347 12.7 * 1.24mm igwe anaghị agba nchara tubing, Molecular usoro nke synchronous electrostatic condensation na coaggregation nke α-synuclein na tau

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347 Igwe anaghị agba nchara nkọwapụta

347 12.7 * 1.24mm igwe anaghị agba nchara tubing

Dayameta dị n'èzí: 6.00 mm OD ruo 914.4 mm OD, nha ruo 24 "NB dị Ex-stock, OD Size Steel Tubes dị Ex-stock

SS 347 Ọkpụrụkpụ Ọkpụrụkpụ: 0.3mm - 50 mm, SCH 5, SCH10, SCH 40, SCH 80, SCH 80S, SCH 160, SCH XXS, SCH XS
WT: SCH5S, SCH10S, SCH40S, SCH80S, SCH160S, wdg (0.5-12mm) ma ọ bụ na-abụghị oge nha ka ahaziri dị ka achọrọ.

Ụdị: SS 347 Ọkụ na-enweghị ntụpọ |SS 347 ERW ọkpọkọ |SS 347 Welded ọkpọkọ |SS 347 ọkpọkọ emepụtara |SS 347 CDW Tubes, Ọkụ LSAW / Seam-welded / Redrawn

Ụdị: SS 347 Round Pipes / tubes, SS 347 Square Pipes / Tubes, SS 347 Pipe / tubes, SS 347 Coiled tubes, SS 347 "U" Ụdị, SS 347 Pan Cake Coils, SS 347 Hydraulic Tubes

Ogologo: Otu Random, Ugboro abụọ Random & Ọgwụgwụ Ogologo achọrọ: Ọgwụgwụ Larịị, Ọgwụgwụ gbagoro agbagọ, Azọkwasịrị

Nchedo ngwụcha: Okpu plastik |N'èzí imecha: 2B, No.4, No.1, No.8 mirror imecha maka igwe anaghị agba nchara Pipes, Mechaa dị ka kwa ahịa chọrọ

Ọnọdụ nnyefe: Annealed na pickled, a na-egbu maramara, na-egbuke egbuke, adọtara oyi

Nyocha, Akụkọ Ule: Asambodo Nnwale Mill, EN 10204 3.1, Akụkọ Chemical, Akụkọ Mechanical, Akụkọ Nnwale PMI, Akụkọ Nlebaanya Anya, Akụkọ Nlele Ndị Ọzọ, Akụkọ Nleba Nleba A kwadoro NABL, Akuko Ule Na-emebi emebi, Akụkọ Nnwale na-adịghị emebi emebi.

Mbukota: Akwajuru n'ime igbe osisi, akpa rọba, akwa akwa akwa, ma ọ bụ dị ka arịrịọ ndị ahịa si dị.

Ọpụrụiche: nha na nkọwapụta ndị ọzọ karịa n'elu nwere ike ịmepụta na arịrịọ

Oke SS 347 Pipe nha: 1/2 inch NB, OD ruo 24 inch

ASTM A312 347: Ọkpụkpọ ọkpọkọ austenitic na-enweghị nkebi na kwụ ọtọ, nke ezubere maka oke okpomọkụ na ọrụ mmebi niile.A naghị anabata ígwè ndochi n'oge ịgbado ọkụ.

ASTM A358 347 Ngwakọta eletrik welded austenitic ọkpọkọ maka ọrụ corrosive na/ma ọ bụ ọrụ okpomọkụ dị elu.A na-emepụta naanị ọkpọkọ ruo inch asatọ na nkọwapụta a.A na-anabata mgbakwunye nke metal filler n'oge ịgbado ọkụ.

ASTM A790 347: enweghị nkebi na kwụ ọtọ welded ferritic/austenitic (duplex) ọkpọkọ echere maka ọrụ corrosive izugbe, na-emesi ike na nguzogide nrụgide corrosion.

ASTM A409 347: Igwe kwụ ọtọ ma ọ bụ spiral-seam electric fusion welded nnukwu dayameta austenitic ọkụ-wall ọkpọkọ na nha 14 "ruo 30" na mgbidi Sch5S na Sch 10S maka corrosive na / ma ọ bụ elu.

ASTM A376 347 ọkpọkọ austenitic enweghị nkebi maka ngwa okpomọkụ dị elu.

ASTM A813 347: Otu onuete, otu-ma ọ bụ abụọ-welded austenitic ọkpọkọ maka oke okpomọkụ na ngwa na-emebi emebi.

ASTM A814 347 Oyi na-arụ ọrụ welded austenitic ọkpọkọ maka oke okpomọkụ na ọrụ corrosive n'ozuzu.

Ihe mejupụtara kemịkalụ 347H igwe anaghị agba nchara

Igwe anaghị agba nchara 347H Pipe Mechanical Properties

Igwe anaghị agba nchara 347H Pipes Njirimara

Ihe akara akara maka 347H igwe anaghị agba nchara

Amyloid alpha-synuclein (αS) aggregation bụ akara nke ọrịa Parkinson na ndị ọzọ synucleinopathies.N'oge na-adịbeghị anya, protein tau nke na-ejikọta ya na ọrịa Alzheimer na-ejikọta ya na αS pathology wee chọpụta na ọ na-ejikọta ya na αS-ọgaranya inclusions, ọ bụ ezie na usoro molecular nke nchịkọta nke protein abụọ ahụ ka edoghị anya.Anyị na-akọ ebe a na usoro αS na-ekewa n'ime condensates mmiri site na condensation mgbagwoju anya electrostatic nwere ebubo polypeptides nke ọma dị ka tau.Dabere na njikọ nke αS maka polycations na ọnụego nke valence depletion nke netwọk coagulation, clots na-enweta ngwa ngwa gelation ma ọ bụ coalescence na-esote ngwa ngwa amyloid aggregation.Site na ijikọta otu ụlọ nke usoro biophysical dị elu, anyị nwere ike ịkọwa nkewa nkewa mmiri-mmiri αS/Tau ma chọpụta ihe ndị bụ isi na-eduga n'ịmepụta mkpokọta dị iche iche nke nwere protein abụọ na protein condensate mmiri.
Na mgbakwunye na akụkụ akpụkpọ ahụ, enwere ike nweta nkewa oghere n'ime sel site na nguzobe nke protein bara ụba, mmiri mmiri dị ka ahụ siri ike nke a na-akpọ biomolecular condensates ma ọ bụ ụmụ irighiri mmiri, site na usoro a maara dị ka nkewa mmiri-mmiri mmiri (LLPS).A na-etolite ụmụ irighiri mmiri ndị a site na mmekọrịta dị ogologo oge, na-abụkarị n'etiti protein ma ọ bụ protein na RNA, ma na-arụ ọrụ dị iche iche n'ihe fọrọ nke nta ka ọ bụrụ usoro ndụ niile.Ọnụ ọgụgụ dị ukwuu nke protein ndị nwere ike LLP na-egosipụta usoro nke mgbagwoju anya dị ala nke na-enwe mgbagwoju anya na ọdịdị na nhazi nke condensates biomolecular3,4,5.Ọtụtụ nchọpụta nnwale ekpughere mgbanwe, na-enwekarị ọgba aghara, na ọdịdị dị iche iche nke protein ndị na-emejupụta mmiri mmiri ndị a dị ka condensates, ọ bụ ezie na a maghị obere ihe gbasara ihe ndị na-achọpụta ihe dị iche iche nke na-achịkwa uto na maturation nke condensates ndị a ka ọ bụrụ ihe siri ike karị. steeti..
Ihe omuma ohuru a na-akwado echiche nke LLPS na-akpata protein na-adighi nma na ngbanwe nke umu mmiri n'ime owuwu siri ike nwere ike bu uzo cellular bara uru nke na-eduga n'inwe nchikota nsi na-adighi ike nke na-abụkarị akara nke ọrịa mmebi.Ọtụtụ LLPS-metụtara intrinsically disordered proteins (IDPs), na-enwekarị nnukwu ebubo na mgbanwe, na ogologo oge ejikọrọ na neurodegeneration site na usoro nke amyloid aggregation.Karịsịa, biomolecular IDP condensates dị ka FUS7 ma ọ bụ TDP-438 ma ọ bụ protein ndị nwere nnukwu mgbagwoju anya ngalaba dị ka hnRNPA19 egosila na ha na-etolite n'ime gel-dị ka ma ọ bụ ọbụna ụdị siri ike site na usoro a na-akpọ fluidization.ngwakọta.na mgbanwe usoro siri ike (LSPT) dị ka ọrụ nke oge ma ọ bụ na nzaghachi na ụfọdụ mgbanwe ntụgharị okwu ma ọ bụ mgbanwe dị ịrịba ama nke pathologically1,7.
IDP ọzọ jikọtara ya na LLPS na vivo bụ Tau, protein na-akpata microtubule na-ejikọta ya na amyloid aggregation na-emetụta na ọrịa Alzheimer10 ma na nso nso a metụtara ọrịa Parkinson (PD) na synaptic nuclear proteinopathies 11, 12, 13 metụtara.E gosiputara Tau ka ọ kewapụ onwe ya na ngwọta/cytoplasm n'ihi mmekọrịta dị mma nke electrostatic14, na-eme ka e guzobe ụmụ irighiri mmiri nke tau na-eme ka a mara dị ka electrostatic coacervates.A chọpụtakwara na ụdị mmekọrịta a na-enweghị isi bụ ihe na-akpata ọtụtụ condensates biomolecular na ọdịdị15.N'ihe banyere protein tau, enwere ike ịmepụta nchịkọta electrostatic site na nchịkọta dị mfe, nke mpaghara protein ndị na-emegiderịta onwe ha na-ebute usoro nkwụsị, ma ọ bụ site na nchịkọta mgbagwoju anya site na mmekọrịta ya na polymers ebubo ebubo dị ka RNA.
N'oge na-adịbeghị anya, α-synuclein (αS), amyloid IDP metụtara na PD na ọrịa ndị ọzọ na-akpata neurodegenerative nke a na-akpọ synucleinopathy17,18, egosipụtara na cellular na ụdị anụmanụ19,20 na-etinye uche na protein condensates na omume yiri mmiri.Ọmụmụ ihe ọmụmụ vitro egosila na αS na-enweta LLPS site na nchịkọta dị mfe site na mmekọrịta hydrophobic kachasị, ọ bụ ezie na usoro a chọrọ ntinye protein dị elu nke ukwuu yana oge ntinye ogologo oge19,21.Ma condensates nwere αS hụrụ na vivo bụ nke a ma ọ bụ usoro LLPS ndị ọzọ ka bụ isi okwu edozibeghị.N'otu aka ahụ, ọ bụ ezie na αS amyloid aggregation ka achọpụtara na neurons na PD na synucleinopathies ndị ọzọ, kpọmkwem usoro nke αS na-enweta nchịkọta amyloid intracellular na-anọgide na-edoghị anya, n'ihi na oke oke nke protein a apụtaghị na ọ na-ebute usoro a n'onwe ya.A na-achọkarị mmebi cellular ọzọ, na-atụ aro na a chọrọ ụfọdụ ebe cellular ma ọ bụ microenvironments maka mweghachi nke intracellular αS amyloid mgbakọ.Otu gburugburu cellular nke na-adịkarị mfe nchịkọta nwere ike ịbụ n'ime ime protein condensates 23.
N'ụzọ na-akpali mmasị, αS na tau achọpụtala na-ejikọta na njirimara ọrịa inclusions na ụmụ mmadụ na ọrịa Parkinson na ndị ọzọ synucleinopathies 24,25 na nnwale akọwo a synergistic pathological mmekọrịta n'etiti abụọ proteins 26,27 na-atụ aro a nwere mmekọrịta dị n'etiti nchịkọta αS na na ọrịa neurodegenerative.ọrịa.Achọpụtala αS na tau ka ha na-emekọrịta ma na-akwalite nchịkọta nke onye ọ bụla na vitro na na vivo 28,29 na nchịkọta dị iche iche nke mejupụtara protein abụọ a ka achọpụtara na ụbụrụ ndị ọrịa na synucleinopathies 30.Otú ọ dị, a maghị ntakịrị ihe gbasara ntọala molekụla nke mmekọrịta dị n'etiti αS na tau na usoro nke nchịkọta ya.A kọwala αS ka ya na tau na-emekọrịta ihe site na mma electrostatic n'etiti mpaghara C-terminal nke αS nke ebubo ebubo na-adịghị mma na mpaghara proline bara ụba nke tau, bụ nke na-emewanyewanye nke ọma na ihe fọdụrụ.
N'ime ọmụmụ ihe a, anyị na-egosi na αS nwere ike n'ezie dissociate n'ime ụmụ irighiri mmiri site electrostatic mgbagwoju condensation na ọnụnọ nke tau protein, dị iche na ya mmekọrịta ya na ndị ọzọ mma ebubo polypeptides dị ka poly-L-lysine (pLK), na na usoro a.αS na-arụ ọrụ dị ka molekul scaffold maka netwọk droplet.Anyị achọpụtala ọdịiche dị ịrịba ama na usoro maturation nke electrostatic αS coacervates, nke jikọtara ya na ọdịiche dị na valency na ike nke mmekọrịta nke protein na-etinye aka na netwọk coacervate.N'ụzọ na-akpali mmasị, anyị hụrụ nchikota nke αS na tau amyloid proteins na ogologo ndụ mmiri mmiri coacervates wee chọpụta ụfọdụ isi ihe na-eduga na-ịchịkọta nke abụọ ndị a proteins na ndị dị otú ahụ coacervates.N'ebe a, anyị na-akọwa n'ụzọ zuru ezu usoro a, nke bụ usoro ihe omimi nke nwere ike ime ka colocalization nke protein abụọ na-agụnye ọrịa kpọmkwem.
αS nwere ọdụ ọdụ C-terminal nke ukwuu na pH na-anọpụ iche (Fig. 1a), anyị chere na ọ nwere ike ịnweta LLPS site na condensation nke ogige electrostatic na polycationic disordered polypeptide molecules.Anyị na-eji 100-residue poly-L-lysine (pLK) dị ka ihe nlereanya mmalite n'ihi ọdịdị polymeric ya nke ọma na nke na-adịghị mma na pH 32 na-anọpụ iche. Nke mbụ, anyị kwadoro na pLK na-emekọrịta na ngalaba Ct nke αS site na Ngwọta NMR spectroscopy. (Foto 1b) na-eji 13C/15N-akara αS n'ebe ọnụnọ αS na-abawanye: pLK molar ratios.Mmekọrịta nke pLK na Ct-ngalaba nke αS na-egosipụta onwe ya na nhụsianya nke mgbanwe kemịkalụ na mbelata n'ogo kacha elu na mpaghara protein a.N'ụzọ na-akpali mmasị, mgbe anyị gwakọtara αS na pLK na mkpokọta αS nke ihe ruru.5-25 µM n'ihu polyethylene glycol (5-15% PEG-8) (ihe nchekwa LLPS a na-ahụkarị: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) anyị gafere ozugbo n'ọhịa sara mbara nke nhazi protein. .A na-ahụ ụmụ irighiri mmiri site na iji fluorescence (WF) na-egbuke egbuke (BF) microscopy (Fig 1c).1-5 µm ụmụ irighiri mmiri nwere αS gbanyere mkpọrọgwụ (agbakwunyere 1 µM AlexaFluor488-labeled αS, AF488-αS), akụrụngwa elektrọn ha nwere ike nweta site na nguzogide ha na 10% 1,6-hexanediol (1,6-HD) na uche ya na ya. mmụba na ntinye uche NaCl (Fig 1c).A na-egosipụta ọdịdị nke mmiri dị ka coacervates nke αS/pLK electrostatic complex site n'ikike ha na-ejikọta n'ime milliseconds (Fig 1d).N'iji turbidimetry, anyị na-atụle nhazi nke ụmụ irighiri mmiri n'okpuru ọnọdụ ndị a, kwadoro ọdịdị electrostatic nke isi mmekọrịta ya na nkwụsi ike ya (Fig 1e), ma nyochaa mmetụta nke ọnụọgụ polymer dị iche iche na usoro LLPS (Fig 1f).Ọ bụ ezie na a na-ahụ nhazi droplet n'ọtụtụ ọnụọgụ polymer, usoro a dị mma nke ukwuu mgbe pLK karịrị αS.A hụkwara LLPs site na iji dextran-70 (70kDa) na-atụgharị kemịkalụ dị iche iche ma ọ bụ na-eji ụdị nlele dị iche iche, gụnyere mmiri mmịfe iko, olulu mmiri microplate nke ihe dị iche iche, Eppendorf ma ọ bụ quartz capillaries.
Ihe nnochi anya nke mpaghara protein dị iche iche na WT-αS na ΔCt-αS dị iche iche ejiri mee ihe n'ọmụmụ ihe a.A na-egosipụta ngalaba amphipathic N-terminal, mpaghara hydrophobic amyloid-forming (NAC), na ngalaba C-terminal na-adịghị mma na-egosi na-acha anụnụ anụnụ, oroma, na uhie, n'otu n'otu.Egosiri maapụ Net Charge Per Residual (NCPR) nke WT-αS.b NMR nyocha nke mmekọrịta αS / pLK na-anọghị na clumps macromolecular.Ka pLK ntinye uche na-abawanye (αS: pLK molar ratios nke 1:0.5, 1:1.5, na 1:10 na-egosi na ìhè akwụkwọ ndụ akwụkwọ ndụ, akwụkwọ ndụ akwụkwọ ndụ, na ọchịchịrị akwụkwọ ndụ akwụkwọ ndụ, n'otu n'otu).c Coacervate αS/pLK (molar ratio 1:10) na 25 µM (1 µM AF488-labeled αS ma ọ bụ Atto647N-labeled pLK maka WF imaging) na LLPS buffer (n'elu) ma ọ bụ gbakwunyere na 500 mm NaCl (n'okpuru 1) % 1,6-hexanediol (1,6-HD; ala aka nri).Ogwe ọnụ ọgụgụ = 20µm.d Ihe nnochite anya ihe onyonyo nke BF droplet fusion nke αS/pLK (molar ratio 1:10) na ntinye nke 25 μM;Àkú na-egosi nchikota nke mwụda onye ọ bụla (akụ uhie na odo odo) n'ime dobe ọhụrụ (akụ oroma) n'ime 200 ms) .Ogwe ọnụ ọgụgụ = 20µm.e Mgbasa ọkụ (na 350 nm) αS / pLK nchịkọta na LLPS buffer tupu na mgbe mgbakwunye nke 500 mM NaCl ma ọ bụ 10% 1,6-HD na 25 µM αS (N = 3 sample replicates, pụtara na ọkọlọtọ egosikwa).f BF oyiyi (n'elu) na nyocha mgbasa ozi ọkụ (na 350 nm, ala) nke nchịkọta αS / pLK na 25 μM αS na-abawanye αS: pLK molar ratio (N = 3 sample replicates, mean and standard deviation also gosiri).Ogwe ọnụ ọgụgụ = 10µm.Ogwe ihe ọ̀tụ̀tụ̀ dị n’otu foto na-egosi ọ̀tụ̀tụ̀ onyonyo niile dị n’otu panel.A na-enye data raw n'ụdị faịlụ data raw.
Dabere na nyocha anyị nke αS/pLK electrostatic complex condensation na nyocha gara aga nke αS dị ka molekul ndị ahịa nke tau / RNA condensate site na mmekọrịta ya na tau31, anyị chere na αS na tau nwere ike jikọta ya na ihe mgbaze na enweghị RNA. condensation.site na ogige electrostatic, na αS bụ protein scaffold na αS/Tau coacervates (lee tau charge nkesa na Figure 2e).Anyị hụrụ na mgbe 10 μM αS na 10 μM Tau441 (nwere 1 μM AF488-αS na 1 μM Atto647N-Tau, n'otu n'otu) jikọtara ọnụ na nchekwa LLPS, ha na-etolite ngwa ngwa nchịkọta protein nwere protein abụọ ahụ, dị ka WF microscopy hụrụ.(Fig 2a).A kwadoro colocalization nke protein abụọ dị na ụmụ irighiri mmiri site na confocal (CF) microscopy (Mgbakwunye Fig. 1a).A hụrụ omume yiri nke ahụ mgbe ejiri dextran-70 mee ihe dị ka onye nchịkọta nchịkọta (Fig 1c).N'iji ma ọ bụ FITC akara PEG ma ọ bụ dextran, anyị chọpụtara na a na-ekesa ndị mmadụ abụọ na-eme mkpọtụ n'ofe sample, na-egosi na ọ bụghị nkewa ma ọ bụ mkpakọrịta (Mgbakwunye 1d).Kama, ọ na-atụ aro na n'ime usoro a, ha na-akwalite nkewa nke usoro site na mmetụta macromolecular igwe mmadụ, ebe PEG bụ onye na-ahụ maka igwe mmadụ kwụsiri ike, dị ka a hụrụ na sistemụ LLP ndị ọzọ33,34.Mmụba mmiri ndị a bara ụba na protein nwere mmetụta na NaCl (1 M) mana ọ bụghị na 1,6-HD (10% v / v), na-akwado njirimara electrostatic ha (Foto mgbakwunye 2a, b).Ekwenyere omume mmiri ha site na ikiri ihe omume droplet jikọrọ ọnụ nke millisecond site na iji microscopy BF (Fig 2b).
ihe onyonyo Confocal (CF) nke αS/Tau441 na-akwado na nchekwa LLPS (10 μM nke protein ọ bụla, 0.5 μM nke AF488-labeled αS na Atto647N-labeled Tau441).b Ihe nnochi anya nnyonye anya di iche (DIC) onyonyo nke mmemme αS/Tau441 droplet fusion (10 μM maka protein ọ bụla).c Usoro eserese nke dabere na ịgbasa ọkụ (na 350 nm) nke Tau441 LLPS (0-15 µM) na enweghị (aka ekpe) ma ọ bụ ọnụnọ (nri) nke 50 µM αS.Agba na-ekpo ọkụ na-egosi mgbasawanye karịa.d Mgbasa ọkụ nke αS / Tau441 LLPS sample na-abawanye ụba αS (Tau441 na 5 µM, N = 2-3 nlele ugboro ugboro dị ka egosiri).e Ngosipụta atụmatụ nke ụfọdụ ụdị protein tau na mpaghara dị iche iche nke protein a na-eji n'ọmụmụ ihe a: ebubo N-terminal ngalaba (red), mpaghara proline bara ụba (acha anụnụ anụnụ), ngalaba microtubule-binding (MTBD, nke e mere na oroma), na amyloid-akpụ ụzọ okirikiri.mpaghara filament (PHF) dị n'ime MTBD (isi awọ).Egosiri maapụ Net Charge Per Residue (NCPR) nke Tau441.f Iji 1 µM AF488-labeled αS na Atto647N-labeled ΔNt-, na-eji 1 µM AF488-labeled αS ma ọ bụ ΔCt-αS n'ihu ΔNt-Tau (n'elu, 10 µM kwa protein) ma ọ bụ K18 (n'okpuru, 5M kwa protein). ))) micrographs nke WF condensed na LLPS ma ọ bụ K18 nchekwa.Ogwe ihe ọ̀tụ̀tụ̀ dị n'otu onyonyo na-anọchi anya ọ̀tụ̀tụ̀ onyonyo niile dị n'otu panel (20µm maka akụkụ a, b na f).A na-enye data raw maka panel c na d dị ka faịlụ data raw.
Iji nwalee ọrụ αS na usoro LLPS a, anyị buru ụzọ nyochaa mmetụta αS na nkwụsi ike nke droplet site na nephelometry na-eji mmụba nke NaCl (Fig 2c).N'ihe dị elu na ntinye nnu dị na ihe atụ ndị nwere αS, nke a na-eme ka ụkpụrụ ịgbasa ọkụ dị elu (na 350 nm), nke na-egosi ọrụ nkwụsị nke αS na usoro LLPS a.Enwere ike ịhụ mmetụta yiri nke ahụ site n'ịba ụba nke αS (na ya mere αS:Tau441 ratio) ruo ihe ruru.Mmụba okpukpu 10 n'ihe gbasara mkpokọta tau (5 µM) (Fig. 2d).Iji gosi na αS bụ protein scaffold na coacervates, anyị kpebiri iji nyochaa omume nke LLPS-disrupted Tau mutant, nke na-enweghị mpaghara N-terminal ebubo na-ezighị ezi (ihe fọdụrụ 1-150, lee Fig. 2e) nke a na-akpọ ΔNt-Tau.WF microscopy na nephelometry kwadoro na ΔNt-Tau n'onwe ya emeghị LLPS (Fig. 2f na Fig. 2d), dị ka a kọrọ na mbụ 14. Otú ọ dị, mgbe αS agbakwunyere na ngwọta mgbasa ozi nke ụdị Tau a truncated, usoro LLPS bụ kpamkpam. eweghachiri ya na njupụta droplet dị nso na njupụta droplet nke ngwọta zuru oke nke Tau na αS n'okpuru ọnọdụ ndị yiri ya na ntinye protein.A pụkwara ịhụ usoro a n'okpuru ọnọdụ nke mkpokọta macromolecular dị ala (Mgbakwunye Fig. 2c).Ọrụ nke mpaghara C-terminal αS, ma ọ bụghị ogologo ya dum, na usoro LLPS gosipụtara site na mgbochi nke nhazi droplet site na iji C-terminal truncated αS variant enweghị ihe fọdụrụ 104-140 (Fig. 1a) nke (ΔCt- αS) protein (Fig. 2f na Mgbakwunye Fig. 2d).Ekwenyere na colocalization nke αS na ΔNt-Tau site na confocal fluorescence microscopy (Mgbakwunye Fig. 1b).
Iji nwalee usoro LLPS n'etiti Tau441 na αS, a na-eji ihe ọzọ Tau variant mee ihe, ya bụ peried helical filament core (PHF) na microtubule-binding domain (MTBD), nke ma ọ bụrụ na ọ nwere ngalaba nkwụghachi njirimara anọ, nke a na-akpọkarị. dị ka iberibe K18 (lee fig. 2e).A kọwapụtala ya n'oge na-adịbeghị anya na αS na-ejikọta ya na protein tau nke dị na ngalaba bara ụba nke proline n'usoro nke na-aga n'ihu ngalaba microtubule.Otú ọ dị, mpaghara PHF nwekwara ọgaranya na ihe nkwụsị nke ọma (lee Figure 2e), karịsịa lysine (15% residues), nke kpaliri anyị ịnwale ma mpaghara a na-enyekwa aka na condensation nke mgbagwoju αS / Tau.Anyị chọpụtara na naanị K18 enweghị ike ịkpalite LLPS na ntinye aka ruru 100 μM n'okpuru ọnọdụ anwalere (LLPS buffer na 15% PEG ma ọ bụ 20% dextran) (Nyocha 2f).Otú ọ dị, mgbe anyị gbakwunyere 50 µM αS na 50 µM K18, a na-ahụ ngwa ngwa nke protein ụmụ irighiri mmiri nwere K18 na αS site na nephelometry (Mgbakwunye Fig. 2d) na WF microscopy (Fig. 2f).Dị ka a tụrụ anya, ΔCt-αS enweghị ike iweghachi omume LLPS nke K18 (Fig 2f).Anyị na-achọpụta na nchịkọta αS / K18 chọrọ ntinye protein dị elu dị elu iji mee ka LLPS tụnyere αS / ΔNt-Tau ma ọ bụ αS / Tau441, ihe ndị ọzọ hà nhata.Nke a kwekọrọ na mmekọrịta siri ike nke mpaghara αS C-terminal na mpaghara proline-rich Tau ma e jiri ya tụnyere ngalaba microtubule-binding, dị ka akọwara na mbụ 31.
Nyere na ΔNt-Tau enweghị ike ịrụ LLPS na enweghị αS, anyị họọrọ ụdị Tau dị ka ihe atụ maka ịkọwa αS/Tau LLPS nyere ịdị mfe ya na sistemụ LLPS nwere Tau ogologo (isotype, Tau441/Tau441).na mgbagwoju anya (heterotypic, αS/Tau441) usoro nchịkọta.Anyị tụnyere ogo nke nchịkọta αS (dị ka akụkụ nke protein condensed frasi, fαS, c) na αS / Tau na αS / ΔNt-Tau usoro site centrifugation na gbasasịa agba SDS-PAGE analysis (lee 2e), hụrụ nnọọ yiri ụkpụrụ. maka protein niile n'otu ntinye uche.Karịsịa, anyị nwetara fαS, c 84 ± 2% na 79 ± 7% maka αS / Tau na αS / ΔNt-Tau, n'otu n'otu, na-atụ aro na mmekọrịta heterotypic n'etiti αS na tau dị elu karịa mmekọrịta dị n'etiti tau molecules.n'etiti.
Mmekọrịta ya na polycations dị iche iche na mmetụta nke usoro condensation na αS kinetics bụ nke mbụ mụrụ site na mgbake fluorescence mgbe usoro photobleaching (FRAP) gasịrị.Anyị nwalere αS/Tau441, αS/ΔNt-Tau na αS/pLK coacervates (100 μM αS gbakwunyere na 2 μM αS AF488-αS na 100 μM Tau441 ma ọ bụ ΔNt-Tau ma ọ bụ 1 mM pLK).Enwetara data n'ime nkeji 30 mbụ mgbe agwakọta ihe nlele.Site na ihe oyiyi FRAP nnọchiteanya (Fig. 3a, αS / Tau441 condensation) na usoro oge ha kwekọrọ (Fig 3b, Fig. 3), enwere ike ịhụ na αS kinetics dị nnọọ ka nke Tau441 coacervates.na ΔNt-Tau, nke na-adị ngwa ngwa na pLK.Ọnụ ọgụgụ mgbasa ozi gbakọọ maka αS n'ime coacervate dị ka FRAP (dị ka Kang et al. 35 kọwara) bụ D = 0.013 ± 0.009 µm2/s na D = 0.026 ± 0.008 µm2/s maka αS/Tau441 na αS/Tau441 Sistemụ / αS.pLK, Tau, na D = 0.18 ± 0.04 µm2/s, n'otu n'otu (Fig. 3c).Otú ọ dị, ọnụọgụ nke mgbasa ozi αS na oge a gbasasịrị bụ ọtụtụ iwu nke ịdị elu dị elu karịa akụkụ niile nke condensed, dị ka Fluorescence Correlation Spectroscopy kpebisiri ike (FCS, lee Mgbakwunye 3) n'okpuru otu ọnọdụ (LLPS buffer) ma na-enweghị polycations. (D = 8 ± 4 µm2/s).Ya mere, kinetics nke nsụgharị αS na-ebelata nke ukwuu na coacervates ma e jiri ya tụnyere ndị na-edozi ahụ na oge a gbasasịrị n'ihi mmetụta nchịkọta mkpụrụ ndụ a na-akpọ molecular, ọ bụ ezie na ihe niile coacervates na-ejigide ihe ndị dị ka mmiri mmiri n'oge ọkara nke mbụ mgbe e guzobere ha, n'ụzọ dị iche na nke tau.Kinetics ngwa ngwa na pLK condensate.
a-c FRAP nyocha nke αS dynamics (2% AF488-labeled αS) na electrostatic coacervates.E gosipụtara onyonyo nnọchite anya nke αS/Tau441 FRAP assays na atọ na (a), ebe okirikiri uhie na-egosi mpaghara agbachapụrụ agbachapụ.Oghere ọnụ ọgụgụ dị 5µm.b Nkezi FRAP curves na (c) gbakọọ ọnụọgụ mgbasa (D) maka 5-6 (N) ụmụ irighiri mmiri dị iche iche sitere na nnwale atọ na-eji 100 µM αS na nha nhata nke Tau441 (red) ma ọ bụ ΔNt-Tau (acha anụnụ anụnụ) ma ọ bụ pLK (green) na ugboro iri ntinye uche nke LLPS.A na-egosi ngbanwe ọkọlọtọ nke akụkụ FRAP na agba ndò.Maka ntụnyere, a na-ekpebi ọnụọgụ ọnụọgụ αS n'ime oge a gbasasịrị n'ime ugboro atọ site na iji fluorescence correlation spectroscopy (FCS) (lee eserese 3 na mgbakwunye maka ozi ndị ọzọ).d Ihe ngosi X-band EPR na-aga n'ihu nke 100 μM TEMPOL-122-αS na nchekwa LLPS na-enweghị polycation (nwa) ma ọ bụ n'ihu 100 μM Tau441 (red) ma ọ bụ ΔNt-Tau (acha anụnụ anụnụ) ma ọ bụ 1 mM pLK (green).Ntinye ahụ na-egosi nleba anya nke ahịrị ubi siri ike ebe mgbanwe kachasị dị egwu na-eme.e Nchikota nke 50 μM TEMPOL-122-αS nwere polycations dị iche iche na enweghị LLPS (enweghị PEG).Mbelata njupụta nke band III ma e jiri ya tụnyere band II (IIII/III) nke ụdịdị EPR a na-ahụkarị ka egosiri na ọ na-abawanye oke molar nke Tau441 (red), ΔNt-Tau (acha anụnụ anụnụ) na pLK (green).Ahịrị ndị nwere agba na-egosi dabara na data na-eji ụdị njigide siri ike nwere saịtị njide nwere otu na nke kwụụrụ onwe ya n'akụkụ ọ bụla.A na-enye data raw n'ụdị faịlụ data raw.
Dị ka ihe mmeju, anyị nyochara ike nke αS na coacervates dị iche iche site na iji akara akara ngosi (SDSL) na resonance paramagnetic na-aga n'ihu (CW-EPR).Usoro a egosila na ọ bara uru nke ukwuu n'ịkọ mgbanwe na mgbanwe dị iche iche nke IDP na mkpebi dị mma 36,37,38.Iji mezuo nke a, anyị wuru ihe fọdụrụ na cysteine ​​​​na otu Cys mutants wee jiri 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Mwepụta nke Maleimide na-akpọ aha ha.Kpọmkwem, anyị tinyere nyocha TEMPOL na ọnọdụ 122 ma ọ bụ 24 αS (TEMPOL-122-αS na TEMPOL-24-αS).N'okwu nke mbụ, anyị na-elekwasị anya na mpaghara C-terminal nke protein, nke na-etinye aka na mmekọrịta ya na polycations.Kama nke ahụ, ọnọdụ 24 nwere ike inye anyị ozi gbasara mkpokọta protein dị na condensate.N'okwu abụọ ahụ, akara EPR nwetara maka protein nke oge a chụsasịrị na-adaba na nitroxide radicals na ọnọdụ na-aga ngwa ngwa.Mgbe nkewa nkewa na ọnụnọ tau ma ọ bụ pLK (100 μM TEMPOL-αS, Tau441 ma ọ bụ ΔNt-Tau na nha 1: 1 ma ọ bụ pLK na nha nke 1: 10), a na-ahụ mmụba nke ọnụ ọgụgụ kachasị elu. Ụdịdị EPR nke αS.Ahịrị ọnwụ ahụ gbasaara, na-egosi mbelata αS reorientation kinetics na ụmụ irighiri mmiri ma e jiri ya tụnyere protein n'ime oge itughari (Fig. 3d, Fig. 4a).Mgbanwe ndị a na-apụtawanye ìhè na ọnọdụ 122. Ọ bụ ezie na n'ọnọdụ 24 ọnụnọ pLK emetụtaghị kinetics nke nyocha ahụ, na ọnọdụ 122 ọdịdị ahịrị spectral gbanwere nke ukwuu (Ngbakwunye Fig. 4a).Mgbe anyị nwara ịmegharị ihe ngosi ahụ na ọnọdụ 122 nke usoro abụọ αS / polycation site na iji ụdị isotropic (Ngbakwunye 5a) nke a na-ejikarị akọwa ihe dị iche iche nke IDP38,39 nke a na-akpọ spin-labeled, anyị enweghị ike ịmegharị ihe ngosi nnwale ahụ..Spectral ịme anwansị nke ọnọdụ nke 24 spins iche (Mgbakwunye Fig. 5a).Nke a na-egosi na enwere ọkwá dị mma na oghere nhazi nhazi nke mpaghara C-terminal nke αS n'ihu polycations.Mgbe ị na-atụle akụkụ nke αS na mpaghara condensed n'okpuru ọnọdụ EPR nnwale (84 ± 2%, 79 ± 7%, na 47 ± 4% maka αS/Tau441, αS/ΔNt-Tau, na αS/pLK, n'otu n'otu-lee Mgbakwụnye. Fig. 2e nke nyocha data c), enwere ike ịhụ na mgbasawanye nke usoro EPR achọpụtara na-egosipụtakarị mmekọrịta nke mpaghara C-terminal nke αS na polycations dị iche iche na mpaghara condensed (isi mgbanwe mgbe ị na-eji TEMPOL-122- αS), na ọ bụghị protein condensation.A na-ahụ mmụba nke microviscosity na nyocha.Dị ka a tụrụ anya ya, e weghachiri ụdịdị EPR nke protein n'okpuru ọnọdụ ndị ọzọ karịa LLPS mgbe agbakwunyere 1 M NaCl na ngwakọta (Fig. 4b).N'ozuzu, data anyị na-atụ aro na mgbanwe ndị CW-EPR achọpụtara na-egosipụtakarị mmekọrịta nke mpaghara C-terminal nke αS na polycations dị iche iche na mpaghara condensed, na mmekọrịta a na-egosi na ọ siri ike na pLK karịa na Tau.
Iji nwetakwuo ozi nhazi gbasara protein ndị dị na coacervate, anyị kpebiri ịmụ usoro LLPS site na iji NMR na ngwọta.Otú ọ dị, naanị anyị nwere ike ịchọpụta akụkụ αS nke fọdụrụ na oge a chụsasịrị, nke nwere ike ịbụ n'ihi mbelata protein dynamics n'ime coacervate na nnukwu akụkụ na ala nke ngwọta na NMR nyocha.Mgbe anyị tụlere usoro na ike nke protein fọdụrụ na agbasasịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịsịị) na-eji NMR (Mgbakwunye 5c. 5c, d), anyị chọpụtara na protein ahụ mere ihe fọrọ nke nta ka ọ bụrụ n'ihu pLK na ΔNt-Tau, ha abụọ. nke dị na nhazi nke abụọ na mgbanwe nke ọkpụkpụ azụ protein, kpughere site na nnwale na mgbanwe kemịkalụ nke abụọ na ntụrụndụ R1ρ.Data NMR na-egosi na C-terminus nke αS na-enwe nnukwu mfu nke mgbanwe mgbanwe mgbe ọ na-ejigide ọdịdị ya mebiri emebi, dị ka usoro protein ndị ọzọ, n'ihi mmekọrịta ya na polycations.
Ebe ọ bụ na mgbasawanye akara CW-EPR hụrụ na TEMPOL-122-αS condensed na-egosipụta mmekọrịta nke protein na polycations, anyị mere EPR titration iji nyochaa njikọ njikọ nke αS na polycations dị iche iche na enweghị LLPS (enweghị nchịkọta nke Buffer LLPS), na-atụ aro na mmekọrịta ahụ bụ otu n'ime usoro ntughari na ntinye uche (nke data anyị kwadoro, Fig. 4a na mgbakwunye Fig. 6).Ebumnobi bụ ịhụ ma ọ bụrụ na ihe niile na-enyere aka, n'agbanyeghị ihe ha na-ahụkarị dị ka mmiri mmiri, na-egosipụta omume ọ bụla dị iche iche na ọkwa molecular.Dị ka a tụrụ anya ya, ụdịdị EPR na-agbasawanye site na ntinye uche nke polycation na-abawanye, na-egosipụta mbelata nke mgbanwe mkpụrụ ndụ n'ihi mkparịta ụka molecular nke ndị mmekọ niile na-emekọrịta ihe fọrọ nke nta ka ọ bụrụ saturation (Fig 3e, Fig. 6).pLK nwetara saturation a na oke molar dị ala (polycation: αS) ma e jiri ya tụnyere ΔNt-Tau na Tau441.N'ezie, ntụnyere data ahụ na ihe nkwekọ nke njide dị nso na-eche na saịtị njide yiri ya na nke onwe ya gosiri na nkesa pụtara ìhè mgbe niile nke pLK (~ 5 μM) bụ usoro nke dị ala karịa nke Tau441 ma ọ bụ ΔNt-Tau (~ 50 μM). ).µM).Ọ bụ ezie na nke a bụ atụmatụ siri ike, nke a na-egosi na αS nwere mmekọrịta dị elu maka polycations dị mfe na mpaghara ụgwọ dị mma na-aga n'ihu.N'iburu nke a dị iche na mmekọrịta dị n'etiti αS na polycations dị iche iche, anyị chere na ihe onwunwe mmiri ha nwere ike gbanwee dị iche iche ka oge na-aga ma si otú a na-ata ahụhụ site na usoro LSPT dị iche iche.
N'iburu n'uche na gburugburu ebe mmadụ juru n'ime protein coacervate na ọdịdị amyloid nke protein, anyị hụrụ omume nke coacervate n'ime oge iji chọpụta usoro LSPT nwere ike ime.N'iji BF na CF microscopy (Njirimara 4), anyị hụrụ na αS / Tau441 na-agbakọta ruo n'ókè dị ukwuu na ngwọta, na-eme nnukwu ụmụ irighiri mmiri na-akpọtụrụ ma na-agba mmiri n'elu ala nke olulu mmiri / slide dị ka ụmụ irighiri mmiri zuru ezu, dị ka a tụrụ anya ya (Fig agbakwunyere). .7d);anyị na-akpọ ihe owuwu ndị a dị n'okpuru "protein raft".Ihe owuwu ndị a nọgidere bụrụ mmiri mmiri ka ha na-ejigide ikike ijikọ (Foto mgbakwunye 7b) na enwere ike ịhụ ya ruo ọtụtụ awa mgbe LLPS kpalitere (Fig 4 na Fig. 7c).Anyị chọpụtara na usoro wetting na-amasị n'elu hydrophilic karịa ihe hydrophobic (Mgbakwunye Fig. 7a), dị ka a na-atụ anya ya maka electrostatic coacervates na-ezighị ezi ụgwọ na otú elu electrostatic elu potentials.N'ụzọ doro anya, αS / ΔNt-Tau coalescence na rafting belatara nke ukwuu, ebe αS / pLK condensates belatara nke ukwuu (Fig 4).N'ime oge ntinye oge dị mkpirikpi, αS / pLK ụmụ irighiri mmiri na-enwe ike ikpokọta ma mee ka mmiri dị n'elu hydrophilic, ma usoro a kwụsịrị ngwa ngwa na mgbe awa 5 gasịrị, ọ bụ naanị ihe omume nchịkọta dị oke na enweghị mmiri ọ bụla.- mgbanwe gel-drip.
Onye nnọchi anya BF (panel greyscale) na CF (panel aka nri, AF488-labeled αS na akwụkwọ ndụ akwụkwọ ndụ) nke ihe atụ coacervate nwere 100 µM αS (1% fluorescent labelụ) na nchekwa LLPS n'ihu 100 µM Tau441 (ΔN) microscoores foto. -Tau (n'etiti) ma ọ bụ 1 mM pLK (ala) na oge incubation dị iche iche na ebe dị elu (z, anya site na ala nke efere efere).Emere nnwale ndị ahụ ugboro 4-6 n'adabereghị onwe ha na otu nsonaazụ ahụ.A na-ete mmiri αS/Tau441 coacervates mgbe awa 24 gachara, na-akpụ rafts buru ibu karịa onyonyo a.Ogwe nha maka onyonyo niile bụ 20µm.
Anyị jụrụ ma nnukwu ọdọ mmiri protein dị ka mmiri etolite na αS/Tau441 LLPS ga-eduga na mkpokọta amyloid nke protein ọ bụla a mụrụ.Anyị na-agbaso maturation nke αS / Tau441 ụmụ irighiri mmiri na oge na WF microscopy n'okpuru otu ọnọdụ dị n'elu, ma na-eji 1 μM AF488-labeled αS na Atto647N-labeled Tau441 (Fig. 5a).Dị ka a tụrụ anya ya, anyị hụrụ nhazi protein zuru oke n'oge usoro ntozu okè.N'ụzọ na-akpali mmasị, site na ca.Mgbe awa 5 gachara, a na-ahụ ihe ndị ọzọ siri ike na-abụghị okirikiri n'ime rafts, nke anyị na-akpọ "isi ihe", ụfọdụ n'ime ha na-ejikọta ya na αS, na ụfọdụ na-eme ka ọ dịkwuo mma na Tau441 (Fig 5a, akụ na-acha ọcha).A na-ahụta ntụpọ ndị a mgbe niile n'ime rafts ruo n'ókè dị ukwuu maka αS/ΔNt-Tau karịa maka αS/ΔNt-Tau.Enweghị ntụpọ dị iche na ụmụ irighiri mmiri nke pLK na sistemụ Tau na-erughị eru maka ngwakọta/wetting.Iji nwalee ma ntụpọ ndị a nwere αS na Tau441 bụ mkpokọta amyloid, anyị mere nnwale yiri nke ahụ site na iji CF microscopy nke akpọrọ Tau441 na Atto647N na 12.5 μM amyloid-specific thioflavin-T (ThT) agbakwunyere site na mmalite.agba agba.Ọ bụ ezie na achọpụtaghị ThT-staining αS / Tau441 droplets ma ọ bụ rafts ọbụna mgbe 24 h nke incubation (Fig. 5b, n'elu ahịrị - fọdụrụ ụmụ irighiri mmiri n'elu protein rafts), ThT-positive Ọdịdị nke nwere Atto647N-Tau441 n'ime rafts adịghị ike.nke a na-emegharị nha, ọdịdị, na ọnọdụ nke ntụpọ ndị a kọwara na mbụ (Fig 5b, ahịrị etiti na nke ala), na-atụ aro na ntụpọ ahụ nwere ike ịdabere na nchịkọta amyloid dị ka nke a na-emepụta na mmiri ịka nká coacervates.
WF 25 μM αS n'oge ntinye dị iche iche na ebe dị elu (z, anya site na mgbapụ ala) n'ihu 25 μM Tau441 (1 μM AF488-labeled αS na Atto647N-labeled Tau441) n'ime olulu mmiri microscope nwere ihe nchekwa LLPS) .Emegharịrị nnwale isii n'onwe ya nwere nsonaazụ yiri ya.b CF ihe onyonyo microscopic nke 25 μM αS n'ihu 25 μM Tau441 (1 μM Atto647N-labeled Tau441) na 12.5 μM thioflavin-T (ThT).A na-egosi ụmụ irighiri mmiri dị arọ na protein rafts na ntụpọ n'ahịrị elu na etiti, n'otu n'otu.Ahịrị dị n'okpuru na-egosi onyonyo nke rafts na ọdịda sitere na 3 nke nwere onwe ha.Àkú na-acha ọcha na-egosi ntụpọ nwere ezi ThT n'akụkụ abụọ ahụ.Ogwe nha maka onyonyo niile bụ 20µm.
Iji nyochaa n'ụzọ zuru ezu mgbanwe na netwọk protein coacervate n'oge mgbanwe site na mmiri mmiri na siri ike, anyị na-eji fluorescence lifetime imaging (FLIM) na Förster resonance energy transfer microscopy (FRET) (Nyocha 6 na Mgbakwunye 8 na 9).Anyị na-eche na coacervate maturation nke oyi akwa n'ime ihe condensed ma ọ bụ ọbụna siri ike-dị ka a gbakọtara n'usoro protein na-eduga na nso nso n'etiti protein na fluorescent nyocha jikọtara ya, nwere ike na-emepụta a quenching mmetụta pụtara ìhè na mkpirisi nyocha oge ndụ (τ) , dị ka akọwara na mbụ40.,41,42.Tụkwasị na nke ahụ, maka ihe nlele abụọ akpọrọ (AF488 na Atto647N dị ka onye na-enye onyinye FRET na ihe ndị na-anabata ya, n'otu n'otu), mbelata a na τ nwekwara ike iso ya na condensation coacervate na mmụba na arụmọrụ FRET (E) n'oge LSPT.Anyị na-enyocha raft na ntụpọ nhazi oge na LLPS αS / Tau441 na αS / ΔNt-Tau samples (25 µM nke protein ọ bụla na nchekwa LLPS nwere 1 µM AF488 nke akpọrọ αS na / ma ọ bụ Atto647N akpọrọ Tau441 ma ọ bụ ΔNt-Tau).Anyị hụrụ a n'ozuzu omume na fluorescence ndụ nke AF488 (τ488) na Atto647N (τ647N) nyocha ebelata ntakịrị ka coacervates tozuru okè (Fig. 6 na mgbakwunye fig. 8c).N'ụzọ na-akpali mmasị, a kwalitere mgbanwe a nke ọma maka ntụpọ n'ime rafts (Fig 6c), na-egosi na njupụta protein ọzọ mere na ntụpọ.N'ịkwado nke a, ọ dịghị mgbanwe dị ịrịba ama na ndụ ndụ nke fluorescence hụrụ maka αS / ΔNt-Tau ụmụ irighiri mmiri maka 24 h (Mgbakwunye 8d), na-atụ aro na gelation droplet bụ usoro dị iche na ịchọta na nke ahụ anaghị eso ya na nhazigharị ihe omimi dị ịrịba ama. n'ime coacervates.Ekwesiri ighota na ntụpọ nwere nha di iche iche na ihe di iche iche di iche iche na αS, karia maka usoro αS/Tau441 (Mgbakwunye 8e).Mbelata nke ntụpọ fluorescence ndụ oge na-esonyere na mmụba nke ike, karịsịa maka Atto647N nke akpọrọ Tau441 (Mgbakwunye Fig. 8a), na arụmọrụ FRET dị elu maka ma αS / Tau441 na αS / ΔNt-Tau usoro, na-egosi n'ihu condensation na LLPS awa ise. mgbe ọ kpalitere, protein ndị dị n'ime ọkụ eletrik static gbakọtara.E jiri ya tụnyere αS/ΔNt-Tau, anyị hụrụ τ647N dị ala na ụkpụrụ τ488 dị elu na ntụpọ αS/Tau441, tinyere ụkpụrụ FRET dị ala na nke na-adịghị mma.Ikekwe, nke a nwere ike jikọta ya na eziokwu ahụ bụ na na usoro αS / Tau441, ihe a na-ahụ anya na nke a na-atụ anya na αS n'ụba na nchịkọta bụ ihe dị iche iche, na-abụkarị substoichiometric tụnyere Tau, ebe ọ bụ na Tau441 n'onwe ya nwekwara ike ịnweta LLPS na nchịkọta (Mgbakwunye Fig. 8e) .Agbanyeghị, ogo nke coalescence droplet, nhazi raft, yana, nke dị mkpa, nchịkọta protein n'ime mmiri mmiri dị ka coacervates bụ nke kachasị mgbe Tau441 na αS dị.
ihe onyonyo nke αS/Tau441 na αS/ΔNt-Tau na 25 μM nke protein ọ bụla (1 μM AF488-labeled αS na 1 μM Atto647N-labeled Tau441 ma ọ bụ ΔNt-Tau) na LLPS.Ogidi ndị a na-egosi onyonyo nnọchite anya nke nlele LLPS n'oge ntozu oke dị iche iche (nkeji 30, awa 5 na awa 24).Okpokoro uhie na-egosi mpaghara nwere ntụpọ αS/Tau441.A na-egosi ogologo ndụ dị ka ogwe agba.Ogwe ọnụ ọgụgụ = 20µm maka onyonyo niile.b Ihe onyonyo FLIM bugoro n'ime mpaghara ahọpụtara, egosiri na igbe uhie dị na panel a.A na-egosi ọkwa ndụ site na iji otu nha agba dị na panel a.Ogwe ọnụ ọgụgụ = 5µm.c Histograms na-egosi AF488 (nke jikọtara αS) ma ọ bụ Atto647N (nke jikọtara na Tau) maka ụdị protein dị iche iche (droplets-D-, raft-R- na speckle-P) chọpụtara na foto FLIM edekọ maka αS-) nkesa oge ndụ nke Tau441 na αS/ΔNt-Tau coacervate samples (N = 17-32 ROI maka D, 29-44 ROI maka R, na 21-51 ROI maka isi).A na-egosi ọnụ ahịa nke etiti na nke etiti dị ka square edo edo na ahịrị ojii n'ime igbe, n'otu n'otu.Oke ala na nke elu nke igbe ahụ na-anọchi anya nke mbụ na nke atọ, n'otu n'otu, na nke kacha nta na ụkpụrụ kachasị n'ime 1.5-fold interquartile range (IQR) ka egosiri dị ka afụ.A na-egosi ndị na-apụ apụ dị ka diamond ojii.Achọpụtara ihe ndekọ ọnụ ọgụgụ dị n'etiti ụzọ abụọ nke nkesa site na iji nlele t-ule abụọ, na-eche na ndịiche enweghị nha.A na-egosi ụkpụrụ p-ule t-ụdụ abụọ na akara mmuke maka ụzọ abụọ nke data atụnyere (* p-uru> 0.01, ** p-uru> 0.001, *** p-uru> 0.0001, **** p-uru> 0.00001), ns Na-egosi nleghara anya (p-uru> 0.05).A na-enye ụkpụrụ p kpọmkwem na tebụl mgbakwunye 1, na-egosikwa data mbụ dị ka faịlụ data raw.
Iji gosikwuo ọdịdị amyloid-dị ka speckles / aggregates, anyị na-emeso ndị na-enweghị ntụpọ coacervate samples maka 24 awa na elu mkpokọta nke (1 M) NaCl, nke mere ka nkewa nke aggregates si protein coacervates.Mgbe a na-ahụ mkpokọta ndị dịpụrụ adịpụ (ntụgharị, ngwọta a chụsasịrị nke mkpokọta) site na iji microscopy atomiki (AFM), anyị hụrụ morphology nke na-abụkarị ihe dị elu nke ihe dị ka 15 nm, nke na-eme ka ọ na-akpakọrịta n'okpuru ọnọdụ nke ntinye nnu dị elu, dị ka omume nke fibrils amyloid ahụkarị n'ihi mmetụta hydrophobic siri ike n'elu (rịba ama na fibrils na-enwekarị elu nke ~ 10 nm) (Mgbakwunye 10a).N'ụzọ na-akpali mmasị, mgbe a na-ejikọta mkpokọta ndị dịpụrụ adịpụ na ThT na ọkọlọtọ ThT fluorescence assay, anyị hụrụ mmụba dị ịrịba ama na ThT fluorescence quantum mkpụrụ, tụnyere nke a hụrụ mgbe agbaji agbaji na-ahụkarị αS amyloid fibrils (Mgbakwunye Fig. 10b), na-atụ aro na. mkpokọta coacervate nwere ihe owuwu amyloid..N'ezie, nchịkọta ahụ na-anabata ya na nnukwu nnu nnu ma na-enwe mmetụta na 4 M guanidine chloride (GdnHCl), dị ka ụdị amyloid fibrils (Mgbakwunye 10c).
Ọzọ, anyị nyochara nhazi nke mkpokọta site na iji otu molecule fluorescence, kpọmkwem fluorescence correlation/cross-correlation spectroscopy (FCS/FCCS), na gbawara nyocha nke nchọpụta nhụta ndakọrịta agba abụọ (TCCD).Iji mezuo nke a, anyị kewapụrụ mkpokọta guzobere mgbe awa 24 nke incubation gasịrị na 100 μl LLPS sample nwere αS na Tau441 (ma 25 μM) yana 1 μM AF488-labeled αS na 1 μM Atto647N-labeled Tau441.Gwakọta ihe ngwọta nchịkọta agbasasị agbasa na steeti monomolecular site na iji otu ihe nchekwa PEG na-enweghị na 1 M NaCl (otu ihe nchekwa a na-eji ekewa mkpokọta na coacervate) iji gbochie mmekọrịta ọ bụla electrostatic n'etiti LLPS na protein.Enwere ike ịhụ ihe atụ nke usoro oge nke otu molekul na Fig. 7a.Nyocha FCCS/FCS (cross-correlation, CC na autocorrelation, AC) gosiri na mkpokọta nwere αS na tau dị ukwuu na nlele (lee CC curve na fig. 7b, ogwe aka ekpe), na ngafe nke protein monomeric fọdụrụnụ bilitere dị ka a Nsonaazụ nke usoro mgbaze (lee akụkụ AC na eserese 7b, akụkụ aka ekpe).Nnwale nchịkwa a na-eme n'okpuru otu ọnọdụ ngwọta ahụ site na iji ihe atụ nwere nanị protein monomeric egosighi ọ bụla CC curves, na AC curves dabara nke ọma na otu akụkụ mgbasa ụdị (Eq. 4), ebe protein monomeric nwere atụmanya mgbasa mgbasa (Fig 7b). ), panel aka nri).Ọnụọgụ mgbasa nke chịkọtara erughị 1 µm2/s, na nke protein monomeric bụ ihe dịka 1 µm2/s.50–100 µm/s;ụkpụrụ dị ka ụkpụrụ e bipụtara na mbụ maka sonicated αS amyloid fibrils na monomeric αS iche n'okpuru ọnọdụ ngwọta yiri nke ahụ44.Mgbe anyị nyochachara nchịkọta na nyocha TCCD gbawara (Fig 7c, panel top), anyị chọpụtara na n'ime nchịkọta ọ bụla dịpụrụ adịpụ (αS / Tau heteroaggregate), ihe dịka 60% nke nchịkọta achọpụtara nwere ma αS na tau, ihe dịka 30% nwere naanị. Tau, ihe dị ka 10% αS naanị.Nyocha Stoichiometric nke αS / Tau heteroaggregates gosiri na ọtụtụ n'ime heteroaggregates na-eme ka ọ dịkwuo mma na tau (stoichiometry n'okpuru 0.5, nkezi ọnụ ọgụgụ nke tau molecules kwa mkpokọta bụ 4 ugboro karịa αS molecules), nke kwekọrọ na ọrụ anyị hụrụ na FLIM na situ. nnwale..Nyocha FRET gosiri na nchịkọta ndị a nwere protein abụọ ahụ, ọ bụ ezie na ụkpụrụ FRET n'ezie na nke a abụghị ihe dị mkpa, ebe ọ bụ na nkesa nke fluorophores na mkpokọta ọ bụla bụ ihe na-enweghị usoro n'ihi oke protein na-enweghị aha ejiri mee nyocha ahụ.N'ụzọ na-akpali mmasị, mgbe anyị mere otu nyocha ahụ site na iji 45,46 tozuru okè amyloid aggregation-deficient Tau variant (lee Mgbakwunye Fig. 11a,b), anyị chọpụtara na ọ bụ ezie na αS electrostatic aggregation bụ otu (Mgbakwunye Fig. 11c, d), ikike imepụta mkpokọta n'ime coacervate belatara nke ukwuu yana FLIM chọtara ọtụtụ ntụpọ n'ime nnwale ọnọdụ, yana a na-ahụ akụkụ nke njikọ na-adịghị ike maka mkpokọta mkpokọta dịpụrụ adịpụ.Otú ọ dị, maka ọnụ ọgụgụ dị nta nke nchịkọta achọpụtara (naanị otu ụzọ n'ụzọ iri nke Tau441), anyị hụrụ na nchịkọta nke ọ bụla nwere ụbara na αS karịa nke a Tau variant, yana ihe dịka 50% nke nchịkọta achọpụtara nwere naanị αS molecules, na αS dị iche iche na oke. .aggregates (lee Mgbakwunye Fig. 11e), n'ụzọ dị iche na nchịkọta dị iche iche nke Tau441 mepụtara (Fig 6f).Nsonaazụ nke nnwale ndị a gosiri na ọ bụ ezie na αS n'onwe ya nwere ike ịgbakọta na tau n'ime coacervate, tau nucleation dị mma karịa n'okpuru ọnọdụ ndị a, na nchịkọta amyloid nke na-esi na ya pụta nwere ike ime dị ka ụdị αS na tau.Otú ọ dị, ozugbo e hiwere isi-ọgaranya tau, mmekọrịta dị iche iche dị n'etiti αS na tau na-amasị na nchịkọta karịa mmekọrịta nwoke na nwanyị n'etiti tau molecules;anyị na-ahụkwa netwọk protein na mmiri mmiri αS/tau coacervates.
ihe nnochite anya fluorescence nwa oge nke otu molekul nke mkpọkọta ndị dịpụrụ adịpụ guzobere na αS/Tau441 electrostatic coacervates.Ahụrụ mgbawa kwekọrọ na αS / Tau441 coaggregates (gbawara n'elu ọnụ ụzọ egosipụtara) na ọwa nchọpụta atọ (AF488 na Atto647N emission mgbe mkpali ozugbo, ahịrị anụnụ anụnụ na uhie, Atto647N emission after indirect excitation), FRET, violet line).b FCS/FCCS nyocha nke ihe nlele nke mkpokọta αS/Tau441 dịpụrụ adịpụ enwetara na LLPS (panel aka ekpe).A na-egosi akụkụ nke autocorrelation (AC) maka AF488 na Atto647N na-acha anụnụ anụnụ na ọbara ọbara, n'otu n'otu, na akụkụ nke njikọ cross-correlation (CC) jikọtara ya na nchịkọta nwere ma esiji na-acha odo odo.Oghere AC na-egosipụta ọnụnọ nke ụdị monomeric akpọrọ na ụdị protein a chịkọtara, ebe akụkụ CC na-egosi naanị mgbasa nke mkpokọta akpọrọ aha abụọ.Otu nyocha ahụ, mana n'okpuru otu ọnọdụ ngwọta dị ka ọ dị na ebe dịpụrụ adịpụ, a na-egosi ihe nlele nwere naanị monomeric αS na Tau441 dị ka njikwa na panel ziri ezi.c Fluorescence flash analysis nke otu ụmụ irighiri ihe nke mkpọkọta ndị dịpụrụ adịpụ guzobere na αS/Tau441 electrostatic coacervates.A na-akpa nkata maka mkpokọta ọ bụla achọtara na nkwugharị anọ dị iche iche (N = 152) megide stoichiometry ha, ụkpụrụ S na arụmọrụ FRET (panel dị elu, ogwe agba na-egosipụta ihe omume).Enwere ike ịmata ụdị nchịkọta atọ: -αS-naanị nchịkọta na S ~ 1 na FRET ~ 0, Tau-only aggregates na S ~ 0 na FRET ~ 1, na ihe dị iche iche Tau / αS aggregates na etiti S na FRET Atụmatụ nke ego. A na-egosipụta protein protein abụọ a na-egosi na mkpokọta dị iche iche (N = 100) na obere panel (ọnụ ọgụgụ agba na-egosipụta ihe omume ahụ).A na-enye data raw n'ụdị faịlụ data raw.
Ntụle ma ọ bụ ịka nká nke protein mmiri mmiri na-abanye n'ime gel-dị ka ma ọ bụ ihe siri ike ka oge na-aga ka akọọrọ na ọ na-etinye aka n'ọtụtụ ọrụ physiological nke condensate47 yana ọrịa, dị ka usoro na-adịghị mma nke na-ebute amyloid aggregation 7, 48, 49. Ebe a. anyị na-amụ nkewa nkewa na omume n'ụzọ zuru ezu.LSPT αS n'ihu polycations na-enweghị usoro na gburugburu ebe a na-achịkwa na obere micromolar na ọnọdụ dị mkpa gbasara ahụ ike (rịba ama na mkpokọta physiological gbakọọ nke αS bụ> 1 µM50), na-agbaso omume ọkụ ọkụ ọkụ nke LPS.Anyị chọpụtara na αS, nke nwere mpaghara C-terminal ebubo na-adịghị mma na pH physiological, na-enwe ike ịmepụta ụmụ irighiri mmiri nwere protein na ngwọta mmiri site na LLPS n'ihu ọnụnọ nke peptides nwere nsogbu dị ukwuu dị ka pLK ma ọ bụ Tau site na usoro electrostatic. mgbagwoju condensation na ọnụnọ nke aggregation macromolecules.Usoro a nwere ike inwe mmetụta dị mkpa na gburugburu cellular ebe αS na-ezute ihe dị iche iche polycationic molecules jikọtara ya na ọrịa ya na-ejikọta ya na in vitro na vivo51,52,53,54.
N'ọtụtụ ọmụmụ, a na-ahụta mgbanwe protein n'ime ụmụ irighiri mmiri dị ka otu n'ime isi ihe na-ekpebi usoro ntozu55,56.Na electrostatic αS coacervates na polycations, usoro maturation doro anya na-adabere na ike nke mmekọrịta ya na polycations, valence, na ịba ụba nke mmekọrịta ndị a.Echiche nha nhata na-atụ aro na nha nhata nke steeti mmiri mmiri abụọ ga-abụ ọnụnọ nke nnukwu ntụpọ mmiri bara ụba na biopolymers na-anya LLPS57,58.Enwere ike nweta uto mkpọda site na Ostwald maturation59, coalescence60 ma ọ bụ oriri nke monomer n'efu n'oge agbasasịsa61.Maka αS na Tau441, ΔNt-Tau ma ọ bụ pLK, ọtụtụ n'ime protein ahụ na-etinye uche na condensate n'okpuru ọnọdụ ndị e ji mee ihe n'ọmụmụ ihe a.Otú ọ dị, ka ụmụ irighiri mmiri na-agbakọta ngwa ngwa n'elu wetting, droplet coalescence na wetting siri ike maka ΔNt-Tau na pLK, na-atụ aro mfu ngwa ngwa nke mmiri mmiri n'ime usoro abụọ a.Dị ka nyocha FLIM-FRET anyị si kwuo, pLK na ΔNt-Tau ụmụ irighiri mmiri gosipụtara ogo nke nchịkọta protein (oge ndụ fluorescence yiri ya) dị ka ụmụ irighiri mmiri mbụ, na-atụ aro na a na-ejigide netwọk protein mbụ, n'agbanyeghị na ọ siri ike karị.
Anyị na-akọwapụta nsonaazụ nnwale anyị n'ụdị na-esonụ (Foto 8).Ụmụ irighiri mmiri na-amalite nwa oge na-abụkarị netwọk protein na-enweghị nkwụghachi ụgwọ electrostatic, ya mere enwere akụkụ nke enweghị ahaghị nhata, karịsịa na oghere droplet, na-ebute ụmụ irighiri mmiri nwere ikike elu electrostatic.Iji kwụọ ụgwọ maka ụgwọ (ihe a na-akpọkarị valence depletion) ma belata ikike elu nke ụmụ irighiri mmiri ndị ahụ, ụmụ irighiri mmiri ahụ nwere ike ịgụnye polypeptides ọhụrụ sitere na akụkụ nke dilute, hazie netwọk protein iji kwalite mmekọrịta ụgwọ-ụgwọ, yana mmekọrịta ya na ụmụ irighiri mmiri ndị ọzọ.na elu (wetting).Akpụkpọ anụ αS / pLK, n'ihi netwọk protein ha dị mfe (naanị mmekọrịta heterotypic n'etiti αS na pLK) na mmekọrịta dị ukwuu maka mmekọrịta protein-protein, yiri ka ọ ga-edozi ụgwọ nke condensate ngwa ngwa;N'ezie, anyị hụrụ protein kinetics ngwa ngwa na mbido αS/pLK coacervates karịa na αS/Tau.Mgbe valence depletion gasịrị, mmekọrịta ahụ na-aghọ obere ephemeral na ụmụ irighiri mmiri na-efunahụ ihe onwunwe mmiri ha wee ghọọ gel-dị ka ụmụ irighiri mmiri na-ere ọkụ na-enweghị ọkụ na-enweghị ike ime elu electrostatic dị ala (ya mere enweghị ike ime ka mmiri dị n'elu).N'ụzọ dị iche, ụmụ irighiri mmiri αS/Tau adịghị arụ ọrụ nke ọma n'ịkwalite nguzozi ụgwọ droplet n'ihi netwọkụ protein dị mgbagwoju anya (ya na mmekọrịta nke homotypic na heterotypic) na ọdịdị adịghị ike nke mmekọrịta protein.Nke a na-ebute ụmụ irighiri mmiri na-ejigide omume mmiri ruo ogologo oge ma na-egosipụta ikike dị elu nke electrostatic nke na-achọ ka a na-ebelata ya site na nchịkọta na-eto eto (si otú ahụ na-ebelata oke elu / oke nke ụmụ irighiri mmiri) na site n'iwepu chem hydrophilic elu.Nke a na-emepụta nnukwu ọba akwụkwọ protein gbanyere mkpọrọgwụ nke na-ejigide akụrụngwa mmiri ka mmekọrịta ahụ na-adịte aka nke ukwuu n'ihi na-achọkarị njikarịcha ụgwọ na netwọk protein.N'ụzọ na-akpali mmasị, ụdị Tau nke N-n'ikpeazụ, gụnyere ụfọdụ isoforms62 na-emekarị, na-egosipụta omume etiti, yana ụfọdụ na-akwado ịka nká na αS n'ime ụmụ irighiri mmiri dị ogologo ndụ, ebe ndị ọzọ na-agbanwe ghọọ nnukwu condensates mmiri.Ihe abụọ a na maturation nke αS electrostatic coacervates kwekọrọ na usoro ihe ọmụma LLPS na nso nso a na nnwale nke achọpụtala njikọ dị n'etiti mbelata valence na electrostatic sieving na condensates dị ka isi ihe na-achịkwa nha condensate na akụrụngwa mmiri.Mechanism 58.61.
Atụmatụ a na-egosi ụzọ nchịkọta amyloid putative maka αS na Tau441 site na LLPS na LSPT.Na mpaghara anion-ọgaranya (red) na cation-ọgaranya (acha anụnụ anụnụ), αS na tau electrostatic coacervates na valence na-eju afọ nwere ike elu ala ya mere ọ na-adịchaghị mma, na-ebute ịka nká ngwa ngwa.A na-enweta steeti gel kwụsiri ike na-abụghị agglomerated..Ọnọdụ a dị ezigbo mma n'ihe gbasara usoro αS / pLK n'ihi njikọ dị elu na njikọ njikọ protein-pair dị mfe, nke na-enye ohere maka mgbanwe ngwa ngwa gel.N'ụzọ megidere nke ahụ, ụmụ irighiri mmiri na valence na-adịghị mma na, ya mere, mpaghara ndị na-ebu protein dị maka mmekọrịta, na-eme ka ọ dịkwuo mfe maka coacervate fuse na mmiri mmiri nke hydrophilic iji belata ike elu ya.Ọnọdụ a dị mma maka αS / Tau441 coacervates, nke nwere netwọk dị mgbagwoju anya nke nwere mmekọrịta Tau-Tau na αS-Tau na-adịghị ike.N'aka nke ya, nnukwu coacervates ga-ejigide ngwa ngwa ha dị ka mmiri mmiri, na-ekwe ka mmekọrịta protein-na-protein ndị ọzọ mee.N'ikpeazụ, amyloid heterogeneous aggregates nwere ma αS na tau form n'ime mmiri coacervate, nke nwere ike jikọta ya na ndị ahụ dị n'ime ahụ gụnyere, nke bụ akara nke ọrịa neurodegenerative.
Nnukwu ihe owuwu dị ka mmiri nke e guzobere n'oge maturation nke αS / Tau441 nwere nnukwu congested ma dị ike na gburugburu ebe obibi protein na, ruo n'ókè dị nta, αS / ΔNt-Tau coacervates bụ ezigbo reservoirs maka nucleation nke protein nchịkọta.Anyị ahụwo n'ezie nhazi nke nchịkọta protein siri ike na ụdị protein coacervates, na-enwekarị ma αS na tau.Anyị egosila na heteroaggregates ndị a na-emeziwanye site na mmekọrịta ndị na-abụghị electrostatic, na-enwe ike ijikọta amyloid-kpọmkwem ThT agba n'otu ụzọ ahụ dị ka amyloid fibrils na-ahụkarị, na n'ezie nwere mmetụta yiri nke ahụ na mmetụta dị iche iche.E gosiputara mkpokọta αS/tau nke LLPS hibere nwere ihe ndị yiri amyloid.N'ezie, variant tozuru okè nke Tau erughi na amyloid aggregation na-emebi nke ọma na nhazi nke a heterogeneous αS aggregates n'ime mmiri mmiri electrostatic coacervate.A na-ahụ nhazi nke αS / Tau441 aggregates naanị n'ime coacervates, nke na-ejigide ihe ndị dị ka mmiri mmiri, ọ dịghịkwa mgbe, ma ọ bụrụ na coacervates / droplets eruteghị steeti gel.N'okwu nke ikpeazụ, ike na-abawanye nke mmekọrịta electrostatic na, n'ihi ya, isi ike nke netwọk protein na-egbochi nhazi nhazi nke protein dị mkpa iji guzobe mmekọrịta protein ọhụrụ dị mkpa maka amyloid nucleation.Otú ọ dị, nke a nwere ike ime na mgbanwe ndị ọzọ, mmiri mmiri-dị ka coacervates, nke n'aka nke ya yikarịrị ka ọ ga-anọgide na mmiri ka ha na-abawanye na nha.
Eziokwu ahụ bụ na nhazi nke mkpokọta n'ime oge a na-eme ka ọ dị mma na nnukwu αS / Tau condensates karịa na obere ụmụ irighiri mmiri na-agba ọsọ ngwa ngwa, na-egosipụta mkpa ọ dị iji chọpụta ihe ndị na-achịkwa nchịkọta mmiri.Ya mere, ọ bụghị nanị na enwere ọchịchọ maka nkewa nke oge, ma a ghaghị ịchịkwa nha nke condensate maka ịrụ ọrụ kwesịrị ekwesị yana mgbochi ọrịa58,61.Nsonaazụ anyị gosikwara mkpa nguzozi n'etiti LLPS na LSPT maka sistemụ αS/Tau.Ọ bụ ezie na nhazi droplet nwere ike ichebe megide nchịkọta amyloid site na ibelata ọnụọgụ protein monomers dị n'okpuru ọnọdụ saturation, dị ka ekwuru na usoro ndị ọzọ63,64, nchịkọta droplet na ọkwa dị elu nwere ike iduga nchịkọta protein dị n'ime site na nhazi nhazi ngwa ngwa.netwọk protein..
N'ozuzu, data anyị na-emesi ike mkpa nke ịdị n'otu valence na afọ ojuju/enweghị afọ ojuju mmekọrịta na netwọk dobe na ọnọdụ nke LSPT.Karịsịa, anyị na-egosi na condensates αS / Tau441 zuru ezu na-enwe ike ijikọ nke ọma na nucleate iji mepụta amyloid-dị ka heteroaggregates nke na-agụnye ma protein ma na-atụ aro usoro mkpụrụ ndụ dabere na nsonaazụ nnwale anyị.Nchikota nke protein abuo na αS/Tau fluid coacervate nke anyi na-akuko ebe a nwere ike n'ezie metụta nchikota nke protein abuo na ntinye, nke bu akara nke oria a, ma nwee ike inye aka n'ighota mmekorita di n'etiti LLPS na amyloid aggregation, na-emepe ụzọ maka IDP dị oke ụgwọ na neurodegeneration.
Monomeric WT-αS, cysteine ​​mutants (Q24C-αS, N122C-αS) na ΔCt-αS variants (Δ101-140) gosipụtara na E. coli ma dị ọcha dị ka akọwara na mbụ.5 mM DTT gụnyere n'ime usoro niile na-adị ọcha nke αS cysteine ​​mutants iji gbochie nguzobe disulfide.Tau441 isoform (plasmid enwetara site na Addgene #16316), ΔNt-Tau variant (Δ1-150, nwetara site na cloning IVA na primers CTTTAAGAAGGAGATACATATGATCGCCACACACCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) na AggDef-Tau7 variant (150) variant-GCC na AggDef-Tau7-Premier. omenala ndị toro na OD600 = 0.6-0.7 na 37°C na 180 rpm, wee jiri IPTG mee ka okwu pụta maka awa 3 na 37°C.Mkpụrụ ndụ owuwe ihe ubi na 11,500 xg maka 15 min na 4 Celsius ma saa ya na saline buffer nwere 150 mM NaCl.Weghachite pellet n'ime ihe nchekwa lysis (20 ml kwa 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).Emere nzọụkwụ sonication na ice na njupụta nke 80% maka pulses 10 (1 min na, 1 min kwụsịrị).Agafela 60 ml na otu ultrasound.E. coli lysates na-ekpo ọkụ na 95 ° C. maka nkeji 20, wee mee ka ọ dị jụụ na ice na centrifuged na 127,000 × g maka 40 nkeji.A na-etinye ihe mkpuchi ahụ doro anya na akpụkpọ ahụ 3.5 kDa (Spectrum ™ Thermo Fisher Scientific, UK) wee chọpụta ya megide 4 L nke dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM. , PMSF 0.1 mM) maka awa 10.Ejiri kọlụm mgbanwe cation 5 ml (HiTrap SPFF, Cytiva, MA, USA) jikọtara ya na nchekwa nha (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).A na-enyocha tau lysate site na nzacha 0.22 μm PVDF wee tinye ya n'ime kọlụm n'ọsọ ọsọ nke 1 ml/min.A na-eji nwayọọ nwayọọ mee ihe ngosi, tau jiri 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).SDS-PAGE nyochara akụkụ nke irighiri ihe, na akụkụ ọ bụla nke nwere otu band nwere ibu arọ nke tau a na-atụ anya ya ka etinyere ya site na iji nzacha centrifuge 10 kDa wee dochie ya na nchekwa nwere 10 mM HEPES, pH 7.4, NaCl 500 mM na DTT 2 mM maka. ntinye protein ikpeazụ bụ 100 μM.A gafechara ngwọta protein ahụ site na nzacha 0.22 μm PVDF, jụrụ oyi ngwa ngwa ma chekwaa ya na -80 Celsius.Prọfesọ Alberto Boffi jiri obiọma nye Protein K18.Ịdị ọcha nke nkwadebe bụ> 95% dị ka SDS-PAGE na MALDI-TOF/TOF kwadoro.Cysteine ​​​​dị iche iche ka ejiri akara AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) ma ọ bụ TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).kwadoro site na nnabata na MALDI-TOF/TOF.A na-akpọ Tau441, ΔNt-Tau, AggDef-Tau na K18 na ihe fọdụrụ nke cysteine ​​​​na ọnọdụ 191 na 322 site na iji Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Germany) na-agbaso otu usoro ahụ.Ejiri CIDER66 nweta ụgwọ net maka maapụ fọdụrụ maka αS na Tau441.
Poly-L-lysine siri ike (pLK DP 90-110 dị ka NMR si soplaya, Alamanda Polymers Inc, Huntsville, Alabama, USA) etisasịwo na 10 mM HEPES, 100 mM NaCl, pH 7.4 ka 10 mM ịta, usoro sonicated maka 5. nkeji na mmiri mmiri ultrasonic na-echekwa na -20 Celsius.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) na FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) bụ mmiri soluble na nkesa ebe niile na nchekwa LLPS.Dialysis na-ewepụ nnu na-emerụ emerụ.A na-enyocha ha site na nzacha sirinji nwere oghere nke 0.22 μm, wee gbakọọ mkpokọta ha site na iji refractometer (Mettler Toledo, Columbus, Ohio, USA).A kwadebere ihe nlele LLPS na ụlọ okpomọkụ n'usoro a: a na-ejikọta ihe nchekwa na extrusion na 1 mM tris (2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2- (Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) na ngwakọta nke 1% protease inhibitor (PMSF 100 mM, benzamide 1 mM, leupeptin 5 μM).Mgbe ahụ, a na-agbakwunye αS na polycations (nhọrọ pLK ma ọ bụ Tau).Maka nnwale usoro usoro oge nke thioflavin-T (ThT, Carbosynth, Compton, UK), jiri mkpokọta ThT ka ọ bụrụ ọkara mkpokọta αS.Jiri nwayọ ma gwakọta ihe nlele ahụ nke ọma iji hụ na ha bụ otu.Nleba nke akụrụngwa ọ bụla dịgasị iche site na nnwale ruo nnwale, dịka akọwara na ngalaba Nsonaazụ.Ejiri Azide mee ihe na ntinye nke 0.02% (w / v) mgbe ọ bụla oge nnwale ahụ gafere awa 4.Maka nyocha niile site na iji nlele LLPS, kwe ka ngwakọta ahụ kwekọọ maka nkeji 5 tupu nyocha.Maka nyocha nke ịgbasa ọkụ, 150 µl nke ihe atụ ka etinyere na 96-nke ọma microplates (µClear®, ojii, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) ma kpuchie ya na ihe nkiri nrapado.A na-enyocha LLP site na ịlele nnabata na 350 nm n'etiti ngwọta na onye na-agụ efere CLARIOstar (BMG Labtech, Ortenberg, Germany).Emere nnwale ndị ahụ ugboro atọ na 25 Celsius C, a na-agbakọkwa mmejọ ndị ahụ dị ka ụkpụrụ ọkọlọtọ si n'aka.A na-atụle usoro ntule site na nlele centrifugation na SDS-PAGE gel analysis, na akụkụ αS dị na usoro ntughari na itinye uche n'ihe ngwọta LLPS dị iche iche.A kwadebere ihe nlele 100 μl LLPS nke nwere 1 μM AF488-labeled αS site na ngwakọta nke ọma na-esote centrifugation na 9600 × g maka nkeji 30, mgbe nke ahụ gasịrị, a na-ahụkarị ihe na-asọ oyi.A na-eji 50 μl kacha elu nke ihe dị elu mee ihe maka nhazi protein site na iji SDS-PAGE gel.A na-enyocha gels na nzacha AF488 site na iji sistemu chemiDoc gel imaging (Bio-Rad Laboratories, Hercules, CA, USA) ma ọ bụ nwee ntụpọ Coomassie wee were nzacha kwesịrị ekwesị were anya ya.A na-enyocha ìgwè ndị a sitere na iji ImageJ version 1.53i (National Institute of Health, USA).Emere nnwale ndị ahụ na oyiri na nnwale abụọ dị iche iche nwere nsonaazụ yiri ya.
A na-etinye 150 μl nke ihe nlele na microplates 96-nke na-adịghị ejikọta ma na-ahụ anya na ọnụ ụlọ okpomọkụ na Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Germany).Maka nnwale ntụpọ, µ-Slide Angiogenesis plates (Ibidi GmbH, Gräfelfing, Germany) ma ọ bụ 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts).A na-eji EL6000 halogen ma ọ bụ mercury metal halide oriọna dị ka isi mmalite ọkụ (maka foto BF/DIC na WF, n'otu n'otu).Maka microscopy WF, a na-eji ebumnobi ikuku 40x magnification (Leica Microsystems, Germany) mee ka ìhè dị na nlele ahụ wee nakọta ya.Maka ihe nlele AF488 na ThT nwere akara, yochaa mkpali na ikuku ya na ihe nzacha GFP ọkọlọtọ, mkpali na ihe nzacha bandpass, n'otu n'otu, 460-500 nm na 512-542 nm bandpass filter, yana enyo dichroic 495 nm.Maka ihe nlele nke akpọrọ Atto647N, a na-eji ihe nzacha Cy5 ọkọlọtọ nwere mkpali na nzacha bandpass 628-40 nm na 692-40 nm n'otu n'otu, yana enyo dichroic 660 nm.Maka microscopy BF na DIC, jiri otu ebumnobi nchịkọta ọkụ egosipụtara.Edere ọkụ anakọtara na igwefoto Leica DFC7000 CCD (Leica Microsystems, Germany).Oge mkpughe bụ 50 ms maka onyonyo onyonyo BF na DIC yana 20-100 ms maka onyonyo microscopy WF.Maka ntụnyere, oge mkpughe maka nnwale niile na ThT bụ 100 ms.Emere nnwale na-agafeghị oge iji hụta nhụta nke njikọta ọnụ mmiri, ebe a na-anakọta onyonyo ọ bụla 100 ms ruo ọtụtụ nkeji.Ejiri ImageJ (NIH, USA) mee nyocha onyonyo.Emere nnwale ndị ahụ ugboro atọ nwere nsonaazụ yiri ya.
Maka nnwale colocalization, FRAP na 3D nrụzigharị, enwetara onyonyo na Zeiss LSM 880 confocal mikroskopu na-eji mbipụta ZEN 2 blue (Carl Zeiss AG, Oberkochen, Germany).A na-etinye ihe atụ nke 50 µl na µ-Slide Angiogenesis Petri efere (Ibidi GmbH, Gröfelfing, Germany), na-emeso ya na polymer hydrophilic (ibiTreat) ma tinye ya na 63 × mmanụ immersion ebumnobi (Plan-Apochromat 63 × / NA 1.4 Mmanụ) na DIC).Enwetara onyonyo site na iji 458 nm, 488 nm, na 633 nm argon laser ahịrị nwere mkpebi nke 0.26 µm/pixel na oge ikpughe nke 8 µs/pixel maka mkpali na nhụta ikuku nke 470-600 nm, 493-628 nm, na 638–755 nm ka ejiri anya hụ ThT, AF488 na Atto647N, n'otu n'otu.Maka nnwale FRAP, eserese oge nke ihe nlele ọ bụla ka edekọtara na 1 frame kwa nkeji.Emere nnwale ndị ahụ ugboro atọ na okpomọkụ ụlọ nwere nsonaazụ yiri ya.A tụlere onyonyo niile site na iji sọftụwia mbipụta blue blue Zen 2 (Carl Zeiss AG, Oberkochen, Germany).A na-ahazi akụkụ FRAP ahụ nke ọma, kpara nkata ma dabara na ike/data oge ewepụtara na onyonyo site na iji Zen 2 site na iji OriginPro 9.1.A na-etinye akụkụ mgbake ahụ n'ụdị mono-exponential iji kọwaa mgbasa ozi molekụla yana okwu nkọwa agbakwunyere iji nweta nsonaazụ ịcha ọcha nke nnweta.Anyị wee gbakọọ D na-eji radius ịcha ọcha ọnụ na ọkara ndụ mgbake kpebiburu dịka n'usoro nke Kang et al.5 35 egosiri.
A na-ejikọta ụdị cysteine ​​​​dị iche iche nke αS na 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) na ọnọdụ 24 (TEMPOL-24-αS) na 122 (TEMPOL-122-αS), n'otu n'otu.Ntugharị Spin Labeling Maka nnwale EPR, αS etinyere na 100 μM yana itinye uche PEG bụ 15% (w/v).Maka ọnọdụ nchịkọta dị iche iche, αS: pLK ruru 1:10, ebe αS: ΔNt-Tau na αS: Tau441 ruru ka edobere na 1: 1.Maka ijikọ nnwale titration na enweghị igwe mmadụ, TEMPOL-122-αS ka edobere na 50 μM na polycations ka etinyere na mkpokọta na-abawanye ụba, na-akwadebe ọnọdụ ọ bụla iche.A na-eme nha CW-EPR site na iji Bruker ELEXSYS E580 X-band spectrometer nke nwere Bruker ER4118 SPT-N1 resonator na-arụ ọrụ na ngwa ndakwa nri (SHF) ugboro ~9.7 GHz.Edobere ọnọdụ okpomọkụ na 25 Celsius ma na-achịkwa mmiri mmiri nitrogen cryostat.Enwetara ihe nkiri ahụ n'okpuru ọnọdụ enweghị ike na ike MW nke 4 mW, njupụta nke 0.1 mT, na oge mgbanwe nke 100 kHz.A na-emeziwanye ike dị iche iche iji zere ọdịiche dị n'ichepụta ntugharị n'etiti ihe atụ na mbelata ọkpụkpụ nwere ike ịbụ n'ihi nkwụsị nke mbelata ndị ọrụ na nlele nwere Tau441 ma ọ bụ ΔNt-Tau (nke dị ugbu a na ngwọta protein mbụ).E nwetara ụkpụrụ g nke enyere n'ihi nlegharị anya EPR spectral emere site na iji ngwa Easyspin (v. 6.0.0-dev.34) etinyere na Matlab®67.A na-eji ụdị isotropic otu/abụọ mee ihe iji ṅomie data ahụ.Ka emechara akara ngosi niile, gbakọọ ihe fọdụrụ site na iwepụ simulation nke ọ bụla na ụdị nnwale dabara adaba.Maka nyocha njide titration, a na-eji ike ikwu ike nke otu nke atọ gaa na nke abụọ nke ụdịdị EPR ahazi (IIII/III) iji nyochaa njide nke polycations na αS.Iji tụọ nhụsianya oge niile (Kd), usoro nke arụpụta dabara adaba na ihe nlere anya na-eche n' saịtị njide yiri ya na nke kwụụrụ onwe ya.
Emere nnwale spectroscopy NMR site na iji Bruker Neo 800 MHz (1H) NMR spectrometer nke nwere cryoprobe na Z-gradient.Emere nyocha niile site na iji 130-207 µM αS na αS / ΔNt-Tau na pLK kwekọrọ na 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 ma mee ya na 15 ° C.Iji nyochaa LPS site n'aka NMR, 10% PEG agbakwunyere na nlele agwakọtara.Ihe ngbanwe mgbanwe kemịkalụ perturbation (Fig 1b) na-egosi nkezi mgbanwe kemịkalụ 1H na 15N.E kenyere ihe ngosi αS 2D1H-15N HSQC dabere na ọrụ gara aga (ntinye BMRB #25227) wee kwado ya site na ịdekọ na nyochaa ụdịdị 3D nke HNCA, HNCO na CBCAcoNH.A na-agbakọ mgbanwe mgbanwe kemịkalụ 13Cα na 13Cβ n'ihu ΔNt-Tau ma ọ bụ pLK iji tụọ mgbanwe enwere ike na usoro nhazi nke abụọ ma e jiri ya tụnyere mgbanwe kemịkalụ αS na ngbanwe dị ọcha na-agbanwe agbanwe 68 (Ngbakwunye 5c).A tụrụ ọnụego R1ρ site na ịdekọ nnwale hsqctretf3gpsi (enwetara na ọbá akwụkwọ Bruker) na igbu oge nke 8, 36, 76, 100, 156, 250, 400, na 800 ms, na arụ ọrụ exponential ka agbanwere ka ọ dị ogologo oge dị iche iche. oge iji chọpụta R1ρ na ejighị n'aka nnwale ya.
A na-eme nnwale microscopy fluorescence agba agba abụọ agbagoro na oge azụmahịa MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) nwere ngwa ngụkọ foton (TCSPC) jikọtara oge.A na-eji isi diode laser eme ihe maka pulsed interleaved excitation (PIE), beam na-agafe n'otu ọnọdụ ebili mmiri ma na-ekiri ya na ike laser nke 10 ruo 100 nW maka ahịrị laser 481 nm na 637 nm laser tụrụ mgbe enyo dichroic.Nke a na-eme ka ọnụ ọgụgụ foton dị mma, na-ezere mmetụta nke foton aliasing, photobleaching na saturation.μ-Slide angiogenesis cover ma ọ bụ efere (Ibidi GmbH, Gräfelfing, Germany) ka etinyere ozugbo na mmiri imikpu n'elu oghere Super Apochromat 60x NA 1.2 nwere olu mgbazi (Olympus Life Sciences, Waltham, USA).A na-eji mirror dichroic 488/640 nm (Semrock, Lake Forest, IL, USA) mee ihe dị ka onye na-ekesa isi ọkụ.A na-egbochi radieshon na-enweghị uche site na oghere nwere dayameta nke 50 microns, mgbe ahụ, a na-ekewa radieshon a na-elekwasị anya n'ime ụzọ nchọpụta 2 site na 50/50 beam splitter.A na-eji ihe nzacha ikuku nke Bandpass (Semrock, Lake Forest, IL, USA) 520/35 maka agba ndụ ndụ ndụ (AF488) na 690/70 maka ịcha ọbara ọbara (Atto647N) n'ihu onye nchọpụta ahụ.Otu foto avalanche diodes (SPAD) (Micro Photon Devices, Bolzano, Italy) ka ejiri mee ihe nchọpụta.Ejiri ngwanrọ SymphoTime64 dị na azụmaahịa (PicoQuant GmbH, Berlin, Germany) were nchịkọta data na nyocha abụọ ahụ.
A na-etinye microliter iri ise nke ihe nlele LLPS na olulu mmiri μ-Slide angiogenesis (Ibidi GmbH, Gräfelfing, Germany).Onyonyo a na-esi na ya pụta lekwasịrị anya na 20µm n'elu ala nke ọma maka ebumnuche ọrụ kacha mma maka ụmụ irighiri mmiri kwụsịtụrụ na ~ 1 µm maka rafts na ntụpọ nwere mkpebi axial opekata mpe 0.25 µm/pixel na oge igbu oge nke 400 µs/pixel.Họrọ data site n'itinye ọnụ ụzọ siri ike dabere na nkezi mgbama mgbaama ndabere ndabere (PBG, mean + 2σ) maka ọwa ọ bụla ka ahọpụtara naanị mmiri mmiri protein ụmụ irighiri mmiri, rafts, rafts, ma ọ bụ ntụpọ, na-enyocha ebe ọ bụla enwere ike isi na nke a gbasasịrị.Iji nyochaa ogologo ndụ nke ụdị ọ bụla (τ) nke ọwa ọ bụla (green, "g" maka AF488 na uhie, "r" maka Atto647N), anyị ahọrọla mpaghara mmasị (ROI) nwere ụmụ irighiri mmiri, rafts, ma ọ bụ ntụpọ (Ntinye mgbakwunye 1). ).8b) ma wepụta ha site n'itinye ire ere nke ndụ ha (τD, τR na τP maka ụmụ irighiri mmiri, rafts ma ọ bụ ntụpọ, n'otu n'otu, lee Mgbakwunye Fig. 8c) na ọwa ọ bụla na-eji nyocha ọdụ ọdụ na ụdị ire ere abụọ.Nkezi τ sitere na τ .ROI ndị rụpụtara foton ole na ole maka nrụpụta ọtụtụ ihe ka ewepụrụ na nyocha ahụ.Mbipu ejiri bụ <104 foton maka rafts na ntụpọ yana 103 maka ọdịda.Mkpụrụ mmiri ndị ahụ nwere ọnụ ụzọ dị ala n'ihi na ọ na-esiri ike ịnweta akụkụ ire ere na ụkpụrụ dị elu dị elu, ebe ọ bụ na ụmụ irighiri mmiri dị n'ọhịa ihe oyiyi na-adịkarị obere ma dị ntakịrị.A tụfuru ROI nwere ọnụ ọgụgụ photon karịa oke mkpokọta foton (nke edobere na> 500 counts/pixel) maka nyocha.Dakọtara usoro ire ere ike nke enwetara site na mpaghara mmasị na ike na 90% nke kacha (obere mgbe oke ire ere) site na mmalite nke ndụ ọrụ iji hụ na nnyonye anya IRF pere mpe ka ị na-edobe otu ihe ahụ maka ire ere niile. ntọala mpio oge ikwu E nyochara 25 ruo 50 ROI maka rafts na ntụpọ yana 15-25 ROI maka ọdịda, onyonyo ahọpụtara site na ihe karịrị 4 oyiri edere site na opekata mpe nnwale 3 nwere onwe.Ejila t-ule ọdụdụ abụọ iji nyochaa ọdịiche ndekọ ọnụ ọgụgụ dị n'etiti ụdị ma ọ bụ n'etiti sistemu coacervate.Maka nyocha pikselụ-site-pixel nke ndụ ndụ (τ), a gbakọrọ ngụkọta mbelata nke oge ndụ niile n'ọhịa maka ọwa ọ bụla yana ihe dị ka akụkụ 2/3 nke attenuation ihe atụ.Emebere ntugharị ndụ oge ndụ maka pikselụ ọ bụla site na iji ụkpụrụ τ agbakọrọ na mbụ, na-ebute pseudocolor FLIM oyiyi dabara.Ogologo oge ndụ nke ọdụdụ dabara adaba bụ otu n'ofe niile nke otu ọwa, na ire ere nke ọ bụla na-emepụta foton zuru oke iji nye ihe dabara adaba.Maka nyocha FRET, ahọpụtara pikselụ site n'itinye ọnụ ụzọ ike dị ala nke 100 photon, bụ nke chịkọtara mgbama ndabere (FBG) nke foton 11.A na-edozi ike ọkụ ọkụ nke ọwa ọ bụla site na nyocha nyocha nke ihe mgbazi: 69 spectral crosstalk α bụ 0.004, mkpali kpọmkwem β bụ 0.0305, nchọpụta nchọpụta γ bụ 0.517.A na-agbakọ arụmọrụ FRET na ọkwa pikselụ site na iji nhata ndị a:
ebe FDD bụ ọkụ ọkụ na-ahụ anya na ọwa inye onyinye (acha akwụkwọ ndụ akwụkwọ ndụ), FDA bụ ọkụ ọkụ na-ahụ anya na ọwa nnabata (red) n'okpuru mkpali na-apụtaghị ìhè, na FAA bụ ọkụ ọkụ na-ahụ anya na ọwa nnabata (red) n'okpuru mkpali kpọmkwem ( PIE).A na-ahụ ọkụ ọkụ ọkụ ọkụ na ọwa).
Debe 100 µl nke azịza mmeghachi omume LLPS nwere 25 µM monomeric Tau441 na-enweghị akara (ya na ma ọ bụ na-enweghị 25 µM αS) na nchekwa LLPS (agbakwunyere dị ka nke dị n'elu) na microplates 96-nke na-adịghị ejikọta ya na mkpuchi mkpuchi na nrapado na ntinye mmiri nke WF microscopy enyochala ya. nha nhata.n'ime 10 min.Mgbe awa 48 nke incubation na ụlọ okpomọkụ, a kwadoro ọnụnọ nke rafts protein na ntụpọ.Mgbe ahụ jiri nlezianya wepụ mmiri mmiri n'elu rafts site na olulu mmiri, wee tinye 50 L nke dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) na incubate maka 10 min.Ntụnye nnu dị elu na-achọpụta na LLPS agaghị emeghachi ya n'ihi PEG fọdụrụnụ, a ga-ekewasịkwa mgbakọ protein nwere naanị site na mmekọrịta electrostatic.A na-ejizi micropipette kpochapụ ala nke olulu mmiri ahụ wee bufee ihe ngwọta nke si na ya pụta na olulu mmiri efu.Mgbe incubation nke samples na 50 μM ThT maka 1 h, ọnụnọ nke dịpụrụ adịpụ ntụpọ enyocha site WF microscopy.Kwadebe αS fibrils sonicated site na incubating 300 µl nke a 70-µM αS ngwọta na PBS na pH 7.4, sodium azide 0.01% na 37 Celsius C na 200 rpm na orbital shaker maka 7 ụbọchị.A na-emezi ihe ngwọta ahụ na 9600 × g maka 30 min, a na-emegharị pellet na PBS pH 7.4 na sonicated (1 min, 50% cycle, 80% amplitude in a Vibra-Cell VC130 sonicator, Sonics, Newton, USA) fibril samples. na nkesa nha nha nha nke obere fibrils.
A na-eme nyocha FCS/FCCS na nchọpụta ihe nhụta nhụta nke agba abụọ (TCCD) n'otu oge MT200 na-edozi fluorescent confocal microscope (Pico-Quant, Berlin, Germany) ejiri mee ihe nyocha microscopy FLIM-FRET site na iji ọnọdụ PIE.Agbakwunyere ike laser maka nnwale ndị a na 6.0 µW (481 nm) na 6.2 µW (637 nm).Nchikota nke ike laser ndị a ka ahọpụtara iji mepụta nchapụta yiri nke ahụ maka ụzọ abụọ nke fluorophores ejiri mee ihe mgbe ha na-enweta ọnụ ọgụgụ kacha mma yana zere ịchacha foto na saturation.Ejiri ngwanro SymphoTime64 dị na-azụmaahịa 2.3 (PicoQuant, Berlin, Germany) chịkọta mkpokọta data na nyocha abụọ ahụ.
A na-agwakọta ihe atụ nke mkpokọta αS/Tau dịpụrụ adịpụ nke enwetara site na iji LLPS na nchekwa dịpụrụ adịpụ na ntinye monomolecular kwesịrị ekwesị (nke a bụ 1: 500 dilution, ebe ọ bụ na mkpokọta adịlarị na mkpokọta dị ala ma ọ bụrụ na e kewapụrụ ya na sample coacervate).A na-etinye ihe nlele ozugbo na mkpuchi mkpuchi (Corning, USA) ejiri ngwọta BSA kpuchie ya na ntinye nke 1 mg / ml.
Maka nyocha PIE-smFRET na ọwa akwụkwọ ndụ akwụkwọ ndụ na ọbara ọbara, etinyere ntakịrị ike nke photons 25 iji wepụta akara ngosi dị ala nke ihe omume monomeric kpatara (rịba ama na ndị monomers karịrị mkpokọta mkpokọta ma e jiri ya tụnyere mkpokọta ndị dịpụrụ adịpụ).A gbakọrọ ọnụ ụzọ a dị ka okpukpu ise nke nkezi ike monomeric αS nwetara site na nyocha nke ụdị monomer dị ọcha iji họrọ kpọmkwem mkpokọta maka nyocha.Sekit mbanye PIE, yana nnweta data TSCPC, enyerela aka itinye nzacha ịdị arọ ndụ oge niile nke na-enyere aka iwepụ ndabere na mkparịta ụka spectral.A na-edozi ike ọkụ ahọpụtara site na iji ọnụ ụzọ ndị a dị n'elu site na iji mgbama n'azụ ekpebisi ike site na histogram nke ihe omume yana ike/bin nke ihe nlere naanị.Ihe mgbawa jikọtara ya na nnukwu mkpokọta na-ejikarị ọtụtụ bins na-esochi n'usoro oge (nke edobere na 1 ms).N'okwu ndị a, a họọrọ bin nke ike kachasị.Maka nyocha FRET na stoichiometric, ejiri usoro gamma factor γ (0.517) kpebisie ike.Spectral crosstalk na onyinye mkpali ozugbo bụ nke a na-eleghara anya (ekpebie na nnwale) na ike laser mkpali ejiri.A na-agbakọ arụmọrụ na stoichiometry nke FRET na mgbawa dị ka ndị a.