347 12.7 * 1.24mm Tubing ea tšepe e sa hloekang e kopantsoeng, mochine oa molek'hule oa synchronous electrostatic condensation le coaggregation ea α-synuclein le tau

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347 Tlhaloso ea Pipe ea tšepe e sa hloekang

347 12.7 * 1.24mm tšupa e entsoeng ka tšepe e sa hloekang

Kantle Diameter: 6.00 mm OD ho fihla ho 914.4 mm OD, Boholo bo fihlang ho 24” NB e fumaneha Ex-stock, OD Size Steel Tubes available Ex-stock

SS 347 Botenya ba Pipe: 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, joalo-joalo (0.5-12mm) Kapa boholo bo sa tloaelehang bo lokisoe kamoo ho hlokahalang.

Mofuta: SS 347 Liphaephe tse se nang Mokotla |SS 347 ERW Liphaephe |SS 347 Welded Pipes |SS 347 Fabricated Pipes |SS 347 CDW Tubes, LSAW Pipes / Seam-welded / Redrawn

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

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Tšireletso ea ho qetela: Likotlolo tsa Plastiki |Kantle ho Qetella: 2B, No.4, No.1, No.8 Mirror Finish bakeng sa Liphaephe tsa Stainless Steel, Qetella ho ea ka Litlhoko tsa bareki

Boemo ba thomello: E kolobisitsoe ebile e khethiloe, e hloekisitsoe, e Khanyang e Khanyang, e Khaotsoe ka Cold

Tlhahlobo, Litlaleho tsa Teko: Litlaleho tsa Teko ea Mill, EN 10204 3.1, Litlaleho tsa Lik'hemik'hale, Litlaleho tsa Mechini, Litlaleho tsa Tlhahlobo ea PMI, Litlaleho tsa Tlhahlobo ea Pono, Litlaleho tsa Tlhahlobo ea Mokha oa Boraro, Litlaleho tse Amoheletsoeng tsa Lab tsa NABL, Tlaleho ea Teko e Senyehileng, Litlaleho tsa Tlhahlobo tse sa Senyeheng.

Ho paka: E pakiloe ka Mabokoseng a Mapolanka, Mekotla ea polasetiki, Melapo ea tšepe e bokeletsoeng, kapa ho latela likopo tsa bareki.

Tse khethehileng: Boholo le Lintlha tse ling ntle le ka holimo li ka etsoa ka kopo

SS 347 Pipe Size Range:1/2 inch NB, OD ho isa ho 24 inch

ASTM A312 347: Peipi ea austenitic e se nang seam le e otlolohileng e etselitsoeng mocheso o phahameng le ts'ebeletso e senyang ka kakaretso.Filler tšepe ha e lumelloe nakong ea tjheseletsa.

ASTM A358 347: Motlakase o kopantsoeng le peipi ea austenitic e cheselitsoeng bakeng sa ts'ebeletso e senyang le / kapa mocheso o phahameng.Ka tloaelo, ke pipe e fihlang ho 8 inch feela e hlahisoang ho latela tlhaloso ena.Ho eketsoa ha tšepe ea filler ho lumelloa nakong ea welding.

ASTM A790 347: Phaephe e se nang moroko le e otlolohileng e cheselitsoeng ka ferritic/austenitic (duplex) e etselitsoeng ts'ebeletso e senyang ka kakaretso, e toboketsa haholo ho hanyetsa khatello ea maikutlo ho phatloha.

ASTM A409 347: Motsoako o otlolohileng oa seam kapa spiral-seam fusion ea motlakase e cheselitsoeng bophara bo boholo ba austenitic light-wall pipe ka boholo ba 14” ho isa ho 30” e nang le mabota a Sch5S le Sch 10S bakeng sa ho senya le/kapa holimo.

ASTM A376 347: Pipe ea austenitic e se nang moeli bakeng sa lisebelisoa tse phahameng tsa mocheso.

ASTM A813 347: Peipi ea austenitic e nang le sekoti e le 'ngoe, e nang le mochini o le mong kapa habeli bakeng sa mocheso o phahameng le lits'ebetso tse senyang ka kakaretso.

ASTM A814 347: Pipe ea austenitic e sebetsang ka mokhoa o batang bakeng sa mocheso o phahameng le tšebeletso e senyang ka kakaretso.

347H Liphaephe tsa tšepe tse sa hloekang tse entsoeng ka lik'hemik'hale

Lisebelisoa tsa Mechanical tsa Pipe ea 347H e se nang tšepe

Lipeipi tsa Stainless 347H tsa 'Mele

Mephato e lekanang bakeng sa Pipe ea tšepe e sa hloekang ea 347H

Amyloid alpha-synuclein (αS) aggregation ke letšoao la lefu la Parkinson le li-synucleinopathies tse ling.Haufinyane tjena, protheine ea tau e atisang ho amahanngoa le lefu la Alzheimer e 'nile ea amahanngoa le lefu la αS' me ea fumanoa e le sebakeng se kopanetsoeng ho αS-rich inclusions, le hoja mokhoa oa limolek'hule oa ho kopana ha liprotheine tse peli o ntse o sa hlaka.Re tlaleha mona hore karolo ea αS e arohana ka li-condensate tsa mokelikeli ka condensation e rarahaneng ea electrostatic e nang le polypeptide e qosoang hantle joalo ka tau.Ho itšetlehile ka kamano ea αS bakeng sa li-polycations le tekanyo ea ho fokotseha ha valence ea marang-rang a coagulation, li-clots li kena ka potlako kapa ho kopana ho lateloa ke ho kopanya butle-butle ha amyloid.Ka ho kopanya sehlopha sa mekhoa e tsoetseng pele ea biophysical, re ile ra khona ho khetholla karolo ea mokelikeli-mokelikeli αS / Tau le ho khetholla lintlha tsa bohlokoa tse lebisang ho thehoeng ha lihlopha tse fapaneng tse nang le liprotheine ka bobeli ka mokelikeli oa protheine condensate.
Ntle le li-membrane compartments, karohano ea sebaka ka har'a lisele e ka boela ea finyelloa ka ho thehoa ha 'mele e teteaneng e nang le protheine, e kang metsi e bitsoang biomolecular condensates kapa marotholi, ka mokhoa o tsejoang e le liquid-liquid phase separation (LLPS).Marotholi ana a thehoa ke litšebelisano tse ngata tsa nakoana, hangata pakeng tsa liprotheine kapa liprotheine le RNA, 'me li sebeletsa mesebetsi e sa tšoaneng hoo e batlang e le lits'ebetsong tsohle tse phelang.Palo e kholo ea liprotheine tse nang le bokhoni ba LLP li bonts'a tatellano ea ho rarahana ho tlase ho nang le mathata a mangata a tlhaho le ho thehoeng ha biomolecular condensates3,4,5.Liphuputso tse ngata tsa liteko li senotse hore liprotheine tse etsang li-condensate tse kang mokelikeli hangata lia tenyetseha, tse atisang ho senyeha, le tse ngata-ngata, le hoja ho tsejoa ho fokolang ka limolek'hule tse laolang kholo le kholo ea li-condensate tsena hore li be thata haholoanyane. naha..
Lintlha tse ncha li tšehetsa khopolo ea hore LLPS e tsamaisoang ke protheine e sa laoleheng le phetoho ea marotholi hore e be mehaho e tiileng e ka 'na ea e-ba litsela tse nepahetseng tsa lisele tse lebisang ho thehoeng ha likarolo tse chefo tse sa qhibiliheng tseo hangata e leng matšoao a mafu a senyehang.Liprotheine tse ngata tse amanang le LLPS tse amanang le intrinsically disordered (IDPs), tse atisang ho qosoa haholo ebile li fetoha habonolo, esale li amahanngoa le neurodegeneration ka ts'ebetso ea amyloid aggregation.Haholo-holo, biomolecular IDP condensates joalo ka FUS7 kapa TDP-438 kapa liprotheine tse nang le likarolo tse kholo tse rarahaneng joalo ka hnRNPA19 li bonts'itsoe hore li tsofala ho ba liforomo tse kang gel kapa tse tiileng ka ts'ebetso e bitsoang fluidization.motsoako.ho ea ho solid phase transition (LSPT) e le ts'ebetso ea nako kapa karabelo ho liphetoho tse itseng tsa morao-rao tsa phetolelo kapa liphetoho tse kholo tsa pathologically1,7.
IDP e 'ngoe e amanang le LLPS ka har'a vivo ke Tau, protheine e amanang le microtubule e sa sebetseng eo amyloid aggregation e kentsoeng lefung la Alzheimer10 empa hape e sa tsoa ameha ho lefu la Parkinson (PD) le li-synaptic nuclear proteinopathies 11, 12, 13 li amana.Tau e bonts'itsoe hore e ikarola ka boithatelo ho tsoa ho tharollo/cytoplasm ka lebaka la tšebelisano e ntle ea electrostatic14, e entseng hore ho thehoe marotholi a nang le tau a nonneng a tsejoang ka hore ke electrostatic coacervates.Ho boetse ho hlokometsoe hore mofuta ona oa tšebelisano e sa tsitsang ke matla a susumetsang a mangata a biomolecular condensates ka tlhaho15.Tabeng ea protheine ea tau, electrostatic aggregation e ka thehoa ka ho kopanya ho bonolo, moo libaka tse fapaneng tse qosoang tsa protheine li bakang ts'ebetso ea cleavage, kapa ka ho kopanya ho rarahaneng ka ho sebelisana le lipolymer tse mpe tse kang RNA.
Haufinyane tjena, α-synuclein (αS), IDP ea amyloid e amehang ho PD le mafu a mang a neurodegenerative ka kakaretso a tsejoang e le synucleinopathy17,18, e bontšitsoe ka mehlala ea lisele le liphoofolo19,20 e tsepamisitsoeng ka protheine condensates ka boitšoaro bo kang ba mokelikeli.Liphuputso tsa in vitro li bontšitse hore αS e kena LLPS ka ho kopanya habonolo ka ho sebelisana ha metsi haholo, le hoja ts'ebetso ena e hloka hore ho be le liprotheine tse phahameng ka ho khetheha le linako tse telele tsa ho incubation19,21.Hore na li-condensate tse nang le αS tse bonoang ka har'a vivo li thehoa ke ts'ebetso ena kapa tse ling tsa LLPS e ntse e le taba ea bohlokoa e sa rarolloang.Ka mokhoa o ts'oanang, le hoja αS amyloid aggregation e 'nile ea hlokomeloa ka li-neurone ho PD le li-synucleinopathies tse ling, mokhoa o tobileng oo ka oona αS e kenang ka har'a intracellular amyloid aggregation e ntse e sa hlaka, kaha ho fetella ha protheine ena ha ho bonahale ho baka ts'ebetso ena ka boeona.Hangata ho hlokahala tšenyo e eketsehileng ea lisele, ho fana ka maikutlo a hore libaka tse itseng tsa cellular kapa microenvironments li hlokeha bakeng sa ho tsosolosa likopano tsa intracellular αS amyloid.Tikoloho e le 'ngoe ea cellular e atisang ho kopana e ka' na ea e-ba ka hare ho protheine condensates 23.
Hoa thahasellisa hore αS le tau li fumanoe li sebelisana sebakeng sa batho ba nang le lefu la Parkinson le li-synucleinopathies tse ling 24,25 'me liteko li tlalehile kamano ea synergistic pathological pakeng tsa liprotheine tse peli tsa 26,27 tse fanang ka maikutlo a kamano e ka bang teng pakeng tsa aggregation αS le. tau mafung a neurodegenerative.bokudi.αS le tau li fumanoe li sebelisana 'me li khothalletsa ho bokellana ha e mong ka har'a vitro le ka vivo 28,29 le lihlopha tse fapaneng tse entsoeng ka liprotheine tsena tse peli li hlokometsoe bokong ba bakuli ba nang le synucleinopathies 30.Leha ho le joalo, ha ho tsejoe hanyane ka motheo oa limolek'hule oa tšebelisano lipakeng tsa αS le tau le mokhoa oa ho kopanya ha eona.αS e tlalehile hore e sebelisana le tau ka khoheli ea motlakase pakeng tsa sebaka sa C-terminal se qosoang hampe haholo sa αS le sebaka se bohareng se ruileng sa proline sa tau, seo hape se ruisitsoeng ka masala a qosoang hantle.
Thutong ena, re bontša hore αS ka sebele e ka ikarola ka marotholi ka electrostatic complex condensation ka pel'a protheine tau, ho fapana le ho sebelisana ha eona le polypeptide tse ling positively qosoa tse kang poly-L-lysine (pLK), 'me tshebetsong ena .αS e sebetsa joalo ka molek'hule ea sekala bakeng sa marang-rang a marotholi.Re hlokometse phapang e hlokomelehang nakong ea kholo ea li-coacervates tsa electrostatic αS, tse amanang le phapang ea valency le matla a ho sebelisana ha liprotheine tse amehang marang-rang a coacervate.Hoa thahasellisa hore ebe re hlokometse ho kopanngoa ha liprotheine tsa αS le tau amyloid ka li-coacervate tsa metsi tse phetseng nako e telele 'me ra tseba lintlha tse ling tsa bohlokoa tse lebisang ho kopanngoa ha liprotheine tsena tse peli ho li-coacervates tse joalo.Mona re hlalosa ka ho qaqileng ts'ebetso ena, e leng mokhoa o ka khonehang oa limolek'hule o itšetlehileng ka colocalization ea liprotheine tse peli ka li-inclusions tse khethehileng tsa mafu.
αS e na le mohatla oa C-terminal o nang le anionic haholo ho pH e sa nke lehlakore (setšoantšo sa 1a), 'me re nahana hore e ka kena LLPS ka condensation ea electrostatic complexes le polycationic disordered polypeptide molecules.Re sebelisitse 100-residue poly-L-lysine (pLK) e le molek'hule oa mohlala oa ho qala ka lebaka la tlhaho ea eona e ntle le e sa tsitsang ea polymeric ka ho se nke lehlakore pH 32. Ntlha ea pele, re netefalitse hore pLK e sebelisana le Ct domain ea αS ka Tharollo ea NMR spectroscopy. (Setšoantšo sa 1b) ho sebelisa 13C / 15N-e ngotsoeng αS boteng ba ho eketseha ha αS: pLK molar ratios.Tšebelisano ea pLK le Ct-domain ea αS e iponahatsa ka ho ferekanngoa ha phetoho ea lik'hemik'hale le ho fokotseha ha tlhōrō ea tlhōrō sebakeng sena sa protheine.Ho khahlisang, ha re ne re kopanya αS le pLK sebakeng sa αS sa hoo e ka bang.5–25 µM boteng ba polyethylene glycol (5–15% PEG-8) (e tloaelehileng LLPS buffer: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) hang-hang re ile ra feta tšimong e pharaletseng ea ho thehoa ha liprotheine. .marotholi a ile a hlokomeloa ho sebelisa fluorescence (WF) le e khanyang-tšimo (BF) microscopy (sa feiga. 1c).1-5 µm marotholi a nang le concentrated αS (e kenyelelitsoe 1 µM AlexaFluor488-labeled αS, AF488-αS), thepa ea bona ea electrostatic e ka fumanoa ho hanyetsa ha bona ho 10% 1,6-hexanediol (1,6-HD) le kutloisiso ea eona ho keketseho ea mahloriso a NaCl (setšoantšo sa 1c).Sebopeho sa mokelikeli o kang oa li-coacervates tsa αS / pLK electrostatic complex e bontšoa ke bokhoni ba bona ba ho fuse ka har'a milliseconds (setšoantšo sa 1d).Ho sebelisa turbidimetry, re quantified sebopeho sa marotholi tlas'a maemo ana, o ile a tiisa tlhaho electrostatic tsa ho sebelisana ka sehloohong amanang le botsitso ba eona (sa feiga. 1e), 'me hlahlojoe phello ea likarolelano tse sa tšoaneng polymer ka tshebetso LLPS (sa feiga. 1f).Le hoja sebopeho sa marotholi se hlokomeloa holim'a mefuta e mengata ea likarohano tsa polymer, ts'ebetso e ntle haholo ha pLK e feta αS.Li-LLP li boetse li hlokometsoe ho sebelisoa mochine o fapaneng oa lik'hemik'hale oa dextran-70 (70 kDa) kapa ho sebelisoa mefuta e fapaneng ea lifomate, ho kenyeletsoa marotholi a li-slide tsa khalase, liliba tsa microplate tsa lisebelisoa tse fapaneng, Eppendorf kapa quartz capillaries.
kemelo ea Schematic ea libaka tse fapaneng tsa protheine mefuteng ea WT-αS le ΔCt-αS e sebelisitsoeng thutong ena.Sebaka sa amphipathic N-terminal domain, sebaka sa hydrophobic amyloid-forming (NAC), le sebaka sa C-terminal se qosoang hampe li bontšoa ka boputsoa, ​​​​orange, le bofubelu, ka ho latellana.'Mapa oa Net Charge Per Residual (NCPR) oa WT-αS oa bontšoa.b NMR tlhahlobo ea tšebelisano ea αS / pLK ha ho se na macromolecular clumps.Ha mahloriso a pLK a ntse a eketseha (αS: pLK molar ratios ea 1: 0.5, 1: 1.5, le 1:10 li bontšoa ka botala bo khanyang, botala le botala bo lefifi, ka ho latellana).c Coacervate αS/pLK (molar ratio 1:10) ho 25 µM (1 µM AF488-lebeled αS or Atto647N-labeled pLK for WF imaging) in LLPS buffer (top) kapa e tlatselletsoa ka 500 mM NaCl (tlase 10 kapa ka morao) % 1,6-hexanediol (1,6-HD; tlase ka ho le letona).Scale bar = 20 µm.d Moemeli oa litšoantšo tsa microscopic tsa BF droplet fusion ea αS / pLK (molar ratio 1: 10) ka bongata ba 25 μM;metsu e bontša ho kopanngoa ha marotholi ka bomong (metsu e khubelu le e mosehla) ho lerotholi le lecha (motsu oa lamunu) ka hare ho 200 ms).Sekhahla = 20 µm.e Ho hasanya ha leseli (ho 350 nm) αS/pLK aggregation ho LLPS buffer pele le ka mor'a ho eketsoa ha 500 mM NaCl kapa 10% 1,6-HD ho 25 µM αS (N = 3 sampole replicates, ho bolela le ho kheloha ho tloaelehileng ho boetse ho bontšitsoe).f setšoantšo sa BF (holimo) le tlhahlobo ea ho hasanya ha leseli (ho 350 nm, ka tlase) ea αS / pLK aggregation ho 25 μM αS ka ho eketseha αS: pLK molar ratio (N = 3 sampuli replicates, ho bolela le ho kheloha ho tloaelehileng ho boetse ho bontšitsoe).Sekhahla = 10 µm.Sekala se setšoantšong se le seng se bontša boholo ba litšoantšo tsohle phanele e le 'ngoe.Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
Ho ipapisitsoe le litebello tsa rona tsa αS/pLK electrostatic complex condensation le litebello tse fetileng tsa αS joalo ka molek'hule ea bareki ea tau/RNA condensate ka tšebelisano e tobileng le tau31, re ile ra nahana hore αS le tau li ka arola hammoho le solvent ha RNA e le sieo. condensation.ka li-electrostatic complexes, 'me αS ke protheine ea scaffold ho αS/Tau coacervates (sheba kabo ea tefiso ea tau ho Figure 2e).Re hlokometse hore ha 10 μM αS le 10 μM Tau441 (e nang le 1 μM AF488-αS le 1 μM Atto647N-Tau, ka ho latellana) li ne li kopantsoe hammoho ka LLPS buffer, li ile tsa theha likaroloana tsa protheine tse nang le liprotheine ka bobeli, joalokaha li bonoa ke microscopy ea WF.(Setšoantšo sa 2a).Colocalization ea liprotheine tse peli ka marotholi e ile ea tiisoa ke confocal (CF) microscopy (Supplementary Fig. 1a).Boitšoaro bo tšoanang bo ile ba hlokomeloa ha dextran-70 e sebelisoa e le moemeli oa ho kopanya (Supplementary Fig. 1c).Re sebelisa PEG kapa dextran e ngotsoeng ke FITC, re fumane hore mahlahana a mabeli a mangata a ne a arolelitsoe ka ho lekana ho pholletsa le lisampole, a sa bontše karohano kapa mokhatlo (Supplementary Fig. 1d).Ho e-na le hoo, e fana ka maikutlo a hore tsamaisong ena ba khothalletsa karohano ea mekhahlelo ka liphello tse ngata tsa macromolecular, kaha PEG ke moemeli ea tsitsitseng ea tsitsitseng, joalokaha ho bonoa lits'ebetsong tse ling tsa LLP33,34.Marotholi ana a nang le liprotheine tse ngata a ne a e-na le boikutlo bo botle ho NaCl (1 M) empa eseng ho 1,6-HD (10% v / v), e tiisang thepa ea bona ea motlakase (Supplementary Fig. 2a, b).Boitšoaro ba bona ba mokelikeli bo tiisitsoe ka ho shebella liketsahalo tsa marotholi a millisecond tse kopanyang ho sebelisa BF microscopy (setšoantšo sa 2b).
a Confocal (CF) litšoantšo tsa microscopy tsa αS/Tau441 coacervates ho LLPS buffer (10 μM ea protheine ka 'ngoe, 0.5 μM ea AF488 e ngotsoeng αS le Atto647N-e ngotsoeng Tau441).b Lits'oants'o tse fapaneng tsa tšitiso e fapaneng (DIC) tsa liketsahalo tsa αS/Tau441 tsa fusion ea marotholi (10 μM bakeng sa protheine ka 'ngoe).c Setšoantšo sa mohato se ipapisitseng le khaso e khanyang (ho 350 nm) ea Tau441 LLPS (0–15 µM) bosieong (ka ho le letšehali) kapa boteng (ka ho le letona) ba 50 µM αS.Mebala e futhumetseng e bontša ho hasana ho eketsehileng.d Ho hasana ha leseli la disampole tsa αS/Tau441 LLPS ka ho eketseha ha αS concentration (Tau441 at 5 µM, N = 2-3 sampole repetitions joalokaha ho bontšitsoe).e Boemeli ba boqapi ba mefuta e meng ea liprotheine tsa tau le libaka tse fapaneng tsa protheine e sebelisitsoeng thutong ena: sebaka se se nang molato sa N-terminal (e khubelu), sebaka se ruileng sa proline (buluu), sebaka se tlamang sa microtubule (MTBD, se totobalitsoeng ka lamunu), le Amyloid-forming pair.libaka tsa filament (PHF) tse ka hare ho MTBD (grey).'Mapa oa Net Charge Per Residue (NCPR) oa Tau441 oa bontšoa.f Ho sebelisa 1 µM AF488-lebeled αS le Atto647N-lebeled ΔNt-, ho sebelisa 1 µM AF488-leibole αS kapa ΔCt-αS boteng ba ΔNt-Tau (holimo, 10 µM ka protheine) kapa K18 (0 µM5 ka tlase, protheine ka 'ngoe ) ) ) li-micrograph tsa WF tse koafalitsoeng ho LLPS kapa buffer ea K18.Likarolo tsa sekala setšoantšong se le seng li emela boholo ba litšoantšo tsohle phanele e le 'ngoe (20 µm bakeng sa liphanele a, b le f).Lintlha tse tala bakeng sa liphanele c le d li fanoa e le lifaele tsa data tse tala.
E le ho hlahloba karolo ea αS ts'ebetsong ena ea LLPS, re ile ra qala ho batlisisa phello ea αS holim'a botsitso ba marotholi ka nephelometry ho sebelisa likhahla tse ntseng li eketseha tsa NaCl (Fig. 2c).Ha letsoai le ntse le phahama lisampoleng tse nang le αS, ho na le boleng bo phahameng ba ho hasanya ha leseli (ho 350 nm), e bonts'ang karolo ea botsitso ea αS tsamaisong ena ea LLPS.Phello e ts'oanang e ka bonoa ka ho eketsa khatello ea αS ('me ka hona αS: Tau441 ratio) ho ea ho hoo e ka bang.Keketseho ea makhetlo a 10 e amanang le mohopolo oa tau (5 µM) (setšoantšo sa 2d).E le ho bontša hore αS ke protheine ea scaffold ka li-coacervates, re ile ra etsa qeto ea ho batlisisa boitšoaro ba LLPS-stirupted Tau mutant, e se nang sebaka se fosahetseng sa N-terminal (masala a 1-150, bona setšoantšo sa 2e) se bitsoang ΔNt-Tau.WF microscopy le nephelometry li netefalitse hore ΔNt-Tau ka boeona ha ea ka ea kena LLPS (Fig. 2f le Supplementary Fig. 2d), joalokaha ho tlalehiloe pele 14. Leha ho le joalo, ha αS e ne e kenyelletsoa ho litharollo tsa ho qhalakanngoa ha mofuta ona oa Tau o fokolitsoeng, ts'ebetso ea LLPS e ne e le ka ho feletseng. e tsosolositsoe ka ho teteana ha marotholi haufi le ho teteana ha marotholi a litharollo tsa boholo bo feletseng tsa Tau le αS tlas'a maemo a tšoanang le a mangata a protheine.Tshebetso ena e ka boela ea hlokomeloa tlas'a maemo a tlaase a mangata a macromolecular (Supplementary Fig. 2c).Karolo ea sebaka sa C-terminal αS, empa eseng bolelele bohle ba eona, ts'ebetsong ea LLPS e ile ea bontšoa ka ho thibela ho thehoa ha marotholi ho sebelisa mofuta oa C-terminal truncated αS o haellang masala 104-140 (Fig. 1a) ea (ΔCt- αS) protheine (Fig. 2f le Supplementary Fig. 2d).Colocalization ea αS le ΔNt-Tau e tiisitsoe ke confocal fluorescence microscopy (Supplementary Fig. 1b).
Ho tsoela pele ho lekola mochini oa LLPS lipakeng tsa Tau441 le αS, ho ile ha sebelisoa mofuta o mong oa Tau, e leng sekhechana sa helical filament core (PHF) sebakeng sa "microtubule-binding domain" (MTBD), seo haeba se na le likarolo tse 'ne tse pheta-phetoang, tse tsejoang hape. joalo ka sekhechana sa K18 (sheba setšoantšo sa 2e).Haufinyane tjena ho tlalehiloe hore αS ka ho khetheha e tlama protheine ea tau e sebakeng se ruileng sa proline ka tatellano e etellang pele sebaka se tlamang sa microtubule.Leha ho le joalo, sebaka sa PHF se boetse se na le masala a nang le chelete e ntle (sheba Setšoantšo sa 2e), haholo-holo lysine (masala a 15%), e leng se ileng sa re susumelletsa hore re leke hore na sebaka sena se boetse se kenya letsoho ho fokotseng motsoako oa αS / Tau.Re hlokometse hore K18 feela ha e khone ho qala LLPS maemong a ho fihla ho 100 μM tlas'a maemo a lekiloeng (LLPS buffer ka 15% PEG kapa 20% dextran) (Setšoantšo sa 2f).Leha ho le joalo, ha re eketsa 50 µM αS ho 50 µM K18, ho thehoa ka potlako ha marotholi a protheine a nang le K18 le αS ho ile ha hlokomeloa ke nephelometry (Supplementary Fig. 2d) le WF microscopy (Fig. 2f).Joalokaha ho lebeletsoe, ΔCt-αS ha ea ka ea khona ho tsosolosa boitšoaro ba LLPS ba K18 (Setšoantšo sa 2f).Rea hlokomela hore αS/K18 aggregation e hloka hore ho be le protheine e phahameng hanyane ho kenya LLPS ha e bapisoa le αS/ΔNt-Tau kapa αS/Tau441, lintho tse ling li lekana.Sena se lumellana le tšebelisano e matla ea sebaka sa αS C-terminal le sebaka sa Tau se ruileng sa proline ha se bapisoa le sebaka se tlamang sa microtubule, joalokaha se hlalositsoe pele 31.
Ka lebaka la hore ΔNt-Tau ha e khone ho etsa LLPS ha αS e le sieo, re khethile mofuta ona oa Tau e le mohlala oa ho khetholla αS/Tau LLPS ka lebaka la ho nolofatsa ha eona lits'ebetsong tsa LLPS tse nang le Tau ea bolelele bo felletseng (isotype, Tau441/Tau441).ka mekhoa ea ho kopanya e rarahaneng (heterotypic, αS/Tau441).Re bapisitse tekanyo ea αS aggregation (e le karolo ea protheine ea karolo e khutsufalitsoeng, fαS, c) tsamaisong ea αS / Tau le αS / ΔNt-Tau ka centrifugation le mokhoa o qhalakaneng oa tlhahlobo ea SDS-PAGE (bona 2e), e fumane litekanyetso tse tšoanang haholo. bakeng sa liprotheine tsohle ka nako e le 'ngoe.Haholo-holo, re fumane fαS, c 84 ± 2% le 79 ± 7% bakeng sa αS/Tau le αS/ΔNt-Tau, ka ho latellana, ho fana ka maikutlo a hore tšebelisano ea heterotypic pakeng tsa αS le tau e phahametse tšebelisano pakeng tsa limolek'hule tsa tau.pakeng tsa.
Tšebelisano le li-polycation tse fapaneng le phello ea ts'ebetso ea condensation ho αS kinetics e ile ea qala ho ithutoa ke ho hlaphoheloa ha fluorescence ka mor'a mokhoa oa photobleaching (FRAP).Re ile ra leka li-coacervates tsa αS/Tau441, αS/ΔNt-Tau le αS/pLK (100 μM αS e tlatsitsoeng ka 2 μM αS AF488-αS le 100 μM Tau441 kapa ΔNt-Tau kapa 1 mML pLK).Lintlha li ile tsa fumanoa nakong ea metsotso ea pele ea 30 ka mor'a ho kopanya likarolo tsa mohlala.Ho tsoa ho baemeli ba litšoantšo tsa FRAP (setšoantšo sa 3a, αS / Tau441 condensation) le nako ea bona e tsamaisanang le nako (setšoantšo sa 3b, Supplementary Fig. 3), ho ka bonoa hore αS kinetics e tšoana haholo le ea Tau441 coacervates.le ΔNt-Tau, e potlakileng haholo ka pLK.Li-coefficients tse baliloeng tsa diffusion bakeng sa αS ka hare ho coacervate ho latela FRAP (joalokaha ho hlalositsoe ke Kang et al. 35) ke D = 0.013 ± 0.009 µm2/s le D = 0.026 ± 0.008 µm2/s bakeng sa αS-1 le αS4 for αS4 tsamaiso ea αS/.pLK, Tau, le D = 0.18 ± 0.04 µm2 / s, ka ho latellana (Setšoantšo sa 3c).Leha ho le joalo, coefficient ea phallo ea αS karolong e qhalakaneng ke litaelo tse 'maloa tsa boholo bo phahameng ho feta mekhahlelo eohle e khutsufalitsoeng, joalokaha ho khethiloe ke Fluorescence Correlation Spectroscopy (FCS, bona Supplementary Fig. 3) tlas'a maemo a tšoanang (LLPS buffer) empa ha ho se na polycations (D = 8 ± 4 µm2/s).Ka hona, li-kinetics tsa phetolelo ea αS li fokotsehile haholo ka li-coacervates ha li bapisoa le liprotheine tse karolong e qhalakaneng ka lebaka la liphello tse boletsoeng tsa limolek'hule, le hoja li-coacervates kaofela li boloka thepa e kang ea metsi nakong ea halofo ea pele ea hora ka mor'a ho thehoa ha bona, ho fapana le karolo ea tau.kinetics e potlakileng ho pLK condensate.
a–c tlhahlobo ea FRAP ea αS dynamics (2% AF488-labeled αS) ho li-electrostatic coacervates.Lits'oants'o tse emetseng tsa liteko tsa αS/Tau441 FRAP ka makhetlo a mararo li bonts'oa ho (a), moo lidikadikwe tse khubelu li bonts'ang libaka tse senyehileng.Sekala sa sekala ke 5µm.b Karolelano ea li-curve tsa FRAP le (c) li-coefficients tsa diffusion (D) tse baliloeng bakeng sa 5–6 (N) marotholi a fapaneng ho tsoa litekong tse tharo tse sebelisang 100 µM αS le likhahla tse lekanang tsa Tau441 (khubelu) kapa ΔNt-Tau (putsoa) kapa pLK (e tala) ka makhetlo a leshome khatello ea LLPS.Phapang e tloaelehileng ea FRAP curve e bontšoa ka 'mala o nang le moriti.Ha ho bapisoa, coefficient ea phallo ea αS karolong e qhalakaneng e ile ea khethoa ka makhetlo a mararo ho sebelisoa fluorescence correlation spectroscopy (FCS) (sheba Supplementary Figure 3 le mekhoa ea boitsebiso bo eketsehileng).d Tšebeliso e tsoelang pele ea X-band EPR ea 100 μM TEMPOL-122-αS ka LLPS buffer ntle le polycation leha e le efe (e ntšo) kapa ka pel'a 100 μM Tau441 (e khubelu) kapa ΔNt-Tau (putsoa) kapa 1 mM pLK (e tala).Sets'oants'o se bonts'a pono e phahamisitsoeng ea mela e matla ea masimo moo ho hlahang liphetoho tse makatsang.e Li-curve tse tlamang tsa 50 μM TEMPOL-122-αS tse nang le li-polycation tse fapaneng ha ho se na LLPS (ha ho PEG).Bophahamo bo fokotsehileng ba sehlopha sa III ha se bapisoa le sehlopha sa II (IIII/III) sa spectrum se tloaelehileng sa EPR se bontšoa ho eketsa tekanyo ea molar ea Tau441 (e khubelu), ΔNt-Tau (putsoa) le pLK (e tala).Mela e mebala e bonts'a ho lekana le data ho sebelisoa mohlala o thata o tlamang o nang le libaka tse tlamang tse ts'oanang le tse ikemetseng mokoting ka mong.Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
E le tlatsetso, re ile ra batlisisa matla a αS ho li-coacervates tse fapaneng re sebelisa spin labeling (SDSL) le continuous electron paramagnetic resonance (CW-EPR).Mokhoa ona o ipakile o thusa haholo ho tlaleha ho feto-fetoha ha maemo le matla a IDP ka qeto ea sebele ea masala36,37,38.Ho fihlela sena, re thehile masala a cysteine ​​​​ka li-cys mutants tse le 'ngoe mme ra sebelisa 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Lintho tse tsoang ho Maleimide li li tšoaea.Ka ho khetheha, re kentse lipatlisiso tsa TEMPOL sebakeng sa 122 kapa 24 αS (TEMPOL-122-αS le TEMPOL-24-αS).Tabeng ea pele, re lebisa tlhokomelo sebakeng sa C-terminal ea protheine, e amehang ho sebelisana le polycations.Sebakeng seo, boemo ba 24 bo ka re fa leseli mabapi le matla a akaretsang a liprotheine tse ho condensate.Maemong ana ka bobeli, matšoao a EPR a fumanoeng bakeng sa liprotheine tsa mokhahlelo o qhalakaneng a tsamaisana le li-radicals tsa nitroxide sebakeng se tsamaeang ka potlako.Ka mor'a karohano ea karolo ka pel'a tau kapa pLK (100 μM TEMPOL-αS, Tau441 kapa ΔNt-Tau ka karolelano ea 1: 1 kapa pLK ka karolelano ea 1: 10), keketseho ea tlhōrō ea tlhōrō e ile ea bonoa palo ea EPR ea αS.Mohala oa tahlehelo o ile oa atolosoa, o bontšang ho fokotseha ha αS reorientation kinetics ka marotholi ha a bapisoa le protheine ka karolo ea dilute (Fig. 3d, Supplementary Fig. 4a).Liphetoho tsena li totobetse haholoanyane boemong ba 122. Ha e ntse e le boemong ba 24 boteng ba pLK ha boa ka ba ama kinetics ea probe, sebakeng sa 122 sebopeho sa line sa spectral se fetohile haholo (Supplementary Fig. 4a).Ha re ne re leka ho etsa mohlala oa spectra boemong ba 122 ba litsamaiso tse peli tsa αS / polycation ho sebelisa mohlala oa isotropic (Supplementary Figure 5a) e atisang ho sebelisoa ho hlalosa matla a li-spin-labeled IDP38,39, ha rea ​​ka ra khona ho tsosolosa ponahalo ea liteko..Spectral papiso ea boemo ba 24 spins phapang (Supplementary Fig. 5a).Sena se fana ka maikutlo a hore ho na le maemo a khethehileng sebakeng sa litlhophiso tsa spin ea sebaka sa C-terminal sa αS boteng ba polycations.Ha ho nahanoa karoloana ea αS karolong e khutsufalitsoeng tlas'a maemo a liteko tsa EPR (84 ± 2%, 79 ± 7%, le 47 ± 4% bakeng sa αS/Tau441, αS/ΔNt-Tau, le αS/pLK, ka ho latellana-bona Tlatsetso. Setšoantšo sa 2e sa tlhahlobo ea data c), ho ka bonoa hore ho atolosoa ho fumanoeng ke mokhoa oa EPR ho bonahatsa haholo-holo ho sebelisana ha sebaka sa C-terminal sa αS le li-polycation tse sa tšoaneng karolong e khutsufalitsoeng (phetoho e kholo ha u sebelisa TEMPOL-122- αS), eseng ho fokotsa protheine.Keketseho ea microviscosity e bonoa ka probe.Joalokaha ho lebeletsoe, palo ea EPR ea protheine tlas'a maemo a mang ntle le LLPS e ile ea tsosolosoa ka ho feletseng ha 1 M NaCl e kenngoa motsoako (Supplementary Fig. 4b).Ka kakaretso, lintlha tsa rona li fana ka maikutlo a hore liphetoho tse fumanoeng ke CW-EPR haholo-holo li bonahatsa ho sebelisana ha sebaka sa C-terminal sa αS le li-polycation tse sa tšoaneng karolong e khutsufalitsoeng, 'me tšebelisano ena e bonahala e le matla le pLK ho feta le Tau.
Bakeng sa ho fumana tlhaiso-leseling e batsi ka liprotheine tse ka har'a coacervate, re nkile qeto ea ho ithuta sistimi ea LLPS re sebelisa NMR ka tharollo.Leha ho le joalo, re ne re ka lemoha feela karolo ea αS e setseng karolong e qhalakaneng, e ka 'nang ea e-ba ka lebaka la ho fokotseha ha protheine dynamics ka hare ho coacervate le karolo e teteaneng e ka tlaase ho tharollo tlhahlobo ea NMR.Ha re sekaseka sebopeho le matla a protheine e setseng karolong e qhalakaneng ea sampole ea LLPS re sebelisa NMR (Supplementary Fig. 5c, d), re hlokometse hore protheine e itšoere ka mokhoa o batlang o tšoana ka pel'a pLK le ΔNt-Tau, ka bobeli. tse neng li le ka har'a sebopeho sa bobeli le matla a mokokotlo oa protheine, tse senotsoeng ke liteko tsa phetoho ea bobeli ea lik'hemik'hale le ho phomola ha R1ρ.Lintlha tsa NMR li bontša hore C-terminus ea αS e na le tahlehelo e kholo ea ho feto-fetoha ha maemo ha e ntse e boloka tlhaho ea eona e sa tsitsang, joalo ka tatellano ea protheine, ka lebaka la ho sebelisana ha eona le polycations.
Kaha ho atolosoa ha lets'oao la CW-EPR ho bonoa karolong e khutsufalitsoeng ea TEMPOL-122-αS ho bonts'a tšebelisano ea protheine le polycations, re entse tlhahiso ea EPR ho lekola kamano e tlamang ea αS ho li-polycation tse fapaneng ha LLPS e le sieo (ha ho pokello ea Buffer LLPS), e fana ka maikutlo a hore litšebelisano li ts'oana ka mekhahlelo e hlapollang le e tsepamisitsoeng (e netefalitsoeng ke data ea rona, Supplementary Fig. 4a le Supplementary Fig. 6).Sepheo e ne e le ho bona hore na li-coacervate tsohle, leha li na le thepa e tloaelehileng e kang mokelikeli, li bonts'a boitšoaro bofe kapa bofe bo fapaneng boemong ba limolek'hule.Joalokaha ho lebeletsoe, palo ea EPR e atolohile ka ho eketseha ha mahloriso a polycation, ho bonahatsa ho fokotseha ha ho feto-fetoha ha limolek'hule ka lebaka la litšebelisano tsa limolek'hule tsa balekane bohle ba litšebelisano hoo e ka bang ho tlala (setšoantšo sa 3e, Supplementary Fig. 6).pLK e fihletse ho tlala hona ka tekanyo e tlase ea molar (polycation:αS) ha e bapisoa le ΔNt-Tau le Tau441.Ebile, papiso ea data le mohlala o hakanyetsoang o tlamang ho nka hore libaka tse tlamang tse ts'oanang le tse ikemetseng li bonts'itse hore nako e telele ea ho arohana ea pLK (~5 μM) ke taelo ea boholo bo tlase ho feta ea Tau441 kapa ΔNt-Tau (~50 μM). ).µM).Leha ena e le khakanyo e mpe, sena se fana ka maikutlo a hore αS e na le kamano e phahameng bakeng sa lipolycation tse bonolo tse nang le libaka tsa litefiso tse tsoelang pele.Ka lebaka la phapang ena ea kamano pakeng tsa αS le li-polycation tse fapaneng, re ile ra nahana hore thepa ea bona ea mokelikeli e ka fetoha ka tsela e fapaneng ha nako e ntse e ea 'me kahoo e tšoaroa ke mekhoa e fapaneng ea LSPT.
Ka lebaka la tikoloho e nang le batho ba bangata haholo ka har'a protheine ea coacervate le tlhaho ea amyloid ea protheine, re ile ra bona boitšoaro ba coacervate ha nako e ntse e ea ho lemoha mekhoa e ka bang teng ea LSPT.Re sebelisa BF le CF microscopy (Setšoantšo sa 4), re hlokometse hore αS/Tau441 e kopanya ka tekanyo e kholo ka tharollo, e etsa marotholi a maholo a kopanang le ho kolobisa bokaholimo ba seliba / slide e le marotholi a feletseng, joalokaha ho lebeletsoe (Fig ea Tlatsetso. 7d);re bitsa meaho ena e ka tlase e bitsoang "protein rafts".Mehaho ena e ile ea lula e le metsi ha e ntse e boloka bokhoni ba ho fuse (Supplementary Fig. 7b) 'me e ka bonoa lihora tse' maloa ka mor'a hore LLPS e qale (setšoantšo sa 4 le sa Supplementary Fig. 7c).Re hlokometse hore mokhoa oa ho kolobisa o ratoa holim'a lisebelisoa tsa hydrophilic ho e-na le lisebelisoa tsa hydrophobic (Supplementary Fig. 7a), joalokaha ho lebeletsoe bakeng sa li-coacervates tsa electrostatic tse nang le litefello tse sa leka-lekaneng 'me kahoo li na le menyetla e phahameng ea electrostatic holim'a metsi.Ka ho hlakileng, αS / ΔNt-Tau coalescence le rafting li ile tsa fokotseha haholo, ha αS / pLK condensates e fokotsehile haholo (setšoantšo sa 4).Nakong ea nako e khutšoanyane ea ho qhoqhoa, marotholi a αS / pLK a ne a khona ho kopanya le ho kolobisa holim'a metsi a hydrophilic, empa ts'ebetso ena e ile ea khaotsa ka potlako 'me ka mor'a lihora tsa 5 tsa ho qhoqhoa, liketsahalo tse fokolang tsa coalescence feela' me ha ho metsi a ileng a bonoa.- phetoho ea gel-drip.
Moemeli oa BF (liphanele tse mosoeu) le CF (liphanele tse ka ho le letona, AF488-e ngotsoeng αS ka botala) ea lisampole tsa coacervate tse nang le 100 µM αS (1% fluorescent label) ka har'a LLPS buffer ka pel'a 100 µM Tau441nce Δt microscopic litšoantšo -Tau (bohareng) kapa 1 mM pLK (ka tlaase) ka linako tse fapaneng tsa ho qhoqhoa le bophahamo ba tsepamiso (z, sebaka ho tloha tlase ho poleiti hantle).Liteko li ile tsa phetoa ka makhetlo a 4-6 ka ntle ho tse ling ka liphello tse tšoanang.Li-coacervate tsa αS/Tau441 li kolobisoa ka mor'a lihora tse 24, li etsa li-rafts tse kholo ho feta setšoantšo.Sekala sa litšoantšo tsohle ke 20 µm.
Ka mor'a moo re ile ra botsa hore na matamo a maholo a protheine a kang mokelikeli a thehiloeng ho αS/Tau441 LLPS a ka lebisa ho amyloid aggregation ea leha e le efe ea liprotheine tse ithutoang.Re ile ra latela ho hōla ha marotholi a αS / Tau441 ka nako le microscopy ea WF tlas'a maemo a tšoanang le a ka holimo, empa re sebelisa 1 μM AF488-e ngotsoeng αS le Atto647N-e ngotsoeng Tau441 (Setšoantšo sa 5a).Joalo ka ha ho ne ho lebelletsoe, re bone protheine e felletseng nakong eohle ea kholo.Hoa thahasellisa hore, ho tloha ho ca.Ka mor'a lihora tsa 5, mehaho e matla e sa pota-potileng e ile ea hlokomeloa ka har'a li-rafts, tseo re li bitsitseng "lintlha", tse ling tsa tsona li ne li kopantsoe le αS, 'me tse ling li ruisitsoe ka Tau441 (setšoantšo sa 5a, metsu e tšoeu).Matheba ana esale a hlokometsoe ka har'a rafts ho isa tekanyong e kholo bakeng sa αS/ΔNt-Tau ho feta bakeng sa αS/ΔNt-Tau.Ho ne ho se na matheba a ikhethileng marotholing a pLK le Tau tsamaiso e sa tsebeng ho kopanya / ho kolobisa.Ho lekola hore na matheba ana a nang le αS le Tau441 ke li-amyloid-like aggregates, re entse teko e tšoanang re sebelisa CF microscopy moo Tau441 e neng e ngotsoe ka Atto647N le 12.5 μM amyloid-specific thioflavin-T (ThT) e kenyelitsoeng ho tloha qalong.dae.Le hoja ThT-staining ea αS / Tau441 marotholi kapa li-rafts e ne e sa hlokomeloe esita le ka mor'a lihora tse 24 tsa incubation (setšoantšo sa 5b, marotholi a ka holimo-a setseng holim'a li-protein rafts), mehaho ea ThT-positive e nang le Atto647N-Tau441 ka har'a rafts e ne e fokola haholo.sena se pheta boholo, sebōpeho, le sebaka sa matheba a hlalositsoeng pele (setšoantšo sa 5b, mela e bohareng le e ka tlase), e fana ka maikutlo a hore matheba a ka 'na a lumellana le li-amyloid-like aggregates tse entsoeng ka li-coacervates tsa mokelikeli oa botsofali.
WF 25 μM αS ka linako tse fapaneng tsa ho qhoqhoa le bophahamo ba tsepamiso (z, sebaka ho tloha tlase e sa tlamisoang) ka pel'a 25 μM Tau441 (1 μM AF488-e ngotsoeng αS le Atto647N-e ngotsoeng Tau441) selibeng sa poleiti ea microscope e nang le LLPS buffer) .Liteko tse tšeletseng li ile tsa phetoa ka boikemelo ka liphello tse tšoanang.b CF microscopic setšoantšo sa 25 μM αS ka pel'a 25 μM Tau441 (1 μM Atto647N-e ngotsoeng Tau441) le 12.5 μM thioflavin-T (ThT).Marotholi a protheine a boima le matheba a liprotheine a kentsoeng le matheba a bontšoa meleng e ka holimo le e bohareng, ka ho latellana.Mola o ka tlase o bonts'a litšoantšo tsa rafts le marotholi ho tsoa ho likopi tse 3 tse ikemetseng.Metsu e tšoeu e bonts'a matheba a nepahetseng a ThT liphanele ka bobeli.Sekala sa litšoantšo tsohle ke 20 µm.
Ho hlahloba ka ho qaqileng liphetoho tsa marang-rang a liprotheine tsa coacervate nakong ea phetoho ho tloha ho metsi ho ea ho tiileng, re sebelisitse litšoantšo tsa bophelo bohle ba fluorescence (FLIM) le Förster resonance energy transfer microscopy (FRET) (Figure 6 le Supplementary Figures 8 le 9).Re ile ra nahana hore coacervate maturation ea lera hore e be sebopeho sa protheine se kopantsoeng haholoanyane kapa se tiileng ho lebisa kamanong e haufi pakeng tsa protheine le probe ea fluorescent e khomaretsoeng ho eona, e ka hlahisang phello ea ho tima e bonahalang ka nako e khutšoanyane ea bophelo ba probe (τ) , joalo ka ha ho hlalositsoe pejana40.,41 ,42.Ho phaella moo, bakeng sa disampole tse ngotsoeng ka makhetlo a mabeli (AF488 le Atto647N e le FRET mofani le lidae tse amohelang, ka ho latellana), ho fokotseha hona ho τ ho ka boela ha tsamaisana le coacervate condensation le ho eketseha ha katleho ea FRET (E) nakong ea LSPT.Re ile ra beha leihlo sebopeho sa raft le matheba ha nako e ntse e ea ho LLPS αS/Tau441 le αS/ΔNt-Tau disampole (25 µM ea protheine e 'ngoe le e 'ngoe ho LLPS buffer e nang le 1 µM AF488 e ngotsoeng αS le/kapa Atto647N e ngotsoeng Tau441 kapa ΔNt-Tau).Re hlokometse mokhoa o akaretsang oa hore nako ea bophelo ea fluorescence ea AF488 (τ488) le Atto647N (τ647N) e fokotsehile hanyenyane ha li-coacervates li ntse li hōla (setšoantšo sa 6 le sa Tlatsetso sa feiga. 8c).Hoa thahasellisa hore phetoho ena e ile ea ntlafatsoa haholo bakeng sa matheba ka har'a li-rafts (Setšoantšo sa 6c), e leng se bontšang hore ho feta ha protheine condensation e etsahetse ka matheba.Ho tšehetsa sena, ha ho phetoho e kholo ea bophelo ba fluorescence e ileng ea hlokomeloa bakeng sa marotholi a αS / ΔNt-Tau a lilemo li 24 h (Supplementary Fig. 8d), a fana ka maikutlo a hore lerotholi la marotholi ke mokhoa o fapaneng le oa ho bona 'me o sa tsamaisane le ho hlophisoa bocha ha limolek'hule. ka hare ho li-coacervates.Hoa lokela ho hlokomeloa hore matheba a na le boholo bo fapaneng le litaba tse fapaneng ho αS, haholo-holo bakeng sa tsamaiso ea αS / Tau441 (Supplementary Fig. 8e).Ho fokotseha ha nako ea bophelo ba fluorescence ho ne ho tsamaisana le ho eketseha ha matla, haholo-holo bakeng sa Atto647N e ngotsoeng Tau441 (Supplementary Fig. 8a), le matla a phahameng a FRET bakeng sa tsamaiso ea αS/Tau441 le αS/ΔNt-Tau ka bobeli, e bontšang ho fokotseha ho eketsehileng ho LLPS lihora tse hlano. ka mor'a ho qalisa, liprotheine tse ka hare ho motlakase o tsitsitseng li fokotsehile.Ha ho bapisoa le αS/ΔNt-Tau, re bone boleng bo tlase ba τ647N le bo phahameng bo itseng ba τ488 libakeng tsa αS/Tau441, bo tsamaea le boleng bo tlase le bo sa tsitsang ba FRET.Mohlomong, sena se ka 'na sa amana le taba ea hore tsamaisong ea αS/Tau441, bongata ba αS bo hlokometsoeng le bo lebelletsoeng ka kakaretso bo fapane haholo, hangata bo le substoichiometric ha bo bapisoa le Tau, kaha Tau441 ka boeona e ka boela ea feta LLPS le ho bokellana (Supplementary Fig. 8e) .Leha ho le joalo, tekanyo ea ho kopana ha marotholi, sebopeho sa raft, 'me, sa bohlokoa, ho bokellana ha protheine ka har'a li-coacervate tse kang metsi ke tse kholo ha Tau441 le αS li le teng.
a Lifetime fluorescence microscopy (FLIM) litšoantšo tsa αS/Tau441 le αS/ΔNt-Tau tse 25 μM tsa protheine ka 'ngoe (1 μM AF488-tse ngotsoeng αS le 1 μM Atto647N-tse ngotsoeng Tau441 kapa ΔNt-Tau) ho buffer ea LLPS.Likholomo li bontša litšoantšo tse emelang lisampole tsa LLPS ka linako tse fapaneng tsa kholo (30 mets, 5 h le 24 h).Foreimi e khubelu e bonts'a sebaka se nang le matheba a αS/Tau441.Nako ea bophelo e bonts'oa joalo ka mebala.Sekala = 20 µm bakeng sa litšoantšo tsohle.b Setšoantšo sa FLIM se atomelitsoeng sa sebaka se khethiloeng, se bontšitsoeng lebokoseng le lefubelu phanele a.Mekhahlelo ea bophelo e bontšoa ho sebelisoa sekala sa mebala se tšoanang le sa phanele a.Scale bar = 5 µm.c Histograms e bontšang AF488 (e khomaretsoeng ho αS) kapa Atto647N (e khomaretsoeng ho Tau) bakeng sa mefuta e fapaneng ea liprotheine (marotholi-D-, raft-R- le speckle-P) tse khetholloang litšoantšong tsa FLIM tse rekotiloeng bakeng sa αS-) kabo ea nako bophelo bohle ba Tau441 le αS/ΔNt-Tau coacervate disampole (N = 17-32 ROI bakeng sa D, 29-44 ROI bakeng sa R, le 21-51 ROI bakeng sa lintlha).Maemo a mahareng le a mahareng a bonts'oa e le lisekoere tse mosehla le mela e ntšo ka har'a mabokose, ka ho latellana.Meeli e ka tlase le e kaholimo ea lebokose e emela li-quartile tsa pele le tsa boraro, ka ho latellana, 'me boleng bo tlase le bo phahameng ka har'a 1.5-fold interquartile range (IQR) e bonts'oa joalo ka litelu.Li-outliers li bontšoa e le litaemane tse ntšo.Bohlokoa ba lipalo lipakeng tsa lipara tsa kabo bo ile ba khethoa ho sebelisoa tlhahlobo ea t ea disampole tse peli, ho nahanoa hore ho na le phapang e sa lekanang.Liteko tsa p-test tse nang le mehatla e 'meli li hlahisoa ka linaleli bakeng sa para e 'ngoe le e 'ngoe ea data e bapisitsoeng (* p-value > 0.01, ** p-value > 0.001, *** p-value > 0.0001, **** p-value > 0.00001), ns E bontša ho se tsotelle (p-value > 0.05).Lintlha tse nepahetseng tsa p li fanoe ho Letlapa la 1 la Tlatsetso, 'me lintlha tsa mantlha li hlahisoa e le lifaele tsa data tse tala.
Ho tsoela pele ho bonts'a mofuta oa li-amyloid-joaloka li-speckles/aggregates, re ile ra tšoara lisampole tse sa tsitsang tsa coacervate bakeng sa lihora tse 24 tse nang le likhahla tse phahameng tsa (1 M) NaCl, e leng se ileng sa fella ka karohano ea li-aggregates ho tloha ho li-protein coacervates.Ha li-aggregates tse arohaneng (ke hore, tharollo e qhalakaneng ea li-aggregates) li ne li bonoa ho sebelisoa atomic force microscopy (AFM), re ile ra bona morphology e nang le boholo ba spherical e nang le bolelele bo tloaelehileng ba hoo e ka bang 15 nm, e atisang ho amahanngoa tlas'a maemo a letsoai le phahameng, joalo ka boitšoaro ba li-amyloid fibrils tse tloaelehileng ka lebaka la phello e matla ea hydrophobic holim'a metsi (hlokomela hore li-fibrils hangata li na le bolelele ba ~ 10 nm) (Supplementary Fig. 10a).Hoa thahasellisa hore ha li-aggregates tse arohaneng li ne li kenngoa ka ThT ka mokhoa o tloaelehileng oa ThT fluorescence assay, re ile ra bona keketseho e tsotehang ea tlhahiso ea ThT fluorescence quantum, e bapisoang le e ileng ea hlokomeloa ha dae e ne e kenngoa ka mokhoa o tloaelehileng oa αS amyloid fibrils (Supplementary Fig. 10b), e fana ka maikutlo a hore 10b) li-aggregate tsa coacervate li na le meaho e kang amyloid..Ha e le hantle, li-aggregates li ne li mamella letsoai le lengata empa li le hlokolosi ho 4 M guanidine chloride (GdnHCl), joaloka li-fibrils tse tloaelehileng tsa amyloid (Supplementary Fig. 10c).
Ka mor'a moo, re ile ra sekaseka sebopeho sa li-aggregates ho sebelisa molek'hule e le 'ngoe ea fluorescence, fluorescence correlation / cross-correlation spectroscopy (FCS/FCCS), le tlhahlobo e phatlohileng ea ho lemoha ka tsietsi ea mebala e 'meli (TCCD).Ho fihlela sena, re ile ra arola lihlopha tse entsoeng ka mor'a lihora tse 24 tsa incubation ka 100 μl LLPS disampole tse nang le αS le Tau441 (ka bobeli 25 μM) hammoho le 1 μM AF488-e ngotsoeng αS le 1 μM Atto647N-e ngotsoeng Tau441.Hlapolla sephetho se qhalakantsoeng aggregate tharollo ho boemo monomolecular sebelisa e tšoanang PEG-free buffer le 1 M NaCl (e tšoanang buffer sebediswa ho arola aggregates ho tloha coacervate) ho thibela leha e le efe ho ka etsahala ho sebelisa electrostatic pakeng tsa LLPS le protheine.Mohlala oa tsela ea nako ea molek'hule e le 'ngoe e ka bonoa setšoantšong sa 7a.Tlhahlobo ea FCCS/FCS (cross-correlation, CC le autocorrelation, AC) e bontšitse hore li-aggregates tse nang le αS le tau li ne li le ngata ka lisampole (sheba CC curve ho Fig. 7b, lehlakoreng le letšehali), 'me ho ile ha hlaha protheine e ngata e setseng ea monomeric. sephetho sa ts'ebetso ea ho hlapolla (sheba li-curve tsa AC ho Setšoantšo sa 7b, phanele e ka letsohong le letšehali).Liteko tsa ho laola tse entsoeng tlas'a maemo a ts'oanang a tharollo ho sebelisa li-sampuli tse nang le liprotheine tsa monomeric feela li bontšitse hore ha ho na li-curve tsa CC, 'me li-curve tsa AC li lumellana hantle le mohlala oa motsoako oa karolo e le' ngoe (Eq. 4), moo liprotheine tsa monomeric li nang le li-coefficients tse lebeletsoeng (setšoan 7b). ), phanele e nepahetseng).Coefficient ea phano ea likaroloana tse kopaneng e ka tlase ho 1 µm2/s, 'me ea protheine ea monomeric e ka ba 1 µm2/s.50–100 µm/s;boleng bo tšoana le boleng bo phatlalalitsoeng pele bakeng sa sonicated αS amyloid fibrils le monomeric αS ka thoko tlasa maemo a tšoanang a tharollo44.Ha re hlahloba lihlopha ka tlhahlobo ea ho phatloha ha TCCD (Setšoantšo sa 7c, karolo e ka holimo), re fumane hore karolong e 'ngoe le e' ngoe e arohaneng (αS / Tau heteroaggregate), hoo e ka bang 60% ea lihlopha tse fumanoeng li na le αS le tau, hoo e ka bang 30% e na le feela. tau, e ka bang 10% αS feela.Tlhahlobo ea stoichiometric ea αS/Tau heteroaggregates e bontšitse hore boholo ba li-heteroaggregate li ruisitsoe ka tau (stoichiometry ka tlase ho 0.5, palo e tloaelehileng ea limolek'hule tsa tau ka kakaretso ke makhetlo a 4 ho feta limolek'hule tsa αS), e lumellanang le mosebetsi oa rona o hlokometsoeng ho FLIM in situ. liteko..Tlhahlobo ea FRET e bonts'itse hore likarolo tsena li na le liprotheine ka bobeli, leha boleng ba 'nete ba FRET ntlheng ena bo se bohlokoa haholo, kaha kabo ea fluorophores ka kakaretso e ne e le ntho e sa reroang ka lebaka la protheine e ngata e sa ngoloang e sebelisitsoeng tekong.Hoa thahasellisa hore ha re etsa tlhahlobo e tšoanang ho sebelisa 45,46 mature amyloid aggregation-deficient Tau variant (bona Supplementary Fig. 11a,b), re hlokometse hore le hoja αS electrostatic aggregation e ne e tšoana (Supplementary Fig. 11c, d), bokhoni ba ho theha li-aggregate ka har'a coacervate bo fokotsehile haholo 'me FLIM e ile ea lemoha libaka tse' maloa litekong tsa situ, 'me ho ile ha hlokomeloa li-curve tse fokolang tse amanang le li-cross-correlation bakeng sa lisampole tse arohaneng.Leha ho le joalo, bakeng sa palo e fokolang ea lihlopha tse fumanoeng (karolo e le 'ngoe feela ea leshome ea Tau441), re hlokometse hore kakaretso e' ngoe le e 'ngoe e ruisitsoe ka αS ho feta mofuta ona oa Tau, ka hoo e ka bang 50% ea lihlopha tse fumanoeng tse nang le limolek'hule tsa αS feela,' me αS e ne e fapane ka ho feteletseng. .aggregates (bona Supplementary Fig. 11e), ho fapana le heterogeneous aggregates hlahisoang ke Tau441 (sa feiga. 6f).Liphello tsa liteko tsena li bontšitse hore le hoja αS ka boeona e khona ho bokella le tau ka hare ho coacervate, tau nucleation e molemo haholoanyane tlas'a maemo ana, 'me lihlopha tse kang li-amyloid tse hlahisoang li khona ho sebetsa e le mofuta oa αS le tau.Leha ho le joalo, hang ha ho thehoa motheo oa tau-rich, litšebelisano tsa heterotypic pakeng tsa αS le tau li ratoa ka lihlopha ho feta ho sebelisana homotypic pakeng tsa limolek'hule tsa tau;re boetse re sheba marang-rang a protheine ka har'a li-coacervate tsa αS/tau.
e Moemeli oa fluorescence mesaletsa ea nakoana ea limolek'hule tse le 'ngoe tsa likarolo tse ka thōko tse entsoeng ka αS/Tau441 electrostatic coacervates.Ho phatloha ho lumellanang le li-coaggregates tsa αS/Tau441 (ho phatloha ka holimo ho moeli o bontšitsoeng) ho ile ha hlokomeloa ka litsela tse tharo tsa ho lemoha (AF488 le Atto647N emission ka mor'a ho hlasimolla ka ho toba, mela e putsoa le e khubelu, ho tsoa ha Atto647N ka mor'a ho tsosoa ka tsela e sa tobang), FRET, violet line).b Tlhahlobo ea FCS/FCCS ea sampole ea likarolo tse ka thoko tsa αS/Tau441 tse fumanoeng ho tsoa ho LLPS (phanele e ka ho le letšehali).Li-curve tsa Autocorrelation (AC) bakeng sa AF488 le Atto647N li bonts'oa ka boputsoa le bokhubelu, ka ho latellana, 'me li-curve tsa cross-correlation (CC) tse amanang le li-aggregate tse nang le lidae ka bobeli li bonts'oa ka bopherese.Li-curve tsa AC li bonts'a boteng ba mefuta ea protheine e ngotsoeng e le monomeric le e kopaneng, ha li-curve tsa CC li bonts'a feela phallo ea likarolo tse ngotsoeng habeli.Tlhahlobo e ts'oanang, empa tlas'a maemo a tšoanang a tharollo le libakeng tse ka thōko, lisampole tse nang le monomeric αS le Tau441 feela li bontšoa e le li-control phanele e nepahetseng.c Tlhahlobo ea Fluorescence flash ea limolek'hule tse le 'ngoe tsa likarolo tse ka thōko tse entsoeng ka αS/Tau441 electrostatic coacervates.Tlhahisoleseding bakeng sa kakaretso e 'ngoe le e' ngoe e fumanoang ka makhetlo a mane a fapaneng (N = 152) e reriloe khahlanong le stoichiometry ea bona, litekanyetso tsa S, le katleho ea FRET (phanele e ka holimo, mebala ea mebala e bonahatsa ketsahalo).Mefuta e meraro ea li-aggregate e ka khetholloa: -αS-aggregates feela ka S~1 le FRET~0, Tau-feela aggregates le S~0 le FRET~1, le heterogeneous Tau/αS aggregates le mahareng S le FRET Khakanyo ea chelete. ea liprotheine tsa matšoao ka bobeli tse fumanoeng ka bongata bo fapaneng (N = 100) li bonts'oa phanele e ka tlase (sekala sa mebala se bonts'a ketsahalo).Lintlha tse tala li fanoa ka mokhoa oa lifaele tsa data tse tala.
Ho tsofala kapa botsofali ba protheine ea metsi e kopanya ka har'a li-gel-like kapa mehaho e tiileng ha nako e ntse e feta ho tlalehoa hore e ameha mesebetsing e mengata ea fisioloji ea condensate47 hammoho le maloetse, e le mokhoa o sa tloaelehang o etellang pele amyloid aggregation 7, 48, 49. Mona re ithuta karohano ea mekhahlelo le boitšoaro ka botlalo.LSPT αS boteng ba li-polycations tse sa reroang sebakeng se laoloang maemong a tlase a micromolar le maemo a amanang le 'mele (hlokomela hore palo e baloang ea fisioloji ea αS ke> 1 µM50), ho latela boits'oaro bo tloaelehileng ba LPS ba thermodynamically.Re fumane hore αS, e nang le sebaka sa C-terminal se nang le boemo bo bobe haholo ho pH ea fisioloji, e khona ho theha marotholi a nang le protheine ka tharollo ea metsi ka LLPS ka pel'a li-peptide tse nang le cationic tse ngata tse kang pLK kapa Tau ka mokhoa oa electrostatic. condensation e rarahaneng ka boteng ba macromolecules aggregation.Ts'ebetso ena e ka 'na ea e-ba le liphello tse loketseng tikolohong ea cellular moo αS e kopanang le limolek'hule tse sa tšoaneng tsa polycationic tse amanang le ho kopana ha eona ho amanang le mafu ka bobeli ka vitro le ka vivo51,52,53,54.
Lithutong tse ngata, matla a protheine ka har'a marotholi a 'nile a nkoa e le e' ngoe ea lintlha tsa bohlokoa tse khethollang mokhoa oa ho hōla55,56.Ho li-electrostatic αS coacervates le polycations, ts'ebetso ea kholo ka ho hlakileng e itšetlehile ka matla a ho sebelisana le polycations, valence, le bongata ba litšebelisano tsena.Khopolo ea tekano e fana ka maikutlo a hore sebaka se lekanang sa linaha tse peli tsa mokelikeli e ka ba boteng ba lerotholi le leholo le ruileng ka li-biopolymers tse tsamaisang LLPS57,58.Khōlo ea Droplet e ka finyelloa ka Ostwald maturation59, coalescence60 kapa tšebeliso ea monomer ea mahala karolong ea61 e qhalakaneng.Bakeng sa αS le Tau441, ΔNt-Tau kapa pLK, boholo ba protheine e ne e tsepamisitsoe ka har'a condensate tlas'a maemo a sebelisitsoeng thutong ena.Leha ho le joalo, le ha marotholi a boholo bo felletseng a tau a hokahana ka potlako ha a koloba, ho kopana ha marotholi le ho koloba ho ne ho le thata ho ΔNt-Tau le pLK, ho fana ka maikutlo a tahlehelo e potlakileng ea thepa ea mokelikeli litsamaisong tsena tse peli.Ho latela tlhahlobo ea rona ea FLIM-FRET, marotholi a khale a pLK le ΔNt-Tau a bonts'itse tekanyo e ts'oanang ea protheine aggregation (e ts'oanang bophelo bohle ba fluorescence) joalo ka marotholi a mantlha, a fana ka maikutlo a hore marang-rang a protheine ea mantlha a bolokoa, leha a le thata ho feta.
Re lokisa liphetho tsa rona tsa liteko ka mokhoa o latelang (setšoantšo sa 8).The marotholi qalong ea nakoana thehoa hangata protheine marangrang ntle le matšeliso electrostatic, 'me kahoo ho na le libaka tsa tefiso imbalance, haholo-holo ka segokanyimmediamentsi sa sebolokigolo, ho fella ka marotholi a nang le matla a phahameng a electrostatic holim'a metsi.Ho lefella tefiso (ketsahalo eo hangata e bitsoang depletion ea valence) le ho fokotsa matla a holimo a marotholi, marotholi a ka kenyelletsa li-polypeptide tse ncha ho tloha mothating oa ho hlapolla, ho hlophisa bocha marang-rang a liprotheine ho ntlafatsa tšebelisano ea tefiso, le ho sebelisana le marotholi a mang.e nang le bokahodimo (ho koloba).Marotholi a αS / pLK, ka lebaka la marang-rang a bona a bonolo a protheine (feela litšebelisano tsa heterotypic pakeng tsa αS le pLK) le kamano e kholoanyane bakeng sa litšebelisano tsa protheine-protein, li bonahala li khona ho leka-lekanya tefiso ea condensate ka potlako;ka sebele, re ile ra bona li-protein kinetics tse potlakileng ha li qala ho thehoa αS / pLK coacervates ho feta αS / Tau.Ka mor'a ho fokotseha ha valence, litšebelisano li fetoha tse fokolang 'me marotholi a lahleheloa ke thepa ea' ona ea mokelikeli 'me a fetoha marotholi a kang gel, a sa cheng a nang le matla a tlaase a electrostatic (ka hona a sitoa ho kolobisa bokaholimo).Ka lehlakoreng le leng, marotholi a αS/Tau ha a sebetse hantle ho beheng tekano ea tefiso ea marotholi ka lebaka la marang-rang a rarahaneng a protheine (a nang le likamano tsa homotypic le heterotypic) le mofuta o fokolang oa likamano tsa protheine.Sena se fella ka marotholi a bolokang boits'oaro ba mokelikeli nako e telele mme a bonts'a matla a holimo a electrostatic a atisang ho fokotsoa ka ho kopana le ho hola (ka tsela eo ho fokotsa sebaka sa bokaholimo ba marotholi) le ka ho kolobisa chem ea hydrophilic surface.Sena se theha lilaebrari tse kholo tsa protheine tse bolokang thepa ea mokelikeli kaha litšebelisano li lula li le nakoana ka lebaka la ho batloa khafetsa bakeng sa ntlafatso ea tefiso marang-rang a protheine.Hoa thahasellisa hore mefuta ea N-terminally truncated ea Tau, ho kenyelletsa le li-isoform62 tse hlahang ka tlhaho, li bonts'a boits'oaro bo mahareng, botsofali bo bong bo nang le αS ho ba marotholi a kang gel a phetseng nako e telele, ha a mang a fetoha li-condensate tse kholo tsa mokelikeli.Bobeli bona ba ho butsoitseng ha αS electrostatic coacervates bo lumellana le lithuto tsa morao-rao tsa LLPS tsa khopolo-taba le liteko tse hlokometseng kamano pakeng tsa ho fokotseha ha valence le electrostatic sieving ka li-condensate e le senotlolo sa ho laola boholo ba condensate le thepa ea mokelikeli.Mokhoa oa 58.61.
Morero ona o bonts'a tsela ea ho kopanya amyloid ea αS le Tau441 ka LLPS le LSPT.Ka libaka tse eketsehileng tse nang le anion-rich (red) le cation-rich (blue), αS le tau electrostatic coacervates tse nang le valence e khotsofatsang li na le matla a tlaase a holim'a metsi 'me ka hona li kopana hanyenyane, tse bakang botsofali ba marotholi ka potlako.Boemo ba gel bo tsitsitseng bo se nang agglomerated boa finyelloa..Boemo bona bo monate haholo tabeng ea tsamaiso ea αS / pLK ka lebaka la kamano ea eona e phahameng le marang-rang a bonolo a ho sebelisana le protheine-pair, e lumellang hore ho be le phetoho e potlakileng ea gel.Ho fapana le hoo, marotholi a nang le valence e sa khotsofatseng, ka hona, libaka tse nang le liprotheine tse fumanehang bakeng sa ho sebelisana, li etsa hore ho be bonolo hore coacervate e fuse le ho kolobisa bokaholimo ba hydrophilic e le ho fokotsa matla a eona a holimo.Boemo bona bo molemo ho li-coacervates tsa αS/Tau441, tse nang le marang-rang a mangata a rarahaneng a nang le litšebelisano tse fokolang tsa Tau-Tau le αS-Tau.Ka lehlakoreng le leng, li-coacervate tse khōloanyane li tla boloka thepa ea tsona e kang ea mokelikeli habonolo, e leng se lumellang hore litšebelisano tse ling tsa protheine-to-protein li etsahale.Qetellong, li-amyloid heterogeneous aggregates tse nang le αS le tau ka bobeli li theha ka har'a mokelikeli oa coacervate, e ka 'nang ea amana le tse fumanoang ka har'a lihlopha tse kenyelletsoeng, e leng matšoao a mafu a neurodegenerative.
Mehaho e meholo e kang mokelikeli e entsoeng nakong ea kholo ea αS/Tau441 e nang le tikoloho ea protheine e nang le tšubuhlellano e matla empa e matla, 'me ho isa bohōleng bo fokolang, li-coacervate tsa αS/ΔNt-Tau ke matamo a loketseng bakeng sa ho kopanya liprotheine.Ka sebele re hlokometse ho thehoa ha lihlopha tsa protheine tse tiileng mofuteng ona oa li-coacervate tsa protheine, tse atisang ho ba le αS le tau ka bobeli.Re bonts'itse hore li-heteroaggregates tsena li tsitsisoa ke litšebelisano tse se nang motlakase, li khona ho tlama li-dae tse khethehileng tsa amyloid tsa ThT ka tsela e tšoanang le ea li-amyloid fibrils tse tloaelehileng, 'me ka sebele li na le khanyetso e tšoanang ho litšusumetso tse sa tšoaneng.Li-aggregates tsa αS/tau tse entsoeng ke LLPS li bontšitsoe li na le thepa e kang ea amyloid.Ha e le hantle, phapang e hōlileng tsebong ea Tau e haelloang ke amyloid aggregation e fokotsehile haholo ho thehoeng ha lihlopha tsena tse sa tšoaneng tsa αS ka har'a mokelikeli electrostatic coacervate.Ho thehoa ha lihlopha tsa αS / Tau441 ho ile ha hlokomeloa feela ka hare ho li-coacervates, tse ileng tsa boloka thepa e kang ea metsi, 'me ha ho mohla, haeba li-coacervates / marotholi a sa fihle boemong ba gel.Boemong ba ho qetela, matla a eketsehileng a litšebelisano tsa electrostatic 'me, ka lebaka leo, ho thatafala ha marang-rang a protheine ho thibela ho hlophisoa bocha ha conformational ea liprotheine ho theha litšebelisano tse ncha tsa protheine tse hlokahalang bakeng sa nucleation ea amyloid.Leha ho le joalo, sena se ka finyelloa ka li-coacervate tse feto-fetohang haholoanyane, tse kang mokelikeli, tseo le tsona li ka 'nang tsa lula li le metsi ha li ntse li eketseha ka boholo.
Taba ea hore ho thehoa ha lihlopha ka har'a karolo e khutsufalitsoeng ho molemo ho li-condensate tse kholo tsa αS / Tau ho feta marotholi a manyenyane a phallang ka potlako, e totobatsa bohlokoa ba ho khetholla lintlha tse laolang ho kopana ha marotholi.Ka hona, hase feela hore ho na le tšekamelo ea ho arohana ha mekhahlelo, empa boholo ba condensate bo tlameha ho laoloa bakeng sa ts'ebetso e nepahetseng hammoho le thibelo ea mafu58,61.Liphetho tsa rona li boetse li totobatsa bohlokoa ba ho leka-lekana pakeng tsa LLPS le LSPT bakeng sa tsamaiso ea αS/Tau.Le hoja sebopeho sa marotholi se ka sireletsa khahlanong le ho kopana ha li-amyloid ka ho fokotsa palo ea li-protein monomers tse fumanehang tlas'a maemo a saturation, joalokaha ho 'nile ha etsoa tlhahiso ea mekhoa e meng ea 63,64, ho kopana ha marotholi ka maemo a phahameng a marotholi ho ka lebisa ho kopanngoeng ha protheine ka hare ka ho lokisoa butle-butle.marang-rang a protheine..
Ka kakaretso, lintlha tsa rona li hatisa ka matla bohlokoa ba valence e kopanetsoeng le litšebelisano tse khotsofatsang / tse sa khotsofalang ho marang-rang a theohang moelelong oa LSPT.Haholo-holo, re bonts'a hore bolelele bo felletseng ba αS/Tau441 li-condensate li khona ho fuse le nucleate hantle ho theha li-heteroaggregate tse kang li-amyloid tse kenyelletsang liprotheine ka bobeli mme li sisinya mokhoa oa limolek'hule ho latela liphetho tsa rona tsa liteko.Khokahano ea liprotheine tse peli ho αS/Tau fluid coacervate eo re e tlalehang mona e kanna ea amana le ho kopanngoa ha liprotheine tse peli ka har'a li-inclusions, e leng matšoao a lefu lena, 'me e ka thusa ho utloisisa kamano lipakeng tsa LLPS le amyloid aggregation, ho bula tsela bakeng sa IDP e qosoang haholo ho neurodegeneration.
Monomeric WT-αS, cysteine ​​​​mutants (Q24C-αS, N122C-αS) le mefuta e fapaneng ea ΔCt-αS (Δ101-140) e hlahisitsoe ho E. coli 'me ea hloekisoa joalokaha ho hlalositsoe pele.5 mM DTT e kenyelelitsoe mehatong eohle ea tlhoekiso ea αS cysteine ​​​​mutants ho thibela ho thehoa ha bond ea disulfide.Tau441 Isoft (Plassmid e tsoa ho eketsa # 16316), ΔTN-Tau Vertataagg er) E. Bokooa ba Coli ba ne ba e holile ho fihla ho OD600 = 0.6–0.7 ho 37°C le 180 rpm, mme polelo e ile ea hlahisoa ka IPTG ka lihora tse 3 ho 37°C.Kotula lisele ka 11,500 xg bakeng sa metsotso e 15 ka 4 °C 'me u hlatsoe ka saline buffer e nang le 150 mM NaCl.Phahamisa pellet ka lysis buffer (20 ml ka 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 1).Mohato oa sonication o ne o etsoa ka leqhoa ka amplitude ea 80% bakeng sa pulses ea 10 (1 min on, 1 min off).U se ke ua feta 60 ml ka ultrasound e le 'ngoe.E. coli lysates e ne e futhumetse ka 95 ° C. ka metsotso e 20, ebe e pholile ka leqhoa 'me e kenngoa ka centrifuged ho 127,000 × g bakeng sa metsotso e 40.Supernatant e hlakisitsoeng e ile ea sebelisoa ho membrane ea 3.5 kDa (Spectrum™ Thermo Fisher Scientific, UK) 'me ea sebelisoa khahlanong le 4 L ea dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) bakeng sa lihora tse 10.Kholomo ea phapanyetsano ea cation ea 5 ml (HiTrap SPFF, Cytiva, MA, USA) e ne e kopantsoe le buffer ea tekano (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF) .Tau lysate e ne e tlhotliloeng ka 0.22 μm PVDF filthara 'me e kenngoa ka har'a kholomo ka tekanyo ea ho phalla ea 1 ml / min.Elution e ile ea etsoa butle-butle, tau e ile ea hlakoloa ka 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).Likaroloana li ile tsa hlahlobjoa ke SDS-PAGE, 'me likaroloana life kapa life tse nang le sehlopha se le seng se nang le boima ba molek'hule ba tau li ne li tsepamisitsoe ho sebelisoa filtara ea 10 kDa centrifuge 'me ea nkeloa sebaka ke buffer e nang le 10 mM HEPES, pH 7.4, NaCl 500 mM le DTT 2 mM bakeng sa motsoako oa ho qetela oa protheine e ne e le 100 μM.Tharollo ea protheine e ile ea fetisoa ka sefe ea 0.22 μm PVDF, ea hoamisoa ka potlako le ho bolokoa ho -80 ° C.Protheine ea K18 e fanoe ka mosa ke Prof. Alberto Boffi.Ho hloeka ha litokisetso e ne e le> 95% joalokaha ho netefalitsoe ke SDS-PAGE le MALDI-TOF/TOF.Li-cysteine ​​​​tse fapaneng li ne li ngotsoe ka lik'hemik'hale ka AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) kapa TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).li netefalitsoe ke ho monya le MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau le K18 li ne li ngotsoe ka masala a matsoalloa a cysteine ​​​​maemong a 191 le 322 a sebelisa Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Jeremane) ho latela mokhoa o tšoanang.Tefiso ea chelete bakeng sa limmapa tse setseng tsa αS le Tau441 li entsoe ho sebelisoa CIDER66.
Solid poly-L-lysine (pLK DP 90-110 ho latela NMR ho tsoa ho mofani oa thepa, Alamanda Polymers Inc, Huntsville, Alabama, USA) e ile ea qhaloa ka 10 mM HEPES, 100 mM NaCl, pH 7.4 ho 10 mM concentration, process sonicated for 5 metsotso ka bateng ea metsi ea ultrasonic 'me u boloke ho -20°C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) le FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) li qhibiliha ka metsing 'me li ajoa haholo LLPS buffer.Dialysis e tlosa matswai a silafatsang.Ka mor'a moo, li ne li sefshoa ka sesefo sa syringe se boholo ba pore ea 0.22 μm, 'me likhahla tsa tsona li ne li baloa ho sebelisoa refractometer (Mettler Toledo, Columbus, Ohio, USA).Mehlala ea LLPS e ile ea lokisoa mocheso oa kamore ka tatellano e latelang: buffer le extrusion li ne li tsoakane le 1 mM tris(2-carboxyethyl)phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) le motsoako oa 1% protease inhibitor (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).Ebe αS le li-polycation tse fused (likhetho pLK kapa Tau) lia eketsoa.Bakeng sa liteko tsa letoto la thioflavin-T (ThT, Carbosynth, Compton, UK), sebelisa kakaretso ea mahloriso a ThT ho ba halofo ea αS.Ka bonolo empa ka botlalo kopanya lisampole ho netefatsa hore li homogeneous.Kholiso ea karolo ka 'ngoe e ne e fapana ho ea ho liteko ho ea ho liteko, joalo ka ha ho hlalositsoe karolong ea Liphetho.Azide e ne e sebelisoa ka bongata ba 0.02% (w / v) neng kapa neng ha nako ea teko e feta lihora tsa 4.Bakeng sa litlhahlobo tsohle tse sebelisang lisampole tsa LLPS, lumella motsoako hore o lekane metsotso e 5 pele ho tlhahlobo.Bakeng sa tlhahlobo ea ho hasana ha bobebe, 150 µl ea lisampole li ile tsa laeloa holim'a li-microplates tse sa tlameng tse 96 (µClear®, black, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) 'me li koahetsoe ka filimi e khomarelang.Li-LLP li ile tsa shejoa ka ho lekanya ho monya ho 350 nm bohareng ba tharollo ho sebali sa poleiti ea CLARIOstar (BMG Labtech, Ortenberg, Jeremane).Liteko li entsoe ka makhetlo a mararo ho 25 ° C, 'me liphoso li ile tsa baloa e le ho kheloha ho tloaelehileng ho tloha ho moelelo.Mokhahlelo oa ho hlapolla o ile oa lekanyetsoa ka mohlala oa centrifugation le tlhahlobo ea gel ea SDS-PAGE, 'me karoloana ea αS ka mekhahlelo ea ho hlapolla le e tsepamisitsoeng e ile ea lekanyetsoa ka litharollo tse fapaneng tsa LLPS.Mohlala oa 100 μl LLPS o nang le 1 μM AF488-labeled αS o ile oa lokisoa ka ho kopanya ka ho feletseng ho lateloa ke centrifugation ho 9600 × g bakeng sa metsotso e 30, ka mor'a moo pula e ne e atisa ho bonahala.50 μl e kaholimo ea matla a holimo e ne e sebelisetsoa ho lekanya liprotheine ho sebelisoa gel ea SDS-PAGE.Li-gel li ile tsa hlahlojoa ka li-filters tsa AF488 ho sebelisoa mochine oa litšoantšo oa gel oa ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) kapa tsa silafatsoa ka letheba la Coomassie 'me tsa bonoa ka li-filters tse loketseng.Lihlopha tse hlahisitsoeng li ile tsa hlahlojoa ho sebelisoa ImageJ version 1.53i (Mekhatlo ea Sechaba ea Bophelo, USA).Liteko li entsoe ka makhetlo a mabeli litekong tse peli tse fapaneng tse nang le liphetho tse tšoanang.
Ka tloaelo, 150 μl ea disampole e ne e sebelisoa ho li-microplate tse sa tlameng tse 96-liliba 'me li bonoa mocheso oa kamore ho Leica DMI6000B microscope e inverted (Leica Microsystems, Wetzlar, Germany).Bakeng sa liteko tsa sebaka, lipoleiti tsa µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Germany) kapa li-microplates tsa polystyrene tse 96 (Corning Costar Corp., Acton, Massachusetts) le tsona li ile tsa sebelisoa.EL6000 halogen kapa mercury metal halide mabone a ne a sebelisoa e le mehloli ea leseli (bakeng sa BF/DIC le WF imaging, ka ho latellana).Bakeng sa microscopy ea WF, sepheo sa moea sa 40x se holisang (Leica Microsystems, Jeremane) se ile sa sebelisoa ho tsepamisa leseli lisampoleng le ho li bokella.Bakeng sa lisampole tse ngotsoeng ka AF488 le ThT, ho hlasimolla le ho ntšoa ha li-filter tse nang le li-filters tse tloaelehileng tsa GFP, li-filters tsa li-bandpass tse hlasimollang le tse tsoang, ka ho latellana, 460-500 nm le 512-542 nm bandpass filters, le seipone sa 495 nm dichroic.Bakeng sa lisampole tse ngotsoeng ka Atto647N, ho ile ha sebelisoa sete e tloaelehileng ea li-filters tsa Cy5 tse nang le li-filter tsa "excitation le emission bandpass filters" tse 628-40 nm le 692-40 nm, ka ho latellana, le seipone sa 660 nm dichroic.Bakeng sa microscope ea BF le DIC, sebelisa sepheo se tšoanang sa pokello ea leseli.Leseli le bokelletsoeng le ne le ngotsoe ho khamera ea Leica DFC7000 CCD (Leica Microsystems, Jeremane).Nako ea ho pepeseha e ne e le 50 ms bakeng sa BF le DIC imaging microscopy le 20-100 ms bakeng sa setšoantšo sa microscopy sa WF.Ha ho bapisoa, nako ea ho pepeseha bakeng sa liteko tsohle tse nang le ThT e ne e le 100 ms.Liteko tsa ho felloa ke nako li ile tsa etsoa ho bona ka mahlo a kelello ho kopana ha marotholi, 'me litšoantšo li bokelloa ka 100 ms metsotso e mengata.ImageJ (NIH, USA) e ne e sebelisetsoa ho hlahloba litšoantšo.Liteko li entsoe ka makhetlo a mararo ka liphello tse tšoanang.
Bakeng sa liteko tsa colocalization, FRAP le 3D reconstruction, litšoantšo li ile tsa fumanoa ka Zeiss LSM 880 inverted confocal microscope ho sebelisoa khatiso e putsoa ea ZEN 2 (Carl Zeiss AG, Oberkochen, Germany).Mehlala ea 50 µl e ne e sebelisoa ho lijana tsa µ-Slide Angiogenesis Petri (Ibidi GmbH, Gröfelfing, Jeremane), e tšoaroang ka hydrophilic polymer (ibiTreat) 'me e kenngoa ka sepheo sa ho qoelisoa ka oli ea 63× (Plan-Apochromat 63×/NA 1.4 Oil) ho DIC).Litšoantšo li fumanoe ho sebelisoa lithapo tsa laser tsa 458 nm, 488 nm le 633 nm argon tse nang le qeto ea 0.26 µm/pixel le nako ea ho pepeseha ea 8 µs/pixel bakeng sa lifensetere tsa 470–600 nm, 493–628 nm, 'me 638-755 nm e ne e sebelisetsoa ho bona ka mahlo a kelello ThT, AF488 le Atto647N, ka ho latellana.Bakeng sa liteko tsa FRAP, ho nka lifoto tsa nako e fetileng tsa sampole ka 'ngoe ho tlalehiloe ka foreimi e le 1 motsotsoana.Liteko li entsoe ka makhetlo a mararo mocheso oa kamore ka liphello tse tšoanang.Litšoantšo tsohle li ile tsa hlahlojoa ho sebelisoa software ea khatiso e putsoa ea Zen 2 (Carl Zeiss AG, Oberkochen, Germany).Li-curve tsa FRAP li ne li tloaelehile, li raliloe 'me tsa kenngoa ho data e matla / nako e nkiloeng litšoantšong tse sebelisang Zen 2 ho sebelisa OriginPro 9.1.Li-curve tsa ho hlaphoheloa li ne li kentsoe mohlala oa mono-exponential ho ikarabella bakeng sa phallo ea limolek'hule ka nako e eketsehileng ea exponential ho ikarabella bakeng sa phello ea ho fumana bleaching.Ka mor'a moo re ile ra bala D ka ho sebelisa radius ea "bleaching radius" le "half-life" e neng e reriloe nakong e fetileng joalo ka ha ho bapisoa le Kang et al.5 35 e bontšitsoeng.
Mefuta e le 'ngoe ea cysteine ​​​​ea αS e entsoe ka 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) maemong a 24 (TEMPOL-24-αS) le 122 (TEMPOL-122-αS), ka ho latellana.Spin Labeling Bakeng sa liteko tsa EPR, mahloriso a αS a behiloe ho 100 μM mme khatello ea PEG e ne e le 15% (w / v).Bakeng sa maemo a fapa-fapaneng a ho kopanya, karolelano ea αS:pLK e ne e le 1:10, athe αS:ΔNt-Tau le αS:Tau441 li ne li bolokiloe ho 1:1.Bakeng sa liteko tse tlamang tsa titration ha ho se na batho ba bangata, TEMPOL-122-αS e ile ea bolokoa ho 50 μM mme li-polycations li ne li lekoa ka likhakanyo tse ntseng li eketseha, li lokisetsa boemo bo bong le bo bong ka thoko.Litekanyo tsa CW-EPR li ne li etsoa ho sebelisoa Bruker ELEXSYS E580 X-band spectrometer e nang le resonator ea Bruker ER4118 SPT-N1 e sebetsang ka maqhubu a microwave (SHF) a ~ 9.7 GHz.Thempereichara e ne e behiloe ho 25°C mme e laoloa ke mokelikeli oa nitrogen cryostat.Pono e fumanoe tlas'a maemo a sa tsitsang ka matla a MW a 4 mW, amplitude ea modulation ea 0.1 mT, le frequency ea modulation ea 100 kHz.Matla a spectral a ne a tloaeleha ho qoba phapang pakeng tsa lisampole le phokotso e ka bang teng ea spin ka lebaka la likhakanyo tse setseng tsa lisebelisoa tse fokotsang lisampole tse nang le Tau441 kapa ΔNt-Tau (e teng litharollong tsa pele tsa protheine).Litekanyetso tse fanoeng tsa g li fumanoe ka lebaka la mohlala oa spectral oa EPR o entsoeng ho sebelisoa software ea Easyspin (v. 6.0.0-dev.34) e kenngoeng tšebetsong ho Matlab®67.Ho ile ha sebelisoa mefuta e le 'ngoe / tse peli tsa isotropic ho etsa mohlala oa data.Ka mor'a ho tloaeleha matšoao 'ohle, mesaletsa e ile ea baloa ka ho tlosa papiso e' ngoe le e 'ngoe ho tloha mofuteng o lumellanang oa liteko.Bakeng sa tlhahlobo e tlamang ea titration, matla a lekanyelitsoeng a sehlopha sa boraro ho sehlopha sa bobeli sa spectrum se tloaelehileng sa EPR (IIII/III) se ile sa sebelisoa ho beha leihlo ho tlama ha polycations ho αS.Ho hakanya "dissociation constant" (Kd), sephetho se phethiloeng se ne se kentsoe moetsong o lekantsoeng ho nka libaka tse tlamang tse ts'oanang le tse ikemetseng.
Liteko tsa spectroscopy tsa NMR li ile tsa etsoa ka Bruker Neo 800 MHz (1H) NMR spectrometer e nang le cryoprobe le Z-gradient.Liteko tsohle li entsoe ho sebelisoa 130-207 µM αS le tse lekanang le αS/ΔNt-Tau le pLK ho 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 mme li entsoe ka 15°C.Ho beha leihlo LPS ka NMR, 10% PEG e kentsoe lisampoleng tse tsoakiloeng pele.The chemical shift perturbation plot (Fig. 1b) e bonts'a karolelano ea 1H le 15N ea lik'hemik'hale tsa lik'hemik'hale.Lipontšo tsa αS 2D1H-15N HSQC li ile tsa abeloa ho latela mosebetsi o fetileng (BMRB ho kena #25227) 'me ea tiisoa ka ho rekota le ho hlahloba ponahalo ea 3D ea HNCA, HNCO le CBCAcoNH.Liphetoho tsa lik'hemik'hale tsa 13Cα le 13Cβ li ne li baloa ka pel'a ΔNt-Tau kapa pLK ho lekanya liphetoho tse ka bang teng mekhoeng ea bobeli ea sebopeho ha li bapisoa le liphetoho tsa lik'hemik'hale tsa αS ka mokhoa o hloekileng oa coil conformation 68 (Supplementary Figure 5c).Litekanyetso tsa R1ρ li lekantsoe ka ho rekota liteko tsa hsqctretf3gpsi (tse fumanoeng laebraring ea Bruker) ka tieho ea 8, 36, 76, 100, 156, 250, 400, le 800 ms, 'me lits'ebetso tsa exponential li ile tsa lokisoa ho fihlela sehlohlolong sa tieho e fapaneng. linako tsa ho fumana R1ρ le ho se kholisehe ha eona ha liteko.
Liteko tse entsoeng ka nako ea mebala e 'meli tsa fluorescence microscopy li ile tsa etsoa ka nako ea khoebo ea MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) ka sesebelisoa se amanang le nako se le seng sa photon counting (TCSPC).Hlooho ea laser diode e sebelisetsoa pulsed interleaved excitation (PIE), beam e feta ka mokhoa o le mong oa waveguide 'me e hlophisitsoe ho matla a laser a 10 ho 100 nW bakeng sa 481 nm le 637 nm laser mela e lekantsoeng ka mor'a seipone sa dichroic.Sena se tiisa sekhahla se nepahetseng sa ho bala, ho qoba litlamorao tsa photon aliasing, photobleaching le saturation.μ-Slide angiogenesis likoahelo kapa lipoleiti (Ibidi GmbH, Gräfelfing, Jeremane) li ile tsa kenngoa ka ho toba metsing a qoelisoang holim'a lense ea Super Apochromat 60x NA 1.2 e nang le molaleng oa ho lokisa (Olympus Life Sciences, Waltham, USA).Seipone sa 488/640 nm dichroic (Semrock, Lake Forest, IL, USA) se ne se sebelisoa e le sekhetho se seholo sa marako.Mahlaseli a sa tsitsang a koetsoe ke sekoti se nang le bophara ba li-microns tse 50, ebe mahlaseli a tsepamisitsoeng a aroloa ka litsela tse 2 tsa ho lemoha ka sekhahla sa 50/50.Li-filters tsa Bandpass emission (Semrock, Lake Forest, IL, USA) 520/35 bakeng sa dae e tala (AF488) le 690/70 bakeng sa dae e khubelu (Atto647N) li ne li sebelisoa ka pel'a mochine.Li-avalanche diode tse nang le photon e le 'ngoe (SPAD) (Lisebelisoa tse nyenyane tsa Photon, Bolzano, Italy) li ne li sebelisoa e le lisebelisoa.Ka bobeli pokello ea lintlha le tlhahlobo li entsoe ho sebelisoa software e fumanehang khoebong ea SymphoTime64 (PicoQuant GmbH, Berlin, Jeremane).
Li-microliter tse mashome a mahlano tsa lisampole tsa LLPS li ile tsa sebelisoa ho liliba tsa μ-Slide angiogenesis (Ibidi GmbH, Gräfelfing, Jeremane).Litšoantšo tse hlahisoang li lebisitsoe ho 20 µm ka holimo ho tlase ho seliba bakeng sa sebaka se nepahetseng sa ho sebetsa bakeng sa marotholi a emisitsoeng le ho ~ 1 µm bakeng sa rafts le matheba a nang le axial resolution ea bonyane 0.25 µm/pixel le nako ea ho lieha ea 400 µs/pixel.Khetha lintlha ka ho sebelisa moeli oa matla o ipapisitseng le boholo ba lets'oao le tloaelehileng (PBG, e bolelang + 2σ) bakeng sa kanale e 'ngoe le e 'ngoe e le hore ho khethoe feela marotholi a protheine ea metsi, lirafshoa kapa matheba, ho sefa tšimoloho efe kapa efe e ka bang teng ho tloha karolong e qhalakaneng.Ho sekaseka nako ea bophelo ea mofuta o mong le o mong (τ) oa kanale ka 'ngoe (e tala, "g" bakeng sa AF488 le khubelu, "r" bakeng sa Atto647N), re khethile libaka tse khahlang (ROIs) tse nang le marotholi, rafts, kapa matheba (Supplementary Figure 1 ).8b) 'me ba li fumane ka ho lumellana le ho bola ha bona bophelo bohle (τD, τR le τP bakeng sa marotholi, lirapa kapa matheba, ka ho latellana, bona Supplementary Fig. 8c) mocha ka mong o sebelisa tlhahlobo ea mohatla le mohlala oa likarolo tse peli tsa ho bola.Karolelano ea τ ho tsoa ho τ.Li-ROI tse hlahisitseng li-photon tse fokolang haholo bakeng sa mefuta e mengata ea li-exponential li ne li sa kenyelletsoa ho hlahlojoa.The cutoff e sebelisitsoeng e ne e le <104 photon bakeng sa rafts le matheba le 103 bakeng sa marotholi.Marotholi a na le moeli o tlase hobane ho thata ho fumana li-curve tse bolileng tse nang le litekanyetso tse phahameng tse matla, kaha marotholi a lebaleng la setšoantšo a atisa ho ba manyane le a mangata.Li-ROI tse nang le lipalo tsa photon ka holimo ho moeli oa ho bokella photon (tse behiloeng ho> 500 counts / pixel) le tsona li ile tsa lahloa bakeng sa tlhahlobo.Bapisa sekhahla sa ho bola ho matla se fumanoeng sebakeng sa thahasello ka matla ho 90% ea boholo (hanyane ka mor'a ho bola ho matla) ho tloha qalong ea bophelo ba ts'ebeletso ho netefatsa hore ho na le tšitiso e fokolang ea IRF ha e ntse e boloka e ts'oana bakeng sa ho senyeha ha matla. litlhophiso Fensetere ea nako e amanang Li ile tsa hlahlojoa 25 ho 50 ROI bakeng sa li-rafts le matheba le 15-25 ROI bakeng sa marotholi, litšoantšo tse khethiloeng ho feta likopi tsa 4 tse tlalehiloeng bonyane litekong tse ikemetseng tsa 3.Liteko tsa t-tailed tse peli li sebelisitsoe ho lekola phapang ea lipalo lipakeng tsa mefuta kapa lipakeng tsa litsamaiso tsa coacervate.Bakeng sa tlhahlobo ea pixel-by-pixel ea bophelo bohle (τ), kakaretso ea nako ea bophelo holim'a lebala bakeng sa mocha ka mong e ile ea baloa 'me ho hakanngoa le 2/3-component ea mohlala oa exponential attenuation.Nako ea bophelo ea pixel e 'ngoe le e 'ngoe e ile ea kenngoa ho sebelisoa litekanyetso tsa τ tse neng li balletsoe pele, ho fella ka setšoantšo se lekanang sa pseudocolor FLIM.Nako ea bophelo ba mohatla e ne e tšoana ho pholletsa le litšoantšo tsohle tsa mocha o le mong, 'me ho bola ka' ngoe ho ne ho hlahisa li-photon tse lekaneng ho fana ka mokhoa o tšepahalang.Bakeng sa tlhahlobo ea FRET, lipikselse li khethiloe ka ho sebelisa moeli o tlase oa li-photon tse 100, tse neng li lekanya letšoao la ka morao (FBG) la 11 photon.Matla a fluorescence a mocha ka mong a ile a lokisoa ke lintlha tsa ho lokisa tse entsoeng ka liteko: 69 spectral crosstalk α e ne e le 0.004, thabo e tobileng β e ne e le 0.0305, ts'ebetso ea ho lemoha γ e ne e le 0.517.Ts'ebetso ea FRET boemong ba pixel e baloa ho sebelisoa equation e latelang:
moo FDD e leng matla a fluorescence a hlokometsoeng mocha oa mofani (o motala), FDA ke matla a fluorescence a bonoang mocha o amohelang (o khubelu) tlas'a ts'oaetso e sa tobang, 'me FAA ke matla a fluorescence a bonoang mocha o amohelang (o khubelu) tlas'a thabo e tobileng ( PIE).Maqhubu a matla a fluorescence a bonoa ka kanaleng).
Beha 100 µl ea litharollo tsa karabelo ea LLPS e nang le 25 µM e sa ngolisoang monomeric Tau441 (e nang le kapa ntle le 25 µM αS) ka har'a LLPS buffer (e tlatselletsoa joalo ka holimo) ho li-microplate tse sa tlameng tse 96 tse nang le foil e khomarelang le sebopeho sa marotholi se hlahlobiloeng ke WF. tekatekano.nakong ea 10 min.Ka mor'a lihora tse 48 tsa ho futhumala mocheso oa kamore, boteng ba liprotheine le matheba bo ile ba tiisoa.Ebe u tlosa mokelikeli ka holim'a lirafshoa ho tloha lilibeng, ebe u eketsa 50 L ea dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) 'me u e kenye metsotso e 10.Letsoai le phahameng le tiisa hore LLPS e ke ke ea phetoa ka lebaka la masala a PEG, 'me likopano tse ka khonehang tsa protheine tse entsoeng feela ka ho sebelisana ha motlakase li tla qhaqhoa.Botlaaseng ba seliba bo ile ba hlajoa ka hloko ka ntlha ea micropipette mme tharollo e hlahisoang e fetisetsoa selibeng se se nang letho.Ka mor'a ho kenngoa ha lisampole ka 50 μM ThT bakeng sa 1 h, ho ba teng ha libaka tse ka thōko ho ile ha hlahlojoa ke WF microscopy.Lokisetsa li-sonicated αS fibrils ka ho kenya 300 µl ea 70-µM αS tharollo ho PBS e nang le pH 7.4, sodium azide 0.01% ho 37 °C le 200 rpm ka orbital shaker bakeng sa matsatsi a 7.Ka nako eo tharollo e ne e le centrifuged ho 9600 × g bakeng sa 30 min, pellet e ile ea tsosolosoa ka PBS pH 7.4 'me ea sonicated (1 min, 50% cycle, 80% amplitude ka Vibra-Cell VC130 sonicator, Sonics, Newton, USA) lisebelisoa tsa fibril. ka kabo e batlang e lekana ea boholo ba li-fibrils tse nyane.
Litlhahlobo tsa FCS/FCCS le tšibollo ea mebala e 'meli e iketsahalletseng (TCCD) li entsoe ka maekerosekoupo ea fluorescent confocal e rarollotsoeng ka nako ea MT200 (Pico-Quant, Berlin, Germany) e sebelisitsoeng bakeng sa liteko tsa microscopy ea FLIM-FRET ho sebelisoa mokhoa oa PIE.Matla a laser bakeng sa liteko tsena a kentsoe ho 6.0 µW (481 nm) le 6.2 µW (637 nm).Motsoako oa matla ana a laser a khethiloe ho hlahisa khanya e tšoanang bakeng sa lipara tsa fluorophores tse sebelisoang ha ho ntse ho fihleloa litekanyetso tse nepahetseng tsa ho bala le ho qoba photobleaching le saturation.Ka bobeli pokello ea lintlha le tlhahlobo li entsoe ho sebelisoa software e fumanehang khoebong ea SymphoTime64 version 2.3 (PicoQuant, Berlin, Germany).
Mehlala ea likarolo tse arohaneng tsa αS/Tau tse fumanoeng ho sebelisoa LLPS li hlapollotsoe ka thoko ho "monomolecular concentration" e nepahetseng (hangata ke dilution ea 1:500, kaha likakaretso li se li le maemong a tlase ha li qheletsoe ka thoko ho lisampole tsa coacervate).Mehlala e ile ea sebelisoa ka kotloloho ho li-coverslips (Corning, USA) tse koahetsoeng ka motsoako oa BSA ka har'a motsoako oa 1 mg/mL.
Bakeng sa tlhahlobo ea PIE-smFRET likanaleng tse tala le tse khubelu, ho ile ha sebelisoa sekhahla se tlase sa li-photon tse 25 ho sefa matšoao a matla a tlase a bakoang ke liketsahalo tsa monomeric (hlokomela hore li-monomers li feta lisampole tse kopaneng ha li bapisoa le likarolo tse arohaneng).Moeli ona o ne o baloa e le makhetlo a mahlano a matla a karolelano ea αS ea monomeric e fumanoang ho tsoa tlhahlobisong ea lisampole tse hloekileng tsa monoma e le hore ho khethoe ka ho khetheha likakaretso bakeng sa tlhahlobo.Potoloho ea koloi ea PIE, hammoho le ts'ebeliso ea data ea TSCPC, e nolofalelitse ts'ebeliso ea filthara ea boima ba bophelo bohle e thusang ho tlosa bokamorao le spectral crosstalk.Bophahamo bo kgethilweng ho sebediswa menyako e ka hodimo bo ile ba lokiswa ho sebediswa letshwao le tlwaelehileng la bokamorao le kgethilweng ho tswa ho histograms ya ketsahalo khahlano le matla/bin tsa disampole tsa buffer-feela.Ho phatloha ho amanang le li-aggregate tse kholo hangata ho nka meqomo e 'maloa e latellanang ka nako (e behiloeng ho 1 ms).Maemong ana, ho ile ha khethoa bin ea matla a holimo.Bakeng sa tlhahlobo ea FRET le stoichiometric, ho ile ha sebelisoa gamma factor γ (0.517).Spectral crosstalk le litlatsetso tse hlasimollang ka kotloloho ha li na letho (li lekantsoe ka liteko) ho matla a laser a hlasimollang a sebelisitsoeng.Ho sebetsa hantle le stoichiometry ea FRET ka ho phatloha ho baloa ka tsela e latelang.