubera ingaruka zikomeye za hydrophobique hejuru (menya ko fibrile ifite uburebure bwa ~ 10 nm) (Ishusho yinyongera 10a).Igishimishije, mugihe igiteranyo cyitaruye cyashyizwemo na ThT mubisubizo bisanzwe bya ThT fluorescence, twabonye ubwiyongere bukabije bwumusaruro wa Quant fluorescence wa ThT fluorescence, ugereranije niyagaragaye mugihe irangi ryashyizwemo fibrile isanzwe ya αS amyloide (Ishusho yinyongera 10b), byerekana ko. coacervate igiteranyo kirimo amyloide imeze..Mubyukuri, igiteranyo cyihanganiraga umunyu mwinshi ariko ukumva chloride ya 4 M ya guanidine (GdnHCl), nka fibrile isanzwe ya amyloide (Ishusho yinyongera 10c).
Ibikurikira, twasesenguye ibice bigize igiteranyo dukoresheje molekile imwe ya fluorescence, isano ya fluorescence ihuriro / kwambukiranya imipaka ya sprosroscopi (FCS / FCCS), no gusesengura guturika byerekana amabara abiri ahuye (TCCD).Kugira ngo ibyo bishoboke, twatandukanije igiteranyo cyakozwe nyuma yamasaha 24 yubushakashatsi muri 100 μl LLPS ntangarugero zirimo αS na Tau441 (byombi 25 μM) hamwe na 1 μM AF488 yanditseho αS na 1 μM Atto647N yanditseho Tau441.Koresha igisubizo cyavuyeho gikusanyirijwe hamwe kuri leta ya monomolecular ukoresheje buffer imwe idafite PEG na 1 M NaCl (buffer imwe ikoreshwa mugutandukanya igiteranyo na coacervate) kugirango wirinde imikoranire ishoboka ya electrostatike hagati ya LLPS na proteyine.Urugero rwigihe cyerekezo cya molekile imwe irashobora kugaragara kumashusho 7a.Isesengura rya FCCS / FCS (kwambukiranya, CC na autocorrelation, AC) ryerekanye ko igiteranyo kirimo αS na tau cyari kinini mu byitegererezo (reba umurongo wa CC ku gishushanyo cya 7b, ikibaho cy’ibumoso), kandi harenze urugero rwa poroteyine zisigaye za monomeric zavutse nka a ibisubizo byokugabanuka (reba umurongo wa AC mumashusho 7b, ikibaho cyibumoso).Ubushakashatsi bwo kugenzura bwakozwe muburyo bumwe bwo gukemura hakoreshejwe ingero zirimo poroteyine za monomeric gusa zerekanye ko nta murongo wa CC, kandi umurongo wa AC uhuza neza na moderi imwe yo gukwirakwiza ibice (Eq. 4), aho poroteyine za monomeric zifite coefficient ziteganijwe gukwirakwizwa (Ishusho 7b) ), ikibaho cy'iburyo).Coefficient de diffuzione yibice byegeranijwe biri munsi ya 1 µm2 / s, naho proteine ​​za monomeric ni nka 1 µm2 / s.50–100 µm / s;indangagaciro zisa nizindi ndangagaciro zasohotse mbere ya sonic αS amyloid fibrile na monomeric αS ukwayo mubihe bisa nkibisubizo44.Mugihe twasesenguye igiteranyo hamwe nisesengura rya TCCD (Igicapo 7c, panne yo hejuru), twasanze muri buri giterane cyitaruye (αS / Tau heteroaggregate), hafi 60% byateranijwe byagaragaye birimo αS na tau, hafi 30% birimo gusa tau, hafi 10% αS gusa.Isesengura rya Stoichiometric ryakozwe na αS / Tau heteroaggregates ryerekanye ko ibyinshi muri heteroaggregates byakungahajwe muri tau (stoichiometry iri munsi ya 0.5, impuzandengo ya molekile ya tau kuri agregate ikubye inshuro 4 kurenza molekile ya αS), ibyo bikaba bihuye nakazi kacu kagaragaye muri FLIM mubihe ubushakashatsi..Isesengura rya FRET ryerekanye ko ibyo byegeranyo byose birimo poroteyine zombi, nubwo indangagaciro nyazo za FRET muri uru rubanza zidafite akamaro kanini, kubera ko ikwirakwizwa rya fluorofore muri buri giterane ryabaye impanuka bitewe n’ikirenga cya poroteyine zitemewe zikoreshwa mu bushakashatsi.Igishimishije, mugihe twakoze isesengura rimwe dukoresheje 45,46 ikuze ya amyloide yegeranya-idafite Tau variant (reba Ishusho yinyongera 11a, b), twabonye ko nubwo igiteranyo cya αS electrostatike cyari kimwe (Ishusho yinyongera 11c, d), ubushobozi bwo gukora igiteranyo muri coacervate cyaragabanutse cyane kandi FLIM yatahuye ahantu henshi mubushakashatsi bwakozwe, kandi intege nke zambukiranya imipaka zagaragaye kuburugero rwihariye.Nyamara, ku mubare muto wabonetse (kimwe cya cumi gusa cya Tau441), twabonye ko buri cyegeranyo cyakungahajwe muri αS kurenza iyi variant ya Tau, hamwe na 50% bya agregate zagaragaye zirimo molekile ya αS gusa, kandi αS yari itandukanye cyane birenze .igiteranyo (reba Ishusho yinyongera. 11e), bitandukanye nuburinganire butandukanye bwakozwe na Tau441 (Ishusho 6f).Ibyavuye muri ubwo bushakashatsi byagaragaje ko nubwo αS ubwayo ishoboye kwegeranya na tau muri coacervate, tau nucleation nibyiza cyane muribi bihe, kandi ibivuyemo bisa na amyloide bishobora gukora nkuburyo bwa αS na tau.Nyamara, iyo hamaze gushingwa intungamubiri ya tau, imikoranire itandukanye hagati ya αS na tau itoneshwa hamwe muguhuza imikoranire ya homotypic hagati ya molekile ya tau;twitegereza kandi imiyoboro ya poroteyine mumazi αS / tau coacervates.
uhagarariye fluorescence ibimenyetso byigihe gito bya molekile imwe yingingo zegeranye zakozwe muri αS / Tau441 coacervates ya electrostatike.Ibisasu bihuye na αS / Tau441 coaggregates (guturika hejuru yurugero rwerekanwe) byagaragaye mumiyoboro itatu yo gutahura (AF488 na Atto647N yoherejwe nyuma yo kwishima bitaziguye, imirongo yubururu numutuku, ibyuka bya Atto647N nyuma yo kwishima bitaziguye), FRET, umurongo wa violet).b FCS / FCCS isesengura ryikitegererezo cya αS / Tau441 yegeranye yakuwe muri LLPS (ikibaho cyibumoso).Imirongo ya Autocorrelation (AC) ya AF488 na Atto647N irerekanwa mubururu nubururu, naho umurongo uhuza (CC) ujyanye na agregate irimo amarangi yombi yerekanwa mubururu.Imirongo ya AC yerekana ko hariho ubwoko bwa poroteyine bwitwa monomeric hamwe na poroteyine zegeranijwe, mugihe imirongo ya CC yerekana gusa ikwirakwizwa ryibice bibiri byanditse.Isesengura rimwe, ariko mugihe kimwe cyibisubizo nkibibanza byitaruye, ingero zirimo monomeric αS na Tau441 gusa zerekanwa nkigenzura muburyo bwiza.c Fluorescence flash isesengura ya molekile imwe ya agregate yihariye yakozwe muri αS / Tau441 coacervates ya electrostatike.Ibisobanuro kuri buri giterane kiboneka mubisubiramo bine bitandukanye (N = 152) byateguwe kurwanya stoichiometry, S indangagaciro, hamwe na FRET ikora neza (panne yo hejuru, ibara ryerekana ibara ryerekana).Ubwoko butatu bwo guteranya bushobora gutandukanywa: -αS-gusa igiteranyo hamwe na S ~ 1 na FRET ~ 0, Tau-yegeranye gusa hamwe na S ~ 0 na FRET ~ 1, hamwe na Tau / αS itandukanye hamwe hamwe na S hamwe na FRET Ikigereranyo cyamafaranga za poroteyine zombi zerekana muri buri giterane (N = 100) cyerekanwe mugice cyo hasi (igipimo cyamabara kigaragaza ibibaho).Amakuru mabi atangwa muburyo bwa dosiye yibanze.
Gukura cyangwa gusaza kwa poroteyine y'amazi ikomatanya mu buryo busa na gel cyangwa ibintu bikomeye mu gihe cyagenwe byavuzwe ko bigira uruhare mu mikorere myinshi ya physiologique ya kondensate47 kimwe no mu ndwara, nk'inzira idasanzwe ibanziriza guteranya amyloide 7, 48, 49. Hano twiga gutandukanya icyiciro nimyitwarire muburyo burambuye.LSPT αS imbere ya polycations zidasanzwe mubidukikije bigenzurwa na micromolar nkeya hamwe nibihe bifitanye isano na physiologique (menya ko ibarwa rya physiologique yabazwe ya αS ari> 1 µM50), ukurikije imyitwarire isanzwe ya termodinamike ya LPS.Twabonye ko αS, ikubiyemo akarere ka C-terminal gashizwemo nabi cyane kuri physiologique pH, ishoboye gukora ibitonyanga bikungahaye kuri proteyine mubisubizo byamazi binyuze muri LLPS imbere ya peptide ya cationic idahwitse cyane nka pLK cyangwa Tau binyuze muburyo bwa electrostatike guhuza ibintu bigoye imbere ya macromolecules.Iyi nzira irashobora kugira ingaruka zingirakamaro mubidukikije bya selile aho αS ihura na molekile zitandukanye za polycationique zifitanye isano nindwara ziterwa no guteranya indwara haba muri vitro ndetse no muri vivo51,52,53,54.
Mu bushakashatsi bwinshi, imbaraga za poroteyine ziri mu bitonyanga byafashwe nkimwe mubintu byingenzi bigena inzira yo gukura 55,56.Muri electrostatike αS ikorana na polycations, inzira yo gukura bigaragara ko iterwa nimbaraga zimikoranire na polycasiyo, agaciro, hamwe nubwinshi bwimikoranire.Ihame rya Equilibrium ryerekana ko imiterere yuburinganire bwibihugu bibiri byamazi yaba ahari igitonyanga kinini gikungahaye kuri biopolymers itwara LLPS57.58.Gukura kw'ibitonyanga birashobora kugerwaho no gukura kwa Ostwald59, coalescence60 cyangwa gukoresha monomer yubusa mugice cyatatanye61.Kuri αS na Tau441, ΔNt-Tau cyangwa pLK, proteine ​​nyinshi zibanze muri kondensate mubihe byakoreshejwe muri ubu bushakashatsi.Nyamara, mugihe ibitonyanga byuzuye bya tau byahurijwe hamwe hejuru yubushyuhe bwo hejuru, ibitonyanga bitonyanga hamwe no guhanagura byari bigoye kuri ΔNt-Tau na pLK, byerekana gutakaza vuba ibintu byamazi muri sisitemu zombi.Dukurikije isesengura ryakozwe na FLIM-FRET, ibitonyanga bishaje bya pLK na ΔNt-Tau byerekanaga urugero rumwe rwo gukusanya poroteyine (ubuzima bwa fluorescence ubuzima busa) nkibitonyanga byumwimerere, byerekana ko umuyoboro wa poroteyine wambere wagumishijwe, nubwo byakomera.
Dushyira mu gaciro ibisubizo byubushakashatsi muburyo bukurikira (Ishusho 8).Ibitonyanga byambere byakozwe byigihe gito ni imiyoboro ya poroteyine idafite indishyi za electrostatike, bityo hakaba hari uduce twinshi two kutaringaniza amafaranga, cyane cyane kuri interineti, bigatuma habaho ibitonyanga bifite ubushobozi bwo hejuru bwa electrostatike.Kugirango yishyure amafaranga (phenomenon ikunze kwitwa valence depletion) no kugabanya ubushobozi bwubuso bwibitonyanga, ibitonyanga birashobora gushiramo polypeptide nshya kuva mugice cya dilute, kuvugurura imiyoboro ya poroteyine kugirango hongerwe imbaraga zishyurwa, kandi zikorane nibindi bitonyanga.hamwe n'ubuso (wetting).Ibitonyanga bya αS / pLK, kubera urusobe rworoshye rwa poroteyine (gusa imikoranire ya heterotypic hagati ya αS na pLK) hamwe no kurushaho guhuza imikoranire ya poroteyine-proteyine, bisa nkaho bishobora kuringaniza amafaranga ya kondensate vuba;mubyukuri, twabonye protein kinetics yihuse muburyo bwambere αS / pLK coacervates kuruta muri αS / Tau.Nyuma yo kugabanuka kwa valence, imikoranire iba mike cyane kandi ibitonyanga bitakaza ibintu byamazi hanyuma bigahinduka nka geli, ibitonyanga bidashya kandi bifite ubushobozi buke bwa electrostatike (bityo ntibishobora guhanagura hejuru).Ibinyuranye, ibitonyanga bya αS / Tau ntibikora neza mugutezimbere ingano yumutwaro bitewe nurusobe rwinshi rwa poroteyine (hamwe n’imikoranire ya homotypic na heterotypic) hamwe na kamere idakomeye yimikoranire ya poroteyine.Ibi bivamo ibitonyanga bigumana imyitwarire yamazi mugihe kinini kandi bikerekana ubushobozi bwa electrostatike yubuso bushobora kugabanuka no guhuriza hamwe no gukura (bityo bikagabanya ubuso bwubuso / igipimo cyibipimo byibitonyanga) no guhanagura chem ya hydrophilique.Ibi birema amasomero manini yibitseho amasomero agumana ibintu byamazi nkuko imikoranire ikomeza kuba iyigihe gito bitewe nubushakashatsi burigihe bwo gushakisha uburyo bwiza bwa neti.Igishimishije, uburyo bwa N-bwaciwe bwa Tau, harimo na bimwe mubisanzwe bibaho isoforms62, byerekana imyitwarire hagati, hamwe na coacervates gusaza hamwe na αS mubitonyanga byigihe kirekire bisa nkibindi, mugihe ibindi bihinduka kanseri nini.Ubu buryo bubiri mu gukura kwa αS electrostatike coacervates ihuye nubushakashatsi bwa LLPS buherutse gukorwa nubushakashatsi bwerekanye isano iri hagati yo kugabanuka kwa valence hamwe no gushungura electrostatike muri kondensate nk'urufunguzo rwo kugenzura ingano ya kondine hamwe n’imiterere y’amazi.Urwego 58.61.
Iyi gahunda yerekana gushira amyloide yo guteranya inzira ya αS na Tau441 ukoresheje LLPS na LSPT.Hamwe n'utundi turere dukungahaye kuri anion (umutuku) hamwe na cation-ikungahaye (ubururu), αS na tau electrostatike coacervates hamwe na valence ishimishije bifite imbaraga zo hasi bityo rero ntibishobora guhurira hamwe, bikaviramo gusaza vuba.Imiterere ihamye idahwitse ya gel igerwaho..Iki kibazo ni cyiza cyane kubijyanye na sisitemu ya αS / pLK bitewe nubusabane bwayo bwinshi hamwe numuyoboro woroshye wa poroteyine-byombi, ibyo bikaba byemerera inzibacyuho yihuta.Ibinyuranye nibyo, ibitonyanga bifite agaciro kadashimishije, bityo rero, uturere twuzuyemo proteyine tuboneka kugirango dusabane, byorohereze coacervate guhuza no guhanagura hejuru ya hydrophilique kugirango igabanye ingufu zayo zo hejuru.Ibi bintu nibyiza kuri αS / Tau441 coacervates, zifite urusobe rwinshi rugizwe na Tau-Tau idakomeye na αS-Tau.Na none, coacervates nini izagumana byoroshye kugumana ibintu bimeze nkamazi, bituma izindi poroteyine-kuri-proteine ​​zibaho.Amaherezo, amyloide heterogeneous agregate irimo αS na tau byombi mumazi ya coacervate, bishobora kuba bifitanye isano nibisangwa mumibiri irimo, ibyo bikaba aribyo biranga indwara zifata ubwonko.
Ibikoresho binini bisa n'amazi byakozwe mugihe cyo gukura kwa αS / Tau441 hamwe na proteine ​​zuzuye cyane ariko zifite imbaraga kandi, ku rugero ruto, αS / ΔNt-Tau coacervates ni ikigega cyiza cyo guteranya poroteyine.Twabonye rwose ishyirwaho rya poroteyine zikomeye muri ubu bwoko bwa protein coacervates, akenshi zirimo αS na tau.Twerekanye ko izo heteroaggregates zihagarikwa n’imikoranire idafite amashanyarazi, zishobora guhuza amarangi yihariye ya ThT amyloide kimwe na fibrile isanzwe ya amyloide, kandi rwose ikaba ifite imbaraga zo kurwanya ingaruka zitandukanye.Igiteranyo cya αS / tau cyakozwe na LLPS cyerekanwe ko gifite imiterere isa na amyloide.Mubyukuri, variant ikuze ya Tau ibuze muguteranya amyloide irabangamiwe cyane mugushinga ayo moko atandukanye ya αS muri coacervate y'amazi ya electrostatike.Ihinduka rya αS / Tau441 igiteranyo cyagaragaye gusa imbere muri coacervates, yagumanye ibintu bimeze nkamazi, kandi ntanarimwe, niba coacervates / ibitonyanga bitageze kuri leta ya gel.Mugihe cyanyuma, imbaraga ziyongereye zikorana na electrostatike kandi, nkigisubizo, gukomera kwurusobe rwa poroteyine birinda ihinduka rikenewe rya poroteyine kugirango habeho imikoranire mishya ya poroteyine ikenewe muri nucleaire ya amyloide.Nyamara, ibi birashobora kugerwaho muburyo bworoshye, busa na coacervates, nazo zikaba zishobora gukomeza kuba amazi uko ziyongera mubunini.
Kuba ishyirwaho rya agregate mugice cyegeranye gikundwa cyane muri kondine nini ya αS / Tau kuruta mu bitonyanga bito byihuta cyane, byerekana akamaro ko kumenya ibintu bigenzura ibitonyanga.Kubwibyo, ntabwo hariho imyumvire yo gutandukanya ibyiciro gusa, ahubwo ingano ya kondensate igomba kugenzurwa kugirango ikore neza kimwe no kwirinda indwara 58,61.Ibisubizo byacu birerekana kandi akamaro k'uburinganire hagati ya LLPS na LSPT kuri sisitemu ya αS / Tau.Mugihe ibitonyanga bishobora kurinda amyloide yegeranya mukugabanya ingano ya protein monomers ziboneka mugihe cyo kwiyuzuzamo, nkuko byasabwe mubindi bikoresho 63,64, guhuza ibitonyanga kurwego rwo hejuru bishobora gutuma poroteyine yimbere ikusanywa buhoro buhoro.imiyoboro ya poroteyine..
Muri rusange, amakuru yacu ashimangira cyane akamaro ka valence ihuriweho hamwe no kunyurwa / kutanyurwa mumikoranire yabatonyanga murwego rwa LSPT.By'umwihariko, twerekana ko uburebure bwuzuye αS / Tau441 bushobora guhuza neza no nucleate kugirango bibe amiloide imeze nka heteroaggregates irimo poroteyine zombi kandi itanga uburyo bwa molekile bushingiye kubisubizo byubushakashatsi.Guhuriza hamwe poroteyine ebyiri muri coacervate ya αS / Tau twavuze hano birashobora rwose kuba bifitanye isano no guhuza poroteyine ebyiri zirimo, ibyo bikaba aribyo biranga indwara, kandi bishobora kugira uruhare mu gusobanukirwa isano iri hagati ya LLPS na guteranya amyloide, guha inzira IDP yishyuwe cyane muri neurodegeneration.
Monomeric WT-αS, ihinduka rya sisitemu (Q24C-αS, N122C-αS) na ΔCt-αS (Δ101-140) byagaragaye muri E. coli kandi bisukurwa nkuko byasobanuwe mbere.5 mM DTT yashyizwe mu ntambwe zose mugusukura mutant αS cysteine ​​kugirango hirindwe isano ya disulfide.Tau441 isoform (plasmid yakuwe muri Addgene # 16316), ΔNt-Tau variant (Δ1–150, wabonye ukoresheje clon IVA hamwe na primers CTTTAAGAAGGAGATACATATGATCGCCACACCGCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) primer) Imico ya E. coli yari gukura kuri OD600 = 0,6–0.7 kuri 37 ° C na 180 rpm, kandi imvugo yatanzwe na IPTG amasaha 3 kuri 37 ° C.Gusarura selile 11.500 xg muminota 15 kuri 4 ° C hanyuma ukarabe hamwe na saline irimo mM 150 NaM.Hagarika pellet muri buffer ya lysis (ml 20 kuri 1 L LB: MES 20 mM, pH 6.8, NaCl 500 mM, EDTA 1 mM, MgCl2 0.2 mM, DTT 5 mM, PMSF 1 mM, benzamidine 50 μM, copeptin 100 μM).Intambwe ya sonication yakorewe ku rubura hamwe na amplitude ya 80% kuri pulses 10 (1 min kuri, 1 min off).Ntukarenge ml 60 muri ultrasound imwe.E. coli lysates yashyutswe kuri 95 ° C. muminota 20, hanyuma ikonjesha kurubura hanyuma igashyirwa kuri 127.000 × g muminota 40.Indengakamere isobanutse yakoreshejwe kuri 3,5 kDa membrane (Spectrum ™ Thermo Fisher Scientific, UK) hanyuma ihamagarwa kuri 4 L ya buffer ya dialyse (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) mu masaha 10.Inkingi ya ml 5 yo guhanahana amakuru (HiTrap SPFF, Cytiva, MA, USA) yagereranijwe na buffer iringaniza (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Tau lysate yayungurujwe binyuze kuri 0.22 μ m PVDF muyungurura hanyuma yinjizwa mu nkingi ku kigero cya 1 ml / min.Gukuraho byakozwe buhoro buhoro, tau yavuzweho na 15-30% ya buffer yo gukuraho (20 mM MES, pH 6.8, 1 M NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Ibice byasesenguwe na SDS-PAGE, kandi uduce twose turimo umurongo umwe ufite uburemere bwa molekile buteganijwe bwa tau wibanze cyane ukoresheje akayunguruzo ka 10 kDa centrifuge hanyuma usimbuzwa buffer irimo 10 mM HEPES, pH 7.4, NaCl 500 mM na DTT 2 mM kuri intungamubiri za poroteyine zanyuma zari 100 μM.Igisubizo cya poroteyine cyanyujijwe kuri 0.22 μ m PVDF muyunguruzi, gikonjeshwa vuba kandi kibikwa kuri -80 ° C.Protein K18 yatanzwe neza na Prof. Alberto Boffi.Isuku yo kwitegura yari> 95% nkuko byemejwe na SDS-PAGE na MALDI-TOF / TOF.Sisitemu zitandukanye zanditswemo imiti na AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) cyangwa TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Kanada).byemejwe no kwinjiza hamwe na MALDI-TOF / TOF.Tau441, ΔNt-Tau, AggDef-Tau na K18 byanditseho ibisigisigi bya sisitemu kavukire ku mwanya wa 191 na 322 ukoresheje Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Ubudage) ukurikije inzira imwe.Amafaranga yishyurwa ku ikarita asigaye kuri αS na Tau441 yakozwe hakoreshejwe CIDER66.
Poly-L-lysine ikomeye (pLK DP 90-110 nkuko NMR ibivuga kubitanga, Alamanda Polymers Inc, Huntsville, Alabama, USA) yasheshwe muri 10 mM HEPES, 100 mM NaCl, pH 7.4 kugeza 10 mM, inzira yatanzwe kuri 5 iminota mu bwogero bwamazi ya ultrasonic hanyuma ubike kuri -20 ° C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) na FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) ni amazi ashonga kandi akwirakwizwa cyane muri buffer ya LLPS.Dialysis ikuraho imyunyu yanduye.Baca barungururwa biciye mu kayunguruzo ka syringe ifite ubunini bwa 0.22 mm, kandi intumbero zabo zabazwe hakoreshejwe reta (Mettler Toledo, Columbus, Ohio, USA).Ingero za LLPS zateguwe ku bushyuhe bwicyumba muburyo bukurikira: buffer na extrusion byavanze hamwe na mM tris 1 (2-carboxyethyl) fosifine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2- (Ethane- 1, 2-diyldinitrile) acide tetraacetic (EDTA, carboxynth) hamwe nuruvange rwa 1% inhibitor ya protease (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).Noneho αS hamwe na polycations zahujwe (amahitamo pLK cyangwa Tau) byongeweho.Kubushakashatsi bwa thioflavin-T bwigihe (ThT, Carbosynth, Compton, UK), koresha ThT yose hamwe kugirango ube kimwe cya kabiri cya αS.Witonze ariko uvange neza ibyitegererezo kugirango urebe ko ari kimwe.Ubwinshi bwa buri kintu cyatandukanijwe mubigeragezo, nkuko byasobanuwe mugice cyibisubizo.Azide yakoreshwaga kuri 0,02% (w / v) igihe cyose igeragezwa ryarenze amasaha 4.Kubisesengura byose ukoresheje LLPS ntangarugero, emera kuvanga kuringaniza muminota 5 mbere yo gusesengura.Kugira ngo isesengura risakaye, 150 µl by'icyitegererezo zashyizwe kuri microplate 96 idahuza neza (µClear®, umukara, F-Bottom / Chimney Iriba, Greiner bio-one, Kremsmünster, Otirishiya) kandi yuzuyeho firime ifata.LLPs yakurikiranwe no gupima iyinjizwa rya 350 nm hagati yumuti mu musomyi wa plaque ya CLARIOstar (BMG Labtech, Ortenberg, Ubudage).Ubushakashatsi bwakorewe muri bitatu kuri 25 ° C, kandi amakosa yabazwe nko gutandukana bisanzwe.Icyiciro cya dilute cyagereranijwe nicyitegererezo cya centrifugation hamwe na SDS-PAGE isesengura rya gel, kandi αS agace mubice byoroheje kandi byibanze byagereranijwe mubisubizo bitandukanye bya LLPS.Icyitegererezo cya 100 μl LLPS kirimo 1 μM AF488 yanditseho αS yateguwe no kuvanga neza bikurikirwa na centrifugation kuri 9600 × g muminota 30, nyuma yimvura ikunze kugaragara.Hejuru ya 50 μl ya supernatant yakoreshejwe mukugereranya poroteyine ukoresheje gel SDS-PAGE.Gels yashizwemo akayunguruzo ka AF488 ikoresheje sisitemu yo gufata amashusho ya ChemiDoc (Laboratoire ya Bio-Rad, Hercules, CA, USA) cyangwa yandujwe irangi rya Coomassie hanyuma igaragara n'amashusho akwiye.Amatsinda yavuyemo yasesenguwe hifashishijwe verisiyo ya ImageJ 1.53i (Ikigo cyigihugu cyubuzima, USA).Ubushakashatsi bwakozwe muburyo bubiri mubushakashatsi bubiri butandukanye hamwe nibisubizo bisa.
Ubusanzwe, μl 150 z'icyitegererezo zashyizwe kuri microplate 96 idahuza kandi igaragara ku bushyuhe bw'icyumba kuri microscope ya Leica DMI6000B (Leica Microsystems, Wetzlar, Ubudage).Kubushakashatsi bwakozwe, µ-Slide Angiogenezi isahani (Ibidi GmbH, Gräfelfing, Ubudage) cyangwa microplates 96-nziza ya polystirene (Corning Costar Corp., Acton, Massachusetts) nayo yakoreshejwe.EL6000 halogen cyangwa mercure ibyuma bya halide amatara yakoreshejwe nkisoko yo kumurika (kumashusho ya BF / DIC na WF).Kuri microscopi ya WF, intego ya 40x yo gukuza ikirere (Leica Microsystems, Ubudage) yakoreshejwe kugirango yerekane urumuri kurugero no kurukusanya.Kuri AF488 na ThT byanditseho ingero, gushungura no gusohora hamwe na sisitemu isanzwe ya GFP, gushungura no gusohora ibyuka byungurura, 460-500 nm na 512-542 nm byungurura, hamwe nindorerwamo ya 495 nm.Kuburugero rwanditseho Atto647N, urwego rusanzwe rwa Cy5 muyunguruzi hamwe no kwishima no gusohora ibyuka byungurura 628-40 nm na 692-40 nm, hamwe nindorerwamo ya 660 nm.Kuri microscopi ya BF na DIC, koresha intego imwe yo gukusanya urumuri.Itara ryegeranijwe ryanditswe kuri kamera ya Leica DFC7000 CCD (Leica Microsystems, Ubudage).Igihe cyo kumurika cyari ms 50 kuri BF na DIC microscopi yerekana na 20-100 ms yo gufata amashusho ya WF microscopi.Kugereranya, igihe cyo kwerekana ubushakashatsi bwose hamwe na ThT cyari ms 100.Ubushakashatsi bwatinze bwakozwe kugirango berekane igitonyanga cya coescence, amashusho akusanywa buri ms 100 muminota mike.ImageJ (NIH, USA) yakoreshejwe mu gusesengura amashusho.Ubushakashatsi bwakozwe muri bitatu hamwe nibisubizo bisa.
Kubushakashatsi bwa colocalisation, FRAP no kwiyubaka kwa 3D, amashusho yabonetse kuri Zeiss LSM 880 ya microscope ihindagurika ikoresheje verisiyo yubururu ya ZEN 2 (Carl Zeiss AG, Oberkochen, Ubudage).Ingero za 50 µl zashyizwe kuri µ-Slide Angiogenezi Petri ibiryo (Ibidi GmbH, Gröfelfing, Ubudage), bivurwa na polymer hydrophilique (ibiTreat) hanyuma bigashyirwa mubintu 63 × byo kwibiza amavuta (Plan-Apochromat 63 × / NA 1.4 Amavuta) kuri DIC).Amashusho yabonetse hakoreshejwe 458 nm, 488 nm, na 633 nm ya argon laser imirongo ifite ibyemezo bya 0.26 µm / pigiseli hamwe nigihe cyo kwerekana 8 µs / pigiseli yo kwishima no kwerekana imyuka ya windows ya 470-600 nm, 493–628 nm, na 638–755 nm yakoreshejwe mu kwerekana amashusho ya ThT, AF488 na Atto647N.Kubushakashatsi bwa FRAP, gufotora umwanya-wa buri sample byanditswe kumurongo 1 kumasegonda.Ubushakashatsi bwakorewe muri bitatu kubushyuhe bwicyumba hamwe nibisubizo bisa.Amashusho yose yasesenguwe hakoreshejwe software ya Zen 2 yubururu (Carl Zeiss AG, Oberkochen, Ubudage).Imirongo ya FRAP yari isanzwe, yateguwe kandi ihuzwa nuburemere / igihe cyakuwe mumashusho ukoresheje Zen 2 ukoresheje OriginPro 9.1.Imirongo yo kugarura yashyizwe kumurongo wa mono-exponential kugirango ubare ikwirakwizwa rya molekile hamwe nijambo ryiyongera ryerekana kugirango ubone ingaruka zo guhumeka.Twahise tubara D dukoresheje radiyo yo guhumeka nominal hamwe mbere yo kugarura gukira igice cyubuzima nkuko muburinganire bwa Kang nabandi.5 35 herekanwa.
Impinduka imwe ya sisitemu imwe ya αS yahujwe na 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) kumwanya wa 24 (TEMPOL-24-αS) na 122 (TEMPOL-122-αS), bikurikiranye.Kuzenguruka Kuzenguruka Kubushakashatsi bwa EPR, intumbero ya αS yashyizwe kuri 100 μM naho PEG yibanze kuri 15% (w / v).Kubintu bitandukanye byo guteranya, igipimo cya αS: pLK cyari 1:10, mugihe αS: ΔNt-Tau na αS: Tau441 igipimo cyagumishijwe kuri 1: 1.Kubushakashatsi bwo guhuza titre mugihe hatabariwemo abantu benshi, TEMPOL-122-αS yagumishijwe kuri 50 μM kandi polycations zahawe titre zo kongera imbaraga, zitegura buri kintu ukwacyo.Ibipimo bya CW-EPR byakozwe hifashishijwe Bruker ELEXSYS E580 X-band ya spekrometrike ifite ibikoresho bya Bruker ER4118 SPT-N1 resonator ikorera kuri microwave (SHF) ya ~ 9.7 GHz.Ubushyuhe bwashyizwe kuri 25 ° C kandi bugenzurwa na azote ya azote.Ikirangantego cyabonetse mubihe bidahagije kuri MW MW 4 mW, amplitude ya modulation ya 0.1 mT, hamwe na modulisiyo ya 100 kHz.Imbaraga za Spectral zari zisanzwe kugirango hirindwe itandukaniro ryimiterere ya spin hagati yintangarugero no kugabanuka kuzunguruka bitewe nubusigisigi bwo kugabanya ibintu mubitegererezo birimo Tau441 cyangwa ΔNt-Tau (biboneka mubisubizo bya poroteyine byumwimerere).Indangagaciro zatanzwe za g zabonetse nkibisubizo bya EPR yerekanwe byakozwe hakoreshejwe software ya Easyspin (umurongo wa 6.0.0-dev.34) yashyizwe mubikorwa muri Matlab®67.Ikintu kimwe / bibiri bigize isotropic moderi yakoreshejwe mugushushanya amakuru.Nyuma yo guhuza ibimenyetso byose, ibisigisigi byabazwe mugukuramo buri simulation kuva murwego rwo kugerageza.Kugirango uhuze titre yisesengura, ugereranije ubukana bwumurongo wa gatatu kumurongo wa kabiri wumurongo wa EPR usanzwe (IIII / III) wakoreshejwe mugukurikirana guhuza polycations kuri αS.Kugereranya gutandukana guhoraho (Kd), umurongo wavuyemo washyizwe kumurongo ugereranije ugereranije nimbuga zihuza kandi zigenga.
Ubushakashatsi bwa NMR spectroscopy bwakozwe hifashishijwe Bruker Neo 800 MHz (1H) NMR spectrometer ifite ibikoresho bya kiroprobe na Z-gradient.Ubushakashatsi bwose bwakozwe hakoreshejwe 130–207 µM αS hamwe na αS / ΔNt-Tau hamwe na pLK bihwanye na 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 kandi byakorewe kuri 15 ° C.Kugenzura LPS na NMR, 10% PEG yongewe kubiteganijwe mbere.Ikibanza cyo guhinduranya imiti (Igishusho 1b) cyerekana impuzandengo ya 1H na 15N ihinduranya imiti.Ikirangantego αS 2D1H-15N HSQC cyahawe hashingiwe ku nshingano zabanjirije iyi (BMRB yinjira # 25227) kandi byemejwe no gufata amajwi no gusesengura 3D ya HNCA, HNCO na CBCAcoNH.13Cα na 13Cβ ihindagurika ryimiti yabazwe imbere ya ΔNt-Tau cyangwa pLK kugirango bapime impinduka zishoboka muburyo bwimiterere ya kabiri ugereranije na αS ihindagurika ryimiti ihindagurika rya coil idasanzwe 68 (Ishusho yinyongera 5c).Igipimo cya R1ρ cyapimwe no gufata ubushakashatsi bwa hsqctretf3gpsi (bwakuwe mu isomero rya Bruker) bukererewe bwa ms 8, 36, 76, 100, 156, 250, 400, na 800, kandi imikorere yerekanwe ihindurwa nubukererwe bukabije bwimpanuka zitandukanye. ibihe kugirango tumenye R1ρ nubushakashatsi bwayo budashidikanywaho.
Ubushakashatsi bwibara ryamabara abiri yakemuwe na fluorescence microscopi yakorewe mugihe cyubucuruzi cyakemuwe na MT200 fluorescence confocal microscope (PicoQuant, Berlin, Ubudage) hamwe nigikoresho cyo kubara fotone imwe (TCSPC).Umutwe wa laser diode ukoreshwa muburyo bwo kwishongora (PIE), urumuri runyura muburyo bumwe bwoguhindura umurongo hanyuma ugahuzwa nimbaraga za laser zingana na 10 kugeza 100 nW kuri 481 nm na 637 nm imirongo ya laser yapimwe nyuma yindorerwamo ya dicroic.Ibi byemeza igipimo cyiza cyo kubara fotone, wirinda ingaruka ziterwa na fotone, gufotora no kwiyuzuzamo.μIndorerwamo ya 488/640 nm (Semrock, Ishyamba ryikiyaga, IL, USA) yakoreshejwe nkigice kinini cyo gutandukanya ibiti.Imirasire idakoreshwa ihagarikwa numwobo ufite diameter ya microne 50, hanyuma imirasire yibanze igabanijwemo inzira 2 zo gutahura na 50/50 itandukanya ibiti.Akayunguruzo kohereza imyanda (Semrock, Ishyamba ry'ikiyaga, IL, Amerika) 520/35 ku irangi ry'icyatsi (AF488) na 690/70 ku irangi ritukura (Atto647N) ryakoreshejwe imbere ya detector.Dode imwe ya fotone imwe (SPAD) (Micro Photon Devices, Bolzano, Ubutaliyani) yakoreshejwe nka detector.Ikusanyamakuru hamwe nisesengura byombi byakozwe hifashishijwe porogaramu ya SymphoTime64 iboneka mu bucuruzi (PicoQuant GmbH, Berlin, Ubudage).
Microliter 50 z'icyitegererezo cya LLPS zashyizwe ku mariba ya μ-Slide angiogenezi (Ibidi GmbH, Gräfelfing, Ubudage).Amashusho yavuyemo yibanze kuri 20 µm hejuru yuriba hepfo kugirango habeho intera nziza yo gukora kubitonyanga byahagaritswe no kuri ~ 1 µm kuri rafts nududomo hamwe nibisubizo bya axial byibuze 0.25 µm / pigiseli hamwe nubukererwe bwa 400 µs / pigiseli.Hitamo amakuru ukoresheje ubukana bwimbaraga ukurikije impuzandengo yerekana ibimenyetso byerekana ibimenyetso (PBG, bivuze + 2σ) kuri buri muyoboro kugirango hatorwe gusa ibitonyanga bya poroteyine byamazi, ibiti, cyangwa ibibanza byatoranijwe, bishungura inkomoko iyo ari yo yose ituruka ku cyiciro cyatatanye.Kugira ngo dusesengure ubuzima bwa buri bwoko (τ) bwa buri muyoboro (icyatsi, “g” kuri AF488 n'umutuku, “r” kuri Atto647N), twahisemo uturere dushimishije (ROIs) turimo ibitonyanga, ibiti, cyangwa ibibanza (Ishusho yinyongera 1 ).8b) kandi yabikomotse muguhuza ubuzima bwabo bwose (τD, τR na τP kubitonyanga, ibiti cyangwa ibibara, reba Ishusho yinyongera 8c) muri buri muyoboro ukoresheje isesengura rihuje umurizo hamwe nuburyo bubiri bwo kubora.Impuzandengo τ kuva τ.ROIs zakoze fotone nkeya cyane kugirango zibe nyinshi-zitandukanijwe zasesenguwe.Igabanywa ryakoreshejwe ryari <104 fotone kuri rafts nududomo na 103 kubitonyanga.Ibitonyanga bifite urwego rwo hasi kuko biragoye kubona ibora ryangirika hamwe nuburemere bwikirenga, kubera ko ibitonyanga mumashusho mubisanzwe ari bito kandi bitari byinshi.ROI hamwe numubare wa foton hejuru yumubare ntarengwa wo gukusanya foton (ushyizwe kuri> 500 ibara / pigiseli) nayo yajugunywe kugirango isesengurwe.Huza ubukana bwangirika bwakuwe mukarere gashishikajwe nuburemere kuri 90% yikirenga (gato nyuma yuburemere bukabije bwo kubora) kuva ubuzima bwumurimo butangira kugirango habeho kwivanga kwa IRF mugihe ukomeje kimwe kubintu byose byangirika. igenamiterere Idirishya rifitanye isano Hasesenguwe ROI 25 kugeza kuri 50 ROI kubitereko hamwe na 15-25 ROI kubitonyanga, amashusho yatoranijwe muri barenga 4 yigana yanditswe byibuze byibuze 3 byigenga.Ibizamini bibiri umurizo byakoreshejwe mugusuzuma itandukaniro ryibarurishamibare hagati yubwoko cyangwa hagati ya sisitemu ya coacervate.Kuri pigiseli-kuri-pigiseli yisesengura ryubuzima (τ), igiteranyo cyuzuye cyubuzima hejuru yumurima kuri buri muyoboro cyabazwe kandi hagereranijwe ikigereranyo cya 2/3 bigize icyerekezo cyerekana icyerekezo.Ubuzima bwa attenuation kuri buri pigiseli noneho yashyizwemo ukoresheje τ agaciro kabaruwe mbere, bivamo pseudocolor FLIM ishusho ikwiye.Umurizo ukwiranye nubuzima bwarimeze kimwe mumashusho yose yumuyoboro umwe, kandi buri kubora byatanze fotone ihagije kugirango itange neza.Kubisesengura rya FRET, pigiseli zatoranijwe mukoresha imbaraga nkeya zingana na fotone 100, wagereranije ikimenyetso cyinyuma (FBG) cya fotone 11.Ubwinshi bwa fluorescence ya buri muyoboro bwakosowe nimpamvu zagenwe zageragejwe: 69 kwambukiranya imipaka α yari 0.004, kwishima bitaziguye β byari 0.0305, gukora neza γ byari 0.517.Imikorere ya FRET kurwego rwa pigiseli noneho ibarwa ukoresheje ikigereranyo gikurikira:
aho FDD nuburemere bwa fluorescence igaragara kumuyoboro wabatanga (icyatsi), FDA nuburemere bwa fluorescence bugaragara kumuyoboro wakira (umutuku) mubyishimo bitaziguye, naho FAA nuburemere bwa fluorescence bugaragara kumuyoboro wakira (umutuku) mubyishimo bitaziguye ( PIE).Fluorescence ubukana bwa pulses bugaragara kumuyoboro).
Shyira 100 µl ya reaction ya LLPS irimo 25 µM idashyizweho ikimenyetso cya monomeric Tau441 (hamwe na 25 µM αS cyangwa idafite 25 µM αS) muri buffer ya LLPS (yongeweho nkuko byavuzwe haruguru) kuri microplates 96 idahuza neza hamwe na fayili yometseho ifu hamwe nigitonyanga cyagenzuwe na microscopi ya WF nyuma kuringaniza.mu minota 10.Nyuma yamasaha 48 yubushuhe mubushyuhe bwicyumba, byemejwe ko hariho intungamubiri za poroteyine nuduce.Noneho kura witonze ukureho amazi hejuru yiziba mumariba, hanyuma wongereho 50 L ya buffer yo gutandukana (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) hanyuma ushire muminota 10.Ubwinshi bwumunyu buremeza ko LLPS itazongera kubera PEG isigaye, kandi intungamubiri za poroteyine zakozwe gusa n’imikoranire ya electrostatique zizasenywa.Hasi y'iriba noneho harahanaguwe neza hifashishijwe micropipette hanyuma igisubizo cyavuyemo cyimurirwa ku iriba ryubusa.Nyuma yubushakashatsi bwintangarugero hamwe na 50 μM ThT kuri 1 h, kuba hari ibibanza byitaruye byagenzuwe na microscopi ya WF.Tegura sonrike αS fibrile ushiramo 300 µl yumuti wa 70-µM αS muri PBS hamwe na pH 7.4, sodium azide 0.01% kuri 37 ° C na 200 rpm kuri shitingi ya orbital muminsi 7.Igisubizo cyahise gishyirwa kuri 9600 × g muminota 30, pellet yahagaritswe muri PBS pH 7.4 hanyuma ihabwa sonic (1 min, 50% cycle, 80% amplitude muri sonicator ya Vibra-Cell VC130, Sonics, Newton, USA) hamwe nubunini buringaniye bwo gukwirakwiza fibrile nto.
Isesengura rya FCS / FCCS hamwe no gutahura amabara abiri (TCCD) byakorewe kuri microscope imwe ya MT200 yakemuwe na fluorescent (Pico-Quant, Berlin, Ubudage) yakoreshejwe mubushakashatsi bwa microscopi FLIM-FRET hakoreshejwe uburyo bwa PIE.Imbaraga za laser kuri ubu bushakashatsi zongerewe kuri 6.0 µW (481 nm) na 6.2 µW (637 nm).Ihuriro ryizo mbaraga za laser ryatoranijwe kugirango ritange urumuri rusa kuri joriji ya fluor ikoreshwa mugihe ugeze ku gipimo cyiza cyo kubara no kwirinda gufotora no kwiyuzuzamo.Ikusanyamakuru hamwe nisesengura byombi byakozwe hifashishijwe ubucuruzi bwa SymphoTime64 verisiyo ya 2.3 (PicoQuant, Berlin, Ubudage).
Ingero ziterwa na αS / Tau zegeranijwe zabonetse ukoresheje LLPS zivangwa muri bffer yo kwigunga kugirango ibone monomolecular ikwiye (mubisanzwe 1: 500, kubera ko igiteranyo kimaze kuba muke cyane iyo gitandukanijwe nicyitegererezo cya coacervate).Ingero zashyizwe muburyo butaziguye (Corning, USA) zabanjirijwe nigisubizo cya BSA kuri concentration ya 1 mg / mL.
Kubisesengura rya PIE-smFRET mumurongo wicyatsi nicyatsi gitukura, hashyizweho ingufu nkeya ya fotone 25 kugirango ushungure ibimenyetso byerekana ubukana buke buterwa nibintu byabayeho (menya ko monomers iruta ingero zegeranijwe ugereranije na agregate yihariye).Uru rugabano rwabazwe nkinshuro eshanu impuzandengo yuburemere bwa monomeric αS yabonetse mu isesengura ryurugero rwiza rwa monomer kugirango uhitemo neza igiteranyo cyo gusesengura.Umuzunguruko wa PIE, hamwe na TSCPC yo kubona amakuru, washoboje gukoresha progaramu yubuzima buremereye ubuzima bwose bufasha gukuraho inyuma no kwambukiranya imipaka.Imbaraga za flare zatoranijwe ukoresheje imbibi zavuzwe haruguru zarakosowe hifashishijwe impuzandengo yerekana ibimenyetso byagenwe uhereye kuri histogrammes yibibaho hamwe nimbaraga / bin ya buffer gusa.Ibisasu bifitanye isano nini nini mubisanzwe bifata bin bikurikiranye mugihe cyagenwe (shyira ms 1).Muri ibi bihe, hatoranijwe binini yimbaraga nini.Kubisesengura bya FRET na stoichiometric, ikoreshwa rya tewolojiya ya gamma factor (0.517) yakoreshejwe.Imisaraba ya Spectral hamwe nintererano yo kwishima itaziguye ni ntangarugero (byagenwe mubigeragezo) kumashanyarazi ya laser yakoreshejwe.Imikorere na stoichiometrie ya FRET muguturika ibarwa kuburyo bukurikira.