347 12.7*1.24mm Ishubhu ehlanganisiwe yensimbi engagqwali, Indlela ye-molecular yokufingqa kwe-electrostatic condensation kanye nokuhlanganisa kwe-α-synuclein ne-tau

Siyabonga ngokuvakashela i-Nature.com.Usebenzisa inguqulo yesiphequluli enosekelo olulinganiselwe lwe-CSS.Ukuze uthole ulwazi olungcono kakhulu, sincoma ukuthi usebenzise isiphequluli esibuyekeziwe (noma ukhubaze i-Compatibility Mode ku-Internet Explorer).Ngaphezu kwalokho, ukuze siqinisekise ukwesekwa okuqhubekayo, sibonisa isayithi ngaphandle kwezitayela ne-JavaScript.
Izilayidi ezibonisa izindatshana ezintathu zesilayidi ngasinye.Sebenzisa izinkinobho ezingemuva nezilandelayo ukuhamba phakathi kwamaslayidi, noma izinkinobho zokulawula ama-slide ekugcineni ukuze uhambe kuslayidi ngasinye.

347 Ukucaciswa Kwepayipi Lensimbi Engagqwali

347 12.7*1.24mm Insimbi engagqwali eboshiwe yamashubhu

Ngaphandle Ububanzi: 6.00 mm OD kufika ku-914.4 mm OD, Osayizi abangafika ku-24” NB abatholakalayo Ex-stock, OD Size Steel Tubes atholakalayo Ex-stock

SS 347 Ibanga Lokuqina Kwepayipi: 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, njll. (0.5-12mm) Noma usayizi ongajwayelekile uzolungiswa njengoba kudingeka

Uhlobo: SS 347 Amapayipi Angenamthungo |SS 347 ERW Amapayipi |SS 347 Welded Pipes |SS 347 Fabricated Pipes |Amashubhu e-SS 347 CDW, Amapayipi e-LSAW / Athungelwe ngomthungo / Adwetshwe kabusha

Ifomu: 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: Okungahleliwe Okukodwa, Okungahleliwe Okuphindwe Kabili & Ukuphela Kobude Obudingekayo: Isiphetho Esingenalutho, Isiphetho Esihlanganisiwe, Kunyatheliwe

Ukuvikela Kokugcina: Ofeleba Bepulasitiki |Ngaphandle Finish: 2B, No.4, No.1, No.8 Mirror Finish for Stainless Steel Pipes, Qeda ngokwezimfuneko zekhasimende ngalinye

Isimo Sokulethwa: I-Annealed and Pickled, Polishiwe, I-Bright Annealed, Cold Drawn

Ukuhlolwa, Imibiko Yokuhlola: Izitifiketi Zokuhlola Imishini, EN 10204 3.1, Imibiko Yekhemikhali, Imibiko Yemishini, Imibiko Yokuhlola Ye-PMI, Imibiko Yokuhlola Okubonakalayo, Imibiko Yokuhlola Yenkampani Yangaphandle, Imibiko Yelebhu Egunyazwe I-NABL, Umbiko Wokuhlola Okulimazayo, Imibiko Yokuhlola Engabhidli

Ukupakisha: Ipakishwe Emabhokisini Okhuni, Izikhwama Zepulasitiki, Imichilo Yensimbi Ehlanganisiwe, noma ngokwezicelo zeKhasimende

Okukhethekile: Osayizi kanye Nemininingwane engaphandle kwalokhu okungenhla kungenziwa ngesicelo

I-SS 347 Ibanga Losayizi Wepayipi:1/2 intshi NB, OD ukuya kumayintshi angama-24

I-ASTM A312 347: Ipayipi le-austenitic elishiselwe elingenamthungo neliqondile elihloselwe izinga lokushisa eliphezulu kanye nensizakalo yokugqwala evamile.I-filler metal ayivunyelwe ngesikhathi sokushisela.

I-ASTM A358 347: Ukuhlanganiswa kukagesi okushiselwe ipayipi le-austenitic lesevisi ebolayo kanye/noma izinga lokushisa eliphezulu.Ngokuvamile ipayipi kuphela elifika kumayintshi angu-8 akhiqizwa kulokhu kucaciswa.Ukwengezwa kwe-filler metal kuvunyelwe ngesikhathi sokushisela.

I-ASTM A790 347: Ipayipi elishiselwe elingenamthungo neliqondile le-ferritic/austenitic (duplex) elihloselwe isevisi yokugqwala evamile, egcizelela ngokukhethekile ukumelana nokuqhekeka kokugqwala kwengcindezi.

I-ASTM A409 347: Ukuhlanganisa kagesi okunomthungo oqondile noma okuvunguzayo kushiselwe ipayipi elikhulu lodonga olukhanyayo lwe-austenitic ngobukhulu obungu-14” ukuya ku-30” elinezindonga i-Sch5S kanye ne-Sch 10S yokubola kanye/noma ukuphakama.

I-ASTM A376 347: Ipayipi le-austenitic elingenamthungo lezicelo zokushisa okuphezulu.

I-ASTM A813 347: Umthungo owodwa, ipayipi le-austenitic elishiselwe olulodwa noma kabili izinga lokushisa eliphezulu kanye nezinhlelo ezijwayelekile zokugqwala.

I-ASTM A814 347: Ipayipi le-austenitic elishiselwe elibandayo elishiselwe izinga lokushisa eliphezulu kanye nesevisi yokugqwala evamile.

I-347H I-Stainless Steel Pipes Ukwakheka Kwamakhemikhali

Insimbi Engagqwali 347H Ipayipi Mechanical Properties

I-Stainless Steel 347H Amapayipi Izakhiwo Zomzimba

Amabanga alinganayo 347H Stainless Steel Pipe

Ukuhlanganiswa kwe-Amyloid alpha-synuclein (αS) kuwuphawu lwesifo sikaParkinson kanye namanye ama-synucleinopathies.Muva nje, iphrotheni ye-tau evame ukuhlotshaniswa nesifo se-Alzheimer iye yahlotshaniswa ne-αS pathology futhi yatholakala ihlanganisa indawo ekufakweni okucebile kwe-αS, nakuba indlela yamangqamuzana yokuhlanganisa amaprotheni amabili ingakacaci.Sibika lapha ukuthi isigaba se-αS sihlukana sibe ama-condensate anguketshezi nge-electrostatic complex condensation nama-polypeptides ashajwe kahle njenge-tau.Ngokuya ngokuhambisana kwe-αS ye-polycations kanye nezinga lokuncipha kwe-valence yenethiwekhi ye-coagulation, amahlule adlula i-gelation esheshayo noma ukuhlangana okulandelwa ukuhlanganisa kancane kwe-amyloid.Ngokuhlanganisa iqoqo lamasu athuthukile e-biophysical, sikwazile ukuveza ukuhlukaniswa kwesigaba se-αS/Tau esiwuketshezi oluwuketshezi futhi sihlonze izici ezibalulekile eziholela ekwakhekeni kwama-aggregate ahlukahlukene aqukethe womabili amaprotheni ku-condensate yephrotheni ewuketshezi.
Ngaphezu kwezingxenye zemembrane, ukuhlukaniswa kwendawo kumaseli kungazuzwa ngokwakhiwa kwemizimba eminyene enamaprotheni, efana noketshezi ebizwa ngokuthi ama-biomolecular condensates noma amaconsi, ngenqubo eyaziwa ngokuthi i-liquid-liquid phase separation (LLPS).Lawa maconsi akhiwa ukusebenzisana kwesikhashana kwe-multivalent, ngokuvamile phakathi kwamaprotheni noma amaprotheni kanye ne-RNA, futhi asebenza imisebenzi ehlukahlukene cishe kuzo zonke izinhlelo eziphilayo.Inani elikhulu lamaprotheni anamandla e-LLP abonisa ukulandelana kobunzima obuphansi obuphazamisekile kakhulu emvelweni nasekubunjweni kwe-biomolecular condensates3,4,5.Ucwaningo oluningi lwembula ukuguquguquka, okuvame ukuphazamiseka, kanye nemvelo ehlukahlukene yamaphrotheni akha lawa ma-condensate afana noketshezi, nakuba kuncane okwaziwayo ngezinqumo ezithile zamangqamuzana ezilawula ukukhula nokuvuthwa kwalawa ma-condensate abe aqine kakhulu. isimo..
Idatha entsha isekela umbono wokuthi i-LLPS eqhutshwa ngamaprotheni ehlanekezelwe kanye nokuguqulwa kwamaconsi abe izakhiwo eziqinile kungase kube izindlela ezifanele zamaselula eziholela ekwakhekeni kwezinhlanganisela ezinobuthi ezingancibilikiyo ezivame ukuba yizimpawu zezifo eziwohlokayo.Amaprotheni amaningi ahlotshaniswa ne-LLPS-intrinsically disordered (IDPs), avame ukushajwa kakhulu futhi aguquguqukayo, sekuyisikhathi eside ehlotshaniswa ne-neurodegeneration ngenqubo ye-amyloid aggregation.Ikakhulukazi, ama-biomolecular IDP condensates afana ne-FUS7 noma i-TDP-438 noma amaprotheni anezizinda ezinkulu eziyinkimbinkimbi eziphansi njenge-hnRNPA19 akhonjiswe ukuguga abe amafomu afana nejeli noma aqinile ngenqubo ebizwa ngokuthi i-fluidization.inhlanganisela.kunguquko yesigaba esiqinile (LSPT) njengomsebenzi wesikhathi noma ekuphenduleni ukuguqulwa okuthile kwangemva kokuhumusha noma izinguquko ezibalulekile ze-pathologically1,7.
Enye i-IDP ehlotshaniswa ne-LLPS ku-vivo i-Tau, iphrotheni ephazamisekile ehlobene ne-microtubule okuhlanganiswe kwayo kwe-amyloid kuye kwathinteka ku-Alzheimer's disease10 kodwa futhi muva nje kufakwe i-Parkinson's disease (PD) kanye namanye ama-synaptic nuclear proteinopathies 11, 12, 13 ahlobene.I-Tau iboniswe ukuthi iyazihlukanisa ngokuzenzakalelayo nesisombululo/i-cytoplasm ngenxa yokusebenzisana okuhle kwe-electrostatic14, okuholele ekwakhekeni kwamaconsi anothiswe i-tau aziwa ngokuthi ama-electrostatic coacervates.Kuphinde kwaphawulwa ukuthi lolu hlobo lokusebenzelana okungaqondile luyimbangela yabaningi be-biomolecular condensates emvelweni15.Esimeni sephrotheni ye-tau, ukuhlanganisa kwe-electrostatic kungakhiwa ngokuhlanganisa okulula, lapho izifunda ezishaje ngokuphambene zeprotheni zicupha inqubo yokuqhekeka, noma ngokuhlanganisa okuyinkimbinkimbi ngokusebenzisana namapholima ashajwe kabi njenge-RNA.
Muva nje, i-α-synuclein (αS), i-IDP ye-amyloid ehilelekile ku-PD nezinye izifo ze-neurodeergenerative ezaziwa ngokuhlanganyela ngokuthi i-synucleinopathy17,18, iboniswe kumamodeli weselula nezilwane19,20 egxilwe ku-protein condensates ngokuziphatha okufana noketshezi.Ucwaningo lwe-in vitro lubonise ukuthi i-αS idlula i-LLPS ngokuhlanganisa okulula ngokusebenzisana ngokuyinhloko kwe-hydrophobic, nakuba le nqubo idinga ukugxila kwamaprotheni aphezulu kakhulu kanye nezikhathi zokufukamela ezinde ngokungajwayelekile19,21.Ukuthi ama-alpha-condensate aqukethwe ku-vivo akhiwa yilokhu noma ezinye izinqubo ze-LLPS kuhlala kuyindaba eyinhloko engaxazululiwe.Ngokufanayo, nakuba ukuhlanganiswa kwe-αS amyloid kuye kwabonwa kuma-neurons ku-PD nakwamanye ama-synucleinopathies, indlela eqondile lapho i-αS ithola i-intracellular amyloid aggregation ihlala ingacacile, njengoba ukucindezeleka okukhulu kwaleli phrotheni kungabonakali kubangela le nqubo ngokwayo.Ukulimala okwengeziwe kweselula kuvame ukudingeka, okuphakamisa ukuthi izindawo ezithile zamaselula noma imvelo encane iyadingeka ukuze kukhishwe kabusha ama-intracellular αS amyloid assemblies.Indawo eyodwa yamaselula ejwayele ukuhlanganisana ingase ibe ingaphakathi lama-protein condensates 23.
Kuyathakazelisa ukuthi i-αS ne-tau zitholakale zihlangene ekufakweni kwesifo kubantu abane-Parkinson's disease kanye nezinye i-synucleinopathies 24,25 kanye nokuhlolwa kubike ubudlelwano be-synergistic pathological phakathi kwamaprotheni amabili i-26,27 ephakamisa ubuhlobo obungaba khona phakathi kwe-aggregation αS kanye. izifo ze-neurodegenerative.ukugula.I-αS ne-tau zitholakale zisebenzisana futhi zikhuthaza ukuhlanganiswa komunye nomunye ku-vitro kanye ne-vivo 28,29 kanye ne-heterogeneous aggregates eyakhiwe ngalawa maprotheni amabili abonwe ebuchosheni beziguli ezine-synucleinopathies 30.Nokho, kuncane okwaziwayo ngesisekelo samangqamuzana okusebenzelana phakathi kwe-αS ne-tau kanye nendlela yokuhlanganisa kwayo.I-αS kubikwe ukuthi isebenzisana ne-tau ngokukhangwa kwe-electrostatic phakathi kwesifunda se-C-theminali egcwele kabi kakhulu ye-αS kanye nesifunda esimaphakathi esicebile se-proline ye-tau, ebuye inothiswe ngezinsalela ezishajelwe kahle.
Kulolu cwaningo, sibonisa ukuthi i-αS ingakwazi ngempela ukuhlukana ibe amaconsi ngokusebenzisa ukufingqa kwe-electrostatic complex condensation lapho kukhona amaprotheni e-tau, ngokungafani nokusebenzelana kwayo namanye ama-polypeptide ane-positively charged njenge-poly-L-lysine (pLK), futhi kule nqubo .I-αS isebenza njenge-molecule ye-scaffold yenethiwekhi yamaconsi.Sihlonze umehluko obonakalayo enqubweni yokuvuthwa kwama-coacervate e-alphas e-electrostatic, ahlotshaniswa nokwehluka kwe-valency namandla okusebenzelana kwamaprotheni ahilelekile kunethiwekhi ye-coacervate.Kuyathakazelisa ukuthi sibonile ukuhlanganisa amaprotheni e-αS kanye ne-tau amyloid kuma-coacervate oketshezi ahlala isikhathi eside futhi sahlonza izici ezithile ezibalulekile eziholela ekuhlanganisweni kwalawa maprotheni amabili ku-coacervates enjalo.Lapha sichaza ngokuningiliziwe le nqubo, okungase kube indlela yamangqamuzana engaphansi kwe-colocalization yamaprotheni amabili ekufakweni okuqondene nezifo.
I-αS inomsila we-C-terminal one-anionic kakhulu ku-pH engathathi hlangothi (Fig. 1a), futhi sacabanga ukuthi ingase ibhekane ne-LLPS ngokusebenzisa ukufingqa kwezakhiwo ze-electrostatic ezinama-polycationic disordered polypeptide molecule.Sisebenzise i-poly-L-lysine (pLK) eyi-100-residue njengemodeli yokuqala ngenxa yemvelo yayo ye-polymeric egcwele icala futhi ephazamisekile ku-pH 32 engathathi hlangothi. Okokuqala, siqinisekise ukuthi i-pLK isebenzisana nesizinda se-Ct se-αS nge-Solution NMR spectroscopy. (Umfanekiso 1b) usebenzisa i-13C / 15N-ebhalwe αS phambi kokwanda kwe-αS: pLK izilinganiso ze-molar.Ukusebenzisana kwe-pLK nesizinda se-Ct se-αS kuzibonakalisa ekuphazamisekeni kokushintsha kwamakhemikhali kanye nokuncipha komthamo omkhulu kule ndawo yamaprotheni.Kuyathakazelisa ukuthi lapho sixuba i-αS ne-pLK ekugxiliseni kwe-αS cishe.5–25 µM ebukhoneni be-polyethylene glycol (5–15% PEG-8) (ibhafa ye-LLPS ejwayelekile: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) ngokushesha sidlule emkhakheni obanzi wokwakheka kwamaprotheni .amaconsi abonwa kusetshenziswa i-fluorescence (WF) kanye ne-bright-field (BF) microscopy (Fig. 1c).Amaconsi angu-1-5 µm aqukethe i-αS egxilile (ingezwe i-1 µM AlexaFluor488-ebhalwe ukuthi αS, AF488-αS), izakhiwo zawo ze-electrostatic zingatholakala ekuphikisweni kwawo ku-10% 1,6-hexanediol (1,6-HD) kanye nokuzwela kwayo ukwanda kwe-NaCl concentration (Fig. 1c).Imvelo efana ne-fluid ye-coacervates ye-αS/pLK electrostatic complex iboniswa ikhono layo lokuhlanganisa ngaphakathi kwama-millisecond (Fig. 1d).Ngokusebenzisa i-turbidimetry, silinganise ukwakheka kwamaconsi ngaphansi kwalezi zimo, saqinisekisa imvelo ye-electrostatic yokuxhumana okuyinhloko okuhlobene nokuzinza kwayo (Fig. 1e), futhi sahlola umphumela wezilinganiso ezihlukahlukene ze-polymer kunqubo ye-LLPS (Fig. 1f).Nakuba ukwakheka kwamaconsi kubonakala phezu kohlu olubanzi lwezilinganiso ze-polymer, inqubo ivuma kakhulu uma i-pLK ingaphezu kwe-αS.Ama-LLP aphinde abonwa kusetshenziswa i-ejenti ekhipha amakhemikhali ehlukile i-dextran-70 (70 kDa) noma kusetshenziswa amafomethi esampula ahlukahlukene, okuhlanganisa amaconsi amaslayidi engilazi, imithombo ye-microplate yezinto ezihlukahlukene, i-Eppendorf noma i-quartz capillaries.
ukumelwa okuhleliwe kwezifunda ezihlukene zamaprotheni kokuhluka kwe-WT-αS kanye ne-ΔCt-αS okusetshenziswe kulolu cwaningo.Isizinda se-amphipathic N-terminal, isifunda se-hydrophobic amyloid-forming (NAC), kanye nesizinda se-C-terminal esikhokhiswe kabi kuboniswa ngokuluhlaza okwesibhakabhaka, okuwolintshi, nokubomvu, ngokulandelana.I-Net Charge Per Residual (NCPR) imephu ye-WT-αS iyaboniswa.b Ukuhlaziywa kwe-NMR kokusebenzisana kwe-αS/pLK ngokungabikho kwama-macromolecular clumps.Njengoba i-pLK ikhula (izilinganiso ze-pLK ze-molar ezingu-1:0.5, 1:1.5, no-1:10 ziboniswa ngokuluhlaza okukhanyayo, okuluhlaza okotshani, nokuluhlaza okumnyama, ngokulandelana).c I-Coacervate αS/pLK (isilinganiso se-molar 1:10) ku-25 µM (1 µM AF488-enelebula αS noma i-Atto647N-enelebula i-pLK ye-WF imaging) kubhafa ye-LLPS (phezulu) noma ingezwe ngo-500 mM NaCl (phansi kwesokunxele 1) noma ngemva kwalokho) % 1,6-hexanediol (1,6-HD; ngezansi kwesokudla).Ibha yesikali = 20 µm.d Izithombe ezimele ezincane ze-BF droplet fusion ye-αS/pLK (i-molar ratio 1:10) ekuhlanganiseni okungu-25 μM;imicibisholo ibonisa ukuhlangana kwamaconsi angawodwana (imicibisholo ebomvu nephuzi) ibe ukuwa okusha (umcibisholo owolintshi) phakathi kuka-200 ms).Ibha yesikali = 20 µm.e Ukusabalalisa ukukhanya (ku-350 nm) ukuhlanganisa kwe-αS/pLK kubhafa ye-LLPS ngaphambi nangemuva kokwengezwa kwe-500 mM NaCl noma u-10% 1,6-HD ku-25 µM αS (N = 3 amasampula wokuphindaphinda, ukuchezuka okumaphakathi nakho kubonisiwe).f isithombe se-BF (phezulu) nokuhlaziywa kokuhlakazeka okukhanyayo (ku-350 nm, phansi) kokuhlanganisa kwe-αS/pLK ku-25 μM αS ngokukhuphuka kwe-αS:pLK isilinganiso se-molar (N = 3 isampula yokuphindaphinda, ukuchezuka kwencazelo nokujwayelekile nakho kubonisiwe).Ibha yesikali = 10 µm.Ibha yesikali esithombeni esisodwa ibonisa isikali sazo zonke izithombe kuphaneli eyodwa.Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Ngokusekelwe ekuqapheleni kwethu kwe-αS/pLK ye-electrostatic condensation eyinkimbinkimbi kanye nokubhekwa kwangaphambilini kwe-αS njenge-molecule yeklayenti le-tau/RNA condensate ngokusebenzisana okuqondile ne-tau31, sicabange ukuthi i-αS ne-tau zingase zihlukanise ngokuhlanganyela ne-solvent ingekho i-RNA. ukujiya.ngokusebenzisa ama-electrostatic complexes, futhi i-αS iyiphrotheni eyisikafula ku-αS/Tau coacervates (bona ukusatshalaliswa kwe-tau kwenkokhelo kuMfanekiso 2e).Siqaphele ukuthi lapho i-10 μM αS kanye ne-10 μM Tau441 (equkethe i-1 μM AF488-αS kanye ne-1 μM Atto647N-Tau, ngokulandelanayo) ixutshwa ndawonye kubhafa ye-LLPS, kwakheka kalula ukuhlanganisa amaprotheni aqukethe womabili amaprotheni, njengoba kubonwa imakroskopu ye-WF.(Umdwebo 2a).Ukuhlanganiswa kwamaprotheni amabili kumaconsi kwaqinisekiswa yi-confocal (CF) microscopy (Supplementary Fig. 1a).Ukuziphatha okufanayo kwabonwa lapho i-dextran-70 isetshenziswa njenge-aggregation agent (Supplementary Fig. 1c).Sisebenzisa i-PEG enelebula ye-FITC noma i-dextran, sithole ukuthi womabili ama-ejenti aminyene asatshalaliswa ngokulinganayo kuwo wonke amasampula, angabonisi ukuhlukaniswa noma ukuhlotshaniswa (I-Supplementary Fig. 1d).Kunalokho, kuphakamisa ukuthi kulolu hlelo bakhuthaza ukuhlukaniswa kwesigaba ngokusebenzisa imiphumela yokugcwala kwama-macromolecular, njengoba i-PEG iyi-ejenti yokugcwala ekhethwayo ezinzile, njengoba kubonakala kwezinye izinhlelo ze-LLP33,34.Lawa maconsi anothe ngamaprotheni abezwela ku-NaCl (1 M) kodwa hhayi ku-1,6-HD (10% v/v), eqinisekisa izici zawo ze-electrostatic (Supplementary Fig. 2a, b).Ukuziphatha kwabo okuwuketshezi kwaqinisekiswa ngokubheka imicimbi yokuhlanganisa i-millisecond kusetshenziswa i-BF microscopy (Fig. 2b).
izithombe ezincane ze-Confocal (CF) ze-αS/Tau441 coacervates kubhafa ye-LLPS (10 μM yeprotheni ngayinye, 0.5 μM ye-AF488 enelebula ethi αS kanye ne-Atto647N enelebula ethi Tau441).b Izithombe ezimele umehluko wokuphazamiseka (i-DIC) zemicimbi yokuhlanganisa amaconsi e-αS/Tau441 (10 μM ngephrotheni ngayinye).c Umdwebo wesigaba osuselwe ekuhlakazekeni kokukhanya (ku-350 nm) kwe-Tau441 LLPS (0–15 µM) lapho kungabikho (kwesokunxele) noma ukuba khona (kwesokudla) kuka-50 µM αS.Imibala efudumele ibonisa ukuhlakazeka okwengeziwe.d Ukuhlakazwa okukhanyayo kwamasampuli e-αS/Tau441 LLPS anokwanda kokugxilisana kwe-αS (Tau441 ku-5 µM, N = 2–3 isampula yokuphindaphinda njengoba kukhonjisiwe).e Ukumelwa okuhleliwe kokunye okuhlukile kwamaprotheni e-tau kanye nezifunda ezihlukene zephrotheni esetshenziswe kulolu cwaningo: isizinda se-N-terminal esikhokhiswe kabi (bomvu), isifunda esicebile se-proline (eluhlaza okwesibhakabhaka), isizinda esibophezela i-microtubule (MTBD, esigqanyiswe ngowolintshi), kanye I-amyloid-forming pair spiral.izifunda ze-filament (PHF) ezitholakala ngaphakathi kwe-MTBD (grey).I-Net Charge Per Residue (NCPR) imephu ye-Tau441 iyaboniswa.f Kusetshenziswa i-1 µM AF488 enelebula ethi αS ne-Atto647N enelebula ethi ΔNt-, kusetshenziswa i-1 µM AF488 enelebula ethi αS noma ΔCt-αS phambi kwe-ΔNt-Tau (phezulu, 10 µM ngeprotheni ngayinye) noma i-K18 (phansi µM5 ngephrotheni ngayinye, 0 µM5 phansi, ) ) ) ama-micrographs e-WF afingqiwe ku-LLPS noma kubhafa ye-K18.Amabha esikali esithombeni esisodwa amelela isikali sazo zonke izithombe kuphaneli eyodwa (20 µm wamaphaneli a, b kanye no-f).Idatha eluhlaza yamaphaneli u-c no-d anikezwa njengamafayela edatha eluhlaza.
Ukuhlola indima ye-αS kule nqubo ye-LLPS, siqale saphenya umthelela we-αS ekuzinzeni kwamaconsi nge-nephelometry sisebenzisa ukugxila okwandayo kwe-NaCl (Fig. 2c).Uma uphakeme ukugxiliswa kukasawoti kumasampuli aqukethe i-αS, ayanda amanani okuhlakazeka kokukhanya (ku-350 nm), okubonisa indima yokuzinza ye-αS kulolu hlelo lwe-LLPS.Umphumela ofanayo ungabonwa ngokwandisa ukugxila kwe-αS (ngakho-ke isilinganiso se-αS:Tau441) sifinyelele cishe.Ukukhuphuka okuphindwe ka-10 ngokuhlobene nokugxilisa kwe-tau (5 µM) (Fig. 2d).Ukuze sibonise ukuthi i-αS iyiprotheyini ye-scaffold kuma-coacervates, sinqume ukuphenya ukuziphatha kwe-LLPS-iphazamise i-Tau mutant, engenayo indawo ye-N-terminal enecala elibi (izinsalela 1-150, bheka i-Fig. 2e) ebizwa ngokuthi i-ΔNt-Tau.I-WF microscopy ne-nephelometry iqinisekise ukuthi i-ΔNt-Tau ngokwayo ayizange ingene kwi-LLPS (Fig. 2f kanye ne-Supplementary Fig. 2d), njengoba kubikwe ngaphambilini 14. Nokho, lapho i-αS yengezwa ezixazululweni zokuhlakazwa kwalokhu kwehluka kwe-Tau encishisiwe, inqubo ye-LLPS yayiphelele. kubuyiselwa ngokuminyana kwamaconsi eduze nokuminyana kwamaconsi ezixazululo ezinosayizi ogcwele we-Tau kanye ne-αS ngaphansi kwezimo ezifanayo nokugxila kwamaprotheni.Le nqubo ingabuye ibonwe ngaphansi kwezimo zokugcwala okuphansi kwe-macromolecular (Supplementary Fig. 2c).Iqhaza lesifunda se-C-terminal αS, kodwa hhayi ubude baso bonke, kunqubo ye-LLPS yaboniswa ngokuvimbela ukwakheka kwamaconsi kusetshenziswa okuhlukile kwe-C-terminal encishisiwe ye-αS entula izinsalela 104–140 (Fig. 1a) ye (ΔCt- αS) amaprotheni (Fig. 2f kanye Supplementary Fig. 2d).I-colocalization ye-αS ne-ΔNt-Tau yaqinisekiswa yi-confocal fluorescence microscopy (I-Supplementary Fig. 1b).
Ukuze kuqhutshekwe kuhlolwe indlela ye-LLPS phakathi kwe-Tau441 ne-αS, kwasetshenziswa okuhlukile kwe-Tau okwengeziwe, okuwucezu olubhanqiwe lwe-helical filament core (PHF) kusizinda esibophayo se-microtubule (MTBD), okuthi uma siqukethe izizinda ezine eziphindaphindayo, ezivame ukwaziwa futhi. njengocezu lwe-K18 (bheka umfanekiso 2e).Kusanda kubikwa ukuthi i-αS ibophezela ngokukhethekile kuphrotheni ye-tau etholakala kusizinda esicebile se-proline ngokulandelana okwandulela isizinda esibophezelayo se-microtubule.Kodwa-ke, isifunda se-PHF sicebile futhi ngezinsalela ezikhokhiswa kahle (bheka uMdwebo 2e), ikakhulukazi i-lysine (izinsalela ezingu-15%), okusishukumisele ukuba sihlole ukuthi lesi sifunda siphinde sibe nesandla yini ekujikeni kwenkimbinkimbi ye-αS/Tau.Siqaphele ukuthi i-K18 iyodwa ayikwazanga ukuqalisa i-LLPS ekugxilweni okufika ku-100 μM ngaphansi kwezimo ezihloliwe (i-LLPS buffer eno-15% PEG noma 20% dextran) (Umfanekiso 2f).Kodwa-ke, lapho sengeza i-50 µM αS ku-50 µM K18, ukwakheka okusheshayo kwamaconsi amaprotheni aqukethe i-K18 ne-αS kwabonwa yi-nephelometry (I-Supplementary Fig. 2d) kanye ne-WF microscopy (Fig. 2f).Njengoba bekulindelekile, i-ΔCt-αS ayikwazanga ukubuyisela ukuziphatha kwe-LLPS kwe-K18 (Fig. 2f).Siyaqaphela ukuthi ukuhlanganisa kwe-αS/K18 kudinga ukugxiliswa kwamaprotheni aphezulu kancane ukuze kunxenxe i-LLPS uma kuqhathaniswa ne-αS/ΔNt-Tau noma i-αS/Tau441, ezinye izinto ziyalingana.Lokhu kuhambisana nokusebenzisana okunamandla kwesifunda se-αS C-terminal nesizinda se-Tau esicebile se-proline uma kuqhathaniswa nesizinda sokubopha i-microtubule, njengoba kuchazwe ngaphambilini i-31.
Njengoba kunikezwe ukuthi i-ΔNt-Tau ayikwazi ukwenza i-LLPS ingekho i-αS, sikhethe lokhu kwehluka kwe-Tau njengemodeli yokubeka i-αS/Tau LLPS uma kubhekwa ubulula bayo ezinhlelweni ze-LLPS ezinobude obugcwele be-Tau (isotype, Tau441/Tau441).ngezinqubo zokuhlanganisa eziyinkimbinkimbi (i-heterotypic, αS/Tau441).Siqhathanise idigri ye-αS aggregation (njengengxenye yeprotheyini yesigaba esifingqiwe, i-fαS,c) ezinhlelweni ze-αS/Tau kanye ne-αS/ΔNt-Tau ngokubeka i-centrifugation nesigaba esihlakaziwe sokuhlaziywa kwe-SDS-PAGE (bona 2e), kutholwe amanani afanayo kakhulu. kuwo wonke amaprotheni ekugxilweni okufanayo.Ikakhulukazi, sithole i-faS,c 84 ± 2% kanye ne-79 ± 7% ye-αS/Tau kanye ne-αS/ΔNt-Tau, ngokulandelana, iphakamisa ukuthi ukusebenzisana kwe-heterotypic phakathi kwe-αS ne-tau kuphakeme kunokusebenzelana phakathi kwama-molecule e-tau.phakathi.
Ukusebenzisana nama-polycation ahlukahlukene kanye nomthelela wenqubo yokufingqa ku-αS kinetics kwaqale kwafundwa ngokutholwa kwe-fluorescence ngemva kwendlela ye-photobleaching (FRAP).Sihlole i-αS/Tau441, αS/ΔNt-Tau kanye ne-αS/pLK coacervates (100 μM αS engezwe ngo-2 μM αS AF488-αS kanye no-100 μM Tau441 noma ΔNt-Tau noma 1 mML pLK).Idatha itholwe phakathi kwemizuzu yokuqala ye-30 ngemuva kokuxuba izingxenye zesampula.Kusuka emifanekisweni ye-FRAP emele (I-Fig. 3a, αS/Tau441 condensation) kanye namajika esifundo sesikhathi esihambisanayo (Fig. 3b, Supplementary Fig. 3), kungabonakala ukuthi i-αS kinetics ifana kakhulu naleyo ye-Tau441 coacervates.kanye ne-ΔNt-Tau, eshesha kakhulu nge-pLK.Ama-coefficients okusabalalisa abaliwe we-αS ngaphakathi kwe-coacervate ngokuya nge-FRAP (njengoba kuchazwe u-Kang et al. 35) angu-D = 0.013 ± 0.009 µm2/s kanye no-D = 0.026 ± 0.008 µm2/s kokuthi αS/Tau ne-αS4 uhlelo lwe-αS/.pLK, Tau, kanye no-D = 0.18 ± 0.04 µm2/s, ngokulandelana (Fig. 3c).Kodwa-ke, i-diffusion coefficient αS esigabeni esihlakaziwe iyimiyalo eminingana yobukhulu ephakeme kunazo zonke izigaba ezifingqiwe, njengoba kunqunywa i-Fluorescence Correlation Spectroscopy (FCS, bheka i-Supplementary Fig. 3) ngaphansi kwezimo ezifanayo (ibhafa ye-LLPS) kodwa uma kungekho polycations. ( D = 8 ± 4 µm2/s).Ngakho-ke, i-kinetics yokuhumusha kwe-αS incishiswe kakhulu kuma-coacervates uma kuqhathaniswa namaprotheni esigabeni esihlakazekile ngenxa yemiphumela yokugcwala kwamangqamuzana, nakuba wonke ama-coacervates agcina izakhiwo ezifana noketshezi phakathi nengxenye yokuqala yehora ngemva kokubunjwa kwazo, ngokungafani nesigaba se-tau.kinetics esheshayo ku-pLK condensate.
a–c Ukuhlaziywa kwe-FRAP kwe-αS dynamics (2% AF488-ilebulwe αS) kuma-coacervates e-electrostatic.Izithombe ezimele ze-αS/Tau441 FRAP zokuhlola okuphindwe kathathu ziboniswa kokuthi (a), lapho imibuthano ebomvu ibonisa izindawo ezisuswe umbala.Ibha yesikali ingu-5 µm.b Amajika e-FRAP amaphakathi kanye (c) nama-diffusion coefficients (D) abaliwe angu-5–6 (N) amaconsi ahlukene avela ekuhlolweni okuthathu kusetshenziswa i-100 µM αS nokugxilisa okulinganayo kwe-Tau441 (bomvu) noma i-ΔNt-Tau (eluhlaza okwesibhakabhaka) noma i-pLK (eluhlaza) izikhathi eziyishumi ukugxila kwe-LLPS.Ukuchezuka okujwayelekile kwejika le-FRAP kuboniswa ngombala onomthunzi.Ukuze uqhathanise, i-diffusion coefficient αS kusigaba esihlakaziwe yanqunywa ngokuphindwe kathathu kusetshenziswa i-fluorescence correlation spectroscopy (FCS) (bona Umfanekiso Owengeziwe 3 nezindlela ukuze uthole ulwazi olwengeziwe).d I-spectra ye-X-band EPR eqhubekayo engu-100 μM TEMPOL-122-αS kubhafa ye-LLPS ngaphandle kwe-polycation (emnyama) noma phambi kuka-100 μM Tau441 (obomvu) noma i-ΔNt-Tau (eluhlaza okwesibhakabhaka) noma 1 mM pLK (eluhlaza).Isingeniso sibonisa ukubuka okukhulisiwe kwemigqa yenkambu eqinile lapho kwenzeka khona izinguquko ezinkulu kakhulu.e Amajika abophezelayo angu-50 μM TEMPOL-122-αS ane-polycation ehlukahlukene lapho ingekho i-LLPS (ayikho i-PEG).I-amplitude encishisiwe yebhendi III uma iqhathaniswa nebhendi II (IIII/III) ye-spectrum ye-EPR evamile iboniswa ukuze ikhulise izilinganiso ze-molar ye-Tau441 (ebomvu), i-ΔNt-Tau (eluhlaza okwesibhakabhaka) kanye ne-pLK (eluhlaza okotshani).Imigqa enombala ikhombisa ukufaneleka kwedatha isebenzisa imodeli ebophayo eqinile enamasayithi abophezelayo n afanayo nazimele ejikeni ngalinye.Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Njengomphelelisi, siphenye ukuguquguquka kwe-αS kuma-coacervate ahlukahlukene sisebenzisa i-directed spin labeling (SDSL) kanye ne-continuous electron paramagnetic resonance (CW-EPR).Le ndlela ibonakale iwusizo kakhulu ekubikeni ukuguquguquka nokuguquguquka kwe-IDP ngokulungiswa okuyinsalela okungokoqobo36,37,38.Kuze kube manje, sakhe izinsalela ze-cysteine ​​​​eziguquguqukayo ze-Cys eyodwa futhi sasebenzisa i-4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Okuphuma kokuphuma ku-Maleimide kuilebula.Ngokuqondile, sifake ama-TEMPOL probes endaweni 122 noma 24 αS (TEMPOL-122-αS ne-TEMPOL-24-αS).Esimweni sokuqala, siqondise indawo ye-C-terminal yeprotheni, ehilelekile ekusebenzisaneni nama-polycations.Esikhundleni salokho, isikhundla sama-24 singasinika ulwazi mayelana nokuguquguquka okuphelele kwamaphrotheni ku-condensate.Kuzo zombili izimo, amasignali e-EPR atholwe amaprotheni esigaba esihlakazekile ahambisana nama-nitroxide radicals esimweni esihamba ngokushesha.Ngemuva kokuhlukaniswa kwesigaba phambi kwe-tau noma i-pLK (100 μM TEMPOL-αS, Tau441 noma ΔNt-Tau ngesilinganiso esingu-1:1 noma pLK ngesilinganiso esingu-1:10), ukwanda kokuqina kwesiqongo esihlobene kwabonwa I-spectrum ye-EPR ye-αS.Umugqa wokulahlekelwa unwetshiwe, okubonisa ukunciphisa i-αS reorientation kinetics kumaconsi uma kuqhathaniswa namaprotheni esigabeni sokunciphisa (Fig. 3d, Supplementary Fig. 4a).Lezi zinguquko zigqama kakhulu endaweni ye-122. Ngenkathi i-24 ukuba khona kwe-pLK akuzange kuthinte i-kinetics ye-probe, endaweni ye-122 i-spectral line shape yashintsha kakhulu (I-Supplementary Fig. 4a).Ngenkathi sizama ukwenza imodeli ye-spectra endaweni engu-122 yezinhlelo ezimbili ze-αS/i-polycation sisebenzisa imodeli ye-isotropic (Umfanekiso Owengeziwe 5a) ovame ukusetshenziswa ukuchaza amandla e-spin anelebuli ye-IDP38,39, asikwazanga ukwakha kabusha i-spectra yokuhlola..Ukulingiswa kwe-Spectral kwesikhundla se-24 spins umehluko (I-Supplementary Fig. 5a).Lokhu kuphakamisa ukuthi kunezikhundla ezincanyelwayo endaweni yokucushwa kwe-spin yesifunda se-C-terminal ye-αS lapho kukhona ama-polycations.Uma kucutshungulwa ingxenye ye-αS esigabeni esifingqiwe ngaphansi kwezimo ze-EPR yokuhlola (84 ± 2%, 79 ± 7%, kanye ne-47 ± 4% ye-αS/Tau441, αS/ΔNt-Tau, kanye ne-αS/pLK, ngokulandelana—bona Okungeziwe Umdwebo 2e wokuhlaziywa kwedatha c), kungabonakala ukuthi ukunwetshwa okutholwe indlela ye-EPR kukhombisa ngokuyinhloko ukusebenzisana kwesifunda se-C-terminal ye-αS nama-polycation ahlukahlukene esigabeni esifingqiwe (ushintsho oluyinhloko lapho usebenzisa i-TEMPOL-122- αS), hhayi ukujiya kwamaprotheni.Ukwanda kwe-microviscosity kubonakala ku-probe.Njengoba kulindelekile, i-spectrum ye-EPR yeprotheyini ngaphansi kwezimo ngaphandle kwe-LLPS ibuyiselwe ngokuphelele lapho i-1 M NaCl yengezwe kungxube (I-Supplementary Fig. 4b).Sekukonke, idatha yethu iphakamisa ukuthi izinguquko ezitholwe yi-CW-EPR ikakhulukazi zibonisa ukusebenzisana kwesifunda se-C-terminal ye-αS nama-polycation ahlukahlukene esigabeni esifingqiwe, futhi lokhu kusebenzisana kubonakala kunamandla nge-pLK kune-Tau.
Ukuze sithole ulwazi lwesakhiwo olwengeziwe mayelana namaprotheni ku-coacervate, sinqume ukutadisha uhlelo lwe-LLPS sisebenzisa i-NMR ekuxazululeni.Kodwa-ke, singathola kuphela ingxenyenamba ye-αS esele esigabeni esihlakazekile, okungenzeka kube ngenxa yokuncipha kwamandla amaprotheni ngaphakathi kwe-coacervate kanye nesigaba esiminyene phansi kwesixazululo ekuhlaziyeni kwe-NMR.Lapho sihlaziya ukwakheka nokuguquguquka kwephrotheni esele esigabeni esihlakazekile sesampula ye-LLPS kusetshenziswa i-NMR (I-Supplementary Fig. 5c, d), saqaphela ukuthi iphrotheni iziphathe cishe ngendlela efanayo phambi kwe-pLK ne-ΔNt-Tau, kokubili ebezisesimweni sesibili kanye nokuguquguquka komgogodla wephrotheni, okuvezwe ukuhlolwa kokushintshwa kwamakhemikhali okwesibili kanye nokukhululeka kwe-R1ρ.Idatha ye-NMR ibonisa ukuthi i-C-terminus ye-αS ibhekana nokulahlekelwa okukhulu kokuvumelana nezimo kuyilapho igcina imvelo yayo ephazamisekile, njengawo wonke amanye amaprotheni alandelanayo, ngenxa yokusebenzelana kwawo nama-polycations.
Njengoba ukunwetshwa kwesiginali ye-CW-EPR okubonwe esigabeni esifingqiwe se-TEMPOL-122-αS kukhombisa ukusebenzisana kwephrotheni nama-polycations, senze i-EPR titration ukuze sihlole ukuhambisana okubophezelayo kwe-αS kuma-polycations ahlukahlukene lapho ingekho i-LLPS (akukho qoqo I-Buffer LLPS), ephakamisa ukuthi ukusebenzisana kuyafana ezigabeni ezihlanjululwayo nezigxilile (okuqinisekiswa idatha yethu, i-Supplementary Fig. 4a kanye ne-Supplementary Fig. 6).Umgomo bekuwukubona ukuthi ingabe wonke ama-coacervate, naphezu kwezakhiwo zawo ezivamile ezifana noketshezi, abonisa noma yikuphi ukuziphatha okuhlukile okucashile ezingeni lamangqamuzana.Njengoba kulindelekile, i-spectrum ye-EPR yanda ngokugxila kwe-polycation okwandayo, okubonisa ukwehla kokuguquguquka kwamangqamuzana ngenxa yokusebenzisana kwamangqamuzana abo bonke abalingani bokusebenzelana cishe ku-saturation (Fig. 3e, Supplementary Fig. 6).I-pLK izuze lokhu kugcwaliswa kwesikhala ngenani eliphansi le-molar (i-polycation:αS) uma iqhathaniswa ne-ΔNt-Tau ne-Tau441.Eqinisweni, ukuqhathaniswa kwedatha nemodeli ebophezelayo elinganiselwe kucatshangwa ukuthi n amasayithi abophezelayo afanayo nazimele kubonise ukuthi ukungaguquguquki okusobala kwe-pLK (~5 μM) kuwuhlelo lobukhulu obuphansi kunalelo lwe-Tau441 noma i-ΔNt-Tau (~50 μM ).µM).Nakuba lokhu kuyisilinganiso esinzima, lokhu kuphakamisa ukuthi i-αS inokuhlobana okuphezulu kwama-polycations alula anezifunda zokushaja okuhle eziqhubekayo.Ngokunikezwa lo mehluko ebudlelwaneni phakathi kwe-αS nama-polycations ahlukahlukene, sicabange ukuthi izakhiwo zabo eziwuketshezi zingashintsha ngokuhlukile ngokuhamba kwesikhathi futhi ngaleyo ndlela zihlupheke ngenxa yezinqubo ezahlukene ze-LSPT.
Uma kubhekwa indawo egcwele kakhulu ngaphakathi kwe-protein coacervate kanye nemvelo ye-amyloid yeprotheni, siqaphele ukuziphatha kwe-coacervate ngokuhamba kwesikhathi ukuze sithole izinqubo ze-LSPT ezingenzeka.Sisebenzisa i-BF kanye ne-CF microscopy (Umfanekiso 4), siqaphele ukuthi i-αS/Tau441 ihlangana ngezinga elikhulu esixazululweni, yenze amaconsi amakhulu athintanayo futhi amanzise indawo engaphansi komthombo/slayidi njengamaconsi agcwele, njengoba kulindelekile (Fig Supplementary Fig. . 7d);lezi zakhiwo ezakhiwe phansi sizibiza ngokuthi "ama-protein rafts".Lezi zakhiwo zahlala zimanzi njengoba zigcina ikhono lokuhlanganisa (I-Supplementary Fig. 7b) futhi ingabonwa amahora ambalwa ngemva kokuba i-LLPS iqalisiwe (Fig. 4 kanye ne-Supplementary Fig. 7c).Siqaphele ukuthi inqubo yokumanzisa ithandwa kakhulu endaweni ye-hydrophilic kune-hydrophobic materials (I-Supplementary Fig. 7a), njengoba kulindelekile kuma-coacervates ka-electrostatic anezindleko ezingalingani kanjalo namandla aphezulu e-electrostatic surface.Ngokuphawulekayo, i-αS / ΔNt-Tau coalescence kanye ne-rafting yancishiswa kakhulu, kuyilapho ama-condensate e-αS / pLK ancishiswa kakhulu (Fig. 4).Ngesikhathi esifushane sokufukamela, amaconsi e-αS/pLK akwazi ukuhlanganisa futhi amanzise indawo engaphansi kwe-hydrophilic, kodwa le nqubo yema ngokushesha futhi ngemva kwamahora angu-5 okufukamela, izenzakalo ezilinganiselwe zokuhlanganisa kuphela futhi akukho ukumanzisa okubonwayo.- ukushintshwa kwe-gel-drip.
Omele i-BF (amaphaneli esikali esimpunga) kanye ne-CF (amaphaneli angakwesokudla, AF488-anelebuli engu-AF488 αS ngokuluhlaza) yamasampuli e-coacervate aqukethe i-100 µM αS (1% ilebula ye-fluorescent) kubhafa ye-LLPS phambi kwezithombe eziyi-100 µM Tau441nce (phezulu) Δfluores -Tau (maphakathi) noma 1 mM pLK (phansi) ngezikhathi ezihlukene zokufukamela kanye nokuphakama okugxile (z, ibanga ukusuka phansi kwepuleti kahle).Ukuhlolwa kwaphindwa izikhathi ezingu-4-6 ngokuzimela komunye nomunye ngemiphumela efanayo.Ama-coacervate e-αS/Tau441 aba manzi ngemva kwamahora angu-24, enza izihlenga ezinkulu kunesithombe.Ibha yesikali yazo zonke izithombe ngu-20 µm.
Sibe sesibuza ukuthi ingabe amachibi amakhulu amaprotheni afana noketshezi akhiwe ku-αS/Tau441 LLPS azoholela ekuhlanganisweni kwe-amyloid kwanoma yimaphi amaprotheni afundwayo.Silandele ukuvuthwa kwamaconsi e-αS/Tau441 ngokuhamba kwesikhathi nge-WF microscopy ngaphansi kwezimo ezifanayo nezingenhla, kodwa sisebenzisa i-1 μM AF488 enelebula ethi αS kanye ne-Atto647N enelebula ethi Tau441 (Fig. 5a).Njengoba bekulindelekile, sibone ukwenziwa kwasendaweni okuphelele kwamaprotheni kuyo yonke inqubo yokuvuthwa.Ngokuthakazelisayo, kusukela ku-ca.Ngemuva kwamahora angu-5, izakhiwo eziqinile ezingezona eziyisiyingi zabonwa ngaphakathi kwe-raft, esiyibiza ngokuthi "amaphuzu", amanye awo ahlanganiswe ne-αS, kanti amanye acetshiswa ku-Tau441 (Fig. 5a, imicibisholo emhlophe).Lawa machashazi ahlala ebonwa phakathi kwama-raft ngokwezinga elikhulu ku-αS/ΔNt-Tau kune-αS/ΔNt-Tau.Awekho amachashaza ahlukile kumaconsi e-pLK kanye nezinhlelo ze-Tau ezingakwazi ukuhlanganisa/ukumanzisa.Ukuhlola ukuthi ingabe lawa mabala aqukethe i-αS ne-Tau441 ayizibalo ezifana ne-amyloid, senze ukuhlolwa okufanayo sisebenzisa i-CF microscopy lapho i-Tau441 ifakwe ilebuli ethi Atto647N kanye ne-12.5 μM eqondene ne-amyloid-specific thioflavin-T (ThT) yengezwe kusukela ekuqaleni.udayi.Nakuba i-ThT-staining yamaconsi e-αS/Tau441 noma ama-raft ayizange ibonwe ngisho nangemva kwamahora angu-24 wokufukamela (Umfanekiso we-5b, umugqa ophezulu-amaconsi asele phezu kwama-protein rafts), izakhiwo ze-ThT-positive eziqukethe i-Atto647N-Tau441 ngaphakathi kwezihlenga zazibuthakathaka kakhulu.lokhu kubuyisela usayizi, umumo, kanye nendawo yezindawo ezichazwe ngaphambilini (Umfanekiso 5b, imigqa ephakathi nephansi), okusikisela ukuthi amachashaza angase ahambisane nama-amyloid-like aggregates akhiwe kuma-coacervate oketshezi oluguga.
I-WF 25 μM αS ngezikhathi ezihlukahlukene zokufukamela kanye nokuphakama okugxilile (z, ibanga ukusuka phansi okungabophekile) phambi kuka-25 μM Tau441 (1 μM AF488-okubhalwe ukuthi αS kanye ne-Atto647N-ebhalwe ukuthi Tau441) emthonjeni wepuleti le-microscope elinebhafa ye-LLPS) .Ukuhlolwa okuyisithupha kwaphindwa ngokuzimela ngemiphumela efanayo.b Isithombe se-CF microscopic esingu-25 μM αS ebukhoneni buka-25 μM Tau441 (1 μM Atto647N-ebhalwe Tau441) kanye no-12.5 μM thioflavin-T (ThT).Amaconsi amaprotheni anesisindo kanye nama-protein raft afakwe namachashaza aboniswa emigqeni engaphezulu nephakathi, ngokulandelanayo.Umugqa ongezansi ubonisa izithombe zama-raft neziconsi zisuka kuzimpinda ezi-3 ezizimele.Imicibisholo emhlophe ibonisa amachashazi ane-ThT kuwo womabili amaphaneli.Ibha yesikali yazo zonke izithombe ngu-20 µm.
Ukuhlola ngokuningiliziwe izinguquko kunethiwekhi ye-coacervate protein ngesikhathi soshintsho kusuka oketshezini kuya kokuqinile, sisebenzise i-fluorescence lifetime imaging (FLIM) kanye ne-Förster resonance energy transfer microscopy (FRET) (Figure 6 and Supplementary Figures 8 and 9).Salinganisa ukuthi ukuvuthwa kwe-coacervate kwesendlalelo kube esakhiweni sephrotheni esihlanganisiwe noma esiqinile njengasesiqinile siholela ekuxhumaneni okuseduze phakathi kwephrotheni kanye ne-fluorescent probe enamathiselwe kuso, okungase kukhiqize umphumela wokucisha oboniswa esikhathini esifushane sempilo ye-probe (τ) , njengoba kuchazwe ngaphambilini40., 41 ,42 .Ngaphezu kwalokho, kumasampula anamalebula aphindwe kabili (AF488 kanye ne-Atto647N njengabanikezeli be-FRET odayi abamukelayo, ngokulandelana), lokhu kuncipha ku-τ kungabuye kuhambisane nokufihlwa kwe-coacervate kanye nokwanda kokusebenza kahle kwe-FRET(E) ngesikhathi se-LSPT.Siqaphe ukwakheka kwe-raft namabala ngokuhamba kwesikhathi kumasampuli e-LLPS αS/Tau441 kanye ne-αS/ΔNt-Tau (25 µM wephrotheni ngayinye kubhafa ye-LLPS equkethe i-1 µM AF488 enelebula ngokuthi αS kanye/noma i-Atto647N enelebula ngokuthi Tau441 noma ΔNt-Tau).Sibone inkambiso evamile ukuthi impilo yonke ye-fluorescence ye-AF488 (τ488) kanye ne-Atto647N (τ647N) yehla kancane njengoba ama-coacervates ekhula (Fig. 6 kanye ne-Supplementary Fig. 8c).Kuyathakazelisa ukuthi lolu shintsho luthuthukiswe kakhulu kumachashazi ngaphakathi kwama-raft (Fig. 6c), okubonisa ukuthi ukufiphala okwengeziwe kwamaprotheni kwenzeke kumachashazi.Ukweseka lokhu, alukho ushintsho olubalulekile empilweni ye-fluorescence olubonwe kumaconsi e-αS/ΔNt-Tau aneminyaka engu-24 h (I-Supplementary Fig. 8d), ephakamisa ukuthi i-droplet gelation iyinqubo ehlukile ekubonakaleni futhi ayihambisani nokuhlelwa kabusha kwamangqamuzana abalulekile. ngaphakathi kwama-coacervates.Kumele kuqashelwe ukuthi amachashazi anobukhulu obuhlukahlukene nokuqukethwe okuguquguqukayo ku-αS, ikakhulukazi ohlelweni lwe-αS/Tau441 (I-Supplementary Fig. 8e).Ukuncipha kwesikhathi sokuphila se-spot fluorescence kuhambisane nokwenyuka kokuqina, ikakhulukazi ku-Atto647N enelebuli engu-Tau441 (I-Supplementary Fig. 8a), kanye nempumelelo ephezulu ye-FRET yakho kokubili amasistimu e-αS/Tau441 kanye ne-αS/ΔNt-Tau, okubonisa ukufiphala okwengeziwe ku-LLPS amahora amahlanu. ngemva kokucupha, amaprotheni angaphakathi kukagesi omile ayafingqa.Uma kuqhathaniswa ne-αS/ΔNt-Tau, siqaphele amanani aphansi angu-τ647N kanye namanani aphezulu angu-τ488 ezindaweni ezingu-αS/Tau441, ephelezelwa amanani aphansi futhi ahluke kakhulu e-FRET.Mhlawumbe, lokhu kungase kuhlobane nokuthi ohlelweni lwe-αS/Tau441, ukuchichima kwe-αS okuphawuliwe nokulindelwe kuma-aggregates kuhluke kakhulu, ngokuvamile kuvame ukuba yi-substoichiometric uma kuqhathaniswa ne-Tau, njengoba i-Tau441 ngokwayo ingase futhi ibhekane ne-LLPS nokuhlanganisa (I-Supplementary Fig. 8e) .Kodwa-ke, izinga lokuhlangana kwamaconsi, ukwakheka kwezihlenga, futhi, okubalulekile, ukuhlanganisa amaprotheni ngaphakathi kwama-coacervate afana noketshezi kukhulu uma kokubili i-Tau441 ne-αS kukhona.
izithombe ze-Lifetime fluorescence microscopy (FLIM) ze-αS/Tau441 kanye ne-αS/ΔNt-Tau ku-25 μM wephrotheni ngayinye (1 μM AF488 enelebula ngokuthi αS kanye no-1 μM Atto647N-ebhalwe ukuthi Tau441 noma ΔNt-Tau) kubhafa ye-LLPS.Amakholomu abonisa izithombe ezimele amasampula e-LLPS ngezikhathi ezihlukene zokuvuthwa (30 min, 5 h kanye 24 h).Uhlaka olubomvu lubonisa isifunda esiqukethe izindawo ezingu-αS/Tau441.Ubude bempilo buboniswa njengamabha ombala.Ibha yesikali = 20 µm yazo zonke izithombe.b Isithombe se-FLIM esondeziwe sendawo ekhethiwe, eboniswe ebhokisini elibomvu kuphaneli a.Ububanzi bokuphila buboniswa kusetshenziswa isikali sombala esifanayo naso esikuphaneli A.Ibha yesikali = 5 µm.c Ama-Histograms abonisa i-AF488 (enamathiselwe ku-αS) noma i-Atto647N (enamathiselwe ku-Tau) ezinhlobo ezahlukene zamaprotheni (amaconsi-D-, i-raft-R- nama-speckle-P) ahlonzwe ezithombeni ze-FLIM ezirekhodwe i-αS-) isikhathi sokusatshalaliswa kwesikhathi sokuphila kwe-Tau441 kanye Amasampuli e-αS/ΔNt-Tau coacervate (N = 17-32 ROI ye-D, 29-44 ROI ye-R, kanye ne-21-51 ROI yamaphoyinti).Amanani aphakathi nendawo aboniswa njengezikwele eziphuzi nemigqa emnyama ngaphakathi kwamabhokisi, ngokulandelana.Imingcele engezansi nengaphezulu yebhokisi imele i-quartiles yokuqala neyesithathu, ngokulandelana, futhi amanani amancane futhi aphezulu ngaphakathi kwe-1.5-fold interquartile range (IQR) aboniswa njengentshebe.Ama-outliers aboniswa njengamadayimane amnyama.Ukubaluleka kwezibalo phakathi kwamapheya okusabalalisa kwanqunywa kusetshenziswa ukuhlolwa kuka-t okunamasampuli amabili, kucatshangelwa ukuhlukahluka okungalingani.Amanani we-p wokuhlola okunemisila emibili aboniswa ngezinkanyezi kupheya ngayinye yedatha eqhathaniswayo (* inani le-p > 0.01, ** p-value > 0.001, *** p-value > 0.0001, **** p-value > 0.00001), ns Ibonisa ukunganaki (inani-p > 0.05).Amanani aqondile we-p anikezwa kuThebula Lokwengeza 1, futhi idatha yangempela yethulwa njengamafayela edatha eluhlaza.
Ukuze siqhubeke sibonise imvelo efana ne-amyloid yamachashaza/ama-aggregate, siphathe amasampula e-coacervate angangcolisiwe amahora angu-24 ngokugxila okuphezulu kwe-(1 M) NaCl, okuholele ekuhlukaniseni ama-aggregate kusuka kuma-protein coacervate.Lapho ama-aggregate ahlukanisiwe (okungukuthi, isixazululo esihlakazekile sama-aggregate) abonwa kusetshenziswa i-atomic force microscopy (AFM), sabona i-morphology eyindilinga kakhulu enobude obuvamile obungaba ngu-15 nm, ejwayele ukuhlotshaniswa ngaphansi kwezimo zokugcwala kukasawoti omningi, okufanayo ukuziphatha kwe-amyloid fibrils evamile ngenxa yomphumela onamandla we-hydrophobic ebusweni (qaphela ukuthi ama-fibrils ngokuvamile anobude obungu-~10 nm) (I-Supplementary Fig. 10a).Kuyathakazelisa ukuthi lapho ama-aggregate ahlukanisiwe ehlanganiswa ne-ThT ekuhlolweni okujwayelekile kwe-ThT fluorescence, sabona ukwanda okumangazayo kwesivuno se-ThT fluorescence quantum, uma kuqhathaniswa nalokho okuphawulwe lapho udayi ufakwa nge-αS amyloid fibrils ejwayelekile (I-Supplementary Fig. 10b), ephakamisa ukuthi ama-coacervate aggregate aqukethe izakhiwo ezinjenge-amyloid..Eqinisweni, ama-aggregate abekezelela ukugcwala kukasawoti omningi kodwa azwela ku-4 M guanidine chloride (GdnHCl), njengama-amyloid fibrils ajwayelekile (I-Supplementary Fig. 10c).
Okulandelayo, sihlaziye ukwakheka kwama-aggregate sisebenzisa i-molecule eyodwa i-fluorescence, i-fluorescence correlation ethile/i-cross-correlation spectroscopy (FCS/FCCS), kanye nokuhlaziya okuqhumayo kokutholwa kokuhlangana kwemibala emibili (TCCD).Kulokhu, sihlukanise ama-aggregate akhiwe ngemva kwamahora angu-24 okufukamela kumasampuli angu-100 μl LLPS aqukethe i-αS ne-Tau441 (kokubili i-25 μM) kanye ne-1 μM AF488 enelebula ethi αS kanye ne-1 μM Atto647N enelebula ethi Tau441.Nciphisa isixazululo se-aggregate esisabalele esiphumela kusimo se-monomolecular usebenzisa isibhafa esingena-PEG esifanayo kanye no-1 M NaCl (isibhafa esifanayo esisetshenziselwa ukuhlukanisa ama-aggregate ku-coacervate) ukuze kuvinjelwe noma yikuphi ukusebenzisana okungenzeka kwe-electrostatic phakathi kwe-LLPS neprotheni.Isibonelo se-trajectory yesikhathi se-molecule eyodwa singabonakala ku-Fig. 7a.Ukuhlaziywa kwe-FCCS/FCS (cross-correlation, CC and autocorrelation, AC) kubonise ukuthi izilinganiso eziqukethe i-αS ne-tau zaziningi kumasampula (bona ijika le-CC ku-Fig. 7b, iphaneli yesokunxele), kanye nenani elidlulele le-monomeric eliyinsalela lavela njenge umphumela wenqubo yokuhlanjululwa (bona amajika e-AC kumfanekiso 7b, iphaneli yesokunxele).Ukuhlolwa kokulawula okwenziwe ngaphansi kwezimo zesixazululo ezifanayo kusetshenziswa amasampuli aqukethe amaprotheni e-monomeric kuphela abonisa ukuthi awekho amajika e-CC, futhi amajika e-AC ahambisana kahle nemodeli yokusabalalisa ingxenye eyodwa (Eq. 4), lapho amaprotheni e-monomeric anama-coefficients okusabalalisa alindelekile (Fig. 7b ), iphaneli yesokudla).I-coefficient yokusabalalisa yezinhlayiya ezihlanganisiwe ingaphansi kuka-1 µm2/s, futhi eyamaprotheni e-monomeric cishe i-1 µm2/s.50–100 µm/s;amanani ayafana namanani ashicilelwe ngaphambilini we-sonicated αS amyloid fibrils kanye ne-monomeric αS ngokuhlukana ngaphansi kwezimo zesixazululo ezifanayo44.Lapho sihlaziya ama-aggregate ngokuhlaziywa kokuqhuma kwe-TCCD (Fig. 7c, panel top), sithole ukuthi ku-aggregate ngayinye ehlukanisiwe (αS/Tau heteroaggregate), cishe u-60% we-aggregate etholiwe iqukethe kokubili i-αS ne-tau, cishe u-30% equkethwe kuphela. tau, cishe u-10% αS kuphela.Ukuhlaziywa kwe-Stoichiometric kwama-αS/Tau heteroaggregates kubonise ukuthi iningi lama-heteroaggregates acetshiswe ku-tau (i-stoichiometry engaphansi kuka-0.5, isilinganiso senani lama-molecule e-tau ngeqoqo ngalinye liphindwe izikhathi ezingu-4 ngaphezu kwama-molecule e-αS), okuhambisana nomsebenzi wethu obonwa ku-FLIM in situ. ukuhlola..Ukuhlaziywa kwe-FRET kubonise ukuthi lezi zibalo ziqukethe womabili amaprotheni, nakuba amanani wangempela e-FRET kulesi simo engabalulekile kakhulu, njengoba ukusatshalaliswa kwama-fluorophore kuqoqo ngalinye kwakungahleliwe ngenxa yokweqisa kwephrotheni engenamalebula esetshenziswe ocwaningweni.Kuyathakazelisa ukuthi lapho senza ukuhlaziya okufanayo sisebenzisa i-45,46 ekhulile ye-amyloid aggregation-Deficie Tau okuhlukile (bona I-Supplementary Fig. 11a,b), siqaphele ukuthi nakuba i-αS electrostatic aggregation yayifana (Supplementary Fig. 11c, d), ikhono lokwakha ama-aggregate ngaphakathi kwe-coacervate lehliswa kakhulu futhi i-FLIM ithole izindawo ezimbalwa ekuhloleni kwe-situ, futhi amajika abuthaka ahlotshaniswa ne-cross-correlation aqashelwa kumasampula ahlanganisiwe angawodwa.Kodwa-ke, ngenani elincane lama-aggregate atholiwe (ingxenye eyodwa kuphela kweshumi ye-Tau441), siqaphele ukuthi isamba ngasinye sacetshiswa ngo-αS kunalokhu okuhlukile kwe-Tau, cishe ngo-50% wezibalo ezitholiwe eziqukethe kuphela ama-molecule e-αS, futhi i-αS yayihluke ngokweqile. .ama-aggregates (bona i-Supplementary Fig. 11e), ngokungafani ne-heterogeneous aggregates ekhiqizwa yi-Tau441 (Fig. 6f).Imiphumela yalezi zivivinyo ibonise ukuthi nakuba i-αS ngokwayo ikwazi ukuqongelela nge-tau ngaphakathi kwe-coacervate, i-tau nucleation ithandeka kakhulu ngaphansi kwalezi zimo, futhi ama-amyloid-like aggregates angumphumela akwazi ukusebenza njengendlela ye-αS ne-tau.Kodwa-ke, uma i-tau-rich core yakhiwe, ukusebenzisana kwe-heterotypic phakathi kwe-αS ne-tau kukhethwa ngama-aggregate ngaphezu kokusebenzelana kwe-homotypic phakathi kwama-molecule e-tau;siphinde sibheke amanethiwekhi amaprotheni ku-liquid αS/tau coacervates.
i-representative fluorescence traces yesikhashana ye-molecule eyodwa ye-aggregate ehlukanisiwe eyakhelwe ku-αS/Tau441 electrostatic coacervates.Ukuqhuma okuhambisana nama-coaggregates e-αS/Tau441 (ukuqhuma ngaphezu komkhawulo obonisiwe) kubonwe eziteshini ezintathu zokutholwa (AF488 kanye nokukhishwa kwe-Atto647N ngemva kokuthakazelisa okuqondile, imigqa eluhlaza okwesibhakabhaka nokubomvu, ukukhishwa kwe-Atto647N ngemva kwesasasa elingaqondile), i-FRET, umugqa we-violet).b Ukuhlaziywa kwe-FCS/FCCS kwesampula yezilinganiso ezihlukanisiwe ze-αS/Tau441 ezitholwe ku-LLPS (iphaneli yesokunxele).Amajika e-Autocorrelation (AC) we-AF488 kanye ne-Atto647N aboniswa ngokuluhlaza okwesibhakabhaka nokubomvu, ngokulandelanayo, futhi amajika e-cross-correlation (CC) ahlotshaniswa nezibalo eziqukethe womabili odayi aboniswa ngokunsomi.Amajika e-AC abonisa ukuba khona kwezinhlobo zeprotheyini ezilebulwe ngokuthi i-monomeric ne-aggregated, kuyilapho amajika e-CC abonisa kuphela ukusatshalaliswa kwama-aggregate anamalebula amabili.Ukuhlaziywa okufanayo, kodwa ngaphansi kwezimo ezifanayo zesixazululo njengasezindaweni ezingazodwa, amasampuli aqukethe kuphela i-αS ye-monomeric ne-Tau441 aboniswa njengezilawuli kuphaneli elungile.c Ukuhlaziywa kwe-Fluorescence flash ye-molecule eyodwa ye-aggregate ehlukanisiwe eyakhelwe ku-αS/Tau441 electrostatic coacervates.Ulwazi lwesamba ngasinye esitholakala kuzimpinda ezine ezihlukene (N = 152) luhlelwe ngokumelene ne-stoichiometry yazo, amanani we-S, nokusebenza kahle kwe-FRET (iphaneli ephezulu, ibha yombala ibonisa ukwenzeka).Izinhlobo ezintathu zama-aggregate zingahlukaniswa: -αS-kuphela ama-aggregates nge-S~1 kanye ne-FRET~0, i-Tau-only aggregates nge-S~0 kanye ne-FRET~1, kanye ne-Tau/αS ehlanganisiwe enezilinganiso ezimaphakathi zika-S kanye ne-FRET yenani womabili amaphrotheni omaka atholwe ekuhlanganiseni ngakunye kwe-heterogeneous (N = 100) aboniswa kuphaneli engezansi (isikali sombala sibonisa ukwenzeka).Idatha eluhlaza inikezwa ngohlobo lwamafayela edatha eluhlaza.
Ukuvuthwa noma ukuguga kweprotheyini ewuketshezi ifinyela ezinhlakeni ezifana nejeli noma eziqinile ngokuhamba kwesikhathi kuye kwabikwa ukuthi kuhileleke emisebenzini eminingana ye-physiological ye-condensate47 kanye nesifo, njengenqubo engavamile eyandulela ukuhlanganisa kwe-amyloid 7, 48, 49. Lapha sifunda ngokuhlukana kwesigaba nokuziphatha ngokuningiliziwe.I-LSPT αS ebukhoneni be-polycations engahleliwe endaweni elawulwayo ekugxilweni kwe-micromolar ephansi kanye nezimo ezifanele zomzimba (qaphela ukuthi ukuhlushwa komzimba okubaliwe kwe-αS ngu>1 µM50), kulandela ukuziphatha okuvamile kwe-LPS okuqhutshwa yi-thermodynamically.Sithole ukuthi i-αS, equkethe indawo ye-C-terminal enecala elibi kakhulu ku-pH yokuphila, iyakwazi ukwakha amaconsi anamaprotheni esixazululweni samanzi nge-LLPS lapho kukhona ama-peptides aphazamiseke kakhulu anjenge-pLK noma i-Tau ngenqubo ye-electrostatic. i-condensation eyinkimbinkimbi phambi kwama-macromolecules ahlanganisiwe.Le nqubo ingase ibe nemiphumela efanele endaweni yeselula lapho i-αS ihlangabezana nama-molecule e-polycationic ahlukahlukene ahlotshaniswa ne-aggregation yayo ehambisana nesifo kokubili ku-vitro kanye ne-vivo51,52,53,54.
Ezifundweni eziningi, ukuguquguquka kwamaprotheni ngaphakathi kwamaconsi kuye kwabhekwa njengenye yezici ezibalulekile ezinquma inqubo yokuvuthwa55,56.Ku-electrostatic αS coacervates with polycations, inqubo yokuvuthwa ngokusobala incike emandleni okusebenzelana nama-polycations, i-valence, kanye nokuphindaphindeka kwalokhu kusebenzisana.Ithiyori yokulinganisa iphakamisa ukuthi indawo elinganayo yezifunda ezimbili eziwuketshezi kungaba ukuba khona kweconsi elikhulu elicebile ngama-biopolymers ashayela i-LLPS57,58.Ukukhula kwe-Droplet kungafinyelelwa ngokuvuthwa kwe-Ostwald59, i-coalescence60 noma ukusetshenziswa kwe-monomer yamahhala kusigaba esihlakazekile61.Ku-αS ne-Tau441, i-ΔNt-Tau noma i-pLK, iningi lamaprotheni laligxilwe ku-condensate ngaphansi kwezimo ezisetshenziswe kulolu cwaningo.Kodwa-ke, ngenkathi amaconsi e-tau anosayizi ogcwele ehlangana ngokushesha lapho kumanziswa phezulu, ukuhlangana kwamaconsi kanye nokumanzisa kwakunzima ku-ΔNt-Tau ne-pLK, okuphakamisa ukulahleka okusheshayo kwezakhiwo eziwuketshezi kulezi zinhlelo ezimbili.Ngokusho kokuhlaziywa kwethu kwe-FLIM-FRET, amaconsi asebekhulile e-pLK kanye ne-ΔNt-Tau abonise izinga elifanayo lokuhlanganisa amaprotheni (impilo efanayo ye-fluorescence) njengamaconsi okuqala, okuphakamisa ukuthi inethiwekhi yamaprotheni yokuqala yagcinwa, nakuba iqinile kakhulu.
Silungisa imiphumela yethu yokuhlola kumodeli elandelayo (Umfanekiso 8).Amaconsi aqale akheke okwesikhashana ngokuvamile amanethiwekhi amaprotheni ngaphandle kwesinxephezelo se-electrostatic, futhi ngaleyo ndlela kunezindawo zokungalingani kwezindleko, ikakhulukazi esibonakalayo samaconsi, okuholela kumaconsi anamandla aphezulu e-electrostatic surface.Ukunxephezela inkokhiso (into evame ukubizwa ngokuthi ukuncipha kwe-valence) kanye nokunciphisa amandla angaphezulu kwamaconsi, amaconsi angabandakanya ama-polypeptide amasha asuka esigabeni sokuhlanjululwa, ahlele kabusha amanethiwekhi amaprotheni ukuze agcwalise ukusebenzisana kwe-charge, futhi ahlanganyele namanye amaconsi.ngezindawo (ezimanzisa).Amaconsi e-αS/pLK, ngenxa yenethiwekhi yawo ye-protein elula (ukusebenzelana kwe-heterotypic kuphela phakathi kwe-αS ne-pLK) kanye nokuhlangana okukhulu kokusebenzelana kwamaprotheni-amaprotheni, kubonakala kukwazi ukulinganisa ukukhokhiswa kwe-condensate ngokushesha;ngempela, siqaphele ama-protein kinetics asheshayo kuma-coacervates e-αS/pLK ekuqaleni akha kunaku-αS/Tau.Ngemuva kokuncipha kwe-valence, ukusebenzisana kuba yi-ephemeral kancane futhi amaconsi alahlekelwa izinto zawo eziwuketshezi futhi aphenduke abe amaconsi afana nejeli, angashi anamandla aphansi e-electrostatic surface (ngakho-ke angakwazi ukumanzisa indawo engaphezulu).Ngokuphambene, amaconsi e-αS/Tau awasebenzi kahle ekuthuthukiseni ibhalansi yokushaja amaconsi ngenxa yamanethiwekhi amaprotheni ayinkimbinkimbi (anakho kokubili ukusebenzisana kwe-homotypic kanye ne-heterotypic) kanye nemvelo ebuthakathaka yokusebenzisana kwamaprotheni.Lokhu kubangela amaconsi agcina ukuziphatha koketshezi isikhathi eside futhi abonise amandla aphezulu e-electrostatic surface avame ukuncishiswa ngokuhlangana nokukhula (ngaleyo ndlela kuncishiswe indawo engaphezulu/isilinganiso sevolumu yamaconsi) kanye nokumanzisa ikhemikhali ye-hydrophilic surface.Lokhu kudala amalabhulali amakhulu amaphrotheni agxilile agcina izinto eziwuketshezi njengoba ukusebenzisana kuhlala kudlula isikhathi eside ngenxa yokusesha okuqhubekayo kokuthuthukisa ukushaja kunethiwekhi yamaprotheni.Kuyathakazelisa ukuthi amafomu e-Tau ancishiswe nge-N, okuhlanganisa nama-isoform62 enziwa ngokwemvelo, abonisa ukuziphatha okuphakathi, namanye ama-coacervates aguga nge-αS abe amaconsi aphila isikhathi eside afana nejeli, kuyilapho amanye eshintsha abe ama-condensate amakhulu.Lokhu okubili ekuvuthweni kwama-alphas electrostatic coacervates kuyahambisana nezifundo zamuva nje zethiyori nezokuhlola ze-LLPS ezihlonze ukuhlobana phakathi kokuncipha kwe-valence nokusefa nge-electrostatic kuma-coacervate njengokhiye wokulawula usayizi we-condensate kanye nezakhiwo zoketshezi.Indlela 58.61.
Lolu hlelo lubonisa indlela yokuhlanganisa i-amyloid putative ye-αS ne-Tau441 nge-LLPS ne-LSPT.Ngezifunda ezengeziwe ezicebile i-anion (ebomvu) kanye ne-cation-rich (eluhlaza okwesibhakabhaka), i-αS kanye ne-tau electrostatic coacervates ene-valence egculisayo inamandla aphansi angaphezulu futhi ngenxa yalokho ayahlangana kancane, okuholela ekugugeni kwamaconsi ngokushesha.Isimo sejeli esizinzile esingahlanganisiwe siyafinyelelwa..Lesi simo sihle kakhulu esimweni sesistimu ye-αS/pLK ngenxa yokuhlobana kwayo okuphezulu kanye nenethiwekhi elula yokusebenzisana kwamaprotheni-pair, okuvumela uguquko olusheshayo olufana nejeli.Ngokuphambene nalokho, amaconsi ane-valence engagculisi futhi, ngakho-ke, izifunda ezikhokhiswa amaprotheni ezitholakalayo ukuze kuxhunyanwe nazo, zenza kube lula nge-coacervate ukuthi ihlanganise futhi imanzise indawo ye-hydrophilic ukuze kwehliswe amandla ayo aphezulu.Lesi simo sinconyelwa kuma-coacervates e-αS/Tau441, anenethiwekhi eyinkimbinkimbi enamavali amaningi ahlanganisa ukusebenzisana okubuthakathaka kwe-Tau-Tau kanye ne-αS-Tau.Ngokulandelayo, ama-coacervate amakhulu azogcina kalula izinto zawo ezifana noketshezi, okuvumela okunye ukusebenzisana kwamaprotheni kuya kuphrotheni ukuthi kwenzeke.Ekugcineni, ama-amyloid heterogeneous aggregates aqukethe kokubili i-αS kanye nefomu ye-tau ngaphakathi koketshezi lwe-coacervate, okungenzeka luhlobane nalezo ezitholakala emizimbeni ehlanganisiwe, okuyizimpawu zezifo ze-neurodegenerative.
Izakhiwo ezinkulu ezinjengoketshezi ezakheka ngesikhathi sokuvuthwa kwe-αS/Tau441 enendawo yamaprotheni eminyene kakhulu kodwa eshukumisayo futhi, ngokwezinga elincane, ama-coacervate e-αS/ΔNt-Tau angamadamu afanelekile okuhlanganisa amaprotheni.Ngempela sikubonile ukwakheka kwama-protein aggregates aqinile kulolu hlobo lwama-protein coacervates, ngokuvamile aqukethe kokubili i-αS ne-tau.Sibonise ukuthi lawa ma-heteroaggregates aqiniswa ngokusebenzisana okungewona ugesi, ayakwazi ukubopha odayi be-amyloid ethize be-ThT ngendlela efanayo nama-amyloid fibrils avamile, futhi ngempela anokumelana okufanayo namathonya ahlukahlukene.Izibalo ze-αS/tau ezakhiwe yi-LLPS ziboniswe zinezakhiwo ezifana ne-amyloid.Ngempela, okuhlukile okuvuthiwe kwe-Tau entula ekuhlanganisweni kwe-amyloid kulimaze kakhulu ekwakhekeni kwalezi zibalo ze-αS ezihlukene ngaphakathi kwe-liquid electrostatic coacervate.Ukwakheka kwe-αS/Tau441 aggregates kwabonwa kuphela ngaphakathi kwama-coacervates, agcina izakhiwo ezinjengoketshezi, futhi ungalokothi, uma ama-coacervates/amaconsi engazange afinyelele kusimo sejeli.Esimeni sakamuva, amandla anda okusebenzelana kwe-electrostatic futhi, ngenxa yalokho, ukuqina kwenethiwekhi yamaprotheni kuvimbela ukuhlelwa kabusha kwe-conformational of proteins ukuze kusungulwe ukusebenzisana kwamaprotheni okusha okudingekayo ku-amyloid nucleation.Kodwa-ke, lokhu kungafezwa ngama-coacervate aguquguqukayo, afana noketshezi, okungenzeka ukuthi azohlala ewuketshezi njengoba ekhula ngosayizi.
Iqiniso lokuthi ukwakheka kwama-aggregate ngaphakathi kwesigaba esijiyile kuyathandeka kumacondensate amakhulu e-αS/Tau kunamaconsi amancane ajiya ngokushesha, agqamisa ukubaluleka kokuhlonza izici ezilawula ukuhlangana kwamaconsi.Ngakho-ke, akukhona nje kuphela ukuthambekela kokuhlukaniswa kwesigaba, kodwa ubukhulu be-condensate kufanele bulawule ukusebenza kahle kanye nokuvimbela izifo58,61.Imiphumela yethu iphinde igqamise ukubaluleka kwebhalansi phakathi kwe-LLPS ne-LSPT kusistimu ye-αS/Tau.Nakuba ukwakheka kwamaconsi kungase kuvikele ekuhlanganisweni kwe-amyloid ngokunciphisa inani lama-protein monomers atholakalayo ngaphansi kwezimo zokugcwala, njengoba kuye kwahlongozwa kwezinye izinhlelo63,64, ukuhlanganiswa kwamaconsi emazingeni aphezulu amaconsi kungase kuholele ekuhlanganisweni kwamaprotheni angaphakathi ngokuhlelwa kabusha kwe-conformational kancane.amanethiwekhi amaprotheni..
Sekukonke, idatha yethu igcizelela kakhulu ukuhlobana kwe-valence ehlangene kanye nokusebenzisana okwanelisayo/okunganeliseki kumanethiwekhi okwehla kumongo we-LSPT.Ikakhulukazi, sibonisa ukuthi ama-condensate e-αS/Tau441 anobude obugcwele ayakwazi ukuhlanganisa kahle futhi ahlanganise i-nucleate ukuze akhe ama-heteroaggregate afana ne-amyloid ahlanganisa womabili amaprotheni futhi aphakamise indlela yamangqamuzana esekelwe emiphumeleni yethu yokuhlola.Ukuhlanganiswa kwamaprotheni amabili ku-αS/Tau fluid coacervate esiyibika lapha kungase kuhlobane ngempela nokuhlanganiswa kwamaprotheni amabili ku-inclusions, okuyizimpawu eziphawulekayo zesifo, futhi kungase kube nomthelela ekuqondeni ubudlelwano phakathi kwe-LLPS ne-LLPS. i-amyloid aggregation, evula indlela ye-IDP ekhokhiswe kakhulu ekuwohlokeni kwemizwa.
I-Monomeric WT-αS, i-cysteine ​​​​mutants (Q24C-αS, N122C-αS) kanye nezinhlobonhlobo ze-ΔCt-αS (Δ101-140) zichazwe ku-E. coli futhi zahlanzwa njengoba kuchazwe ngaphambilini.I-5 mM DTT ifakiwe kuzo zonke izinyathelo zokuhlanzwa kwe-αS cysteine ​​​​mutants ukuvimbela ukwakheka kwebhondi ye-disulfide.I-Tau441 isoform (i-plasmid etholakala ku-addgene # 16316), i-ΔNT-Tau Variant (Δ1-150 (Δ1-150, etholakala ngokutholwa kwe-Iva nge-PTTTTAAGAAGGAAGCAAGTCTTaAAC) kanye ne-AGGDEF-TAU (Δ275-311, kuhlanjululwe nge-GGCTC5 Primer) E. Coli Cults kwakukhona ikhule yaba ngu-OD600 = 0.6–0.7 ku-37°C kanye no-180 rpm, futhi inkulumo yakhelwe nge-IPTG amahora angu-3 ku-37°C.Vuna amaseli ku-11,500 xg imizuzu engu-15 ku-4 °C futhi ugeze nge-saline buffer equkethe u-150 mM NaCl.Misa kabusha i-pellet ku-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 senziwa eqhweni nge-amplitude ye-80% ye-pulses ye-10 (i-1 iminithi ivuliwe, i-1 min off).Ungadluli ku-60 ml ku-ultrasound eyodwa.I-E. coli lysates yashiswa ku-95 ° C. imizuzu engu-20, bese ipholiswa eqhweni futhi i-centrifuged ku-127,000 × g imizuzu engu-40.I-supernatant ecacisiwe isetshenziswe kulwelwesi lwe-3.5 kDa (Spectrum™ Thermo Fisher Scientific, UK) futhi yacolwa ngokumelene no-4 L we-dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) amahora angu-10.Ikholomu yokushintshisana ye-cation engu-5 ml (HiTrap SPFF, Cytiva, MA, USA) yahlanganiswa nesibhafa sokulinganisa (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 yahlungwa ngesihlungi se-PVDF esingu-0.22 μm futhi yajovwa kukholamu ngenani lokugeleza elingu-1 ml/min.I-Elution yenziwa kancane kancane, i-tau yakhishwa 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).Izingxenyana zihlaziywe yi-SDS-PAGE, futhi noma yiziphi izingxenyana eziqukethe ibhande elilodwa elinesisindo esilindelekile semolekyuli ye-tau zagxiliswa kusetshenziswa isihlungi se-centrifuge esingu-10 kDa futhi kwathathelwa indawo isigcinalwazi esiqukethe 10 mM HEPES, pH 7.4, NaCl 500 mM kanye ne-DTT 2 mM ye ukugxila kokugcina kwamaprotheni kwaba ngu-100 μM.Isixazululo samaphrotheni sabe sesidluliswa ngesihlungi se-PVDF esingu-0.22 μm, siqandiswe ngokushesha futhi sigcinwe ku -80°C.Iphrotheni K18 yanikezwa ngomusa nguProf. Alberto Boffi.Ukuhlanzeka kwamalungiselelo kwaku> 95% njengoba kuqinisekiswe i-SDS-PAGE kanye ne-MALDI-TOF/TOF.Ama-cysteines ahlukahlukene afakwe amakhemikhali alebulwe ngokuthi i-AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) noma i-TEMPOL-maleimide (i-Toronto Research Chemicals, i-Toronto, Canada).kuqinisekiswe ukumuncwa kanye ne-MALDI-TOF/TOF.I-Tau441, i-ΔNt-Tau, i-AggDef-Tau kanye ne-K18 zilebulwe ngezinsalela zomdabu ze-cysteine ​​​​ezikhundleni 191 kanye ne-322 zisebenzisa i-Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Germany) ngokulandela inqubo efanayo.Isamba sesamba semephu eyinsalela ngayinye ye-αS ne-Tau441 yenziwe kusetshenziswa i-CIDER66.
I-Solid poly-L-lysine (pLK DP 90-110 ngokusho kwe-NMR evela kumphakeli, i-Alamanda Polymers Inc, Huntsville, Alabama, USA) yahlakazwa ngo-10 mM HEPES, 100 mM NaCl, pH 7.4 kuya ku-10 mM, inqubo ye-sonicated 5 imizuzu kubhavu wamanzi we-ultrasonic futhi ugcine ku -20°C.I-PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) kanye ne-FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) ancibilika emanzini futhi asakazwa kabanzi kubhafa ye-LLPS.I-Dialysis isusa usawoti ongcolisayo.Zabe sezihlungwa ngesisefo sesirinji esinosayizi wembotshana ongu-0.22 μm, futhi ukugxila kwawo kubalwa kusetshenziswa i-refractometer (Mettler Toledo, Columbus, Ohio, USA).Amasampula e-LLPS alungiselelwa ekamelweni lokushisa ngendlela elandelayo: i-buffer ne-extrusion yaxutshwa kanye ne-1 mM tris(2-carboxyethyl)phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) i-tetraacetic acid (EDTA, carboxynth) kanye nengxube ye-1% ye-protease inhibitor (PMSF 100 mM, i-benzimide 1 mM, i-leupeptin 5 μM).Bese kwengezwa i-αS kanye nama-polycations ahlanganisiwe (izinketho pLK noma i-Tau).Ocwaningweni lochungechunge lwesikhathi lwe-thioflavin-T (i-ThT, i-Carbosynth, i-Compton, e-UK), sebenzisa ingqikithi yokugxilisa ingqondo ye-ThT ukuze ube uhhafu wokugxiliswa kwe-αS.Hlanganisa ngobumnene kodwa ngokucophelela amasampula ukuze uqinisekise ukuthi afana ne-homogeneous.Ukugxiliswa kwengxenye ngayinye kuyahluka kusukela ekuhlolweni kuya ekuhlolweni, njengoba kuchazwe esigabeni Semiphumela.I-Azide isetshenziswe ekugxiliseni okungu-0.02% (w/v) noma nini lapho ubude bokuhlolwa budlula amahora angu-4.Kukho konke ukuhlaziya kusetshenziswa amasampula e-LLPS, vumela ingxube ukuthi ilingane imizuzu emi-5 ngaphambi kokuhlaziya.Ukuze uthole ukuhlaziya okuhlakazeka okuncane, amasampula angu-150 µl alayishwa kuma-microplates angabophi angu-96 (µClear®, black, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) futhi embozwe ngefilimu enamathelayo.Ama-LLP aqashwe ngokulinganisa ukumunca ku-350 nm maphakathi nesixazululo kusifundi sepuleti se-CLARIOstar (BMG Labtech, Ortenberg, Germany).Ukuhlolwa kwenziwe ngokuphindwe kathathu ku-25°C, futhi amaphutha abalwa njengokuchezuka okujwayelekile kuncazelo.Isigaba sokuhlanjululwa salinganiswa ngesampula ye-centrifugation kanye nokuhlaziywa kwejeli ye-SDS-PAGE, futhi ingxenyenamba ye-αS esigabeni sokuhlanjululwa nokugxilile yalinganiswa kuzixazululo ezihlukahlukene ze-LLPS.Isampula engu-100 μl LLPS equkethe 1 μM AF488-ilebula αS yalungiswa ngokuxutshwa okuphelele okulandelwa yi-centrifugation ku-9600×g imizuzu engu-30, ngemva kwalokho imvula yayivame ukubonakala.I-50 μl ephezulu ye-supernatant yasetshenziselwa ukulinganisa amaprotheni kusetshenziswa ijeli ye-SDS-PAGE.Amajeli askeniwe ngezihlungi ze-AF488 kusetshenziswa isistimu yokuthwebula ijeli ye-ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) noma angcoliswe ibala le-Coomassie futhi abonwa ngezihlungi ezifanele.Amabhendi angumphumela ahlaziywa kusetshenziswa inguqulo ye-ImageJ 1.53i (Izikhungo Zezempilo Zikazwelonke, e-USA).Izivivinyo zenziwe ngokuphindwe kabili ezivivinyweni ezimbili ezihlukene ezinemiphumela efanayo.
Ngokujwayelekile, amasampula angu-150 μl asetshenziswa kuma-microplates angabophi angu-96 futhi abonwa kuzinga lokushisa legumbi kusibonakhulu esihlanekezelwe se-Leica DMI6000B (Leica Microsystems, Wetzlar, Germany).Ngokuhlolwa kwendawo, amapuleti e-µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Germany) noma ama-microplates e-polystyrene angama-96 (Corning Costar Corp., Acton, Massachusetts) nawo asetshenziswa.I-EL6000 halogen noma izibani ze-mercury metal halide zisetshenziswe njengemithombo yokukhanyisa (ye-BF/DIC ne-WF imaging, ngokulandelana).Ngemakroskopu ye-WF, inhloso yomoya yokukhulisa engu-40x (Leica Microsystems, Germany) yasetshenziswa ukuze kugxiliswe ukukhanya kusampula futhi kuqoqwe.Kumasampula anelebuli ye-AF488 kanye ne-ThT, izihlungi zokuhlunga kanye nokuphumayo okunamasethi okuhlunga e-GFP ajwayelekile, izihlungi ze-bandpass ezivusa amadlingozi neziphumayo, ngokulandelana kwazo, izihlungi ze-bandpass ezingu-460–500 nm nezingu-512–542 nm, nesibuko se-dichroic esingu-495 nm.Kumasampuli alebulwe ngokuthi i-Atto647N, isethi evamile yezihlungi ze-Cy5 ezinezihlungi ze-bandpass ezivusa amadlingozi ezingu-628–40 nm no-692–40 nm, ngokulandelana, kanye nesibuko se-dichroic esingu-660 nm.Ngemakroskopu ye-BF ne-DIC, sebenzisa inhloso efanayo yokuqoqwa kokukhanya ebonisiwe.Ukukhanya okuqoqiwe kuqoshwe kukhamera ye-Leica DFC7000 CCD (Leica Microsystems, Germany).Isikhathi sokuchayeka bekungu-50 ms we-BF ne-DIC imaging microscopy kanye no-20-100 ms we-WF imaging microscopy.Uma kuqhathaniswa, isikhathi sokuchayeka sazo zonke izivivinyo nge-ThT sasingu-100 ms.Ukuhlolwa kokudlula kwesikhathi kwenziwa ukuze kubonakale ukuhlangana kwamaconsi, izithombe ziqoqwa njalo ngo-100 ms imizuzu embalwa.I-ImageJ (NIH, USA) isetshenziselwe ukuhlaziya isithombe.Izivivinyo zenziwa ngokuphindwe kathathu ngemiphumela efanayo.
Ngokuhlolwa kwe-colocalization, i-FRAP kanye nokwakhiwa kabusha kwe-3D, izithombe zitholwe kusibonakhulu esihlanekezelwe se-Zeiss LSM 880 kusetshenziswa i-ZEN 2 edition eluhlaza okwesibhakabhaka (Carl Zeiss AG, Oberkochen, Germany).Amasampula e-50 µl asetshenziswe ezitsheni ze-µ-Slide Angiogenesis Petri (Ibidi GmbH, Gröfelfing, Germany), eziphathwe nge-hydrophilic polymer (ibiTreat) futhi zifakwe kunhloso yokucwiliswa kawoyela engu-63× (Plan-Apochromat 63×/NA 1.4 Oil) ku-DIC).Izithombe zitholwe kusetshenziswa imigqa ye-laser engu-458 nm, 488 nm, kanye ne-633 nm argon laser enokulungiswa okungu-0.26 µm/pixel nesikhathi sokuchayeka esingu-8 µs/pixel ukuze uthole amafasitela enjabulo nokuphuma kwawo angu-470–600 nm, 493–628 nm, kanye ne-638–755 nm yasetshenziselwa ukubona ngeso lengqondo i-ThT, AF488 kanye ne-Atto647N, ngokulandelana.Ocwaningweni lwe-FRAP, izithombe ezidlula isikhathi zesampula ngayinye zarekhodwa ngohlaka olu-1 ngesekhondi.Izivivinyo zenziwa ngokuphindwe kathathu ezingeni lokushisa legumbi nemiphumela efanayo.Zonke izithombe zahlaziywa kusetshenziswa isofthiwe ye-Zen 2 blue edition (Carl Zeiss AG, Oberkochen, Germany).Amajika e-FRAP enziwa ajwayelekile, ahlelwa futhi afakwa kudatha eqinile/isikhathi ekhishwe ezithombeni kusetshenziswa i-Zen 2 kusetshenziswa i-OriginPro 9.1.Amajika okubuyisela afakwe kumodeli ye-mono-exponential ukuze kulandiswe ngokusatshalaliswa kwamangqamuzana kanye nethemu elingeziwe le-exponential ukuze kulandiswe ngomthelela wokutholwa kokufiphala.Sabe sesibala u-D sisebenzisa irediyasi yokufiphala okuzisholo kanye nengxenye yempilo yokutakula enqunywe ngaphambili njengakwisibalo sika-Kang et al.5 35 kukhonjisiwe.
Okuhlukile kwe-cysteine ​​​​okukodwa kwe-αS kwahlanganiswa ne-4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) ezikhundleni 24 (TEMPOL-24-αS) kanye ne-122 (TEMPOL-122-αS), ngokulandelana.I-Spin Ilebula Ngokuhlolwa kwe-EPR, ukugxiliswa kwe-αS kwamiswa ku-100 μM futhi ukugxiliswa kwe-PEG kwaba ngu-15% (w/v).Ezimweni ezihlukahlukene zokuhlanganisa, isilinganiso se-αS:pLK sasingu-1:10, kuyilapho izilinganiso ze-αS:ΔNt-Tau ne-αS:Tau441 zigcinwe kokuthi 1:1.Ngokuhlolwa kwe-titration okubophayo lapho kungekho ukuminyana, i-TEMPOL-122-αS yagcinwa ku-50 μM futhi ama-polycations afakwe ama-titrated ekugxilweni okukhulayo, kulungiselelwa isimo ngasinye ngokwehlukana.Izilinganiso ze-CW-EPR zenziwa kusetshenziswa i-spectrometer ye-Bruker ELEXSYS E580 X-band efakwe i-Bruker ER4118 SPT-N1 resonator esebenza kuma-microwave (SHF) angu-~9.7 GHz.Izinga lokushisa lalibekwe ku-25°C futhi lilawulwa yi-nitrogen cryostat ewuketshezi.I-spectra itholwe ngaphansi kwezimo ezingenalutho kumandla we-MW angu-4 mW, i-modulation amplitude engu-0.1 mT, kanye ne-modulation frequency engu-100 kHz.Ukuqina kwe-Spectral kwenziwe kwajwayelekile ukuze kugwenywe umehluko ekugxilweni kwe-spin phakathi kwamasampuli kanye nokwehliswa kwe-spin okungenzeka ngenxa yokugxilisa okusele kwama-ejenti anciphisayo kumasampuli aqukethe i-Tau441 noma i-ΔNt-Tau (ekhona kuzixazululo zamaprotheni zangempela).Amanani anikeziwe we-g atholwe ngenxa yemodeli ye-spectral ye-EPR eyenziwe kusetshenziswa isofthiwe ye-Easyspin (v. 6.0.0-dev.34) esetshenziswe kokuthi Matlab®67.Amamodeli e-isotropic engxenye eyodwa/ezimbili asetshenziswe ukwenza imodeli yedatha.Ngemva kokwenza wonke amasignali abejwayelekile, izinsalela zibalwe ngokukhipha ukulingisa ngakunye ku-spectrum yokuhlola ehambisanayo.Ukuze kuhlaziywe i-titration ebophezelayo, ukushuba okuhlobene kwebhendi yesithathu kubhendi yesibili ye-spectrum ye-EPR evamile (IIII/III) kwasetshenziswa ukuqapha ukubophezela kwe-polycations ku-αS.Ukuze ulinganisele i-dissociation constant (Kd), ijika eliwumphumela lafakwa kumodeli elinganiselwe kucatshangwa ukuthi n amasayithi abophezelayo afanayo nazimele.
Ukuhlolwa kwe-NMR spectroscopy kwenziwa kusetshenziswa i-spectrometer ye-Bruker Neo 800 MHz (1H) NMR efakwe i-cryoprobe ne-Z-gradient.Konke ukuhlola kwenziwe kusetshenziswa i-130–207 µM αS nokuhambisanayo kwe-αS/ΔNt-Tau nokulingana kwe-pLK ku-10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 futhi kwenziwa ku-15°C.Ukuqapha i-LPS nge-NMR, u-10% we-PEG wengezwe kumasampuli axutshwe ngaphambilini.Isakhiwo samakhemikhali esiphazamisayo (Umfanekiso 1b) sibonisa isilinganiso sokushintsha kwamakhemikhali okungu-1H no-15N.I-spectra ye-αS 2D1H-15N HSQC yabelwe ngokusekelwe kumsebenzi owabelwe ngaphambili (ukufakwa kwe-BMRB #25227) futhi yaqinisekiswa ngokurekhoda nokuhlaziya i-3D spectra ye-HNCA, i-HNCO ne-CBCAcoNH.I-13Cα kanye ne-13Cβ amashifu amakhemikhali abalwe phambi kwe-ΔNt-Tau noma i-pLK ukuze kukalwe izinguquko ezingaba khona kumathrendi esakhiwo sesibili uma kuqhathaniswa namashifu amakhemikhali e-αS ekuhlanganiseni okungahleliwe kwekhoyili okungahleliwe 68 (Umfanekiso Owengeziwe 5c).Amanani e-R1ρ akalwa ngokurekhoda ukuhlolwa kwe-hsqctretf3gpsi (etholwe kumtapo wolwazi we-Bruker) nokubambezeleka okungu-8, 36, 76, 100, 156, 250, 400, kanye no-800 ms, futhi imisebenzi yomchazi yalungiswa ukuze ibe sezingeni eliphakeme ngokulibaziseka okuhlukile. izikhathi zokunquma i-R1ρ kanye nokungaqiniseki kokuhlolwa kwayo.
Ukuhlolwa kwe-fluorescence microscopy okuxazululwe ngemibala emibili okuxazululwe ngesikhathi kwenziwa kusibonakhulu esixazululwe isikhathi sezentengiso se-MT200 fluorescence confocal (PicoQuant, Berlin, Germany) ngedivayisi yokubala ye-photon eyodwa ehlobene nesikhathi (TCSPC).Ikhanda le-laser diode lisetshenziselwa i-pulsed interleaved excitation (PIE), i-beam idlula i-waveguide yemodi eyodwa futhi ishunwe emandleni e-laser angu-10 kuya ku-100 nW ku-481 nm kanye nemigqa ye-laser engu-637 nm elinganiswa ngemva kwesibuko se-dichroic.Lokhu kuqinisekisa izinga elilungile lokubala le-photon, ukugwema imiphumela ye-photon aliasing, i-photobleaching kanye nokugcwaliswa kwesikhala.μ-Amasembozo e-angiogenesis yesilayidi noma amapuleti (Ibidi GmbH, Gräfelfing, Germany) abekwe ngokuqondile emanzini acwiliswa phezu kwelensi ye-Super Apochromat 60x NA 1.2 enekhola yokulungisa (Olympus Life Sciences, Waltham, USA).Isibuko se-dichroic esingu-488/640 nm (Semrock, Lake Forest, IL, USA) sisetshenziswe njengesihlukanisi esikhulu semishayo.Imisebe engagxilile ivinjwe imbobo enobubanzi obungama-microns angu-50, khona-ke imisebe egxilile ihlukaniswa ibe izindlela ezi-2 zokutholwa nge-splitter ye-50/50 ye-beam.Izihlungi ze-Bandpass emission (Semrock, Lake Forest, IL, USA) 520/35 zikadayi oluhlaza (AF488) kanye no-690/70 wodayi obomvu (Atto647N) zisetshenziswe phambi komtshina.I-single-photon avalanche diode (SPAD) (Amadivayisi amancane we-Photon, e-Bolzano, e-Italy) asetshenziswe njengezitholi.Kokubili ukuqoqwa kwedatha nokuhlaziya kwenziwe kusetshenziswa isofthiwe ye-SymphoTime64 etholakalayo kwezohwebo (PicoQuant GmbH, Berlin, Germany).
Kwasetshenziswa ama-microlitre angamashumi amahlanu amasampula e-LLPS emithonjeni ye-angiogenesis ye-μ-Slide (Ibidi GmbH, Gräfelfing, Germany).Izithombe eziwumphumela zigxile ku-20 µm ngaphezu komthombo ophansi webanga elilungile lokusebenza lenjongo yamaconsi amisiwe kanye nokuya ku-~1 µm yama-raft namachashazi anokulungiswa kwe-axial okungenani okungu-0.25 µm/pixel kanye nesikhathi sokulibaziseka esingu-400 µs/pixel.Khetha idatha ngokusebenzisa umkhawulo wokuqina ngokusekelwe kubukhulu besignali yangemuva emaphakathi (i-PBG, isho + 2σ) yesiteshi ngasinye ukuze kukhethwe amaconsi amaprotheni awuketshezi kuphela, ama-raft, noma amachashazi, kuhlungwe noma iyiphi imvelaphi engaba khona esigabeni esihlakaziwe.Ukuze sihlaziye isikhathi sokuphila sohlobo ngalunye (τ) lwesiteshi ngasinye (okuluhlaza, “g” kwe-AF488 nokubomvu, “r” kwe-Atto647N), sikhethe izifunda ezinentshisekelo kuzo (ama-ROI) aqukethe amaconsi, ama-raft, noma amachashazi (Umfanekiso Owengeziwe 1 ).8b) futhi yawathola ngokufaka ukubola kwawo kwesikhathi sonke sokuphila (τD, τR kanye ne-τP yamaconsi, ama-raft noma amachashaza, ngokulandelana, bheka i-Supplementary Fig. 8c) kushaneli ngayinye usebenzisa ukuhlaziya okulingana nomsila kanye nemodeli yokubola enezingxenye ezimbili.Isilinganiso esingu-τ sisuka ku-τ.Ama-ROI akhiqize ama-photon ambalwa kakhulu ukuze afane ne-multi-exponential awafakwanga ekuhlaziyweni.I-cutoff esetshenzisiwe yayingu-<104 photon yama-raft namachashazi kanye no-103 okokwehla.Amaconsi anomkhawulo ophansi ngenxa yokuthi kunzima ukuthola amajika okubola anamanani aphezulu okuqina, njengoba amaconsi endimeni yesithombe avamise ukuba mancane futhi abe maningi kancane.Ama-ROI anezibalo ze-photon ezingaphezu komkhawulo wokuqongelela ama-photon (amiswe ku->500 count/pixel) nawo alahlwa ukuze ahlaziywe.Qondanisa ijika lokubola kokuqina elitholwe endaweni enentshisekelo ngamandla angu-90% wobukhulu (kancane ngemva kobukhulu bokubola) kusukela ekuqaleni kwempilo yesevisi ukuze kuqinisekiswe ukuthikamezeka okuncane kwe-IRF kuyilapho kugcinwa okufanayo kukho konke ukubola okukhulu. izilungiselelo Iwindi lesikhathi esihlobene Lahlaziywa i-25 kuya ku-50 ROI yama-raft namabala kanye ne-15-25 ROI ukuze uthole amaconsi, izithombe ezikhethwe kuzimpinda ezingaphezu kwezi-4 eziqoshwe okungenani ekuhlolweni okuzimele okungu-3.Ukuhlola kuka-t okunemisila emibili kusetshenziswe ukuhlola umehluko wezibalo phakathi kwezinhlobo zezilwane noma phakathi kwezinhlelo ze-coacervate.Ukuze kube nokuhlaziywa kwephikseli ngephikseli kwesikhathi sonke sempilo (τ), isamba sokuncipha kwempilo yonke enkambu yesiteshi ngasinye sibaliwe futhi ukulinganiselwa kwemodeli ye-exponential engu-2/3-ingxenye ye-attenuation kwenziwa.Ukuncishiswa kwesikhathi sonke sempilo kwephikseli ngayinye bese kufakwe kusetshenziswa amanani angu-τ abalwe ngaphambilini, okuholela esithombeni esilingana ne-pseudocolor FLIM.Ibanga lokuphila elilingana nomsila lalifana kuzo zonke izithombe zesiteshi esifanayo, futhi ukubola ngakunye kukhiqize ama-photon anele ukuze kunikeze ukulingana okuthembekile.Ukuze kuhlaziywe i-FRET, amaphikseli akhethwe ngokusebenzisa umkhawulo ophansi wokuqina wama-photon angu-100, okulinganisa isignali yangemuva (i-FBG) yama-photon angu-11.Ukuqina kwe-fluorescence yesiteshi ngasinye kwalungiswa yizici zokulungisa ezinqunywe ngokuhlolwa: i-69 spectral crosstalk α yayiyi-0.004, i-excitation eqondile β yayingu-0.0305, ukusebenza kahle kokutholwa γ kwakuyi-0.517.Ukusebenza kahle kwe-FRET ezingeni lephikseli bese kubalwa kusetshenziswa isibalo esilandelayo:
lapho i-FDD iwumfutho we-fluorescence obonwa esiteshini somnikeli (esiluhlaza), i-FDA iwumfutho we-fluorescence obonwa esiteshini esivumayo (obomvu) ngaphansi kwesasasa elingaqondile, futhi i-FAA iwumfutho we-fluorescence obonwa esiteshini sokwamukela (obomvu) ngaphansi kwenjabulo eqondile ( I-PIE).I-fluorescence intensity pulses ibonwa esiteshini).
Beka i-100 µl yezixazululo zokusabela ze-LLPS eziqukethe i-25 µM engalebuli ye-monomeric Tau441 (ene-25 µM αS noma ngaphandle kwayo) kubhafa ye-LLPS (yengezwe njengenhla) kuma-microplates angabophi angu-96-well anembombo ye-foil enamathelayo kanye nokwakheka kwamaconsi kuhlolwe nge-WF ukulinganisa.phakathi kwemizuzu eyi-10.Ngemva kwamahora angu-48 ekufukamela ekamelweni lokushisa, ukuba khona kwama-protein rafts namachashaza kwaqinisekiswa.Bese ukhipha ngokucophelela uketshezi phezu kwezihlenga ezivela emithonjeni, bese ufaka u-50 L we-dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) bese ufukamela imizuzu eyi-10.Ukugcwala kukasawoti omningi kuqinisekisa ukuthi i-LLPS ngeke iphinde ngenxa ye-PEG eyinsalela, futhi kungenzeka ukuthi amaphrotheni ahlangane akhiwe kuphela ngokusebenzisana kwe-electrostatic azohlakazwa.Iphansi lomthombo labe selisulwa ngokucophelela ngethiphu ye-micropipette futhi isixazululo esiwumphumela sadluliselwa emthonjeni wokubuka ongenalutho.Ngemva kokufakwa kwamasampula nge-50 μM ThT ihora elingu-1, ukuba khona kwamabala angawodwa kwahlolwa nge-WF microscopy.Lungiselela ama-sonicated αS fibrils ngokufaka i-300 µl yesisombululo se-70-µM αS ku-PBS nge-pH 7.4, i-sodium azide 0.01% ku-37 °C kanye no-200 rpm ku-orbital shaker izinsuku ezingu-7.Isixazululo sabe se-centrifuged ku-9600 × g ngamaminithi angu-30, i-pellet yaphinde yamiswa ku-PBS pH 7.4 futhi yafakwa i-sonicated (1 min, 50% cycle, 80% amplitude ku-Vibra-Cell VC130 sonicator, Sonics, Newton, USA) amasampula e-fibril. ngokusatshalaliswa kosayizi ofanayo kwama-fibrils amancane.
Ukuhlaziywa kwe-FCS/FCCS kanye nokutholwa kokuqondana kwemibala emibili (TCCD) kwenziwe kusibonakhulu esixazululwe isikhathi se-fluorescent confocal MT200 (Pico-Quant, Berlin, Germany) esisetshenziselwa ukuhlolwa kwe-FLIM-FRET microscopy kusetshenziswa imodi ye-PIE.Amandla e-laser alokhu kuhlolwa engezwe ku-6.0 µW (481 nm) no-6.2 µW (637 nm).Inhlanganisela yala mandla e-laser yakhethwa ukuze kukhiqizwe ukukhanya okufanayo kwamapheya e-fluorophores asetshenziswa ngenkathi kufinyelelwa amanani okubala alungile futhi kugwenywa ukwenza izithombe nokugcwaliswa kwesikhala.Kokubili ukuqoqwa kwedatha nokuhlaziya kwenziwa kusetshenziswa isofthiwe ye-SymphoTime64 version 2.3 etholakalayo kwezohwebo (PicoQuant, Berlin, Germany).
Amasampuli okuhlanganisiwe okukodwa kwe-αS/Tau atholwe kusetshenziswa i-LLPS ahlanjululwa kubhafa yokuhlukanisa ukuya ekugxiliseni okufanelekile kwe-monomolecular (imvamisa i-dilution engu-1:500, njengoba ama-aggregate asevele ekugxilweni okuphansi uma ehlukanisiwe kumasampula e-coacervate).Amasampuli asetshenziswe ngokuqondile kuma-coverlips (Corning, USA) agcotshwe ngaphambili ngesisombululo se-BSA ekuhlanganiseni okungu-1 mg/mL.
Ekuhlaziyweni kwe-PIE-smFRET eziteshini eziluhlaza nokubomvu, kusetshenziswe umkhawulo wokuqina ophansi wama-photon angu-25 ukuze kuhlungwe amasiginali anamandla aphansi abangelwa izehlakalo ze-monomeric (qaphela ukuthi ama-monomers amasampula ahlanganisiwe aqhathaniswa nama-aggregate angawodwana ngenani).Lo mkhawulo ubalwe njengokuphindwe kahlanu kokuqina kwesilinganiso se-αS ye-monomeric etholwe ekuhlaziyweni kwamasampuli e-monomer emsulwa ukuze kukhethwe ngokukhethekile izilinganiso zokuhlaziywa.Isekethe yedrayivu ye-PIE, kanye nokutholwa kwedatha ye-TSCPC, inikeze amandla ukusetshenziswa kwesihlungi sesisindo sokuphila konke esiza ukuqeda ingemuva ne-spectral crosstalk.Amandla okuvuleka akhethwe kusetshenziswa ama-threshold angenhla alungisiwe kusetshenziswa isignali yangemuva emaphakathi enqunywe kusukela kuma-histograms wesenzeko ngokumelene nokuqina/umgqomo wamasampuli webhafa kuphela.Ukuqhuma okuhlotshaniswa nama-aggregate amakhulu ngokuvamile kuthatha imigqomo eminingana elandelanayo ekulandeleni kwesikhathi (kusethwe ku-1 ms).Kulezi zimo, kukhethwe umgqomo wamandla amakhulu.Ukuze kuhlaziywe i-FRET ne-stoichiometric, i-gamma factor γ (0.517) enqunywe ngokomqondo isetshenzisiwe.Iminikelo ye-Spectral crosstalk ne-direct excitation ayinakwa (inqunywa ngokokuhlola) kumandla e-laser evusa amadlingozi asetshenzisiwe.Ukusebenza kahle kanye ne-stoichiometry ye-FRET ekuqhumeni kubalwa ngale ndlela elandelayo.