347 12.7 * 1.24mm Stainless hlau coiled tubing, Molecular mechanism ntawm synchronous electrostatic condensation thiab coaggregation ntawm α-synuclein thiab tau

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347 Stainless hlau yeeb nkab Specification

347 12.7 * 1.24mm Stainless hlau coiled tubing

Sab nraud txoj kab uas hla: 6.00 hli OD txog 914.4 hli OD, Qhov ntau thiab tsawg mus txog 24 "NB muaj Ex-Tshuag, OD Loj Hlau Tubes muaj Ex-Tshuag

SS 347 Yeeb Nkab Thickness: 0.3mm - 50mm, SCH 5, SCH10, SCH 40, SCH 80, SCH 80S, SCH 160, SCH XXS, SCH XS
WT: SCH5S, SCH10S, SCH40S, SCH80S, SCH160S, thiab lwm yam. (0.5-12mm) Los yog tsis tu ncua qhov loj me kom haum raws li qhov xav tau

Hom: SS 347 Seamless Pipes |SS 347 ERW Pipes |SS 347 Welded Pipes |SS 347 Fabricated Pipes |SS 347 CDW Tubes, LSAW Pipes / Seam-welded / Redrawn

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

Length: Ib qho Random, Ob Chav Random & Yuav Tsum Tau Ntev Ntev: Plain End, Beveled End, Treaded

Kev Tiv Thaiv Kawg: Yas Caps |Sab Nraud Tag Nrho: 2B, No.4, No.1, No.8 Daim iav tiav rau cov kav hlau stainless, Ua tiav raws li cov neeg siv khoom xav tau

Kev Xa Khoom: Annealed thiab Pickled, Polished, Bright Annealed, Txias Drawn

Kev tshuaj xyuas, Kev Tshaj Tawm Kev Xeem: Mill Test Certificate, EN 10204 3.1, Chemical Reports, Mechanical Reports, PMI Test Reports, Visual Inspection Reports, Third Party Inspection Reports, NABL Approved Lab Reports, Destructive Test Reports, Non Destructive Test Reports

Ntim: Ntim rau hauv cov thawv ntoo, hnab yas, cov hlua hlau, lossis raws li cov neeg siv khoom thov

Tshwj xeeb: Qhov ntau thiab tsawg thiab Specifications uas tsis yog saum toj no tuaj yeem tsim tau raws li kev thov

SS 347 Yeeb Nkab Loj: 1/2 nti NB, OD txog 24 nti

ASTM A312 347: Seamless thiab ncaj-seam welded austenitic yeeb nkab npaj rau kev kub siab thiab kev pabcuam corrosive.Filler hlau tsis tso cai thaum lub sij hawm vuam.

ASTM A358 347: Hluav taws xob fusion welded austenitic yeeb nkab rau corrosive thiab / lossis kub kev pab cuam.Feem ntau tsuas yog cov yeeb nkab mus txog 8 nti yog tsim rau qhov kev qhia no.Ntxiv cov hlau muab tub lim raug tso cai thaum lub sij hawm vuam.

ASTM A790 347: Seamless thiab ncaj-seam welded ferritic / austenitic (duplex) yeeb nkab npaj rau kev pabcuam corrosive, nrog rau qhov tshwj xeeb ntawm kev tiv thaiv kev ntxhov siab corrosion.

ASTM A409 347: Ncaj-seam lossis kauv-seam hluav taws xob fusion welded loj txoj kab uas hla austenitic lub teeb-phab ntsa yeeb nkab hauv qhov ntau thiab tsawg 14 "mus rau 30" nrog phab ntsa Sch5S thiab Sch 10S rau corrosive thiab / lossis siab

ASTM A376 347 Seamless austenitic yeeb nkab rau kev siv kub.

ASTM A813 347: Ib leeg-seam, ib leeg-los yog ob-welded austenitic yeeb nkab rau kev kub siab thiab kev siv corrosive.

ASTM A814 347: Cold-worked welded austenitic yeeb nkab rau kev kub siab thiab kev pabcuam corrosive.

347H Stainless Steel Pipes Chemical Composition

Stainless hlau 347H yeeb nkab Mechanical Properties

Stainless hlau 347H Pipes Lub cev muaj zog

Qib sib npaug rau 347H Stainless Hlau Yeeb Nkab

Amyloid alpha-synuclein (αS) aggregation yog ib qho cim ntawm Parkinson tus kab mob thiab lwm yam kab mob.Tsis ntev los no, tau cov protein uas feem ntau cuam tshuam nrog Alzheimer's kab mob tau cuam tshuam nrog αS pathology thiab pom tias muaj kev sib koom ua ke hauv αS-nplua nuj suav nrog, txawm hais tias cov txheej txheem molecular ntawm coaggregation ntawm ob cov protein tseem tsis meej.Peb qhia ntawm no tias αS theem sib cais ua kua condensates ntawm electrostatic complex condensation nrog zoo them polypeptides xws li tau.Nyob ntawm seb qhov kev sib raug zoo ntawm αS rau polycations thiab tus nqi ntawm valence depletion ntawm coagulation network, cov hlab ntsha tau nrawm nrawm los yog coalescence tom qab qeeb amyloid aggregation.Los ntawm kev sib koom ua ke ntawm cov txheej txheem biophysical siab heev, peb tuaj yeem ua tus yam ntxwv ntawm cov kua-kua αS / Tau theem sib cais thiab txheeb xyuas cov ntsiab lus tseem ceeb uas ua rau tsim cov heterogeneous aggregates uas muaj ob qho proteins hauv cov kua protein condensate.
Ntxiv nrog rau daim nyias nyias, kev sib cais hauv cov hlwb kuj tuaj yeem ua tiav los ntawm kev tsim cov protein-nplua nuj, kua zoo li lub cev ntom ntom hu ua biomolecular condensates lossis cov tee dej, los ntawm cov txheej txheem hu ua kua-kua theem sib cais (LLPS).Cov tee dej no yog tsim los ntawm ntau yam kev sib cuam tshuam ntawm lub cev, feem ntau ntawm cov proteins lossis cov proteins thiab RNA, thiab ua haujlwm ntau yam hauv yuav luag txhua lub neej nyob.Ib tug loj tus naj npawb ntawm LLP-muaj peev xwm proteins muaj ib tug sequences ntawm tsawg complexity uas yog heev tsis meej nyob rau hauv cov xwm thiab nyob rau hauv tsim ntawm biomolecular condensates3,4,5.Ntau cov kev tshawb fawb sim tau qhia txog qhov hloov pauv tau, feem ntau tsis zoo, thiab ntau yam ntawm cov proteins uas ua rau cov kua zoo li cov condensates, txawm tias me ntsis paub txog cov kev txiav txim siab molecular tshwj xeeb uas tswj kev loj hlob thiab kev loj hlob ntawm cov condensates mus rau ib qho khoom zoo li ntau. xeev..
Cov ntaub ntawv tshiab txhawb nqa qhov kev xav tias qhov tsis txaus ntseeg ntawm cov protein-tsav LLPS thiab kev hloov pauv ntawm cov tee rau hauv cov qauv khoom yuav yog cov txheej txheem ntawm tes uas ua rau tsim cov tshuaj lom tsis sib xws uas feem ntau yog cov cim ntawm cov kab mob degenerative.Ntau tus LLPS-txuas nrog cov proteins uas tsis muaj kev cuam tshuam (IDPs), feem ntau raug them thiab hloov tau, tau ntev tau cuam tshuam nrog neurodegeneration los ntawm cov txheej txheem ntawm amyloid aggregation.Tshwj xeeb, biomolecular IDP condensates xws li FUS7 lossis TDP-438 lossis cov proteins uas muaj qhov tsis sib xws loj xws li hnRNPA19 tau pom tias muaj hnub nyoog mus rau hauv gel-zoo li lossis cov ntaub ntawv tseem ceeb los ntawm cov txheej txheem hu ua fluidization.compound.mus rau cov theem kev hloov pauv (LSPT) raws li kev ua haujlwm ntawm lub sijhawm lossis hauv kev teb rau qee qhov kev hloov pauv tom qab lossis kev hloov pauv tseem ceeb ntawm pathologically1,7.
Lwm qhov IDP cuam tshuam nrog LLPS hauv vivo yog Tau, microtubule-txuas cuam tshuam cov protein uas nws cov amyloid aggregation tau cuam tshuam rau Alzheimer's disease10 tab sis kuj tsis ntev los no tau cuam tshuam nrog Parkinson's disease (PD) thiab lwm yam synaptic nuclear proteinopathies 11, 12, 13 muaj feem xyuam.Tau tau pom tias muaj kev sib cais tsis tu ncua los ntawm kev daws teeb meem / cytoplasm vim muaj kev cuam tshuam zoo ntawm electrostatic kev sib cuam tshuam14, uas ua rau tsim cov tau-enriched droplets hu ua electrostatic coacervates.Nws kuj tau raug pom tias hom kev sib cuam tshuam uas tsis yog tshwj xeeb yog lub zog tsav tom qab ntau yam biomolecular condensates hauv xwm15.Nyob rau hauv cov ntaub ntawv ntawm ib tug tau protein, electrostatic aggregation yuav tsim los ntawm yooj yim aggregation, nyob rau hauv uas oppositely them cheeb tsam ntawm cov protein ua rau lub cleavage txheej txheem, los yog complex aggregation los ntawm kev sib cuam tshuam nrog tsis zoo nqi polymers xws li RNA.
Tsis ntev los no, α-synuclein (αS), ib qho amyloid IDP cuam tshuam hauv PD thiab lwm yam kab mob neurodegenerative sib sau ua ke hu ua synucleinopathy17,18, tau pom nyob rau hauv cellular thiab tsiaj qauv19,20 concentrated nyob rau hauv protein condensates nrog dej zoo li tus cwj pwm.Cov kev tshawb fawb hauv vitro tau pom tias αS tau txais LLPS los ntawm kev sib sau yooj yim los ntawm kev sib cuam tshuam ntawm hydrophobic feem ntau, txawm hais tias cov txheej txheem no yuav tsum muaj cov protein ntau tshwj xeeb thiab atypically incubation lub sij hawm ntev 19,21.Txawm hais tias αS-muaj condensates pom nyob rau hauv vivo yog tsim los ntawm qhov no lossis lwm yam txheej txheem LLPS tseem yog qhov tseem ceeb tsis tau daws qhov teeb meem.Ib yam li ntawd, txawm hais tias αS amyloid aggregation tau pom nyob rau hauv cov neurons hauv PD thiab lwm yam synucleinopathies, qhov tseeb mechanism uas αS nkag mus rau intracellular amyloid aggregation tseem tsis meej, vim hais tias overexpression ntawm cov protein no tsis tshwm sim ua rau cov txheej txheem no los ntawm nws tus kheej.Kev puas tsuaj ntxiv ntawm tes yog feem ntau xav tau, qhia tias qee qhov chaw ntawm tes lossis microenvironments yuav tsum tau ua kom rov ua dua tshiab ntawm intracellular αS amyloid cov rooj sib txoos.Ib puag ncig ntawm tes uas tshwj xeeb tshaj yog rau kev sib sau ua ke tuaj yeem yog sab hauv ntawm cov protein condensates 23 .
Interestingly, αS thiab tau tau pom muaj kev sib koom ua ke hauv cov kab mob hauv cov tib neeg nrog Parkinson's disease thiab lwm yam synucleinopathies 24,25 thiab cov kev sim tau qhia txog kev sib koom ua ke ntawm kev sib raug zoo ntawm ob cov proteins 26,27 qhia txog kev sib raug zoo ntawm kev sib sau ua ke αS thiab tau nyob rau hauv cov kab mob neurodegenerative.ua mob.αS thiab tau tau pom muaj kev cuam tshuam thiab txhawb kev sib sau ua ke hauv vitro thiab hauv vivo 28,29 thiab cov sib xyaw ua ke sib xyaw ua ke ntawm ob cov proteins no tau pom nyob rau hauv lub hlwb ntawm cov neeg mob synucleinopathies 30 .Txawm li cas los xij, me ntsis paub txog lub hauv paus molecular ntawm kev sib cuam tshuam ntawm αS thiab tau thiab cov txheej txheem ntawm nws cov co-aggregation.αS tau tshaj tawm tias muaj kev cuam tshuam nrog tau los ntawm electrostatic attraction ntawm qhov tsis zoo ntawm C-terminal cheeb tsam ntawm αS thiab lub hauv paus proline-nplua nuj cheeb tsam ntawm tau, uas yog tseem enriched nyob rau hauv zoo them nqi residues.
Nyob rau hauv txoj kev tshawb no, peb qhia tau hais tias αS muaj tseeb dissociate rau hauv cov tee dej ntawm electrostatic complex condensation nyob rau hauv lub xub ntiag ntawm tau protein, nyob rau hauv sib piv rau nws kev sib raug zoo nrog rau lwm yam zoo them polypeptides xws li poly-L-lysine (pLK), thiab nyob rau hauv cov txheej txheem no.αS ua raws li scaffold molecule rau lub droplet network.Peb tau txheeb xyuas qhov sib txawv ntawm cov txheej txheem ntawm kev loj hlob ntawm electrostatic αS coacervates, uas cuam tshuam nrog qhov sib txawv ntawm qhov muaj txiaj ntsig thiab lub zog ntawm kev sib cuam tshuam ntawm cov proteins koom nrog hauv coacervate network.Interestingly, peb pom kev sib sau ua ke ntawm αS thiab tau amyloid proteins nyob rau hauv cov kua coacervates ntev thiab txheeb xyuas qee yam tseem ceeb uas ua rau kev sib sau ua ke ntawm ob cov proteins hauv cov coacervates.Ntawm no peb piav qhia meej txog cov txheej txheem no, uas yog qhov ua tau molecular mechanism hauv qab ntawm colocalization ntawm ob lub proteins hauv cov kab mob tshwj xeeb suav nrog.
αS muaj ib tug heev anionic C-terminal tus Tsov tus tw ntawm nruab nrab pH (Daim duab 1a), thiab peb hypothesized tias nws yuav undergo LLPS los ntawm cov condensation ntawm electrostatic complexes nrog polycationic disordered polypeptide molecules.Peb siv 100-residue poly-L-lysine (pLK) raws li cov qauv pib molecule vim nws qhov zoo thiab tsis zoo polymeric xwm ntawm nruab nrab pH 32. Ua ntej, peb paub tseeb tias pLK cuam tshuam nrog Ct domain ntawm αS ntawm Solution NMR spectroscopy (Daim duab 1b) siv 13C/15N-labeled αS nyob rau hauv lub xub ntiag ntawm nce αS: pLK molar ratios.Kev sib cuam tshuam ntawm pLK nrog Ct-domain ntawm αS manifests nws tus kheej nyob rau hauv perturbations ntawm cov tshuaj hloov thiab ib tug txo nyob rau hauv lub ncov siv nyob rau hauv lub cheeb tsam ntawm cov protein.Interestingly, thaum peb sib tov αS nrog pLK ntawm ib qho αS concentration ntawm approx.5–25 µM nyob rau hauv lub xub ntiag ntawm polyethylene glycol (5–15% PEG-8) (ib txwm LLPS tsis: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) peb tam sim ntawd mus dhau ntawm qhov dav ntawm kev tsim cov protein. .Cov tee dej tau pom los ntawm kev siv cov tshuaj fluorescence (WF) thiab lub teeb pom kev zoo (BF) microscopy (Fig. 1c).1-5 µm cov tshuaj uas muaj cov concentrated αS (ntxiv 1 µM AlexaFluor488-labeled αS, AF488-αS), lawv cov khoom electrostatic tuaj yeem muab tau los ntawm lawv qhov tsis kam mus rau 10% 1,6-hexanediol (1,6-HD) thiab nws rhiab heev rau nce hauv NaCl concentration (Fig. 1c).Cov dej zoo li cov coacervates ntawm αS/pLK electrostatic complex yog pom los ntawm lawv lub peev xwm mus fuse nyob rau hauv milliseconds (Fig. 1d).Siv turbidimetry, peb ntsuas qhov tsim ntawm cov tee dej nyob rau hauv cov kev mob no, paub tseeb tias electrostatic xwm ntawm lub ntsiab kev cuam tshuam nrog nws stability (Fig. 1e), thiab ntsuam xyuas cov nyhuv ntawm ntau yam polymer ratios ntawm cov txheej txheem LLPS (Fig. 1f).Txawm hais tias kev tsim cov tee dej tau pom nyob rau ntau qhov sib txawv ntawm cov polymer, cov txheej txheem yog qhov zoo heev thaum pLK yog ntau tshaj αS.LLPs kuj tau raug soj ntsuam siv cov tshuaj sib txawv ntawm cov neeg sawv cev dextran-70 (70 kDa) los yog siv ntau hom qauv, suav nrog cov iav swb, microplate qhov dej ntawm ntau yam ntaub ntawv, Eppendorf lossis quartz capillaries.
ib tug Schematic sawv cev ntawm txawv cheeb tsam protein nyob rau hauv WT-αS thiab ΔCt-αS variants siv nyob rau hauv txoj kev tshawb no.Lub amphipathic N-terminal domain, thaj av hydrophobic amyloid-forming (NAC), thiab qhov tsis zoo C-terminal domain tau qhia hauv xiav, txiv kab ntxwv, thiab liab, raws li.Net Charge Per Residual (NCPR) daim ntawv qhia ntawm WT-αS tau qhia.b NMR tsom xam ntawm αS / pLK kev sib cuam tshuam hauv qhov tsis muaj macromolecular clumps.Raws li pLK concentration nce (αS: pLK molar ratios ntawm 1:0.5, 1:1.5, thiab 1:10 yog qhia nyob rau hauv lub teeb ntsuab, ntsuab, thiab tsaus ntsuab, feem).c Coacervate αS/pLK (molar ratio 1:10) ntawm 25 µM (1 µM AF488-labeled αS lossis Atto647N-labeled pLK rau WF imaging) hauv LLPS tsis (sab saum toj) lossis ntxiv nrog 500 mM NaCl (hauv qab 10) lossis tom qab % 1,6-hexanediol (1,6-HD; hauv qab sab xis).Luas bar = 20 µm.d Cov neeg sawv cev ntawm microscopic dluab ntawm BF droplet fusion ntawm αS / pLK (molar ratio 1:10) ntawm ib tug concentration ntawm 25 μM;Cov xub qhia txog kev sib koom ua ke ntawm ib tus neeg tee (xov liab thiab daj) rau hauv qhov poob tshiab (txiv kab ntxwv xub) hauv 200 ms).Luas bar = 20 µm.e Lub teeb tawg (ntawm 350 nm) αS / pLK aggregation nyob rau hauv LLPS tsis ua ntej thiab tom qab ntxiv ntawm 500 mM NaCl los yog 10% 1,6-HD ntawm 25 µM αS (N = 3 qauv replicates, txhais tau tias thiab tus qauv sib txawv kuj qhia).f BF duab (sab saum toj) thiab lub teeb tawg tsom (ntawm 350 nm, hauv qab) ntawm αS / pLK aggregation ntawm 25 μM αS nrog nce αS: pLK molar ratio (N = 3 qauv replicates, txhais tau tias thiab tus qauv sib txawv kuj qhia).Luas bar = 10 µm.Qhov ntsuas ntsuas ntawm ib daim duab qhia qhov ntsuas ntawm txhua cov duab hauv ib lub vaj huam sib luag.Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Raws li peb qhov kev soj ntsuam ntawm αS / pLK electrostatic complex condensation thiab kev soj ntsuam yav dhau los ntawm αS raws li tus neeg siv khoom molecule ntawm tau / RNA condensate los ntawm kev sib cuam tshuam ncaj qha nrog tau31, peb xav tias αS thiab tau tuaj yeem sib cais nrog cov kuab tshuaj thaum tsis muaj RNA. condensation.los ntawm electrostatic complexes, thiab αS yog scaffold protein nyob rau hauv αS/Tau coacervates (saib tau them tis nyob rau hauv daim duab 2e).Peb pom tias thaum 10 μM αS thiab 10 μM Tau441 (muaj 1 μM AF488-αS thiab 1 μM Atto647N-Tau, feem) tau sib xyaw ua ke hauv LLPS tsis, lawv yooj yim tsim cov protein sib xyaw uas muaj ob qho protein, raws li pom los ntawm WF microscopy.(Daim duab 2a).Lub colocalization ntawm ob lub proteins nyob rau hauv cov tee dej tau lees paub los ntawm confocal (CF) microscopy (Supplementary Fig. 1a).Cov cwj pwm zoo sib xws tau pom thaum dextran-70 tau siv los ua tus neeg sawv cev sib sau ua ke (Ntxiv daim duab 1c).Siv FITC-labeled PEG los yog dextran, peb pom tias ob qho tib si neeg coob coob tau muab faib sib npaug sib npaug ntawm cov qauv, tsis qhia tias tsis muaj kev sib cais lossis kev koom ua ke (Ntxiv daim duab 1d).Xwb, nws qhia tias nyob rau hauv cov kab ke no lawv txhawb cov theem sib cais los ntawm macromolecular crowding los, vim PEG yog ib tug nyiam nyob ruaj khov neeg sawv cev, raws li pom nyob rau hauv lwm yam LLP systems33,34.Cov kua dej uas muaj protein ntau no tau nkag siab rau NaCl (1 M) tab sis tsis yog rau 1,6-HD (10% v/v), lees paub lawv cov khoom siv hluav taws xob (Cov duab ntxiv 2a, b).Lawv tus cwj pwm ua kua dej tau lees paub los ntawm kev soj ntsuam millisecond merging droplet txheej xwm siv BF microscopy (Fig. 2b).
Confocal (CF) microscopy dluab ntawm αS/Tau441 coacervates nyob rau hauv LLPS tsis (10 μM ntawm txhua protein, 0.5 μM ntawm AF488-labeled αS thiab Atto647N-labeled Tau441).b Cov neeg sawv cev sib txawv ntawm qhov sib txawv ntawm qhov sib txawv (DIC) cov duab ntawm αS/Tau441 droplet fusion txheej xwm (10 μM rau txhua cov protein).c Phase diagram raws li lub teeb tawg (ntawm 350 nm) ntawm Tau441 LLPS (0–15 µM) nyob rau hauv qhov tsis muaj (sab laug) lossis muaj (txoj cai) ntawm 50 µM αS.Cov xim sov dua qhia tias tawg ntau dua.d Lub teeb tawg ntawm αS/Tau441 LLPS cov qauv nrog nce αS concentration (Tau441 ntawm 5 µM, N = 2-3 cov qauv rov ua raws li qhia).e Schematic sawv cev ntawm qee qhov tau protein ntau yam sib txawv thiab thaj chaw sib txawv ntawm cov protein siv hauv txoj kev tshawb no: tsis zoo them N-terminal domain (liab), thaj av nplua nuj proline (xiav), microtubule-binding domain (MTBD, tseem ceeb hauv txiv kab ntxwv), thiab amyloid-forming khub kauv.filament regions (PHF) nyob hauv MTBD (grey).The Net Charge Per Residue (NCPR) map of Tau441 is showed.f Siv 1 µM AF488-labeled αS thiab Atto647N-labeled ΔNt-, siv 1 µM AF488-labeled αS los yog ΔCt-αS nyob rau hauv lub xub ntiag ntawm ΔNt-Tau (sab saum toj, 10 µM ib protein) los yog K18 (hauv qab, 50 µM protein) ) ) ) micrographs ntawm WF condensed hauv LLPS lossis K18 tsis.Cov nplai tuav hauv ib daim duab sawv cev rau qhov ntsuas ntawm tag nrho cov duab hauv ib lub vaj huam sib luag (20 µm rau panels a, b thiab f).Cov ntaub ntawv nyoos rau panels c thiab d yog muab raws li cov ntaub ntawv nyoos.
Txhawm rau kuaj lub luag haujlwm ntawm αS hauv cov txheej txheem LLPS no, peb thawj zaug tshawb xyuas cov txiaj ntsig ntawm αS ntawm kev ruaj khov ntawm cov kua dej los ntawm nephelometry siv cov kev nce ntxiv ntawm NaCl (Fig. 2c).Qhov siab dua cov ntsev concentration nyob rau hauv cov qauv uas muaj αS, qhov siab dua lub teeb tawg qhov tseem ceeb (ntawm 350 nm), uas qhia txog lub luag haujlwm ruaj khov ntawm αS hauv LLPS system no.Cov txiaj ntsig zoo sib xws tuaj yeem pom los ntawm kev nce αS concentration (thiab li no αS: Tau441 piv) rau kwv yees.10-fold nce txheeb ze rau tau concentration (5 µM) (Fig. 2d).Txhawm rau ua kom pom tias αS yog scaffold protein nyob rau hauv coacervates, peb tau txiav txim siab los tshawb xyuas tus cwj pwm ntawm LLPS- cuam tshuam Tau mutant, uas tsis muaj qhov tsis zoo rau thaj tsam N-terminal (residues 1-150, saib daim duab 2e) hu ua ΔNt-Tau.WF microscopy thiab nephelometry tau lees paub tias ΔNt-Tau nws tus kheej tsis tau txais LLPS (Daim duab 2f thiab Supplementary Fig. 2d), raws li yav dhau los tau tshaj tawm 14. Txawm li cas los xij, thaum αS tau ntxiv rau cov kev daws teeb meem ntawm qhov truncated Tau variant, cov txheej txheem LLPS tau ua tiav. Rov qab nrog cov qauv hluav taws xob kom ze rau lub sijhawm muaj kev daws teeb meem ntawm TAU thiab cov xwm txheej zoo sib xws thiab cov protein ntau.Cov txheej txheem no tseem tuaj yeem pom nyob rau hauv cov xwm txheej ntawm cov neeg coob coob uas tsis muaj macromolecular (Supplementary Fig. 2c).Lub luag hauj lwm ntawm C-terminal αS cheeb tsam, tab sis tsis yog nws tag nrho ntev, nyob rau hauv cov txheej txheem LLPS tau pom los ntawm inhibition of droplet formation siv C-terminal truncated αS variant tsis muaj residues 104-140 (Fig. 1a) ntawm (ΔCt- αS) protein (Fig. 2f thiab Supplementary Fig. 2d).Lub colocalization ntawm αS thiab ΔNt-Tau tau lees paub los ntawm confocal fluorescence microscopy (Ntxiv Fig. 1b).
Txhawm rau kuaj ntxiv LLPS cov txheej txheem ntawm Tau441 thiab αS, tau siv Tau ntau yam ntxiv, uas yog qhov sib xyaw ua ke helical filament core (PHF) fragment hauv microtubule-binding domain (MTBD), uas yog tias nws muaj plaub tus yam ntxwv rov ua dua, feem ntau kuj paub. raws li K18 fragment (saib daim duab 2e).Nws tau tsis ntev los no tau tshaj tawm tias αS nyiam khi rau cov protein ntau nyob rau hauv ib qho proline-nplua nuj sau nyob rau hauv ib theem uas ua ntej lub microtubule-binding domain.Txawm li cas los xij, thaj tsam PHF kuj tseem nplua nuj nyob hauv cov khoom seem zoo (saib daim duab 2e), tshwj xeeb tshaj yog cov lysine (15% residues), uas tau ua rau peb sim seb thaj av no puas muaj txiaj ntsig rau qhov condensation ntawm αS / Tau complex.Peb pom tias K18 ib leeg tsis tuaj yeem ua rau LLPS ntawm qhov siab txog 100 μM nyob rau hauv cov xwm txheej kuaj (LLPS tsis nrog 15% PEG lossis 20% dextran) (Daim duab 2f).Txawm li cas los xij, thaum peb ntxiv 50 µM αS rau 50 µM K18, kev tsim sai ntawm cov kua dej protein uas muaj K18 thiab αS tau pom los ntawm nephelometry (Ntxiv daim duab 2d) thiab WF microscopy (Fig. 2f).Raws li qhov xav tau, ΔCt-αS tsis tuaj yeem rov qab kho LLPS tus cwj pwm ntawm K18 (Daim duab 2f).Peb nco ntsoov tias αS / K18 aggregation yuav tsum muaj cov protein ntau dua me ntsis kom ntxias LLPS piv rau αS / ΔNt-Tau lossis αS / Tau441, lwm yam sib npaug.Qhov no yog ua raws li kev sib raug zoo ntawm αS C-terminal cheeb tsam nrog cov proline-nplua nuj Tau sau piv rau microtubule-binding domain, raws li tau piav qhia yav dhau los 31.
Muab hais tias ΔNt-Tau tsis tuaj yeem ua LLPS thaum tsis muaj αS, peb xaiv Tau qhov sib txawv no ua tus qauv rau tus yam ntxwv ntawm αS / Tau LLPS muab nws qhov yooj yim hauv LLPS systems nrog tag nrho-ntev Tau (isotype, Tau441 / Tau441).nrog complex (heterotypic, αS/Tau441) aggregation processes.Peb piv cov degree ntawm αS aggregation (raws li ib feem ntawm condensed theem protein, fαS, c) nyob rau hauv lub αS/Tau thiab αS/ΔNt-Tau systems los ntawm centrifugation thiab dispersed theem SDS-PAGE tsom xam (saib 2e), pom muaj txiaj ntsig zoo sib xws. rau tag nrho cov protein nyob rau tib lub concentration.Tshwj xeeb, peb tau txais fαS, c 84 ± 2% thiab 79 ± 7% rau αS / Tau thiab αS / ΔNt-Tau, feem, qhia tias kev sib cuam tshuam heterotypic ntawm αS thiab tau yog superior rau kev sib cuam tshuam ntawm tau molecules.nruab nrab.
Kev sib cuam tshuam nrog ntau yam polycations thiab cov nyhuv ntawm cov txheej txheem condensation ntawm αS kinetics yog thawj zaug kawm los ntawm fluorescence rov qab tom qab photobleaching (FRAP) txoj kev.We tested αS/Tau441, αS/ΔNt-Tau and αS/pLK coacervates (100 μM αS supplemented with 2 μM αS AF488-αS and 100 μM Tau441 or ΔNt-Tau or 1 mM pLK).Cov ntaub ntawv tau txais hauv thawj 30 feeb tom qab sib xyaw cov qauv piv txwv.Los ntawm tus neeg sawv cev FRAP cov duab (Fig. 3a, αS/Tau441 condensation) thiab lawv cov sij hawm sib txuas lus nkhaus (Fig. 3b, Supplementary Fig. 3), nws tuaj yeem pom tias αS kinetics zoo ib yam li cov ntawm Tau441 coacervates.thiab ΔNt-Tau, uas nrawm dua nrog pLK.Lub laij lej diffusion coefficients rau αS hauv coacervate raws li FRAP (raws li tau piav qhia los ntawm Kang li al. 35) yog D = 0.013 ± 0.009 µm2 / s thiab D = 0.026 ± 0.008 µm2 / s rau αS / Δt-4 / s lub αS/system.pLK, Tau, and D = 0.18 ± 0.04 µm2/s, respectively (Fig. 3c).Txawm li cas los xij, qhov diffusion coefficient αS nyob rau hauv cov theem dispersed yog ntau qhov kev txiav txim siab ntau dua li tag nrho cov theem condensed, raws li kev txiav txim los ntawm Fluorescence Correlation Spectroscopy (FCS, saib ntxiv daim duab 3) nyob rau hauv tib yam mob (LLPS tsis) tab sis nyob rau hauv tsis muaj polycations. (D = 8 ± 4 µm2/s).Yog li ntawd, kinetics ntawm αS kev txhais lus yog txo qis hauv coacervates piv rau cov proteins nyob rau hauv cov theem dispersed vim hais tias cov nyhuv molecular crowding, txawm hais tias tag nrho cov coacervates khaws cov khoom zoo li cov kua hauv thawj ib nrab teev tom qab lawv tsim, nyob rau hauv sib piv rau theem tau.ceev kinetics hauv pLK condensate.
a–c FRAP tsom xam ntawm αS dynamics (2% AF488-labeled αS) hauv electrostatic coacervates.Cov duab sawv cev ntawm αS/Tau441 FRAP kev soj ntsuam hauv triplicate tau pom nyob rau hauv (a), qhov twg lub voj voog liab qhia cov cheeb tsam decolorized.Qhov ntsuas bar yog 5 µm.b Qhov nruab nrab FRAP nkhaus thiab (c) xam diffusion coefficients (D) rau 5–6 (N) cov tee dej sib txawv los ntawm peb qhov kev sim siv 100 µM αS thiab equimolar concentrations ntawm Tau441 (liab) lossis ΔNt-Tau (xiav) lossis pLK (ntsuab) ntawm kaum npaug ntawm qhov concentration ntawm LLPS.Tus qauv sib txawv ntawm FRAP nkhaus yog pom hauv cov xim ntxoov ntxoo.Rau kev sib piv, diffusion coefficient αS nyob rau hauv lub dispersed theem tau txiav txim nyob rau hauv triplicate siv fluorescence correlation spectroscopy (FCS) (saib Daim duab ntxiv 3 thiab cov txheej txheem rau cov lus qhia ntxiv).d Nruam X-band EPR spectra ntawm 100 μM TEMPOL-122-αS hauv LLPS tsis muaj polycation (dub) lossis nyob rau hauv muaj 100 μM Tau441 (liab) lossis ΔNt-Tau (xiav) lossis 1 mM pLK (ntsuab).Lub inset qhia pom qhov loj ntawm cov kab hauv cheeb tsam uas muaj kev hloov pauv loj tshaj plaws.e Binding nkhaus ntawm 50 μM TEMPOL-122-αS nrog ntau yam polycations thaum tsis muaj LLPS (tsis muaj PEG).Qhov txo qis ntawm band III piv rau band II (IIII / III) ntawm qhov normalized EPR spectrum yog pom tias yuav ua rau kom cov molar ratios ntawm Tau441 (liab), ΔNt-Tau (xiav) thiab pLK (ntsuab).Cov kab uas muaj xim zoo nkauj qhia tau hais tias haum rau cov ntaub ntawv siv cov qauv sib khi nrog n zoo tib yam thiab kev ywj pheej khi qhov chaw ntawm txhua qhov nkhaus.Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Raws li ib qho ntxiv, peb tau tshawb xyuas qhov kev hloov pauv ntawm αS nyob rau hauv ntau yam coacervates siv cov ntawv qhia spin labeling (SDSL) thiab nruam electron paramagnetic resonance (CW-EPR).Txoj kev no tau ua pov thawj tias muaj txiaj ntsig zoo hauv kev tshaj tawm qhov hloov pauv tau yooj yim thiab qhov muaj zog ntawm IDP nrog rau qhov muaj tseeb residual daws teeb meem36,37,38.Txog rau qhov kawg no, peb tsim cov cysteine ​​​​ residues hauv ib Cys mutants thiab siv 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin sojntsuam.Maleimide derivatives sau npe rau lawv.Tshwj xeeb tshaj yog, peb tau ntxig TEMPOL sojntsuam ntawm txoj haujlwm 122 lossis 24 αS (TEMPOL-122-αS thiab TEMPOL-24-αS).Hauv thawj kis, peb tsom rau thaj tsam C-terminal ntawm cov protein, uas koom nrog kev cuam tshuam nrog polycations.Hloov chaw, txoj hauj lwm 24 tuaj yeem muab peb cov ntaub ntawv hais txog tag nrho cov dynamics ntawm cov proteins hauv cov condensate.Nyob rau hauv ob qho tib si, EPR cov cim tau txais rau cov proteins ntawm cov txheej txheem tawg tau sib raug rau nitroxide radicals hauv lub xeev ceev ceev.Tom qab theem sib cais nyob rau hauv lub xub ntiag ntawm tau los yog pLK (100 μM TEMPOL-αS, Tau441 los yog ΔNt-Tau ntawm ib tug ratio ntawm 1: 1 los yog pLK ntawm ib tug ratio ntawm 1:10), ib qho kev nce nyob rau hauv cov txheeb ze ncov siv tau pom nyob rau hauv EPR spectrum ntawm αS.Cov kab poob tau nthuav dav, qhia tias txo qis αS reorientation kinetics hauv cov tee dej piv rau cov protein hauv cov theem dilute (Fig. 3d, Ntxiv Fig. 4a).Cov kev hloov no tau tshaj tawm ntawm txoj hauj lwm 122. Thaum nyob rau ntawm txoj hauj lwm 24 lub xub ntiag ntawm pLK tsis cuam tshuam rau lub kinetics ntawm kev sojntsuam, ntawm txoj hauj lwm 122 lub spectral kab duab hloov pauv loj heev (Ntxiv daim duab 4a).Thaum peb sim ua qauv ntawm spectra ntawm txoj hauj lwm 122 ntawm ob lub αS / polycation systems siv cov qauv isotropic (Ntxiv daim duab 5a) feem ntau siv los piav qhia txog cov dynamics ntawm spin-labeled IDP38,39, peb tsis tuaj yeem tsim kho qhov kev sim spectra..Spectral simulation ntawm txoj hauj lwm ntawm 24 spin contrasts (Ntxiv daim duab 5a).Qhov no qhia tau hais tias muaj qhov tshwj xeeb tshaj plaws nyob rau hauv qhov chaw ntawm spin configurations ntawm C-terminal cheeb tsam ntawm αS nyob rau hauv lub xub ntiag ntawm polycations.Thaum xav txog cov feem ntawm αS nyob rau hauv lub condensed theem nyob rau hauv kev sim EPR tej yam kev mob (84 ± 2%, 79 ± 7%, thiab 47 ± 4% rau αS / Tau441, αS / ΔNt-Tau, thiab αS / pLK, ntsig txog-saib Ntxiv Daim duab 2e ntawm cov ntaub ntawv tsom xam c), nws tuaj yeem pom tau tias qhov kev nthuav dav pom los ntawm txoj kev EPR feem ntau qhia txog kev sib cuam tshuam ntawm C-terminal cheeb tsam ntawm αS nrog ntau yam polycations nyob rau hauv lub condensed theem (lub ntsiab hloov thaum siv TEMPOL-122- αS), thiab tsis yog protein condensation.Kev nce hauv microviscosity yog pom hauv kev sojntsuam.Raws li qhov xav tau, EPR spectrum ntawm cov protein nyob rau hauv cov xwm txheej uas tsis yog LLPS tau rov qab ua tiav thaum 1 M NaCl tau ntxiv rau qhov sib xyaw (Cov Lus Qhia Ntxiv 4b).Zuag qhia tag nrho, peb cov ntaub ntawv qhia tias cov kev hloov pauv tau pom los ntawm CW-EPR feem ntau cuam tshuam txog kev sib cuam tshuam ntawm C-terminal cheeb tsam ntawm αS nrog ntau yam polycations nyob rau hauv cov theem condensed, thiab qhov kev sib cuam tshuam no zoo li muaj zog nrog pLK dua nrog Tau.
Txhawm rau kom tau txais cov ntaub ntawv ntau ntxiv txog cov proteins hauv coacervate, peb txiav txim siab los kawm LLPS system siv NMR hauv kev daws teeb meem.Txawm li cas los xij, peb tsuas tuaj yeem tshawb xyuas qhov αS feem seem nyob rau hauv cov theem tawg, uas tej zaum yuav yog vim txo cov protein ntau hauv cov coacervate thiab cov theem ntom ntom ntawm cov tshuaj hauv NMR tsom xam.Thaum peb txheeb xyuas cov qauv thiab cov kev hloov pauv ntawm cov protein uas tseem tshuav nyob rau hauv cov txheej txheem tawg ntawm LLPS cov qauv siv NMR (Supplementary Fig. 5c, d), peb pom tias cov protein tau coj zoo ib yam ntawm pLK thiab ΔNt-Tau, ob qho tib si ntawm uas tau nyob rau hauv cov qauv theem nrab thiab kev ua kom muaj zog ntawm cov pob txha protein, qhia los ntawm kev sim ntawm kev hloov tshuaj lom neeg theem nrab thiab R1ρ so.NMR cov ntaub ntawv qhia tau hais tias C-terminus ntawm αS raug kev txom nyem loj ntawm kev hloov pauv yooj yim thaum khaws nws cov xwm txheej tsis zoo, zoo li lwm cov protein ntau, vim nws cuam tshuam nrog polycations.
Txij li thaum CW-EPR teeb liab nthuav dav pom nyob rau hauv TEMPOL-122-αS condensed theem qhia txog kev sib cuam tshuam ntawm cov protein nrog polycations, peb tau ua EPR titration los ntsuas qhov kev sib raug zoo ntawm αS rau ntau yam polycations thaum tsis muaj LLPS (tsis muaj kev sib txuam ntawm Buffer LLPS), tawm tswv yim tias cov kev sib cuam tshuam yog tib yam hauv cov theem dilute thiab concentrated (uas tau lees paub los ntawm peb cov ntaub ntawv, Ntxiv Fig. 4a thiab Supplementary Fig. 6).Lub hom phiaj yog txhawm rau saib yog tias txhua tus coacervates, txawm tias lawv cov khoom zoo li cov kua dej sib txawv, pom muaj tus cwj pwm sib txawv ntawm qib molecular.Raws li qhov xav tau, EPR spectrum nthuav dav nrog kev nce polycation concentration, qhia txog qhov txo qis hauv molecular yooj vim kev sib cuam tshuam ntawm molecular ntawm txhua tus neeg koom tes sib cuam tshuam yuav luag mus rau saturation (Fig. 3e, Ntxiv Fig. 6).pLK ua tiav qhov saturation ntawm qis molar piv (polycation: αS) piv rau ΔNt-Tau thiab Tau441.Qhov tseeb, kev sib piv ntawm cov ntaub ntawv nrog ib qho piv txwv binding qauv piv txwv li n zoo tib yam thiab ywj siab binding qhov chaw pom tau hais tias qhov pom tseeb dissociation tas li ntawm pLK (~ 5 μM) yog ib qho kev txiav txim ntawm qhov loj tshaj ntawm Tau441 los yog ΔNt-Tau (~ 50 μM. ).µM).Txawm hais tias qhov no yog qhov kev kwv yees ntxhib, qhov no qhia tau tias αS muaj kev sib raug zoo dua rau cov polycations yooj yim nrog cov cheeb tsam zoo txuas ntxiv.Muab qhov sib txawv ntawm qhov sib txawv ntawm αS thiab ntau yam polycations, peb xav tias lawv cov khoom ua kua tuaj yeem hloov pauv txawv raws sijhawm thiab yog li raug kev txom nyem los ntawm cov txheej txheem LSPT sib txawv.
Muab qhov chaw muaj neeg coob coob nyob rau hauv cov protein coacervate thiab cov amyloid yam ntawm cov protein, peb tau soj ntsuam tus cwj pwm ntawm coacervate dhau sij hawm los xyuas txog cov txheej txheem LSPT.Siv BF thiab CF microscopy (Daim duab 4), peb tau pom tias αS/Tau441 coacervates mus rau qhov loj hauv kev daws, tsim cov tee dej loj uas sib cuag thiab ntub dej saum npoo hauv qab ntawm lub qhov dej / swb raws li cov tee tag nrho, raws li xav tau (Cov duab ntxiv 7d); ib.peb hu cov qauv hauv qab no "protein rafts".Cov qauv no tseem muaj dej raws li lawv khaws lub peev xwm los sib txuas (Ntxiv daim duab 7b) thiab tuaj yeem pom tau ob peb teev tom qab LLPS tau tshwm sim (Fig. 4 thiab Ntxiv Fig. 7c).Peb tau pom tias cov txheej txheem ntub dej yog nyiam nyob rau saum npoo ntawm hydrophilic es tsis yog cov khoom siv hydrophobic (Supplementary Fig. 7a), raws li xav tau rau electrostatic coacervates nrog cov nqi tsis sib npaug thiab yog li qhov muaj peev xwm electrostatic siab.Qhov tseem ceeb, αS / ΔNt-Tau coalescence thiab rafting tau txo qis, thaum αS / pLK condensates tau txo qis (Daim duab 4).Thaum lub sij hawm luv luv incubation, αS / pLK cov tee dej tau tuaj yeem sib koom ua ke thiab ntub dej ntawm qhov dej, tab sis cov txheej txheem no sai sai thiab tom qab 5 teev ntawm incubation, tsuas yog txwv coalescence txheej xwm thiab tsis muaj ntub dej.- gel-drip kev hloov pauv.
Tus neeg sawv cev BF (greyscale panels) thiab CF (txoj cai vaj huam sib luag, AF488-labeled αS hauv ntsuab) ntawm cov qauv coacervate uas muaj 100 µM αS (1% fluorescent daim ntawv lo) hauv LLPS tsis nyob rau hauv lub xub ntiag ntawm 100 µM Tau441 (sab saum toj) fluorescence microspicence) -Tau (center) los yog 1 mM pLK (hauv qab) ntawm qhov sib txawv incubation lub sij hawm thiab focal heights (z, deb ntawm lub hauv qab ntawm lub phaj zoo).Cov kev sim tau rov ua dua 4-6 zaug ntawm nws tus kheej ntawm ib leeg nrog tib cov txiaj ntsig.αS/Tau441 coacervates yog wetted tom qab 24 teev, tsim rafts loj dua daim duab.Qhov ntsuas bar rau tag nrho cov duab yog 20 µm.
Tom qab ntawd peb nug seb puas muaj cov kua dej loj zoo li cov protein ntau tsim hauv αS/Tau441 LLPS yuav ua rau amyloid aggregation ntawm ib qho ntawm cov proteins uas tau kawm.Peb tau ua raws li kev loj hlob ntawm αS/Tau441 cov tee dej nyob rau lub sijhawm nrog WF microscopy raws li cov xwm txheej saum toj no, tab sis siv 1 μM AF488-labeled αS thiab Atto647N-labeled Tau441 (Fig. 5a).Raws li kev cia siab, peb tau pom ua tiav cov protein hauv zos thoob plaws hauv txoj kev loj hlob.Interestingly, los ntawm ca.Tom qab 5 teev, cov qauv tsis sib thooj ntau dua tau pom hauv cov rafts, uas peb hu ua "cov ntsiab lus", qee qhov tau colocalized nrog αS, thiab qee qhov tau ntxiv rau Tau441 (Fig. 5a, dawb xub).Cov chaw no ib txwm tau pom nyob rau hauv rafts kom ntau dua rau αS / ΔNt-Tau dua li αS / ΔNt-Tau.Tsis muaj qhov sib txawv ntawm cov tee dej ntawm pLK thiab Tau systems tsis muaj peev xwm rau fusion/wetting.Txhawm rau kuaj seb cov stains uas muaj αS thiab Tau441 yog cov sib xyaw zoo li amyloid, peb tau ua ib qho kev sim zoo sib xws siv CF microscopy uas Tau441 tau sau nrog Atto647N thiab 12.5 μM amyloid-specific thioflavin-T (ThT) tau ntxiv txij thaum pib.xim.Txawm hais tias ThT-staining ntawm αS/Tau441 cov tee dej los yog cov rafts tsis tau pom txawm tias tom qab 24 h ntawm incubation (Fig. 5b, sab saum toj kab-tseem tshuav cov tee dej ntawm cov rafts), ThT-zoo cov qauv uas muaj Atto647N-Tau441 hauv cov rafts tsis muaj zog heev.qhov no rov ua dua qhov loj, zoo li, thiab qhov chaw ntawm qhov chaw tau piav qhia yav dhau los (Daim duab 5b, kab nruab nrab thiab hauv qab), qhia tias cov pob yuav sib xws rau amyloid-zoo li aggregates tsim nyob rau hauv cov laus cov kua coacervates.
WF 25 μM αS ntawm ntau yam incubation lub sij hawm thiab focal heights (z, deb ntawm unbound hauv qab) nyob rau hauv lub xub ntiag ntawm 25 μM Tau441 (1 μM AF488-labeled αS thiab Atto647N-labeled Tau441) nyob rau hauv ib lub qhov dej ntawm ib tug microscope phaj nrog LLPS tsis) .Rau qhov kev sim tau rov ua dua nrog cov txiaj ntsig zoo sib xws.b CF microscopic duab ntawm 25 μM αS nyob rau hauv muaj 25 μM Tau441 (1 μM Atto647N-labeled Tau441) thiab 12.5 μM thioflavin-T (ThT).Cov kua dej uas muaj protein ntau thiab tso cov protein rafts thiab cov pob tau pom nyob rau hauv cov kab saum toj thiab nruab nrab, feem.Cov kab hauv qab qhia cov duab ntawm rafts thiab tee los ntawm 3 tus neeg ua haujlwm ywj pheej.Cov xub dawb qhia ThT-zoo dots hauv ob lub vaj huam sib luag.Qhov ntsuas bar rau tag nrho cov duab yog 20 µm.
Txhawm rau tshuaj xyuas kom meej ntxiv txog cov kev hloov pauv hauv coacervate protein network thaum lub sijhawm hloov pauv ntawm cov kua mus rau cov khoom, peb siv fluorescence lifetime imaging (FLIM) thiab Förster resonance zog hloov microscopy (FRET) (Daim duab 6 thiab Cov duab ntxiv 8 thiab 9).Peb xav tias cov coacervate maturation ntawm txheej rau hauv ib tug ntau condensed los yog ib yam khoom zoo li aggregated protein qauv ua rau kom muaj kev sib cuag ntawm cov protein thiab cov fluorescent sojntsuam txuas rau nws, muaj peev xwm ua tau ib tug quenching nyhuv tshwm sim nyob rau hauv ib tug shortened sojntsuam lub neej (τ) , raws li tau piav ua ntej 40., 41, 42 ib.Tsis tas li ntawd, rau ob daim ntawv sau ua qauv (AF488 thiab Atto647N raws li FRET pub dawb thiab txais dyes, feem), qhov kev txo qis hauv τ kuj tuaj yeem nrog coacervate condensation thiab nce hauv FRET (E) efficiency thaum LSPT.Peb tau soj ntsuam cov khoom tsim thiab qhov chaw nyob rau lub sijhawm hauv LLPS αS / Tau441 thiab αS / ΔNt-Tau cov qauv (25 µM ntawm txhua cov protein hauv LLPS tsis muaj 1 µM AF488 sau αS thiab / lossis Atto647N sau tau Tau441 lossis ΔNt-Tau).Peb tau pom ib qho kev sib txawv nyob rau hauv uas lub fluorescence lifetime ntawm AF488 (τ488) thiab Atto647N (τ647N) probes poob me ntsis raws li cov coacervates matured (Fig. 6 thiab Supplementary Fig. 8c).Qhov zoo siab, qhov kev hloov pauv no tau ua kom muaj txiaj ntsig zoo rau cov dots hauv rafts (Fig. 6c), qhia tias ntxiv cov protein condensation tshwm sim ntawm cov dots.Hauv kev txhawb nqa ntawm qhov no, tsis muaj kev hloov pauv tseem ceeb hauv fluorescence lub neej tau pom rau αS / ΔNt-Tau cov tee uas muaj hnub nyoog rau 24 h (Ntxiv Fig. 8d), qhia tias cov dej gelation yog cov txheej txheem txawv ntawm qhov pom thiab qhov ntawd tsis nrog los ntawm qhov tseem ceeb ntawm kev tsim kho molecular. hauv coacervates.Nws yuav tsum raug sau tseg tias cov dots muaj ntau qhov sib txawv thiab cov ntsiab lus sib txawv hauv αS, tshwj xeeb tshaj yog rau αS/Tau441 system (Ntxiv daim duab 8e).Qhov txo qis hauv qhov chaw fluorescence lub neej tau nrog rau kev siv zog ntxiv, tshwj xeeb tshaj yog rau Atto647N sau tias Tau441 (Cov Lus Ntxiv 8a), thiab FRET ntau dua rau ob qho tib si αS / Tau 441 thiab αS / ΔNt-Tau systems, qhia ntxiv cov xuab moos hauv FLL. Tom qab ua tiav, cov proteins hauv cov hluav taws xob static condensed.Piv rau αS / ΔNt-Tau, peb pom qis τ647N thiab me ntsis siab dua τ488 qhov tseem ceeb hauv αS / Tau441 qhov chaw, nrog rau qis dua thiab ntau dua inhomogeneous FRET qhov tseem ceeb.Tej zaum, qhov no yuav cuam tshuam rau qhov tseeb tias nyob rau hauv αS / Tau441 system, qhov pom thiab xav tias αS abundance nyob rau hauv aggregates yog ntau heterogeneous, feem ntau substoichiometric piv rau Tau, txij thaum Tau441 nws tus kheej kuj yuav undergo LLPS thiab aggregation (Supplementary Fig. 8e) .Txawm li cas los xij, qhov degree ntawm cov kua dej coalescence, raft tsim, thiab, qhov tseem ceeb, protein aggregation nyob rau hauv cov kua zoo li coacervates yog qhov siab tshaj plaws thaum ob leeg Tau441 thiab αS nyob.
Lifetime fluorescence microscopy (FLIM) images of αS/Tau441 and αS/ΔNt-Tau at 25 μM of each protein (1 μM AF488-labeled αS and 1 μM Atto647N-labeled Tau441 or ΔNt-Taubuff) in LLPSCov kab ntawv qhia cov duab sawv cev ntawm LLPS cov qauv ntawm cov sijhawm sib txawv (30 feeb, 5 teev thiab 24 teev).Cov ncej liab qhia thaj tsam uas muaj αS/Tau441 me ntsis.Lub neej ncua yog qhia raws li cov xim tuav.Scale bar = 20 µm rau tag nrho cov duab.b Zoomed-hauv FLIM duab ntawm thaj chaw xaiv, pom hauv lub thawv liab hauv vaj huam sib luag a.Lub neej ntau yam tau qhia siv tib xim nplai raws li hauv vaj huam sib luag a.Luas bar = 5 µm.c Histograms qhia AF488 (txuas ntxiv rau αS) lossis Atto647N (nrog rau Tau) rau ntau hom protein ntau (droplets-D-, raft-R- thiab speckle-P) tau txheeb xyuas hauv FLIM cov duab sau tseg rau αS-) lub sijhawm xa tawm lub neej ntawm Tau441 thiab αS/ΔNt-Tau coacervate samples (N = 17-32 ROI for D, 29-44 ROI for R, and 21-51 ROI for points).Qhov nruab nrab thiab nruab nrab qhov tseem ceeb yog pom raws li cov squares daj thiab cov kab dub hauv cov thawv, feem.Qhov qis thiab sab sauv ciam teb ntawm lub thawv sawv cev rau thawj thiab thib peb quartiles, feem, thiab qhov tsawg kawg nkaus thiab siab tshaj qhov tseem ceeb nyob rau hauv lub 1.5-fold interquartile ntau yam (IQR) yog qhia raws li whiskers.Outliers tau pom zoo li pob zeb diamond dub.Kev txheeb xyuas qhov tseem ceeb ntawm cov khub ntawm kev faib khoom tau txiav txim siab siv ob qho qauv t-test, piv txwv li qhov sib txawv tsis sib xws.Ob-tailed t-test p-values ​​yog qhia nrog asterisks rau txhua khub ntawm cov ntaub ntawv piv (* p-tus nqi> 0.01, ** p-tus nqi> 0.001, *** p-tus nqi> 0.0001, **** p-tus nqi> 0.00001), ns Qhia qhov tsis saib xyuas (p-tus nqi> 0.05).Qhov tseeb p qhov tseem ceeb tau muab rau hauv Cov Lus Ntxiv 1, thiab cov ntaub ntawv qub tau nthuav tawm raws li cov ntaub ntawv nyoos.
Txhawm rau ua kom pom qhov zoo li amyloid zoo li cov speckles/aggregates, peb tau kho cov qauv coacervate uas tsis muaj stained rau 24 teev nrog siab concentrations (1 M) NaCl, uas ua rau kev sib cais ntawm aggregates los ntawm protein coacervates.Thaum cov aggregates cais tawm (piv txwv li, ib qho kev sib cais ntawm cov aggregates) tau pom los ntawm kev siv atomic force microscopy (AFM), peb tau pom qhov feem ntau cov duab puab kheej kheej nrog qhov siab li ntawm 15 nm, uas nyiam koom nrog hauv cov xwm txheej ntawm cov ntsev ntau, zoo ib yam li. tus cwj pwm ntawm amyloid fibrils vim muaj zog hydrophobic nyhuv ntawm qhov chaw (nco ntsoov tias fibrils feem ntau muaj qhov siab ntawm ~ 10 nm) (Cov duab ntxiv. 10a).Interestingly, thaum cais aggregates raug incubated nrog ThT nyob rau hauv ib tug qauv ThT fluorescence assay, peb pom ib tug loj heev nce nyob rau hauv ThT fluorescence quantum yield, piv rau cov uas pom thaum lub dye yog incubated nrog ib txwm αS amyloid fibrils (Supplementary Fig. 10b). cov coacervate aggregates muaj cov qauv zoo li amyloid..Qhov tseeb, cov aggregates tau ua siab ntev rau siab ntsev ntau, tab sis rhiab rau 4 M guanidine chloride (GdnHCl), zoo li cov amyloid fibrils (Ntxiv daim duab 10c).
Tom ntej no, peb txheeb xyuas qhov sib xyaw ntawm cov khoom sib xyaw uas siv ib qho fluorescence molecule, tshwj xeeb fluorescence correlation / cross-correlation spectroscopy (FCS / FCCS), thiab tawg tsom xam ntawm ob-xim coincidence detection (TCCD).Txog qhov kawg no, peb cais cov aggregates tsim tom qab 24 teev ntawm incubation hauv 100 μl LLPS cov qauv uas muaj αS thiab Tau441 (ob leeg 25 μM) ua ke nrog 1 μM AF488-labeled αS thiab 1 μM Atto647N-labeled Tau441.Dilute qhov tshwm sim dispersed aggregate tov rau lub xeev monomolecular siv tib lub PEG-dawb tsis muaj thiab 1 M NaCl (tib yam tsis siv los cais cov aggregates los ntawm coacervate) los tiv thaiv kev sib cuam tshuam electrostatic ntawm LLPS thiab cov protein.Ib qho piv txwv ntawm lub sij hawm trajectory ntawm ib molecule tuaj yeem pom hauv daim duab 7a.FCCS/FCS tsom xam (kev sib raug zoo, CC thiab autocorrelation, AC) qhia tau hais tias cov aggregates uas muaj αS thiab tau muaj ntau nyob rau hauv cov qauv (saib CC nkhaus nyob rau hauv daim duab 7b, sab laug vaj huam sib luag), thiab ib tug ntau tshaj ntawm residual monomeric protein tshwm sim raws li ib tug tshwm sim. tshwm sim ntawm cov txheej txheem dilution (saib AC nkhaus hauv daim duab 7b, sab laug vaj huam sib luag).Cov kev sim tswj tau ua raws li cov kev daws teeb meem tib yam uas siv cov qauv uas tsuas muaj cov proteins monomeric pom tsis muaj CC nkhaus, thiab AC curves haum zoo nrog cov qauv sib txawv ntawm ib qho (Eq. 4), qhov twg cov proteins monomeric muaj qhov xav tau diffusion coefficients (Fig. 7b. ), txoj cai vaj huam sib luag).Lub diffusion coefficient ntawm aggregated hais yog tsawg dua 1 µm2 / s, thiab cov proteins monomeric yog li 1 µm2 / s.50-100 μm / s;cov txiaj ntsig zoo ib yam li yav tas los qhov tseem ceeb tshaj tawm rau sonicated αS amyloid fibrils thiab monomeric αS cais raws li cov kev daws teeb meem zoo sib xws44.Thaum peb txheeb xyuas cov sib sau ua ke nrog TCCD kev soj ntsuam tawg (Daim duab 7c, sab saum toj vaj huam sib luag), peb pom tias nyob rau hauv txhua qhov sib cais sib cais (αS / Tau heteroaggregate), kwv yees li 60% ntawm cov khoom kuaj pom muaj αS thiab tau, txog 30% tsuas muaj. tau, txog 10% αS nkaus xwb.Stoichiometric tsom xam ntawm αS / Tau heteroaggregates pom tias feem ntau ntawm cov heteroaggregates tau enriched nyob rau hauv tau (stoichiometry hauv qab no 0.5, qhov nruab nrab tus naj npawb ntawm tau molecules ib aggregate yog 4 npaug ntau tshaj li αS molecules), uas yog raws li peb cov hauj lwm pom nyob rau hauv FLIM nyob rau hauv qhov chaw. kev sim..FRET tsom xam pom tias cov aggregates no muaj ob qho proteins, txawm hais tias qhov tseeb FRET qhov tseem ceeb hauv qhov no tsis yog qhov tseem ceeb, txij li kev faib cov fluorophores hauv txhua qhov sib xyaw yog random vim muaj ntau tshaj ntawm cov protein tsis tau siv hauv kev sim.Interestingly, thaum peb ua tib yam kev ntsuam xyuas siv 45,46 mature amyloid aggregation-deficient Tau variant (saib Daim duab ntxiv. 11a, b), peb pom tias txawm tias αS electrostatic aggregation yog tib yam (Supplementary Fig. 11c, d), Lub peev xwm los tsim cov aggregates nyob rau hauv lub coacervate tau txo qis thiab FLIM tau kuaj pom ntau qhov chaw hauv qhov kev sim, thiab qhov tsis muaj zog cross-correlation curves raug soj ntsuam rau cov qauv sib cais.Txawm li cas los xij, rau qee qhov kev kuaj pom me me (tsuas yog ib feem kaum ntawm Tau441), peb pom tias txhua qhov sib xyaw ua ke tau nplua nuj nyob rau hauv αS dua li Tau qhov sib txawv no, nrog kwv yees li 50% ntawm cov khoom kuaj pom muaj tsuas yog αS molecules, thiab αS tau ntau dhau lawm. .aggregates (saib Daim Duab Ntxiv. 11e), sib piv rau cov sib sau ua ke uas tsim los ntawm Tau441 (Fig. 6f).Cov txiaj ntsig ntawm cov kev sim no tau pom tias txawm tias αS nws tus kheej muaj peev xwm sib sau nrog tau hauv coacervate, tau nucleation yog qhov zoo dua nyob rau hauv cov xwm txheej no, thiab cov txiaj ntsig zoo li amyloid aggregates tuaj yeem ua raws li daim ntawv ntawm αS thiab tau.Txawm li cas los xij, ib zaug tau-nplua nuj core yog tsim, heterotypic kev sib cuam tshuam ntawm αS thiab tau yog nyiam nyob rau hauv aggregates tshaj homotypic kev sib cuam tshuam ntawm tau molecules;peb kuj soj ntsuam cov tes hauj lwm protein hauv kua αS/tau coacervates.
Tus Neeg Sawv Cev fluorescence ib ntus ntawm ib qho molecules ntawm kev sib cais aggregates tsim nyob rau hauv αS/Tau441 electrostatic coacervates.Kev tawg ua ke rau αS / Tau 441 coaggregates (bursts saum toj no qhov chaw pib) tau pom nyob rau hauv peb txoj kev tshawb nrhiav (AF488 thiab Atto647N emission tom qab ncaj qha excitation, xiav thiab liab kab, Atto647N emission tom qab indirect excitation), FRET, violet kab).b FCS/FCCS tsom xam ntawm ib qho piv txwv ntawm cais αS/Tau441 aggregates tau los ntawm LLPS (sab laug vaj huam sib luag).Autocorrelation (AC) nkhaus rau AF488 thiab Atto647N tau pom hauv xiav thiab liab, feem, thiab hla kev sib raug zoo (CC) nkhaus cuam tshuam nrog cov sib sau ua ke uas muaj ob qho xim muaj xim liab.Cov AC nkhaus qhia txog qhov muaj cov cim npe monomeric thiab cov protein sib sau ua ke, thaum CC nkhaus qhia tsuas yog qhov sib txawv ntawm ob daim ntawv sau ua ke.Tib qhov kev tshuaj xyuas, tab sis nyob rau hauv tib txoj kev daws teeb meem xws li hauv cov chaw sib cais, cov qauv uas tsuas muaj monomeric αS thiab Tau441 tau pom tias yog kev tswj hwm hauv vaj huam sib luag txoj cai.c Fluorescence flash tsom ntawm ib qho molecules ntawm kev sib cais aggregates tsim nyob rau hauv αS/Tau441 electrostatic coacervates.Cov ntaub ntawv rau txhua qhov aggregate pom nyob rau hauv plaub qhov sib txawv rov ua dua (N = 152) yog npaj tawm tsam lawv cov stoichiometry, S qhov tseem ceeb, thiab FRET efficiency (sab saum toj vaj huam sib luag, xim bar qhia txog qhov tshwm sim).Peb hom aggregates tuaj yeem paub qhov txawv: -αS-tsuas yog sib sau nrog S ~ 1 thiab FRET ~ 0, Tau-tsuas aggregates nrog S ~ 0 thiab FRET ~ 1, thiab heterogeneous Tau / αS aggregates nrog nruab nrab S thiab FRET Kev kwv yees ntawm tus nqi ntawm ob qho tib si marker proteins kuaj pom nyob rau hauv txhua heterogeneous aggregate (N = 100) yog qhia nyob rau hauv lub qis vaj huam sib luag (cov xim teev qhia txog qhov tshwm sim).Cov ntaub ntawv nyoos yog muab rau hauv daim ntawv ntawm cov ntaub ntawv raw cov ntaub ntawv.
Kev loj hlob los yog kev laus ntawm cov kua protein condensates rau hauv gel-zoo li lossis cov qauv khoom nyob rau lub sijhawm tau tshaj tawm tias tau koom nrog ntau lub zog ntawm lub cev condensate47 nrog rau cov kab mob, raws li cov txheej txheem txawv txav ua ntej amyloid aggregation 7, 48, 49. peb kawm txog theem kev sib cais thiab kev coj cwj pwm kom ntxaws.LSPT αS nyob rau hauv lub xub ntiag ntawm random polycations nyob rau hauv ib tug tswj ib puag ncig ntawm tsawg micromolar concentrations thiab physiologically cuam tshuam tej yam kev mob (nco ntsoov hais tias cov xam physiological concentration ntawm αS yog> 1 µM50), raws li ib txwm thermodynamically tsav tus cwj pwm ntawm LPS.Peb pom tias αS, uas muaj qhov tsis zoo rau C-terminal cheeb tsam ntawm lub cev pH, muaj peev xwm tsim cov kua dej muaj protein ntau hauv cov kua dej ntawm LLPS hauv qhov muaj cov cationic disordered peptides xws li pLK lossis Tau los ntawm cov txheej txheem ntawm electrostatic. complex condensation nyob rau hauv lub xub ntiag ntawm aggregation macromolecules.Cov txheej txheem no yuav muaj feem cuam tshuam rau hauv ib puag ncig ntawm tes uas αS ntsib ntau yam polycationic molecules cuam tshuam nrog nws cov kab mob sib xyaw ua ke hauv vitro thiab hauv vivo51,52,53,54.
Hauv ntau cov kev tshawb fawb, protein dynamics nyob rau hauv cov tee dej tau raug suav hais tias yog ib qho ntawm cov ntsiab lus tseem ceeb uas txiav txim siab txog kev loj hlob ntawm 55,56.Hauv electrostatic αS coacervates nrog polycations, cov txheej txheem maturation pom meej nyob ntawm lub zog ntawm kev sib cuam tshuam nrog polycations, valence, thiab ntau ntau ntawm cov kev sib cuam tshuam.Equilibrium txoj kev xav qhia tias thaj chaw sib npaug ntawm ob lub xeev ua kua yuav muaj cov tee dej loj uas nplua nuj nyob hauv biopolymers uas tsav LLPS57,58.Droplet kev loj hlob tuaj yeem ua tiav los ntawm Ostwald maturation59, coalescence60 lossis noj dawb monomer nyob rau hauv dispersed phase61.Rau αS thiab Tau441, ΔNt-Tau lossis pLK, feem ntau ntawm cov protein tau concentrated nyob rau hauv cov condensate raws li cov kev siv hauv txoj kev tshawb no.Txawm li cas los xij, thaum puv loj tau cov tee dej coalesced sai sai rau ntawm qhov chaw ntub dej, cov tee dej coalescence thiab ntub dej yog qhov nyuaj rau ΔNt-Tau thiab pLK, qhia txog kev poob sai ntawm cov kua hauv ob lub cev.Raws li peb qhov kev soj ntsuam FLIM-FRET, cov laus pLK thiab ΔNt-Tau cov tee dej tau pom zoo ib yam ntawm cov protein sib sau ua ke (zoo li fluorescence lub neej) raws li cov tee dej qub, qhia tias thawj cov protein network tau khaws cia, txawm tias nruj dua.
Peb rationalize peb cov txiaj ntsig kev sim hauv cov qauv hauv qab no (Daim duab 8).Thawj cov tee ib ntus feem ntau yog cov protein sib txuas uas tsis muaj cov khoom siv hluav taws xob hluav taws xob, thiab yog li muaj cov cheeb tsam ntawm kev tsis sib haum xeeb, tshwj xeeb tshaj yog nyob rau hauv cov tee dej interface, uas ua rau cov tee dej uas muaj qhov muaj peev xwm electrostatic siab.Txhawm rau them tus nqi (ib qho tshwm sim feem ntau hu ua valence depletion) thiab txo qhov muaj peev xwm ntawm cov tee dej, cov tee dej tuaj yeem suav nrog cov polypeptides tshiab los ntawm cov theem dilute, rov txhim kho cov kev sib txuas ntawm cov protein kom ua kom zoo dua cov nqi sib cuam tshuam, thiab cuam tshuam nrog lwm cov tee dej.nrog rau qhov chaw (wetting).Cov αS / pLK cov tee dej, vim lawv txoj kev sib txuas ntawm cov protein yooj yim (tsuas yog kev sib cuam tshuam ntawm heterotypic ntawm αS thiab pLK) thiab kev sib raug zoo ntau dua rau cov protein-protein sib cuam tshuam, zoo li muaj peev xwm sib npaug ntawm tus nqi ntawm cov condensate sai dua;Tseeb tiag, peb tau pom cov protein kinetics sai dua nyob rau hauv pib tsim αS/pLK coacervates dua li hauv αS/Tau.Tom qab valence depletion, cov kev sib cuam tshuam ua rau tsis tshua muaj ephemeral thiab cov tee dej poob lawv cov khoom ua kua thiab tig mus rau hauv cov gel zoo li, cov tee dej uas tsis muaj hluav taws xob nrog lub peev xwm qis electrostatic deg (thiab yog li tsis tuaj yeem ntub dej).Hauv qhov sib piv, αS / Tau cov tee dej tsis tshua muaj txiaj ntsig ntawm kev ua kom zoo dua cov dej tshuav nyiaj li cas vim muaj kev sib txuas ntawm cov protein ntau (nrog rau kev sib cuam tshuam homotypic thiab heterotypic) thiab qhov tsis muaj zog ntawm cov protein sib cuam tshuam.Qhov no ua rau cov tee dej uas khaws cov cwj pwm ua kua rau lub sijhawm ntev thiab muaj peev xwm ua kom muaj hluav taws xob zoo heev uas zoo li txo qis los ntawm coalescing thiab loj hlob (yog li txo qhov chaw saum npoo / ntim qhov sib piv ntawm cov tee dej) thiab los ntawm ntub dej hydrophilic deg chem.Qhov no tsim cov tsev qiv ntawv loj muaj protein ntau uas khaws cov kua dej vim qhov kev sib cuam tshuam tseem nyob ntev heev vim qhov kev tshawb nrhiav tas li rau kev them nqi kho kom zoo hauv cov protein ntau.Interestingly, N-terminally truncated forms of Tau, suav nrog qee qhov tshwm sim ntawm isoforms62, nthuav tawm tus cwj pwm nruab nrab, nrog qee qhov coacervates aging nrog αS mus rau hauv lub neej ntev gel-zoo li tee dej, thaum lwm tus hloov mus rau hauv cov kua dej loj.Qhov no duality nyob rau hauv maturation ntawm αS electrostatic coacervates yog raws li tsis ntev los no LLPS theoretical thiab kev tshawb fawb soj ntsuam uas tau txheeb xyuas ib tug correlation ntawm valence depletion thiab electrostatic sieving nyob rau hauv condensates raws li tus yuam sij rau kev tswj condensate loj thiab kua zog.Mechanism 58.61.
Cov tswv yim no qhia txog kev sib sau ua ke ntawm amyloid rau αS thiab Tau441 ntawm LLPS thiab LSPT.Ntxiv nrog rau cov anion-nplua nuj (liab) thiab cation-nplua nuj (xiav) cheeb tsam, αS thiab tau electrostatic coacervates nrog txaus siab valence muaj qis zog saum npoo thiab yog li ntawd coalescence tsawg, ua rau poob sai sai.Qhov ruaj khov uas tsis yog-agglomerated gel lub xeev tau ua tiav..Qhov teeb meem no yog qhov zoo heev nyob rau hauv cov ntaub ntawv ntawm αS / pLK system vim nws cov affinity ntau dua thiab yooj yim protein-pair sib cuam tshuam network, uas tso cai rau ib tug ceev ceev gel zoo li kev hloov.Ntawm qhov tsis sib xws, cov tee dej uas tsis txaus siab valence thiab, yog li ntawd, cov cheeb tsam uas muaj protein ntau muaj rau kev sib cuam tshuam, ua rau nws yooj yim dua rau coacervate rau fuse thiab ntub dej hydrophilic nto kom txo tau nws lub zog saum npoo.Qhov xwm txheej no yog qhov zoo dua rau αS/Tau441 coacervates, uas muaj ntau qhov sib txuas ua ke uas muaj zog Tau-Tau thiab αS-Tau kev sib cuam tshuam.Nyob rau hauv lem, loj coacervates yuav yooj yim khaws lawv cov khoom zoo li kua, cia lwm cov protein-rau-protein kev sib cuam tshuam tshwm sim.Nws thiaj li, amyloid heterogeneous aggregates uas muaj ob qho tib si αS thiab tau daim ntawv nyob rau hauv cov kua coacervate, uas tej zaum yuav muaj feem xyuam rau cov uas pom nyob rau hauv lub cev suav nrog, uas yog cov cim ntawm cov kab mob neurodegenerative.
Cov dej loj zoo li cov qauv tsim thaum lub sij hawm loj hlob ntawm αS / Tau 441 nrog ib tug heev congested tab sis dynamic protein ib puag ncig thiab, rau ib tug tsawg dua, αS / ΔNt-Tau coacervates yog zoo tagnrho reservoirs rau lub nucleation ntawm protein aggregation.Peb tau pom tseeb qhov tsim ntawm cov khoom sib xyaw ua ke ntawm cov protein coacervates, feem ntau muaj αS thiab tau.Peb tau pom tias cov heteroaggregates ruaj khov los ntawm kev sib cuam tshuam uas tsis yog hluav taws xob, muaj peev xwm khi amyloid-specific ThT dyes zoo ib yam li amyloid fibrils, thiab qhov tseeb muaj qhov zoo sib xws rau ntau yam cuam tshuam.Cov αS/tau aggregates tsim los ntawm LLPS tau pom tias muaj cov khoom zoo li amyloid.Tseeb tiag, qhov sib txawv ntawm Tau tsis txaus hauv amyloid aggregation yog qhov cuam tshuam loj heev hauv kev tsim cov heterogeneous αS aggregates hauv cov kua electrostatic coacervate.Kev tsim ntawm αS / Tau 441 aggregates tau pom tsuas yog hauv cov coacervates, uas khaws cov khoom zoo li cov kua dej, thiab tsis yog, yog tias cov coacervates / droplets tsis ncav cuag lub gel.Hauv qhov kawg, qhov muaj zog ntawm kev sib cuam tshuam ntawm electrostatic thiab, vim li ntawd, qhov nruj ntawm cov protein network tiv thaiv qhov tsim nyog hloov kho ntawm cov proteins los tsim cov protein sib cuam tshuam tshiab tsim nyog rau amyloid nucleation.Txawm li cas los xij, qhov no tuaj yeem ua tiav hauv kev hloov pauv tau yooj yim dua, ua kua zoo li coacervates, uas feem ntau yuav nyob twj ywm kua thaum lawv loj tuaj.
Qhov tseeb hais tias qhov tsim ntawm aggregates nyob rau hauv lub condensed theem yog nyiam nyob rau hauv loj αS / Tau condensates tshaj nyob rau hauv me me droplets uas sai gel, qhia txog qhov tseem ceeb ntawm kev txheeb xyuas cov yam ntxwv uas tswj cov droplet coalescence.Yog li, tsis tsuas yog muaj kev nyiam rau theem kev sib cais, tab sis qhov loj ntawm cov condensate yuav tsum tau tswj kom ua haujlwm kom raug raws li kev tiv thaiv kab mob 58,61.Peb cov txiaj ntsig kuj tseem qhia txog qhov tseem ceeb ntawm qhov sib npaug ntawm LLPS thiab LSPT rau αS / Tau system.Thaum kev tsim cov tee dej tuaj yeem tiv thaiv cov amyloid aggregation los ntawm kev txo cov protein monomers muaj nyob rau hauv cov kev mob saturation, raws li tau hais nyob rau hauv lwm yam systems63,64, droplet fusion ntawm high droplet theem yuav ua rau muaj protein ntau aggregation los ntawm qeeb conformational rearrangements.protein networks..
Zuag qhia tag nrho, peb cov ntaub ntawv hais txog qhov tseem ceeb ntawm kev sib koom ua ke valence thiab kev txaus siab / tsis txaus siab kev sib cuam tshuam hauv cov tes hauj lwm poob hauv cov ntsiab lus ntawm LSPT.Tshwj xeeb, peb qhia tau hais tias tag nrho-ntev αS/Tau441 condensates muaj peev xwm ua tau zoo fuse thiab nucleate los tsim amyloid-zoo li heteroaggregates uas suav nrog ob qho tib si proteins thiab tawm tswv yim molecular mechanism raws li peb qhov kev sim.Kev sib sau ua ke ntawm ob lub proteins hauv αS / Tau kua coacervate uas peb tshaj tawm ntawm no tuaj yeem muaj feem cuam tshuam nrog kev sib koom ua ke ntawm ob lub proteins hauv kev suav nrog, uas yog cov cim ntawm tus kab mob, thiab tuaj yeem ua rau nkag siab txog kev sib raug zoo ntawm LLPS thiab amyloid aggregation, paving txoj kev rau siab heev IDP nyob rau hauv neurodegeneration.
Monomeric WT-αS, cysteine ​​​​mutants (Q24C-αS, N122C-αS) thiab ΔCt-αS variants (Δ101-140) tau qhia hauv E. coli thiab huv si raws li tau piav qhia yav dhau los.5 mM DTT tau suav nrog hauv txhua kauj ruam hauv kev ua kom huv ntawm αS cysteine ​​​​mutants los tiv thaiv kev tsim cov nyiaj disulfide.Tau441 isoform (plasmid tau los ntawm Addgene #16316), ΔNt-Tau variant (Δ1–150, tau los ntawm cloning IVA nrog primers CTTTAAGAAGGAGAATACATATGATCGCCACACCGCGG, CATATGTATATCCTCTCTTTAAAGTTAAAC) thiab Aggpur5-TCAGATAACATATGATCGCCACACCGGG, CATATGTATATCCTCTCTTTAAAGTTAAAC) thiab Aggpur5-TCAGTTAAC . coli kab lis kev cai tau loj hlob mus rau OD600 = 0.6-0.7 ntawm 37 ° C thiab 180 rpm, thiab kev qhia tau raug ntxias nrog IPTG rau 3 teev ntawm 37 ° C.Sau cov hlwb ntawm 11,500 xg rau 15 feeb ntawm 4 ° C thiab ntxuav nrog saline tsis muaj 150 mM NaCl.Tshem tawm cov pellet nyob rau hauv lysis buffer (20 ml ib 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 10 cope.Cov kauj ruam sonication tau ua rau cov dej khov nrog qhov amplitude ntawm 80% rau 10 pulses (1 min rau, 1 min tawm).Tsis txhob ntau tshaj 60 ml hauv ib qho ultrasound.E. coli lysates tau rhuab ntawm 95 ° C. rau 20 feeb, ces txias rau dej khov thiab centrifuged ntawm 127,000 × g rau 40 feeb.Lub clarified supernatant tau siv rau 3.5 kDa membrane (Spectrum ™ Thermo Fisher Scientific, UK) thiab dialyzed tawm tsam 4 L ntawm kev lim ntshav tsis (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT , PMSF 0.1 mM) rau 10 teev.A 5 ml cation txauv kem (HiTrap SPFF, Cytiva, MA, USA) tau sib npaug nrog equilibration buffer (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, SF) 0.1 mMCov tau lysate tau lim los ntawm 0.22 μm PVDF lim thiab txhaj rau hauv kem ntawm tus nqi ntws ntawm 1 ml / min.Elution tau ua tiav maj mam, tau raug eluted nrog 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).Cov feem cuam tshuam tau txheeb xyuas los ntawm SDS-PAGE, thiab txhua feem uas muaj ib pawg nrog qhov xav tau molecular hnyav ntawm tau tau muaj zog siv 10 kDa centrifuge lim thiab hloov nrog cov tsis muaj 10 mM HEPES, pH 7.4, NaCl 500 mM thiab DTT 2 mM rau Qhov kawg protein concentration yog 100 μM.Cov kua protein tau dhau los ntawm 0.22 μm PVDF lim, khov sai thiab khaws cia ntawm -80 ° C.Protein K18 tau ua siab zoo los ntawm Prof. Alberto Boffi.Lub purity ntawm kev npaj yog> 95% raws li tau lees paub los ntawm SDS-PAGE thiab MALDI-TOF / TOF.Ntau yam cysteine ​​​​tau raug sau npe nrog AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) lossis TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).tau lees paub los ntawm absorbance thiab MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau thiab K18 tau sau npe nrog haiv neeg cysteine ​​​​residues ntawm txoj haujlwm 191 thiab 322 siv Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Lub teb chaws Yelemees) ua raws li cov txheej txheem tib yam.Net nqi ib daim ntawv qhia seem rau αS thiab Tau441 tau tsim siv CIDER66.
Khoom poly-L-lysine (pLK DP 90-110 raws li NMR los ntawm tus neeg muag khoom, Alamanda Polymers Inc, Huntsville, Alabama, USA) tau yaj hauv 10 mM HEPES, 100 mM NaCl, pH 7.4 txog 10 mM concentration, txheej txheem sonicated rau 5 feeb hauv dej da dej ultrasonic thiab khaws cia ntawm -20 ° C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) thiab FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) yog dej soluble thiab dav faib nyob rau hauv LLPS tsis.Dialysis tshem tawm cov kab mob ntsev.Tom qab ntawd lawv tau lim los ntawm lub syringe lim nrog lub pore loj ntawm 0.22 μm, thiab lawv cov concentrations tau suav nrog siv lub refractometer (Mettler Toledo, Columbus, Ohio, USA).LLPS cov qauv tau npaj nyob rau hauv chav tsev kub hauv qhov kev txiav txim hauv qab no: tsis muaj thiab extrusion tau tov thiab 1 mM tris (2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) thiab sib tov ntawm 1% protease inhibitor (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).Tom qab ntawd αS thiab fused polycations (options pLK lossis Tau) raug ntxiv.Rau thioflavin-T lub sij hawm series thwmsim (ThT, Carbosynth, Compton, UK), siv tag nrho ThT concentration yog ib nrab ntawm αS concentration.Maj mam tab sis ua tib zoo sib xyaw cov qauv kom ntseeg tau tias lawv yog homogeneous.Qhov concentration ntawm txhua yam sib txawv los ntawm kev sim mus rau kev sim, raws li tau piav qhia hauv Cov Lus Qhia.Azide tau siv ntawm qhov concentration ntawm 0.02% (w / v) thaum twg lub sijhawm ntawm kev sim siab tshaj 4 teev.Rau txhua qhov kev ntsuam xyuas siv LLPS cov qauv, tso cai rau qhov sib xyaw kom sib npaug li 5 feeb ua ntej kev tshuaj xyuas.Rau kev tsom xam lub teeb pom kev zoo, 150 µl ntawm cov qauv raug thauj mus rau qhov tsis yog-binding 96-zoo microplates (µClear®, dub, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) thiab npog nrog zaj duab xis nplaum.LLPs tau saib xyuas los ntawm kev ntsuas qhov nqus ntawm 350 nm ntawm qhov nruab nrab ntawm kev daws teeb meem hauv CLARIOstar phaj nyeem ntawv (BMG Labtech, Ortenberg, Lub teb chaws Yelemees).Cov kev sim tau ua nyob rau hauv triplicate ntawm 25 ° C, thiab qhov yuam kev raug xam raws li tus qauv sib txawv ntawm qhov nruab nrab.Cov theem dilute tau suav los ntawm cov qauv centrifugation thiab SDS-PAGE gel tsom xam, thiab αS feem hauv cov theem dilute thiab cov concentrated tau suav rau hauv ntau yam kev daws teeb meem LLPS.Ib qho piv txwv 100 μl LLPS uas muaj 1 μM AF488-labeled αS tau npaj los ntawm kev sib xyaw ua ke tom qab los ntawm centrifugation ntawm 9600 × g rau 30 feeb, tom qab ntawd cov nag lossis daus feem ntau pom.Sab saum toj 50 μl ntawm cov supernatant tau siv rau cov protein kom muaj nuj nqis siv SDS-PAGE gel.Gels tau raug tshuaj xyuas nrog AF488 cov ntxaij lim dej siv ChemiDoc gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA) lossis stained nrog Coomassie stain thiab pom nrog cov lim dej tsim nyog.Cov qhabnias tau raug tshuaj xyuas siv ImageJ version 1.53i (National Institutes of Health, USA).Cov kev sim tau ua tiav hauv qhov sib npaug hauv ob qhov kev sim sib txawv nrog cov txiaj ntsig zoo sib xws.
Feem ntau, 150 μl ntawm cov qauv tau siv rau cov tsis-binding 96-zoo microplates thiab pom ntawm chav sov ntawm Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Lub teb chaws Yelemees).Rau qhov chaw sim, µ-Slide Angiogenesis daim hlau (Ibidi GmbH, Gräfelfing, Lub teb chaws Yelemees) lossis 96-zoo polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) kuj tau siv.EL6000 halogen lossis mercury hlau halide teeb tau siv los ua qhov chaw teeb pom kev zoo (rau BF / DIC thiab WF imaging, ntsig txog).Rau WF microscopy, lub hom phiaj 40x magnification huab cua (Leica Microsystems, Lub teb chaws Yelemees) tau siv los tsom lub teeb ntawm tus qauv thiab sau nws.Rau AF488 thiab ThT sau cov qauv, lim excitation thiab emission nrog cov qauv GFP lim teeb, excitation thiab emission bandpass lim, feem, 460–500 nm thiab 512–542 nm bandpass lim, thiab 495 nm dichroic daim iav.Rau cov qauv sau nrog Atto647N, txheej txheem Cy5 lim nrog excitation thiab emission bandpass lim 628-40 nm thiab 692-40 nm, raws li, thiab 660 nm dichroic iav tau siv.Rau BF thiab DIC microscopy, siv tib lub hom phiaj kev sib sau lub teeb.Lub teeb sib sau tau kaw rau ntawm lub koob yees duab Leica DFC7000 CCD (Leica Microsystems, Lub teb chaws Yelemees).Lub sij hawm raug yog 50 ms rau BF thiab DIC microscopy imaging thiab 20-100 ms rau WF microscopy imaging.Rau kev sib piv, lub sijhawm raug rau txhua qhov kev sim nrog ThT yog 100 ms.Lub sij hawm-lapse kev sim tau ua kom pom cov dej sib txuas, nrog cov duab tau sau txhua 100 ms rau ob peb feeb.ImageJ (NIH, USA) tau siv rau kev tsom xam duab.Cov kev sim tau ua tiav hauv triplicate nrog cov txiaj ntsig zoo sib xws.
Rau kev sim colocalization, FRAP thiab 3D reconstruction, dluab tau txais ntawm Zeiss LSM 880 inverted confocal microscope siv ZEN 2 xiav tsab (Carl Zeiss AG, Oberkochen, Lub teb chaws Yelemees).Cov qauv ntawm 50 µl tau siv rau µ-Slide Angiogenesis Petri cov tais diav (Ibidi GmbH, Gröfelfing, Lub teb chaws Yelemees), kho nrog hydrophilic polymer (ibiTreat) thiab mounted hauv 63 × roj immersion lub hom phiaj (Plan-Apochromat 63 × / NA 1.4 Roj) ntawm DIC).Cov duab tau txais kev siv 458 nm, 488 nm, thiab 633 nm argon laser kab nrog kev daws teeb meem ntawm 0.26 µm / pixel thiab lub sijhawm raug ntawm 8 µs / pixel rau excitation thiab emission detection windows ntawm 470-600 nm, 493-628 nm. thiab 638-755 nm tau siv los ua kom pom ThT, AF488 thiab Atto647N, feem.Rau qhov kev sim FRAP, lub sij hawm-lapse yees duab ntawm txhua tus qauv raug kaw ntawm 1 thav duab ib ob.Cov kev sim tau ua nyob rau hauv triplicate ntawm chav tsev kub nrog cov txiaj ntsig zoo sib xws.Tag nrho cov duab tau soj ntsuam siv Zen 2 xiav tsab software (Carl Zeiss AG, Oberkochen, Lub teb chaws Yelemees).Cov FRAP nkhaus tau zoo ib yam, npaj thiab haum rau cov ntaub ntawv siv / sijhawm muab rho tawm los ntawm cov duab siv Zen 2 siv OriginPro 9.1.Cov curves rov qab tau haum rau tus qauv mono-exponential los suav rau molecular diffusion nrog rau lub sij hawm ntxiv exponential kom suav rau qhov tau txais cov tshuaj dawb huv.Peb mam li xam D siv lub nominal bleaching radius thiab yav tas los txiav txim rov qab ib nrab-lub neej raws li nyob rau hauv qhov sib npaug ntawm Kang li al.5 35 ua pa.
Ib leeg cysteine ​​variants ntawm αS tau ua ke nrog 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) ntawm txoj haujlwm 24 (TEMPOL-24-αS) thiab 122 (TEMPOL-122-αS), raws.Spin Labeling Rau EPR kev sim, αS concentration tau teeb tsa ntawm 100 μM thiab PEG concentration yog 15% (w / v).Rau ntau yam kev sib sau ua ke, αS: pLK piv yog 1: 10, thaum lub αS: ΔNt-Tau thiab αS: Tau441 ratios tau khaws cia ntawm 1: 1.Rau kev sib khi titration sim nyob rau hauv qhov tsis muaj neeg coob coob, TEMPOL-122-αS tau khaws cia ntawm 50 μM thiab polycations tau titrated ntawm kev nce siab, npaj txhua yam sib cais.Kev ntsuas CW-EPR tau ua tiav siv Bruker ELEXSYS E580 X-band spectrometer nruab nrog Bruker ER4118 SPT-N1 resonator ua haujlwm ntawm microwave (SHF) zaus ntawm ~ 9.7 GHz.Qhov kub tau teem rau ntawm 25 ° C thiab tswj los ntawm cov kua nitrogen cryostat.Lub spectrum tau txais nyob rau hauv unsaturated tej yam kev mob ntawm ib tug MW hwj chim ntawm 4 mW, ib tug modulation amplitude ntawm 0.1 mT, thiab ib tug modulation zaus ntawm 100 kHz.Spectral intensities tau normalized kom tsis txhob muaj qhov sib txawv ntawm spin concentrations ntawm cov qauv thiab muaj peev xwm tig tau vim qhov seem seem ntawm txo cov neeg ua hauj lwm nyob rau hauv cov qauv uas muaj Tau441 los yog ΔNt-Tau (tam sim no nyob rau hauv thawj cov ntsiab lus protein).Qhov muab qhov tseem ceeb ntawm g tau txais los ntawm EPR spectral modeling ua tau siv Easyspin software (v. 6.0.0-dev.34) siv hauv Matlab®67.Ib / ob feem ntawm isotropic qauv tau siv los ua qauv cov ntaub ntawv.Tom qab normalizing tag nrho cov teeb liab, cov seem raug xam los ntawm kev rho tawm txhua qhov simulation los ntawm cov kev sim spectrum sib thooj.Rau kev khi titration tsom xam, qhov txheeb ze ntawm qhov thib peb band mus rau qhov thib ob band ntawm normalized EPR spectrum (IIII / III) tau siv los saib xyuas kev khi ntawm polycations rau αS.Txhawm rau kwv yees qhov tsis sib xws ntawm qhov tsis tu ncua (Kd), qhov txiaj ntsig nkhaus tau haum rau qhov kwv yees tus qauv piv txwv li n zoo tib yam thiab qhov chaw sib txuas ywj pheej.
NMR spectroscopy thwmsim tau ua tiav siv Bruker Neo 800 MHz (1H) NMR spectrometer nruab nrog lub cryoprobe thiab Z-gradient.Txhua qhov kev sim tau ua tiav siv 130–207 µM αS thiab sib thooj αS / ΔNt-Tau thiab pLK sib npaug hauv 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 thiab tau ua ntawm 15 ° C.Txhawm rau saib xyuas LPS los ntawm NMR, 10% PEG tau ntxiv rau cov qauv sib xyaw ua ntej.Cov tshuaj hloov pauv perturbation plot (Fig. 1b) qhia qhov nruab nrab 1H thiab 15N tshuaj hloov pauv.Lub αS 2D1H-15N HSQC spectra raug muab tso rau raws li txoj haujlwm dhau los (BMRB nkag #25227) thiab tau lees paub los ntawm kev sau thiab tshuaj xyuas 3D spectra ntawm HNCA, HNCO thiab CBCAcoNH.13Cα thiab 13Cβ cov tshuaj hloov pauv tau suav nrog hauv lub xub ntiag ntawm ΔNt-Tau lossis pLK los ntsuas qhov muaj peev xwm hloov pauv hauv cov qauv theem nrab piv rau αS cov tshuaj hloov pauv hauv cov ntshiab random coil conformation 68 (Cov duab ntxiv 5c).Cov nqi R1ρ tau ntsuas los ntawm kev kaw cov kev sim hsqctretf3gpsi (tau los ntawm lub tsev qiv ntawv Bruker) nrog kev ncua ntawm 8, 36, 76, 100, 156, 250, 400, thiab 800 ms, thiab cov haujlwm exponential tau hloov kho rau qhov sib txawv ntawm qhov sib txawv. sij hawm los txiav txim R1ρ thiab nws qhov kev sim tsis paub meej.
Ob-xim lub sij hawm daws teeb meem fluorescence microscopy thwmsim tau ua nyob rau hauv lub lag luam lub sij hawm daws teeb meem MT200 fluorescence confocal microscope (PicoQuant, Berlin, lub teb chaws Yelemees) nrog ib lub sij hawm-correlated ib photon suav (TCSPC) ntaus ntawv.Lub taub hau laser diode yog siv rau kev sib cuam tshuam kev sib tw (PIE), lub nqaj hla dhau ntawm ib hom waveguide thiab yog tuned rau lub zog laser ntawm 10 mus rau 100 nW rau 481 nm thiab 637 nm laser kab ntsuas tom qab ib daim iav dichroic.Qhov no ua kom muaj kev pom zoo photon suav tus nqi, zam qhov cuam tshuam ntawm photon aliasing, photobleaching thiab saturation.μ-Slide angiogenesis npog npog los yog daim hlau (Ibidi GmbH, Gräfelfing, Lub teb chaws Yelemees) tau muab tso ncaj qha rau hauv dej immersion hla Super Apochromat 60x NA 1.2 lo ntsiab muag nrog lub dab tshos kho (Olympus Life Sciences, Waltham, USA).A 488/640 nm dichroic daim iav (Semrock, Lake Forest, IL, USA) tau siv los ua lub ntsiab nqaj splitter.Unfocused hluav taws xob yog thaiv los ntawm ib lub qhov nrog ib txoj kab uas hla ntawm 50 microns, ces lub tsom teeb pom kev zoo muab faib ua 2 txoj kev nrhiav pom los ntawm ib tug 50/50 beam splitter.Bandpass emission filters (Semrock, Lake Forest, IL, USA) 520/35 rau xim ntsuab (AF488) thiab 690/70 rau xim liab (Atto647N) tau siv rau pem hauv ntej ntawm lub ntes.Ib leeg-photon avalanche diodes (SPAD) (Micro Photon Devices, Bolzano, Ltalis) tau siv los ua cov khoom ntes.Ob qhov kev sau thiab kev tshuaj xyuas tau ua tiav siv SymphoTime64 software (PicoQuant GmbH, Berlin, Lub teb chaws Yelemees).
Tsib caug microliters ntawm LLPS cov qauv tau siv rau μ-Slide angiogenesis qhov dej (Ibidi GmbH, Gräfelfing, Lub teb chaws Yelemees).Cov duab tshwm sim tau tsom mus rau 20 µm saum lub qhov dej hauv qab rau lub hom phiaj ua haujlwm zoo tshaj plaws rau kev tshem tawm cov tee dej thiab mus rau ~ 1 µm rau rafts thiab dots nrog axial daws teeb meem tsawg kawg yog 0.25 µm / pixel thiab ncua sij hawm ntawm 400 µs / pixel.Xaiv cov ntaub ntawv los ntawm kev siv qhov pib siv zog raws li qhov nruab nrab ntawm qhov teeb meem tom qab siv (PBG, txhais tau tias + 2σ) rau txhua qhov channel kom tsuas yog cov kua dej muaj protein ntau, cov rafts, lossis cov pob zeb raug xaiv, lim tawm cov keeb kwm yav dhau los ntawm cov theem tawg.Txhawm rau txheeb xyuas lub neej ntawm txhua hom (τ) ntawm txhua qhov channel (ntsuab, “g” rau AF488 thiab liab, “r” rau Atto647N), peb xaiv thaj chaw txaus siab (ROIs) uas muaj cov tee dej, cov rafts, lossis cov chaw (Cov duab ntxiv 1 ).8b) thiab muab tau los ntawm kev ua kom haum rau lawv lub neej kev lwj (τD, τR thiab τP rau cov tee dej, rafts los yog me ntsis, raws li, saib Ntxiv Fig. 8c) nyob rau hauv txhua txoj kab uas siv ib tug tail-haum tsom xam thiab ib tug ob-component decay qauv.Nruab nrab τ los ntawm τ .ROIs uas tsim tawm tsawg dhau photons rau ntau qhov kev nthuav dav tau raug tshem tawm los ntawm kev tshuaj xyuas.Qhov kev txiav tawm siv yog <104 photons rau rafts thiab dots thiab 103 rau tee.Cov tee dej muaj qhov qis dua vim tias nws tsis yooj yim kom tau txais qhov nkhaus nkhaus nrog qhov muaj txiaj ntsig ntau dua, vim tias cov tee dej hauv daim duab teb feem ntau me dua thiab tsawg dua.ROIs nrog photon suav saum toj no photon accumululation txwv (teeb rau> 500 suav/pixel) kuj tau muab pov tseg rau kev tsom xam.Sib piv qhov kev siv dag zog ntawm qhov nkhaus uas tau txais los ntawm thaj tsam ntawm kev txaus siab nrog qhov kev siv ntawm 90% ntawm qhov siab tshaj plaws (me ntsis tom qab qhov siab tshaj plaws ntawm kev lwj) txij thaum pib ntawm lub neej kev pab cuam kom ntseeg tau tias IRF cuam tshuam tsawg kawg nkaus thaum tuav tib yam rau txhua qhov kev siv dag zog. chaw txheeb ze lub sij hawm qhov rais tau soj ntsuam 25 mus rau 50 ROI rau rafts thiab me ntsis thiab 15-25 ROI rau tee, dluab xaiv los ntawm ntau tshaj 4 replicates kaw los ntawm tsawg kawg yog 3 kev sim ywj siab.Ob-tailed t-tests tau siv los ntsuas qhov sib txawv ntawm cov tsiaj lossis nruab nrab ntawm coacervate systems.Rau kev tsom xam pixel-by-pixel ntawm lub neej (τ), tag nrho cov attenuation ntawm lub neej dhau ntawm daim teb rau txhua qhov channel raug xam thiab kwv yees kwv yees li 2/3-tus qauv exponential attenuation qauv.Lub neej attenuation rau txhua pixel ces haum siv yav tas los xam τ qhov tseem ceeb, ua rau pseudocolor FLIM haum duab.Tus Tsov tus tw-haum lub neej ntev yog tib yam thoob plaws txhua cov duab ntawm tib lub channel, thiab txhua qhov kev lwj ua kom muaj cov duab txaus txaus los muab qhov haum zoo.Rau kev tsom xam FRET, pixels raug xaiv los ntawm kev siv lub zog qis dua ntawm 100 photons, uas nruab nrab lub teeb liab tom qab (FBG) ntawm 11 photons.Qhov kev siv fluorescence ntawm txhua channel raug kho los ntawm kev sim txiav txim siab kho yam tseem ceeb: 69 spectral crosstalk α yog 0.004, direct excitation β yog 0.0305, nrhiav kom tau efficiency γ yog 0.517.Qhov kev ua tau zoo ntawm FRET ntawm qib pixel yog suav nrog kev sib npaug hauv qab no:
qhov twg FDD yog qhov kev siv fluorescence pom nyob rau hauv tus neeg pub dawb (ntsuab) channel, FDA yog qhov kev siv tshuaj fluorescence pom nyob rau hauv tus neeg txais (liab) channel nyob rau hauv kev tsis ncaj qha excitation, thiab FAA yog qhov fluorescence siv pom nyob rau hauv tus txais (liab) channel nyob rau hauv ncaj qha excitation ( PIB).Fluorescence siv pulses tau pom hauv cov channel).
Muab 100 µl ntawm LLPS cov tshuaj tiv thaiv kev daws teeb meem uas muaj 25 µM unlabeled monomeric Tau441 (nrog lossis tsis muaj 25 µM αS) hauv LLPS tsis (supplemented li saum toj no) ntawm non-binding 96-zoo microplates nrog cov nplaum nplaum txheej thiab cov xau tsim tau kuaj xyuas los ntawm WF kev sib npaug.hauv 10 min.Tom qab 48 teev ntawm incubation nyob rau hauv chav tsev kub, lub xub ntiag ntawm protein rafts thiab me ntsis tau lees paub.Tom qab ntawd ua tib zoo tshem tawm cov kua dej hla cov rafts los ntawm qhov dej, tom qab ntawd ntxiv 50 L ntawm qhov tsis sib haum xeeb (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) thiab incubate rau 10 min.Cov ntsev ntsev ntau ua kom ntseeg tau tias LLPS yuav tsis rov ua dua vim qhov seem PEG, thiab cov khoom sib xyaw ua ke ntawm cov protein uas tsim los ntawm kev sib cuam tshuam electrostatic nkaus xwb yuav raug tshem tawm.Hauv qab ntawm lub qhov dej tau ua tib zoo khawb tawm nrog lub micropipette taub thiab cov txiaj ntsig daws tau raug xa mus rau qhov chaw pom qhov khoob.Tom qab incubation ntawm cov qauv nrog 50 μM ThT rau 1 h, lub xub ntiag ntawm cais me ntsis tau soj ntsuam los ntawm WF microscopy.Npaj sonicated αS fibrils los ntawm incubating 300 µl ntawm 70-µM αS tov nyob rau hauv PBS nrog pH 7.4, sodium azide 0.01% ntawm 37 ° C thiab 200 rpm ntawm lub orbital shaker rau 7 hnub.Cov tshuaj yog ces centrifuged ntawm 9600 × g rau 30 min, lub pellet tau resuspended nyob rau hauv PBS pH 7.4 thiab sonicated (1 min, 50% voj voog, 80% amplitude nyob rau hauv Vibra-Cell VC130 sonicator, Sonics, Newton, USA) fibril qauv nrog ib tug kuj zoo sib xws loj faib ntawm me me fibrils.
FCS/FCCS tsom xam thiab ob-xim coincidence detection (TCCD) tau ua nyob rau tib lub sijhawm MT200 daws teeb meem fluorescent confocal microscope (Pico-Quant, Berlin, Germany) siv rau FLIM-FRET microscopy thwmsim siv PIE hom.Lub zog laser rau cov kev sim no tau ntxiv rau 6.0 µW (481 nm) thiab 6.2 µW (637 nm).Kev sib xyaw ua ke ntawm cov laser fais fab no tau raug xaiv los tsim qhov ci zoo sib xws rau cov khub ntawm fluorophores siv thaum ua tiav cov txiaj ntsig zoo thiab tsis txhob photobleaching thiab saturation.Ob qho kev sau thiab kev tshuaj xyuas tau ua tiav siv cov khoom lag luam muaj SymphoTime64 version 2.3 software (PicoQuant, Berlin, Lub teb chaws Yelemees).
Cov qauv ntawm cov αS/Tau aggregates cais tau siv LLPS yog diluted nyob rau hauv kev sib cais tsis mus rau qhov tsim nyog monomolecular concentration (feem ntau yog 1: 500 dilution, txij li cov aggregates twb tsis muaj concentrations thaum cais los ntawm coacervate kuaj).Cov qauv raug siv ncaj qha rau cov ntawv npog (Corning, USA) precoated nrog cov tshuaj BSA ntawm qhov concentration ntawm 1 mg / mL.
Rau kev tsom xam PIE-smFRET hauv cov kab ntsuab thiab liab, qhov qis qis ntawm 25 photons tau siv los lim tawm cov teeb liab siv tsawg los ntawm cov xwm txheej monomeric (nco ntsoov tias monomers ntau dua cov qauv sib xyaw piv rau cov sib cais sib cais).Qhov chaw pib no tau suav tias yog tsib npaug ntawm qhov nruab nrab siv ntawm monomeric αS tau los ntawm kev tshuaj xyuas ntawm cov qauv ntshiab monomer txhawm rau txhawm rau xaiv cov sib sau ua ke rau kev tshuaj xyuas.Lub PIE tsav Circuit Court, ua ke nrog TSCPC cov ntaub ntawv tau, tau ua rau daim ntawv thov ntawm lub neej hnyav lim uas pab tshem tawm keeb kwm yav dhau thiab spectral crosstalk.Qhov kev siv hluav taws xob tau xaiv los ntawm cov theem saum toj no tau raug kho siv qhov nruab nrab keeb kwm yav dhau los txiav txim siab los ntawm cov histograms ntawm qhov tshwm sim piv rau qhov siv / hauv qhov ntawm cov qauv tsis-tsuas yog.Kev tawg cuam tshuam nrog cov aggregates loj feem ntau nyob hauv ob peb qhov sib law liag hauv lub sijhawm taug qab (teem rau 1 ms).Nyob rau hauv cov ntaub ntawv no, ib tug bin ntawm lub siab tshaj plaws lub zog raug xaiv.Rau FRET thiab stoichiometric tsom xam, theoretically txiav txim siab gamma yam γ (0.517) tau siv.Spectral crosstalk thiab ncaj qha excitation contributions yog negligible (txiav txim siab sim) ntawm excitation laser zog siv.Kev ua tau zoo thiab stoichiometry ntawm FRET hauv kev tawg yog suav raws li hauv qab no.