347 12.7 * 1.24mm Chitsulo chosapanga dzimbiri chophimbidwa ndi chubu, Molecular limagwirira wa synchronous electrostatic condensation ndi coaggregation wa α-synuclein ndi tau

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347 Tsatanetsatane wa Chitoliro Chachitsulo chosapanga dzimbiri

347 12.7 * 1.24mm Chitsulo chosapanga dzimbiri chophimbidwa ndi machubu

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Amyloid alpha-synuclein (αS) aggregation ndi chizindikiro cha matenda a Parkinson ndi ma synucleinopathies ena.Posachedwapa, mapuloteni a tau omwe nthawi zambiri amagwirizanitsidwa ndi matenda a Alzheimer's akhala akugwirizana ndi matenda a αS ndipo akupezeka kuti akugwirizana ndi αS-rich inclusions, ngakhale kuti njira ya ma molekyulu yogwirizanitsa mapuloteni awiriwa sakudziwika bwino.Tikunena pano kuti gawo αS alekanitsa mu madzi condensates kudzera electrostatic zovuta condensation ndi polypeptides zabwino mlandu monga tau.Kutengera kuyanjana kwa αS kwa polycations ndi kuchuluka kwa valence kutha kwa netiweki ya coagulation, ma clots amapita mwachangu kapena kuphatikizana ndikutsatiridwa ndi kuphatikizika kwapang'onopang'ono kwa amyloid.Pophatikiza gulu la njira zapamwamba za biophysical, tinatha kuwonetsa kulekana kwa gawo lamadzi-lamadzi αS/Tau ndikuzindikira zinthu zofunika kwambiri zomwe zimapangitsa kuti pakhale magulu osiyanasiyana omwe ali ndi mapuloteni onse mu protein yamadzimadzi condensate.
Kuphatikiza pa zigawo za nembanemba, kulekana kwa malo m'maselo kungathenso kutheka popanga matupi ochuluka a protein, owundana ngati madzi otchedwa biomolecular condensates kapena droplets, kudzera mu njira yotchedwa liquid-liquid phase separation (LLPS).Madonthowa amapangidwa ndi kuyanjana kwakanthawi kochepa, nthawi zambiri pakati pa mapuloteni kapena mapuloteni ndi RNA, ndipo amagwira ntchito zosiyanasiyana pafupifupi machitidwe onse amoyo.Mapuloteni ambiri omwe ali ndi LLP amawonetsa zovuta zochepa zomwe zimasokonezeka kwambiri m'chilengedwe komanso kupanga biomolecular condensates3,4,5.Kafukufuku wambiri woyesera awonetsa kusinthika, kosasinthika, komanso kuchuluka kwa mapuloteni omwe amapanga ma condensate amadzimadziwa, ngakhale zochepa zomwe zimadziwika pazomwe zimadziwika ndi mamolekyulu omwe amawongolera kukula ndi kukhwima kwa ma condensatewa kukhala olimba ngati. boma..
Deta yatsopanoyi imathandizira malingaliro akuti LLPS yoyendetsedwa ndi mapuloteni komanso kusintha kwa madontho kukhala zinthu zolimba kungakhale njira zama cell zomwe zimatsogolera kupanga magulu akupha osasungunuka omwe nthawi zambiri amakhala zizindikiro za matenda osachiritsika.Mapuloteni ambiri okhudzana ndi LLPS (IDPs), omwe nthawi zambiri amakhala okwera mtengo komanso osinthika, akhala akugwirizana ndi neurodegeneration kudzera pakuphatikiza kwa amyloid.Makamaka, biomolecular IDP condensates monga FUS7 kapena TDP-438 kapena mapuloteni okhala ndi madera akuluakulu ocheperako monga hnRNPA19 awonetsedwa kuti amakalamba kukhala mawonekedwe ngati gel kapena olimba kudzera munjira yotchedwa fluidization.palimodzi.to solid phase transition (LSPT) monga ntchito ya nthawi kapena poyankha zosintha zina pambuyo pa kumasulira kapena kusintha kwakukulu kwa pathologically1,7.
IDP ina yolumikizidwa ndi LLPS mu vivo ndi Tau, mapuloteni osagwirizana ndi ma microtubule omwe kuphatikizika kwake kwa amyloid kumakhudzidwa ndi matenda a Alzheimer's10 koma posachedwapa adakhudzidwa ndi matenda a Parkinson (PD) ndi ma synaptic nuclear proteinopathies 11, 12, 13 amagwirizana.Tau yasonyezedwa kuti imasiyanitsidwa ndi yankho/cytoplasm chifukwa cha kuyanjana kwabwino kwa electrostatic14, zomwe zinapangitsa kupanga madontho owonjezera a tau otchedwa electrostatic coacervates.Zawonedwanso kuti kuyanjana kosagwirizana kumeneku ndiko kumayambitsa ma biomolecular condensates ambiri mu chilengedwe15.Pankhani ya mapuloteni tau, electrostatic aggregation akhoza kupangidwa ndi yosavuta aggregation, imene oppositely mlandu zigawo za mapuloteni kuyambitsa ndondomeko cleavage, kapena ndi aggregation zovuta mwa kucheza ndi ma polima zoipa mlandu monga RNA.
Posachedwapa, α-synuclein (αS), IDP ya amyloid yomwe imakhudzidwa ndi PD ndi matenda ena a neurodegenerative omwe amadziwika kuti synucleinopathy17,18, asonyezedwa m'maselo amtundu ndi zinyama19,20 yokhazikika mu mapuloteni osakanikirana ndi khalidwe lamadzimadzi.Kafukufuku wa in vitro awonetsa kuti αS imakumana ndi LLPS mwa kuphatikizika kosavuta kudzera mu kuyanjana kwakukulu kwa hydrophobic, ngakhale kuti njirayi imafunikira kuchuluka kwa mapuloteni ochulukirapo komanso nthawi yayitali yoyambira19,21.Kaya ma condensate okhala ndi αS omwe amawonedwa mu vivo amapangidwa ndi izi kapena njira zina za LLPS ikadali nkhani yayikulu yosathetsedwa.Mofananamo, ngakhale kuti αS amyloid aggregation yakhala ikuwonetsedwa mu neurons mu PD ndi ma synucleinopathies ena, njira yeniyeni yomwe αS imayendera intracellular amyloid aggregation imakhalabe yosadziwika bwino, chifukwa kuwonjezereka kwa mapuloteniwa sikukuwoneka kuti kumayambitsa ndondomekoyi yokha.Kuwonongeka kwa ma cell kumafunika nthawi zambiri, kutanthauza kuti malo ena am'manja kapena ma microenvironments amafunikira kuti abwezeretsenso misonkhano ya intracellular αS amyloid.Malo amodzi a ma cell omwe amatha kuphatikizika makamaka amakhala mkati mwa protein condensates 23.
Chochititsa chidwi n'chakuti, αS ndi tau zapezeka kuti zimagwirizanitsa m'magulu a matenda omwe amapezeka mwa anthu omwe ali ndi matenda a Parkinson ndi ma synucleinopathies 24,25 ndipo kuyesera kwawonetsa mgwirizano wapakati pa mapuloteni awiriwa 26,27 akusonyeza mgwirizano womwe ungakhalepo pakati pa kuphatikizira αS ndi tau mu matenda a neurodegenerative.kudwala.αS ndi tau zapezeka kuti zimagwirizana ndikulimbikitsana wina ndi mzake mu vitro ndi mu vivo 28,29 ndi magulu osakanikirana omwe amapangidwa ndi mapuloteni awiriwa awonedwa mu ubongo wa odwala omwe ali ndi synucleinopathies 30.Komabe, ndi zochepa zomwe zimadziwika ponena za maziko a mamolekyu a mgwirizano pakati pa αS ndi tau ndi makina ake ophatikizana.αS yanenedwa kuti ilumikizana ndi tau kudzera mu kukopa kwa electrostatic pakati pa chigawo cha C-terminal cha αS chomwe chili ndi vuto lalikulu la αS ndi dera lapakati lolemera la proline la tau, lomwe lilinso ndi zotsalira zabwino.
Mu phunziro ili, timasonyeza kuti αS akhozadi dissociate mu m'malovu kudzera electrostatic complex condensation pamaso pa tau mapuloteni, mosiyana ndi kugwirizana kwake ndi ma polypeptides ena abwino chaji monga poly-L-lysine (pLK), ndipo potero .αS imagwira ntchito ngati scaffold molekyulu ya network droplet.Tazindikira kusiyana noticeable m`kati kusasitsa electrostatic αS coacervates, amene amagwirizana ndi kusiyana valency ndi mphamvu ya mogwirizana mapuloteni nawo coacervate maukonde.Chochititsa chidwi n'chakuti, tinawona kuphatikizika kwa mapuloteni a αS ndi tau amyloid m'mapuloteni amadzimadzi omwe akhalapo kwa nthawi yaitali ndipo tinazindikira zinthu zina zomwe zimapangitsa kuti mapuloteni awiriwa asagwirizane ndi ma coacervates.Apa tikufotokoza mwatsatanetsatane ndondomekoyi, yomwe ndi njira yotheka ya maselo yomwe imayambitsa colocalization ya mapuloteni awiri m'magulu okhudzana ndi matenda.
αS ali kwambiri anionic C-pothera mchira pa ndale pH (mkuyu. 1a), ndipo ife hypothesized kuti akhoza kudutsa LLPS kudzera condensation wa electrostatic complexes ndi polycationic disordered polypeptide mamolekyu.Tinagwiritsa ntchito 100-residue poly-L-lysine (pLK) monga molekyulu yachitsanzo chifukwa cha khalidwe lake labwino komanso losokonezeka la polymeric pa pH 32. Choyamba, tinatsimikizira kuti pLK imagwirizana ndi Ct domain ya αS kudzera mu Solution NMR spectroscopy. (Chithunzi 1b) pogwiritsa ntchito 13C / 15N-yotchedwa αS pamaso pa kuwonjezeka kwa αS: pLK molar ratios.Kuyanjana kwa pLK ndi Ct-domain ya αS kumadziwonetsera mwa kusokonezeka kwa kusintha kwa mankhwala ndi kuchepa kwapamwamba kwambiri m'chigawo chino cha mapuloteni.Chosangalatsa ndichakuti, titasakaniza αS ndi pLK pamlingo wa αS wa pafupifupi.5–25 µM pamaso pa polyethylene glycol (5–15% PEG-8) (chotchinga cha LLPS: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) nthawi yomweyo tinadutsa gawo lalikulu la mapangidwe a mapuloteni .m'malovu ankaona ntchito fluorescence (WF) ndi kuwala-munda (BF) maikulosikopu (mkuyu. 1c).1-5 µm m'malovu okhala ndi αS yokhazikika (yowonjezera 1 µM AlexaFluor488-yolembedwa αS, AF488-αS), mphamvu zawo zama electrostatic zitha kutengedwa kukana kwawo kwa 10% 1,6-hexanediol (1,6-HD) ndi kukhudzika kwake kuwonjezeka kwa chiwerengero cha NaCl (mkuyu 1c).Chikhalidwe chamadzimadzi cha coacervates cha αS / pLK electrostatic complex chimasonyezedwa ndi kuthekera kwawo kusakaniza mkati mwa milliseconds (mkuyu 1d).Kugwiritsa turbidimetry, ife quantified mapangidwe m'malovu pansi zinthu izi, anatsimikizira electrostatic chikhalidwe cha mogwirizana waukulu kugwirizana ndi kukhazikika kwake (mkuyu. 1e), ndi kuwunika zotsatira zosiyanasiyana polima chiŵerengero pa ndondomeko LLPS (mkuyu. 1f).Ngakhale kupangika kwa madontho kumawonedwa pamitundu yosiyanasiyana ya ma polima, njirayi ndi yabwino kwambiri pamene pLK ikupitilira αS.Ma LLP adawonedwanso pogwiritsa ntchito chosinthira chamitundu yosiyanasiyana cha dextran-70 (70 kDa) kapena kugwiritsa ntchito mitundu ingapo, kuphatikiza madontho a galasi, zitsime za microplate zazinthu zosiyanasiyana, Eppendorf kapena quartz capillaries.
chiwonetsero cha Schematic cha zigawo zosiyanasiyana zamapuloteni mumitundu ya WT-αS ndi ΔCt-αS yomwe imagwiritsidwa ntchito mu kafukufukuyu.Dera la amphipathic N-terminal domain, hydrophobic amyloid-forming (NAC) dera, ndi dera loyipa la C-terminal amawonetsedwa mu buluu, lalanje, ndi wofiira, motsatana.Mapu a Net Charge Per Residual (NCPR) a WT-αS akuwonetsedwa.b NMR kusanthula kwa αS / pLK kuyanjana popanda ma macromolecular clumps.Pamene ndende ya pLK ikuwonjezeka (αS: pLK molar ratios ya 1: 0.5, 1: 1.5, ndi 1:10 ikuwonetsedwa mu kuwala kobiriwira, kobiriwira, ndi kobiriwira, motsatira).c Coacervate αS/pLK (molar chiŵerengero 1:10) pa 25 µM (1 µM AF488-yolembedwa αS kapena Atto647N-yolembedwa pLK ya kujambula kwa WF) mu LLPS buffer (pamwamba) kapena kuwonjezeredwa ndi 500 mM NaCl (pansi kumanzere) kapena pambuyo pake) % 1,6-hexanediol (1,6-HD; pansi kumanja).Scale bar = 20 µm.d Zithunzi zoyimilira zazing'ono za BF droplet fusion ya αS / pLK (molar ratio 1:10) pamagulu a 25 μM;mivi imawonetsa kuphatikiza kwa madontho amodzi (mivi yofiira ndi yachikasu) kukhala dontho latsopano (muvi walalanje) mkati mwa 200 ms).Scale bar = 20 µm.e Kubalalitsa kowala (pa 350 nm) αS/pLK aggregation mu LLPS buffer isanayambe komanso itatha kuwonjezera 500 mM NaCl kapena 10% 1,6-HD pa 25 µM αS (N = 3 zitsanzo zotsatizana, kutanthawuzira ndi kupatuka kokhazikika kumasonyezedwanso).f chithunzi cha BF (pamwamba) ndi kusanthula kufalikira kwa kuwala (pa 350 nm, pansi) kwa kuphatikizika kwa αS / pLK pa 25 μM αS ndi kuwonjezeka kwa αS: pLK molar ratio (N = 3 zitsanzo zotsatizana, kutanthawuzira ndi kupatuka kokhazikika kumasonyezedwanso).Scale bar = 10 µm.Sikelo pa chithunzi chimodzi imasonyeza kukula kwa zithunzi zonse mu gulu limodzi.Data yaiwisi imaperekedwa ngati mafayilo amtundu wa data.
Kutengera zomwe tawonera za αS/pLK electrostatic complex condensation ndi zomwe taziwona m'mbuyomu za αS ngati molekyulu yamakasitomala a tau/RNA condensate polumikizana mwachindunji ndi tau31, tidaganiza kuti αS ndi tau zitha kulekanitsa ndi zosungunulira pakalibe RNA. condensation.kudzera m'ma electrostatic complexes, ndipo αS ndi mapuloteni a scaffold mu αS/Tau coacervates (onani kugawa kwa tau mu Chithunzi 2e).Tidawona kuti 10 μM αS ndi 10 μM Tau441 (yokhala ndi 1 μM AF488-αS ndi 1 μM Atto647N-Tau, motsatana) idasakanizidwa pamodzi mu buffer ya LLPS, idapanga mosavuta ma protein omwe ali ndi mapuloteni onsewa, monga momwe tawonera ndi ma microscopy a WF.(Mku. 2a).The colocalization wa mapuloteni awiri m'malovu anatsimikiziridwa ndi confocal (CF) microscopy (Supplementary mkuyu. 1a).Khalidwe lofananalo lidawonedwa pamene dextran-70 idagwiritsidwa ntchito ngati aggregation agent (Supplementary Fig. 1c).Pogwiritsa ntchito PEG yolembedwa ndi FITC kapena dextran, tidapeza kuti magulu awiriwa adagawidwa mofanana pazitsanzo, osawonetsa tsankho kapena kuyanjana (Supplementary Fig. 1d).M'malo mwake, zikuwonetsa kuti m'dongosolo lino amalimbikitsa kupatukana kwa gawo kudzera muzotsatira za kuchuluka kwa macromolecular, popeza PEG ndi njira yokhazikika yokhazikika, monga momwe zimawonera muzinthu zina za LLP33,34.Madontho olemera a mapuloteniwa anali okhudzidwa ndi NaCl (1 M) koma osati 1,6-HD (10% v / v), kutsimikizira mphamvu zawo zamagetsi (Supplementary Fig. 2a, b).Makhalidwe awo amadzimadzi adatsimikiziridwa ndikuwona zochitika za millisecond zomwe zimagwirizanitsa madontho pogwiritsa ntchito maikulosikopu a BF (mkuyu 2b).
a Confocal (CF) zithunzi za microscopy za αS/Tau441 coacervates mu LLPS buffer (10 μM ya puloteni iliyonse, 0.5 μM ya AF488 yolembedwa αS ndi Atto647N-yolembedwa Tau441).b Zithunzi zoyimira zosiyana zosokoneza (DIC) za zochitika za αS/Tau441 droplet fusion (10 μM pa puloteni iliyonse).c Chithunzi chagawo chotengera kuwala (pa 350 nm) kwa Tau441 LLPS (0–15 µM) pakalibe (kumanzere) kapena kupezeka (kumanja) kwa 50 µM αS.Mitundu yotentha imasonyeza kubalalika kwambiri.d Kubalalika kowala kwa zitsanzo za αS/Tau441 LLPS ndi kuchuluka kwa αS ndende (Tau441 pa 5 µM, N = 2-3 kubwereza kwachitsanzo monga zasonyezedwa).e Kuyimira kwadongosolo kwa mitundu ina ya mapuloteni a tau ndi zigawo zosiyanasiyana za mapuloteni omwe amagwiritsidwa ntchito mu kafukufukuyu: malo owopsa a N-terminal (wofiira), dera lolemera kwambiri la proline (buluu), domain-binding domain (MTBD, yowonetsedwa mu lalanje), ndi amyloid-forming pair.zigawo za filament (PHF) zomwe zili mkati mwa MTBD (grey).Mapu a Net Charge Per Residue (NCPR) a Tau441 akuwonetsedwa.f Pogwiritsa ntchito 1 µM AF488-yolembedwa αS ndi Atto647N-yotchedwa ΔNt-, pogwiritsa ntchito 1 µM AF488-yolembedwa αS kapena ΔCt-αS pamaso pa ΔNt-Tau (pamwamba, 10 µM pa protein) kapena K18 (pansi pa µM5 pa puloteni iliyonse ) )) ma micrograph a WF ofupikitsidwa mu LLPS kapena K18 buffer.Mipiringidzo ya sikelo pachithunzi chimodzi imayimira kukula kwa zithunzi zonse mu gulu limodzi (20 µm pa mapanelo a, b ndi f).Zambiri zamapanelo c ndi d zimaperekedwa ngati mafayilo amtundu wa data.
Kuti tiyese ntchito ya αS mu ndondomeko ya LLPS, tinayamba tafufuza zotsatira za αS pa kukhazikika kwa droplet ndi nephelometry pogwiritsa ntchito kuchuluka kwa NaCl (mkuyu 2c).Kuchuluka kwa mchere m'miyeso yomwe ili ndi αS, kumapangitsanso kuwala kwakuya (pa 350 nm), zomwe zimasonyeza kukhazikika kwa αS mu dongosolo la LLPS.Zotsatira zofananazo zikhoza kuwonedwa mwa kuonjezera chiwerengero cha αS (ndipo chifukwa chake αS: Tau441 chiŵerengero) mpaka pafupifupi.Kuwonjezeka kwa 10 poyerekeza ndi ndende ya tau (5 µM) (mkuyu 2d).Kusonyeza kuti αS ndi scaffold mapuloteni mu coacervates, tinaganiza kufufuza khalidwe la LLPS-kusokoneza Tau mutant, amene alibe zoipa mlandu N-terminal dera (zotsalira 1-150, onani Mkuyu 2e) wotchedwa ΔNt-Tau.WF microscopy ndi nephelometry inatsimikizira kuti ΔNt-Tau mwiniwakeyo sanachite LLPS (mkuyu 2f ndi Supplementary Fig. 2d), monga momwe tafotokozera kale 14. kubwezeretsedwa ndi kachulukidwe ka madontho pafupi ndi kachulukidwe ka madontho a mayankho athunthu a Tau ndi αS pansi pamikhalidwe yofanana ndi kuchuluka kwa mapuloteni.Njirayi imathanso kuwonedwa pansi pamikhalidwe ya kuchuluka kwa ma macromolecular (Zowonjezera mkuyu. 2c).Udindo wa C-terminal αS dera, koma osati kutalika kwake konse, mu ndondomeko ya LLPS inasonyezedwa ndi kuletsa mapangidwe a madontho pogwiritsa ntchito C-terminal truncated αS kusiyana kopanda zotsalira 104-140 (Mkuyu 1a) wa (ΔCt- αS) mapuloteni (mkuyu 2f ndi Supplementary Fig. 2d).Colocalization ya αS ndi ΔNt-Tau inatsimikiziridwa ndi confocal fluorescence microscopy (Supplementary Fig. 1b).
Kuti mupitirize kuyesa njira ya LLPS pakati pa Tau441 ndi αS, mtundu wina wa Tau unagwiritsidwa ntchito, womwe ndi chidutswa cha helical filament core (PHF) mu microtubule-binding domain (MTBD), yomwe ngati ili ndi madera anayi obwerezabwereza, omwe amadziwikanso bwino. monga chidutswa cha K18 (onani mkuyu 2e).Zanenedwa posachedwa kuti αS imakonda kumangiriza puloteni ya tau yomwe ili mu dera lolemera kwambiri la proline motsatizana lomwe limatsogola dera lomanga ma microtubule.Komabe, dera la PHF limakhalanso ndi zotsalira zabwino (onani Chithunzi 2e), makamaka lysine (15% zotsalira), zomwe zinatipangitsa kuti tiyese ngati derali likuthandiziranso kuti αS / Tau iwonongeke.Tidawona kuti K18 yokha sichitha kuyambitsa LLPS pakukhazikika mpaka 100 μM pansi pamikhalidwe yoyesedwa (LLPS buffer ndi 15% PEG kapena 20% dextran) (Chithunzi 2f).Komabe, pamene tidawonjezera 50 µM αS ku 50 µM K18, mapangidwe ofulumira a madontho a mapuloteni okhala ndi K18 ndi αS adawonedwa ndi nephelometry (Supplementary Fig. 2d) ndi WF microscopy (mkuyu 2f).Monga kuyembekezera, ΔCt-αS sinathe kubwezeretsa khalidwe la LLPS la K18 (Mkuyu 2f).Tikuwona kuti kuphatikiza kwa αS/K18 kumafuna kuchuluka kwa mapuloteni okwera pang'ono kuti apangitse LLPS poyerekeza ndi αS/ΔNt-Tau kapena αS/Tau441, zinthu zina kukhala zofanana.Izi zikugwirizana ndi kuyanjana kwamphamvu kwa chigawo cha αS C-terminal ndi proline-rich Tau domain poyerekeza ndi microtubule-binding domain, monga momwe tafotokozera kale 31.
Popeza kuti ΔNt-Tau sangathe kuchita LLPS pakalibe αS, tinasankha mtundu wa Tau ngati chitsanzo chodziwika ndi αS/Tau LLPS potengera kuphweka kwake m'makina a LLPS okhala ndi Tau wautali (isotype, Tau441/Tau441).ndi zovuta (heterotypic, αS/Tau441) njira zophatikizira.Tinayerekezera mlingo wa αS aggregation (monga gawo la mapuloteni a condensed phase, fαS, c) mu αS / Tau ndi αS / ΔNt-Tau machitidwe ndi centrifugation ndi dispersed gawo SDS-PAGE kusanthula (onani 2e), anapeza makhalidwe ofanana kwambiri kwa mapuloteni onse pamlingo womwewo.Makamaka, tinapeza faS, c 84 ± 2% ndi 79 ± 7% ya αS/Tau ndi αS/ΔNt-Tau, motero, kutanthauza kuti kuyanjana kwapakati pa αS ndi tau ndikopambana kuposa kuyanjana pakati pa mamolekyu a tau.pakati.
Kuyanjana ndi ma polycations osiyanasiyana ndi zotsatira za ndondomeko ya condensation pa αS kinetics inayamba kuphunziridwa ndi fluorescence recovery after photobleaching (FRAP) njira.Tinayesa αS/Tau441, αS/ΔNt-Tau ndi αS/pLK coacervates (100 μM αS yowonjezeredwa ndi 2 μM αS AF488-αS ndi 100 μM Tau441 kapena ΔNt-Tau kapena 1 mM pLK).Deta inapezedwa mkati mwa maminiti oyambirira a 30 mutatha kusakaniza zigawo zachitsanzo.Kuchokera kuzithunzi za FRAP zoimira (Mkuyu 3a, αS / Tau441 condensation) ndi maulendo awo a nthawi yofanana (mkuyu 3b, Supplementary Fig. 3), zikhoza kuwoneka kuti αS kinetics ndi yofanana kwambiri ndi ya Tau441 coacervates.ndi ΔNt-Tau, yomwe imathamanga kwambiri ndi pLK.Ma coefficients owerengeka a αS mkati mwa coacervate molingana ndi FRAP (monga momwe Kang et al. 35) akuwerengera) ndi D = 0.013 ± 0.009 µm2/s ndi D = 0.026 ± 0.008 µm2/s kwa αS-Ntchito αS4/Tau dongosolo la αS /.pLK, Tau, ndi D = 0.18 ± 0.04 µm2 / s, motero (mkuyu 3c).Komabe, kufalikira kwa coefficient αS mu gawo lobalalika ndi maulamuliro angapo apamwamba kuposa magawo onse okhazikika, monga momwe Fluorescence Correlation Spectroscopy (FCS, onani Supplementary Fig. 3) pansi pazikhalidwe zomwezo (LLPS buffer) koma popanda polycations (D = 8 ± 4 µm2/s).Choncho, kinetics ya kumasulira kwa αS imachepetsedwa kwambiri mu coacervates poyerekeza ndi mapuloteni omwe amamwazikana chifukwa cha kuchulukitsidwa kwa maselo, ngakhale kuti coacervates onse amasunga zinthu zamadzimadzi mu theka loyamba la ola pambuyo pa mapangidwe awo, mosiyana ndi gawo la tau.kuthamanga kinetics mu pLK condensate.
a-c FRAP kusanthula kwa αS dynamics (2% AF488-yotchedwa αS) mu electrostatic coacervates.Zithunzi zoyimira za αS/Tau441 FRAP zoyesa katatu zikuwonetsedwa mu (a), pomwe zozungulira zofiira zimawonetsa madera osasinthika.Sikelo ya bar ndi 5 µm.b Avereji yokhotakhota ya FRAP ndi (c) kuwerengetsera ma coefficients (D) a 5-6 (N) madontho osiyanasiyana kuchokera pazoyeserera zitatu pogwiritsa ntchito 100 µM αS ndi kuchuluka kwa equimolar kwa Tau441 (red) kapena ΔNt-Tau (buluu) kapena pLK (wobiriwira) nthawi khumi kuchuluka kwa LLPS.Kupatuka kokhazikika kwa FRAP curve kumawonetsedwa mumtundu wamthunzi.Poyerekeza, kufalikira kwa coefficient αS mu gawo lobalalika kunatsimikiziridwa katatu pogwiritsa ntchito fluorescence correlation spectroscopy (FCS) (onani Supplementary Figure 3 ndi njira kuti mudziwe zambiri).d Mawonekedwe a X-band EPR osalekeza a 100 μM TEMPOL-122-αS mu LLPS buffer popanda polycation (yakuda) kapena pamaso pa 100 μM Tau441 (yofiira) kapena ΔNt-Tau (buluu) kapena 1 mM pLK (yobiriwira).Tsambali likuwonetsa mawonekedwe okulirapo amizere yolimba yamunda pomwe kusintha kwakukulu kumachitika.e Kumanga ma curve a 50 μM TEMPOL-122-αS okhala ndi ma polycations osiyanasiyana pakalibe LLPS (palibe PEG).Kuchepetsedwa kwa matalikidwe a gulu III poyerekeza ndi gulu II (IIII/III) la mawonekedwe a EPR okhazikika akuwonetsedwa kuti akuwonjezera ma molar ratios a Tau441 (wofiira), ΔNt-Tau (buluu) ndi pLK (wobiriwira).Mizere yamitundu imasonyeza kuti ikugwirizana ndi deta pogwiritsa ntchito chitsanzo chomangirira chokhala ndi n malo ofanana ndi odziyimira pawokha pamapindikira aliwonse.Zambiri zimaperekedwa m'mafayilo a data yaiwisi.
Monga chothandizira, tidafufuza zamphamvu za αS m'magulu osiyanasiyana ophatikizira pogwiritsa ntchito ma spin labeling (SDSL) ndi continuous electron paramagnetic resonance (CW-EPR).Njirayi yatsimikizira kuti ndi yothandiza kwambiri pofotokoza za kusinthasintha ndi mphamvu za IDP ndi chigamulo chotsalira chenicheni36,37,38.Kuti izi zitheke, tidapanga zotsalira za cysteine ​​​​mu ma Cys mutants amodzi ndikugwiritsa ntchito 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Zochokera ku Maleimide zimazilemba.Mwachindunji, tidayika ma probe a TEMPOL pamalo 122 kapena 24 αS (TEMPOL-122-αS ndi TEMPOL-24-αS).Pachiyambi choyamba, timayang'ana dera la C-terminal la mapuloteni, omwe amakhudzidwa ndi kuyanjana ndi ma polycations.M'malo mwake, malo 24 angatipatse chidziwitso chokhudza mphamvu zonse za mapuloteni mu condensate.Muzochitika zonsezi, zizindikiro za EPR zopezedwa zamapuloteni a gawo lobalalika zimafanana ndi ma radicals a nitroxide omwe akuyenda mwachangu.Pambuyo pa kupatukana kwa gawo pamaso pa tau kapena pLK (100 μM TEMPOL-αS, Tau441 kapena ΔNt-Tau pa chiŵerengero cha 1: 1 kapena pLK pa chiŵerengero cha 1:10), kuwonjezeka kwa chiwerengero chapamwamba kwambiri kunawonedwa mu Mtengo wa EPR wa αS.Mzere wotayika unakula, kusonyeza kuchepetsedwa kwa αS reorientation kinetics mu madontho poyerekeza ndi mapuloteni mu gawo lochepetsetsa (mkuyu 3d, Supplementary Fig. 4a).Kusintha kumeneku kumawonekera kwambiri pa malo a 122. Pamene ali pa udindo wa 24 kukhalapo kwa pLK sikunakhudze kinetics ya kafukufuku, pa malo a 122 mawonekedwe a mzere wa spectral anasintha kwambiri (Supplementary Fig. 4a).Pamene tinkayesa kutengera mawonekedwe a spectra pa malo a 122 a machitidwe awiri a αS / polycation pogwiritsa ntchito chitsanzo cha isotropic (Supplementary Figure 5a) yomwe imagwiritsidwa ntchito pofotokozera zochitika za spin-labeled IDP38,39, sitinathe kukonzanso mawonekedwe oyesera..Kuyerekezera kwapadera kwa malo a 24 spins kusiyana (Supplementary Fig. 5a).Izi zikusonyeza kuti pali malo osankhidwa mu malo osinthika amtundu wa C-terminal ya αS pamaso pa polycations.Poganizira gawo la αS mu gawo lofupikitsidwa pansi pa kuyesa kwa EPR (84 ± 2%, 79 ± 7%, ndi 47 ± 4% ya αS/Tau441, αS/ΔNt-Tau, ndi αS/pLK, motsatana-onani Supplementary Chithunzi cha 2e cha kusanthula deta c), zikhoza kuwoneka kuti kufalikira komwe kumadziwika ndi njira ya EPR makamaka kumasonyeza kuyanjana kwa dera la C-terminal la αS ndi ma polycations osiyanasiyana mu gawo lofupikitsidwa (kusintha kwakukulu pogwiritsa ntchito TEMPOL-122- αS), osati mapuloteni.Kuwonjezeka kwa microviscosity kumawonedwa mu kafukufuku.Monga momwe zimayembekezeredwa, mawonekedwe a EPR a mapuloteni pansi pa zinthu zina osati LLPS adabwezeretsedwa kwathunthu pamene 1 M NaCl inawonjezeredwa kusakaniza (Supplementary Fig. 4b).Ponseponse, deta yathu imasonyeza kuti zosintha zomwe zapezedwa ndi CW-EPR makamaka zimasonyeza kuyanjana kwa dera la C-terminal la αS ndi ma polycations osiyanasiyana mu gawo lofupikitsidwa, ndipo kuyanjana kumeneku kumawoneka ngati kolimba ndi pLK kusiyana ndi Tau.
Kuti tidziwe zambiri zama protein omwe ali mu coacervate, tidaganiza zophunzira kachitidwe ka LLPS pogwiritsa ntchito NMR yankho.Komabe, titha kuzindikira gawo la αS lomwe latsala mu gawo lobalalika, lomwe lingakhale chifukwa cha kuchepa kwa mphamvu zamapuloteni mkati mwa coacervate ndi gawo lolimba pansi pa yankho mu kusanthula kwa NMR.Pamene tidasanthula mawonekedwe ndi mphamvu za mapuloteni otsala mu gawo lobalalika la LLPS chitsanzo pogwiritsa ntchito NMR (Supplementary Fig. 5c, d), tinawona kuti puloteniyo imachita pafupifupi mofanana pamaso pa pLK ndi ΔNt-Tau, onse awiri. zomwe zinali mu kapangidwe yachiwiri ndi mphamvu ya mapuloteni msana, zowululidwa ndi zoyeserera pa yachiwiri mankhwala kusintha ndi R1ρ kumasuka.Deta ya NMR ikuwonetsa kuti C-terminus ya αS imakhala ndi kutayika kwakukulu kwa kusinthasintha kwa conformational pamene ikusunga chikhalidwe chake chosokonezeka, monga machitidwe ena onse a mapuloteni, chifukwa cha kugwirizana kwake ndi polycations.
Popeza kufalikira kwa siginecha ya CW-EPR komwe kumawonedwa mu gawo la TEMPOL-122-αS kofupikitsa kumawonetsa kuyanjana kwa mapuloteni ndi polycations, tidapanga EPR titration kuti tiwunikire kugwirizana kwa αS ndi ma polycations osiyanasiyana pakalibe LLPS (palibe kudzikundikira kwa Buffer LLPS), kutanthauza kuti kuyanjana kuli kofanana mu magawo ochepa komanso okhazikika (omwe amatsimikiziridwa ndi deta yathu, Supplementary Fig. 4a ndi Supplementary Fig. 6).Cholinga chake chinali kuwona ngati ma coacervates onse, ngakhale ali ndi mawonekedwe ofanana ndi madzimadzi, amawonetsa kusiyana kulikonse pamlingo wa maselo.Monga momwe zimayembekezeredwa, mawonekedwe a EPR adakula ndi kuwonjezeka kwa ndende ya polycation, kusonyeza kuchepa kwa kusinthasintha kwa maselo chifukwa cha kuyanjana kwa maselo a mabwenzi onse ogwirizana pafupifupi kukhutitsidwa (mkuyu 3e, Supplementary Fig. 6).pLK inakwaniritsa kukhuta uku pamlingo wochepa wa molar (polycation: αS) poyerekeza ndi ΔNt-Tau ndi Tau441.M'malo mwake, kuyerekeza kwa datayo ndi mtundu womangirira woyerekeza potengera kuti n malo ofanana komanso odziyimira pawokha adawonetsa kuti mawonekedwe owoneka bwino a pLK (~5 μM) ndi dongosolo lotsika kwambiri kuposa la Tau441 kapena ΔNt-Tau (~50 μM) ).µM).Ngakhale izi ndizongoyerekeza, izi zikuwonetsa kuti αS ili ndi kuyanjana kwakukulu kwa ma polycations osavuta okhala ndi zigawo zolipiritsa nthawi zonse.Poganizira kusiyana kumeneku pakati pa αS ndi ma polycations osiyanasiyana, tidaganiza kuti katundu wawo wamadzimadzi amatha kusintha mosiyana pakapita nthawi ndipo amavutika ndi njira zosiyanasiyana za LSPT.
Popeza malo odzaza kwambiri mkati mwa protein coacervate ndi chikhalidwe cha amyloid cha mapuloteni, tidawona machitidwe a coacervate pakapita nthawi kuti tizindikire njira za LSPT.Pogwiritsa ntchito ma microscopy a BF ndi CF (Chithunzi 4), tidawona kuti αS/Tau441 imalumikizana kwambiri ndi yankho, kupanga madontho akulu omwe amalumikizana ndikunyowetsa pamwamba pa chitsime / slide ngati madontho athunthu, monga momwe amayembekezeredwa (Mkuyu Wowonjezera). 7d;;timatcha nyumba zopangidwa pansi izi "mapuloteni okwera".Zomangamangazi zinakhalabe zamadzimadzi pamene zinkakhalabe ndi mphamvu zowonongeka (Supplementary Fig. 7b) ndipo zikhoza kuwonedwa kwa maola angapo pambuyo poyambitsa LLPS (mkuyu 4 ndi Supplementary Fig. 7c).Tinaona kuti kunyowetsa ndondomeko amakonda padziko hydrophilic m'malo hydrophobic zipangizo (Supplementary mkuyu. 7a), monga kuyembekezera coacervates electrostatic ndi mlandu wosagwirizana motero mkulu electrostatic pamwamba zingayambitse.Makamaka, αS / ΔNt-Tau coalescence ndi rafting zinachepetsedwa kwambiri, pamene αS / pLK condensates anachepetsedwa kwambiri (mkuyu 4).Pakanthawi kochepa, madontho a αS / pLK adatha kugwirizanitsa ndi kunyowa pamwamba pa hydrophilic, koma ndondomekoyi inasiya mwamsanga ndipo pambuyo pa maola a 5 a incubation, zochitika zochepa za coalescence ndipo palibe kunyowa komwe kunawonedwa.- kusintha kwa gel-drip.
Woyimira BF (mapanelo otuwa) ndi CF (mapanelo akumanja, AF488-olembedwa αS obiriwira) a zitsanzo za coacervate zomwe zili ndi 100 µM αS (1% label fulorosenti) mu LLPS buffer pamaso pa 100 µM Tau441nce (pamwamba) zithunzi zazing'onoting'ono -Tau (pakati) kapena 1 mM pLK (pansi) pa nthawi zoyankhulirana zosiyanasiyana ndi utali wolunjika (z, mtunda kuchokera pansi pa mbale bwino).Kuyesera kunabwerezedwa nthawi 4-6 popanda wina ndi mzake ndi zotsatira zofanana.Ma coacervates a αS/Tau441 amanyowetsedwa pambuyo pa maola 24, kupanga ma raft akulu kuposa chithunzicho.Sikelo yazithunzi zonse ndi 20 µm.
Kenako tidafunsa ngati maiwe akulu ngati mapuloteni amadzimadzi opangidwa mu αS/Tau441 LLPS angatsogolere kuphatikizika kwa ma amyloid a mapuloteni aliwonse omwe amaphunziridwa.Tinatsatira kusasitsa kwa madontho a αS / Tau441 pakapita nthawi ndi WF microscopy pansi pazikhalidwe zomwe zili pamwambapa, koma pogwiritsa ntchito 1 μM AF488-yotchedwa αS ndi Atto647N-yotchedwa Tau441 (Mkuyu 5a).Monga momwe zimayembekezeredwa, tinawona kuchuluka kwa mapuloteni athunthu panthawi yonse yokhwima.Chochititsa chidwi, kuchokera ku ca.Pambuyo pa maola a 5, zowonongeka kwambiri zomwe sizinali zozungulira zinkawoneka mkati mwa rafts, zomwe tinazitcha "mfundo", zina zomwe zinapangidwa ndi colocalized ndi αS, ndipo zina zinalemeretsedwa mu Tau441 (mkuyu 5a, mivi yoyera).Mawangawa akhala akuwonedwa nthawi zonse mkati mwa ma rafts mokulirapo kwa αS/ΔNt-Tau kuposa αS/ΔNt-Tau.Panalibe mawanga odziwika m'malovu a pLK ndi machitidwe a Tau osakwanira kuphatikizika / kunyowetsa.Kuti tiwone ngati madontho omwe ali ndi αS ndi Tau441 ali ophatikizana ngati amyloid, tinayesanso chimodzimodzi pogwiritsa ntchito CF microscopy momwe Tau441 idalembedwa ndi Atto647N ndi 12.5 μM amyloid-specific thioflavin-T (ThT) idawonjezedwa kuyambira pachiyambi.dye.Ngakhale kuti ThT-staining ya αS / Tau441 madontho kapena ma rafts sanawonedwe ngakhale pambuyo pa 24 h ya incubation (Mkuyu 5b, mzere wapamwamba-otsalira madontho otsalira pa mapuloteni), mapangidwe a ThT omwe ali ndi Atto647N-Tau441 mkati mwazitsulo anali ofooka kwambiri.izi replicates kukula, mawonekedwe, ndi malo amene tafotokozazi mawanga (mkuyu. 5b, pakati ndi pansi mizere), kusonyeza kuti mawanga angagwirizane ndi amyloid ngati aggregates opangidwa mu ukalamba madzimadzi coacervates.
WF 25 μM αS pa nthawi zosiyanasiyana zamakulitsidwe ndi utali wolunjika (z, mtunda kuchokera pansi osamangidwa) pamaso pa 25 μM Tau441 (1 μM AF488-otchedwa αS ndi Atto647N-otchedwa Tau441) mu chitsime cha mbale maikulosikopu ndi LLPS buffer) .Zoyeserera zisanu ndi chimodzi zidabwerezedwa paokha ndi zotsatira zofanana.b CF chithunzi cha microscopic cha 25 μM αS pamaso pa 25 μM Tau441 (1 μM Atto647N-chotchedwa Tau441) ndi 12.5 μM thioflavin-T (ThT).Madontho olemera a protein ndi ma rafts oyikidwa ndi mawanga amawonetsedwa pamwamba ndi pakati, motsatana.Mzere wapansi ukuwonetsa zithunzi za ma rafts ndi madontho kuchokera pazithunzi 3 zodziyimira pawokha.Mivi yoyera imawonetsa madontho abwino a ThT m'magulu onse awiri.Sikelo yazithunzi zonse ndi 20 µm.
Kuti tifufuze mwatsatanetsatane kusintha kwa coacervate mapuloteni network pa kusintha kuchokera madzi kukhala olimba, ife ntchito fluorescence lifetime imaging (FLIM) ndi Förster resonance mphamvu kutengerapo microscopy (FRET) (Figure 6 ndi Supplementary Figures 8 ndi 9).Tinkaganiza kuti kusasitsa kwa coacervate kwa wosanjikiza kukhala wokhazikika kwambiri kapena wokhazikika ngati mapuloteni ophatikizana kumabweretsa kuyandikana kwambiri pakati pa puloteni ndi kafukufuku wa fulorosenti yomwe imalumikizidwa nayo, zomwe zimapangitsa kuti pakhale kuzizimitsa komwe kumawonetsedwa mu kafukufuku wamfupi wamoyo wonse (τ) , monga tafotokozera kale40., 41 , 42 .Kuphatikiza apo, kwa zitsanzo zolembedwa pawiri (AF488 ndi Atto647N monga FRET wopereka ndi utoto wolandila, motsatana), kuchepa uku kwa τ kumathanso kutsagana ndi coacervate condensation komanso kuwonjezeka kwa FRET (E) kugwira ntchito pa LSPT.Tidayang'anira mapangidwe a raft ndi mawanga pakapita nthawi mu LLPS αS/Tau441 ndi αS/ΔNt-Tau zitsanzo (25 µM ya puloteni iliyonse mu LLPS buffer yomwe ili ndi 1 µM AF488 yolembedwa αS ndi/kapena Atto647N yolembedwa kuti Tau441 kapena ΔNt-Tau).Tinaona zimene zimachitika ambiri kuti fluorescence moyo wa AF488 (τ488) ndi Atto647N (τ647N) kafukufuku utachepa pang'ono pamene coacervates okhwima (mkuyu. 6 ndi Supplementary mkuyu. 8c).Chochititsa chidwi n'chakuti, kusintha kumeneku kunalimbikitsidwa kwambiri kwa madontho mkati mwa rafts (mkuyu 6c), kusonyeza kuti kuwonjezereka kwa mapuloteni kunachitika pamadontho.Pothandizira izi, palibe kusintha kwakukulu kwa moyo wa fluorescence komwe kunawonedwa kwa madontho a αS / ΔNt-Tau omwe ali ndi zaka za 24 h (Supplementary Fig. 8d), kutanthauza kuti droplet gelation ndi njira yosiyana ndi mawanga ndipo sagwirizana ndi kukonzanso kwakukulu kwa maselo. mkati mwa coacervates.Tiyenera kuzindikira kuti madontho ali ndi kukula kosiyana ndi zosinthika mu αS, makamaka kwa αS / Tau441 dongosolo (Supplementary Fig. 8e).Kuchepa kwa nthawi ya moyo wa fluorescence kunatsagana ndi kuwonjezeka kwamphamvu, makamaka kwa Atto647N yolembedwa kuti Tau441 (Supplementary Fig. 8a), komanso FRET yapamwamba pamagulu onse a αS/Tau441 ndi αS/ΔNt-Tau, kusonyeza kuwonjezereka kwina mu LLPS maola asanu. pambuyo poyambitsa, mapuloteni mkati mwa magetsi osasunthika amafupikitsidwa.Poyerekeza ndi αS/ΔNt-Tau, tidawona kutsika kwa τ647N komanso kukwezeka kwa τ488 m'malo a αS/Tau441, motsatizana ndi mikhalidwe yotsika komanso yosagwirizana ndi FRET.Mwinamwake, izi zikhoza kukhala zogwirizana ndi mfundo yakuti mu dongosolo la αS/Tau441, kuchuluka kwa αS komwe kumawonedwa komanso kuyembekezera mumagulu kumakhala kosiyana kwambiri, nthawi zambiri kumakhala kocheperako poyerekeza ndi Tau, popeza Tau441 yokha imathanso kukumana ndi LLPS ndi kuphatikizika (Supplementary Fig. 8e) .Komabe, kuchuluka kwa ma droplet coalescence, mapangidwe a raft, ndipo, chofunikira kwambiri, kuphatikiza mapuloteni mkati mwa ma coacervates amadzimadzi ndiabwino pamene onse a Tau441 ndi αS alipo.
a Lifetime fluorescence microscopy (FLIM) zithunzi za αS/Tau441 ndi αS/ΔNt-Tau pa 25 μM za protein iliyonse (1 μM AF488-yolembedwa αS ndi 1 μM Atto647N-yolembedwa Tau441 kapena ΔNt-Tau) mu LLPS buffer.Mizatiyo ikuwonetsa zithunzi zoyimira zitsanzo za LLPS panthawi zosiyanasiyana zakukhwima (30 min, 5 h ndi 24 h).Chojambula chofiyira chikuwonetsa dera lomwe lili ndi mawanga a αS/Tau441.Kutalika kwa moyo kumawonetsedwa ngati mipiringidzo yamitundu.Scale bar = 20 µm pazithunzi zonse.b Chithunzi chojambulidwa cha FLIM cha malo osankhidwa, chowonetsedwa mubokosi lofiira pagawo a.Miyezo ya moyo imawonetsedwa pogwiritsa ntchito sikelo yamitundu yofanana ndi yomwe ili pagawo A.Scale bar = 5 µm.c Histograms yosonyeza AF488 (yophatikizidwa ku αS) kapena Atto647N (yophatikizidwa ku Tau) yamitundu yosiyanasiyana ya mapuloteni (madontho-D-, raft-R- ndi madontho-P) odziwika muzithunzi za FLIM zojambulidwa αS-) kugawa kwanthawi kwa Tau441 ndi αS/ΔNt-Tau coacervate zitsanzo (N = 17-32 ROI ya D, 29-44 ROI ya R, ndi 21-51 ROI ya mfundo).Makhalidwe apakati ndi apakatikati amawonetsedwa ngati mabwalo achikasu ndi mizere yakuda mkati mwamabokosi, motsatana.Malire apansi ndi apamwamba a bokosilo amayimira quartiles yoyamba ndi yachitatu, motsatana, ndipo zotsika komanso zotsika kwambiri mkati mwa 1.5-fold interquartile range (IQR) zikuwonetsedwa ngati ndevu.Kunja kumawonetsedwa ngati diamondi yakuda.Kufunika kwachiwerengero pakati pa magawo awiriwa kunatsimikiziridwa pogwiritsa ntchito kuyesa kwa zitsanzo ziwiri, kutengera kusiyana kosiyana.Ma t-test p-value okhala ndi michira iwiri amawonetsedwa ndi nyenyezi pagulu lililonse la data yofananizidwa (* p-value> 0.01, ** p-value> 0.001, *** p-value> 0.0001, **** p-mtengo> 0.00001), ns Zimasonyeza kusasamala (p-mtengo> 0.05).Miyezo yeniyeni ya p imaperekedwa mu Supplementary Table 1, ndipo zoyambira zimaperekedwa ngati mafayilo aiwisi.
Kuti tiwonetserenso zamtundu wa amyloid wa timadontho / zophatikizika, tidachitira zitsanzo za coacervate zosawoneka bwino kwa maola 24 okhala ndi kuchuluka kwa (1 M) NaCl, zomwe zidapangitsa kulekanitsidwa kwa ma aggregates kuchokera ku protein coacervates.Pamene aggregates olekanitsidwa (ie, yankho omwazika a aggregates) ankaona ntchito atomic mphamvu microscopy (AFM), tinaona morphology makamaka ozungulira ndi kutalika wokhazikika pafupifupi 15 nm, amene amakonda kuyanjana pansi mikhalidwe ya mkulu mchere ndende, ofanana ndi khalidwe la amyloid fibrils chifukwa cha mphamvu ya hydrophobic pamtunda (zindikirani kuti ma fibrils amakhala ndi kutalika kwa ~ 10 nm) (Supplementary Fig. 10a).Chochititsa chidwi n'chakuti, pamene magulu odzipatula adakulungidwa ndi ThT muyeso wamba wa ThT fluorescence, tinawona kuwonjezeka kwakukulu kwa zokolola za ThT fluorescence quantum, zofanana ndi zomwe zinawonedwa pamene utoto unkaphatikizidwa ndi αS amyloid fibrils (Supplementary Fig. 10b), kutanthauza kuti 10b), ma coacervate aggregates ali ndi mapangidwe a amyloid..M'malo mwake, zophatikizirazo zinali zololera kuchulukira kwa mchere wambiri koma kukhudzidwa ndi 4 M guanidine chloride (GdnHCl), monga ma amyloid fibrils (Supplementary Fig. 10c).
Kenako, tidasanthula kaphatikizidwe kamaguluwo pogwiritsa ntchito mamolekyu amodzi a fluorescence, ma fluorescence correlation/cross-correlation spectroscopy (FCS/FCCS), komanso kusanthula kophulika kwa kuzindikira kwamitundu iwiri (TCCD).Kuti izi zitheke, tinalekanitsa magulu omwe adapangidwa pambuyo pa maola 24 a incubation mu 100 μl LLPS zitsanzo zomwe zili ndi αS ndi Tau441 (zonse 25 μM) pamodzi ndi 1 μM AF488-zolembedwa αS ndi 1 μM Atto647N zolembedwa Tau441.Chepetsani njira yophatikizira yomwe imabalalitsidwa ku dziko la monomolecular pogwiritsa ntchito chotchinga chopanda PEG ndi 1 M NaCl (chotchinga chomwechi chomwe chimagwiritsidwa ntchito kulekanitsa zophatikizira kuchokera ku coacervate) kuti mupewe kulumikizana kulikonse pakati pa LLPS ndi mapuloteni.Chitsanzo cha njira ya nthawi ya molekyulu imodzi chikhoza kuwonedwa mu mkuyu 7a.Kusanthula kwa FCCS/FCS (kuphatikizana, CC ndi autocorrelation, AC) kunasonyeza kuti magulu omwe ali ndi αS ndi tau anali ochuluka mu zitsanzo (onani CC curve mu Fig. 7b, gulu lakumanzere), ndipo zowonjezera zowonjezera zotsalira za mapuloteni a monomeric zinayambira monga zotsatira za njira yochepetsera (onani ma curve a AC mu Chithunzi 7b, gulu lakumanzere).Kuyesera koyang'anira kunachitika pansi pazifukwa zomwezo pogwiritsa ntchito zitsanzo zomwe zili ndi mapuloteni okha a monomeric sanawonetsere ma curve a CC, ndipo ma AC ma curve amagwirizana bwino ndi gawo limodzi lophatikizana lachitsanzo (Eq. 4), pomwe mapuloteni a monomeric ali ndi ma coefficients osakanikirana (mkuyu 7b). ), gulu lakumanja).The diffusion coefficient of aggregated particles ndi zosakwana 1 µm2/s, ndipo ya monomeric proteins ndi pafupifupi 1 µm2/s.50-100 µm/s;mfundo ndizofanana ndi zomwe zidasindikizidwa kale za sonicated αS amyloid fibrils ndi monomeric αS padera pamikhalidwe yofananira44.Pamene tidasanthula ma aggregates ndi kusanthula kwa kuphulika kwa TCCD (mkuyu 7c, gulu lapamwamba), tidapeza kuti pagulu lililonse lakutali (αS / Tau heteroaggregate), pafupifupi 60% yamagulu omwe adapezeka omwe ali ndi αS ndi tau, pafupifupi 30% yokha. tau, pafupifupi 10% αS yokha.Kusanthula kwa Stoichiometric kwa αS / Tau heteroaggregates kunawonetsa kuti ambiri mwa ma heteroaggregates adalemeretsedwa mu tau (stoichiometry pansi pa 0.5, kuchuluka kwa mamolekyu a tau pagulu lililonse ndi 4 nthawi zambiri kuposa ma molecule a αS), zomwe zimagwirizana ndi ntchito yathu yomwe ikuwonetsedwa mu FLIM in situ. zoyesera..Kusanthula kwa FRET kunawonetsa kuti zophatikizirazi zinali ndi mapuloteni onsewa, ngakhale kuti mfundo zenizeni za FRET pakadali pano sizofunika kwambiri, chifukwa kugawa kwa fluorophores pagulu lililonse kunali kwachisawawa chifukwa cha kuchuluka kwa mapuloteni osalembedwa omwe amagwiritsidwa ntchito poyesera.Chochititsa chidwi n'chakuti, pamene tinachita kusanthula komweko pogwiritsa ntchito 45,46 okhwima amyloid aggregation-deficient Tau kusiyana (onani Supplementary Fig. 11a, b), tinawona kuti ngakhale kuti αS electrostatic aggregation inali yofanana (Supplementary Fig. 11c, d), Kuthekera kopanga magulu mkati mwa coacervate kunachepetsedwa kwambiri ndipo FLIM idazindikira mawanga angapo mu zoyeserera za situ, ndipo zokhotakhota zofooka zolumikizana zidawonedwa pazosankha zophatikiza.Komabe, pamagulu ochepa opezeka (gawo limodzi mwa magawo khumi a Tau441), tidawona kuti gulu lililonse lidapindula mu αS kuposa mtundu wa Tau, pafupifupi 50% yamagulu omwe adapezeka omwe ali ndi mamolekyu a αS okha, ndipo αS anali ochulukirapo mopitilira muyeso. .aggregates (onani Supplementary Fig. 11e), mosiyana ndi heterogeneous aggregates kwaiye Tau441 (Fig. 6f).Zotsatira za mayeserowa zimasonyeza kuti ngakhale kuti αS yokha imatha kudziunjikira ndi tau mkati mwa coacervate, tau nucleation ndi yabwino kwambiri pansi pazimenezi, ndipo zotsatira za amyloid-ngati aggregates amatha kuchita ngati mawonekedwe a αS ndi tau.Komabe, pamene maziko olemera a tau apangidwa, kugwirizana kwa heterotypic pakati pa αS ndi tau kumayamikiridwa pamagulu ophatikizana ndi ma homotypic pakati pa mamolekyu a tau;timawonanso ma netiweki a protein mumadzimadzi a αS/tau coacervates.
ndi Woimira fluorescence zosakhalitsa kuda kwa mamolekyu amodzi a aggregates akutali opangidwa mu αS/Tau441 electrostatic coacervates.Kuphulika kofanana ndi ma coaggregates a αS/Tau441 (kuphulika pamwamba pa chigawo chosonyezedwa) kunawonedwa m'njira zitatu zodziwikiratu (AF488 ndi Atto647N kutulutsa pambuyo pa kusangalatsa kwachindunji, mizere yabuluu ndi yofiira, kutulutsa kwa Atto647N pambuyo pa chisangalalo chosalunjika), FRET, mzere wa violet).b Kusanthula kwa FCS/FCCS kwa zitsanzo za magulu akutali a αS/Tau441 otengedwa ku LLPS (gulu lakumanzere).Ma curve a Autocorrelation (AC) a AF488 ndi Atto647N amawonetsedwa abuluu ndi ofiira, motsatana, ndipo ma curve ophatikizika (CC) olumikizidwa ndi magulu omwe ali ndi utoto wonsewo amawonetsedwa mu utoto wofiirira.Ma curve a AC amawonetsa kukhalapo kwa mitundu ya mapuloteni otchedwa monomeric ndi aggregated protein, pomwe ma curve a CC amangowonetsa kufalikira kwa magulu olembedwa kawiri.Kusanthula komweko, koma pansi pamikhalidwe yofananira monga momwe zilili m'malo akutali, zitsanzo zomwe zili ndi monomeric αS ndi Tau441 zimawonetsedwa ngati zowongolera pagawo loyenera.c Fluorescence flash kusanthula kwa mamolekyu amodzi a magulu akutali opangidwa mu αS/Tau441 electrostatic coacervates.Zambiri pagulu lililonse lomwe limapezeka kubwereza zinayi kosiyanasiyana (N = 152) limakonzedwa motsutsana ndi ma stoichiometry, ma S values, ndi magwiridwe antchito a FRET (gulu lapamwamba, mipiringidzo yamitundu ikuwonetsa zomwe zimachitika).Mitundu itatu yophatikizika ingasiyanitsidwe: -αS-aggregates yokhala ndi S~1 ndi FRET~0, Tau-yokhayo yophatikiza ndi S~0 ndi FRET~1, ndi mitundu yosiyanasiyana ya Tau/αS yokhala ndi ma S ~ 1 ndi FRET Kuyerekeza kwa ndalamazo. Mapuloteni onse omwe amapezedwa mugulu lililonse lamitundu yosiyanasiyana (N = 100) amawonetsedwa pagawo lapansi (mulingo wamtundu ukuwonetsa zomwe zimachitika).Zambiri zimaperekedwa m'mafayilo a data yaiwisi.
Kusasitsa kapena kukalamba kwa mapuloteni amadzimadzi kumapangitsa kuti gel-ngati kapena zolimba zolimba pakapita nthawi zakhala zikudziwika kuti zimakhudzidwa ndi ntchito zingapo za thupi za condensate47 komanso matenda, monga njira yosadziwika bwino yomwe imatsogolera amyloid aggregation 7, 48, 49. Pano timaphunzira kulekanitsa gawo ndi khalidwe mwatsatanetsatane.LSPT αS pamaso pa ma polycations mwachisawawa m'malo olamulidwa ndi otsika kwambiri a micromolar ndi zochitika zokhudzana ndi thupi (zindikirani kuti kuchuluka kwa thupi kwa αS ndi> 1 µM50), kutsatira zomwe LPS imayendetsedwa ndi thermodynamically.Tidapeza kuti αS, yomwe ili ndi dera loyipa kwambiri la C-terminal pa physiological pH, imatha kupanga madontho olemera a protein mu njira yamadzi kudzera pa LLPS pamaso pa ma peptides osokonezeka kwambiri monga pLK kapena Tau kudzera munjira ya electrostatic. condensation zovuta pamaso pa aggregation macromolecules.Izi zitha kukhala ndi zotsatira zoyenera m'malo am'manja pomwe αS imakumana ndi mamolekyu osiyanasiyana a polycationic okhudzana ndi kuphatikizika kwake kokhudzana ndi matenda onse mu vitro ndi vivo51,52,53,54.
M'maphunziro ambiri, mphamvu zamapuloteni mkati mwa m'malovu zimawonedwa ngati chimodzi mwazinthu zazikulu zomwe zimatsimikizira kusasitsa55,56.Mu electrostatic αS coacervates ndi polycations, ndondomeko kusasitsa mwachionekere zimadalira mphamvu ya kugwirizana ndi polycations, valence, ndi kuchulukira kwa zinthu izi.Chiphunzitso chofananira chikuwonetsa kuti malo ofananirako a zigawo ziwiri zamadzimadzi kungakhale kukhalapo kwa dontho lalikulu lokhala ndi ma biopolymers omwe amayendetsa LLPS57,58.Kukula kwa Droplet kumatha kupezedwa ndi Ostwald maturation59, coalescence60 kapena kugwiritsa ntchito monomer yaulere mu gawo lobalalika61.Kwa αS ndi Tau441, ΔNt-Tau kapena pLK, mapuloteni ambiri adayikidwa mu condensate pansi pa zomwe zimagwiritsidwa ntchito mu phunziroli.Komabe, ngakhale madontho akulu akulu a tau amalumikizana mwachangu pakunyowetsa pamwamba, kulumikizana kwa madontho ndi kunyowetsa kunali kovuta kwa ΔNt-Tau ndi pLK, kutanthauza kutayika mwachangu kwa zinthu zamadzimadzi m'makina awiriwa.Malinga ndi kusanthula kwathu kwa FLIM-FRET, madontho okalamba a pLK ndi ΔNt-Tau adawonetsa kuchuluka kofananira kwa protein aggregation (yofanana ndi moyo wa fluorescence) monga madontho oyambilira, kutanthauza kuti netiweki ya protein yoyambirira idasungidwa, ngakhale yolimba kwambiri.
Timalinganiza zotsatira zathu zoyesera mu chitsanzo chotsatira (Chithunzi 8).The m'malovu poyamba anapangidwa mongoyembekezera nthawi zambiri mapuloteni maukonde popanda electrostatic chipukuta misozi, ndipo motero pali madera mlandu kusamvana, makamaka pa mawonekedwe droplet, chifukwa mu m'malovu ndi mkulu electrostatic padziko angathe.Kulipirira chiwongola dzanja (chodabwitsa chomwe chimatchedwa kuchepa kwa valence) ndikuchepetsa kuthekera kwapamtunda kwa madontho, madontho amatha kuphatikiza ma polypeptides atsopano kuchokera pagawo lochepetsera, kukonzanso ma network a protein kuti akwaniritse kulumikizana kwacharge-charge, ndikulumikizana ndi madontho ena.ndi pamwamba (kunyowetsa).Madontho a αS / pLK, chifukwa cha mapuloteni awo ophweka (okhawo omwe ali ndi heterotypic interactions pakati pa αS ndi pLK) ndi kugwirizana kwakukulu kwa mapuloteni-mapuloteni, akuwoneka kuti amatha kulinganiza malipiro a condensate mofulumira;Zowonadi, tidawona ma kinetics othamanga kwambiri poyambira αS/pLK coacervates kuposa αS/Tau.Pambuyo pa kuchepa kwa valence, kuyanjana kumakhala kochepa kwambiri ndipo madonthowo amataya katundu wawo wamadzimadzi ndikusandulika kukhala ngati gel osakaniza, madontho osayaka omwe ali ndi mphamvu yochepa ya electrostatic pamwamba (ndipo sangathe kunyowetsa pamwamba).Mosiyana ndi izi, madontho a αS/Tau sagwira ntchito bwino pakuwongolera kuchuluka kwa madontho chifukwa cha ma netiweki ovuta kwambiri a mapuloteni (omwe amalumikizana ndi ma homotypic ndi heterotypic) komanso kufooka kwama protein.Izi zimabweretsa madontho omwe amasunga khalidwe lamadzimadzi kwa nthawi yaitali ndikuwonetsa mphamvu yapamwamba ya electrostatic pamwamba yomwe imakhala yochepa kwambiri chifukwa cha coalescing ndi kukula (potero kuchepetsa kuchuluka kwa malo / kuchuluka kwa madontho) ndi kunyowetsa hydrophilic pamwamba chem.Izi zimapanga malaibulale akuluakulu opangidwa ndi mapuloteni omwe amasunga zinthu zamadzimadzi chifukwa kuyanjana kumakhalabe kwakanthawi chifukwa chakusaka kosalekeza kwa kukhathamiritsa kwa ma protein network.Chochititsa chidwi n'chakuti, mitundu ya N-terminally truncated ya Tau, kuphatikizapo ma isoform62 omwe amapezeka mwachibadwa, amawonetsa khalidwe lapakati, ndi ena omwe amachititsa kuti akalamba ndi αS kukhala madontho amtundu wa gel, pamene ena amasintha kukhala ma condensate amadzimadzi.Kuphatikizika kumeneku pakusasitsa kwa ma αS electrostatic coacervates kumagwirizana ndi kafukufuku waposachedwa wa LLPS wamalingaliro ndi oyesera omwe apeza kugwirizana pakati pa kuchepa kwa valence ndi electrostatic sieving mu condensates monga chinsinsi chowongolera kukula kwa condensate ndi katundu wamadzimadzi.Njira 58.61.
Chiwembuchi chikuwonetsa njira yophatikizira amyloid ya αS ndi Tau441 kudzera pa LLPS ndi LSPT.Ndi madera owonjezera a anion (ofiira) ndi cation-rich (buluu), αS ndi tau electrostatic coacervates yokhala ndi valence yokhutiritsa imakhala ndi mphamvu zochepa zapamtunda ndipo motero imachepa kwambiri, zomwe zimapangitsa kuti madontho azikalamba mofulumira.Gulu lokhazikika la gel osagwirizana ndi agglomerated limakwaniritsidwa..Mkhalidwewu ndi wabwino kwambiri pamtundu wa αS/pLK chifukwa cha kuyanjana kwake kwakukulu komanso kulumikizana kosavuta kwa protein-pair, komwe kumalola kusintha kwachangu ngati gel.M'malo mwake, madontho okhala ndi valence osakwanira ndipo, chifukwa chake, madera okhala ndi mapuloteni omwe amapezeka kuti azilumikizana, amapangitsa kuti coacervate ikhale yosavuta kuphatikiza ndikunyowetsa hydrophilic pamwamba kuti achepetse mphamvu zake zapamwamba.Izi ndizabwino kwa ma coacervates a αS/Tau441, omwe ali ndi netiweki yochulukirachulukira yomwe imakhala ndi kusagwirizana kwa Tau-Tau ndi αS-Tau.Komanso, ma coacervates akuluakulu amatha kusunga zinthu zawo ngati zamadzimadzi, zomwe zimapangitsa kuti ma protein ndi mapuloteni azigwirizana.Pamapeto pake, ma amyloid heterogeneous aggregates okhala ndi αS ndi mawonekedwe a tau mkati mwamadzimadzi a coacervate, omwe atha kukhala okhudzana ndi omwe amapezeka m'matupi ophatikizika, omwe ndizizindikiro zamatenda a neurodegenerative.
Mapangidwe akuluakulu amadzimadzi omwe amapangidwa panthawi ya kusasitsa kwa αS/Tau441 yokhala ndi mapuloteni ochuluka kwambiri koma amphamvu kwambiri ndipo, pang'onopang'ono, αS / ΔNt-Tau coacervates ndi malo abwino osungiramo ma nucleation a protein aggregation.Tawonadi kupangidwa kwa magulu olimba a mapuloteni mumtundu uwu wa mapuloteni a coacervates, omwe nthawi zambiri amakhala ndi αS ndi tau.Tawonetsa kuti ma heteroaggregateswa amakhazikika ndi kusagwirizana kwa electrostatic, amatha kumanga utoto wamtundu wa amyloid wa ThT mofanana ndi ma amyloid fibrils, ndipo ali ndi kukana kofanana ndi zochitika zosiyanasiyana.Magulu a αS/tau opangidwa ndi LLPS adawonetsedwa kuti ali ndi zinthu ngati amyloid.Zowonadi, kusiyanasiyana kokhwima kwa Tau komwe kulibe kuphatikizika kwa amyloid kumasokonekera kwambiri pakupangika kwa mitundu yosiyanasiyana ya αS mkati mwamadzimadzi electrostatic coacervate.Mapangidwe a αS/Tau441 aggregates adawonedwa kokha mkati mwa coacervates, omwe adasunga zinthu zamadzimadzi, ndipo ayi, ngati ma coacervates / madontho sanafike ku gel.Pamapeto pake, mphamvu yowonjezereka ya machitidwe a electrostatic ndipo, chifukwa chake, kusasunthika kwa mapuloteni opangira mapuloteni kumateteza kukonzanso koyenera kwa mapuloteni kuti akhazikitse mgwirizano watsopano wa mapuloteni ofunikira kuti amyloid nucleation.Komabe, izi zitha kupezedwa ndi ma coacervates osinthika, omwe amakhala ngati amadzimadzi, omwe amatha kukhala amadzimadzi akamakula kukula.
Mfundo yakuti mapangidwe aggregates mkati mwa gawo condensed ndi bwino mu lalikulu αS/Tau condensates kuposa m'malovu ang'onoang'ono kuti mofulumira gel osakaniza, zimasonyeza kufunika kuzindikira zinthu zimene kulamulira droplet coalescence.Choncho, sikuti pali chizolowezi cholekanitsa gawo, koma kukula kwa condensate kuyenera kuyang'aniridwa kuti zigwire bwino ntchito komanso kupewa matenda58,61.Zotsatira zathu zikuwonetsanso kufunikira kwa mgwirizano pakati pa LLPS ndi LSPT pa dongosolo la αS / Tau.Ngakhale mapangidwe a madontho amatha kuteteza motsutsana ndi kuphatikizika kwa amyloid mwa kuchepetsa kuchuluka kwa mapuloteni omwe amapezeka pansi pa machulukitsidwe, monga momwe tafotokozera m'makina ena63,64, kuphatikizika kwa madontho pamadontho apamwamba kungayambitse kuphatikizika kwa mapuloteni mkati mwa kukonzanso pang'onopang'ono.ma protein network..
Ponseponse, deta yathu imatsindika kwambiri kufunikira kwa valence yogwirizana komanso kukhutitsidwa / kusakhutitsidwa kwapaintaneti mumayendedwe a LSPT.Makamaka, timasonyeza kuti kutalika kwa αS/Tau441 condensates amatha kuphatikizira bwino ndi nucleate kuti apange ma heteroaggregates amyloid omwe amaphatikizapo mapuloteni onse ndikupereka ndondomeko ya maselo kutengera zotsatira zathu zoyesera.Kuphatikizika kwa mapuloteni awiri mu αS/Tau fluid coacervate yomwe tikunena pano ingakhale yokhudzana ndi kuphatikizika kwa mapuloteni awiri mu inclusions, omwe ali zizindikiro za matendawa, ndipo angathandize kumvetsetsa mgwirizano pakati pa LLPS ndi amyloid aggregation, kuyambitsa njira ya IDP yolipitsidwa kwambiri mu neurodegeneration.
Monomeric WT-αS, cysteine ​​​​mutants (Q24C-αS, N122C-αS) ndi mitundu ya ΔCt-αS (Δ101-140) adawonetsedwa mu E. coli ndikuyeretsedwa monga momwe tafotokozera kale.5 mM DTT idaphatikizidwa pamasitepe onse pakuyeretsedwa kwa αS cysteine ​​​​mutants kuteteza mapangidwe a mgwirizano wa disulfide.Tau441 Isoform (plasmid yopezeka kuchokera ku Orgene # 16316), Δnt-tasu (ΔTe-tambala) CRC5 Primer) E. Zikhalidwe za Coli zinali wakula kufika ku OD600 = 0.6–0.7 pa 37°C ndi 180 rpm, ndipo mawuwo anakopeka ndi IPTG kwa maola atatu pa 37°C.Kololani maselo pa 11,500 xg kwa mphindi 15 pa 4 °C ndi kusamba ndi saline buffer yokhala ndi 150 mM NaCl.Kuyimitsanso pellet mu lysis buffer (20 ml pa 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 10 μM1).The sonication sitepe inkachitika pa ayezi ndi matalikidwe a 80% kwa 10 zimachitika (1 min pa, 1 min kuchoka).Musapitirire 60 ml mu ultrasound imodzi.E. coli lysates anatenthedwa pa 95 ° C. kwa mphindi 20, kenako utakhazikika pa ayezi ndi centrifuged pa 127,000 × g kwa mphindi 40.Mphamvu yodziwika bwino idayikidwa pa nembanemba ya 3.5 kDa (Spectrum™ Thermo Fisher Scientific, UK) ndikuyimba motsutsana ndi 4 L ya dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) kwa maola 10.Mzere wa 5 ml cation exchange (HiTrap SPFF, Cytiva, MA, USA) adalumikizidwa ndi buffer yolumikizira (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF) .Tau lysate idasefedwa kudzera mu fyuluta ya 0.22 μm PVDF ndikulowetsa muzakudya pamlingo wothamanga wa 1 ml / min.Kuwunika kunachitika pang'onopang'ono, tau idatulutsidwa ndi 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).Tizigawo ting'onoting'ono tidawunikidwa ndi SDS-PAGE, ndipo tizigawo ting'onoting'ono tomwe timakhala ndi gulu limodzi lolemera mamolekyulu a tau tidakhazikika pogwiritsa ntchito fyuluta ya 10 kDa centrifuge ndikusinthidwa ndi buffer yokhala ndi 10 mM HEPES, pH 7.4, NaCl 500 mM ndi DTT 2 mM ya mapuloteni omaliza anali 100 μM.Njira yothetsera puloteniyo idadutsa mu fyuluta ya 0.22 μm PVDF, yowundana mwachangu ndikusungidwa pa -80 ° C.Protein K18 inaperekedwa mokoma mtima ndi Prof. Alberto Boffi.Kuyera kwa kukonzekera kunali> 95% monga kutsimikiziridwa ndi SDS-PAGE ndi MALDI-TOF/TOF.Ma cysteines osiyanasiyana adalembedwa ndi AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) kapena TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).adatsimikiziridwa ndi kuyamwa ndi MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau ndi K18 adalembedwa ndi zotsalira za cysteine ​​​​pamalo 191 ndi 322 pogwiritsa ntchito Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Germany) kutsatira njira yomweyo.Malipiro onse pamapu otsalira a αS ndi Tau441 adapangidwa pogwiritsa ntchito CIDER66.
Olimba poly-L-lysine (pLK DP 90-110 malinga NMR kuchokera katundu, Alamanda Polymers Inc, Huntsville, Alabama, USA) anali kusungunuka mu 10 mm HEPES, 100 mm NaCl, pH 7.4 kuti 10 mm ndende, ndondomeko sonicated kwa 5 Mphindi mu osamba akupanga madzi ndi kusunga pa -20 °C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) ndi FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) ndizosungunuka m'madzi ndipo zimagawidwa kwambiri mu LLPS buffer.Dialysis imachotsa mchere woipa.Kenako amasefedwa kudzera mu sefa ya syringe yokhala ndi pore kukula kwa 0.22 μm, ndipo kuchuluka kwawo kudawerengedwa pogwiritsa ntchito refractometer (Mettler Toledo, Columbus, Ohio, USA).Zitsanzo za LLPS zinakonzedwa kutentha kwa firiji motere: buffer ndi extrusion zinasakanizidwa ndi 1 mm tris(2-carboxyethyl)phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) ndi chisakanizo cha 1% protease inhibitor (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).Kenako αS ndi ma polycations osakanikirana (zosankha pLK kapena Tau) amawonjezedwa.Pazoyeserera zanthawi ya thioflavin-T (ThT, Carbosynth, Compton, UK), gwiritsani ntchito kuchuluka kwa ThT kukhala theka la ndende ya αS.Pang'onopang'ono koma bwinobwino sakanizani zitsanzo kuti zitsimikizire kuti ndizofanana.Kuchuluka kwa chigawo chilichonse kumasiyana kuchokera pakuyesa mpaka kuyesa, monga tafotokozera mu gawo la Zotsatira.Azide idagwiritsidwa ntchito pagulu la 0.02% (w / v) nthawi iliyonse yoyeserera ikadutsa maola 4.Pazowunikira zonse pogwiritsa ntchito zitsanzo za LLPS, lolani kuti kusakaniza kufanane kwa mphindi 5 musanayambe kusanthula.Kuti tiwunike mowala, 150 µl ya zitsanzo zidakwezedwa pama microplates osamangirira a 96 (µClear®, wakuda, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) ndikukutidwa ndi filimu yomatira.Ma LLP adayang'aniridwa poyesa kuyamwa kwa 350 nm pakati pa yankho mu CLARIOstar plate reader (BMG Labtech, Ortenberg, Germany).Kuyesera kunachitika katatu pa 25 ° C, ndipo zolakwikazo zidawerengedwa ngati kupatuka koyenera kuchokera ku tanthauzo.Gawo la dilute linayesedwa ndi chitsanzo cha centrifugation ndi SDS-PAGE gel kusanthula, ndipo gawo la αS mu magawo ochepetsera ndi okhazikika linayesedwa muzothetsera zosiyanasiyana za LLPS.Chitsanzo cha 100 μl LLPS chokhala ndi 1 μM AF488-cholembedwa αS chinakonzedwa ndikusakaniza bwino ndikutsatiridwa ndi centrifugation pa 9600 × g kwa mphindi 30, pambuyo pake mpweya unkawoneka.50 μl yapamwamba ya supernatant idagwiritsidwa ntchito powerengera mapuloteni pogwiritsa ntchito gel osakaniza a SDS-PAGE.Ma gels adasinthidwa ndi zosefera za AF488 pogwiritsa ntchito makina oyerekeza a gel a ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) kapena odetsedwa ndi banga la Coomassie ndikuwoneka ndi zosefera zoyenera.Magulu omwe adatsatirawo adawunikidwa pogwiritsa ntchito mtundu wa ImageJ 1.53i (National Institutes of Health, USA).Zoyesererazo zidachitika mobwerezabwereza muzoyeserera ziwiri zosiyana zokhala ndi zotsatira zofanana.
Nthawi zambiri, 150 μl ya zitsanzo zidagwiritsidwa ntchito ku ma microplates osamangirira a 96-well ndipo amawonedwa kutentha kwachipinda pa microscope ya Leica DMI6000B (Leica Microsystems, Wetzlar, Germany).Pakuyesa kwaposachedwa, mbale za µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Germany) kapena ma 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) adagwiritsidwanso ntchito.EL6000 halogen kapena mercury metal halide nyali anagwiritsidwa ntchito ngati magwero owunikira (ya BF/DIC ndi WF kujambula, motsatana).Kwa maikulosikopu a WF, cholinga cha mpweya cha 40x (Leica Microsystems, Germany) chinagwiritsidwa ntchito kuyang'ana kuwala pa chitsanzo ndikusonkhanitsa.Pazitsanzo zolembedwa za AF488 ndi ThT, zosefera zosefera ndi zotulutsa zokhala ndi seti zosefera za GFP, zosefera za 460-500 nm ndi 512-542 nm bandpass, ndi galasi la 495 nm dichroic.Pazitsanzo zolembedwa ndi Atto647N, zosefera za Cy5 zokhala ndi zosefera za 628-40 nm ndi 692-40 nm motsatana, ndi galasi la 660 nm dichroic zidagwiritsidwa ntchito.Pa maikulosikopu a BF ndi DIC, gwiritsani ntchito cholinga chotolera chowunikira chomwechi.Kuwala kosonkhanitsidwa kunajambulidwa pa kamera ya Leica DFC7000 CCD (Leica Microsystems, Germany).Nthawi yowonekera inali 50 ms ya kujambula kwa microscope ya BF ndi DIC ndi 20-100 ms ya kujambula kwa microscopy ya WF.Poyerekeza, nthawi yowonekera pazoyeserera zonse ndi ThT inali 100 ms.Kuyesa kwanthawi yayitali kunachitika kuti muwone momwe madonthowo akugwirira ntchito, zithunzi zimasonkhanitsidwa pa 100 ms iliyonse kwa mphindi zingapo.ImageJ (NIH, USA) idagwiritsidwa ntchito posanthula zithunzi.Kuyesera kunachitika katatu ndi zotsatira zofanana.
Pazoyeserera zamakoloni, FRAP ndi kukonzanso kwa 3D, zithunzi zidapezedwa pa Zeiss LSM 880 inverted confocal microscope pogwiritsa ntchito mtundu wa buluu wa ZEN 2 (Carl Zeiss AG, Oberkochen, Germany).Zitsanzo za 50 µl zidagwiritsidwa ntchito ku mbale za µ-Slide Angiogenesis Petri (Ibidi GmbH, Gröfelfing, Germany), zogwiritsidwa ntchito ndi hydrophilic polima (ibiTreat) ndikuyika mu 63 × cholinga chomiza mafuta (Plan-Apochromat 63×/NA 1.4 Oil) pa DIC).Zithunzi zinapezedwa pogwiritsa ntchito mizere ya 458 nm, 488 nm, ndi 633 nm argon laser yokhala ndi 0.26 µm/pixel komanso nthawi yowonekera ya 8 µs/pixel yosangalatsa komanso yozindikira mawindo a 470–600 nm, 493–628 nm, ndi 638-755 nm idagwiritsidwa ntchito kuwona ThT, AF488 ndi Atto647N, motsatana.Pazoyeserera za FRAP, kujambula kwanthawi yayitali kwachitsanzo chilichonse kunajambulidwa pa chimango cha 1 pamphindikati.Kuyesera kunachitika katatu kutentha kwa chipinda ndi zotsatira zofanana.Zithunzi zonse zidawunikidwa pogwiritsa ntchito pulogalamu ya Zen 2 blue edition (Carl Zeiss AG, Oberkochen, Germany).Ma curve a FRAP adasinthidwa, adakonzedwa ndikuyikidwa kuti azitha kuzama / nthawi yochotsedwa pazithunzi pogwiritsa ntchito Zen 2 pogwiritsa ntchito OriginPro 9.1.Mapiritsi obwezeretsawo adayikidwa pamtundu wa mono-exponential kuti awerengere kufalikira kwa ma molekyulu ndi mawu owonjezera owonjezera kuti awerengere zotsatira za blekning.Kenako tidawerengera D pogwiritsa ntchito ma radius otchedwa bleaching radius komanso theka la moyo womwe tidatsimikiziridwa kale monga momwe Kang et al.5 35 ikuwonetsedwa.
Mitundu imodzi ya cysteine ​​​​ya αS idapangidwa ndi 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) pamalo 24 (TEMPOL-24-αS) ndi 122 (TEMPOL-122-αS), motsatana.Spin Labeling Pazoyeserera za EPR, ndende ya αS idayikidwa pa 100 μM ndipo ndende ya PEG inali 15% (w/v).Pazinthu zosiyanasiyana zophatikiza, chiŵerengero cha αS:pLK chinali 1:10, pamene αS:ΔNt-Tau ndi αS:Tau441 chiŵerengero chinasungidwa pa 1:1.Poyesa kuyesa kwa titration pakalibe kuchulukana, TEMPOL-122-αS idasungidwa pa 50 μM ndipo ma polycations adasinthidwa pakuwonjezereka, kukonzekera chikhalidwe chilichonse padera.Miyezo ya CW-EPR idachitika pogwiritsa ntchito chowonera cha Bruker ELEXSYS E580 X-band chokhala ndi cholumikizira cha Bruker ER4118 SPT-N1 chomwe chimagwira ntchito pa microwave (SHF) pafupipafupi ~ 9.7 GHz.Kutentha kumayikidwa pa 25 ° C ndikuyendetsedwa ndi madzi a nitrogen cryostat.Zowonera zidapezedwa pansi pamikhalidwe yopanda unsaturated pa mphamvu ya MW ya 4 mW, matalikidwe osinthika a 0.1 mT, ndi ma frequency a 100 kHz.Kuchuluka kwa Spectral kunali koyenera kuti tipewe kusiyana kwa ma spin pakati pa zitsanzo ndi zotheka kuchepetsa kuchepa chifukwa cha zotsalira zochepetsera zochepetsera mu zitsanzo zomwe zili ndi Tau441 kapena ΔNt-Tau (yomwe ilipo muzoyambira zamapuloteni).Makhalidwe opatsidwa a g adapezedwa chifukwa cha mawonekedwe a EPR opangidwa pogwiritsa ntchito pulogalamu ya Easyspin (v. 6.0.0-dev.34) yokhazikitsidwa ku Matlab®67.Mitundu imodzi/ziwiri ya isotropic idagwiritsidwa ntchito potengera deta.Pambuyo posintha zizindikiro zonse, zotsalirazo zinawerengedwa pochotsa kayeseleledwe kalikonse kuchokera pamayesero ofanana.Pakusanthula kwa titration, kuchulukira kwa gulu lachitatu ku gulu lachiwiri la sipekitiramu yokhazikika ya EPR (IIII/III) idagwiritsidwa ntchito kuyang'anira kumangirira kwa ma polycations ku αS.Kuti muyerekeze kukhazikika kwa dissociation (Kd), njira yokhotakhota idayikidwa ku chifaniziro chotengera n malo ofanana komanso odziyimira pawokha.
Kuyesera kowonera za NMR kunachitika pogwiritsa ntchito chowonera cha Bruker Neo 800 MHz (1H) NMR chokhala ndi cryoprobe ndi Z-gradient.Zoyesera zonse zinachitidwa pogwiritsa ntchito 130-207 µM αS ndi zofanana αS/ΔNt-Tau ndi pLK zofanana mu 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 ndipo zinachitidwa pa 15 ° C.Kuwunika LPS ndi NMR, 10% PEG idawonjezedwa ku zitsanzo zosakanizidwa kale.Kusintha kwa mankhwala kusokoneza chiwembu (mkuyu 1b) kumasonyeza pafupifupi 1H ndi 15N kusintha kwa mankhwala.Mawonekedwe a αS 2D1H-15N HSQC adapatsidwa kutengera ntchito yapitayi (kulowa kwa BMRB #25227) ndikutsimikiziridwa ndi kujambula ndi kusanthula mawonekedwe a 3D a HNCA, HNCO ndi CBCAcoNH.13Cα ndi 13Cβ kusintha kwa mankhwala kunawerengedwa pamaso pa ΔNt-Tau kapena pLK kuti ayese kusintha komwe kungatheke muzochitika zachiwiri poyerekeza ndi αS kusintha kwa mankhwala mu conformation yoyera yosasinthika 68 (Supplementary Figure 5c).Mitengo ya R1ρ idayezedwa pojambulira kuyesa kwa hsqctretf3gpsi (kuchokera ku laibulale ya Bruker) ndikuchedwa kwa 8, 36, 76, 100, 156, 250, 400, ndi 800 ms, ndipo ntchito zofotokozera zidasinthidwa kuti zifike pachimake pakuchedwa kosiyana. nthawi kudziwa R1ρ ndi kukayika experimental.
Kuyesera kwa ma microscopy amitundu iwiri opangidwa ndi nthawi yamtundu wa fluorescence kunachitika pa MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) yokhala ndi nthawi yolumikizana ndi chithunzi chimodzi (TCSPC).Mutu wa laser diode umagwiritsidwa ntchito pa pulsed interleaved excitation (PIE), mtengowo umadutsa mumayendedwe amodzi ndipo umasinthidwa ndi mphamvu ya laser ya 10 mpaka 100 nW kwa 481 nm ndi mizere ya 637 nm laser yoyesedwa pambuyo pa galasi la dichroic.Izi zimatsimikizira kuchuluka kwa kuwerengera kwazithunzi, kupewa zotsatira za Photon aliasing, Photobleaching and saturation.μ-Slide angiogenesis zophimba kapena mbale (Ibidi GmbH, Gräfelfing, Germany) anayikidwa mwachindunji m'madzi omiza pamwamba pa Super Apochromat 60x NA 1.2 lens ndi kolala yokonza (Olympus Life Sciences, Waltham, USA).Galasi la 488/640 nm dichroic (Semrock, Lake Forest, IL, USA) linagwiritsidwa ntchito ngati chogawa chachikulu.Ma radiation osasunthika amatsekedwa ndi dzenje lokhala ndi mainchesi 50, ndiye kuti kuwala kokhazikika kumagawidwa m'njira ziwiri zodziwikiratu ndi choboola cha 50/50.Zosefera za bandpass emission (Semrock, Lake Forest, IL, USA) 520/35 za utoto wobiriwira (AF488) ndi 690/70 wa utoto wofiira (Atto647N) zidagwiritsidwa ntchito kutsogolo kwa chowunikira.Ma photon avalanche diode (SPAD) (Micro Photon Devices, Bolzano, Italy) adagwiritsidwa ntchito ngati zowunikira.Kusonkhanitsa ndi kusanthula deta kunachitika pogwiritsa ntchito pulogalamu yamalonda ya SymphoTime64 (PicoQuant GmbH, Berlin, Germany).
Ma microliters makumi asanu a zitsanzo za LLPS anagwiritsidwa ntchito ku zitsime za μ-Slide angiogenesis (Ibidi GmbH, Gräfelfing, Germany).Zithunzi zomwe zatsatiridwazi zimayang'ana ku 20 µm kumtunda kwa chitsime kuti mupeze mtunda wokwanira wogwirira ntchito wa madontho oyimitsidwa ndi ~1 µm pa ma raft ndi madontho okhala ndi axial resolution ya osachepera 0.25 µm/pixel ndi nthawi yochedwa ya 400 µs/pixel.Sankhani deta pogwiritsa ntchito mphamvu yamphamvu yotengera kulimba kwa siginecha yakumbuyo (PBG, kutanthawuza + 2σ) pa tchanelo chilichonse kuti madontho a protein amadzimadzi okha, ma raft, kapena mawanga amasankhidwe, ndikusefa zomwe zingatheke kuchokera pagawo lobalalitsidwa.Kuti tiwunikenso moyo wa mtundu uliwonse (τ) wa tchanelo chilichonse (chobiriwira, “g” cha AF488 ndi chofiira, “r” cha Atto647N), tinasankha madera osangalatsa (ROI) okhala ndi madontho, ma raft, kapena mawanga (Chithunzi Chowonjezera 1 ).8b) ndipo anawapeza powayenerera kuwonongeka kwa moyo wawo wonse ( τD, τR ndi τP kwa madontho, ma raft kapena mawanga, motsatira, onani Supplementary Fig. 8c) mu njira iliyonse pogwiritsa ntchito kusanthula kwa mchira ndi chitsanzo cha zigawo ziwiri zowola.Avereji τ kuchokera ku τ .Ma ROI omwe adatulutsa ma photon ochepa kwambiri kuti agwirizane ndi ma multi-exponential adachotsedwa pakuwunika.Kudulira komwe kunagwiritsidwa ntchito kunali <104 zithunzi za ma raft ndi madontho ndi 103 za madontho.Madonthowa ali ndi malire otsika chifukwa zimakhala zovuta kupeza ma curve ovunda omwe ali ndi mphamvu zapamwamba kwambiri, popeza madontho omwe ali m'munda wazithunzi nthawi zambiri amakhala ang'onoang'ono komanso ochepa.Ma ROI okhala ndi ma photon omwe ali pamwamba pa malire a photon accumulation (oikidwa ku> 500 count / pixel) adatayidwanso kuti afufuzidwe.Fananizani zopindika zowola zomwe zimapezedwa kuchokera kudera lachidwi ndi mphamvu pa 90% yapakatikati (pang'ono pambuyo pakuwola kwakukulu) kuyambira pachiyambi cha moyo wautumiki kuti muwonetsetse kusokoneza kochepa kwa IRF ndikusunga zomwezo pakuwola konse. makonda Zenera la nthawi yofananira Anawunikidwa 25 mpaka 50 ROI ya rafts ndi mawanga ndi 15-25 ROI ya madontho, zithunzi zosankhidwa kuchokera ku zoposa 4 replicates zolembedwa zosachepera 3 zoyesera zodziimira.Mayeso a t-tailed awiri agwiritsidwa ntchito kuyesa kusiyana kwa chiwerengero pakati pa mitundu kapena pakati pa machitidwe a coacervate.Pakuwunika kwa pixel-by-pixel kwa moyo wonse (τ), kuchepetsedwa kwathunthu kwa moyo wonse pamtunda wa tchanelo chilichonse kunawerengedwa ndipo kuyerekezera kwa 2/3-gawo la exponential attenuation model inachitika.Kuchepetsa kwa moyo wa pixel iliyonse kumayikidwa pogwiritsa ntchito ziwerengero za τ zomwe zidawerengedwa kale, zomwe zimapangitsa kuti pakhale chithunzi chokwanira cha pseudocolor FLIM.Kutalika kwa moyo wa mchira kunali kofanana pazithunzi zonse za njira imodzi, ndipo kuwonongeka kulikonse kunatulutsa ma photon okwanira kuti apereke zoyenera zodalirika.Pakuwunika kwa FRET, ma pixel adasankhidwa pogwiritsa ntchito mawonekedwe otsika kwambiri a ma photon 100, omwe amakhala ndi chizindikiro chakumbuyo (FBG) cha mafoto 11.Kuchulukira kwa fluorescence kwa njira iliyonse kunakonzedwa ndi zoyeserera zoyeserera: 69 spectral crosstalk α inali 0.004, chisangalalo chachindunji β chinali 0.0305, kuzindikira bwino γ kunali 0.517.Kuchita bwino kwa FRET pamlingo wa pixel kumawerengedwa pogwiritsa ntchito equation iyi:
kumene FDD ndi mphamvu ya fluorescence yomwe imawonedwa mu njira yoperekera (yobiriwira), FDA ndi mphamvu ya fluorescence yomwe imawonedwa mu njira yovomerezeka (yofiira) pansi pa chisangalalo chosalunjika, ndipo FAA ndi mphamvu ya fluorescence yomwe imawonedwa mu njira yovomerezeka (yofiira) pansi pa chisangalalo cholunjika ( PIE).Fluorescence intensity pulses amawonedwa mu njira).
Ikani 100 µl ya mayankho a LLPS okhala ndi 25 µM osalembedwa monomeric Tau441 (okhala kapena opanda 25 µM αS) mu LLPS buffer (wowonjezera monga pamwambapa) pa ma microplates osamangirira a 96-well okhala ndi zokutira zomatira komanso kupanga madontho kufufuzidwa pambuyo pa WF. kulinganiza.mkati mwa 10 min.Pambuyo pa maola 48 a incubation kutentha kwa firiji, kupezeka kwa mapuloteni ndi mawanga kunatsimikiziridwa.Kenaka chotsani mosamala madzi omwe ali pamwamba pa rafts kuchokera m'zitsime, kenaka yikani 50 L of dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) ndikuumira kwa mphindi khumi.Kuchuluka kwa mchere wamchere kumatsimikizira kuti LLPS sidzabwerezedwa chifukwa cha PEG yotsalira, ndipo mapuloteni omwe angakhalepo opangidwa ndi ma electrostatic interactions adzaphwanyidwa.Pansi pa chitsimecho adakankhidwa mosamala ndi nsonga ya micropipette ndipo yankho lake linasamutsidwa ku chitsime chopanda kanthu.Pambuyo pa makulitsidwe a zitsanzo ndi 50 μM ThT kwa 1 h, kukhalapo kwa mawanga akutali kunayang'aniridwa ndi microscope ya WF.Konzani sonicated αS fibrils ndi incubating 300 µl wa 70-µM αS njira PBS ndi pH 7.4, sodium azide 0,01% pa 37 °C ndi 200 rpm pa orbital shaker kwa 7 masiku.The njira ndiye centrifuged pa 9600 × g kwa 30 min, pellet anali resuspended mu PBS pH 7.4 ndi sonicated (1 mphindi, 50% mkombero, 80% matalikidwe mu Vibra-Cell VC130 sonicator, Sonics, Newton, USA) fibril zitsanzo. ndi yunifolomu kukula kugawa ang'onoang'ono fibrils.
Kusanthula kwa FCS/FCCS ndi kuzindikira kwa mitundu iwiri mwangozi (TCCD) kudachitika pa MT200 yofanana ndi maikolofoni ya fluorescent confocal microscope (Pico-Quant, Berlin, Germany) yogwiritsidwa ntchito poyeserera pa microscopy ya FLIM-FRET pogwiritsa ntchito mawonekedwe a PIE.Mphamvu ya laser yoyesererayi idawonjezedwa ku 6.0 µW (481 nm) ndi 6.2 µW (637 nm).Kuphatikiza kwa mphamvu za laser izi kunasankhidwa kuti apange kuwala kofananira kwa awiriawiri a fluorophores omwe amagwiritsidwa ntchito ndikukwaniritsa kuwerengera koyenera komanso kupewa kujambula zithunzi ndi machulukitsidwe.Kusonkhanitsa ndi kusanthula deta kunachitika pogwiritsa ntchito pulogalamu ya SymphoTime64 version 2.3 yomwe ilipo malonda (PicoQuant, Berlin, Germany).
Zitsanzo zamagulu akutali a αS/Tau omwe amapezedwa pogwiritsa ntchito LLPS amasungunulidwa m'malo odzipatula kupita kumalo oyenerera a monomolecular (nthawi zambiri 1:500 dilution, popeza zophatikizira zimakhala kale zocheperako zikasiyanitsidwa ndi zitsanzo za coacervate).Zitsanzo zidagwiritsidwa ntchito molunjika ku zophimba (Corning, USA) zopaka kale ndi yankho la BSA pamlingo wa 1 mg/mL.
Pakuwunika kwa PIE-smFRET mumayendedwe obiriwira ndi ofiira, kutsika kwamphamvu kwa ma photon 25 kudayikidwa kuti kusefa ma siginecha otsika kwambiri obwera chifukwa cha zochitika zamtundu wa monomeric (zindikirani kuti ma monomers amaposa zitsanzo zophatikizika poyerekeza ndi magulu akutali).Mpata uwu unawerengedwa ngati kuwirikiza kasanu kuchuluka kwa mphamvu ya monomeric αS yopezedwa kuchokera pakuwunika kwa zitsanzo zoyera za monoma kuti musankhe mwapadera zophatikiza zowunikira.Dongosolo loyendetsa PIE, limodzi ndi kupeza deta ya TSCPC, lathandiza kugwiritsa ntchito fyuluta yolemetsa ya moyo wonse yomwe imathandiza kuthetsa maziko ndi ma spectral crosstalk.Kuchuluka kwa flare komwe kumasankhidwa pogwiritsa ntchito ziwombankhanga zomwe zili pamwambazi zidakonzedwa pogwiritsa ntchito chizindikiro chakumbuyo chakumbuyo komwe kumatsimikiziridwa kuchokera ku histograms of zochitika motsutsana ndi mphamvu/bin ya zitsanzo za buffer-only.Kuphulika komwe kumalumikizidwa ndi magulu akulu nthawi zambiri kumakhala ma bin angapo motsatizana pakanthawi kochepa (kukhazikitsidwa ku 1 ms).Muzochitika izi, bin yamphamvu kwambiri idasankhidwa.Pakuwunika kwa FRET ndi stoichiometric, gamma factor γ (0.517) idagwiritsidwa ntchito.Zopereka za Spectral crosstalk ndi zolimbikitsa zachindunji ndizosafunika (zoyesedwa moyesera) pa mphamvu ya laser yosangalatsa yomwe imagwiritsidwa ntchito.Kuchita bwino ndi stoichiometry ya FRET pakuphulika kumawerengedwa motere.