347 12.7 * 1.24mm Tuubooyinka duuban ee birta ah, Habka molecular ee isku-dhafka korantada iyo isku-ururinta α-synuclein iyo tau

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347 Qeexidda Dhuumaha Bir-la-aanta ah

347 12.7*1.24mm Tuubooyinka duuban ee aan birta lahayn

Dhexroor ka baxsan: 6.00 mm OD ilaa 914.4 mm OD, Cabirrada ilaa 24" NB la heli karo Ex-stock, OD Cabbirka Tuubooyinka birta ah oo diyaar ah

SS 347 Dhumucda Dhumucda Tuubbada: 0.3mm - 50 mm, SCH 5, SCH10, SCH 40, SCH 80, SCH 80S, SCH 160, SCH XXS, SCH XS
WT: SCH5S, SCH10S, SCH40S, SCH80S, SCH160S, iwm.

Nooca: SS 347 Tubooyin aan Xumaan lahayn |SS 347 ERW Tubooyinka |SS 347 tuubooyinka alxanka |SS 347 Tuubooyinka Farsamaynta |SS 347 Tubooyinka CDW, Dhuumaha LSAW / Tolmo-welded / Casriyeyn

Foomka: SS 347 Tuubooyinka Wareegga / Tuubooyinka, SS 347 Tubooyinka / Tubooyinka, SS 347 Tubooyinka layliga ah, SS 347 Tubooyinka duuban, SS 347 "U" Qaabka, SS 347 Gariirada Keega, SS 347 Tubooyinka Haydaruliga

Dhererka: Keli ah Random, Laba Jibbaar random & Dhamaadka Dhererka Loo Baahan Yahay: Dhamaadka Cad, Dhammaadka Go'ay, La Tumaday

Ilaalinta dhamaadka: Koofiyadaha caaga ah |Dhammaystirka Dibadda: 2B, No.4, No.1, No.8 Muraayada u dhammayn Tubooyinka Biraha, U dhammayn sida shuruudaha macaamiisha

Xaaladda gaarsiinta: la miiray oo la miiray, la miiray, la dhalaaliyay, la sawiray Qabow

Kormeerka, Warbixinada Tijaabada: Shahaadooyinka Tijaabada Milli, EN 10204 3.1, Warbixinada Kiimikada, Warbixinada Farsamada, Warbixinada Imtixaanka PMI, Warbixinada Kormeerka Muuqaalka, Warbixinada Kormeerka Dhinacyada Sadexaad, Warbixinta Shaybaarka ee NABL La Ansixiyay

Xirxirida: Waxaa lagu baakadeeyay sanduuqyo alwaax ah, bacaha balaastikada ah, birta birta ah ee la xidhxidhay, ama sida ku cad codsiyada macaamiisha

Gaar ahaan: Cabbirrada iyo Tilmaamaha aan kor ku ahayn waxaa la soo saari karaa haddii la codsado

SS 347 Cabirka Dhuumaha: 1/2 inch NB, OD ilaa 24 inch

ASTM A312 347 Tuubbooyinka austenitic-ka ah ee alxanka ah ee aan xumi lahayn oo toosan ayaa loogu talagalay heerkul sare iyo adeegga daxalka guud.Biraha buuxinta lama ogola inta alxanka la isticmaalayo.

ASTM A358 347 Isku-dhafka korantada ee alxanka tuubada austenitic ee adeega daxalka iyo/ama heerkulka sareCaadi ahaan kaliya tuubo ilaa 8 inch ah ayaa loo soo saaraa qeexitaankan.Ku darida biraha buuxinta waa la oggol yahay inta alxanka la sameynayo.

ASTM A790 347: Biibiile aan kala go 'lahayn oo toosan oo alxanka ferritic/austenitic (duplex) loogu talagalay adeega daxalka guud, iyadoo xooga la saarayo iska caabinta dildilaaca daxalka.

ASTM A409 347: Tol toosan ama fidsan-tol fiilo koronto ah oo alxan leh dhexroor weyn oo tuubo iftiin ah oo austenitic ah oo cabbirkeedu yahay 14 "ilaa 30" oo leh derbiyo Sch5S iyo Sch 10S oo loogu talagalay daxalka iyo/ama sare

ASTM A376 347 Tuubbada austenitic-ka aan xuduud lahayn ee codsiyada heerkulka sare

ASTM A813 347: Hal tol, hal-ama laba- alxanka tuubbada austenitic ee heerkulka sare iyo codsiyada daxalka guud

ASTM A814 347 tuubooyinka austenitic alxanka qabow-ka shaqeeya ee heerkulka sare iyo adeega daxalka guud

347H Tubooyinka aan-Aamiga ahayn ee Kiimikada

Guryaha Makaanikada Tuubooyinka 347H Birta Aan Laheyn

Birta Aan Lahayn 347H Tubooyinka Guryaha Jirka

Heerarka u dhigma ee 347H Dhuumaha birta-la'aanta ah

Amyloid alpha-synuclein (αS) isu geynta waa calaamad lagu garto cudurka Parkinson iyo synucleinopathies kale.Dhowaan, borotiinka tau ee sida caadiga ah lala xiriiriyo cudurka Alzheimers ayaa lala xiriiriyay αS pathology waxaana la ogaaday in ay ka wada shaqeeyaan αS-ka-midaynta, inkastoo habka molecular ee isku-darka labada borotiin aan weli caddayn.Waxaan halkan ku soo sheegnay in wejiga αS uu u kala soocayo kondensates dareere ah iyada oo loo sii marayo uumiga isku dhafan ee korantada leh polypeptides si togan loo dallaco sida tau.Iyada oo ku xidhan xidhiidhka αS ee polycations iyo heerka hoos u dhaca valence ee shabakada xinjirowga, xinjiruhu waxa ay maraan jeexid degdeg ah ama isku xidhid ay ku xigto isu geynta amyloid oo gaabis ah.Marka la isku daro qaybo ka mid ah farsamooyinka biophysical ee horumarsan, waxaan awoodnay inaan tilmaamno dareeraha dareeraha ah ee αS/Tau wejiga oo aan aqoonsanno qodobbada muhiimka ah ee keena samaynta isku-darka isku-dhafka ah ee ka kooban labadaba borotiinnada ku jira kondensate borotiinka dareeraha ah.
Marka laga soo tago qaybaha xuubabka, kala soocida boosaska unugyada sidoo kale waxaa lagu gaari karaa samaynta borotiin qani ah, jidh cufan oo dareere ah oo loo yaqaan kondensates biomolecular ama dhibco, iyada oo loo marayo habka loo yaqaan kala saarista dareeraha dareeraha ah (LLPS).Dhibcahaan waxaa sameeyay isdhexgal ku meel gaar ah oo kala duwan, badiyaa ka dhexeeya borotiinno ama borotiinno iyo RNA, waxayna u adeegaan hawlo kala duwan ku dhawaad ​​dhammaan hababka nolosha.Tiro badan oo borotiinno karti u leh LLP waxay soo bandhigaan taxanaha kakanaanta hoose kuwaas oo aad u khalkhalsan dabeecadda iyo samaynta kondensates biomolecular3,4,5.Daraasado badan oo tijaabo ah ayaa daaha ka qaaday dabacsanaanta, badiyaa khalkhalsan, iyo dabeecadaha kala duwan ee borotiinnada ka kooban kondensates-yada dareeraha ah, in kasta oo wax yar laga og yahay go'aamiyeyaasha molecular ee gaarka ah ee xakameynaya korriinka iyo korriinka kondensooyinkan si ay u noqdaan kuwo adag-u eg. gobol..
Xogta cusubi waxay taageertaa mala-awaalka ah in LLPS ee borotiinku horseedka ka yahay iyo isbeddelka dhibcaha ee qaab-dhismeed adag ay noqon karaan dariiqyada gacanta ee ku habboon taas oo horseedaysa samaynta isku-darka sunta ah ee aan la milmi karin kuwaas oo inta badan calaamad u ah cudurrada xumaaday.Qaar badan oo ka mid ah borotiinada khalkhalsan gudaha LLPS (IDPs), oo inta badan aad loo dallaco oo dabacsan, ayaa muddo dheer lala xiriiriyay neurodegeneration iyada oo loo marayo habka isku-darka amyloid.Gaar ahaan, kondensates biomolecular IDP sida FUS7 ama TDP-438 ama borotiinada leh qaybo kakanaanta hooseeya sida hnRNPA19 ayaa la muujiyay inay da'da u galaan gel-la mid ah ama xitaa qaabab adag iyada oo loo marayo habka loo yaqaan dareeraha.xarun.u gudubka wejiga adag (LSPT) oo ah shaqo waqti ah ama ka jawaabaya qaar ka mid ah isbeddelada tarjumaada ka dambeeya ama isbeddellada cudur-sidaha ee muhiimka ah1,7.
Barokac kale oo la xidhiidha LLPS in vivo waa Tau, borotiinka cilladaysan ee microtubule-ku xiran kaas oo isku-darka amyloid uu ku lug leeyahay cudurka Alzheimers10 laakiin sidoo kale dhawaan ayaa lagu lug yeeshay cudurka Parkinson (PD) iyo borotiinada kale ee nukliyeerka synaptika 11, 12, 13 ayaa la xiriira.Tau waxa la tusay in uu si kedis ah uga go'o xalka/cytobalaasamka iyada oo ay ugu wacan tahay is dhexgalka elektrostatic ee wanaagsan14, taas oo keentay samaynta dhibco tau-hodanka ah oo loo yaqaan electrostatic coacervates.Waxa kale oo la arkay in is dhexgalka noocan oo kale ah uu yahay awoodda ka dambeysa kondensates badan oo biomolecular ah ee dabeecadda15.Xaaladda borotiinka tau, isku-darka korantada waxaa lagu samayn karaa isku-dar fudud, taas oo gobollada ka soo horjeeda ee borotiinku ay kiciyaan habka kala-goynta, ama isku-darka adag iyada oo la falgalayo polymers-ka xun sida RNA.
Dhawaan, α-synuclein (αS), oo ah amyloid IDP oo ku lug leh PD iyo cudurrada kale ee neurodegenerative ee si wadajir ah loo yaqaan synucleinopathy17,18, ayaa lagu muujiyay moodooyinka unugyada iyo xayawaanka19,20 oo ku urursan kondensates borotiinka leh dabeecad dareere ah.Daraasadaha gudaha ee vitro waxay muujiyeen in αS ay ku marto LLPS iskudar fudud iyada oo loo marayo isdhexgalka ugu badan ee hydrophobic, in kasta oo geeddi-socodkani uu u baahan yahay uruurin borotiinno gaar ah oo caadi ah iyo waqtiyo ku-meel-gaar ah oo caadi ah19,21.Haddi kondensates-ka αS-ku jira ee lagu arkay vivo ay sameysteen kan ama hababka kale ee LLPS ayaa ah arin muhim ah oo aan la xalin.Sidoo kale, inkasta oo isku-darka αS amyloid lagu arkay neerfayaasha PD iyo synucleinopathies kale, habka saxda ah ee αS ay ku socoto isugeynta amyloid intracellular ayaa weli ah mid aan caddayn, maaddaama xad-dhaafka borotiinkan uusan u muuqan inuu kicinayo habkan laftiisa.Dhaawaca gacanta ee dheeraadka ah ayaa badanaa loo baahan yahay, taas oo soo jeedinaysa in meelo gaar ah oo gacanta ah ama microenvironments loo baahan yahay dib-u-kicinta unugyada αS amyloid ee gudaha.Mid ka mid ah bay'ada gacanta ee si gaar ah ugu nugul isugeynta ayaa noqon karta gudaha kondensates borotiinka 23.
Waxa xiisaha lihi leh, αS iyo tau ayaa la ogaaday inay ka wada-shaqeeyaan isku-dhafka cudurrada sifo ee bini-aadamka qaba cudurka Parkinson iyo synucleinopathies kale 24,25 iyo tijaabooyinku waxay soo sheegeen xidhiidh is-dhexgal ah oo u dhexeeya labada borotiinada 26,27 oo soo jeedinaya xiriirka ka dhexeeya isugeynta αS cudurada neurodegenerative.jiro.αS iyo tau ayaa la ogaaday in ay is dhexgalaan oo ay kor u qaadaan isku-darka midba midka kale ee vitro iyo in vivo 28,29 iyo isku-darka isku-dhafka ah ee ka kooban labadan borotiinada ayaa lagu arkay maskaxda bukaanka qaba synucleinopathies 30.Si kastaba ha ahaatee, wax yar ayaa laga og yahay saldhigga molecular ee isdhexgalka ka dhexeeya αS iyo tau iyo habka isku-ururintiisa.αS ayaa lagu soo warramey in ay la falgasho tau iyada oo loo marayo soo jiidasho elektrostatic ah oo u dhexeeya gobolka C-terminal ee aadka u xun ee αS iyo gobolka proline-ka dhexe ee tau, kaas oo sidoo kale lagu hodmay hadhaaga si togan loo dallacay.
Daraasaddan, waxaan ku tusineynaa in αS ay dhab ahaantii kala saari karto dhibco iyada oo loo marayo uumiga isku dhafan ee korantada ee joogitaanka borotiinka tau, marka la barbardhigo isdhexgalka kale ee polypeptides kale ee si togan loo soo oogay sida poly-L-lysine (pLK), iyo habkan.αS waxay u shaqeysaa sidii unug-fudud ee shabakada dhibcaha.Waxaan aqoonsanay kala duwanaansho la dareemi karo oo ku saabsan habka korriinka korantada αS coacervates, kuwaas oo lala xiriiriyo kala duwanaanshaha valency iyo xoogga isdhexgalka borotiinnada ku lug leh shabakadda isku-xidhka.Waxa xiisaha lihi leh, waxaanu ku aragnay isku-darka borotiinnada αS iyo tau amyloid ee isku-xidhayaasha dareeraha muddada-dheer waxaanan aqoonsannay arrimo muhiim ah oo horseeda isu-ururinta labadan borotiin ee isku-darka noocan oo kale ah.Halkan waxaan si faahfaahsan ugu sharraxaynaa habkan, kaas oo ah habka molecular ee suurtogalka ah ee ka hooseeya isku-dhafka laba borotiin ee ku jira ka-qaybgalyada gaarka ah ee cudurrada.
αS waxay leedahay dabo C-terminal aad u sarreeya oo pH dhexdhexaad ah (Jaantus. 1a), waxaanan ku qiyaasnay ​​inay mari karto LLPS iyada oo loo marayo uumiga dhismayaasha korantada ee leh unugyo polypeptide-ka ah oo cilladaysan.Waxaan u isticmaalnay 100-hadhaaga poly-L-lysine (pLK) sida molecule-ka bilawga ah sababtoo ah dabeecadeeda si wanaagsan loo dallacay iyo dabeecadda polymeric ee dhexdhexaadka ah pH 32. Marka hore, waxaan xaqiijinay in pLK ay la falgasho qaybta Ct ee αS iyada oo loo marayo Solution NMR spectroscopy (Jaantuska 1b) iyadoo la adeegsanayo 13C/15N-ku-calaamadeysan αS iyadoo ay jirto kororka αS: pLK saamiga molarka.Is dhexgalka pLK ee Ct-domain ee αS waxay isu muujisaa carqaladaynta isbeddelka kiimikada iyo hoos u dhaca xoogga ugu sarreeya ee gobolkan borotiinka.Waxa xiiso leh, markii aan ku qasnay ​​αS iyo pLK marka la eego qiyaasta αS ee qiyaastii.5-25 µM joogitaanka polyetylen glycol (5-15% PEG-8) (caadiga ah LLPS baffer: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) waxaanu isla markiiba soo marnay beer ballaadhan oo samaynta borotiinka .Dhibcaha ayaa lagu arkay iyadoo la isticmaalayo fluorescence (WF) iyo meel dhalaalaya (BF) microscopy (Jaantus. 1c).Dhibcaha 1-5 µm oo ay ku jiraan αS xoog leh (ku daray 1 µM AlexaFluor488-labeled αS, AF488-αS), sifooyinkooda korantada waxaa laga heli karaa caabbinta 10% 1,6-hexanediol (1,6-HD) iyo dareenkooda kororka xoogga NaCl (Jaantus. 1c).Dabeecadda dareeraha u eg ee coacervates ee αS/pLK kakalka elektrostatic waxaa lagu muujiyay awooda ay u leeyihiin in ay ku milmaan gudahooda (Jaantus. 1d).Isticmaalka turbidimetry, waxaanu ku qiyaasnay ​​samaynta dhibco hoos yimaada xaaladahan, waxaanu xaqiijinay dabeecadda elektrostatic ee isdhexgalka ugu weyn ee la xidhiidha xasilloonida (Sawir 1e), waxaanan qiimeynay saameynta kala duwan ee polymers ee habka LLPS (Sawir 1f).In kasta oo samaynta dhibco lagu arkay tiro balaadhan oo ah saamiyo polymer, nidaamku aad buu u wanaagsan yahay marka pLK ay ka badato αS.LLPs ayaa sidoo kale lagu arkay iyadoo la isticmaalayo wakiilka bedelka dextran-70 (70 kDa) ama iyadoo la adeegsanayo noocyo muunado oo kala duwan, oo ay ku jiraan dhibcaha dhalooyinka, ceelasha microplate ee walxo kala duwan, Eppendorf ama quartz capillaries.
matalaad qaabaysan ee gobollada borotiinka ee kala duwan ee WT-αS iyo ΔCt-αS noocyada loo isticmaalo daraasaddan.Qaybta amphipathic N-terminal domain, hydrophobic amyloid-forming (NAC), iyo qaybta C-terminal-ka ee sida xun loo dallacay ayaa lagu muujiyay buluug, oranji, iyo casaan, siday u kala horreeyaan.Khariidada saafiga ah ee Hadhaaga (NCPR) ee WT-αS waa la muujiyay.b NMR ee is dhexgalka αS/pLK ee maqnaanshaha kurumaha makromolecular.Marka fiirsashada pLK ay korodho (αS: pLK saamiga molar ee 1:0.5, 1:1.5, iyo 1:10 ayaa lagu muujiyay cagaar khafiif ah, cagaar, iyo cagaar madow, siday u kala horreeyaan).c Coacervate αS/pLK (samiga molar 1:10) at 25 µM (1 µM AF488-ku calaamadsan αS ama Atto647N-ku calaamadsan pLK ee sawirka WF) ee ku jira LLPS (sare) ama lagu kabay 500 mM NaCl (bidix 1 ka dib) ama % 1,6-hexanediol (1,6-HD; hoose ee midig).Qiyaasta bar = 20 µm.d Sawirrada microscopic ee matalayaasha ee isku-dhafka dhibcaha BF ee αS/pLK (saamiga molar 1:10) ee fiirsashada 25 μM;Fallaaro ayaa tilmaamaya isku darka dhibco gaar ah ( fallaadho guduudan iyo jaale ah ) oo galay dhibco cusub (Falaar oranji) gudaha 200 ms) .Qiyaasta bar = 20 µm.e kala firdhinta iftiinka (oo ah 350 nm) αS/pLK isku-darka LLPS ka hor iyo ka dib marka lagu daro 500 mM NaCl ama 10% 1,6-HD ee 25 µM αS (N = 3 muunadaha muunada, celceliska iyo leexashada caadiga ah ayaa sidoo kale la tilmaamay).f Sawirka BF (korka) iyo falanqaynta iftiinka iftiinka (350 nm, hoose) ee iskudarka αS / pLK ee 25 μM αS oo leh kordhinta αS: pLK molar ratio (N = 3 muunadaha muunada, celceliska iyo leexashada caadiga ah ayaa sidoo kale la tilmaamay).Qiyaasta bar = 10 µm.Bar miisaanka ku yaal hal sawir ayaa tilmaamaya miisaanka dhammaan sawirada hal gole.Xogta ceeriin waxaa lagu bixiyaa qaab faylal xog ceeriin ah.
Iyada oo ku saleysan indha-indhayntayada αS/pLK kondensation-ka korantada ee korantada iyo indho-indhayntii hore ee αS sida molecule-ka macmiilka ee kondensate tau/RNA iyada oo loo marayo isdhexgalka tooska ah ee tau31, waxaanu qiyaasnay ​​in αS iyo tau ay la wadaagi karaan dareeraha maqnaanshaha RNA uumiye.iyada oo loo marayo dhismooyinka korantada, iyo αS waa borotiinka qallafsan ee αS/Tau coacervates (eeg qaybinta lacagta tau ee Jaantuska 2e).Waxaan aragnay in markii 10 μM αS iyo 10 μM Tau441 (oo ay ku jiraan 1 μM AF488-αS iyo 1 μM Atto647N-Tau, siday u kala horreeyaan) lagu daray LLPS kaydinta, waxay si fudud u sameeyeen isku-darka borotiinka oo ay ku jiraan labadaba borotiinno, sida uu arkay WF microscopy.(Jaantuska. 2a).Isku-dhafka labada borotiin ee dhibcaha dhibcaha waxaa lagu xaqiijiyay konfocal (CF) microscopy (Jaantuska Dheeraadka ah. 1a).Dabeecad la mid ah ayaa la arkay markii dextran-70 loo adeegsaday wakiil isku-dar ah (Jaantuska Dheeraadka ah. 1c).Annaga oo adeegsanayna FITC-ku sumadeeyey PEG ama dextran, waxaan ogaanay in labada wakiil ee isku dhafka ah si siman loogu qaybiyey shaybaarrada, oo aan muujinayn kala-soocid iyo urur midna (Jaantuska Dheeraadka ah 1d).Hase yeeshe, waxay soo jeedinaysaa in nidaamkan ay kor u qaadaan kala-soocidda wejiga iyada oo loo marayo saamaynta isku-ururinta makromolecular, maadaama PEG ay tahay wakiilka xasilloonida ee doorbidaya, sida lagu arkay nidaamyada kale ee LLP33,34.Dhibcahaan borotiinka leh ayaa xasaasi u ahaa NaCl (1 M) laakiin aan ahayn 1,6-HD (10% v/v), iyagoo xaqiijinaya sifooyinkooda korantada (Jaantuska Dheeraadka ah 2a, b).Dabeecaddooda dareere waxaa lagu xaqiijiyay in la arkay dhacdooyinka dhibcaha isku-dhafka ee millise-ilbiriqsi iyadoo la adeegsanayo microscopy BF (Jaantus. 2b).
a Confocal (CF) sawirada mikroskoob ee αS/Tau441 waxay isku xidhaa kaydka LLPS (10 μM borotiin kasta, 0.5 μM ee AF488-ku calaamadeysan αS iyo Atto647N-ku calaamadeysan Tau441).b Sawirada kala duwanaanshaha faragelinta kala duwanaanta (DIC) ee sawirada αS/Tau441 dhacdooyinka isku-dhafka dhibcaha (10 μM ee borotiin kasta).Jaantuska wejiga ee ku salaysan kala firdhinta iftiinka (350 nm) ee Tau441 LLPS (0-15 µM) maqnaanshaha (bidix) ama joogitaanka (midig) 50 µM αS.Midabada diiran waxay muujinayaan kala firdhis badan.d Iftiinka kala firdhisan ee αS/Tau441 LLPS muunado leh korodhka αS (Tau441 at 5 µM, N = 2-3 muunad ku celcelin ah sida la tilmaamay).e Metelaad qorshe ah oo ka mid ah noocyada kala duwan ee borotiinka tau iyo gobollada kala duwan ee borotiinka loo isticmaalo daraasaddan: N-terminal domain si xun loo dallacay (casaan), gobolka hodanka ku ah (buluug), microtubule-binding domain (MTBD, oo lagu muujiyey orange), iyo amyloid-samaysa lammaane wareeg ah.gobollada fiilo (PHF) ee ku dhex yaal MTBD (cawlan).Khariidada saafiga ah ee hadhaaga (NCPR) ee Tau441 waa la muujiyay.Isticmaalka 1 µM AF488-calaamadaha αS iyo Atto647N-ku calaamadsan ΔNt-, iyadoo la adeegsanayo 1 µM AF488-labeled αS ama ΔCt-αS iyadoo ay joogaan ΔNt-Tau (sare, 10 µM borotiinkiiba) ama K18 (boot, 5M per protein) ) )) micrographs ee WF oo ku urursan LLPS ama kaydka K18.Baararka cabirka ee ku jira hal sawir ayaa u taagan cabirka dhammaan sawirada hal gole (20 µm ee baalasha a, b iyo f).Xogta ceeriin ee panels c iyo d waxaa loo bixiyaa sidii faylal xog cayriin ah.
Si loo tijaabiyo doorka αS ee geeddi-socodkan LLPS, waxaan marka hore baarnay saameynta αS ku leedahay xasilloonida dhibcaha iyadoo la adeegsanayo nephelometry annagoo adeegsanayna kordhinta NaCl (Jaantus. 2c).Marka ay sare u kacdo milixda muunadaha ay ku jiraan αS, ayaa sare u kacaysa qiyamka kala firdhisanaanta iftiinka (350 nm), taas oo muujinaysa doorka xasilinta ee αS ee nidaamkan LLPS.Saameyn la mid ah ayaa lagu arki karaa iyadoo la kordhinayo fiirsashada αS (oo markaa αS: saamiga Tau441) ilaa qiyaastii.10-laab koror marka loo eego fiirsashada tau (5 µM) (Jaantus. 2d).Si loo muujiyo in αS ay tahay borotiinka qulqulaya ee coacervates, waxaan go'aansanay inaan baarno habdhaqanka LLPS-khasaare ee Tau mutant, kaas oo ka maqan gobolka N-terminal si xun loo soo oogay (haraadiga 1-150, fiiri sawirka 2e) oo loo yaqaan ΔNt-Tau.WF microscopy iyo nephelometry ayaa xaqiijiyay in ΔNt-Tau lafteedu aysan marin LLPS (Sawir 2f iyo Sawirka Dheeraadka 2d), sidii hore loo soo sheegay 14. Si kastaba ha ahaatee, markii αS lagu daray xalalka kala firdhisan ee kala duwanaanshahan Tau ee la gooyay, habka LLPS wuxuu ahaa mid dhamaystiran. lagu soo celiyay cufnaanta dhibicda ee u dhow cufnaanta dhibicda ee xalalka cabbirka buuxa ee Tau iyo αS ee xaaladaha la midka ah iyo uruurinta borotiinka.Nidaamkan waxaa sidoo kale lagu arki karaa xaaladaha ciriiriga makromolecular hoose (Jaantuska Dheeraadka ah. 2c).Doorka gobolka C-terminal αS, laakiin ma aha dhererkiisa oo dhan, habka LLPS waxaa lagu muujiyay xannibaadda samaynta dhibicda iyadoo la adeegsanayo kala duwanaanshaha C-terminal αS ee ka maqan 104-140 (Jaantus 1a) ee (ΔCt- αS) borotiin (Jaantus. 2f iyo Sawirka Dheeraadka ah 2d).Isku-duubnida αS iyo ΔNt-Tau waxaa lagu xaqiijiyay mikroskoobyada konfocal fluorescence (Jaantuska Dheeraadka ah 1b).
Si loo sii tijaabiyo habka LLPS ee u dhexeeya Tau441 iyo αS, kala duwanaansho Tau oo dheeraad ah ayaa la isticmaalay, kuwaas oo ah qaybta la isku daray ee helical filament core (PHF) ee qaybta microtubule-binding (MTBD), taas oo haddii ay ka kooban tahay afar qaybood oo soo noqnoqonaya, caadi ahaan sidoo kale loo yaqaan sida jajabka K18 (eeg sawirka 2e).Waxaa dhawaan la soo wariyay in αS ay doorbidayso inay ku xidho borotiinka tau ee ku yaal aagga proline-hodanka ah ee isku xigxiga ee ka horreeya qaybta microtubule-binding.Si kastaba ha ahaatee, gobolka PHF wuxuu sidoo kale qani ku yahay hadhaaga si togan loo dallaco (fiiri Jaantuska 2e), gaar ahaan lysine (15% hadhaaga), taas oo nagu kalliftay inaan tijaabino bal in gobolkani uu sidoo kale gacan ka geysto uumiga dhismaha αS/Tau.Waxaan aragnay in K18 kaligiis uusan kicin karin LLPS ilaa 100 μM marka la eego shuruudaha la tijaabiyay (LLPS buffer with 15% PEG ama 20% dextran) (Jaantuska 2f).Si kastaba ha ahaatee, markii aan ku darnay 50 µM αS 50 µM K18, samaynta degdega ah ee dhibco borotiin ah oo ay ku jiraan K18 iyo αS ayaa lagu arkay nefelometry (Jaantuska Dheeraadka ah. 2d) iyo microscopy WF (Jaantus. 2f).Sidii la filayey, ΔCt-αS waxay awoodi wayday inay soo celiso hab-dhaqankii LLPS ee K18 (Jaantus. 2f).Waxaan ognahay in isku-darka αS/K18 ay u baahan tahay uruurin borotiin ah oo xoogaa sare u kaca si loo kiciyo LLPS marka la barbar dhigo αS/ΔNt-Tau ama αS/Tau441, waxyaabo kale waa siman yihiin.Tani waxay la socotaa isdhexgalka xooggan ee gobolka αS C-terminal oo leh proline-hodan Tau domain marka la barbardhigo microtubule-binding domain, sida hore loogu sharaxay 31.
Marka la eego in ΔNt-Tau aysan qaban karin LLPS maqnaanshaha αS, waxaan u dooranay kala duwanaanshahan Tau qaab lagu muujinayo αS/Tau LLPS iyada oo la siiyay fududaanteeda nidaamyada LLPS ee leh Tau dhererkiisu dhan yahay (isotype, Tau441/Tau441).oo leh kakan (heterotypic, αS/Tau441) hababka isu geynta.Waxaan is barbar dhignay heerka isku-darka αS (sida qayb ka mid ah borotiinka wajiga isku dhafan, fαS, c) ee nidaamyada αS / Tau iyo αS / ΔNt-Tau iyadoo la adeegsanayo centrifugation iyo falanqaynta wejiga SDS-PAGE ee kala firdhiyey (eeg 2e), waxay heleen qiimeyo isku mid ah. dhammaan borotiinada isku urursan.Gaar ahaan, waxaan helnay fαS, c 84 ± 2% iyo 79 ± 7% ee αS / Tau iyo αS / ΔNt-Tau, siday u kala horreeyaan, iyagoo soo jeedinaya in isdhexgalka heterotypic ee u dhexeeya αS iyo tau uu ka sarreeyo isdhexgalka ka dhexeeya molecules tau.u dhexeeya
Is dhexgalka kala duwanaansho kala duwan iyo saameynta habka uumiga ee kinetics αS ayaa markii ugu horeysay lagu bartay soo kabashada fluorescence ka dib habka sawir-qaadista (FRAP).Waxaan tijaabinay αS/Tau441, αS/ΔNt-Tau iyo αS/pLK coacervates (100 μM αS lagu kabay 2 μM αS AF488-αS iyo 100 μM Tau441 ama ΔNt-Tau ama 1 mM pLK).Xogta ayaa la helay 30-kii daqiiqo ee ugu horreeyay ka dib marka la isku daro qaybaha muunada.Laga soo bilaabo sawirada FRAP wakiilka (Jaantus. 3a, αS/Tau441 uumi) iyo qaloocyada koorsada waqtiga ee u dhigma (Jaantus. 3b, Sawirka Dheeraadka ah. 3), waxaa la arki karaa in αS kinetics ay aad ula mid yihiin kuwa Tau441 coacervates.iyo ΔNt-Tau, oo aad uga dheereeya pLK.Isugeynta faafinta la xisaabiyay ee αS gudaha coacervate sida ku cad FRAP (sida lagu sharaxay Kang et al. 35) waa D = 0.013 ± 0.009 µm2/s iyo D = 0.026 ± 0.008 µm2/s ee αS/Tau441 iyo αS/Tau441 nidaamka αS/.pLK, Tau, iyo D = 0.18 ± 0.04 µm2/s, siday u kala horreeyaan (Jaantus. 3c).Si kastaba ha ahaatee, iskudarka faafinta αS ee marxaladda kala firdhiyey waa dhawr amar oo ka sarreeya dhammaan wejiyada ciriiriga ah, sida lagu go'aamiyo Fluorescence Correlation Spectroscopy (FCS, eeg Sawirka Dheeraadka ah. (D = 8 ± 4 µm2/s).Sidaa darteed, kinetics-ka αS tarjumaadda ayaa si weyn hoos ugu dhacay coacervates marka la barbar dhigo borotiinada ku jira marxaladda kala firdhiyey ay sabab u tahay saamaynta ciriiriga molecular dhawaaqa, in kasta oo dhammaan coacervates ay haystaan ​​guryaha dareere-sida inta lagu jiro qeybtii hore ee saac ka dib markii ay sameysteen, lid ku ah wajiga tau.Kinetics dhaqsaha badan ee kondensate pLK.
Falanqaynta a-c FRAP ee dhaqdhaqaaqa αS (2% AF488-ku calaamadsan αS) ee isku xidhka korantada.Sawirrada matalayaasha ah ee αS/Tau441 FRAP ee qiimaynta saddex geesoodka ah ayaa lagu muujiyey (a), halkaasoo wareegyada guduudan ay muujinayaan meelaha midabkoodu go'ay.Baaxadda cabbirku waa 5 µm.b Celceliska qalooca FRAP iyo (c) isku xidhka fidinta faafinta la xisaabiyay (D) ee 5-6 (N) dhibco kala duwan oo ka yimid saddex tijaabo oo isticmaalaya 100 µM αS iyo isku-dheelitirnaanta isku midka ah ee Tau441 (casaan) ama ΔNt-Tau (buluug) ama pLK (cagaaran) toban jeer fiirsiga LLPS.Isbeddelka caadiga ah ee qalooca FRAP waxaa lagu muujiyey midab hadhsan.Marka la barbardhigo, iskudarka faafinta αS ee marxaladda kala firdhiyey waxaa lagu go'aamiyey saddex jeer iyadoo la isticmaalayo fluorescence correlation spectroscopy (FCS) (eeg Jaantuska Dheeraadka ah 3 iyo hababka macluumaad dheeraad ah).d spectra X-band EPR joogto ah oo ah 100 μM TEMPOL-122-αS oo ku jira kaydka LLPS iyada oo aan la isticmaalin polycation (madow) ama joogitaanka 100 μM Tau441 (casaan) ama ΔNt-Tau (buluug) ama 1 mM pLK (cagaaran).Gelinku wuxuu muujinayaa aragti weyn oo ah xariiqyada garoonka ee xooggan halkaasoo isbeddellada ugu weyni ay dhacaan.e Qalloocyada 50 μM TEMPOL-122-αS oo leh meelo kala duwan marka ay maqan tahay LLPS (aan lahayn PEG).Weynaanta baaxadda ee band III marka la barbar dhigo band II (IIII/III) ee heerka caadiga ah EPR spectrum ayaa lagu muujiyay inay kordhiso saamiga molar ee Tau441 (casaan), ΔNt-Tau (buluug) iyo pLK (cagaaran).Khadadka midabka leh ayaa muujinaya ku habboon xogta iyaga oo isticmaalaya qaab xidhid qallafsan oo leh goobo isku mid ah oo madax-bannaan oo ku dheggan qalooca kasta.Xogta ceeriin waxaa lagu bixiyaa qaab faylal xog ceeriin ah.
Dhamaystir ahaan, waxaanu baarnay dhaqdhaqaaqa αS ee coacervates kala duwan anagoo adeegsanayna calaamadaynta lafdhabarta tooska ah (SDSL) iyo resonance eletroonig ah oo joogto ah (CW-EPR).Habkani waxa uu caddeeyey in uu faa'iido badan u leeyahay ka warbixinta dabacsanaanta iyo dhaqdhaqaaqa barokacayaasha iyada oo la raacayo xallin dhab ah oo hadhaaga ah36,37,38.Si taas loo gaaro, waxaan ku dhisnay hadhaaga cysteine ​​ee hal-mutatari Cys ah waxaana isticmaalnay 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Maleimide derivatives iyagaa calaamadeeyay.Si gaar ah, waxaan gelinay TEMPOL baarayaal booska 122 ama 24 αS (TEMPOL-122-αS iyo TEMPOL-24-αS).Xaaladda ugu horreysa, waxaan bartilmaameedsaneynaa gobolka C-terminal ee borotiinka, kaas oo ku lug leh isdhexgalka polycations.Taa baddalkeeda, booska 24 wuxuu na siin karaa macluumaadka ku saabsan dhaqdhaqaaqa guud ee borotiinnada kondensate-ka.Labada xaaladoodba, calaamadaha EPR ee laga helay borotiinnada wajiga kala firdhiyey waxay u dhigmaan xag-jirrada nitroxide ee xaaladda degdegga ah.Ka dib kala-soocidda wejiga ee joogitaanka tau ama pLK (100 μM TEMPOL-αS, Tau441 ama ΔNt-Tau ee saamiga 1: 1 ama pLK ee saamiga 1: 10), korodhka xoojinta ugu sarreysa ayaa lagu arkay spectrum EPR ee αS.Khadka lumitaanka waa la ballaariyay, taasoo muujineysa hoos u dhac ku yimid dib u habeynta αS ee dhibcaha dhibcaha marka la barbar dhigo borotiinka wajiga walaaqa (Jaantus. 3d, Jaantuska Dheeraadka ah. 4a).Isbeddelladani waxay si aad ah ugu cad yihiin booska 122. Halka booska 24 joogitaanka pLK aysan saameyn ku yeelan kaniiniga baaritaanka, booska 122 qaabka xariiqda muraayadda ayaa si weyn isu beddelay (Jaantuska Dheeraadka ah. 4a).Markii aan isku daynay in aan ku daydo jaantuska booska 122 ee labada nidaam αS / polycation iyadoo la adeegsanayo qaabka isotropic (Jaantuska Dheeraadka ah 5a) sida caadiga ah loo isticmaalo si loo qeexo dhaqdhaqaaqa lafdhabarta IDP38,39, ma awoodno inaan dib u dhisno tijaabada tijaabada..Jilitaanka Spectral ee booska 24 wareegyada isbarbardhiga (Jaantuska Dheeraadka ah. 5a).Tani waxay soo jeedinaysaa in ay jiraan boosas doorbidaya booska qaabaynta lafdhabarta ee gobolka C-terminal ee αS iyada oo ay jiraan meelo badan.Markaad tixgelinayso jajabka αS ee marxaladda isku dhafan ee xaaladaha EPR ee tijaabada ah (84 ± 2%, 79 ± 7%, iyo 47 ± 4% ee αS / Tau441, αS / ΔNt-Tau, iyo αS / pLK, siday u kala horreeyaan-fiiri Dheeraadka Jaantuska 2e ee falanqaynta xogta c), waxaa la arki karaa in ballaarinta lagu ogaaday habka EPR ay inta badan ka tarjumayso isdhexgalka gobolka C-terminal ee αS oo leh noocyo kala duwan oo ka mid ah wejiga isku dhafan (isbeddelka ugu weyn marka la isticmaalayo TEMPOL-122- αS), oo aan ahayn uumi borotiinka.Korodhka microviscosity ayaa lagu arkay baaritaanka.Sida la filayo, spectrum EPR ee borotiinka xaaladaha aan ka ahayn LLPS ayaa si buuxda loo soo celiyay markii 1 M NaCl lagu daray isku dar ah (Jaantuska Dheeraadka ah. 4b).Guud ahaan, xogtayadu waxay soo jeedinaysaa in isbeddelada ay ogaatay CW-EPR ay inta badan ka tarjumayaan isdhexgalka gobolka C-terminal ee αS oo leh qaybo kala duwan oo ku jira marxaladda isku dhafan, iyo isdhexgalkaani wuxuu u muuqdaa mid ka xoog badan pLK marka loo eego Tau.
Si loo helo macluumaad dhismeed oo badan oo ku saabsan borotiinnada ku jira coacervate, waxaan go'aansanay inaan barano nidaamka LLPS anagoo adeegsanayna NMR xalka.Si kastaba ha ahaatee, waxaan kaliya ogaan karnaa jajabka αS ee ku haray marxaladda kala firidhsan, taas oo laga yaabo inay sabab u tahay dhaqdhaqaaqa borotiinka oo yaraaday gudaha coacervate iyo weji cufan oo hoose ee xalka ee falanqaynta NMR.Markii aan falanqeynay qaabka iyo dhaqdhaqaaqa borotiinka ee ku haray marxaladda kala firdhisan ee muunadda LLPS iyadoo la adeegsanayo NMR (Jaantuska Dheeraadka ah. 5c, d), waxaan ogaanay in borotiinku u dhaqmay si isku mid ah marka ay joogaan pLK iyo ΔNt-Tau, labadaba kuwaas oo ku jiray qaab-dhismeedka sare iyo dhaqdhaqaaqa laf-dhabarka borotiinka, oo ay shaaca ka qaadeen tijaabooyinka isbeddelka kiimikada sare iyo nasashada R1ρ.Xogta NMR waxay muujineysaa in C-terminus-ka αS uu leeyahay lumin weyn oo dabacsanaan qaabaysan iyadoo la ilaalinayo dabeecadeeda qallafsan, sida inta soo hartay ee isku xigxiga borotiinka, taasoo ay ugu wacan tahay is dhexgalka uu la leeyahay polycations.
Maaddaama ballaarinta calaamadda CW-EPR ee lagu arkay TEMPOL-122-αS wejiga ciriiriga ah ay ka tarjumayso isdhexgalka borotiinka iyo polycations, waxaan sameynay titration EPR si aan u qiimeyno xiriirka ku xiran αS ee kala duwanaansho kala duwan maqnaanshaha LLPS (wax ururin ah Buffer LLPS), oo soo jeedinaysa in is-dhexgalyadu ay isku mid yihiin marka la eego wejiyada kala milan iyo kuwa xoogga leh (taas oo lagu xaqiijiyay xogtayada, Sawirka Dheeraadka ah. 4a iyo Jaantuska Dheeraadka ah. 6).Ujeeddadu waxay ahayd in la arko haddii dhammaan isku-dubaridyada, inkasta oo alaabtooda caadiga ah ee dareeraha u eg, ay muujiyaan dabeecad kasta oo hoosaysa oo ka duwan heerka molecular.Sidii la filayey, spectrum EPR ayaa la balaariyay iyadoo la kordhinayo xooga-saarista, taasoo ka tarjumaysa hoos u dhaca dabacsanaanta molecular sababtoo ah isdhexgalka molecular ee dhammaan wada-hawlgalayaasha isdhexgalka oo ku dhawaad ​​​​saturation (Jaantus. 3e, Jaantuska Dheeraadka ah. 6).pLK waxay ku gaadhay buuxintan saamiga molar hoose (polycation:αS) marka la barbar dhigo ΔNt-Tau iyo Tau441.Dhab ahaantii, isbarbardhigga xogta oo leh qaab xiritaan qiyaas ah oo loo maleynayo in goobo isku mid ah oo madaxbannaan ayaa muujiyay in kala-baxa joogtada ah ee pLK (~ 5 μM) uu yahay amar ka hooseeya kan Tau441 ama ΔNt-Tau (~ 50 μM) ).µM)In kasta oo tani ay tahay qiyaas adag, tani waxay soo jeedinaysaa in αS ay leedahay xidhiidh sare oo kala-goysyada fudud oo leh gobollo lacag bixineed oo joogto ah.Marka la eego farqiga u dhexeeya xidhiidhka ka dhexeeya αS iyo polycations kala duwan, waxaanu qiyaasnay ​​in hantidooda dareeraha ah ay isbeddeli karaan si ka duwan waqti ka dib oo ay la kulmaan hababka LSPT ee kala duwan.
Marka la eego jawiga aadka u camiran ee ku dhex jira coacervate borotiinka iyo dabeecada amyloid ee borotiinka, waxaanu aragnay habdhaqanka coacervate waqti ka dib si loo ogaado hababka LSPT ee suurtogalka ah.Isticmaalka BF iyo CF microscopy (Jaantuska 4), waxaanu aragnay in αS / Tau441 ay isku xirto ilaa xad weyn oo xal ah, samaynta dhibco waaweyn oo taabta oo qoyan dusha hoose ee ceelka / simbiriirixan dhibco buuxa, sida la filayo (Sawir Dheeraad ah 7d;Waxaan ugu yeernaa kuwan qaab-dhismeedka hoose ee la sameeyay " raafka borotiinka ".Qaab-dhismeedyadani waxay noqdeen kuwo dareere ah maadaama ay awood u leeyihiin in ay isku fidaan (Jaantuska Dheeraadka ah 7b) waxaana la arki karaa dhawr saacadood ka dib markii LLPS ay kicisay (Jaantus. 4 iyo Sawirka Dheeraadka 7c).Waxaan aragnay in geeddi-socodka qoynta uu doorbidayo dusha sare ee hydrophilic halkii laga heli lahaa qalabka hydrophobic (Jaantuska Dheeraadka ah. 7a), sida la filayo coacervates electrostatic oo leh kharashyo aan dheellitirnayn oo sidaas awgeed awood sare oo koronto ah.Waxaa xusid mudan, αS/ΔNt-Tau isku-dhafka iyo rafting si weyn ayaa loo dhimay, halka kondensooyinka αS/pLK si weyn loo dhimay (Jaantus. 4).Inta lagu jiro wakhtiga gaaban ee gaaban, dhibcaha αS / pLK waxay awoodeen in ay isku duubaan oo qoyaan dusha sare ee hydrophilic, laakiin habkani si dhakhso ah ayuu u istaagay iyo ka dib saacadaha 5 ee ku-soo-baxa, kaliya dhacdooyinka is-dhajinta xaddidan iyo wax qoyan lama arkin.- kala-guurka gel-drip.
Wakiilada BF (panels greyscale) iyo CF (panels sax ah, AF488-ku calaamadsan αS cagaaran) ee muunado coacervate ka kooban 100 µM αS (1% sumadda fluorescent) ee LLPS kaydinta iyadoo ay joogaan 100 µM Tau441 (sare) sawirada Δ microscoores -Tau (dhexda) ama 1 mM pLK (hoose) wakhtiyo kala duwan oo kuleej ah iyo dhererka fogaanta (z, fogaanta hoose ee saxanka si fiican).Tijaabooyinku waxay soo noqnoqdeen 4-6 jeer si madaxbannaan midba midka kale natiijooyin isku mid ah.αS/Tau441 coacervates waa la qooyay 24 saacadood ka dib, samaynta rafyo ka weyn sawirka.Baaxadda cabbirka sawirada oo dhan waa 20 µm.
Waxaan markaa waydiinay bal in barkadaha borotiinka ee dareeraha u eg ee laga sameeyay αS/Tau441 LLPS ay u horseedi karaan isu geynta amyloid mid ka mid ah borotiinada la bartay.Waxaan raacnay korriinka dhibcaha αS/Tau441 waqti ka dib iyada oo la adeegsanayo microscopy WF iyadoo la raacayo shuruudaha kor ku xusan, laakiin annagoo adeegsanayna 1 μM AF488-labeled αS iyo Atto647N-ku calaamadeysan Tau441 (Jaantus. 5a).Sidii la filayey, waxaanu aragnay meelaynta borotiinka oo dhammaystiran intii lagu jiray habka korriinka.Waxa xiiso leh, laga bilaabo ca.Saacadaha 5 ka dib, dhismayaal aad u xoog badan oo aan wareeg ahayn ayaa lagu arkay gudaha qulqulka, kuwaas oo aan ugu yeernay "dhibcaha", qaar ka mid ah kuwaas oo la mideeyey αS, qaarna waxay ku hodmeen Tau441 (Jaantus. 5a, fallaadho cad).Dhibcahaan ayaa had iyo jeer lagu arkay rafts dhexdooda ilaa xad ka weyn αS/ΔNt-Tau marka loo eego αS/ΔNt-Tau.Ma jirin dhibco kala duwan oo ku jira dhibcaha pLK iyo nidaamka Tau oo aan karti u lahayn isku-dhafka/qoynta.Si loo tijaabiyo in wasakhooyinkan ay ku jiraan αS iyo Tau441 ay yihiin wadar ahaan amyloid-ka, waxaanu samaynay tijaabo la mid ah anagoo adeegsanayna CF microscopy kaas oo Tau441 lagu calaamadeeyay Atto647N iyo 12.5 μM amyloid-specific thioflavin-T (ThT) ayaa lagu daray bilawgii.dheeh.Inkasta oo ThT-staining αS/Tau441 dhibco ama rafts aan la arkin xitaa ka dib 24 h ee ku-soo-baxa (Jaantus. 5b, safka sare-dhibcaha haray oo ka sarreeya raafka borotiinka), qaababka ThT-positive ee ka kooban Atto647N-Tau441 gudaha rafts aad bay u daciifeen.tani waxay soo celinaysaa cabbirka, qaabka, iyo meesha dhibco hore loo sifeeyay (Jaantus. 5b, safafka dhexe iyo hoose), taasoo soo jeedinaysa in dhibcuhu ay u dhigmi karaan isku darka amyloid-ka oo kale ah oo lagu sameeyay dareeraha gabowga.
WF 25 μM αS waqtiyo kala duwan oo soo-bax ah iyo meelo fogaan ah (z, fogaanta hoose ee aan la xirin) iyadoo ay joogaan 25 μM Tau441 (1 μM AF488-ku calaamadeysan αS iyo Atto647N-ku calaamadeysan Tau441) .Lix tijaabo ayaa si madax banaan loogu soo celiyay natiijooyin la mid ah.b CF sawirka microscopic ee 25 μM αS joogitaanka 25 μM Tau441 (1 μM Atto647N-labeled Tau441) iyo 12.5 μM thioflavin-T (ThT).Dhibcaha borotiinka ee miisaanka leh iyo raafka borotiinka ee la dhigay iyo dhibco ayaa lagu muujiyay safafka sare iyo dhexe, siday u kala horreeyaan.Safka hoose wuxuu muujinayaa sawirada rafts iyo dhibco ka yimid 3 nuqul oo madax banaan.Fallaadhaha cadcad waxay muujinayaan dhibco ThT-positive ah ee labada dhinac.Baaxadda cabbirka sawirada oo dhan waa 20 µm.
Si aan si faahfaahsan u baadho isbeddelada shabakada borotiinka coacervate inta lagu jiro kala guurka dareeraha una gudubno adag, waxaanu isticmaalnay sawirka nolosha ee fluorescence (FLIM) iyo Förster resonance energy transfer microscopy (FRET) (Jaantuska 6 iyo Sawirada Dheeraadka ah 8 iyo 9).Waxaan ku qiyaasnay ​​in korriinka coacervate ee lakabka oo noqda qaab dhismeedka borotiinka la isku daray ee la isku daray ama xitaa adag u eg uu keeno xiriir dhow oo ka dhexeeya borotiinka iyo baaritaanka fluorescent ee ku dheggan, taasoo suurta gal ah inay soo saarto saameyn deminta oo ka muuqata cimriga baaritaanka gaaban (τ) , sida hore loogu sharaxay 40.,41,42.Intaa waxaa dheer, muunado laba-calaamad leh (AF488 iyo Atto647N oo ah deeq bixiyaha FRET iyo midabada aqbalaha, siday u kala horreeyaan), hoos u dhacan τ waxa kale oo la socon kara uumiga coacervate iyo korodhka waxtarka FRET (E) inta lagu jiro LSPT.Waxaan la soconay raft-ka iyo samaynta booska muddo ka dib LLPS αS/Tau441 iyo αS/ΔNt-Tau muunadaha (25 µM borotiin kasta oo ku jira kaydka LLPS oo ka kooban 1 µM AF488 oo ku calaamadsan αS iyo/ama Atto647N oo ku calaamadsan Tau441 ama ΔNt-Tau).Waxaan ku aragnay isbeddel guud oo ah in cimriga iftiimaya ee AF488 (τ488) iyo Atto647N (τ647N) baaritaannada ay wax yar hoos u dhaceen markii coacervates bislaaday (Jaantus. 6 iyo Sawirka Dheeraadka ah 8c).Waxa xiisaha lihi leh, isbeddelkan ayaa si weyn loogu wanaajiyey dhibco ku dhex jira rafts (Jaantus. 6c), taas oo muujinaysa in uumi borotiinka dheeraad ah uu ka dhacay dhibco.Taageerada tan, wax isbeddel ah oo la taaban karo oo ku yimid nolosha fluorescence laguma arag dhibcaha αS/ΔNt-Tau ee da'doodu tahay 24 h (Jaantuska Dheeraadka ah. 8d), taas oo soo jeedinaysa in dhibco-dhicisku uu yahay habraac ka duwan dhibcaha iyo taas oo aan la socon dib-u-habayn molecular oo muhiim ah. gudahood coacervates.Waa in la ogaadaa in dhibcuhu ay leeyihiin cabbirro kala duwan iyo nuxur kala duwan oo ku jira αS, gaar ahaan nidaamka αS/Tau441 (Jaantuska Dheeraadka ah. 8e).Hoos-u-dhaca ku yimid cimri-dhererka dhibcaha ayaa waxaa weheliyay kororka xoogga, gaar ahaan Atto647N oo ku calaamadsan Tau441 (Jaantuska Dheeraadka ah. 8a), iyo waxtarka sare ee FRET ee nidaamyada αS/Tau441 iyo αS/ΔNt-Tau, taasoo muujinaysa uumi dheeraad ah LLPS Shan saacadood kicinta ka dib, borotiinada ku jira korantada taagan ayaa isku urursan.Marka la barbar dhigo αS/ΔNt-Tau, waxaan aragnay τ647N hoose iyo xoogaa sare τ488 qiyamka αS/Tau441, oo ay la socdaan qiimayaasha FRET hoose iyo kuwa aan sinnayn.Waxaa suurtogal ah, tani waxay la xiriiri kartaa xaqiiqda ah in nidaamka αS / Tau441, la arkay iyo la filayo αS tirada badan ee isku-darka ah waa mid aad u kala duwan, inta badan substoichiometric marka la barbardhigo Tau, tan iyo Tau441 lafteedu waxay sidoo kale mari kartaa LLPS iyo isku-darka (Jaantuska Dheeraadka ah 8e) .Si kastaba ha noqotee, heerka isku-dhafka dhibcaha, samaynta raft, iyo, muhiimad ahaan, isku-darka borotiinka ee isku-darka dareeraha u eg waa ugu badnaan marka labadaba Tau441 iyo αS ay joogaan.
Sawirada αS/Tau441 iyo αS/ΔNt-Tau ee 25 μM ee borotiin kastaTiirarku waxay muujinayaan sawiro metelaya shaybaarada LLPS wakhtiyo korriimo kala duwan (30 daqiiqo, 5 saac iyo 24 saac).Qaabka cas wuxuu muujinayaa gobolka ka kooban αS/Tau441 dhibco.Muddada nolosha waxaa lagu muujiyey sida baararka midabka.Bar miisaanka = 20 µm sawirada oo dhan.b Sawirka FLIM ee la soo dhoweeyay ee aagga la doortay, ee lagu muujiyay sanduuqa cas ee guddiga a.Kala duwanaanshaha nolosha ayaa lagu muujiyay iyadoo la isticmaalayo miisaan midab leh oo la mid ah guddiga a.Qiyaasta bar = 5 µm.C Histograms muujinaya AF488 (ku lifaaqan αS) ama Atto647N (ku lifaaqan Tau) ee noocyada borotiinka ee kala duwan (D-, raft-R- iyo speckle-P) oo lagu aqoonsaday sawirada FLIM ee loo duubay αS-) qaybinta wakhtiga nolosha Tau441 iyo αS/ΔNt-Tau muunadaha coacervate (N = 17-32 ROI ee D, 29-44 ROI ee R, iyo 21-51 ROI ee dhibcood).Qiimaha celceliska iyo dhexdhexaadka ah waxaa lagu muujiyay labajibbaaran jaale ah iyo khadadka madow ee sanduuqyada, siday u kala horreeyaan.Xudduudaha hoose iyo kan sare ee sanduuqu waxay ka dhigan yihiin rubucyada koowaad iyo saddexaad, siday u kala horreeyaan, iyo qiimaha ugu yar iyo kan ugu sarreeya ee 1.5-laab dhexda u dhexeeya (IQR) ayaa lagu muujiyay sida sharoobada.Outliers waxa lagu muujiyay sida dheeman madow.Muhiimadda tirakoobka ee udhaxeysa lammaane qaybinta waxaa lagu go'aamiyay iyadoo la adeegsanayo laba-muunado t-tijaab ah, iyada oo loo maleynayo kala duwanaansho aan sinnayn.Qiimaha p-tijaabada laba-dabo leh ayaa lagu muujiyay astaamo la mid ah lammaane kasta oo xogta isbarbardhigga ah (* p-qiimaha> 0.01, ** p-qiimaha> 0.001, *** p-qiimaha> 0.0001, **** p-qiimaha> 0.00001), ns waxay muujinaysaa dayacaad (p-qiimaha> 0.05).Qiimaha saxda ah ee p waxaa lagu bixiyaa shaxda Dheeraadka ah 1, xogta asalka ah waxaa loo soo bandhigay sida faylalka xogta ceeriin.
Si aan u sii muujinno dabeecadda amyloid-ka u eg ee xadhkaha goostay, waxaanu ku daawnay muunado coacervate ah oo aan la nadiifin saacadaha 24 oo leh xaddi sare oo ah (1 M) NaCl, taas oo keentay kala soocida isku-darka isku-darka borotiinka.Marka isku-darka go'doonsan (ie, xal la kala firdhiyey ee isku-darka) ayaa lagu arkay iyadoo la adeegsanayo microscopy xoogga atomikada (AFM), waxaan aragnay qaab-dhismeedka qaab-dhismeedka u badan ee leh dherer joogto ah oo ku saabsan 15 nm, kaas oo u muuqda inuu ku xiran yahay xaaladaha milixda sare, oo la mid ah habdhaqanka fibrilada amyloid ee caadiga ah sababtoo ah saameynta hydrophobic ee xoogga leh ee dusha sare (xusuusnow in fibriilyadu ay caadi ahaan leeyihiin dhererka ~ 10 nm) (Jaantuska Dheeraadka ah 10a).Waxa xiisaha lihi leh, markii isku-darka go'doonsan lagu dhex daray ThT-ga caadiga ah ee ThT fluorescence assay, waxaanu aragnay koror aad u wayn oo ThT fluorescence quantum dhalid ah, oo la mid ah tii la arkay markii dheeha lagu dhex daray αS amyloid fibrils caadiga ah (Jaantuska Dheeraadka ah 10b), isagoo soo jeedinaya in Isku-darka isku-darka waxay ka kooban yihiin qaab-dhismeedka amyloid-ka..Dhab ahaantii, isku-darka ayaa u dulqaadanayay milix badan laakiin waxay u nugul yihiin 4 M guanidine chloride (GdnHCl), sida fibrillada amyloid ee caadiga ah (Jaantuska Dheeraadka ah. 10c).
Marka xigta, waxaanu falanqeynay isku-darka isku-darka anagoo adeegsanayna hal unug oo fluorescence ah, isku xidhka fluorescence gaar ah/isku-xidhka spectroscopy (FCS/FCCS), iyo falanqaynta dillaaca ogaanshaha isku dhaca laba-midab (TCCD).Si taas loo gaaro, waxaanu go'doominay isku-darka la sameeyay kadib 24 saacadood oo ku-tiirsanaan ah 100 μl LLPS muunado ka kooban αS iyo Tau441 (labadaba 25 μM) oo ay weheliso 1 μM AF488-ku calaamadeysan αS iyo 1 μM Atto647N-ku calaamadeysan Tau441Ku mili xalka wadarta kala firidhsan ee ka dhashay xaalad monomolecular ah adiga oo isticmaalaya kayd PEG-la'aan ah iyo 1 M NaCl (isku-xidhka isku midka ah ee loo isticmaalo in lagu kala saaro isku-darka coacervate) si looga hortago wax kasta oo suurtagal ah isdhexgalka korantada ee u dhexeeya LLPS iyo borotiinka.Tusaalaha wakhtiga hal molecule waxa lagu arki karaa sawirka 7a.Falanqaynta FCCS/FCS (isku-xidhka-is-dhaafsiga, CC iyo autocorrelation, AC) waxay muujisay in isku-darka ka kooban αS iyo tau ay ku badan yihiin shaybaarada (eeg qalooca CC ee sawirka 7b, guddiga bidix), iyo xad-dhaafka borotiinka monomeric ee haraaga ah ayaa kacay sida natiijada habka milanka (eeg qalooca AC ee Jaantuska 7b, guddi bidix).Tijaabooyinka xakamaynta ee lagu sameeyay xalal isku mid ah iyada oo la adeegsanayo muunado ay ku jiraan borotiinno monomeric oo keliya ma muujin curyaaminta CC, iyo qaloocyada AC waxay si fiican ugu habboon yihiin qaabka fidinta hal qayb oo ka mid ah (Eq. 4), halkaasoo borotiinnada monomeric ay leeyihiin isku-dhafka fidinta fidinta ee la filayo (Jaantus. 7b). ), guddi midig).Isku-darka faafinta walxaha la isku daray waa in ka yar 1 µm2/s, iyo tan borotiinnada monomeric waa qiyaastii 1 µm2/s.50–100 µm/s;qiyamku waxay la mid yihiin qiyamkii hore loo daabacay ee loogu talagalay αS amyloid fibrils sonicated iyo monomeric αS si gooni gooni ah iyadoo hoos timaad xalalka la midka ah44.Markii aan falanqeynay isku-darka falanqaynta qaraxa TCCD (Jaantus. 7c, guddiga sare), waxaan ogaanay in wadar kasta oo go'doonsan (αS/Tau heteroaggregate), qiyaastii 60% isku darka la ogaaday ayaa ka kooban αS iyo tau labadaba, qiyaastii 30% ayaa ka kooban kaliya tau, qiyaastii 10% αS kaliya.Falanqaynta Stoichiometric ee αS/Tau heteroaggregates waxay muujisay in badi heteroaggregates ay ku hodmeen tau (stoichiometry ka hooseeya 0.5, celceliska tirada molecules tau ee wadar ahaan waa 4 jeer in ka badan αS molecules), taas oo la socota shaqadayada lagu arkay FLIM ee goobta. tijaabooyin..Falanqaynta FRET waxay muujisay in isku-daryadani ay ka kooban yihiin labadaba borotiinno, in kasta oo qiyamka FRET ee kiiskani aanu ahayn muhiimad weyn, maaddaama qaybinta fluorophores ee isku-dar kasta ay ahayd mid aan kala sooc lahayn sababtoo ah xad-dhaafka borotiinka aan la calaamadin ee loo isticmaalo tijaabada.Waxa xiisaha lihi leh, markii aanu samaynay isla falanqaynta anagoo adeegsanayna 45,46 amyloid amyloid aggregation-deficient Tau variant (eeg Sawirka Dheeraadka ah Awoodda lagu sameeyo isku-darka isku-darka gudaha coacervate si weyn ayaa loo yareeyay FLIM waxay ogaatay dhibco dhowr ah oo ku jira tijaabooyinka goobta, iyo qaloocyada isku-xiran ee daciifka ah ayaa lagu arkay muunado wadar ah oo go'doonsan.Si kastaba ha ahaatee, tiro yar oo isku-dar ah oo la ogaaday (kaliya toban meelood meel Tau441), waxaan aragnay in wadar kasta uu ku hodmay αS marka loo eego kala duwanaanshahan Tau, iyada oo ku dhawaad ​​50% isku darka la ogaaday ay ku jiraan unugyada αS kaliya, iyo αS waxay ahayd mid aad u kala duwan. .isku darka (fiiri sawirka Dheeraadka ah 11e), si ka duwan isku darka kala duwan ee ay abuurtay Tau441 (Jaantus. 6f).Natiijooyinka tijaabooyinkan ayaa muujiyay in inkasta oo αS lafteedu ay awood u leedahay in ay ku ururto tau gudaha coacervate, tau nucleation ayaa aad ugu fiican xaaladahan, iyo natiijada isku-darka amyloid-la mid ah waxay awoodaan inay u dhaqmaan qaab αS iyo tau.Si kastaba ha ahaatee, marka xudunta hodanka ah la sameeyo, isdhexgalka heterotypic ee u dhexeeya αS iyo tau ayaa laga door bidaa wadar ahaan isdhexgalka homotypic ee u dhexeeya molecules tau;Waxaan sidoo kale la soconaa shabakadaha borotiinka ee dareeraha αS/tau coacervates.
raadraac ku meel gaadh ah oo matale ah oo ka kooban unugyo keli ah oo isku-darka gooni-gooni ah oo lagu sameeyay αS/Tau441 korantadaBursts u dhiganta αS/Tau441 coaggregates (ka sarreeya heerka la tilmaamay) ayaa lagu arkay saddex channels ogaanshaha (AF488 iyo Atto647N sii daayo ka dib kicin toos ah, khadadka buluug iyo cas, Atto647N sii daayo ka dib kicin dadban), FRET, line violet).b FCS/FCCS ee muunad isku darka αS/Tau441 go'doonsan oo laga helay LLPS (guud bidix).Qalloocyada Autocorrelation (AC) ee AF488 iyo Atto647N waxaa lagu muujiyey buluug iyo casaan, siday u kala horreeyaan, iyo isku-xidhka-isku-xidhka (CC) ee la xidhiidha isku-darka ka kooban labada midabba waxaa lagu muujiyey guduud.Qalloocyada AC waxay ka tarjumayaan joogitaanka noocyada borotiinka monomeric ah ee la isku daray, halka CC-du ay muujinayaan oo kaliya faafinta isku-darka laba-calaamadeysan.Falanqaynta isku midka ah, laakiin xaaladaha xalinta isku midka ah ee meelaha go'doonsan, muunado ka kooban monomeric αS iyo Tau441 oo keliya ayaa lagu muujiyay sida kontaroolada guddiga saxda ah.c Fluorescence flash falanqaynta hal molecules ee isku-darka go'doonsan ee lagu sameeyay αS/Tau441 coacervates electrostatic.Macluumaadka wadar kasta oo lagu helo afar soo noqnoqosho oo kala duwan (N = 152) waxaa loo dejiyay si ka dhan ah stoichiometry, qiyamka S, iyo hufnaanta FRET (guud sare, bar midabka ayaa ka tarjumaysa dhacdo).Saddex nooc oo isku-dar ah ayaa loo kala saari karaa: -αS-kaliya isku-darka S~1 iyo FRET~0, Tau-kaliya isku-darka S~0 iyo FRET~1, iyo isku-darka kala duwan ee Tau/αS oo leh dhexdhexaad S iyo FRET Qiyaasta qaddarka Labada borotiinada calaamadeeyayaasha ee laga helay wadar kasta oo kala duwan (N = 100) ayaa lagu muujiyay guddiga hoose (miisaanka midabku wuxuu ka tarjumayaa dhacdada).Xogta ceeriin waxaa lagu bixiyaa qaab faylal xog ceeriin ah.
Kobcinta ama da'da borotiinka dareeraha ah ee kondhisyada dareeraha ah ee gel-la midka ah ama qaababka adag ee waqti ka dib ayaa lagu soo warramey inay ku lug leeyihiin dhowr hawlood oo nafsi ah oo ka mid ah condensate47 iyo sidoo kale cudur, sida habka aan caadiga ahayn ee ka horreeya isku-darka amyloid 7, 48, 49. Halkan Waxaan si faahfaahsan u baranaynaa kala-tagga wajiga iyo hab-dhaqanka.LSPT αS marka ay jirto kala-baxyo aan kala sooc lahayn oo ka jira deegaan la kantaroolo oo ah uruurinta micromolar-ka hooseeya iyo xaaladaha jireed ee laxiriira (xusuusnow in uruurinta jireed ee la xisaabiyay ee αS ay tahay> 1 µM50), iyadoo la raacayo dabeecadda heerkulbeegga ah ee caadiga ah ee LPS.Waxaan ogaanay in αS, oo ka kooban gobolka C-terminal si xun loogu soo oogay pH physiological, uu awood u leeyahay inuu sameeyo dhibco hodan ku ah borotiinka oo ku jira xal aqueous iyada oo loo marayo LLPS iyadoo ay joogaan peptides aadka u cakiran sida pLK ama Tau iyada oo loo marayo habka elektrostatic. Uumi adag oo ay joogaan macromolecules-ka-ururinta.Nidaamkani waxa laga yaabaa inuu saameyn ku yeesho deegaanka gacanta ee αS uu la kulmo unugyo kala duwan oo polycationic ah oo la xidhiidha isku-darka jirrooyinka la xidhiidha labadaba gudaha vitro iyo gudaha vivo51,52,53,54.
Daraasado badan, dhaqdhaqaaqa borotiinka ee dhibcaha dhibcaha ayaa loo tixgeliyey mid ka mid ah qodobbada muhiimka ah ee go'aaminaya habka korriinka55,56.In electrostatic αS coacervates leh polycations, habka korriinka sida muuqata waxay ku xiran tahay xoogga isdhexgalka ee polycations, valence, iyo badnaanta isdhexgalka kuwan.Aragtida isku dheelitirnaanta waxay soo jeedinaysaa in muuqaalka siman ee laba dawladood oo dareere ah ay noqon doonto joogitaanka dhibic weyn oo hodan ku ah bioopolymers ee wada LLPS57,58.Kobaca dhibicda waxaa lagu gaari karaa Ostwald maturation59, coalescence60 ama isticmaalka monomer bilaash ah ee marxaladda kala firdhiyey61.Wixii αS iyo Tau441, ΔNt-Tau ama pLK, borotiinka intiisa badan waxay ku urursan yihiin kondensate hoos yimaada shuruudaha loo isticmaalo daraasaddan.Si kastaba ha ahaatee, halka dhibcaha buuxa ee tau ay si degdeg ah ugu dhaqmeen qoynta dusha sare, isku-dhafka dhibcaha iyo qoynta ayaa ku adkeyd ΔNt-Tau iyo pLK, taasoo soo jeedinaysa luminta hantida dareeraha ah ee labadan nidaam.Marka loo eego falanqayntayada FLIM-FRET, dhibcaha pLK iyo ΔNt-Tau ee da'da ah waxay muujiyeen heer isku mid ah isku-darka borotiinka (noolaha fluorescence la mid ah) sida dhibcaha asalka ah, oo soo jeedinaya in shabakadda borotiinka asalka ah la hayo, inkastoo ay aad u adag tahay.
Waxaan ku qiimeyneynaa natiijadayada tijaabada qaabka soo socota (Jaantuska 8).Dhibcaha markii hore si ku meel gaadh ah loo sameeyay ayaa badanaa ah shabakado borotiin ah oo aan lahayn magdhow elektrostatic ah, sidaas darteed waxaa jira meelo lacag la'aan ah, gaar ahaan barta dhibcaha dhibcaha, taasoo keentay dhibco leh awood sare oo koronto ah.Si loo magdhabo kharashka ( ifafaale inta badan loo yaqaan valence depletion) oo loo yareeyo awoodda dusha sare ee dhibcaha dhibcaha, dhibcuhu waxay ku dari karaan polypeptides cusub oo ka soo jeeda wajiga walaaqaya, dib-u-habeynta shabakadaha borotiinka si kor loogu qaado isdhexgalka lacag-bixinta, iyo la falgalka dhibcaha kale.oo leh dusha sare (qoynta).Dhibcaha αS / pLK, sababtoo ah shabakadooda borotiinka ee fudud (kaliya isdhexgalka heterotypic ee u dhexeeya αS iyo pLK) iyo isku xirnaanta weyn ee isdhexgalka borotiinka-protein, waxay u muuqdaan inay awood u leeyihiin inay si dhakhso ah u dheelitiraan kharashka kondensate;Runtii, waxaan ku aragnay kinetics borotiinka degdega ah markii hore la sameeyay αS/pLK coacervates marka loo eego αS/Tau.Ka dib dhimista valence, isdhexgalka ayaa noqda mid yar oo dhibco ah waxayna luminayaan hantidooda dareeraha waxayna isu beddelaan dhibco jel u eg, dhibco aan guban oo leh awood hoose oo korontostatic ah (oo sidaas darteed aan awoodin inay qoyaan dusha sare).Taas bedelkeeda, dhibcaha αS/Tau aad bay ugu wanagsan yihiin hagaajinta dheelitirka kharashka dhibcaha sababtoo ah shabakadaha borotiinka ee kakan (labadaba isdhexgalka khaniisnimada iyo heterotypic) iyo dabeecadda daciifka ah ee isdhexgalka borotiinka.Tani waxay keenaysaa dhibco soo ceshanaya dabeecadda dareeraha muddo dheer oo muujiya awoodda korantada sare ee awoodda taas oo u janjeerta in la yareeyo iyadoo la isku duba ridayo oo korayo (sidaas darteed la yareeyo bedka dusha / saamiga dhibcaha dhibcaha) iyo iyada oo qoynaysa kiimikada dusha sare ee hydrophilic.Tani waxay abuurtaa maktabado borotiin ah oo waaweyn kuwaaso xajiya sifooyinka dareeraha maadaama isdhexgalka uu yahay mid aad u kala guur ah sababtoo ah raadinta joogtada ah ee hagaajinta kharashka shabakada borotiinka.Waxa xiiso leh, qaababka N-terminally gooyay ee Tau, oo ay ku jiraan qaar ka mid ah isoforms62 si dabiici ah u dhaca, waxay muujiyaan dabeecad dhexdhexaad ah, iyada oo qaar ka mid ah ay xoojiyaan gabowga oo leh αS oo noqda dhibco jel oo kale ah oo cimri dheer, halka kuwa kalena ay isu beddelaan kondensates dareere ah oo waaweyn.Labadan lammaane ee korriinka koofiyadaha korantada ee αS waxay la jaan qaadayaan LLPS aragtiyaha iyo daraasadaha tijaabada ah ee dhowaan ogaaday xidhiidhka ka dhexeeya valence depletion iyo shaandhaynta korantada ee kondensates oo fure u ah xakamaynta cabbirka kondensate iyo sifooyinka dareeraha.Farsamaynta 58.61.
Nidaamkani wuxuu muujinayaa dariiqa isu geynta amyloid-ka ee αS iyo Tau441 iyada oo loo marayo LLPS iyo LSPT.Iyada oo dheeraad ah gobollada anion-hodanka ah (casaan) iyo cation-hodanka (buluug), αS iyo tau electrostatic coacervates leh valence ku qanacsan waxay leeyihiin tamar hoose oo dusha sare ah oo sidaas darteed isku-duubni yar, taasoo keentay gabowga degdega ah.Xaalad jel ah oo xasilan oo aan gabbasho lahayn ayaa la gaadhay..Xaaladdani waa mid aad u wanaagsan marka loo eego nidaamka αS / pLK sababtoo ah isku xirnaanta sare iyo shabakada isdhexgalka borotiinka-labadaba, kaas oo u oggolaanaya isbeddel degdeg ah oo jel ah.Taas beddelkeeda, dhibco leh valence aan ku qanacsanayn iyo, sidaas darteed, gobollada borotiinka lagu dallaco ee diyaar u ah isdhexgalka, waxay u sahlaysaa coacervate in ay isku dhejiso oo qoyaan dusha sare ee hydrophilic si loo yareeyo tamarta sare ee dusha sare.Xaaladdani waxay u fiican tahay αS/Tau441 coacervates, kuwaas oo leh shabakad isku dhafan oo isku dhafan oo ka kooban isdhexgalka Tau-Tau iyo αS-Tau daciif ah.Taa baddalkeeda, coacervates waaweyn ayaa si aad u diyaarsan u haysan doona sifooyinkooda dareeraha ah, taas oo u oggolaanaysa isdhexgalka borotiinka iyo borotiinka kale inay dhacaan.Ugu dambeyntii, isku-darka amyloid heterogeneous oo ka kooban αS iyo tau labadaba gudaha dareeraha coacervate, kuwaas oo laga yaabo inay la xiriiraan kuwa laga helo jirka ku darida, kuwaas oo ah calaamadaha cudurrada neurodegenerative.
Qaababka waaweyn ee dareeraha u eg ee la sameeyay inta lagu guda jiro qaan-gaarnimada αS/Tau441 oo leh jawi aad u ciriiri badan laakiin firfircoonida borotiinka iyo, ilaa xad ka yar, αS/ΔNt-Tau coacervates waa kayd ku habboon nukleemka isu-ururinta borotiinka.Waxaan runtii aragnay samaynta isku-darka borotiinka adag ee noocaan ah coacervates borotiinka, oo inta badan ka kooban αS iyo tau labadaba.Waxaan tusnay in heteroaggregates-kan ay dejiyeen isdhexgalka aan-electrostatic-ka ahayn, waxay awoodaan inay ku xidhaan amyloid-gaar ah midabada ThT si la mid ah fibrillada amyloid-ka caadiga ah, runtiina waxay leeyihiin iska caabin la mid ah saameyno kala duwan.Isku darka αS/tau ee ay samaysteen LLPS waxa la tusay inay leeyihiin sifooyin amyloid u eg.Runtii, kala duwanaanshaha qaan-gaadhka ah ee Tau ee ku liidata isu-ururinta amyloid ayaa si weyn u liidata samaynta isku-daryadan αS-ga ah ee ku dhex jira dareeraha korantada dareeraha ah.Samaynta isku-darka αS/Tau441 ayaa lagu arkay oo keliya gudaha coacervates, kuwaas oo xajistay guryaha dareeraha u eg, oo waligood, haddii isku-xidhaha/dhibcaha aysan gaarin gobolka gel.Xaaladda dambe, xoogga kordhay ee isdhexgalka elektrostatic iyo, natiijada, adkeynta shabakada borotiinka waxay ka hortagtaa dib-u-habeynta lagama maarmaanka ah ee borotiinnada si loo dhiso isdhexgalka borotiinka cusub ee lagama maarmaanka u ah nucleation amyloid.Si kastaba ha ahaatee, tan waxaa lagu gaari karaa dabacsanaan badan, dareere u eg coacervates, taas oo iyana u badan tahay in ay sii ahaanayaan dareeraha marka ay kordhiyaan cabbirka.
Xaqiiqda ah in samaynta isku-darka gudaha marxaladda isku-duubni ay ka door bidayso kondhisyada waaweyn ee αS/Tau marka loo eego dhibco yaryar oo si degdeg ah u gelaya, waxay muujinaysaa muhiimadda aqoonsiga qodobbada xakameynaya isku-dhafka dhibcaha dhibcaha.Sidaa darteed, ma aha oo kaliya inay jirto u janjeera kala-soocidda wejiga, laakiin cabbirka kondensate waa in la xakameeyaa si ay u shaqeyso habboon iyo sidoo kale ka hortagga cudurrada58,61.Natiijooyinkayagu waxay sidoo kale muujinayaan muhiimada dheelitirka u dhexeeya LLPS iyo LSPT ee nidaamka αS/Tau.Halka samaynta dhibco laga yaabo inay ka hortagto isku-darka amyloid iyadoo la dhimayo qadarka monomers-ka borotiinka ee lagu heli karo xaaladaha saturation, sida lagu soo jeediyay nidaamyada kale63,64, isku-dhafka dhibcaha ee heerarka sare ee dhibcaha waxay u horseedi kartaa isku-darka borotiinka gudaha iyada oo loo marayo dib-u-qaabayn gaabis ah.shabakadaha borotiinka..
Guud ahaan, xogtayadu waxay si adag u xoojinaysaa ku-talogalka wadajirka ah iyo is-dhexgalka ku qanacsan/aan ku qanacsanayn ee shabakadaha hoos u dhaca ee macnaha guud ee LSPT.Gaar ahaan, waxaan ku tusineynaa in dhererka buuxa ee αS / Tau441 kondensates ay awoodaan inay si hufan u dhuuxaan oo ay u sameeyaan amyloid-like heteroaggregates oo ay ku jiraan borotiinnada labadaba waxayna soo jeedinayaan habka molecular ee ku salaysan natiijooyinkayada tijaabada ah.Isku-darka labada borotiin ee isku-dhafka dareeraha αS/Tau ee aan halkan ku soo sheegnay waxaa laga yaabaa inay dhab ahaantii la xiriirto isku-dubarid laba borotiinno oo ku jira isku-darka, kuwaas oo ah calaamadaha cudurka, waxayna gacan ka geysan karaan fahamka xiriirka ka dhexeeya LLPS iyo amyloid aggregation, oo u gogol xaaraya barokac aad u sarreeya oo ku dhaca neerfayaasha.
Monomeric WT-αS, mutants cysteine ​​(Q24C-αS, N122C-αS) iyo ΔCt-αS kala duwanaanshaha (Δ101-140) ayaa lagu muujiyay E. coli oo la nadiifiyey sidii hore loo sharraxay.5 mM DTT ayaa lagu daray dhammaan tillaabooyinka sifaynta αS cysteine ​​mutants si looga hortago samaynta curaarta disulfide.Tau441 isoform (plasmid laga helay Addgene #16316), kala duwanaanshaha ΔNt-Tau (Δ1-150, lagu helay by cloning IVA oo leh furayaasha CTTTAAGAAGGAGATACATATGATCGCCACACACCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) iyo AggDef-Tau7-ka-soo-baxa G5G3. dhaqamada ahaayeen koray ilaa OD600 = 0.6-0.7 at 37°C iyo 180 rpm, iyo muujinta waxaa lagu kiciyay IPTG 3 saacadood at 37°C.Unugyada goosashada 11,500 xg 15 min at 4°C kuna dhaq kaydka cusbada leh ee ka kooban 150 mM NaCl.Ku celi pellet-ka ku jira lysis kayd (20 ml halkii 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, kopeptin).1Tallaabada sonication waxaa lagu sameeyay baraf leh baaxadda 80% ee garaaca garaaca 10 (1 min on, 1 min off).Ha dhaafin 60 ml hal ultrasound.E. coli lysates waxaa lagu kululeeyay 95 ° C. 20 daqiiqo, ka dibna lagu qaboojiyey barafka oo la xakameeyey 127,000 × g 40 daqiiqo.Daawada la caddeeyey ayaa lagu dabaqay 3.5 kDa xuub (Spectrum™ Thermo Fisher Scientific, UK) waxaana laga sifeeyay 4 L ee sifeynta sifeynta (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) ilaa 10 saacadood.Tiirka isweydaarsiga 5 ml cation (HiTrap SPFF, Cytiva, MA, USA) ayaa lagu simay bakhaar dheellitiran (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Tau lysate waxaa lagu sifeeyay 0.22 μm PVDF filter waxaana lagu duray tiirka xadiga socodka 1 ml/min.Elution waxaa loo fuliyay si tartiib tartiib ah, tau waxaa lagu iftiimiyay 15-30% elution buffer (20 mM MES, pH 6.8, 1 M NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Jajabyada waxaa lagu falanqeeyay SDS-PAGE, iyo jajab kasta oo ka kooban hal koox oo leh miisaanka molecular ee la filayo ee tau ayaa lagu uruuriyay iyadoo la adeegsanayo shaandhada 10 kDa centrifuge waxaana lagu bedelay kayd ka kooban 10 mM HEPES, pH 7.4, NaCl 500 mM iyo DTT 2 mM fiirsashada borotiinka ugu dambeeya waxay ahayd 100 μM.Xalka borotiinka ayaa markaa la dhex mariyey shaandhada 0.22 μm PVDF, si degdeg ah loo qaboojiyey oo lagu kaydiyey -80°C.Protein K18 waxaa si naxariis leh u bixiyay Prof. Alberto Boffi.Nadiifinta diyaarintu waxay ahayd> 95% sida ay xaqiijiyeen SDS-PAGE iyo MALDI-TOF/TOF.Sisteeyno kala duwan ayaa kiimiko ahaan lagu calaamadeeyay AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) ama TEMPOL-maleimide (Kimikada Cilmi-baarista Toronto, Toronto, Canada).waxaa lagu xaqiijiyay nuugid iyo MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau iyo K18 ayaa lagu calaamadeeyay hadhaaga cysteine-ka asalka ah ee boosaska 191 iyo 322 iyadoo la adeegsanayo Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Germany) iyadoo la raacayo habraac la mid ah.Kharash saafi ah khariidadaha hadhaaga ah ee αS iyo Tau441 ayaa la soo saaray iyadoo la isticmaalayo CIDER66.
Poly-L-lysine adag (pLK DP 90-110 marka loo eego NMR oo ka socota alaab-qeybiye, Alamanda Polymers Inc, Huntsville, Alabama, USA) ayaa lagu milmay 10 mM HEPES, 100 mM NaCl, pH 7.4 ilaa 10 mM, habka loogu talagalay 5 daqiiqado qubayska biyaha ultrasonic oo ku kaydi -20°C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) iyo FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) waa biyo milmay oo si weyn loogu qaybiyey kaydka LLPS.Sifeyntu waxay meesha ka saartaa milixda wasakhaysan.Ka dib waxaa lagu sifeeyay shaandhada saliingaha oo cabbirkeedu yahay 0.22 μm, xooggooda waxaa la xisaabiyay iyadoo la isticmaalayo refractometer (Mettler Toledo, Columbus, Ohio, USA).Shaybaarada LLPS waxaa lagu diyaariyey heerkulka qolka sida soo socota: bakhaar iyo extrusion ayaa la isku daray iyo 1 mM tris (2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) iyo isku dar ah 1% protease inhibitor (PMSF 100 mM, benzamide 1 mM, leupeptin 5 μM).Kadibna αS iyo polycations (options pLK ama Tau) ayaa lagu daraa.Tijaabooyinka taxanaha wakhtiga thioflavin-T (ThT, Carbosynth, Compton, UK), isticmaal wadarta fiirsashada ThT si ay u noqoto kala badh fiirsiga αS.Si tartiib ah laakiin si fiican isku qas shaybaarada si loo hubiyo inay isku mid yihiin.Isku-duubnaanta qayb kastaa way kala duwanaatay tijaabo ilaa tijaabo, sida lagu qeexay qaybta Natiijooyinka.Azide waxaa loo isticmaalay xaddi dhan 0.02% (w/v) mar kasta oo ay muddada tijaabada ahi dhaaftay 4 saacadood.Dhammaan falanqaynta iyadoo la adeegsanayo muunado LLPS, u oggolow isku-darka inay isku mid noqdaan 5 daqiiqo kahor falanqaynta.Falanqaynta iftiinka iftiinka, 150 µl oo muunado ah ayaa lagu raray 96 microplates oo aan xidhnayn (µClear®, madow, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) waxaana lagu daboolay filim dhejis ah.LLP-yada waxaa lagu kormeeray cabbiraadda nuugista 350 nm ee xarunta dhexe ee xalka ee akhristaha saxanka CLARIOstar (BMG Labtech, Ortenberg, Germany).Tijaabooyinka waxaa lagu sameeyay saddex jeer heerkulka 25 ° C, khaladaadka waxaa loo xisaabiyay sida caadiga ah ka leexashada celceliska.Marxaladda isku-darka ah waxaa lagu qiyaasey muunadda centrifugation iyo SDS-PAGE falanqeynta jel, iyo jajabka αS ee wejiyada milan iyo xoogga la saaray waxaa lagu qiyaasey xalal kala duwan oo LLPS ah.Muunad 100 μl LLPS ah oo ka kooban 1 μM AF488-calaamadeysan αS ayaa la diyaariyey iyadoo la isku qasay si fiican oo ay ku xigto centrifugation at 9600×g 30 daqiiqo, ka dib da'da ayaa caadi ahaan la arki jiray.50 μl ee ugu sarreeya ee supernatant-ka ayaa loo isticmaalay qiyaasida borotiinka iyadoo la isticmaalayo gel SDS-PAGE.Gels waxaa lagu sawiray filtarrada AF488 iyadoo la adeegsanayo nidaamka sawir-qaadista ee ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) ama lagu dheehday wasakh Coomassie oo lagu sawiray shaandhooyin ku habboon.Qaybaha natiijada ayaa lagu falanqeeyay iyadoo la adeegsanayo ImageJ version 1.53i (Macahadyada Qaranka ee Caafimaadka, USA).Tijaabooyinka waxaa lagu sameeyay labo tijaabo oo kala duwan oo leh natiijooyin isku mid ah.
Caadi ahaan, 150 μl oo muunado ah ayaa lagu dabaqay microplates 96-ceel ah oo aan xidhnayn waxaana lagu arkay heerkulka qolka Leica DMI6000B mikroskoob rogan (Leica Microsystems, Wetzlar, Germany).Tijaabooyinka dhibcaha, µ-Slide Angiogenesis taarikada (Ibidi GmbH, Gräfelfing, Jarmalka) ama 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) ayaa sidoo kale la isticmaalay.EL6000 halogen ama nalalka halide birta meerkuri ayaa loo isticmaalay ilo iftiimin (loogu talagalay BF/DIC iyo sawirka WF, siday u kala horreeyaan).Marka loo eego mikroskoobyada WF, ujeeddada hawo weyneynta 40x (Leica Microsystems, Germany) ayaa loo adeegsaday in diiradda la saaro iftiinka muunadda oo la soo ururiyo.Shaybaarada AF488 iyo ThT ee calaamadeysan, shaandheynta xamaasadda iyo sii daynta leh jaangooyooyinka shaandhada GFP ee caadiga ah, xiisaha iyo miirayaasha bandpass, siday u kala horreeyaan, 460-500 nm iyo 512-542 nm filtarrada bandpass, iyo 495 nm muraayad dichroic ah.Shaybaarada lagu calaamadeeyay Atto647N, jaangooyooyin shaandhooyin Cy5 ah oo leh miirayaasha xamaasadda iyo sii daynta 628-40 nm iyo 692-40 nm, siday u kala horreeyaan, iyo muraayad 660 nm dichroic ah ayaa la isticmaalay.BF iyo DIC microscopy, isticmaal ujeeddada ururinta iftiinka oo la mid ah.Iftiinka la ururiyey waxaa lagu duubay kamarada Leica DFC7000 CCD (Leica Microsystems, Germany).Waqtiga soo-gaadhista waxay ahayd 50 ms BF iyo DIC sawir-qaadista mikroskoobyada iyo 20-100 ms ee sawirka mikroskoobyada WF.Marka la barbardhigo, wakhtiga soo-gaadhista ee dhammaan tijaabooyinka ThT waxay ahayd 100 ms.Tijaabooyin waqti-dhaaf ah ayaa la sameeyay si loo arko isku-dhafka dhibcaha dhibcaha, iyadoo sawirada la ururiyay 100 ms kasta dhowr daqiiqo.ImageJ (NIH, USA) ayaa loo isticmaalay falanqaynta sawirka.Tijaabooyinka waxaa lagu sameeyay saddex jeer natiijooyin isku mid ah.
Tijaabooyinka isku-darka, FRAP iyo dib-u-dhiska 3D, sawirada waxaa lagu helay Zeiss LSM 880 mikroskoob konfocal rogan iyadoo la adeegsanayo daabacaadda buluugga ah ee ZEN 2 (Carl Zeiss AG, Oberkochen, Germany).Tusaalooyinka 50 µl ayaa lagu dabaqay µ-Slide Angiogenesis Petri suxuunta (Ibidi GmbH, Gröfelfing, Jarmalka), oo lagu daaweeyay polymer hydrophilic (ibiTreat) oo lagu dhejiyay ujeeddada immersion saliid 63 × (Qorshaha-Apochromat 63 ×/NA 1.4 Saliid) ee DIC).Sawirada waxaa lagu helay iyadoo la adeegsanayo 458 nm, 488 nm, iyo 633 nm khadadka laser argon oo leh xallinta 0.26 µm/pixel iyo wakhtiga soo-gaadhista 8 μs/pixel ee kicinta iyo ogaanshaha daaqadaha 470-600 nm, 493-628 nm, iyo 638-755 nm ayaa loo isticmaalay in lagu sawiro ThT, AF488 iyo Atto647N, siday u kala horreeyaan.Tijaabooyinka FRAP, sawir qaadista muunad kasta oo waqti-dhaaf ah ayaa lagu duubay 1 jir ilbiriqsikii.Tijaabooyinka waxaa lagu sameeyay saddex jeer heerkulka qolka iyadoo natiijooyin isku mid ah ay heleen.Dhammaan sawirada waxaa lagu falanqeeyay iyadoo la isticmaalayo Zen 2 software daabacaadda buluuga ah (Carl Zeiss AG, Oberkochen, Germany).Qalloocyada FRAP waa la caadiyeeyay, la qorsheeyay oo lagu rakibay xoojinta/waqtiga xogta laga soo saaray sawirada iyadoo la adeegsanayo Zen 2 iyadoo la adeegsanayo OriginPro 9.1.Curyaaminta soo kabashada ayaa lagu rakibay qaab hal jibbaaran si loogu xisaabtamo fidinta molecular oo leh erey jibbaaran oo dheeri ah si loogu xisaabtamo saamaynta biliijka iibsiga.Ka dib waxaan xisaabinay D annagoo adeegsanayna radius bleaching magac ahaan iyo noloshii nus ka soo kabashada ee hore loo go'aamiyay sida isla'egta Kang et al.5 35 la tusay.
Kala duwanaanshaha cysteine ​​​​ka ah ee αS ayaa lagu sameeyay 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) ee boosaska 24 (TEMPOL-24-αS) iyo 122 (TEMPOL-122-αS), siday u kala horreeyaan.Labeling Labeling ee tijaabooyinka EPR, fiirsiga αS waxaa lagu dejiyay 100 μM halka PEG-fiirinta ay ahayd 15% (w/v).Shuruudaha isu geynta ee kala duwan, saamiga αS: pLK wuxuu ahaa 1:10, halka αS: ΔNt-Tau iyo αS: Tau441 saamiga lagu hayo 1:1.Si loo xidho tijaabooyinka titration-ka ee maqnaanshaha ciriiriga, TEMPOL-122-αS waxaa lagu hayaa 50 μM iyo polycations ayaa lagu titrateed kor u qaadida uruurinta, diyaarinta xaalad kasta si gaar ah.Cabbiraadaha CW-EPR waxaa lagu fuliyay iyadoo la adeegsanayo spectrometer Bruker ELEXSYS E580 X-band oo ku qalabaysan Bruker ER4118 SPT-N1 resonator oo ku shaqeeya mikrowrow (SHF) inta jeer ee ~9.7 GHz.Heerkulka waxaa la dejiyay 25°C waxaana gacanta ku haya dareeraha nitrogen-cyostat.Muuqaalka ayaa lagu helay xaalado aan sasaturated ahayn oo ah awoodda MW ee 4 mW, baaxadda qaabaynta 0.1 mT, iyo inta jeer ee habaynta 100 kHz.Xoojinta Spectral ayaa la caadiyeeyay si looga fogaado kala duwanaanshiyaha uruurinta lafdhabarta ee udhaxeysa shaybaarada iyo dhimista laf dhabarta suurtagalka ah sababtoo ah uruurinta haraaga yaraynta wakiilada ee muunado ay kujiraan Tau441 ama ΔNt-Tau (oo ku jira xalalka borotiinka asalka ah).Qiimaha g ee la bixiyay waxaa lagu helay natiijadii qaabaynta EPR ee la sameeyay iyadoo la adeegsanayo softiweerka Easyspin (v. 6.0.0-dev.34) ee laga hirgaliyay Matlab®67.Hal/laba nooc oo nooc isotropic ah ayaa loo adeegsaday qaabaynta xogta.Ka dib markii la caadaystay dhammaan calaamadaha, haraaga ayaa la xisaabiyay iyadoo laga jaray sawir kasta oo ka mid ah tijaabada tijaabada ah.Falanqaynta titration xidhidhiyaha, xoojinta qaraabada ee guutada saddexaad ilaa guuta labaad ee heerka caadiga ah ee EPR spectrum (IIII/III) ayaa loo isticmaalay si loola socdo ku xidhka xidhidhiyaha αS.Si loo qiyaaso kala tagga joogtada ah (Kd), qalooca ka dhashay waxaa lagu rakibay qaab qiyaas ah iyadoo loo qaadanayo in goobo isku mid ah oo madaxbannaan.
Tijaabooyin spectroscopy NMR ayaa la sameeyay iyadoo la adeegsanayo spectrometer Bruker Neo 800 MHz (1H) NMR oo ku qalabaysan cryoprobe iyo Z-gradient.Dhammaan tijaabooyinka waxaa lagu sameeyay iyadoo la adeegsanayo 130-207 µM αS iyo αS / ΔNt-Tau iyo pLK u dhiganta 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 waxaana lagu sameeyay 15 ° C.Si loola socdo LPS ee NMR, 10% PEG ayaa lagu daray shaybaarkii hore la isku daray.Goobta khalkhalgelinta isbeddelka kiimikaad (Jaantus. 1b) waxay muujinaysaa celceliska 1H iyo 15N beddelka kiimikaad.Muuqaalka αS 2D1H-15N HSQC waxaa loo qoondeeyay iyadoo lagu salaynayo hawlo hore (BMRB gelitaanka #25227) waxaana lagu xaqiijiyay duubista iyo falanqaynta muuqaalka 3D ee HNCA, HNCO iyo CBCAcoNH.13Cα iyo 13Cβ isbeddelada kiimikaad ayaa lagu xisaabiyay joogitaanka ΔNt-Tau ama pLK si loo cabbiro isbeddelada suurtagalka ah ee isbeddellada qaab dhismeedka labaad marka la barbar dhigo αS isbeddellada kiimikaad ee isugeynta gariiradda random saafiga ah 68 (Jaantuska Dheeraadka ah 5c).Heerarka R1ρ waxaa lagu cabiray duubista hsqctretf3gpsi tijaabooyinka (laga helay maktabadda Bruker) oo leh daahitaanka 8, 36, 76, 100, 156, 250, 400, iyo 800 ms. waqtiyo lagu go'aamiyo R1ρ iyo hubanti la'aanteeda tijaabada ah.
Tijaabooyinka florescence-ka laba-midab leh ee lagu xalliyo mikroskoobiga ayaa lagu sameeyay waqti-ganacsiyeed MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) oo wata qalab tirinta sawir qaade ah (TCSPC).Madaxa diode-ka leysarka waxaa loo isticmaalaa pulsed interleaved excitation (PIE), beam wuxuu dhex maraa hal qaab waveguide waxaana lagu hagaajiyaa awooda leysarka ee 10 ilaa 100 nW ee 481 nm iyo 637 nm xadhkaha leysarka oo lagu cabiray muraayad dichroic ka dib.Tani waxay hubinaysaa heerka ugu wanaagsan ee tirinta sawir-qaadista, iyada oo laga fogaanayo saamaynta sawir-qaadista, sawir-qaadista iyo saturation.μ-Slide angiogenesis daboolka ama taarikada (Ibidi GmbH, Gräfelfing, Germany) ayaa si toos ah loogu dhex geliyey biyaha immersion korka Super Apochromat 60x NA 1.2 lens leh koodhka saxda ah (Olympus Life Sciences, Waltham, USA).Muraayad 488/640 nm dichroic ah (Semrock, Lake Forest, IL, USA) ayaa loo isticmaalay sidii kala qaybinta biraha ugu weyn.Shucaaca aan diirada la saarin waxaa xiray dalool dhexroorkiisu yahay 50 micron, ka dibna shucaaca diirada lagu saaray waxaa loo qaybiyaa 2 waddo oo lagu ogaanayo iyadoo loo qaybinayo 50/50 beam.Shaandhooyinka sii daaya Bandpass (Semrock, Lake Forest, IL, USA) 520/35 ee dheeha cagaaran (AF488) iyo 690/70 ee dheeha cas (Atto647N) ayaa loo isticmaalay baaraha hortiisa.Diodes avalanche (SPAD) oo hal sawir leh (Micro Photon Devices, Bolzano, Italy) ayaa loo adeegsaday wax-sheegayaal ahaan.Xog ururinta iyo falanqaynta labadaba waxaa la sameeyay iyada oo la adeegsanayo software-ka SymphoTime64 ee ganacsi ahaan loo heli karo (PicoQuant GmbH, Berlin, Germany).
Konton microliter oo muunado LLPS ah ayaa lagu dabaqay ceelasha μ-Slide angiogenesis (Ibidi GmbH, Gräfelfing, Germany).Sawirada soo baxay ayaa diirada lagu saaray 20 µm ka sarreeya ceelka hoose ee fogaanta shaqada ee ugu habboon ee dhibcaha la hakiyey iyo ~ 1 µm ee rafts iyo dhibco leh xallinta axial ee ugu yaraan 0.25 µm/pixel iyo dib u dhac 400 µs/pixel.Dooro xogta adiga oo adeegsanaya xadka xoogga ah ee ku salaysan celceliska xoogga calaamada asalka (PBG, celceliska + 2σ) kanaal kasta si kaliya dhibcaha borotiinka dareeraha ah, rafts, ama dhibco loo doorto, sifeynta asal kasta oo suurtagal ah marxaladda kala firdhiyey.Si loo falanqeeyo cimriga nooc kasta (τ) kanaal kasta (cagaaran, "g" ee AF488 iyo casaan, "r" ee Atto647N), waxaan dooranay gobollada xiisaha (ROIs) oo ay ku jiraan dhibco, rafts, ama dhibco (Jaantuska Dheeraadka 1) ).8b) oo ay ka soo saareen iyaga oo ku habboon suuska noloshooda (τD, τR iyo τP ee dhibco, rafts ama dhibco, siday u kala horreeyaan, eeg Sawirka Dheeraadka ah. 8c) ee kanaal kasta iyada oo la adeegsanayo falanqaynta dabada ku habboon iyo qaabka qudhunka ee laba qaybood ka kooban.Celceliska τ laga bilaabo τ .ROIs oo soo saaray sawiro aad u yar oo ku haboon jibbaarada badan ayaa laga saaray falanqaynta.Goynta la isticmaalay waxay ahayd <104 photons oo loogu talagalay rafts iyo dhibco iyo 103 dhibcood.Dhibcuhu waxay leeyihiin marin hoose sababtoo ah way adagtahay in la helo qalooca qudhunka leh ee leh qiime sare oo xoog leh, maadaama dhibcaha ku jira goobta sawirka ay badanaa yar yihiin oo ay ka yar yihiin tirada.ROI-yada leh tirooyinka sawir-qaadista ee ka sarreeya xadka ururinta sawir-qaadista (oo loo dhigay> 500 tirinta/pixel) ayaa sidoo kale la tuuray si loo falanqeeyo.Ku dhig qalooca suuska xoogga leh ee laga helay gobolka xiisaha leh xoojinta 90% ee ugu badnaan (wax yar ka dib xoojinta ugu badan ee suuska) laga bilaabo bilawga nolosha adeegga si loo hubiyo faragelinta ugu yar ee IRF iyadoo la ilaalinayo isku mid ah dhammaan suuska xoojinta habaynta daaqada wakhtiga qaraabada Waxa lagu falanqeeyay 25 ilaa 50 ROI ee rafts iyo dhibco iyo 15-25 ROI ee dhibcaha, sawirada laga soo xushay in ka badan 4 nuqul oo laga duubay ugu yaraan 3 tijaabo oo madax banaan.T-tijaabooyin laba-dabo leh ayaa loo isticmaalay si loo qiimeeyo kala duwanaanshaha tirakoobka ee u dhexeeya noocyada ama inta u dhaxaysa nidaamyada coacervate.Falanqaynta pixel-by-pixel ee inta lagu jiro nolosha (τ), wadarta guud ee cimriga inta lagu jiro garoonka ee kanaal kasta waa la xisaabiyay waxaana la sameeyay qiyaas 2/3-qayb ka kooban qaabka jibbaarada.Hoos u dhigista nolosha ee pixel kasta ayaa markaa lagu rakibay iyadoo la adeegsanayo qiyam hore loo xisaabiyay, taasoo keentay sawir ku habboon FLIM midab been abuur ah.Kala duwanaanshaha nolosha dabada ku habboon waxay ahayd isku mid dhammaan sawirada isla kanaalka, iyo qudhun kastaa wuxuu soo saaray sawiro ku filan si ay u bixiyaan taam la isku halayn karo.Falanqaynta FRET, pixels waxaa lagu doortay iyada oo la adeegsanayo xadka xoogga hoose ee 100 photons, kaas oo celcelis ahaan ahaa calaamada asalka (FBG) ee 11 photons.Xoogga fluorescence ee kanaal kasta waxaa lagu saxay arrimo sixitaan lagu go'aamiyay: 69 spectral crosstalk α wuxuu ahaa 0.004, kicinta tooska ah β waxay ahayd 0.0305, hufnaanta ogaanshaha γ waxay ahayd 0.517.Waxtarka FRET ee heerka pixel ayaa markaa la xisaabiyaa iyadoo la adeegsanayo isla'egta soo socota:
halka FDD ay tahay xoojinta fluorescence ee lagu arkay kanaalka deeq bixiyaha (cagaaran), FDA waa xoojinta fluorescence ee lagu arkay kanaalka aqbalaha (casaanka) ee hoos yimaada kicinta aan tooska ahayn, iyo FAA waa xoojinta fluorescence ee lagu arkay aqbalaha (casaan) kanaalka hoos yimaada kicinta tooska ah (casaanka). PIE).garaaca garaaca fluorescence waxaa lagu arkay kanaalka).
Ku rid 100 µl ee xalalka falcelinta LLPS oo ay ku jiraan 25 µM monomeric Tau441 (oo leh ama la'aan 25 µM αS) gudaha LLPS kaydinta (ku daray sida kor ku xusan) oo aan xidhnayn 96-ceel microplates oo leh daahan foil-dhesive ah iyo samaynta dhibicda waxaa hubiyay ka dib markii WF microscopy isu dheelitirnaan.10 daqiiqo gudahoodKadib 48 saacadood oo ku-soo-bax ah heerkulka qolka, joogitaanka raafka borotiinka iyo dhibco ayaa la xaqiijiyay.Kadibna si taxadar leh uga saar dareeraha ku dul qulqulaya ceelasha, ka dibna ku dar 50 L oo ah bakhaar kala-baxa (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) oo ku rid 10 min.Isku-duubnaanta milixdu waxay hubisaa in LLPS aysan ku soo noqon doonin PEG haraaga ah awgeed, iyo isu imaatinka borotiinka ee suurtagalka ah ee ay sameeyeen isdhexgalka elektrostatic waa la kala diri doonaa.Qeybta hoose ee ceelka ayaa si taxadar leh loo xoqay iyadoo la adeegsanayo caarada micropipette waxaana xalkii ka soo baxay loo wareejiyay ceel maran.Ka dib markii shaybaarada lagu shubay 50 μM ThT muddo 1 saac ah, joogitaanka baro go'doon ayaa lagu hubiyay microscopy WF.U diyaari fibrillada sonicated αS adigoo ku dhex shubaya 300 µl oo ah 70-µM αS xal PBS leh pH 7.4, sodium azide 0.01% at 37 °C iyo 200 rpm orbital ruxul 7 maalmood.Xalka ayaa markaa la xoojiyay 9600 × g 30 min, pellet ayaa dib loogu celiyay PBS pH 7.4 iyo sonicated (1 min, 50% cycle, 80% amplitude in a Vibra-Cell VC130 sonicator, Sonics, Newton, USA) muunadaha fibril oo leh cabbir isku mid ah oo loo qaybiyey fibrillo yaryar.
Falanqaynta FCS/FCCS iyo ogaanshaha isku-dhac laba-midab ah (TCCD) ayaa lagu sameeyay isla MT200 waqti-xalliyeed fluorescent confocal mikroskoob (Pico-Quant, Berlin, Germany) oo loo adeegsaday FLIM-FRET tijaabooyinka microscopy iyadoo la adeegsanayo habka PIE.Awoodda laysarka ee tijaabooyinkan waxaa lagu daray 6.0 µW (481 nm) iyo 6.2 µW (637 nm).Isku darka awoodaha laysarka ayaa loo doortay si ay u soo saaraan dhalaal la mid ah lammaanaha fluorophores ee la isticmaalay iyadoo la gaarayo heerar tirsi oo wanaagsan lagana fogaado sawir-qaadista iyo saturation.Xog ururinta iyo falanqaynta labadaba waxaa la sameeyay iyada oo la adeegsanayo SymphoTime64 nooca 2.3 software ee ganacsi ahaan loo heli karo (PicoQuant, Berlin, Germany).
Tusaalooyinka isku-darka αS/Tau go'doonsan ee la helay iyadoo la isticmaalayo LLPS ayaa lagu qasi karaa si gooni ah u-fiirsashada monomolecular ee ku habboon (sida caadiga ah 1:500 qasi, maadaama isku-darka horeba ay ugu jiraan uruurin hoose marka laga sooco muunadaha coacervate).Muunado ayaa si toos ah loogu dabaqay daboolka daboolka (Corning, USA) oo lagu sii dahaaray xal BSA ah oo lagu uruurinayo 1 mg/mL.
Falanqaynta PIE-smFRET ee kanaalada cagaarka iyo casaanka, heerka xoojinta hoose ee 25 photons ayaa lagu dabaqay si loo shaandheeyo calaamadaha xoogga yar ee ay sababaan dhacdooyinka monomeric (xusuusnow in monomers ay ka badan yihiin muunado la isku daray marka la barbar dhigo isku geynta go'doonsan).Xaddigan waxa loo xisaabiyay shan jeer celceliska xoojinta monomeric αS ee laga helay falanqaynta muunadaha monomer saafiga ah si gaar ahaan loogu xusho isu geynta falanqaynta.Wareegga wadista PIE, oo ay weheliso helitaanka xogta TSCPC, ayaa awood u siisay codsiga shaandhaynta miisaanka nolosha oo gacan ka geysata baabi'inta asalka iyo isdhaafsiga muuqaalka.Xoojinta dabka ee la xushay iyadoo la isticmaalayo marinada sare waa la saxay iyadoo la isticmaalayo celceliska calaamada asalka ee lagu go'aamiyay histogaraamyada dhacdada iyo xoojinta/bin ee muunado kayd-kaliya.Burburka la xidhiidha isu geynta waaweyn waxay caadi ahaan galaan dhawr haamood oo xidhiidh ah wakhtiga raadraaca (oo loo dhigay 1 ms).Xaaladahan, waxaa la doortay biin awoodda ugu badan leh.FRET iyo falanqaynta stoichiometric, aragti ahaan la go'aamiyay gamma factor γ (0.517) ayaa la isticmaalay.Tabarucyada isdhaafsiga tooska ah iyo wax ku biirinta kicinta tooska ah ayaa ah mid dayacan (tijaab ahaan loo go'aamiyay) awoodda laysarka kicinta ee la isticmaalo.Waxtarka iyo stoichiometry ee FRET ee qaraxa waxaa loo xisaabiyaa sida soo socota.