347 12.7 * 1.24mm Ityhubhu yentsimbi engenastainless, imolekyuli ye-synchronous electrostatic condensation kunye nokudityaniswa kwe-α-synuclein kunye ne-tau

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347 iSicaciso sombhobho wentsimbi engatyiwayo

347 12.7 * 1.24mm steel stainless coiled ityhubhu

Ngaphandle kweDiameter: 6.00 mm OD ukuya kuthi ga kwi-914.4 mm OD, ubukhulu ukuya kuma-24” NB ekhoyo Ex-stock, OD Size Steel Tubes ekhoyo Ex-stock

I-SS 347 Uluhlu lokutyeba kwemibhobho: 0.3mm - 50 mm, SCH 5, SCH10, SCH 40, SCH 80, SCH 80S, SCH 160, SCH XXS, SCH XS
I-WT: SCH5S, SCH10S, SCH40S, SCH80S, SCH160S, njl. (0.5-12mm) Okanye ubungakanani obungekho rhoqo ukuba bulungelelaniswe njengoko kufuneka

Uhlobo: SS 347 Imibhobho engenamthungo |SS 347 ERW Imibhobho |SS 347 Welded Imibhobho |SS 347 Imibhobho eyenziweyo |Imibhobho ye-SS 347 ye-CDW, iMibhobho ye-LSAW / i-welded-umthungo / iphinde yenziwe

Ifom: SS 347 Round Pipes / Tubes, SS 347 Square Pipes / Tubes, SS 347 Rectangular Pipe / Tubes, SS 347 Coiled Tubes, SS 347 "U" Shape, SS 347 Pan Cake Coils, SS 347 Hydraulic Tubes

Ubude: I-Single Random, I-Double Random & Isiphelo sobude obufunekayo: Isiphelo esicacileyo, isiphelo esiBeveled, sinyathelwe

Ukuphela koKhuseleko: IiCaps zePlastiki |Ngaphandle kokugqiba: 2B, No.4, No.1, No.8 Mirror Finish for Stainless Steel Pipes, Gqiba ngokweeMfuno zomthengi

Imeko yokuhanjiswa: I-Aneiled kwaye iPickled, ilungisiwe, i-Anneal ekhanyayo, i-Cold Drawn

Ukuhlolwa, iiNgxelo zoVavanyo: IziQinisekiso zoVavanyo lweMill, i-EN 10204 3.1, iiNgxelo zeMichiza, iiNgxelo zeMechanical, iiNgxelo zoVavanyo lwe-PMI, iiNgxelo zokuHlola eziBonwayo, iiNgxelo zokuHlola zeQela lesithathu, iiNgxelo zeLab ezivunyiweyo ze-NABL, iNgxelo yoVavanyo elonakalisayo, iiNgxelo zoVavanyo ezingonakalisi.

Ukupakishwa: Kupakishwe kwiiBhokisi zoMthi, iiBagi zePlastiki, iintambo zentsimbi ezidityanisiweyo, okanye ngokwezicelo zabathengi

Ezizodwa: Ubungakanani kunye neNgcaciso ngaphandle kwalapha ngasentla zinokwenziwa xa ziceliwe

SS 347 Ubungakanani boMbhobho Uluhlu: 1/2 intshi NB, OD ukuya 24 intshi

I-ASTM A312 347: Umbhobho we-austenitic ongenamthungo kunye nomthungo othe tye olungiselelwe ubushushu obuphezulu kunye nenkonzo edlekayo jikelele.Intsimbi yokuzalisa ayivumelekanga ngexesha le-welding.

I-ASTM A358 347: Ukudibanisa kombane umbhobho odityanisiweyo we-austenitic wenkonzo edlekayo kunye/okanye yobushushu obuphezulu.Ngokuqhelekileyo umbhobho kuphela ukuya kwi-intshi ezi-8 uveliswa kolu nkcukacha.Ukongezwa kwesinyithi sokuzalisa kuvunyelwe ngexesha le-welding.

I-ASTM A790 347: Umbhobho odityanisiweyo we-ferritic/austenitic (duplex) ongenamthungo kunye nomthungo othe tye olungiselelwe inkonzo etshabalalisayo jikelele, ngogxininiso olukhethekileyo lokuxhathisa ukuqhekeka komhlwa.

I-ASTM A409 347: Umthungo othe tye okanye umthungo wokudibanisa umbane odityaniswe nedayamitha enkulu iaustenitic yodonga olukhanyayo umbhobho ngobukhulu obuyi-14” ukuya kuma-30” eneendonga iSch5S kunye neSch 10S yokudleka kunye/okanye ukuphakama.

I-ASTM A376 347: Umbhobho we-austenitic ongenamthungo wokusetyenziswa kobushushu obuphezulu.

I-ASTM A813 347: Umthungo omnye, umbhobho we-austenitic odityanisiweyo omnye-okanye kabini wobushushu obuphezulu kunye nezicelo ezidlekayo.

I-ASTM A814 347: Umbhobho we-austenitic odityanisiweyo obandayo wobushushu obuphezulu kunye nenkonzo edlekayo jikelele.

I-347H yeMibhobho yeNtsimbi engagqwali Ukuqulunqwa kweMichiza

iStainless Steel 347H iiPropati zoomatshini boMbhobho

iStainless Steel 347H Imibhobho yePhysical Properties

AmaBanga aLinganayo kwi-347H yePipe yeNsimbi eNgatyiwayo

I-Amyloid alpha-synuclein (αS) i-aggregation luphawu lwesifo sikaParkinson kunye nezinye ii-synucleinopathies.Kungekudala, iprotheni ye-tau edla ngokuhambelana nesifo se-Alzheimer idibene ne-αS pathology kwaye ifunyenwe i-co-localize kwi-αS-rich inclusions, nangona indlela ye-molecular of coaggregation yeeprotheni ezimbini ihlala ingacacanga.Sinika ingxelo apha ukuba isigaba se-αS sahlulahlulwe kwi-condensates engamanzi nge-electrostatic complex condensation ene-polypeptides ene-positive charge efana ne-tau.Ngokuxhomekeke kwi-affinity ye-αS ye-polycations kunye nesantya sokuchithwa kwe-valence yenethiwekhi ye-coagulation, ama-clots ahamba ngokukhawuleza i-gelation okanye i-coalescence elandelwa yi-slow amyloid aggregation.Ngokudibanisa i-suite yeendlela eziphambili ze-biophysical, sakwazi ukubonakalisa ukuhlukana kwesigaba se-liquid αS / Tau kunye nokuchonga izinto eziphambili ezikhokelela ekubunjweni kwee-aggregates ezidibeneyo eziqulethe zombini iiprotheni kwi-protein condensate yolwelo.
Ukongeza kwiikhompatimenti zemembrane, ukwahlukana kwendawo kwiiseli nako kungaphunyezwa ngokwenziwa kwemizimba exineneyo eneprotheyini, efana nolwelo efana ne-biomolecular condensates okanye amathontsi, ngenkqubo eyaziwa ngokuba yi-liquid phase separation (LLPS).La mathontsi akhiwa ngonxibelelwano lwexeshana, ngokuqhelekileyo phakathi kweeprotheyini okanye iiprotheyini kunye ne-RNA, kwaye asebenze imisebenzi eyahlukeneyo phantse kuzo zonke iinkqubo eziphilayo.Inani elikhulu leeprotheyini ezinomthamo we-LLP zibonisa ukulandelelana kobunzima obuphantsi obuphazamiseka kakhulu kwindalo kunye nokwakhiwa kwe-biomolecular condensates3,4,5.Uphononongo oluninzi lovavanyo lubonakalise ukubhetyebhetye, okuhlala kungacwangciswanga, kunye nendalo eninzi yeeproteni ezenza ezi condensates zifana nolwelo, nangona kuncinci okwaziwayo malunga nezithinteli ezithile zeemolekyuli ezilawula ukukhula kunye nokuvuthwa kwezi condensates ukuba ziqine ngakumbi. urhulumente..
Idatha entsha ixhasa i-hypothesis yokuba i-LLPS eqhutywa yiprotheyini egwenxa kunye nokuguqulwa kwamathontsi kwizakhiwo eziqinileyo kunokuba ziindlela ezifanelekileyo zeselula ezikhokelela ekwakhiweni kweeaggregates eziyityhefu ezinganyibilikiyo ezidla ngokuba ziimpawu zezifo eziwohlokayo.Iiproteni ezininzi ezinxulunyaniswa ne-LLPS-ezinxulunyaniswa ne-intrinsically disordered (IDPs), zihlala zihlawuliswa kakhulu kwaye zibhetyebhetye, kudala zinxulunyaniswa ne-neurodegeneration ngenkqubo ye-amyloid aggregation.Ngokukodwa, i-IDP ye-biomolecular i-condensates efana ne-FUS7 okanye i-TDP-438 okanye iiprotheyini ezinemimandla emikhulu ephantsi efana ne-hnRNPA19 iboniswe ukuba iguge ibe yi-gel-like okanye iifom eziqinileyo ngenkqubo ebizwa ngokuba yi-fluidization.ikhompawundi.kwinguqu yesigaba esiqinileyo (LSPT) njengomsebenzi wexesha okanye ekuphenduleni ukuguqulwa okuthile emva kokuguqulelwa okanye ukuguqulwa kwe-pathologically ebalulekileyo1,7.
Enye i-IDP ehambelana ne-LLPS kwi-vivo yi-Tau, i-microtubule-ehambelana ne-protein ephazamisayo ene-amyloid aggregation iye yabandakanyeka kwi-Alzheimer's disease10 kodwa iphinde ifakwe kutshanje kwi-Parkinson's disease (PD) kunye nezinye i-synaptic nuclear proteinopathies 11, 12, 13 zihlobene.I-Tau ibonakaliswe ngokuzenzekela yokwahlukana nesisombululo/i-cytoplasm ngenxa yokusebenzisana okuncomekayo kwe-electrostatic14, okukhokelele ekwenziweni kwamathontsi e-tau-etyetyiswe aziwa ngokuba zii-electrostatic coacervates.Kwakhona kuye kwaphawulwa ukuba olu hlobo lwentsebenziswano olungeyodwa luyimpembelelo emva kwee-condensates ezininzi ze-biomolecular kwindalo15.Kwimeko yeprotein ye-tau, i-electrostatic aggregation inokwakheka ngokudityaniswa okulula, apho imimandla echasayo yeprotheyini ibangela inkqubo yokuqhekeka, okanye ngokudityaniswa okuntsonkothileyo ngokusebenzisana neepolymers ezihlawuliswa kakubi njenge-RNA.
Kungekudala, i-α-synuclein (αS), i-IDP ye-amyloid echaphazelekayo kwi-PD kunye nezinye izifo ze-neurodeergenerative ezihlangeneyo ezibizwa ngokuba yi-synucleinopathy17,18, ibonakaliswe kwiimodeli zeselula kunye nezilwanyana19,20 ezigxininiswe kwiiprotheyini ezidibanisa nokuziphatha okufana nolwelo.Uphononongo lwe-in vitro lubonise ukuba i-αS ifumana i-LLPS ngokudibanisa ngokulula ngokusebenzisa i-hydrophobic kakhulu, nangona le nkqubo idinga ugxininiso lweeprotheyini eziphezulu kunye namaxesha amade atypically incubation19,21.Nokuba i-αS-equlathe i-condensates ebonwe kwi-vivo yenziwa yile okanye ngezinye iinkqubo ze-LLPS ihlala ingumba ongundoqo ongasonjululwanga.Ngokufanayo, nangona i-αS i-amyloid aggregation iye yabonwa kwi-neurons kwi-PD kunye nezinye i-synucleinopathies, indlela echanekileyo eyenziwa ngayo i-αS ye-intracellular amyloid aggregation ihlala ingacacanga, njengoko ukugqithiswa kwale proteni kungabonakali ukuba kubangele le nkqubo ngokwayo.Umonakalo owongezelelweyo weselula usoloko ufuneka, ebonisa ukuba iindawo ezithile zeselula okanye i-microenvironments zifunekayo ukuze kuhlaziywe iindibano ze-intracellular αS amyloid.Enye indawo yeselula ethandwa kakhulu kwi-aggregation ingaba yi-protein condensates 23.
Okubangel 'umdla kukuba, i-αS kunye ne-tau zifunyenwe ngokubambisana kwi-co-localise kwimpawu ezibandakanya izifo kubantu abane-Parkinson's disease kunye nezinye i-synucleinopathies 24,25 kunye novavanyo luye lwabika ubudlelwane be-synergistic pathological phakathi kweeprotheni ezimbini ze-26,27 ezibonisa ubudlelwane obunokwenzeka phakathi kwe-aggregation αS kunye tau kwizifo ze-neurodeergenerative.isigulo.I-αS kunye ne-tau zifunyenwe ukusebenzisana kunye nokukhuthaza ukuhlanganiswa komnye nomnye kwi-vitro kunye ne-vivo 28,29 kunye ne-heterogeneous aggregates eyakhiwe kwezi ziprotheni ezimbini ziye zabonwa kwiingqondo zezigulane ezine-synucleinopathies 30.Nangona kunjalo, kuncinci okwaziwayo malunga nesiseko semolekyuli yentsebenziswano phakathi kwe-αS kunye ne-tau kunye nendlela yokudibanisa kwayo.I-αS kuxelwe ukuba isebenzisane ne-tau ngomtsalane we-electrostatic phakathi kwengingqi ye-C-terminal echajiwe kakhulu kakubi ye-αS kunye nommandla otyebileyo we-proline we-tau, ekwatyetyiswe ngeentsalela ezinentlawulo entle.
Kolu phononongo, sibonisa ukuba i-αS iyakwazi ukwahlukana ibe ngamathontsi nge-electrostatic complex condensation phambi kweprotein ye-tau, ngokungafaniyo nokusebenzelana kwayo nezinye iipolypeptides ezichajiwe kakuhle ezifana ne-poly-L-lysine (pLK), kwaye kule nkqubo.I-αS isebenza njenge-molecule ye-scaffold ye-droplet network.Siye sachonga ukungafani okubonakalayo kwinkqubo yokuvuthwa kwe-electrostatic αS coacervates, ehambelana nokungafani kwi-valency kunye namandla okusebenzisana kweeprotheni ezibandakanyekayo kwinethiwekhi ye-coacervate.Okubangela umdla kukuba, siye sabona ukudityaniswa kwe-αS kunye ne-tau amyloid iiprotheyini kwii-coacervates zolwelo oluhlala ixesha elide kwaye sichonge ezinye izinto eziphambili ezikhokelela ekudityanisweni kwezi proteni zimbini kwi-coacervates enjalo.Apha sichaza ngokweenkcukacha le nkqubo, eyona ndlela inokwenzeka yemolekyuli ephantsi kwe-colocalization yeeprotheni ezimbini kwi-inclusions ethile yesifo.
I-αS inomsila omkhulu we-anionic C-terminal kwi-pH engathathi hlangothi (umzobo 1a), kwaye sicinge ukuba inokungena kwi-LLPS nge-condensation ye-electrostatic complexes kunye ne-polycationic disordered polypeptide molecules.Sasebenzisa i-polymeric eyi-100-residue poly-L-lysine (pLK) njengemodeli yokuqala ye-molecule ngenxa yendalo yayo yepolymeric efanelekileyo kunye ne-disordered kwi-pH engathathi hlangothi 32. Okokuqala, siqinisekisile ukuba i-pLK isebenzisana ne-Ct domain ye-αS nge-Solution NMR spectroscopy. (Umzobo 1b) usebenzisa i-13C / 15N-ebhalwe αS phambi kokunyuka kwe-αS: pLK molar ratios.Ukusebenzisana kwe-pLK kunye ne-Ct-domain ye-αS ibonakalisa ekuphazamisekeni kokutshintshwa kweekhemikhali kunye nokuncipha kwesantya esiphezulu kule ndawo yeprotheni.Okubangela umdla kukuba, xa sixuba i-αS kunye ne-pLK kuxinzelelo lwe-αS malunga.5–25 µM kubukho be-polyethylene glycol (5–15% PEG-8) (isithinteli se-LLPS esiqhelekileyo: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) ngoko nangoko siye saphumela kwinkalo ebanzi yokwenziwa kweprotheyini. .amathontsi aye ajongwa kusetyenziswa i-fluorescence (WF) kunye ne-bright-field (BF) microscopy (Fig. 1c).I-1-5 µm amaconsi aqulethe i-αS egxininisiweyo (eyongeziweyo 1 µM AlexaFluor488-ebhalwe αS, AF488-αS), iipropati zabo ze-electrostatic zinokuthathwa ekuchaseni kwazo kwi-10% 1,6-hexanediol (1,6-HD) kunye novakalelo lwayo ukwanda koxinzelelo lwe-NaCl (Umfanekiso 1c).Ubume obufana ne-fluid ye-coacervates ye-αS / pLK electrostatic complex iboniswa ngokukwazi kwabo ukuxubha kwii-milliseconds (Umfanekiso 1d).Ukusebenzisa i-turbidimetry, silinganise ukubunjwa kwamaconsi phantsi kwezi meko, siqinisekisile ubume be-electrostatic yentsebenziswano ephambili ehambelana nokuzinza kwayo (Umfanekiso we-1e), kunye nokuvavanya umphumo wemilinganiselo eyahlukeneyo ye-polymer kwinkqubo ye-LLPS (Fig. 1f).Nangona ukwakheka kwamathontsi kujongwa kuluhlu olubanzi lweereyishini zepolymer, inkqubo ithandeka kakhulu xa i-pLK ingaphezulu kwe-αS.Ii-LLPs nazo ziye zabonwa kusetyenziswa i-agent yokususa ikhemikhali eyahlukileyo i-dextran-70 (70 kDa) okanye kusetyenziswa iifomathi ezahlukeneyo zesampulu, kubandakanywa i-glass slide drops, i-microplate imithombo yezinto ezahlukeneyo, i-Eppendorf okanye i-quartz capillaries.
ukumelwa okucwangcisiweyo kwemimandla yeprotheyini eyahlukeneyo kwi-WT-αS kunye ne-ΔCt-αS eyahlukileyo esetyenziswe kolu phononongo.I-domain ye-amphipathic N-terminal, i-hydrophobic amyloid-forming (NAC) ummandla, kunye ne-C-terminal domain ehlawuliswa kakubi iboniswe nge-blue, i-orange, kunye nebomvu, ngokulandelanayo.INtlawulelo yeNtlawulo ngeNtsalela (NCPR) yemephu ye-WT-αS iyaboniswa.b Uhlalutyo lwe-NMR lwentsebenziswano ye-αS/pLK ngokungabikho kwama-macromolecular clumps.Njengoko i-pLK i-concentration iyanda (i-αS: pLK i-molar ratios ye-1: 0.5, 1: 1.5, kunye ne-1: 10 iboniswe ngokukhanya okuluhlaza, okuluhlaza, kunye nobumnyama obumnyama, ngokulandelanayo).c Coacervate αS/pLK (molar ratio 1:10) ku-25 µM (1 µM AF488-ilebhile i-αS okanye i-Atto647N-ebhalwe i-pLK yomfanekiso we-WF) kwi-LLPS buffer (phezulu) okanye idityaniswe ne-500 mM NaCl (ezantsi 10 ezantsi okanye emva koko) I-% 1,6-hexanediol (1,6-HD; ezantsi ekunene).Ibar yesikali = 20 µm.d Imifanekiso ye-microscopic emele i-BF ye-droplet fusion ye-αS / pLK (i-molar ratio 1: 10) kwi-concentration ye-25 μM;iintolo zibonisa ukudityaniswa kwamathontsi ngamanye (utolo olubomvu nolutyheli) kwithontsi elitsha (utolo oluorenji) phakathi kwama-200 ms).Ibar yesikali = 20 µm.e Ukusasazwa kokukhanya (kwi-350 nm) αS/pLK aggregation kwi-LLPS buffer ngaphambi nangemva kokongezwa kwe-500 mM NaCl okanye i-10% 1,6-HD kwi-25 µM αS (N = 3 isampula yokuphindaphinda, intsingiselo kunye nokuphambuka okuqhelekileyo kubonisiwe).f umfanekiso we-BF (phezulu) kunye nohlalutyo lokusabalalisa ukukhanya (kwi-350 nm, ezantsi) ye-αS / pLK aggregation kwi-25 μM αS ngokunyuka kwe-αS: pLK i-molar ratio (N = 3 isampula yokuphindaphinda, intsingiselo kunye nokuphambuka okuqhelekileyo kubonisiwe).Ibar yesikali = 10 µm.Ibar yesikali kumfanekiso omnye ibonisa isikali sayo yonke imifanekiso kwiphaneli enye.Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Ngokusekwe kwimigqaliselo yethu ye-αS/pLK ye-electrostatic condensation eyinkimbinkimbi kunye nemigqaliselo yangaphambili ye-αS njengemolekyuli yomxhasi we-tau/RNA condensate ngokusebenzisana ngokuthe ngqo ne-tau31, siqikelele ukuba i-αS kunye ne-tau inokwahlula-hlula kunye ne-solvent xa ingekho i-RNA. ukujiya.ngokusebenzisa ii-electrostatic complexes, kunye ne-αS yiprotheni ye-scaffold kwi-αS/Tau coacervates (jonga ukuhanjiswa kwentlawulo ye-tau kuMfanekiso 2e).Siye saqaphela ukuba xa i-10 μM αS kunye ne-10 μM Tau441 (equlethe i-1 μM AF488-αS kunye ne-1 μM Atto647N-Tau, ngokulandelanayo) zixutywe kunye kwi-buffer ye-LLPS, zenza lula i-protein aggregates equlethe zombini iiprotheni, njengoko kubonwa yi-WF microscopy.(Umfanekiso 2a).I-colocalization yeeprotheni ezimbini kumaconsi kwaqinisekiswa yi-confocal (CF) microscopy (i-Supplementary Fig. 1a).Ukuziphatha okufanayo kwabonwa xa i-dextran-70 isetyenziswe njenge-aggregation agent (i-Supplementary Fig. 1c).Ukusebenzisa i-PEG ebhalwe nge-FITC okanye i-dextran, sifumene ukuba zombini ii-agent ezixutywayo zasasazwa ngokulinganayo kwiisampuli, zingabonakali ulwahlulo okanye ukudibanisa (i-Supplementary Fig. 1d).Kunoko, iphakamisa ukuba kule nkqubo bakhuthaza ukuhlukana kwesigaba ngokusebenzisa imiphumo yokuxinwa kwe-macromolecular, ekubeni i-PEG iyindawo ekhethiweyo yokuxinana, njengoko kubonwa kwezinye iinkqubo ze-LLP33,34.La maconsi aneprotheyini ane-protein ayenovelwano kwi-NaCl (1 M) kodwa kungekhona kwi-1,6-HD (10% v / v), eqinisekisa iimpawu zabo ze-electrostatic (i-Supplementary Fig. 2a, b).Ukuziphatha kwabo kwe-fluid kwaqinisekiswa ngokujonga iziganeko ze-droplet zokudibanisa i-millisecond usebenzisa i-BF microscopy (Umfanekiso we-2b).
imifanekiso ye-Confocal (CF) ye-microscopy ye-αS/Tau441 i-coacervates kwi-LLPS buffer (i-10 μM yeprotheni nganye, i-0.5 μM ye-AF488-ebhalwe ngu-αS kunye ne-Atto647N-ebhalwe Tau441).b Imifanekiso emeleyo yokuphazamiseka kokuphazamiseka (DIC) yemifanekiso ye-αS/Tau441 yeziganeko zokudibanisa i-droplet (10 μM kwiprotheni nganye).c Umzobo wesigaba osekelwe kukusasaza ukukhanya (ku-350 nm) ye-Tau441 LLPS (0–15 µM) ngokungabikho (ekhohlo) okanye ubukho (ekunene) be-50 µM αS.Imibala efudumeleyo ibonisa ukusabalalisa ngakumbi.d Ukusasazwa kokukhanya kweesampuli ze-αS/Tau441 LLPS ngokunyuka koxinzelelo lwe-αS (i-Tau441 kwi-5 µM, N = 2-3 isampula yokuphindaphinda njengoko kubonisiwe).e Ukubonakaliswa kweSchematic kwezinye iiprotheyini ze-tau kunye nemimandla eyahlukeneyo yeprotheyini esetyenziswe kolu phando: i-N-terminal domain ehlawuliswa kakubi (ibomvu), i-proline-rich region (blue), i-microtubule-binding domain (MTBD, igxininiswe kwi-orange), kunye I-amyloid-forming pair spiral.imimandla yefilament (PHF) ebekwe ngaphakathi kweMTBD (grey).I-Net Charge Per Residue (NCPR) imephu ye-Tau441 iyaboniswa.f Usebenzisa i-1 µM AF488-ebhalwe αS kunye ne-Atto647N-ebhalwe ΔNt-, usebenzisa i-1 µM AF488 ebhalwe αS okanye ΔCt-αS kubukho be-ΔNt-Tau (phezulu, 10 µM ngeprotheni) okanye i-K18 (ezantsi µM5 kwiprotheni nganye, ngezantsi kwe-M5 ) ) iimicrographs zeWF ezicutshiweyo kwiLLPS okanye kwi-K18 buffer.Iibar zesikali kumfanekiso omnye zimele isikali sayo yonke imifanekiso kwindawo yolawulo enye (20 µm yeephaneli a, b kunye nof).Idatha ekrwada yeephaneli ze-c kunye no-d zinikezelwa njengeefayili zedatha ekrwada.
Ukuvavanya indima ye-αS kule nkqubo ye-LLPS, saqala saphanda umphumo we-αS kwi-droplet stability nge-nephelometry usebenzisa i-concentrations eyandayo ye-NaCl (Fig. 2c).Ukuphakama koxinzelelo lwetyuwa kwiisampulu eziqulethe i-αS, kokukhona kuphakamileyo kumaxabiso okusasaza ukukhanya (kwi-350 nm), ebonisa indima yokuzinzisa ye-αS kule nkqubo ye-LLPS.Umphumo ofanayo unokubonwa ngokunyusa ugxininiso lwe-αS (kwaye ngoko ke i-αS: i-Tau441 ratio) ukuya kwi-approx.Ukunyuka kwe-10 ngokumalunga nokugxininiswa kwe-tau (5 µM) (Umfanekiso we-2d).Ukubonisa ukuba i-αS yiprotheni ye-scaffold kwi-coacervates, sagqiba ekubeni siphande ukuziphatha kwe-LLPS-iphazamise i-Tau mutant, engenawo ummandla we-N-terminal ohlawulwe kakubi (iintsalela ze-1-150, jonga i-Fig. 2e) ebizwa ngokuba yi-ΔNt-Tau.I-WF microscopy kunye ne-nephelometry yaqinisekisa ukuba i-ΔNt-Tau ngokwayo ayizange ingene kwi-LLPS (Fig. 2f kunye neFig. ibuyiselwe ngoxinaniso lwamathontsi kufutshane noxinano lwamathontsi lwezisombululo ezinobungakanani obupheleleyo be-Tau kunye ne-αS phantsi kweemeko ezifanayo kunye nokugxilwa kweprotheyini.Le nkqubo inokujongwa phantsi kweemeko zokuxinana kwe-macromolecular ephantsi (i-Supplementary Fig. 2c).Indima yommandla we-C-terminal αS, kodwa hayi ubude bayo bonke, kwinkqubo ye-LLPS yabonakaliswa ngokuthintela ukubunjwa kwamathontsi kusetyenziswa i-C-terminal ecuthiweyo ye-αS eyahlukileyo eswele iintsalela 104–140 (Umfanekiso 1a) we (ΔCt- αS) iprotheni (Umfanekiso 2f kunye neFig. 2d).I-colocalization ye-αS kunye ne-ΔNt-Tau yaqinisekiswa yi-confocal fluorescence microscopy (I-Fig. 1b eyoNgezelelweyo).
Ukuvavanya ngakumbi indlela ye-LLPS phakathi kwe-Tau441 kunye ne-αS, ukwahluka kwe-Tau okongeziweyo kusetyenzisiwe, oku kukuthi iqhekeza elidityanisiweyo le-helical filament core (PHF) kwidomeyini yokubopha i-microtubule (MTBD), ethi ukuba iqulethe imimandla ephinda-phindwayo eneempawu, eyaziwayo eyaziwayo. njengeqhekeza le-K18 (bona umfanekiso 2e).Kutshanje kuye kwaxelwa ukuba i-αS ibophelela ngokukhethekileyo kwiprotheyini ye-tau efumaneka kwi-proline-rich domain ngokulandelelana okuhamba phambili kwe-microtubule-binding domain.Nangona kunjalo, ummandla we-PHF ucebile kwiintsalela ezihlawulwe kakuhle (jonga uMfanekiso 2e), ngakumbi i-lysine (i-15% intsalela), eyasishukumisela ukuba sivavanye ukuba lo mmandla nawo unegalelo kwi-condensation ye-αS/Tau complex.Siye saqaphela ukuba i-K18 iyodwa ayinakubangela i-LLPS ekugxininiseni ukuya kuthi ga kwi-100 μM phantsi kweemeko ezivavanyiweyo (i-LLPS buffer ene-15% PEG okanye i-20% dextran) (Umfanekiso 2f).Nangona kunjalo, xa songeza i-50 µM αS kwi-50 µM K18, ukubunjwa ngokukhawuleza kwamaconsi eprotheyini aqukethe i-K18 kunye ne-αS yabonwa nge-nephelometry (i-Supplementary Fig. 2d) kunye ne-WF microscopy (Fig. 2f).Njengoko bekulindelekile, i-ΔCt-αS ayikwazanga ukubuyisela ukuziphatha kwe-LLPS ye-K18 (Umfanekiso 2f).Siyaqaphela ukuba udibaniso lwe-αS/K18 lufuna ugxininiso lweprotheyini ephezulu kancinane ukubangela i-LLPS xa kuthelekiswa ne-αS/ΔNt-Tau okanye i-αS/Tau441, ezinye izinto zilingana.Oku kuhambelana nokusebenzisana okunamandla kwingingqi ye-αS C-terminal kunye ne-proline-rich Tau domain xa kuthelekiswa ne-microtubule-binding domain, njengoko kuchazwe ngaphambili i-31.
Ngenxa yokuba i-ΔNt-Tau ayikwazi ukwenza i-LLPS xa ingekho i-αS, sikhethe oku kwahluka kwe-Tau njengomzekelo wophawu lwe-αS/Tau LLPS inikwe ubulula bayo kwiinkqubo zeLLPS ezinobude obugcweleyo be-Tau (isotype, Tau441/Tau441).kunye neenkqubo ezidibeneyo (i-heterotypic, αS/Tau441) ezidibanisayo.Siqhathanise idigri ye-αS aggregation (njengenxalenye yeprotheyini yesigaba esincitshisiweyo, i-fαS, c) kwiinkqubo ze-αS / Tau kunye ne-αS / ΔNt-Tau nge-centrifugation kunye nesigaba esasasazwa uhlalutyo lwe-SDS-PAGE (jonga i-2e), ifumene amaxabiso afanayo kakhulu. kuzo zonke iiprotheyini ekugxininiseni okufanayo.Ngokukodwa, sifumene i-faS, c 84 ± 2% kunye ne-79 ± 7% ye-αS / Tau kunye ne-αS / ΔNt-Tau, ngokulandelanayo, iphakamisa ukuba i-heterotypic interaction phakathi kwe-αS kunye ne-tau iphezulu kunentsebenziswano phakathi kwee-molecule ze-tau.phakathi.
Ukusebenzisana kunye neepolycations ezahlukeneyo kunye nomphumo wenkqubo ye-condensation kwi-αS kinetics yaqale yafundwa ngokubuyiswa kwe-fluorescence emva kwe-photobleaching (FRAP) indlela.Sivavanye i-αS/Tau441, αS/ΔNt-Tau kunye ne-αS/pLK i-coacervates (i-100 μM αS yongezwa nge-2 μM αS AF488-αS kunye ne-100 μM Tau441 okanye i-ΔNt-Tau okanye i-1 mML pLK).Idatha ifunyenwe kwimizuzu yokuqala ye-30 emva kokuxuba amacandelo esampuli.Ukususela kwimifanekiso ye-FRAP emele (umzobo 3a, αS / Tau441 condensation) kunye neengqungquthela zabo zekhosi ezihambelanayo (umzobo 3b, i-Supplementary Fig. 3), kunokubonwa ukuba i-αS kinetics ifana kakhulu ne-Tau441 coacervates.kunye ne-ΔNt-Tau, ekhawuleza kakhulu nge-pLK.I-diffusion coefficients ebalwayo ye-αS ngaphakathi kwe-coacervate ngokwe-FRAP (njengoko ichazwe ngu-Kang et al. 35) yi-D = 0.013 ± 0.009 µm2/s kunye ne-D = 0.026 ± 0.008 µm2/s ye-αS/ΔαS4 kunye ne-αS4 inkqubo ye-αS.pLK, Tau, kunye no-D = 0.18 ± 0.04 µm2/s, ngokulandelelanayo (umzobo 3c).Nangona kunjalo, i-coefficient yokusabalalisa i-αS kwisigaba esisasaziweyo yimiyalelo emininzi yobukhulu obuphezulu kunazo zonke izigaba ezinqamlekileyo, njengoko kunqunywe yi-Fluorescence Correlation Spectroscopy (FCS, bona i-Supplementary Fig. 3) phantsi kweemeko ezifanayo (i-LLPS buffer) kodwa ngokungabikho kwe-polycations ( D = 8 ± 4 µm2/s).Ngoko ke, i-kinetics yokuguqulelwa kwe-αS iyancipha kakhulu kwi-coacervates xa kuthelekiswa neeprotheyini kwisigaba esisasazwayo ngenxa yemiphumo yokuxinana kweemolekyuli, nangona zonke ii-coacervates zigcina iipropati ezinjengolwelo ngexesha leyure yokuqala emva kokubunjwa kwazo, ngokungafaniyo nesigaba se-tau.kinetics ngokukhawuleza kwi pLK condensate.
a–c Uhlalutyo lwe-FRAP lwe-αS dynamics (2% AF488-ebhalwe αS) kwii-electrostatic coacervates.Imifanekiso emele i-αS/Tau441 FRAP yeemvavanyo kwi-triplicate iboniswe ku-(a), apho izangqa ezibomvu zibonisa iindawo eziguqulwe umbala.Ibar yesikali yi-5 µm.b I-avareji yeegophe ze-FRAP kunye (c) ne-diffusion coefficients (D) ye-5–6 (N) yamathontsi ahlukeneyo asuka kwimifuniselo emithathu esebenzisa i-100 µM αS kunye nokugxilwa kwe-equimolar ye-Tau441 (bomvu) okanye i-ΔNt-Tau (ebhlowu) okanye i-pLK (eluhlaza) ngamaxesha alishumi kuxinzelelo lweLLPS.Ukutenxa okusemgangathweni kwegophe le-FRAP kuboniswe kumbala ofakwe umbala.Ukuthelekisa, i-diffusion coefficient αS kwisigaba esisasaziweyo yamiselwa ngokuphindwe kathathu kusetyenziswa i-fluorescence correlation spectroscopy (FCS) (jonga iSazobe esoNgezelelweyo 3 kunye neendlela zolwazi oluthe vetshe).d Umboniso oqhubekayo we-X-band EPR we-100 μM TEMPOL-122-αS kwi-LLPS buffer ngaphandle kwepolycation (emnyama) okanye phambi kwe-100 μM Tau441 (ebomvu) okanye i-ΔNt-Tau (ebhlowu) okanye i-1 mM pLK (eluhlaza).I-inset ibonisa umbono onyusiweyo wemigca yentsimi eyomeleleyo apho utshintsho oluphawulekayo lwenzeka khona.e Iigophe ezibophelelayo ze-50 μM TEMPOL-122-αS kunye neepolycations ezahlukeneyo ngokungabikho kweLLPS (akukho PEG).Ukunciphisa i-amplitude ye-band III xa kuthelekiswa ne-band II (IIII / III) ye-spectrum ye-EPR eqhelekileyo iboniswa ukwandisa i-molar ratios ye-Tau441 (ebomvu), i-ΔNt-Tau (eluhlaza okwesibhakabhaka) kunye ne-pLK (eluhlaza).Imigca enemibala ibonisa ukufaneleka kwidatha usebenzisa imodeli ebophayo erhabaxa eneendawo ezizibophelelayo ezilinganayo nezizimeleyo kwigophe ngalinye.Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Njengomncedi, siphande amandla e-αS kwii-coacervates ezahlukeneyo zisebenzisa i-directed spin labeling (SDSL) kunye ne-electron paramagnetic resonance eqhubekayo (CW-EPR).Le ndlela ibonakalise ukuba luncedo kakhulu ekuchazeni ukuguquguquka kunye nokuguquguquka kwe-IDP kunye nesisombululo esiyintsalela esiyinyani36,37,38.Ukuza kuthi ga ngoku, sakha i-cysteine ​​​​intsalela kwi-Cys mutants enye kwaye sasebenzisa i-4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Izinto eziphuma kwi-Maleimide zilebhile.Ngokukodwa, sifake i-TEMPOL probes kwindawo ye-122 okanye i-24 αS (TEMPOL-122-αS kunye ne-TEMPOL-24-αS).Kwimeko yokuqala, sijolise kwindawo ye-C-terminal yeprotheni, ebandakanyeka ekusebenzisaneni kunye neepolycations.Endaweni yoko, indawo ye-24 inokusinika ulwazi malunga nokutshintsha kweeprotheni kwi-condensate.Kuzo zombini iimeko, izibonakaliso ze-EPR ezifunyenwe kwiiprotheni zesigaba esisasazekayo zihambelana ne-nitroxide radicals kwimo ehamba ngokukhawuleza.Emva kokwahlulwa kwesigaba phambi kwe-tau okanye i-pLK (i-100 μM TEMPOL-αS, i-Tau441 okanye i-ΔNt-Tau kumlinganiselo we-1: 1 okanye i-pLK kumlinganiselo we-1: 10), ukunyuka kweqondo eliphezulu elihambelanayo liye labonwa I-spectrum ye-EPR ye-αS.Umgca welahleko wandisa, ubonisa ukunciphisa i-αS reorientation kinetics kumaconsi xa kuthelekiswa neprotheyini kwisigaba sokunciphisa (Umfanekiso we-3d, i-Supplementary Fig. 4a).Olu tshintsho lubonakala ngakumbi kwindawo ye-122. Ngelixa kwindawo ye-24 ubukho be-pLK abuzange buchaphazele i-kinetics ye-probe, kwindawo ye-122 i-spectral line shape yatshintsha kakhulu (i-Supplementary Fig. 4a).Xa sizama ukulinganisa i-spectra kwindawo ye-122 yeenkqubo ezimbini ze-αS / i-polycation zisebenzisa imodeli ye-isotropic (i-Supplementary Figure 5a) esetyenziswa ngokuqhelekileyo ukuchaza i-dynamics ye-spin-ilebheli i-IDP38,39, asikwazanga ukuvuselela i-spectra yovavanyo..Ukulinganisa okucacileyo kwesikhundla se-24 spins ukuchasana (i-Supplementary Fig. 5a).Oku kuphakamisa ukuba kukho izikhundla ezikhethiweyo kwisithuba sokucwangciswa kwe-spin yengingqi ye-C-terminal ye-αS phambi kweepolycations.Xa kuqwalaselwa iqhezu le-αS kwisigaba esifinyeziweyo phantsi kweemeko ze-EPR zovavanyo (84 ± 2%, 79 ± 7%, kunye ne-47 ± 4% ye-αS/Tau441, αS/ΔNt-Tau, kunye ne-αS/pLK, ngokulandelanayo-jonga i-Supplementary Umzobo we-2e wohlalutyo lwedatha c), kunokubonwa ukuba ukwandiswa okufunyanwe yindlela ye-EPR ikakhulu kubonisa ukusebenzisana kwendawo ye-C-terminal ye-αS kunye neepolycations ezahlukahlukeneyo kwisigaba esincitshisiweyo (utshintsho oluphambili xa usebenzisa i-TEMPOL-122- αS), kwaye hayi iprotein condensation.Ukunyuka kwe-microviscosity kubonwa kwi-probe.Njengoko kulindeleke, i-EPR spectrum yeprotheni phantsi kweemeko ngaphandle kwe-LLPS yabuyiselwa ngokupheleleyo xa i-1 M NaCl yongezwa kumxube (i-Supplementary Fig. 4b).Ngokubanzi, idatha yethu ibonisa ukuba utshintsho olufunyenwe yi-CW-EPR lubonisa ikakhulu ukusebenzisana kwe-C-terminal yengingqi ye-αS kunye ne-polycations eyahlukeneyo kwisigaba esincitshisiweyo, kwaye oku kusebenzisana kubonakala kunamandla kunye ne-pLK kuneTau.
Ukuze sifumane ulwazi olungakumbi lwesakhiwo malunga neeprotheni kwi-coacervate, sagqiba ekubeni sifunde inkqubo ye-LLPS sisebenzisa i-NMR kwisisombululo.Nangona kunjalo, sinokubona kuphela iqhezu le-αS eliseleyo kwisigaba esisasazwayo, esinokubakho ngenxa yokunciphisa amandla eprotheyini ngaphakathi kwe-coacervate kunye nesigaba esixineneyo phantsi kwesisombululo kuhlalutyo lwe-NMR.Xa sihlalutya ubume kunye ne-dynamics yeprotheyini eseleyo kwisigaba esichithwayo sesampuli ye-LLPS usebenzisa i-NMR (i-Supplementary Fig. 5c, d), saqaphela ukuba iprotheni iziphatha phantse ngokufanayo phambi kwe-pLK kunye ne-ΔNt-Tau, zombini ebezikulwakhiwo lwesibini kunye neentshukumo zomqolo weprotheyini, ezityhilwe luvavanyo kutshintsho lwekhemikhali yesibini kunye nokuphumla kwe-R1ρ.Idatha ye-NMR ibonisa ukuba i-C-terminus ye-αS ifumana ilahleko enkulu yokuguquguquka kwe-conformational ngelixa igcina indalo yayo ephazamisekileyo, njengalo lonke ulandelelwano lweprotheni, ngenxa yokusebenzisana kwayo kunye neepolycations.
Ekubeni i-CW-EPR isignali yokwandisa ibonakaliswe kwisigaba esinqamlekileyo se-TEMPOL-122-αS sibonisa ukusebenzisana kweprotheni kunye ne-polycations, senze i-EPR titration ukuvavanya ubudlelwane obubophezelayo be-αS kwiipolycations ezahlukeneyo ngokungabikho kwe-LLPS (akukho qoqo le-LLPS). I-Buffer LLPS), ebonisa ukuba ukusebenzisana kuyafana kwizigaba ezixubileyo kunye nezigxininisiweyo (eziqinisekiswa yidatha yethu, i-Supplementary Fig. 4a kunye ne-Supplementary Fig. 6).Injongo yayikukubona ukuba zonke ii-coacervates, ngaphandle kweepropathi zazo eziqhelekileyo ezifana nolwelo, zibonisa nayiphi na indlela yokuziphatha eyahlukileyo kwinqanaba le-molecular.Njengoko kulindelekile, i-spectrum ye-EPR yandiswe ngokunyuka kwe-polycation concentration, ebonisa ukuhla kwe-molecular flexibility ngenxa yokusebenzisana kweemolekyuli zabo bonke amaqabane asebenzisanayo malunga nokuzaliseka (Umfanekiso we-3e, i-Supplementary Fig. 6).I-pLK ifezekise oku kuzaliseka kumlinganiselo ophantsi we-molar (ipolycation:αS) xa kuthelekiswa ne-ΔNt-Tau kunye ne-Tau441.Ngapha koko, uthelekiso lwedatha kunye nemodeli eqikelelwayo yokubophelela kucingwa ukuba iindawo ezibophelelayo ezifanayo nezizimeleyo zibonise ukuba ukwahlukana okubonakalayo okungaguqukiyo kwe-pLK (~5 μM) lulandelelwano lobungakanani obusezantsi kunolo lweTau441 okanye ΔNt-Tau (~50 μM) ).µM).Nangona olu luqikelelo olurhabaxa, oku kuphakamisa ukuba i-αS inobudlelwane obuphezulu bepolycations ezilula ezinemimandla yentlawulo eqhubekayo.Ukunika lo mahluko kubudlelwane phakathi kwe-αS kunye neepolycations ezahlukeneyo, sicinge ukuba iipropathi zabo zolwelo zingatshintsha ngokwahlukileyo ngokuhamba kwexesha kwaye ngaloo ndlela zihlupheke kwiinkqubo ezahlukeneyo ze-LSPT.
Ukunikezelwa kwendawo enabantu abaninzi kakhulu ngaphakathi kweprotheni ye-coacervate kunye ne-amyloid nature yeprotheni, siye sabona ukuziphatha kwe-coacervate ngokuhamba kwexesha ukuze sibone iinkqubo ezinokwenzeka ze-LSPT.Ukusebenzisa i-BF kunye ne-CF microscopy (Figure 4), siye saqaphela ukuba i-αS/Tau441 idibanisa kumlinganiselo omkhulu kwisisombululo, isenza amathontsi amakhulu adibanisa kwaye amanzise umphezulu ongezantsi kwequla / isilayidi njengamathontsi agcweleyo, njengoko kulindelekile (Fig Supplementary Fig 7d);sibiza ezi zakhiwo ezisezantsi "iiprotein rafts".Ezi zakhiwo zahlala zimanzi njengoko zigcina amandla okudibanisa (i-Supplementary Fig. 7b) kwaye inokubonwa kwiiyure ezininzi emva kokuba i-LLPS iqhutywe (umzobo 4 kunye neFig. 7c).Siye saqaphela ukuba inkqubo yokumanzisa ithandwa kumphezulu we-hydrophilic kunemathiriyeli ye-hydrophobic (Fig. 7a eyoNgezelelweyo), njengoko kulindelekile kwi-electrostatic coacervates eneendleko ezingalungelelananga kwaye ngaloo ndlela amandla aphezulu e-electrostatic surface.Ngokucacileyo, i-αS / ΔNt-Tau i-coalescence kunye ne-rafting yancitshiswa kakhulu, ngelixa i-αS / pLK condensates yancitshiswa kakhulu (umzobo 4).Ngethuba lexesha elifutshane lokufukamela, amaconsi e-αS / pLK akwazi ukudibanisa kunye nokumanzisa umphezulu we-hydrophilic, kodwa le nkqubo yayeka ngokukhawuleza kwaye emva kweeyure ze-5 zokuxilisa, kuphela iziganeko ezinqamlekileyo zokubambisana kwaye akukho manzi abonwayo.– ijeli-drip utshintsho.
Ummeli we-BF (iipaneli ezingwevu) kunye ne-CF (iiphaneli zasekunene, AF488-ezibhalwe αS kuluhlaza) yeesampuli ze-coacervate eziqulathe i-100 µM αS (1% ileyibhile yefluorescent) kwi-LLPS buffer phambi kwe-100 µM Tau441ncencencencencencencencences zemicroscores (phezulu) -I-Tau (embindini) okanye i-1 mM pLK (ezantsi) ngamaxesha ahlukeneyo okufukamela kunye nokuphakama kwe-focal (z, umgama ukusuka ezantsi kweplate kakuhle).Iimvavanyo ziphindwe ngamaxesha e-4-6 ngokuzimeleyo komnye nomnye kunye neziphumo ezifanayo.I-αS/Tau441 i-coacervates imanziswa emva kweeyure ze-24, zenza iirafti ezinkulu kunomfanekiso.Ibar yesikali sayo yonke imifanekiso ngama-20 µm.
Siye sabuza ukuba ingaba amachibi amakhulu eprotheyini afana nolwelo olwenziwe kwi-αS/Tau441 LLPS angakhokelela ekudityanisweni kwe-amyloid yazo naziphi na iiproteni ezifundiweyo.Silandele ukuvuthwa kwamaconsi e-αS / Tau441 ngexesha kunye ne-WF microscopy phantsi kweemeko ezifanayo nalapha ngasentla, kodwa usebenzisa i-1 μM AF488-ebhalwe αS kunye ne-Atto647N-ebhalwe Tau441 (Fig. 5a).Njengoko bekulindelekile, siye sabona iprotein epheleleyo yasekuhlaleni kuyo yonke inkqubo yokuvuthwa.Okubangel 'umdla kukuba, ukusuka ca.Emva kweeyure ze-5, izakhiwo ezinzulu ezingezizo zesetyhula zabonwa ngaphakathi kwee-rafts, esazibiza ngokuba "ngamanqaku", ezinye zazo zenziwe nge-colocalized kunye ne-αS, kwaye ezinye zixutywe kwi-Tau441 (umzobo 5a, iintolo ezimhlophe).La mabala ahlala eqwalaselwe ngaphakathi kwizihlenga ukuya kumlinganiselo omkhulu we-αS/ΔNt-Tau kune-αS/ΔNt-Tau.Kwakungekho mabala ahlukeneyo kumaconsi e-pLK kunye ne-Tau iinkqubo ezingakwaziyo ukudibanisa / ukumanzisa.Ukuvavanya ukuba ingaba la mabala aqulethe i-αS kunye ne-Tau441 zi-amyloid-like aggregates, senze uvavanyo olufanayo sisebenzisa i-CF microscopy apho i-Tau441 ibhalwe nge-Atto647N kunye ne-12.5 μM i-amyloid-specific thioflavin-T (ThT) yongezwa ukusuka ekuqaleni.idayi.Nangona i-ThT-staining ye-αS / Tau441 yamaconsi okanye i-rafts ayizange ibonwe nangemva kwe-24 h ye-incubation (umzobo we-5b, umgca ophezulu-oseleyo amaconsi phezu kweeprotheni), izakhiwo ze-ThT eziqulethe i-Atto647N-Tau441 ngaphakathi kwee-rafts zazibuthathaka kakhulu.oku kuphindaphinda ubungakanani, imilo, kunye nendawo yeendawo ezichazwe ngaphambili (umzobo 5b, imigca ephakathi kunye nesezantsi), ebonisa ukuba amabala angahambelana ne-amyloid-like aggregates eyenziwe kwi-coacervates egugayo yamanzi.
WF 25 μM αS ngamaxesha ahlukeneyo okufukamela kunye nobude obugxininisiweyo (z, umgama ukusuka ezantsi okungadityaniswanga) kubukho be-25 μM Tau441 (1 μM AF488-ebhalwe αS kunye ne-Atto647N-ebhalwe Tau441) kwiqula lepleyiti yemicroscope ene-LLPS buffer) .Imifuniselo emithandathu yaphindwa ngokuzimeleyo ngeziphumo ezifanayo.b CF umfanekiso we-microscopic we-25 μM αS phambi kwe-25 μM Tau441 (1 μM Atto647N-ebhalwe Tau441) kunye ne-12.5 μM thioflavin-T (ThT).Amaconsi eprotheyini enobunzima kunye neeprotheyini ezifakwe kwi-raft kunye namabala aboniswa kwimiqolo ephezulu kunye nephakathi, ngokulandelanayo.Umqolo ongezantsi ubonisa imifanekiso yee-rafts kunye nehla ukusuka kwi-3 ii-replicates ezizimeleyo.Iintolo ezimhlophe zibonisa amachaphaza e-ThT kuzo zombini iiphaneli.Ibar yesikali sayo yonke imifanekiso ngama-20 µm.
Ukuphonononga ngokubanzi utshintsho kwinethiwekhi yeprotheni ye-coacervate ngexesha lokuguquka ukusuka kwi-liquid ukuya kwi-solid, sasebenzisa umfanekiso we-fluorescence lifetime imaging (FLIM) kunye ne-Förster resonance energy transfer microscopy (FRET) (Umfanekiso 6 kunye neFigures eyongezelelweyo 8 kunye ne-9).Siye saqikelela ukuba ukuvuthwa kwe-coacervate yomaleko ibe kulwakhiwo olujikene ngakumbi okanye oluqinileyo njengeprotheyini edityanisiweyo ikhokelela kunxibelelwano olusondeleyo phakathi kweprotein kunye neprobe yefluorescent edityaniswe kuyo, enokuthi ivelise isiphumo sokucima esibonakaliswa kubomi obufutshane beprobe (τ) , njengoko kuchaziwe ngaphambili40.,41 ,42.Ukongeza, kwiisampuli ezibhalwe kabini (AF488 kunye ne-Atto647N njengomnikeli we-FRET kunye needayi zokumkela, ngokulandelanayo), oku kuncipha kwi-τ kungakhatshwa ne-coacervate condensation kunye nokunyuka kwe-FRET (E) ukusebenza kakuhle ngexesha le-LSPT.Sibeke iliso kwi-raft kunye nokwakhiwa kwamabala ekuhambeni kwexesha kwi-LLPS αS/Tau441 kunye neesampuli ze-αS/ΔNt-Tau (25 µM yeprotheyini nganye kwi-LLPS buffer equlethe i-1 µM AF488 ebhalwe αS kunye/okanye i-Atto647N ebhalwe Tau441 okanye ΔNt-Tau).Siye saqaphela intsingiselo jikelele ekubeni ubomi be-fluorescence be-AF488 (τ488) kunye ne-Atto647N (τ647N) probes zehle kancinane njengoko i-coacervates ikhula (Umfanekiso wesi-6 kunye neFig. 8c).Okubangel 'umdla kukuba, olu tshintsho luye lwaphuculwa kakhulu kumachaphaza angaphakathi kwi-rafts (Umfanekiso we-6c), ebonisa ukuba ukuxinwa kweprotheyini okungaphezulu kwenzeka kumachaphaza.Ukuxhasa oku, akukho tshintsho lubalulekileyo kubomi be-fluorescence yabonwa kwi-αS / ΔNt-Tau yamaconsi aneminyaka eyi-24 (iFig. 8d eyongezelelweyo), ebonisa ukuba i-droplet gelation yinkqubo eyahlukileyo ekubonweni kwaye ayihambisani nokuhlengahlengiswa kwe-molecular. ngaphakathi coacervates.Kufuneka kuqatshelwe ukuba amachaphaza anobukhulu obuhlukeneyo kunye nomxholo oguquguqukayo kwi-αS, ngokukodwa kwi-αS / Tau441 inkqubo (i-Supplementary Fig. 8e).Ukuncipha kubomi be-spot fluorescence kukhatshwa kukunyuka kokuqina, ngakumbi kwi-Atto647N ebhalwe Tau441 (Fig. 8a eyoNgezelelweyo), kunye nocikizeko oluphezulu lweFRET kuzo zombini iinkqubo ze-αS/Tau441 kunye ne-αS/ΔNt-Tau, ebonisa ukuxinana ngakumbi kwiiyure ezintlanu zeLLPS. emva kokuqalisa, iiprotheyini ezingaphakathi kombane omileyo zixutywe.Xa kuthelekiswa ne-αS/ΔNt-Tau, siqaphele amaxabiso asezantsi e-τ647N kunye noko aphezulu τ488 amaxabiso kwi-αS/Tau441 amabala, ehamba kunye namaxabiso asezantsi nangenamhomogeneous ngaphezulu kwe-FRET.Mhlawumbi, oku kunokuhambelana nenyaniso yokuba kwinkqubo ye-αS/Tau441, ubuninzi be-αS obujongwayo kunye nokulindelekileyo kwii-aggregates buninzi, ngokuqhelekileyo i-substoichiometric xa kuthelekiswa ne-Tau, ekubeni i-Tau441 ngokwayo inokuthi iphinde iqhube i-LLPS kunye nokudibanisa (i-Supplementary Fig. 8e) .Nangona kunjalo, iqondo lokuhlangana kwe-droplet, ukubunjwa kwe-raft, kwaye, ngokubalulekileyo, ukuhlanganiswa kweeprotheni ngaphakathi kwe-coacervates efana nolwelo kuphezulu xa zombini i-Tau441 kunye ne-αS zikhona.
i-Lifetime fluorescence microscopy (FLIM) imifanekiso ye-αS/Tau441 kunye ne-αS/ΔNt-Tau kwi-25 μM yeprotheyini nganye (1 μM AF488-ebhalwe αS kunye ne-1 μM Atto647N-ebhalwe Tau441 okanye ΔNt-Tau) kwi-LLPS buffer.Iikholamu zibonisa imifanekiso emele iisampulu zeLLPS ngamaxesha ahlukeneyo okuvuthwa (30 min, 5 h and 24 h).Isakhelo esibomvu sibonisa ummandla oqulethe amabala e-αS/Tau441.Ubude bobomi buboniswa njengemivalo yombala.Ibar yesikali = 20 µm kuyo yonke imifanekiso.b Sondezwe-ngaphakathi umfanekiso weFLIM wendawo ekhethiweyo, eboniswe kwibhokisi ebomvu kwindawo yolawulo a.Uluhlu lobomi luboniswa kusetyenziswa isikali sombala ofanayo nanjengoko kwiphaneli a.Ibar yesikali = 5 µm.c Ihistograms ebonisa i-AF488 (encanyathiselwe kwi-αS) okanye i-Atto647N (encanyatheliswe ku-Tau) kwiindidi zeprotheyini ezahlukeneyo (amathontsi-D-, isihlenga-R- namachaphaza-P) achongiweyo kwimifanekiso yeFLIM erekhodishelwe αS-) unikezelo lwexesha lobomi beTau441 kunye I-αS / ΔNt-Tau iisampulu ze-coacervate (N = 17-32 ROI ye-D, i-29-44 ROI ye-R, kunye ne-21-51 ROI yamanqaku).Amaxabiso aphakathi kunye naphakathi aboniswa njengezikwere ezityheli kunye nemigca emnyama ngaphakathi kweebhokisi, ngokulandelelanayo.Imida esezantsi kunye nephezulu yebhokisi imele i-quartiles yokuqala neyesithathu, ngokulandelanayo, kwaye amaxabiso amancinci kunye nawona aphezulu ngaphakathi kwe-1.5-fold interquartile range (IQR) iboniswa njenge-whiskers.Izinto ezingaphandle ziboniswa njengedayimani ezimnyama.Ukubaluleka kwamanani phakathi kwezibini zonikezelo kwamiselwa kusetyenziswa isampuli ezimbini zovavanyo lwe-t, kucingelwa ukungafani okungalinganiyo.Amaxabiso e-p anemisila emibini aboniswa ngeenkwenkwezi kwipere nganye yedatha ethelekisayo (* ixabiso le-p> 0.01, ** p-ixabiso > 0.001, *** p-value > 0.0001, **** p-value > 0.00001), ns Ibonisa ukungahoyi (p-value > 0.05).Awona maxabiso achanekileyo e-p anikwa kwiTheyibhile eyoNgezelelweyo 1, kwaye idatha yoqobo inikezelwa njengeefayile zedatha ekrwada.
Ukubonisa ngakumbi ubume be-amyloid obufana ne-speckles/aggregates, siye saphatha iisampuli ze-coacervate ezingacocekanga kwiiyure ze-24 kunye noxinzelelo oluphezulu lwe-(1 M) ye-NaCl, ebangele ukuhlukana kwee-aggregates ezivela kwi-protein coacervates.Xa ii-aggregates ezizimeleyo (oko kukuthi, isisombululo esisasaziweyo se-aggregates) sabonwa kusetyenziswa i-atomic force microscopy (AFM), siye saqaphela i-morphology ye-spherical ubukhulu becala enobude obuqhelekileyo obumalunga ne-15 nm, ethanda ukunxulumana phantsi kweemeko zoxinzelelo lwetyuwa ephezulu, efana ukuziphatha kwee-amyloid fibrils eziqhelekileyo ngenxa yempembelelo enamandla ye-hydrophobic kumphezulu (qaphela ukuba iifibrils zihlala zinobude obuyi- ~ 10 nm) (I-Supplementary Fig. 10a).Okubangel 'umdla kukuba, xa ii-aggregates ezizimeleyo zixutywe kunye ne-ThT kuvavanyo oluqhelekileyo lwe-ThT fluorescence, siye sabona ukunyuka okuphawulekayo kwi-ThT fluorescence quantum isivuno, xa kuthelekiswa naloo nto ibonwayo xa idayi ifakwe kwi-fibrils ye-αS amyloid (Fig. i-coacervate aggregates iqulethe izakhiwo ezinjenge-amyloid..Enyanisweni, i-aggregates ikwazi ukunyamezela ukugxilwa kwetyuwa ephezulu kodwa i-4 M guanidine chloride (GdnHCl), njenge-fibrils ye-amyloid eqhelekileyo (umfanekiso owongezelelweyo we-10c).
Emva koko, sihlalutye ukubunjwa kwee-aggregates kusetyenziswa i-molecule ye-fluorescence enye, ukulungelelaniswa kwe-fluorescence ethile / i-spectroscope yokudibanisa (i-FCS / FCCS), kunye nohlalutyo olugqamile lokufunyanwa kwe-coincidence yemibala emibini (TCCD).Ukuza kuthi ga ngoku, siye sahlukanisa iiaggregates ezenziwe emva kweeyure ezingama-24 zokufukanyelwa kwi-100 μl LLPS iisampulu eziqulethe i-αS ne-Tau441 (zombini i-25 μM) kunye ne-1 μM AF488-ebhalwe αS kunye ne-1 μM Atto647N-ebhalwe Tau441.Nciphisa isisombululo esisisiphumo esisasaziweyo se-aggregate kwisimo se-monomolecular usebenzisa i-PEG-free buffer efanayo kunye ne-1 M NaCl (i-buffer efanayo esetyenziselwa ukwahlula i-aggregates kwi-coacervate) ukuthintela naluphi na unxibelelwano lwe-electrostatic olunokwenzeka phakathi kwe-LLPS kunye neprotheni.Umzekelo wexesha lokuhamba kwe-molecule enye ingabonwa kwi-Fig. 7a.Uhlalutyo lwe-FCCS/FCS (i-cross-correlation, i-CC kunye ne-autocorrelation, i-AC) ibonise ukuba i-aggregates equkethe i-αS kunye ne-tau yayininzi kwiisampuli (jonga i-CC curve kwi-Fig. 7b, ipaneli ekhohlo), kunye nokugqithiswa kweprotheni ye-monomeric eseleyo yavela njenge isiphumo senkqubo ye-dilution (jonga i-AC curves kwi-Figure 7b, ipaneli yasekhohlo).Uvavanyo lokulawula olwenziwe phantsi kweemeko zesisombululo esifanayo kusetyenziswa iisampuli eziqulethe iiprotheni ze-monomeric kuphela ezibonisa ukuba akukho curves CC, kwaye i-AC curves ihambelana kakuhle nemodeli yokusabalalisa icandelo elinye (Eq. 4), apho iiprotheni ze-monomeric zinee-coefficients ezilindelekileyo zokusabalalisa (Fig. 7b) ), indawo yolawulo yasekunene).I-diffusion coefficient yamasuntswana adityanisiweyo ingaphantsi kwe-1 µm2/s, kwaye leyo yeeproteni ze-monomeric imalunga ne-1 µm2/s.50–100 µm/s;amaxabiso ayafana namaxabiso apapashiweyo ngaphambili kwi-sonicated αS amyloid fibrils kunye ne-monomeric αS ngokwahlukileyo phantsi kweemeko ezifanayo zesisombululo44.Xa sihlalutya i-aggregates kunye nohlalutyo lokuqhuma kwe-TCCD (umzobo 7c, ipaneli ephezulu), sifumene ukuba kwi-aggregate nganye esecaleni (αS / Tau heteroaggregate), malunga ne-60% ye-aggregates efunyenweyo equlethwe zombini i-αS kunye ne-tau, malunga ne-30% equlethwe kuphela. tau, malunga ne-10% αS kuphela.Uhlalutyo lwe-Stoichiometric lwe-αS / Tau heteroaggregates lubonise ukuba uninzi lwee-heteroaggregates zaye zatyetyiswa kwi-tau (i-stoichiometry engaphantsi kwe-0.5, inani eliqhelekileyo leemolekyuli ze-tau nge-aggregate yi-4 amaxesha angaphezu kwe-αS molecules), ehambelana nomsebenzi wethu obonwa kwi-FLIM kwindawo imifuniselo..Uhlalutyo lwe-FRET lubonise ukuba ezi aggregates ziqulathe zombini iiproteni, nangona awona maxabiso e-FRET enyani kule meko awabalulekanga kakhulu, kuba ukuhanjiswa kwe-fluorophores kwi-aggregate nganye kwakungacwangciswanga ngenxa yobuninzi beprotheyini engabhalwanga esetyenzisiweyo kuvavanyo.Okubangel 'umdla kukuba, xa sisenza uhlalutyo olufanayo sisebenzisa i-45,46 ekhulileyo yokudibanisa i-amyloid-intsilelo yeTau eyahlukileyo (jonga i-Fig. ukukwazi ukwenza ii-aggregates ngaphakathi kwe-coacervate kwancitshiswa kakhulu kwaye i-FLIM yafumanisa iindawo ezininzi kwi-experimenti ye-situ, kwaye i-curves ebuthathaka yokudibanisa i-curves yabonwa kwiisampuli ezizimeleyo ezidibeneyo.Nangona kunjalo, kwinani elincinci lee-aggregates ezichongiweyo (isinye seshumi kuphela se-Tau441), siye saqaphela ukuba i-aggregate nganye yatyetyiswa kwi-αS kunolu tshintsho lwe-Tau, malunga ne-50% ye-aggregates echongiweyo equlethe kuphela ii-molecule ze-αS, kwaye i-αS yayininzi ngokugqithisileyo. .i-aggregates (jonga i-Supplementary Fig. 11e), ngokungafaniyo ne-heterogeneous aggregates eyenziwe yi-Tau441 (umzobo 6f).Iziphumo zolu vavanyo zibonise ukuba nangona i-αS ngokwayo ikwazi ukuqokelela kunye ne-tau ngaphakathi kwe-coacervate, i-tau nucleation ithandeka ngakumbi phantsi kwezi meko, kwaye iziphumo ezinjenge-amyloid-like aggregates ziyakwazi ukusebenza njengohlobo lwe-αS kunye ne-tau.Nangona kunjalo, nje ukuba i-tau-rich core yenziwe, i-heterotypic interactions phakathi kwe-αS kunye ne-tau ithandwa kwii-aggregates ngaphezu kobudlelwane be-homotypic phakathi kwee-molecule ze-tau;sikwajonga uthungelwano lweprotheyini kulwelo αS/tau coacervates.
i-representative fluorescence temporal traces of the molekyuli enye yeeaggregates ezizimeleyo ezenziwe kwi-αS/Tau441 electrostatic coacervates.Ukuqhuma okuhambelana ne-αS / Tau441 coaggregates (ukuqhuma ngaphezu komda obonisiweyo) kubonwe kwiindlela ezintathu zokubona (AF488 kunye nokukhutshwa kwe-Atto647N emva kokukhutshwa ngokuthe ngqo, imigca eluhlaza okwesibhakabhaka kunye nebomvu, ukukhutshwa kwe-Atto647N emva kokutshatyalaliswa ngokungathanga ngqo), i-FRET, umgca we-violet).b Uhlalutyo lweFCS/FCCS lwesampulu yee-aggregates ezikwanti ze-αS/Tau441 ezifunyenwe kwiLLPS (iphaneli yasekhohlo).I-Autocorrelation (AC) iigophe ze-AF488 kunye ne-Atto647N ziboniswa ngombala oluhlaza okwesibhakabhaka nobomvu, ngokulandelelanayo, kunye ne-cross-correlation (CC) iigophe ezinxulumene ne-aggregates equlethe zombini iidayi ziboniswa ngomfusa.Iigophe ze-AC zibonisa ubukho beentlobo zeprotheyini ezibhalwe nge-monomeric kunye ne-aggregated protein, ngelixa i-CC curves ibonisa kuphela ukusasazwa kwee-aggregates ezibhalwe kabini.Uhlalutyo olufanayo, kodwa phantsi kweemeko ezifanayo zesisombululo njengakwiindawo ezizimeleyo, iisampuli eziqukethe kuphela i-αS ye-monomeric kunye ne-Tau441 ziboniswa njengolawulo kwiphaneli elungileyo.c Uhlalutyo lweflash ye-Fluorescence yeemolekyuli enye yee-aggregates ezizimeleyo ezenziwe kwi-αS/Tau441 electrostatic coacervates.Ulwazi lwe-aggregate nganye efunyenwe kwiimpinda ezine ezahlukeneyo (N = 152) zicwangciswe ngokuchasene ne-stoichiometry yazo, amaxabiso e-S, kunye nokusebenza kakuhle kwe-FRET (iphaneli ephezulu, ibha yombala ibonisa ukwenzeka).Zintathu iindidi zeeaggregates ezinokwahlulwa: -αS-kuphela aggregates kunye S~1 kunye FRET~0, Tau-kuphela aggregates kunye S~0 kunye FRET~1, kunye ne-heterogeneous Tau/αS aggregates kunye phakathi S kunye noqikelelo FRET yesixa zombini iiprotheyini ezimakishayo ezichongiweyo kwi-aggregate nganye ye-heterogeneous (N = 100) iboniswe kwiphaneli esezantsi (isikali sombala sibonisa ukwenzeka).Idatha ekrwada inikezelwe ngohlobo lweefayile zedatha ekrwada.
Ukuvuthwa okanye ukuguga kweprotheyini ye-liquid condensates kwi-gel-like okanye izakhiwo eziqinileyo ekuhambeni kwexesha kuye kwabikwa ukuba kubandakanyeka kwimisebenzi emininzi ye-physiological ye-condensate47 kunye nesifo, njengenkqubo engaqhelekanga eyandulela i-amyloid aggregation 7, 48, 49. Apha Apha sifunda ukwahlukana kwesigaba kunye nokuziphatha ngokweenkcukacha.I-LSPT αS kubukho bepolycations engacwangciswanga kwindawo elawulwayo kwindawo ephantsi ye-micromolar kunye neemeko ezifanelekileyo ze-physiologically (qaphela ukuba i-physiological concentration ye-αS yi>1 µM50), ilandela ukuziphatha okuqhelekileyo kwe-LPS ye-thermodynamically.Sifumene ukuba i-αS, equlethe i-C-terminal yengingqi ehlawuliswa kakubi kakhulu kwi-pH ye-physiological, iyakwazi ukwenza amathontsi aneprotheyini ecebileyo kwisisombululo samanzi nge-LLPS phambi kweepeptides eziphazamiseke kakhulu ezifana ne-pLK okanye i-Tau ngenkqubo ye-electrostatic. ukudibanisa okuntsonkothileyo kubukho be-aggregation macromolecules.Le nkqubo ingaba neempembelelo ezifanelekileyo kwindawo yeselula apho i-αS idibana neemolekyuli ezahlukeneyo ze-polycationic ezinxulumene ne-aggregation yayo ehambelana nesifo zombini kwi-vitro kunye ne-vivo51,52,53,54.
Kwizifundo ezininzi, i-protein dynamics ngaphakathi kwamaconsi athathwe njengenye yezinto eziphambili ezimisela inkqubo yokuvuthwa55,56.Kwi-electrostatic αS coacervates kunye nepolycations, inkqubo yokuvuthwa ngokucacileyo ixhomekeke kumandla onxibelelwano kunye neepolycations, i-valence, kunye nobuninzi bolu nxibelelwano.Ithiyori yolungelelwaniso icebisa ukuba imbonakalo-mhlaba elinganayo yeemeko zolwelo ezimbini inokuba bubukho bethontsi elikhulu elityebileyo kwii-biopolymers eziqhuba i-LLPS57,58.Ukukhula kweDroplet kunokufezekiswa yi-Ostwald maturation59, i-coalescence60 okanye ukusetyenziswa kwe-monomer yamahhala kwi-phase61.I-αS kunye ne-Tau441, i-ΔNt-Tau okanye i-pLK, ininzi yeprotheni yayigxininiswe kwi-condensate phantsi kweemeko ezisetyenziswe kolu cwaningo.Nangona kunjalo, ngelixa amathontsi e-tau anobungakanani obugcweleyo adibana ngokukhawuleza phezu kokumanzisa komphezulu, ukuhlangana kwamathontsi kunye nokumanzisa kwakunzima kwi-ΔNt-Tau kunye ne-pLK, ecebisa ilahleko ekhawulezileyo yeempawu zolwelo kwezi nkqubo zimbini.Ngokohlalutyo lwethu lwe-FLIM-FRET, amathontsi e-pLK asele ekhulile kunye ne-ΔNt-Tau abonise inqanaba elifanayo leprotein aggregation (ixesha elifanayo le-fluorescence life) njengamathontsi oqobo, ecebisa ukuba inethiwekhi yeprotein yokuqala yagcinwa, nangona iqinile.
Silungelelanisa iziphumo zethu zovavanyo kwimodeli elandelayo (Umfanekiso 8).Amathontsi enziwe okwethutyana adla ngokuba luthungelwano lweprotheyini ngaphandle kwembuyekezo ye-electrostatic, kwaye ke ngoko kukho iindawo zokungalingani kwentlawulo, ngakumbi kujongano lwamathontsi, okukhokelela kumathontsi anomgangatho ophezulu we-electrostatic surface.Ukuhlawulela intlawulo (into eqhelekileyo ebizwa ngokuba yi-valence depletion) kunye nokunciphisa amandla angaphezulu komphezulu wamathontsi, amathontsi anokubandakanya iipolypeptides ezintsha ukusuka kwinqanaba lokuhlanjululwa, ukulungelelanisa kwakhona uthungelwano lweprotheyini ukuze kwandiswe ukusebenzisana kwentlawulo, kunye nokusebenzisana namanye amathontsi.ngemiphezulu (ukumanzisa).Amaconsi e-αS / pLK, ngenxa yenethiwekhi yabo yeprotheyini elula (kuphela ukusebenzisana kwe-heterotypic phakathi kwe-αS kunye ne-pLK) kunye nobudlelwane obukhulu be-protein-protein interactions, kubonakala kukwazi ukulinganisa umrhumo we-condensate ngokukhawuleza;ngokwenene, siye sabona i-protein kinetics ekhawulezayo kwi-coacervates yokuqala ye-αS / pLK kune-αS / Tau.Emva kokuncipha kwe-valence, ukusebenzisana kuya kuba ngaphantsi kwe-ephemeral kwaye amaconsi alahlekelwa yimpahla yawo yolwelo kwaye ajike abe ngamaconsi afana nejeli, angenakutsha kunye ne-electrostatic surface ephantsi (kwaye ngoko ayikwazi ukumanzisa umphezulu).Ngokwahlukileyo, amathontsi e-αS/Tau akasebenzi kangako ekuphuculeni ibhalansi yentlawulo yethontsi ngenxa yothungelwano lweprotheyini entsonkothileyo (kunye nokudibana kwe-homotypic kunye ne-heterotypic) kunye nobume obubuthathaka bokusebenzisana kweprotheyini.Oku kubangela amathontsi agcina ukuziphatha kolwelo ixesha elongeziweyo kwaye abonise amandla aphezulu omphezulu we-electrostatic athande ukuncitshiswa ngokudibana kunye nokukhula (ngaloo ndlela kucuthwe indawo yomphezulu/umlinganiselo wamathontsi) kunye nokumanzisa i-hydrophilic surface chem.Oku kudala iilayibrari ezinkulu zeprotheyini ezigxininisiweyo ezigcina iipropathi ezinolwelo njengoko unxibelelwano luhlala ludlula kakhulu ngenxa yokukhangela rhoqo ukuphuculwa kwentlawulo kuthungelwano lweprotheyini.Okubangela umdla kukuba, iifom ze-N-terminally truncated ze-Tau, kubandakanywa ezinye ze-isoforms62 ezenzeka ngokwendalo, zibonisa ukuziphatha okuphakathi, kunye nezinye i-coacervates ukuguga kunye ne-αS zibe ngamathontsi afana nejeli ahlala ixesha elide, ngelixa amanye aguquka abe yi-condensates enkulu yolwelo.Oku kubini ekuvuthweni kwe-αS electrostatic coacervates iyahambelana ne-LLPS yamva nje yethiyori kunye nezifundo zovavanyo ezichonge unxulumano phakathi kokuncipha kwe-valence kunye ne-electrostatic sieving kwiicondensates njengesitshixo sokulawula ubungakanani becondensate kunye neempawu zolwelo.Inkqubo 58.61.
Olu dweliso lubonisa i-amyloid edityanisiweyo yokubeka indlela ye-αS kunye ne-Tau441 nge-LLPS kunye ne-LSPT.Ngemimandla eyongezelelweyo ye-anion-rich (ebomvu) kunye ne-cation-rich (blue), i-αS kunye ne-tau electrostatic coacervates ene-valence eyanelisayo inamandla angaphantsi komhlaba kwaye ngoko ke i-coalescence encinci, ekhokelela ekugugeni kwamathontsi ngokukhawuleza.Uzinzo lwejeli olungena-agglomerated luyafezekiswa..Le meko ithandeka kakhulu kwimeko yenkqubo ye-αS / pLK ngenxa yobudlelwane bayo obuphezulu kunye nenethiwekhi ye-protein-pair ye-protein elula, evumela ukuba i-gel-like iguquke ngokukhawuleza.Ngokuchasene noko, amathontsi ane-valence enganelisiyo kwaye, ngenxa yoko, imimandla ehlawuliswa iiprotheyini ekhoyo ukuze isebenze, yenza kube lula kwi-coacervate ukuba idibanise kwaye imanzise umphezulu we-hydrophilic ukwenzela ukunciphisa amandla ayo aphezulu.Le meko ikhethwayo kwi-αS/Tau441 i-coacervates, ene-multivalent network complex equkethe i-Tau-Tau ebuthathaka kunye ne-αS-Tau ukusebenzisana.Ngapha koko, ii-coacervates ezinkulu ziya kuzigcina ngokulula iipropathi zazo ezinjengolwelo, zivumela ezinye iiprotein-to-protein zenzeke.Ekugqibeleni, i-amyloid heterogeneous aggregates equlethe zombini i-αS kunye nefom ye-tau ngaphakathi kwe-coacervate fluid, enokuthi inxulumane naleyo ifunyenwe kwimizimba edibeneyo, eyimpawu zezifo ze-neurodeergenerative.
Izakhiwo ezinkulu ezinjengolwelo ezenziwe ngexesha lokuvuthwa kwe-αS/Tau441 eneprotein exinene kakhulu kodwa eguquguqukayo kwaye, kwinqanaba elingaphantsi, i-αS/ΔNt-Tau i-coacervates yindawo efanelekileyo yokugcina i-nucleation ye-protein aggregation.Siye saqaphela ngokwenene ukubunjwa kweeprotheyini eziqinileyo kolu hlobo lweprotheyini ye-coacervates, ehlala iqulethe zombini i-αS kunye ne-tau.Siye sabonisa ukuba ezi heteroaggregates zizinziswe yi-non-electrostatic interactions, ziyakwazi ukubopha i-amyloid-specific ThT idayi ngendlela efanayo ne-amyloid fibrils eqhelekileyo, kwaye ngokwenene inokuchasana okufanayo kwiimpembelelo ezahlukeneyo.Ii-aggregates ze-αS/tau ezenziwe yiLLPS ziboniswe zineempawu ezifana ne-amyloid.Ngenene, umahluko oqolileyo we-Tau onqongopheleyo kwi-amyloid aggregation yonakele kakhulu ekwenziweni kwezi ngxubevange ze-αS ngaphakathi kolwelo lwe-electrostatic coacervate.Ukuqulunqwa kwe-αS / Tau441 i-aggregates yabonwa kuphela ngaphakathi kwe-coacervates, egcina iipropati ezifana ne-liquid, kwaye ingaze, ukuba i-coacervates / droplets ayizange ifike kwi-gel state.Kwimeko yokugqibela, ukonyuka kwamandla okunxibelelana kwe-electrostatic kwaye, ngenxa yoko, ukuqina kothungelwano lweprotheyini kuthintela uhlengahlengiso oluyimfuneko lweprotheyini ukuseka unxibelelwano lweprotheyini entsha eyimfuneko kwi-amyloid nucleation.Nangona kunjalo, oku kunokufezekiswa ngokuguquguquka ngakumbi, ii-coacervates ezinjengolwelo, ezinokuthi ziphinde zihlale zingamanzi njengoko zisanda ngobukhulu.
Inyaniso yokuba ukubunjwa kwee-aggregates ngaphakathi kwesigaba esinqamlekileyo sikhethwa kwi-condensates enkulu ye-αS/Tau kunamaconsi amancinci akhawuleza ijeli, igxininisa ukubaluleka kokuchonga izinto ezilawula ukuhlangana kwe-droplet.Ngaloo ndlela, kungekhona nje ukuba kukho ukuthambekela kokuhlukana kwesigaba, kodwa ubukhulu be-condensate kufuneka ilawulwe ukuze kusebenze ngokufanelekileyo kunye nokukhusela izifo58,61.Iziphumo zethu zikwabonisa ukubaluleka kokulinganisela phakathi kwe-LLPS kunye ne-LSPT yenkqubo ye-αS / Tau.Ngelixa ukubunjwa kwe-droplet kunokukhusela kwi-amyloid aggregation ngokunciphisa inani le-protein monomers ekhoyo phantsi kweemeko ze-saturation, njengoko kucetywayo kwezinye iinkqubo63,64, i-droplet fusion kumanqanaba aphezulu e-droplet inokukhokelela ekudibaneni kweeprotheyini zangaphakathi ngokuhlengahlengiswa okucothayo.iinethiwekhi protein..
Ngokubanzi, idatha yethu igxininisa kakhulu ukufaneleka kwe-valence edibeneyo kunye nokwaneliseka / ukungoneliseki kokunxibelelana kwi-drop networks kumxholo we-LSPT.Ngokukodwa, sibonisa ukuba ubude obugcweleyo be-αS/Tau441 i-condensates iyakwazi ukudibanisa ngokufanelekileyo kunye ne-nucleate ukwenza i-amyloid-like heteroaggregates ebandakanya zombini iiprotheni kwaye iphakamise indlela ye-molecular esekelwe kwiziphumo zethu zokulinga.Ukudityaniswa kweeprotheyini ezimbini kwi-αS/Tau fluid coacervate esiyibikayo apha inokuthi ngenene inxibelelene nokudityaniswa kweeprotheyini ezimbini ezidityanisiweyo, eziyimpawu zesi sifo, kwaye zinokuba negalelo ekuqondeni ubudlelwane phakathi kwe-LLPS kunye. i-amyloid aggregation, ivula indlela ye-IDP ehlawuliswa kakhulu kwi-neurodegeneration.
I-Monomeric WT-αS, i-cysteine ​​​​mutants (Q24C-αS, N122C-αS) kunye neentlobo ze-ΔCt-αS (Δ101-140) zibonakaliswe kwi-E. coli kwaye zahlanjululwa njengoko kuchaziwe ngaphambili.I-5 mM DTT ifakwe kuwo onke amanyathelo ekuhlanjululweni kwe-αS cysteine ​​​​mutants ukukhusela ukubunjwa kwebhondi ye-disulfide.Tau441 isoform (plasmid obtained from Addgene #16316), ΔNt-Tau variant (Δ1–150, obtained by cloning IVA with primers CTTTAAGAAGGAGATACATATGATCGCCACACCGCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) and AggDef-Tau variant (Δ275–311, purified with GGCTC5 primer) E. coli cultures were ikhule ukuya kwi-OD600 = 0.6-0.7 kwi-37 ° C kunye ne-180 rpm, kwaye inkcazo yenziwe nge-IPTG kwiiyure ze-3 kwi-37 ° C.Ukuvuna iiseli kwi-11,500 xg ye-15 min kwi-4 °C kwaye uhlambe nge-saline buffer equkethe i-150 mM NaCl.Phinda umise i-pellet kwi-lysis buffer (20 ml nge-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 μM1).Isinyathelo se-sonication senziwe kwi-ice kunye ne-amplitude ye-80% ye-pulses ye-10 (i-1 min, i-1 min off).Ungadluli kwi-60 ml kwi-ultrasound enye.I-E. coli lysates yatshiswa kwi-95 ° C. imizuzu engama-20, emva koko ipholile kwi-ice kunye ne-centrifuged kwi-127,000 × g imizuzu engama-40.I-supernatant ecacisiweyo isetyenziswe kwi-3.5 kDa membrane (Spectrum™ Thermo Fisher Scientific, UK) kwaye i-dialysed ngokuchasene ne-4 L ye-dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) iiyure ezili-10.Ikholamu yokutshintshiselana nge-5 ml ye-cation (i-HiTrap SPFF, i-Cytiva, i-MA, i-USA) yafaniswa ne-equilibration buffer (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).I-tau lysate yahluzwa nge-0.22 μm i-PVDF yokucoca kwaye ifakwe kwikholomu kwinqanaba lokuhamba kwe-1 ml / min.Uvavanyo lwaqhutywa ngokuthe ngcembe, i-tau yahluthwa nge-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).Amaqhezu ahlalutyiweyo yi-SDS-PAGE, kwaye nawaphi na amaqhezu aqulathe ibhendi enye enobunzima obulindelekileyo bemolekyuli ye-tau agxininiswe kusetyenziswa i-10 kDa centrifuge filter kwaye endaweni yayo kwafakwa isithinteli esiqulethe 10 mM HEPES, pH 7.4, NaCl 500 mM kunye neDTT 2 mM ye ugxininiso lweprotheni lokugqibela lwaluyi-100 μM.Isisombululo seprotheni saye sagqithiswa kwi-PVDF ye-0.22 μm, ifakwe ngokukhawuleza kwaye igcinwe kwi -80 ° C.Iprotheyini i-K18 ibonelelwe ngobubele nguProf Alberto Boffi.Ukucoceka kwamalungiselelo kwaba> 95% njengoko kuqinisekiswe yi-SDS-PAGE kunye ne-MALDI-TOF/TOF.Ii-cysteine ​​​​ezahlukeneyo zazibhalwe ngamachiza kunye ne-AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) okanye i-TEMPOL-maleimide (i-Toronto Research Chemicals, eToronto, eCanada).zaqinisekiswa yi-absorbence kunye ne-MALDI-TOF/TOF.I-Tau441, i-ΔNt-Tau, i-AggDef-Tau kunye ne-K18 zibhalwe ngeentsalela ze-cysteine ​​​​zendalo kwiindawo ze-191 kunye ne-322 usebenzisa i-Atto647N-maleimide (ATTO-TEC GmbH, Siegen, eJamani) ngokulandela inkqubo efanayo.Intlawulo eseleyo ngokwemephu yentsalela ye-αS kunye ne-Tau441 yenziwe kusetyenziswa i-CIDER66.
I-poly-L-lysine eqinile (i-pLK DP 90-110 ngokwe-NMR evela kumthengisi, i-Alamanda Polymers Inc, i-Huntsville, i-Alabama, i-USA) yachithwa kwi-10 mM HEPES, i-100 mM NaCl, i-pH 7.4 ukuya kwi-10 mM yoxinaniso, inkqubo ye-sonicated ye-5 imizuzu kwindawo yokuhlambela amanzi e-ultrasonic kwaye ugcine kwi -20 ° C.I-PEG-8, i-dextran-70, i-FITC-PEG-10 (i-Biochempeg, i-Watertown, i-MA, i-USA) kunye ne-FITC-dextran-500 (i-Sigma -Aldrich, i-Sant Louis, i-MI, i-USA) i-soluble yamanzi kwaye isasazwa ngokubanzi kwi-buffer ye-LLPS.I-Dialysis isusa iityuwa ezingcolisayo.Emva koko zahluzwa ngesihluzo sesirinji kunye nobukhulu bepore ye-0.22 μm, kwaye ukugxininiswa kwabo kubalwa ngokusebenzisa i-refractometer (Mettler Toledo, Columbus, Ohio, USA).Iisampulu ze-LLPS zalungiswa kwiqondo lobushushu begumbi ngolu hlobo lulandelayo: i-buffer kunye ne-extrusion yaxutywa kunye ne-1 mM tris(2-carboxyethyl)phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- I-1, i-2-diyldinitrile) i-tetraacetic acid (EDTA, i-carboxynth) kunye nomxube we-1% ye-protease inhibitor (PMSF 100 mM, i-benzimide 1 mM, i-leupeptin 5 μM).Emva koko i-αS kunye ne-polycations edibeneyo (ukhetho lwe-pLK okanye i-Tau) yongezwa.Kwiimvavanyo zexesha le-thioflavin-T (i-ThT, iCarbosynth, i-Compton, i-UK), sebenzisa i-concentration ye-ThT iyonke ukuba ibe sisiqingatha soxinzelelo lwe-αS.Ngobunono kodwa ngokucokisekileyo xuba iisampuli ukuqinisekisa ukuba ziyafana.Ugxininiso lwecandelo ngalinye lwahluka ukusuka kumfuniselo ukuya kumfuniselo, njengoko kuchaziwe kwicandelo leZiphumo.I-Azide isetyenziswe kwi-concentration ye-0.02% (w / v) nanini na ixesha lovavanyo lidlula iiyure ze-4.Kulo lonke uhlalutyo usebenzisa iisampuli ze-LLPS, vumela umxube ukuba ulungelelanise imizuzu emi-5 ngaphambi kohlalutyo.Kuphononongo lokusasazwa okukhanyayo, i-150 µl yeesampulu zalayishwa kwii-microplates ezingabophelelanga ezingama-96-well (µClear®, emnyama, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) kwaye yagqunywa ngefilimu encamathelayo.Ii-LLPs zajongwa ngokulinganisa ukufunxa kwi-350 nm embindini wesisombululo kwi-CLARIOstar plate reader (BMG Labtech, Ortenberg, Germany).Iimvavanyo zenziwa ngokuphindwe kathathu kwi-25 ° C, kwaye iimpazamo zibalwe njengokuphambuka okusemgangathweni kwintsingiselo.Isigaba sokuhlanjululwa silinganiswe ngesampulu ye-centrifugation kunye ne-SDS-PAGE yohlalutyo lwejeli, kunye neqhezu le-αS kwizigaba ezixubileyo kunye nezigxininisiweyo zalinganiswa kwiisombululo ezahlukeneyo ze-LLPS.Isampuli ye-100 μl LLPS equlethe i-1 μM AF488-ebhalwe αS yalungiswa ngokuxutywa ngokucokisekileyo kulandelwa yi-centrifugation kwi-9600 × g kwimizuzu engama-30, emva koko imvula yayidla ngokubonakala.I-50 μl ephezulu ye-supernatant yayisetyenziselwa ubungakanani beprotheyini kusetyenziswa ijeli ye-SDS-PAGE.Iigels zaskenwa ngezihluzi ze-AF488 kusetyenziswa inkqubo yemifanekiso yejeli ye-ChemiDoc (i-Bio-Rad Laboratories, i-Hercules, i-CA, i-USA) okanye ifakwe ibala le-Coomassie kwaye yabonwa ngezihluzo ezifanelekileyo.Iibhanti ezibangelwayo zahlalutywa kusetyenziswa i-ImageJ version 1.53i (amaZiko eSizwe ezeMpilo, eU.SA).Imifuniselo yenziwe ngokuphindiweyo kwimifuniselo emibini eyahlukeneyo eneziphumo ezifanayo.
Ngokuqhelekileyo, i-150 μl yeisampulu isetyenziswe kwii-microplates ezingabophiyo ezingama-96 kwaye zabonwa kwiqondo lobushushu begumbi kwi-microscope ye-Leica DMI6000B eguqulweyo (i-Leica Microsystems, i-Wetzlar, eJamani).Kuvavanyo lwamabala, iipleyiti ze-µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Germany) okanye ii-96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) nazo zasetyenziswa.I-EL6000 halogen okanye izibane ze-mercury metal halide zisetyenziswe njengemithombo yokukhanyisa (ye-BF / DIC kunye ne-WF imaging, ngokulandelanayo).Kwi-WF microscopy, injongo yomoya yokukhulisa i-40x (i-Leica Microsystems, eJamani) yayisetyenziselwa ukugxininisa ukukhanya kwisampuli kwaye iqokelele.Iisampulu ze-AF488 kunye ne-ThT ezibhalwe phantsi, ukuvuswa kohluzo kunye nokukhutshwa kunye neeseti zokucoca ezisemgangathweni ze-GFP, izihluzi ze-excitation kunye ne-emission bandpass, ngokulandelelana, i-460-500 nm kunye ne-512-542 nm i-bandpass filters, kunye ne-495 nm dichroic mirror.Kwiisampuli ezibhalwe nge-Atto647N, isethi esemgangathweni yeefilitha ze-Cy5 ezine-excitation kunye ne-emission bandpass filters 628-40 nm kunye ne-692-40 nm, ngokulandelanayo, kunye nesibuko se-dichroic se-660 nm sisetyenzisiwe.Kwi-BF kunye ne-DIC microscopy, sebenzisa injongo efanayo yokuqokelela ukukhanya.Ukukhanya okuqokelelweyo kubhalwe kwikhamera yeLeica DFC7000 CCD (iLeica Microsystems, eJamani).Ixesha lokuvezwa laliyi-50 ms ye-BF kunye ne-DIC yomfanekiso we-microscopy kunye ne-20-100 ms ye-WF imaging microscopy.Ukuthelekisa, ixesha lokuvezwa kwayo yonke imifuniselo nge-ThT yayiyi-100 ms.Uvavanyo lokudlula kwexesha lwenziwa ukujonga ukuhlangana kwe-droplet, kunye nemifanekiso eqokelelwa rhoqo nge-100 ms imizuzu emininzi.I-ImageJ (NIH, USA) yayisetyenziselwa uhlalutyo lomfanekiso.Uvavanyo lwenziwe ngokuphindwe kathathu ngeziphumo ezifanayo.
Kwimifuniselo ye-colocalization, i-FRAP kunye nokwakhiwa kwakhona kwe-3D, imifanekiso yafunyanwa kwi-Zeiss LSM 880 inverted confocal microscope kusetyenziswa i-ZEN 2 blue edition (Carl Zeiss AG, Oberkochen, Germany).Iisampulu ze-50 µl zisetyenziswe kwi-µ-Slide Angiogenesis Petri izitya (Ibidi GmbH, Gröfelfing, eJamani), ziphathwe nge-polymer ye-hydrophilic (ibiTreat) kwaye ifakwe kwi-63 × injongo yokuntywiliselwa kweoli (iPlan-Apochromat 63×/NA 1.4 Oil) kwi-DIC).Imifanekiso yafunyanwa kusetyenziswa i-458 nm, 488 nm, kunye ne-633 nm imigca ye-argon ye-laser enesisombululo se-0.26 µm/pixel kunye nexesha lokuvezwa le-8 µs/pixel lokufumana imincili kunye nokufunyanwa kweefestile ze-470-600 nm, 493-628 nm, kunye ne-638-755 nm yayisetyenziselwa ukujonga i-ThT, AF488 kunye ne-Atto647N, ngokulandelanayo.Kwimifuniselo ye-FRAP, ukufotowa kwexesha lokuthatha ixesha kwisampulu nganye kwarekhodwa kwisakhelo se-1 ngomzuzwana.Uvavanyo lwenziwe ngokuphindwe kathathu kwiqondo lobushushu begumbi elineziphumo ezifanayo.Yonke imifanekiso yahlaziywa kusetyenziswa i-software ye-Zen 2 blue edition (Carl Zeiss AG, Oberkochen, Germany).Iijika ze-FRAP zenziwe ziqhelekileyo, zicwangcisiwe kwaye zifakwe kwi-intensite / data data ekhutshwe kwimifanekiso usebenzisa i-Zen 2 usebenzisa i-OriginPro 9.1.Iigophe zokubuyisela zifakwe kwimodeli ye-mono-exponential ukuhlawula i-molecular diffusion kunye ne-exponential term eyongezelelweyo ukuhlawula i-acquisition bleaching effect.Emva koko sabala i-D sisebenzisa i-radius ye-bleaching ye-nominal kunye ne-half-life enqunywe ngaphambili yokubuyisela njenge-equation ye-Kang et al.5 35 ebonisiweyo.
Iintlobo ze-cysteine ​​​​ezinye ze-αS zenziwe nge-4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) kwiindawo ze-24 (TEMPOL-24-αS) kunye ne-122 (TEMPOL-122-αS), ngokulandelelana.I-Spin Labeling Kwimifuniselo ye-EPR, i-concentration ye-αS yamiselwa kwi-100 μM kwaye i-PEG yoxinaniso yayiyi-15% (w / v).Kwiimeko ezahlukeneyo zokudibanisa, umlinganiselo we-αS:pLK ube ngu-1:10, ngelixa umlinganiselo we-αS:ΔNt-Tau kunye ne-αS:Tau441 ugcinwe ku-1:1.Kwimifuniselo yokubopha i-titration ngokungabikho kokuxinana, i-TEMPOL-122-αS yagcinwa kwi-50 μM kunye ne-polycations yayifakwe kwi-titrated ekugxininiseni okunyukayo, ukulungiselela imeko nganye ngokwahlukileyo.Imilinganiselo ye-CW-EPR yenziwe kusetyenziswa i-Bruker ELEXSYS E580 X-band spectrometer ene-Bruker ER4118 SPT-N1 resonator esebenza kwi-microwave (SHF) frequency ~ 9.7 GHz.Iqondo lobushushu lalibekwe kwi-25°C kwaye lilawulwa yi-nitrogen cryostat engamanzi.Umboniso ufunyenwe phantsi kweemeko ezingaxutywanga kumandla we-MW we-4 mW, i-modulation amplitude ye-0.1 mT, kunye ne-modulation frequency ye-100 kHz.Amandla e-Spectral ayenziwa njengesiqhelo ukuphepha ukungafani koxinzelelo lwe-spin phakathi kweesampulu kunye nokunciphisa okunokwenzeka kwe-spin ngenxa ye-residual concentrations ye-agent zokunciphisa kwiisampuli eziqulethe i-Tau441 okanye i-ΔNt-Tau (ekhoyo kwiisombululo zeprotheni zangaphambili).Amaxabiso anikiweyo e-g afunyenwe ngenxa ye-EPR ye-spectral modeling eyenziwa kusetyenziswa i-software ye-Easyspin (v. 6.0.0-dev.34) ephunyezwe kwiMatlab®67.Imodeli enye/ezimbini ze-isotropic zisetyenziselwe ukulinganisa idatha.Emva kokulungelelanisa yonke imiqondiso, iintsalela zibalwe ngokususa ukulinganisa ngalunye kwi-spectrum yovavanyo oluhambelanayo.Uhlalutyo lwe-titration olubophelelayo, ukuqina okuhambelanayo kwebhanti yesithathu ukuya kwinqanaba lesibini le-EPR yesiqhelo (IIII / III) yayisetyenziselwa ukubeka iliso kwi-polycations kwi-αS.Ukuqikelela i-dissociation constant (Kd), ijika elinesiphumo lifakelwe kumzekelo oqikelelweyo kucingwa ukuba n iziza ezibophelelayo ezifanayo nezizimeleyo.
Imifuniselo ye-NMR ye-spectroscopy yenziwa kusetyenziswa i-Bruker Neo 800 MHz (1H) i-NMR ye-spectrometer exhotyiswe nge-cryoprobe kunye ne-Z-gradient.Zonke iimvavanyo zenziwa kusetyenziswa i-130-207 µM αS kunye ne-αS / ΔNt-Tau ehambelanayo kunye ne-pLK elinganayo kwi-10 mM HEPES, i-100 mM NaCl, i-10% DO, i-pH 7.4 kwaye yenziwa kwi-15 ° C.Ukubeka iliso kwi-LPS yi-NMR, i-10% ye-PEG yongezwa kwiisampuli ezixutywe kwangaphambili.Isicwangciso sokuphazamiseka kweekhemikhali (Umfanekiso 1b) ubonisa umyinge we-1H kunye ne-15N yeekhemikhali zokutshintsha.I-spectra ye-αS 2D1H-15N HSQC yabelwe ngokusekelwe kwisabelo sangaphambili (i-BMRB entry #25227) kwaye iqinisekiswe ngokurekhoda nokuhlalutya i-spectra ye-3D ye-HNCA, i-HNCO kunye ne-CBCAcoNH.I-13Cα kunye ne-13Cβ ukutshintshwa kweekhemikhali kubalwa phambi kobukho be-ΔNt-Tau okanye i-pLK ukulinganisa utshintsho olunokwenzeka kwiindlela zesakhiwo sesibini xa kuthelekiswa nokutshintshwa kweekhemikhali ze-αS kwi-coil random conformation 68 (i-Supplementary Figure 5c).Amazinga e-R1ρ alinganiswa ngokurekhoda imifuniselo ye-hsqctretf3gpsi (efunyenwe kwithala leencwadi laseBruker) ngolibaziseko lwe-8, 36, 76, 100, 156, 250, 400, kunye ne-800 ms, kunye nemisebenzi ye-exponential yahlengahlengiswa ukuya kwincopho yobunzima obahlukeneyo. amaxesha okumisela i-R1ρ kunye nokungaqiniseki kovavanyo.
Imifuniselo ye-fluorescence microscopy enemibala emibini esonjululwe ngexesha lentengiso yenziwa kwi-MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) enesixhobo sokubala ifotoni enye (TCSPC) ehambelana nexesha.Intloko ye-laser diode isetyenziselwa i-pulsed interleaved excitation (PIE), i-beam idlula kwimowudi ye-waveguide enye kwaye iguqulelwe kumandla we-laser we-10 ukuya kwi-100 nW ye-481 nm kunye ne-637 nm imigca ye-laser elinganiswa emva kwesibuko se-dichroic.Oku kuqinisekisa elona nqanaba lokubala lefoton, ukuthintela iziphumo zefoton aliasing, i-photobleaching kunye ne-saturation.μ-Izilayidi ze-angiogenesis ezigubungelayo okanye iipleyiti (Ibidi GmbH, Gräfelfing, eJamani) zafakwa ngokuthe ngqo emanzini antywiliselwa phezu kwe-Super Apochromat 60x NA 1.2 lens enekhola yokulungisa (i-Olympus Life Sciences, Waltham, USA).Isibuko se-488/640 nm dichroic (i-Semrock, iLake Forest, i-IL, i-USA) isetyenziswe njengeyona nto ingundoqo yokuhlukanisa i-boam.I-radiation engagxilwanga ivalwe ngumngxuma onobubanzi be-microns ye-50, emva koko i-radiation egxininisiweyo ihlulwe kwiindlela ezi-2 zokufumanisa nge-50/50 ye-beam splitter.I-Bandpass emission filters (Semrock, Lake Forest, IL, USA) 520/35 yedayi eluhlaza (AF488) kunye ne-690/70 yedayi ebomvu (Atto647N) isetyenziswe phambi komtshini.I-single-photon avalanche diodes (SPAD) (Izixhobo ze-Micro Photon, i-Bolzano, i-Italy) zisetyenziswe njengezixhobo zokubona.Zombini uqokelelo lwedatha kunye nohlalutyo lwenziwa kusetyenziswa isoftware yeSymphoTime64 ekhoyo ngokurhweba (PicoQuant GmbH, Berlin, Germany).
Amashumi amahlanu eemicroliters eisampulu zeLLPS afakwe kwi-μ-Slide angiogenesis imithombo (Ibidi GmbH, Gräfelfing, Germany).Imifanekiso enesiphumo igxininiswe kwi-20 µm ngaphezulu komzantsi wequla ngenjongo yowona mgama wokusebenza wamathontsi amisiweyo kunye ne ~1 µm yokuhlenga kunye namachaphaza anesisombululo se-axial ubuncinane se-0.25 µm/pixel kunye nexesha lokulibaziseka lama-400 µs/pixel.Khetha idata ngokusebenzisa i-intensitem threshold esekelwe kumndilili wobunzulu bomqondiso ongasemva (PBG, ithetha + 2σ) kumjelo ngamnye ukuze kukhethwe kuphela amathontsi eprotheyini yolwelo, iirafti, okanye amabala, ukuhluza nayiphi na imvelaphi enokwenzeka ukusuka kwinqanaba elisasaziweyo.Ukuhlalutya ubomi bohlobo ngalunye (τ) lwetshaneli nganye (eluhlaza, “g” ye-AF488 kunye nebomvu, “r” ye-Atto647N), sikhethe iingingqi zomdla (ROIs) eziqulathe amathontsi, iirafti, okanye amabala (Figure Supplementary Figure 1) ).8b) kwaye zafunyanwa ngokuzifakela ukubola kwazo zonke ubomi (τD, τR kunye ne tP kumathontsi, iirafti okanye amabala, ngokulandelelanayo, bona iFig eyoNgezelelweyo 8c) kumjelo ngamnye usebenzisa uhlalutyo olulungele umsila kunye nemodeli yokubola yamacandelo amabini.Umyinge τ ukusuka ku τ .Ii-ROI ezivelise iifotoni ezimbalwa kakhulu kwi-multi-exponential fit zazingabandakanywa kuhlalutyo.I-cutoff esetyenzisiweyo yayiyi-<104 photons ye-rafts kunye namachaphaza kunye ne-103 yamaconsi.Amathontsi anomda osezantsi ngenxa yokuba kunzima ukufumana iigophe zokubola kunye namaxabiso aphezulu aphezulu, kuba amathontsi kwindawo yomfanekiso aqhele ukuba mancinci kwaye abe maninzi.I-ROIs kunye ne-photon counts ngaphezu komda wokuqokelela i-photon (ukusetha> ukubala kwe-500 / i-pixel) nazo zalahlwa ukuhlalutya.Tshatisa igophe lokubola koxinzelelo olufunyenwe kummandla womdla ngokuqina kwi-90% yowona mgangatho uphezulu (kancinane emva kowona mandla uphezulu wokubola) ukususela ekuqaleni kobomi benkonzo ukuqinisekisa uphazamiseko oluncinci lwe-IRF lo gama lugcinwe lufana kuko konke ukubola okukhulu. izicwangciso Iwindow ixesha Relative zahlalutywa 25 ukuba 50 ROI for rafts kunye namabala kunye 15-25 ROI amathontsi, imifanekiso ekhethiweyo ukusuka ngaphezu 4 replicates erekhodiweyo ubuncinane 3 imifuniselo ezizimeleyo.Uvavanyo lwe-t olunemisila emibini lusetyenziselwe ukuvavanya ukungafani kwezibalo phakathi kweentlobo okanye phakathi kweenkqubo ze-coacervate.Kuhlalutyo lwepixel ngepixel yobomi bonke ( τ ), ukuthotywa ngokupheleleyo kobomi phezu kwendawo yetshaneli nganye kubalwa kwaye uqikelelo lwe-2/3-inxalenye yemodeli yokunciphisa i-exponential yenziwa.Ukunciphisa ubomi bepixel nganye emva koko kwafakwa kusetyenziswa amanani angaphambili abalwe τ, okukhokelela kumfanekiso ofanelekileyo we-FLIM wepseudocolor.Uluhlu lwexesha lokuphila lomsila lwalufana kuyo yonke imifanekiso yetshaneli enye, kwaye ukubola ngakunye kwavelisa iifotoni ezaneleyo zokubonelela ngokuthembekileyo.Uhlalutyo lwe-FRET, iipikseli zikhethwe ngokufaka umyinge osezantsi wokuqina kweefotoni ze-100, ezilinganisela isignali yangasemva (FBG) yeefotoni ze-11.Ukuqina kwe-fluorescence yeshaneli nganye yalungiswa ngokulungiswa kwezinto ezichanekileyo: i-69 spectral crosstalk α yayiyi-0.004, i-excitation ngqo β yayingu-0.0305, ukufumanisa ukusebenza kakuhle γ kwakuyi-0.517.Ukusebenza kwe-FRET kwinqanaba le-pixel emva koko kubalwa ngokusebenzisa le equation ilandelayo:
apho i-FDD i-fluorescence intensity ebonwa kumxhasi (oluhlaza), i-FDA yintambo ye-fluorescence ebonwa kwijelo elamkelekileyo (elibomvu) phantsi kwe-excitation engathanga ngqo, kunye ne-FAA yi-fluorescence intensity ebonwa kwijelo lokwamkela (obomvu) phantsi kwe-excitation ngqo ( I-PIE).I-Fluorescence intensity pulses ibonwa kwitshaneli).
Beka i-100 µl yezisombululo ze-LLPS zokuphendula eziqulathe i-25 µM engabhalwanga i-monomeric Tau441 (ene-25 µM αS okanye ngaphandle kwayo) kwi-LLPS buffer (eyongeziweyo njengasentla) kwii-microplates ezingabophiyo ezingama-96-ezaneleyo ezinefoyile yokuncamathelisa kunye nokwakheka kwe-droplet yakhangelwa emva kwe-WF ulungelelwaniso.phakathi kwe-10 min.Emva kweeyure ezingama-48 zokufukamela kwiqondo lobushushu begumbi, ubukho beprotein rafts kunye namabala buqinisekisiwe.Emva koko susa ngononophelo ulwelo phezu kweerafti ezivela equleni, uze udibanise i-50 L ye-dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) kwaye ufukamele i-10 min.Uxinzelelo lwetyuwa oluphezulu luqinisekisa ukuba i-LLPS ayiyi kuphinda ngenxa ye-PEG eshiyekileyo, kwaye iindibano zeprotheyini ezinokwenzeka ezenziwe kuphela ngokudibana kwe-electrostatic ziya kuchithwa.Umzantsi wequla wawukhutshiwe ngononophelo ngencam ye-micropipette kwaye isisombululo esisiphumo saye sagqithiselwa kwindawo engenanto yokujonga.Emva kokufakwa kweesampulu nge-50 μM ThT kwi-1 h, ubukho beendawo ezizimeleyo zajongwa yi-WF microscopy.Lungiselela i-sonicated αS fibrils ngokufaka i-300 µl yesisombululo se-70-µM αS kwi-PBS kunye ne-pH 7.4, i-sodium azide 0.01% kwi-37 ° C kunye ne-200 rpm kwi-orbital shaker kwiintsuku ze-7.Isisombululo sabe se-centrifuged kwi-9600 × g ye-30 min, i-pellet yabuyiselwa kwi-PBS pH 7.4 kunye ne-sonicated (i-1 min, i-50% umjikelezo, i-80% amplitude kwi-Vibra-Cell VC130 sonicator, Sonics, Newton, USA) iisampulu zefibril. kunye nokuhanjiswa kobungakanani obufanayo beefibrili ezincinci.
Uhlalutyo lwe-FCS/FCCS kunye nobhaqo lwemibala emibini yokudibana (TCCD) lwenziwe kwangexesha leMT200 le-fluorescent confocal microscope (Pico-Quant, Berlin, Germany) esetyenziselwa imifuniselo ye-FLIM-FRET microscopy kusetyenziswa imo ye-PIE.Amandla e-laser kwezi zilingo zongezwa kwi-6.0 µW (481 nm) kunye ne-6.2 µW (637 nm).Indibaniselwano yala mandla e-laser yakhethwa ukuvelisa ukuqaqamba okufanayo kwizibini ze-fluorophores ezisetyenziswayo ngelixa kuphunyezwa awona mazinga afanelekileyo okubala kunye nokuphepha ukufota kunye nokugcwala.Zombini ukuqokelelwa kwedatha kunye nohlalutyo lwenziwa kusetyenziswa isoftware yeSymphoTime64 version 2.3 efumanekayo yorhwebo (PicoQuant, Berlin, Germany).
Iisampulu zeaggregates ezikwanti ze-αS/Tau ezifunyenwe kusetyenziswa i-LLPS zihlanjululwe kwisithinteli sokwahlula ukuya kugxininiso olufanelekileyo lwe-monomolecular (ngokuqhelekileyo i-1:500 dilution, kuba ii-aggregates sele zikwindawo ephantsi xa zibekwe zodwa kwiisampulu ze-coacervate).Iisampulu zisetyenziswe ngokuthe ngqo kwi-coverlips (i-Corning, e-USA) ifakwe ngaphambili kunye nesisombululo se-BSA kwi-concentration ye-1 mg / mL.
Kuhlalutyo lwe-PIE-smFRET kwitshaneli eziluhlaza kunye nezibomvu, umlinganiselo osezantsi wokuqina weefotoni ezingama-25 zasetyenziswa ukuhluza imiqondiso ephantsi ebangelwa ziziganeko ze-monomeric (qaphela ukuba ii-monomers zininzi ukodlula iisampulu ezidityanisiweyo xa kuthelekiswa neeaggregates ezizimeleyo).Lo mda ubalwa ngokuphindwe kahlanu ubukhulu be-avareji ye-αS ye-monomeric efunyenwe kuhlalutyo lweesampulu ze-monomer ecocekileyo ukuze kukhethwe ngokuthe ngqo ii-aggregates zohlalutyo.Isekethe ye-PIE yokuqhuba, kunye ne-TSCPC yokufumana idatha, yenze ukuba usetyenziso lwesihlungi sobunzima bobomi sinceda ukuphelisa imvelaphi kunye ne-spectral crosstalk.Ubunzulu bedangatye obukhethiweyo kusetyenziswa le mida ingasentla bulungiswe kusetyenziswa umqondiso ophakathi oqikelelwe ukusuka kwiihistograms zokwenzeka ngokuchasene nobunzulu/umgqomo weesampulu zesithinteli-kuphela.Ukugqabhuka okunxulumene neeaggregates ezinkulu ngokuqhelekileyo kuhlala imigqomo emininzi elandelelanayo ngexesha lomkhondo (usetelwe ku-1 ms).Kwezi meko, kwakhethwa umgqomo wamandla aphezulu.Uhlalutyo lwe-FRET kunye ne-stoichiometric, i-gamma factor enqunywe ngokwethiyori γ (0.517) isetyenzisiwe.I-Spectral crosstalk kunye neminikelo yokuvuselela ngokuthe ngqo ayinanto (inqunywe ngovavanyo) kumandla e-laser yokuvuselela asetyenziswayo.Ukusebenza kakuhle kunye ne-stoichiometry yeFRET kuqhushumbo kubalwa ngolu hlobo lulandelayo.