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347 Stainless Simbi Pipe Kutsanangurwa
347 12.7 * 1.24mm Stainless simbi yakakoswa tubing
Kunze Diameter: 6.00 mm OD kusvika 914.4 mm OD, Saizi inosvika 24” NB iripo Ex-stock, OD Size Simbi Tubes iripo Ex-stock
SS 347 Kukora Kwepombi: 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, zvichingodaro (0.5-12mm) Kana saizi isiri-yenguva kuti igadziriswe sezvinodiwa
Type: SS 347 Seamless Pipes |SS 347 ERW Pipes |SS 347 Welded Pipes |SS 347 Yakagadzirwa Pipes |SS 347 CDW Machubhu, LSAW Mapaipi / musono-welded / Redrawn
Form: 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
Kureba: Imwechete Random, Kaviri Random & Inodiwa Length End: Plain End, Beveled End, Yakatsikwa
Kugumisa Kudzivirirwa: Plastic Caps |Kunze Kupedzisa: 2B, No.4, No.1, No.8 Mirror Finish yeStainless Steel Pipes, Pedzisa maererano nezvinodiwa nevatengi
Mamiriro Ekutumira: Yakanamirwa uye yakanyungudutswa, yakapushwa, Inopenya yakanyungudutswa, Cold Drawn
Kuongorora, Mishumo Yekuyedza: Zvitupa zveMiri Yekuyedza, EN 10204 3.1, Chemical Reports, Mechanical Reports, PMI Test Reports, Visual Inspection Reports, Wechitatu Bato Rokuongorora Mishumo, NABL Yakatenderwa Lab Mishumo, Inoparadza Test Report, Isiri Inoparadza Mishumo Yekuyedza.
Packing: Yakaiswa muMabhokisi Emapuranga, Mabhegi ePlastiki, Simbi Zvisungo zvakasungwa, kana sezvikumbiro zveVatengi.
Specials: Saizi uye Zvinotsanangurwa kunze kwepamusoro zvinogona kugadzirwa pakukumbira
SS 347 Pipe Size Range: 1/2 inch NB, OD kusvika 24 inch
ASTM A312 347: Yasina musono uye yakatwasuka-seam yakashongedzerwa austenitic pombi yakagadzirirwa kupisa kwakanyanya uye sevhisi inoparadza.Filler simbi haibvumidzwe panguva yewelding.
ASTM A358 347: Electric fusion welded austenitic pombi yekuora uye/kana yakakwirira tembiricha sevhisi.Kazhinji chete pombi inosvika 8 inch inogadzirwa kune iyi yakatarwa.Kuwedzerwa kwesimbi yekuzadza kunobvumirwa panguva yekuputika.
ASTM A790 347: Yasina musono uye yakatwasuka-seam yakasungwa ferritic/austenitic (duplex) pombi inoitirwa kuita corrosion sevhisi, ichinyanya kukoshesa kuramba kushushikana kwegora kutsemuka.
ASTM A409 347: Yakatwasuka-seam kana spiral-seam electric fusion yakasungirirwa dhayamita hombe austenitic light-wall pombi muhukuru 14” kusvika 30” ine madziro Sch5S uye Sch 10S yekuora uye/kana yakakwirira.
ASTM A376 347: Seamless austenitic pombi yemhando yepamusoro yekupisa.
ASTM A813 347: Single-seam, single- kana kaviri- welded austenitic pombi yekushisa kwepamusoro uye zvinosvibisa zvikumbiro.
ASTM A814 347: Cold-worked welded austenitic pombi yekupisa kwakanyanya uye general corrosive service.
347H Stainless Steel Pipes Chemical Composition
Giredhi | C | Mn | Si | P | S | Cr | Mo | Ni | N | |
---|---|---|---|---|---|---|---|---|---|---|
347H | min. | 0.04 | - | - | - | - | 17.0 | 3.00 | 9.0 | - |
max. | 0.10 | 2.0 | 1.00 | 0.045 | 0.030 | 19.0 | 4.00 | 13.0 | - |
Stainless Simbi 347H Pipe Mechanical Properties
Giredhi | Tensile Simba (MPa) min | Goho Simba 0.2% Uchapupu (MPa) min | Elongation (% mu50mm) min | Kuoma | |
---|---|---|---|---|---|
Rockwell B (HR B) max | Brinell (HB) max | ||||
347H | 515 | 205 | 40 | 92 | 201 |
Stainless Simbi 347H Mapaipi Zvenyama Zvivakwa
Giredhi | Density (kg/m3) | Elastic Modulus (GPa) | Zvinoreva Coefficient yeKuwedzera Kwekupisa (m/m/0C) | Thermal Conductivity (W/mK) | Chaicho Kupisa 0-1000C (J/kg.K) | Electrical Resistivity (nm) | |||
---|---|---|---|---|---|---|---|---|---|
0-1000C | 0-3150C | 0-5380C | pa1000C | pa5000C | |||||
347H | 8000 | 193 | 17.2 | 17.8 | 18.4 | 16.2 | 21.5 | 500 | 720 |
Akaenzana Magiredhi e347H Stainless Simbi Pipe
Giredhi | UNS Nha | Old British | Euronorm | Swedish SS | Japanese JIS | ||
---|---|---|---|---|---|---|---|
BS | En | No | Zita | ||||
347H | S34709 | - | - | 1.4961 | - | - | - |
Mitemo | Designation |
ASTM | A312 |
---|---|
ASME | SA 312 |
Amyloid alpha-synuclein (αS) kuunganidzwa chiratidzo chechirwere cheParkinson uye mamwe ma synucleinopathies.Munguva ichangopfuura, puroteni yetau inowanzobatanidzwa nechirwere cheAlzheimer yakabatanidzwa ne αS pathology uye yakawanikwa ichibatanidza mu-αS-rich inclusions, kunyange zvazvo ma molecular maitiro ekubatanidza mapuroteni maviri anoramba asina kujeka.Isu tinoshuma pano kuti αS chikamu chinopatsanurwa kuita mvura condensates kuburikidza ne electrostatic complex condensation ine positive charged polypeptides senge tau.Zvichienderana nekubatana kwe αS yepolycations uye chiyero chekuderera kwevalence ye coagulation network, clots inopinda nekukurumidza gelation kana coalescence inoteverwa neanononoka amyloid aggregation.Nekubatanidza sutu yemhando yepamusoro biophysical tekinoroji, takakwanisa kuratidza iyo yemvura-yemvura αS/Tau chikamu chekuparadzaniswa uye kuona izvo zvakakosha zvinotungamira mukuumbwa kweakasiyana aggregates ane ese mapuroteni mune yemvura protein condensate.
Pamusoro pemamembrane compartments, kupatsanurwa kwenzvimbo mumaseru kunogonawo kuwanikwa nekuumbwa kweprotein-rich, liquid-like dense body inonzi biomolecular condensates kana madonhwe, kuburikidza nemaitiro anozivikanwa se liquid-liquid phase separation (LLPS).Aya madonhwe anoumbwa neakawanda epanguva yekudyidzana, kazhinji pakati pemapuroteni kana mapuroteni neRNA, uye anoshanda akasiyana mabasa munenge ese masystem ehupenyu.Nhamba huru yeLLP-anokwanisa mapuroteni anoratidza kutevedzana kwepasi kuomarara kwakakanganiswa zvakanyanya mune zvakasikwa uye mukuumbwa kwebiomolecular condensates3,4,5.Zvidzidzo zvakawanda zvekuyedza zvakaratidza kuchinjika, kazhinji kusagadzikana, uye kuwanda kwemaitiro emapuroteni anoumba aya emvura-se-condensates, kunyangwe zvishoma zvinozivikanwa nezve chaiwo mamorekuru anotemesa anodzora kukura nekukura kweaya condensates kune akanyanya kusimba-senge. state..
Iyo data nyowani inotsigira fungidziro yekuti aberrant protein-driven LLPS uye shanduko yemadonhwe kuita yakasimba zvimiro inogona kuve yakakosha nzira dzemaserura dzinotungamira mukuumbwa kwezvisinganyunguriki zvine chepfu aggregates izvo zvinowanzova zviratidzo zvezvirwere zvinopera.Mazhinji maLLPS-akabatanidzwa intrinsically disordered mapuroteni (IDPs), anowanzo kubhadhariswa zvakanyanya uye anochinjika, ave kwenguva refu achibatanidzwa neurodegeneration kuburikidza nekuita kweamyloid aggregation.Kunyanya, biomolecular IDP condensates senge FUS7 kana TDP-438 kana mapuroteni ane hombe yakaderera kuomarara madomasi senge hnRNPA19 akaratidzwa zera kuita gel-senge kana mafomu akasimba kuburikidza nemaitiro anonzi fluidization.compound.kushanduko yakasimba yechikamu (LSPT) sechiitiko chenguva kana mukupindura kune dzimwe shanduko mushure mekushandura kana pathologically yakakosha mutations1,7.
Imwe IDP yakabatana neLLPS mu vivo iTau, microtubule-yakabatana neprotein isina kurongeka iyo amyloid aggregation yakave yakabatanidzwa muAlzheimer's disease10 asi zvakare ichangobva kuve yakabatanidzwa muParkinson's disease (PD) uye mamwe synaptic nuclear proteinopathies 11, 12, 13 ane hukama.Tau yakaratidzwa kuti yakangoerekana yasiyana nemhinduro/cytoplasm nekuda kwekudyidzana kwakanaka kwemagetsi14, zvichiita kuti pave nemadonhwe etau-enriched anozivikanwa se electrostatic coacervates.Izvo zvakaonekwawo kuti rudzi urwu rwekusawirirana kusingatarisirwi ndiro simba rinofambisa kuseri kweakawanda biomolecular condensates muzvisikwa15.Panyaya yeprotein yetau, electrostatic aggregation inogona kuumbwa nekuunganidzwa kwakapusa, umo nzvimbo dzakapokana dzeprotein dzinokonzeresa cleavage process, kana nekuunganidzwa kwakaomarara kuburikidza nekudyidzana nemapolymer akashata akadai seRNA.
Munguva ichangopfuura, α-synuclein (αS), amyloid IDP inobatanidzwa muPD uye zvimwe zvirwere zveurourodegenerative pamwe chete zvinozivikanwa se synucleinopathy17,18, yakaratidzwa mumaserura uye mhuka dzemhando19,20 yakakonzerwa muprotein condensates ne-fluid-like behavior.In vitro zvidzidzo zvakaratidza kuti αS inopinda LLPS nekuunganidza nyore kuburikidza nekunyanya kushandiswa kwehydrophobic, kunyange zvazvo izvi zvinoda kunyanya kuwanda kweprotein uye atypically refu incubation times19,21.Kunyangwe iyo αS-ine condensates inocherechedzwa muvivo inoumbwa neiyi kana mamwe maitiro eLLPS inoramba iri chinhu chakakosha chisina kugadziriswa.Saizvozvowo, kunyange zvazvo αS amyloid aggregation yakaonekwa mueuroni muPD uye mamwe ma synucleinopathies, iyo chaiyo nzira iyo αS inopinda nayo intracellular amyloid aggregation inoramba isina kujeka, sezvo kuwedzera kweprotein iyi kusingaoneki kunokonzera izvi pachayo.Kuwedzera kukuvadzwa kweserura kunowanzo kudiwa, zvichiratidza kuti dzimwe nzvimbo dzemaserura kana microenvironments dzinodiwa pakudzoreredza intracellular αS amyloid assemblies.Imwe nharaunda yemasero inonyanya kuwirirana kuunganidza inogona kunge iri mukati meprotein condensates 23.
Sezvineiwo, αS uye tau vakawanikwa vachibatana-nzvimbo mune hunhu hwechirwere inclusions muvanhu vane chirwere cheParkinson uye mamwe ma synucleinopathies 24,25 uye zviedzo zvakashuma hukama hwehukama hwepakati pakati pemapuroteni maviri 26,27 ichiratidza hukama hunogona kuitika pakati peaggregation αS uye. tau mune neurodegenerative zvirwere.urwere.αS uye tau zvakawanikwa kuti zvibatane uye zvinokurudzira kuwirirana kweumwe neumwe mu vitro uye mu vivo 28,29 uye hterogeneous aggregates inoumbwa nemapuroteni maviri aya akaonekwa muuropi yevarwere vane synucleinopathies 30.Nekudaro, zvishoma zvinozivikanwa nezve hwaro hwemamorekuru hwekudyidzana pakati pe αS ne tau uye magadzirirwo ekubatanidzwa kwayo.αS inonzi yaidyidzana netau kuburikidza nekukwezva kwemagetsi pakati peC-terminal dunhu re αS nepakati peproline-rine dunhu retau, iro rakapfumiswawo muzvisaririra zvinochajiswa zvakanaka.
Muchidzidzo ichi, tinoratidza kuti αS inogona kunyatso patsanuka kuita madonhwe kuburikidza ne electrostatic complex condensation pamberi petau protein, mukupesana nekudyidzana kwayo nemamwe mapolypeptides anochajiswa zvakanaka sepoly-L-lysine (pLK), uye mukuita uku.αS inoshanda se scaffold molecule ye droplet network.Takaona misiyano inocherechedzwa mukuita kwekukura kwe electrostatic αS coacervates, iyo inosanganiswa nekusiyana kwevalency uye simba rekudyidzana kwemapuroteni anobatanidzwa mucoacervate network.Sezvineiwo, takaona co-aggregation ye αS uye tau amyloid mapuroteni mune anogara kwenguva refu emvura coacervates uye takaona zvimwe zvakakosha zvinotungamira mukubatanidzwa kweaya mapuroteni maviri mune akadaro coacervates.Pano isu tinotsanangura zvakadzama maitiro aya, inova inogoneka molecular mechanism iri pasi peiyo colocalization yemapuroteni maviri muchirwere-chaiyo inclusions.
αS ine yakanyanya anionic C-terminal muswe pane kwayakarerekera pH (Fig. 1a), uye isu takafungidzira kuti inogona kupinda LLPS kuburikidza ne condensation ye electrostatic complexes ne polycationic disordered polypeptide molecules.Isu takashandisa 100-residue poly-L-lysine (pLK) seyokutanga modhi molecule nekuda kweiyo yakanaka chaja uye yakakanganiswa polymeric hunhu pakusarerekera pH 32. Kutanga, takasimbisa kuti pLK inopindirana neCt domain ye αS kuburikidza neSolution NMR spectroscopy. (Mufananidzo 1b) uchishandisa 13C / 15N-yakanyorwa αS muhupo hwekuwedzera αS: pLK molar ratios.Kubatana kwepLK neCt-domain ye αS inozviratidza mukuvhiringidza kwekuchinja kwemakemikari uye kuderera kwepamusoro-soro munharaunda iyi yeprotini.Sezvineiwo, patakasanganisa αS ne pLK pane iyo αS yekumisikidza approx.5–25 µM pamberi pe polyethylene glycol (5–15% PEG-8) (yakajairika LLPS buffer: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) takabva tangopfuura nemumunda wakakura wekugadzira mapuroteni. .madonhwe akaonekwa achishandisa fluorescence (WF) uye inopenya-munda (BF) microscopy (Fig. 1c).1-5 µm madonhwe ane concentrated αS (yakawedzerwa 1 µM AlexaFluor488-yakanyorwa αS, AF488-αS), maitiro avo emagetsi anogona kutorwa kubva mukupokana kwavo kusvika ku10% 1,6-hexanediol (1,6-HD) uye kunzwisiswa kwayo kuwedzera kweNaCl concentration (Fig. 1c).Iyo yemvura-yakafanana yemhando ye coacervates ye αS / pLK electrostatic complex inoratidzwa nekukwanisa kwavo kusanganisa mukati memilliseconds (Fig. 1d).Tichishandisa turbidimetry, takaongorora kuumbwa kwemadonhwe pasi pemamiriro ezvinhu aya, takasimbisa chimiro che electrostatic chekubatana kukuru kwakabatana nekugadzikana kwayo (Fig. 1e), uye takaongorora maitiro emhando dzakasiyana-siyana dzepolymer pachirongwa cheLLPS (Fig. 1f).Kunyangwe kuumbwa kwemadonhwe kuchicherechedzwa pamusoro pehuwandu hwakasiyana hwepolymer ratios, maitiro acho akanaka kwazvo kana pLK iri kudarika αS.MaLLP akaonekwa zvakare achishandisa makemikari akasiyana-siyana ekudzinga agent dextran-70 (70 kDa) kana kushandisa akasiyana mafomati emuenzaniso, anosanganisira madonhwe egirazi anotsvedza, matsime emicroplate emhando dzakasiyana siyana, Eppendorf kana quartz capillaries.
a Schematic inomiririra yeakasiyana mapuroteni matunhu muWT-αS uye ΔCt-αS akasiyana anoshandiswa muchidzidzo ichi.Iyo amphipathic N-terminal domain, iyo hydrophobic amyloid-forming (NAC) dunhu, uye yakashata inochaja C-terminal domain inoratidzwa mubhuruu, orenji, uye tsvuku, zvichiteerana.Iyo Net Charge Per Residual (NCPR) mepu yeWT-αS inoratidzwa.b NMR kuongororwa kweiyo αS / pLK kupindirana mukusavapo kwemacromolecular clumps.Sezvo pLK concentration inowedzera (αS: pLK molar ratios ye1: 0.5, 1: 1.5, uye 1:10 inoratidzwa muchiedza chegirini, girini, uye rima rakasvibira, zvichiteerana).c Coacervate αS/pLK (molar ratio 1:10) pa25 µM (1 µM AF488-yakanyorwa αS kana Atto647N-yakanyorwa pLK yeWF imaging) muLLPS buffer (kumusoro) kana kuwedzerwa ne500 mM NaCl (pazasi 1 pasi kana mushure) % 1,6-hexanediol (1,6-HD; pasi kurudyi).Scale bar = 20 µm.d Mumiririri microscopic mifananidzo yeBF droplet fusion ye αS / pLK (molar ratio 1: 10) pamusangano we 25 μM;miseve inoratidza kubatanidzwa kwemadonhwe ega ega (miseve yakatsvuka neyero) mudonhwe idzva (museve weorenji) mukati me200 ms).Scale bar = 20 µm.e Kupararira kwechiedza (pa350 nm) αS/pLK aggregation muLLPS buffer pamberi uye mushure mekuwedzera kwe500 mM NaCl kana 10% 1,6-HD pa 25 µM αS (N = 3 sampuli replicates, zvinoreva uye kutsauka kwakajairwa kunoratidzwawo).f BF mufananidzo (kumusoro) uye chiedza kuparadzira kuongorora (pa 350 nm, pasi) ye αS/pLK aggregation pa 25 μM αS nekuwedzera αS: pLK molar ratio (N = 3 sampuli replicates, kureva uye chiyero kutsauka kunoratidzwawo).Scale bar = 10 µm.Scale bar pamufananidzo mumwe inoratidza chiyero chemifananidzo yese mupaneri imwe.Raw data inopiwa nenzira yemafaira e data.
Kubva pane zvatinoona nezve αS/pLK electrostatic complex condensation uye zvakamboonekwa zve αS semutengi molecule ye tau/RNA condensate kuburikidza nekudyidzana kwakananga ne tau31, isu takafungidzira kuti αS uye tau zvinogona kupatsanura pamwe nezvinonyungudutsa kana RNA isipo. condensation.kuburikidza ne electrostatic complexes, uye αS ndiyo scaffold protein mu αS/Tau coacervates (ona tau charge distribution muFigure 2e).Takaona kuti kana 10 μM αS uye 10 μM Tau441 (ine 1 μM AF488-αS uye 1 μM Atto647N-Tau, zvichiteerana) yakasanganiswa pamwe chete muLLPS buffer, vakagadzira nyore mapuroteni akaunganidzwa ane ese mapuroteni, sekuonekwa neWF microscopy.(Mufananidzo 2a).Iyo colocalization yemapuroteni maviri mumadonhwe akasimbiswa neiyo confocal (CF) microscopy (Supplementary Fig. 1a).Maitiro akafanana akaonekwa apo dextran-70 yakashandiswa seaggregation agent (Supplementary Fig. 1c).Tichishandisa FITC-yakanyorwa PEG kana dextran, takaona kuti maajenti maviri ekutsvikinyidzana akagoverwa zvakaenzana pamasampuli, asingaratidzi kusarura kana kushamwaridzana (Supplementary Fig. 1d).Asi, inoratidza kuti muhurongwa uhu vanosimudzira kupatsanurwa kwechikamu kuburikidza nemacromolecular crowding mhedzisiro, sezvo PEG iri yakasarudzika yakagadzikana yakagadzikana mumiriri, sezvinoonekwa mune mamwe maLLP masisitimu33,34.Aya madonhwe eprotein-rich ane hanya neNaCl (1 M) asi kwete ku1,6-HD (10% v / v), achisimbisa maitiro avo emagetsi (Supplementary Fig. 2a, b).Maitiro avo emvura akasimbiswa nekucherechedza millisecond kubatanidza madonhwe zviitiko vachishandisa BF microscopy (Fig. 2b).
a Confocal (CF) microscopy mifananidzo ye αS/Tau441 coacervates muLLPS buffer (10 μM yeprotein yega yega, 0.5 μM yeAF488-yakanyorwa αS uye Atto647N-yakanyorwa Tau441).b Representative differential interference difference (DIC) mifananidzo ye αS/Tau441 droplet fusion zviitiko (10 μM yeprotein imwe neimwe).c Chikamu dhayagiramu chakavakirwa pakupararira kwechiedza (pa350 nm) yeTau441 LLPS (0–15 µM) pasina (kuruboshwe) kana kuvapo (kurudyi) kwe50 µM αS.Mavara anodziya anoratidza kuwedzera kupararira.d Kupararira kwechiedza che αS/Tau441 LLPS samples nekuwedzera αS concentration (Tau441 pa 5 µM, N = 2-3 kudzokorora sampuli sezvakaratidzwa).e Schematic inomiririra yemamwe maprotein etau akasiyana uye matunhu akasiyana eprotein anoshandiswa muchidzidzo ichi: yakashata yakapihwa N-terminal domain (tsvuku), proline-rich dunhu (blue), microtubule-binding domain (MTBD, yakasimbiswa muorenji), uye amyloid-forming pair spiral.filament regions (PHF) iri mukati meMTBD (grey).Iyo Net Charge Per Residue (NCPR) mepu yeTau441 inoratidzwa.f Kushandisa 1 µM AF488-yakanyorwa αS uye Atto647N-yakanyorwa ΔNt-, uchishandisa 1 µM AF488-yakanyorwa αS kana ΔCt-αS pamberi peΔNt-Tau (pamusoro, 10 µM paprotein) kana K18 (pasi µM5 µ ) ) ) mamicrographs eWF akapfupikiswa muLLPS kana K18 buffer.Masikero emumufananidzo mumwe anomiririra kuyerwa kwemifananidzo yese mupaneri imwe (20 µm yemapanera a, b uye f).Raw data yemapanera c uye d anopihwa semafaira edata.
Kuti tiedze basa re αS muchirongwa ichi cheLLPS, takatanga taongorora mhedzisiro ye αS pakudzikama kwemadonhwe nephelometry tichishandisa kuwedzera kweNaCl (Fig. 2c).Iyo yakakwirira yemunyu mumienzaniso ine αS, iyo inokwirisa iyo mwenje yekuparadzira kukosha (pa350 nm), iyo inoratidza kudzikamisa basa re αS mune ino LLPS system.Mhedzisiro yakafanana inogona kucherechedzwa nekuwedzera iyo αS concentration (uye nokudaro iyo αS: Tau441 ratio) kusvika approx.10-kuwedzerwa kuwedzera kune tau concentration (5 µM) (Fig. 2d).Kuti tiratidze kuti αS ipuroteni ye scaffold mu coacervates, takasarudza kuongorora maitiro eLLPS-akavhiringidza Tau mutant, iyo inoshaya nzvimbo yakaipa yeN-terminal (masara 1-150, ona Fig. 2e) inonzi ΔNt-Tau.WF microscopy uye nephelometry yakasimbisa kuti ΔNt-Tau pachayo haina kuita LLPS (Fig. 2f uye Supplementary Fig. 2d), sezvakambotaurwa kare 14. Zvisinei, apo αS yakawedzerwa kune zvigadziriso zvekupararira kweiyo truncated Tau yakasiyana, iyo LLPS nzira yakanga yakakwana zvachose. yakadzoreredzwa nedonhwe density padhuze nedonhwe density yeazere saizi mhinduro dzeTau uye αS pasi pemamiriro akafanana uye maprotein concentrations.Izvi zvinogonawo kuonekwa pasi pemamiriro epasi macromolecular crowding (Supplementary Fig. 2c).Basa renzvimbo yeC-terminal αS, asi kwete urefu hwayo hwose, muchirongwa cheLLPS chakaratidzwa nekudzivisa kuumbwa kwedonhwe uchishandisa C-terminal truncated αS variant inoshaya masara 104-140 (Fig. 1a) ye (ΔCt- αS) mapuroteni (Fig. 2f uye Supplementary Fig. 2d).Iko colocalization ye αS uye ΔNt-Tau yakasimbiswa ne confocal fluorescence microscopy (Supplementary Fig. 1b).
Kuti uwedzere kuyedza nzira yeLLPS pakati peTau441 uye αS, imwe yekuwedzera yeTau yakashandiswa, inova iyo paired helical filament core (PHF) chidimbu muiyo microtubule-binding domain (MTBD), iyo kana iine maitiro mana ekudzokorora madomasi, anowanzo zivikanwa zvakare. seK18 fragment (ona mufananidzo 2e).Yakave ichangobva kutaurwa kuti αS inonyanya kusungira kune puroteni yetau iri munharaunda yeproline-rich munhevedzano inotangira microtubule-binding domain.Zvisinei, dunhu rePHF rakapfumawo mune zvakasara zvinopomerwa zvakanaka (ona Mufananidzo 2e), kunyanya lysine (15% zvakasara), izvo zvakatikurudzira kuti tiongorore kana dunhu iri richibatsirawo pakugadzirisa αS / Tau yakaoma.Takaona kuti K18 chete yaisakwanisa kukonzeresa LLPS pakudzika kusvika ku100 μM pasi pemamiriro akaedzwa (LLPS buffer ne15% PEG kana 20% dextran) (Mufananidzo 2f).Zvisinei, patakawedzera 50 µM αS ku50 µM K18, kukurumidza kuumbwa kweprotein madonhwe ane K18 uye αS yakaonekwa nephelometry (Supplementary Fig. 2d) uye WF microscopy (Fig. 2f).Sezvaitarisirwa, ΔCt-αS haina kukwanisa kudzorera maitiro eLLPS eK18 (Fig. 2f).Isu tinocherekedza kuti αS/K18 aggregation inoda zvishoma yakakwira mapuroteni ekudzika kuti ape LLPS zvichienzaniswa ne αS/ΔNt-Tau kana αS/Tau441, zvimwe zvinhu zvakaenzana.Izvi zvinopindirana nekubatana kwakasimba kwe-αS C-terminal region ine proline-rich Tau domain kana ichienzaniswa ne-microtubule-binding domain, sezvakatsanangurwa kare 31.
Tichifunga kuti ΔNt-Tau haigone kuita LLPS kana αS isipo, takasarudza iyi Tau mutsauko semuenzaniso wekuratidza αS/Tau LLPS yakapihwa kuve nyore muLLPS masisitimu ane yakazara-refu Tau (isotype, Tau441/Tau441).ine yakaoma (heterotypic, αS/Tau441) maitiro ekuunganidza.Isu takaenzanisa dhigirii re αS aggregation (sechikamu cheyakagadziriswa chikamu protein, fαS, c) mune αS / Tau uye αS / ΔNt-Tau masisitimu ne centrifugation uye yakapararira chikamu SDS-PAGE kuongorora (ona 2e), yakawana maitiro akafanana zvakanyanya. kune ese mapuroteni panguva imwechete.Kunyanya, takawana fαS, c 84 ± 2% uye 79 ± 7% ye αS/Tau uye αS/ΔNt-Tau, zvichiteerana, zvichiratidza kuti kuwirirana kweheterotypic pakati pe αS ne tau kwakakwirira kudarika kuwirirana pakati petau molecules.pakati.
Kudyidzana nemapolycation akasiyana-siyana uye mhedzisiro yeiyo condensation process pane αS kinetics yakatanga kudzidzwa nefluorescence kupora mushure mekuita photobleaching (FRAP) nzira.Takaedza αS/Tau441, αS/ΔNt-Tau uye αS/pLK coacervates (100 μM αS yakawedzerwa ne2 μM αS AF488-αS uye 100 μM Tau441 kana ΔNt-Tau kana 1 mM pLK).Dhiyabhorosi yakawanikwa mukati memaminitsi ekutanga e30 mushure mekusanganisa zvikamu zvemuenzaniso.Kubva mumiririri FRAP mifananidzo (Fig. 3a, αS / Tau441 condensation) uye yavo inoenderana nguva kosi curves (Fig. 3b, Supplementary Fig. 3), zvinogona kuonekwa kuti αS kinetics akafanana chaizvo neaya Tau441 coacervates.uye ΔNt-Tau, iyo inokurumidza kukurumidza ne pLK.Iwo akaverengerwa diffusion coefficients e αS mukati mecoacervate maererano neFRAP (sekutsanangurwa kwaKang et al. 35) ari D = 0.013 ± 0.009 µm2/s uye D = 0.026 ± 0.008 µm2/s ye αS/Tau αS/Δ4 iyo αS/ system.pLK, Tau, uye D = 0.18 ± 0.04 µm2/s, maererano (Fig. 3c).Zvisinei, iyo diffusion coefficient αS muchikamu chakaparadzirwa ine mirairo yakawanda yehukuru yakakwirira kudarika zvikamu zvose zvakapfupiswa, sezvinotsanangurwa neFluorescence Correlation Spectroscopy (FCS, ona Supplementary Fig. 3) pasi pemamiriro akafanana (LLPS buffer) asi pasina mapolycations. ( D = 8 ± 4 µm2/s).Nokudaro, iyo kinetics yeshanduro ye αS yakaderedzwa zvakanyanya mu coacervates kana ichienzaniswa nemapuroteni muchikamu chakapararira nekuda kwekududzwa kwema molecular crowding effects, kunyange zvazvo coacervates yose inochengetedza mvura-yakafanana nezvinhu mukati mehafu yekutanga yeawa mushure mekuumbwa kwavo, kusiyana nechikamu chetau.nekukurumidza kinetics mu pLK condensate.
a–c FRAP ongororo ye αS dynamics (2% AF488-yakanyorwa αS) muma electrostatic coacervates.Mifananidzo inomiririra ye αS/Tau441 FRAP assays mune katatu inoratidzwa mu (a), apo madenderedzwa matsvuku anoratidza nzvimbo dzakabviswa.Chikero bhaa i5 µm.b Avhareji yeFRAP curves uye (c) akaverengerwa diffusion coefficients (D) ye5-6 (N) madonhwe akasiyana kubva mukuedza katatu uchishandisa 100 µM αS uye equimolar concentrations yeTau441 (tsvuku) kana ΔNt-Tau (bhuruu) kana pLK (girinhi) kakapetwa kagumi kuwanda kweLLPS.Iyo yakajairwa kutsauka kweFRAP curve inoratidzwa mune ine shaded color.Kuenzanisa, iyo diffusion coefficient αS muchikamu chakapararira yakatemerwa mune katatu uchishandisa fluorescence correlation spectroscopy (FCS) (ona Supplementary Figure 3 uye nzira dzerumwe ruzivo).d Inoenderera mberi X-band EPR spectra ye100 μM TEMPOL-122-αS muLLPS buffer pasina polycation (dema) kana pamberi pe100 μM Tau441 (tsvuku) kana ΔNt-Tau (bhuruu) kana 1 mM pLK (girinhi).Iyo inset inoratidza maonero akakwidziridzwa emitsara yakasimba yemunda apo shanduko dzinoshamisa dzinoitika.e Binding curves ye50 μM TEMPOL-122-αS ine mapolycation akasiyana-siyana mukusavapo kweLLPS (hapana PEG).Iyo yakaderedzwa amplitude yebhendi III ichienzaniswa nebhendi II (IIII/III) yeyakajairwa EPR spectrum inoratidzwa kuwedzera molar ratios yeTau441 (tsvuku), ΔNt-Tau (bhuruu) uye pLK (girinhi).Mitsetse ine mavara inoratidza yakakodzera kune data uchishandisa yakakasharara inosunga modhi ine n yakafanana uye yakazvimirira inosunga nzvimbo pane yega yega.Raw data inopiwa nenzira yemafaira e data.
Semubatsiri, takaongorora mafambisirwo eiyo αS mune akasiyana coacervates tichishandisa yakatungamirwa spin labeling (SDSL) uye inoenderera electron paramagnetic resonance (CW-EPR).Iyi nzira yakaratidza kuva inobatsira zvikuru mukutaura kushanduka uye kushanduka kweIDP nekugadzirisa kwechokwadi kwasara36,37,38.Kuti izvi zviitike, takagadzira cysteine masara mune imwechete Cys mutants uye takashandisa iyo 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Maleimide derivatives anoanyora.Kunyanya, takaisa TEMPOL probes pachinzvimbo 122 kana 24 αS (TEMPOL-122-αS uye TEMPOL-24-αS).Muchiitiko chekutanga, tinotarisa nzvimbo yeC-terminal yeprotini, iyo inobatanidzwa mukubatana nemapolycations.Pane kudaro, chinzvimbo 24 chinogona kutipa ruzivo rwezvese dynamics yemapuroteni ari mu condensate.Muzviitiko zvese izvi, zvikwangwani zveEPR zvakawanikwa zvemapuroteni echikamu chakapararira anoenderana ne nitroxide radicals munzvimbo inokurumidza kufamba.Mushure mekuparadzaniswa kwechikamu muhupo hwetau kana pLK (100 μM TEMPOL-αS, Tau441 kana ΔNt-Tau pachiyero che1: 1 kana pLK pachiyero che1: 10), kuwedzera kwehuwandu hwehuwandu hwepamusoro hwakaonekwa mu iyo EPR spectrum ye αS.Mutsara wekurasikirwa wakawedzera, uchiratidza kuderedzwa αS reorientation kinetics mumadonhwe akaenzaniswa neprotein mu dilute phase (Fig. 3d, Supplementary Fig. 4a).Idzi shanduko dzinonyanya kuoneka panzvimbo ye 122. Paunenge uri panzvimbo ye24 kuvapo kwepLK hakuna kukanganisa kinetics ye probe, panzvimbo ye122 mutsara wemutsara wakashanduka zvikuru (Supplementary Fig. 4a).Patakaedza kuenzanisira spectra panzvimbo 122 yeviri αS / polycation masisitimu tichishandisa isotropic model (Supplementary Figure 5a) inowanzoshandiswa kurondedzera kushandiswa kwe-spin-yakanyorwa IDP38,39, hatina kukwanisa kuvakazve maitiro ekuedza..Spectral simulation yechinzvimbo che 24 spins kusiyana (Supplementary Fig. 5a).Izvi zvinoratidza kuti pane zvinzvimbo zvakasarudzika munzvimbo yekutenderera magadzirirwo eC-terminal dunhu re αS pamberi pepolycations.Kana uchifunga nezvechikamu che αS muchikamu chakapfupikiswa pasi peyedzo EPR mamiriro (84 ± 2%, 79 ± 7%, uye 47 ± 4% ye αS/Tau441, αS/ΔNt-Tau, uye αS/pLK, zvichiteerana-ona Supplementary Mufananidzo 2e wekuongorora data c), zvinogona kuonekwa kuti kuwedzera kunoonekwa neEPR nzira inonyanya kuratidza kupindirana kweC-terminal nharaunda ye αS nemapolycation akasiyana-siyana muchikamu checondensed (kuchinja kukuru pakushandisa TEMPOL-122- αS), uye kwete protein condensation.Kuwedzera kwe microviscosity kunoonekwa mu probe.Sezvaitarisirwa, EPR spectrum yeprotein pasi pemamiriro ezvinhu kunze kweLLPS yakadzorerwa zvachose apo 1 M NaCl yakawedzerwa kumusanganiswa (Supplementary Fig. 4b).Pakazara, data yedu inoratidza kuti shanduko dzakaonekwa neCW-EPR dzinonyanya kuratidza kusangana kweC-terminal nharaunda ye αS nemapolycation akasiyana muchikamu chakapfupikiswa, uye kusangana uku kunoratidzika kunge kwakasimba ne pLK kupfuura neTau.
Kuti tiwane rumwe ruzivo rwekuumbwa nezve mapuroteni ari mucoacervate, takasarudza kudzidza iyo LLPS sisitimu tichishandisa NMR mumhinduro.Zvisinei, taigona kungoona chidimbu che αS chakasara muchikamu chakapararira, chinogona kunge chiri nekuda kwekuderedzwa kweprotein dynamics mukati mecoacervate uye chikamu chakaoma pazasi kwemhinduro muNMR kuongorora.Patakaongorora chimiro uye simba repuroteni yakasara muchikamu chakapararira chemuenzaniso weLLPS tichishandisa NMR (Supplementary Fig. 5c, d), takaona kuti puroteni yakaita zvakafanana pamberi pepLK uye ΔNt-Tau, zvose zviri zviviri. izvo zvaive muchimiro chechipiri uye masimba eprotein musana, zvakaratidzwa nekuyedza kwechipiri kemikari kuchinja uye R1ρ kuzorora.NMR dhata inoratidza kuti C-terminus ye αS inotambura kurasikirwa kukuru kwe conformational flexibility ichiri kuchengetedza chimiro chayo chisina kurongeka, senge yasara yeprotein sequence, nekuda kwekudyidzana kwayo nemapolycation.
Sezvo chiratidzo cheCW-EPR chichiwedzera chinoonekwa muTEMPOL-122-αS chikamu chakapfupika chinoratidza kudyidzana kweprotein nemapolycations, takaita EPR titration kuti tiongorore kusungirirwa kweiyo αS kune akasiyana mapolycation pasina LLPS (hapana kuunganidza Buffer LLPS), zvichiratidza kuti kupindirana kwakafanana muzvikamu zvakasvibiswa uye zvakanyanya (izvo zvinosimbiswa nemashoko edu, Supplementary Fig. 4a uye Supplementary Fig. 6).Chinangwa chaive chekuona kana ese coacervates, kunyangwe iwo akajairwa mvura-senge zvivakwa, achiratidza chero hunhu hwakasiyana pachiyero chemolecular.Sezvinotarisirwa, EPR spectrum yakawedzera nekuwedzera polycation concentration, ichiratidza kuderera kwema molecular flexibility nekuda kwekubatana kwema molecular yevose vanobatana vanobatana kusvika pakuzara (Fig. 3e, Supplementary Fig. 6).pLK yakawana saturation iyi pazasi molar ratio (polycation: αS) ichienzaniswa neΔNt-Tau uye Tau441.Muchokwadi, kufananidzwa kweiyo data neinongedzo inosunga modhi ichifungidzira n yakafanana uye yakazvimirira inosunga nzvimbo yakaratidza kuti iyo inooneka dissociation nguva dzose ye pLK (~ 5 μM) hurongwa hwehukuru hwakaderera pane hwaTau441 kana ΔNt-Tau (~ 50 μM. )µM).Kunyangwe iyi iri fungidziro yakakasharara, izvi zvinoratidza kuti αS ine hukama hwepamusoro hwemapolycations akareruka ane anoenderera achichaja matunhu.Tichifunga uyu musiyano muhukama pakati pe αS uye akasiyana mapolycations, isu takafungidzira kuti yavo yemvura zvinhu inogona kuchinja zvakasiyana nekufamba kwenguva uye nekudaro vanotambura neakasiyana LSPT maitiro.
Tichifunga nezvemhoteredzo yakawandisa mukati meprotein coacervate uye chimiro cheamyloid cheprotein, takacherekedza maitiro eiyo coacervate nekufamba kwenguva kuti tione zvinogona kuitika zveLSPT.Tichishandisa BF uye CF microscopy (Mufananidzo 4), takaona kuti αS/Tau441 inobatana kusvika pamwero mukuru mumhinduro, ichigadzira madonhwe makuru anobata uye anonyorovesa pamusoro pezasi petsime / slide semadonhwe akazara, sezvaitarisirwa (Supplementary Fig. 7d);tinodaidza zvivakwa zvepasi-zvakaumbwa "protein rafts".Izvi zvivako zvakaramba zvakanyunguduka sezvavakachengetedza kukwanisa kusanganisa (Supplementary Fig. 7b) uye inogona kuonekwa kwemaawa akawanda mushure mokunge LLPS yatanga (Fig. 4 uye Supplementary Fig. 7c).Takaona kuti kunyorova kwacho kunofarirwa pamusoro pe hydrophilic pane hydrophobic zvinhu (Supplementary Fig. 7a), sezvingatarisirwa kuti electrostatic coacervates pamwe unbalanced mhosva uye nokudaro yakakwirira electrostatic pamusoro zvinogona.Zvinonyanya kukosha, αS / ΔNt-Tau coalescence uye rafting zvakaderedzwa zvakanyanya, nepo αS / pLK condensates yakaderedzwa zvakanyanya (Fig. 4).Munguva yenguva pfupi yekuputira, madonhwe e-αS / pLK akakwanisa kubatanidza uye kunyorovesa hydrophilic surface, asi iyi nzira yakakurumidza kumira uye mushure memaawa mashanu ekuputira, zviitiko zvishomanana chete zvekubatana uye hapana kunyorova kwakaonekwa.- gel-drip shanduko.
Mumiriri BF (greyscale panels) uye CF (kurudyi mapaneru, AF488-akanyorwa αS mugirinhi) yemacoacervate samples ane 100 µM αS (1% fluorescent label) muLLPS buffer pamberi pe100 µM Tau441nce (kumusoro) makrosikoro -Tau (pakati) kana 1 mM pLK (pasi) panguva dzakasiyana dzekuisa uye nzvimbo dzakakwirira (z, kureba kubva pasi peplate zvakanaka).Miedzo yakadzokororwa 4-6 nguva yakasununguka kune mumwe nemumwe nemigumisiro yakafanana.αS/Tau441 coacervates inonyoroveswa mushure memaawa makumi maviri nemana, ichigadzira mapuranga akakura kupfuura mufananidzo.Chikero bhaa yemifananidzo yese i20 µm.
Isu takazobvunza kana iwo makuru emvura-senge mapuroteni madziva akaumbwa mu αS/Tau441 LLPS aizotungamira kune amyloid aggregation yechero ipi yemapuroteni akadzidzwa.Takatevera maturation e-αS / Tau441 madonhwe nekufamba kwenguva neWF microscopy pasi pemamiriro akafanana nepamusoro, asi kushandisa 1 μM AF488-yakanyorwa αS uye Atto647N-yakanyorwa Tau441 (Fig. 5a).Sezvaitarisirwa, isu takaona yakazara mapuroteni kugariswa mukati menguva yese yekukura.Zvinofadza, kubva ca.Mushure meawa 5, zvivako zvakanyanya zvisina kutenderera zvakaonekwa mukati memapuranga, izvo zvatakadana kuti "mapoinzi", mamwe acho akaiswa colocalized ne αS, uye mamwe akafumiswa muTau441 (Fig. 5a, miseve yakachena).Aya makwapa agara achicherechedzwa mukati memapuranga kusvika pamwero mukuru kune αS/ΔNt-Tau kupfuura αS/ΔNt-Tau.Pakanga pasina mavara akasiyana mumadonhwe epLK uye Tau masisitimu asingakwanisi fusion / wetting.Kuongorora kana aya mavara ane αS neTau441 ari amyloid-seakaunganidzwa, takaita ongororo yakafanana tichishandisa CF microscopy umo Tau441 yakanyorwa neAtto647N uye 12.5 μM amyloid-specific thioflavin-T (ThT) yakawedzerwa kubva pakutanga.dye.Kunyange zvazvo ThT-staining ye αS / Tau441 madonhwe kana rafts haina kucherechedzwa kunyange mushure me24 h ye incubation (Fig. 5b, mutsara wepamusoro-yakasara madonhwe pamusoro peprotein rafts), ThT-positive structures ine Atto647N-Tau441 mukati memakumbo akanga asina simba.izvi zvinodzokorora ukuru, chimiro, uye nzvimbo yezvakambotsanangurwa mavara (Fig. 5b, mitsara yepakati nepasi), zvichiratidza kuti mavara anogona kuenderana neamyloid-like aggregates inoumbwa mukukwegura kwemvura coacervates.
WF 25 μM αS panguva dzakasiyana-siyana dzekudyara uye kureba (z, chinhambwe kubva pasi chisina kusungwa) pamberi pe25 μM Tau441 (1 μM AF488-yakanyorwa αS uye Atto647N-yakanyorwa Tau441) mutsime regirazi remaikorosikopu rine LLPS buffer) .Kuedza kutanhatu kwakadzokororwa zvakasununguka nemigumisiro yakafanana.b CF microscopic mufananidzo we25 μM αS pamberi pe25 μM Tau441 (1 μM Atto647N-yakanyorwa Tau441) uye 12.5 μM thioflavin-T (ThT).Madonhwe eprotein akaremerwa uye akaiswa mapuroteni rafts uye mavara anoratidzwa mumitsetse yepamusoro nepakati, zvichiteerana.Mutsara wepazasi unoratidza mifananidzo yemarafts uye madonhwe kubva ku3 yakazvimirira replicates.Miseve machena inoratidza ThT-positive dots mune ese mapaneru.Chikero bhaa yemifananidzo yese i20 µm.
Kuti uongorore zvakadzama shanduko yecoacervate protein network panguva yekuchinja kubva kumvura kuenda kune yakasimba, takashandisa fluorescence lifetime imaging (FLIM) uye Förster resonance energy transfer microscopy (FRET) (Figure 6 uye Supplementary Figures 8 uye 9).Isu takafungidzira kuti iyo coacervate maturation yedhiri kuita yakawedzera kupfupika kana kunyange yakasimba-yakaita aggregated protein structure inotungamira kuswedera pedyo pakati peprotein uye fluorescent probe yakanamirwa pairi, zvichigona kuburitsa quenching mhedzisiro inoratidzwa mukupfupikiswa kwehupenyu hwese (τ) , sezvakatsanangurwa kare40.,41 ,42.Pamusoro pezvo, kune masampula akanyorwa kaviri (AF488 uye Atto647N seFRET donor uye madhayi anobvuma, zvichiteerana), kudzikira uku mu τ kunogonawo kuperekedzwa necoacervate condensation uye kuwedzera kweFRET (E) kushanda zvakanaka panguva yeLSPT.Isu takatarisa raft uye kuumbwa kwemavara nekufamba kwenguva muLLPS αS/Tau441 uye αS/ΔNt-Tau samples (25 µM yeprotein yega yega muLLPS buffer ine 1 µM AF488 yakanyorwa αS uye/kana Atto647N yakanyorwa Tau441 kana ΔNt-Tau).Takaona zvakajairika pakuti fluorescence lifetime yeAF488 (τ488) uye Atto647N (τ647N) probes yakaderera zvishoma sezvo coacervates yakakura (Fig. 6 uye Supplementary Fig. 8c).Sezvineiwo, shanduko iyi yakawedzerwa zvakanyanya kune madots mukati me rafts (Fig. 6c), zvichiratidza kuti mamwe maprotein condensation akaitika pamadots.Mukutsigira izvi, hapana kuchinja kukuru kwehupenyu hwefluorescence kwakaonekwa kune αS / ΔNt-Tau madonhwe ane makore 24 h (Supplementary Fig. 8d), zvichiratidza kuti droplet gelation inzira yakasiyana nekuona uye iyo isingaperekedzwi nekugadziriswa kwema molecular. mukati macoacervates.Zvinofanira kuonekwa kuti mavara ane hukuru hwakasiyana uye zvinyorwa zvakasiyana mu αS, kunyanya kune αS / Tau441 system (Supplementary Fig. 8e).Kuderera kwehupenyu hwehupenyu hwefluorescence kwaiperekedzwa nekuwedzera kwesimba, kunyanya kuAtto647N yakanyorwa Tau441 (Supplementary Fig. 8a), uye yakakwirira FRET efficiencies kune zvose αS/Tau441 uye αS/ΔNt-Tau masisitimu, zvichiratidza kuwedzera kuwedzeredzwa muLLPS maawa mashanu. mushure mekutangisa, mapuroteni ari mukati memagetsi akadzikama akadzikiswa.Kuenzaniswa ne αS/ΔNt-Tau, takaona yakaderera τ647N uye yakati wandei yakakwirira τ488 tsika mu αS/Tau441 mavara, achiperekedzwa neakaderera uye akawanda inhomogeneous FRET tsika.Zvichida, izvi zvinogona kunge zvakabatana nekuti mu αS/Tau441 system, iyo inocherechedzwa uye inotarisirwa αS kuwanda muaggregates yakawanda heterogeneous, kazhinji substoichiometric kana ichienzaniswa neTau, sezvo Tau441 pachayo inogona zvakare kupinda LLPS uye aggregation (Supplementary Fig. 8e) .Nekudaro, iyo dhigirii rekudonhedza coalescence, raft kuumbwa, uye, zvakakosha, protein aggregation mukati memvura-senge coacervates yakakura kana zvese Tau441 uye αS zviripo.
a Lifetime fluorescence microscopy (FLIM) mifananidzo ye αS/Tau441 uye αS/ΔNt-Tau pa 25 μM yeprotein yega yega (1 μM AF488-yakanyorwa αS uye 1 μM Atto647N-yakanyorwa Tau441 kana ΔNt-Tau) muLLPS buffer.Makoramu anoratidza mifananidzo inomiririra yemasampuli eLLPS panguva dzakasiyana dzekukura (30 min, 5 h uye 24 h).Iyo tsvuku furemu inoratidza dunhu rine αS/Tau441 mavara.Hupenyu hurefu hunoratidzwa semavara emavara.Scale bar = 20 µm yemifananidzo yese.b Yakadhonzwa muFLIM mufananidzo wenzvimbo yakasarudzwa, inoratidzwa mubhokisi dzvuku mupanera a.Hupenyu huzhinji hunoratidzwa pachishandiswa chikero cheruvara chakafanana sepapanera a.Scale bar = 5 µm.c Histograms inoratidza AF488 (yakanamatira ku αS) kana Atto647N (yakanamirwa kuTau) yemhando dzakasiyana dzeprotein (madonhwe-D-, raft-R- uye makwapa-P) akaonekwa muFLIM mifananidzo yakarekodhwa αS-) nguva yekugovera hupenyu hweTau441 uye αS/ΔNt-Tau coacervate samples (N = 17-32 ROI yeD, 29-44 ROI yeR, uye 21-51 ROI yemapoinzi).Mean uye epakati kukosha anoratidzwa seyero masikweya uye mitema mitsetse mukati memabhokisi, zvichiteerana.Iwo ezasi uye ekumusoro miganhu yebhokisi inomiririra yekutanga neyechitatu quartiles, zvichiteerana, uye hudiki uye hwakanyanya kukosha mukati meiyo 1.5-fold interquartile renji (IQR) inoratidzwa sendebvu.Kunze kunoratidzwa semadhaimondi matema.Kukosha kwenhamba pakati pezviviri zvekugovera kwakatemwa pachishandiswa t-sample-t-test, zvichitora misiyano isina kuenzana.Miswe miviri t-test p-value inoratidzwa ine asterisks kune imwe neimwe yedata yakaenzaniswa (* p-value > 0.01, ** p-value > 0.001, *** p-value > 0.0001, **** p-value> 0.00001), ns Inoratidza kuregeredza (p-value> 0.05).Iwo chaiwo p maitiro anopihwa muSupplementary Tafura 1, uye iyo yekutanga data inounzwa seyakaomeswa data mafaira.
Kuti tiwedzere kuratidza maitiro e-amyloid-akafanana nemaspeckles / aggregates, takabata sampuli dzisina kusvibiswa coacervate kwemaawa makumi maviri nemana nehupamhi hwepamusoro hwe (1 M) NaCl, izvo zvakakonzera kuparadzaniswa kwezvikamu kubva kuprotein coacervates.Pakaonekwa maatomic force microscopy (AFM), takaona morphology ine spherical morphology ine hurefu hwenguva dzose hunosvika 15 nm, iyo inowanzobatana pasi pemamiriro emunyu wakawanda, wakafanana maitiro ezvakajairwa amyloid fibrils nekuda kwesimba rakasimba re hydrophobic pamusoro (chinyorwa chekuti fibrils kazhinji ine urefu hwe ~ 10 nm) (Supplementary Fig. 10a).Sezvineiwo, apo zvikwata zvega zvakabatanidzwa neThT mune yakajairwa ThT fluorescence assay, takaona kuwedzera kunoshamisa kweTT fluorescence quantum goho, kufananidzwa neyakacherechedzwa apo dhayi yakaiswa neyakajairwa αS amyloid fibrils (Supplementary Fig. 10b), inoratidza kuti 10b), iyo coacervate aggregate ine amyloid-senge zvimiro..Kutaura zvazviri, zvikamu zvacho zvaishivirira kuhuwandu hwemunyu asi hunonzwisisika kune 4 M guanidine chloride (GdnHCl), kufanana neamyloid fibrils (Supplementary Fig. 10c).
Tevere, takaongorora kuumbwa kweaggregates tichishandisa single molecule fluorescence, chaiyo fluorescence correlation/cross-correlation spectroscopy (FCS/FCCS), uye kuputika kwekuongorora kwemavara-maviri kusangana kusangana (TCCD).Kuti izvi zviitike, isu takazviparadzanisa zvikwata zvakaumbwa mushure memaawa makumi maviri nemana ekuiswa mu100 μl LLPS masampuli ane αS neTau441 (ose 25 μM) pamwe chete ne1 μM AF488-yakanyorwa αS uye 1 μM Atto647N-yakanyorwa Tau441.Deretsa mhinduro yakaparadzirwa yakapararira kune monomolecular state uchishandisa imwechete PEG-isina buffer uye 1 M NaCl (iyo bhafa imwe chete inoshandiswa kupatsanura akaunganidzwa kubva kucoacervate) kudzivirira chero kudyidzana kwe electrostatic pakati peLLPS neprotein.Muenzaniso wenguva yekufamba kwemorekuru imwe chete inogona kuonekwa mumufananidzo 7a.FCCS/FCS analysis (cross-correlation, CC and autocorrelation, AC) yakaratidza kuti aggregates ane αS uye tau akanga akawanda mumasampuli (ona CC curve muFig. 7b, left panel), uye kuwandisa kweprotein yakasara monomeric yakasimuka se mhedzisiro ye dilution process (ona AC curves muFigure 7b, kuruboshwe panhi).Kudzora kuedza kunoitwa pasi pemamiriro akafanana ekugadzirisa uchishandisa sampuli dzine chete monomeric mapuroteni haana kuratidza CC curves, uye AC curves inokodzera zvakanaka ne-one-component diffusion model (Eq. 4), apo monomeric mapuroteni ane anotarisirwa diffusion coefficients (Fig. 7b) ), panhovo yekurudyi).Iyo diffusion coefficient yeaggregated particles ishoma pane 1 µm2/s, uye iyo yemapuroteni emonomeric inenge 1 µm2/s.50–100 µm/s;kukosha kwakafanana neyakambodhindwa tsika dze sonicated αS amyloid fibrils uye monomeric αS zvakasiyana pasi pemamiriro ekugadzirisa akafanana44.Patakaongorora zvikamu neTCCD kuputika kwekuongorora (Fig. 7c, panhizha yepamusoro), takaona kuti mune imwe neimwe yakasarudzika aggregate (αS / Tau heteroaggregate), inenge 60% yezvikamu zvakaonekwa zvine zvose αS uye tau, inenge 30% yaiva chete. Tau, inenge 10% αS chete.Kuongorora kweStoichiometric kwe αS/Tau heteroaggregates kwakaratidza kuti mazhinji eheteroaggregates akafumiswa mu tau (stoichiometry pazasi 0.5, avhareji yenhamba yemamolekemu etau paaggregates ndeye 4 kakapetwa kupfuura αS mamorekuru), izvo zvinoenderana nebasa redu rakaonekwa muFLIM in situ. experiments..Ongororo yeFRET yakaratidza kuti aya akaunganidzwa aive ese mapuroteni, kunyangwe iwo chaiwo FRET hunhu mune iyi nyaya hauna kukosha kukuru, sezvo kugoverwa kwefluorophores muchikamu chega chega kwaive kwakasarudzika nekuda kwekuwanda kweprotein isina kunyorwa yakashandiswa mukuyedza.Sezvineiwo, patakaita ongororo imwechete tichishandisa 45,46 yakakura amyloid aggregation-deficient Tau variant (ona Supplementary Fig. 11a,b), takaona kuti kunyange zvazvo αS electrostatic aggregation yakanga yakafanana (Supplementary Fig. 11c, d), kugona kugadzira aggregate mukati meiyo coacervate kwakadzikiswa zvakanyanya uye FLIM yakaona nzvimbo dzinoverengeka mune in situ kuyedza, uye isina kusimba muchinjiko-yekubatanidza macurves akacherechedzwa kune ega akaunganidzwa samples.Zvisinei, nokuda kwenhamba shoma yezvikamu zvakaonekwa (chegumi chimwe chete cheTau441), takaona kuti imwe neimwe yakaunganidzwa yakafumiswa mu αS kupfuura iyi Tau yakasiyana, iine inenge 50% yezvikamu zvakaonekwa zvine chete αS mamorekuru, uye αS yakanga yakanyanyisa kudarika. .aggregates (ona Supplementary Fig. 11e), kusiyana nehterogeneous aggregates yakagadzirwa neTau441 (Fig. 6f).Migumisiro yezviedzo izvi yakaratidza kuti kunyange zvazvo αS pachayo inokwanisa kuunganidza ne tau mukati me coacervate, tau nucleation inonyanya kufarirwa pasi pemamiriro ezvinhu aya, uye zvinokonzerwa neamyloid-like aggregates inokwanisa kuita sechimiro che αS uye tau.Zvisinei, kana tau-rich core yakaumbwa, heterotypic interactions pakati pe αS ne tau inofarirwa muzvikamu pamusoro pehomotypic interactions pakati pema molecules;isu zvakare tinocherekedza mapuroteni network mune yemvura αS/tau coacervates.
Mumiriri wefluorescence temporal traces yemamorekuru ega ega akaunganidzwa akaumbwa mu αS/Tau441 electrostatic coacervates.Kuputika kunoenderana ne αS/Tau441 coaggregates (kuputika pamusoro pechikumbaridzo chakaratidzwa) kwakaonekwa mumatanho matatu ekuonekwa (AF488 uye Atto647N emission mushure mekunakidzwa kwakananga, mitsara yebhuruu uye tsvuku, Atto647N emission mushure mekunangana kwekunakidzwa), FRET, violet mutsara).b FCS/FCCS ongororo yemuenzaniso weakasarudzika αS/Tau441 aggregates akawanikwa kubva kuLLPS (kuruboshwe panhi).Autocorrelation (AC) macurves eAF488 uye Atto647N anoratidzwa mubhuruu uye tsvuku, zvichiteerana, uye muchinjika-wekubatanidza (CC) macurves ane hukama neaggregates ane ese madhayi anoratidzwa nepepuru.Iwo ma AC curves anoratidza kuvepo kweakanyorwa kuti monomeric uye aggregated mapuroteni marudzi, nepo CC curves ichiratidza chete kupararira kweaviri-akanyorwa aggregates.Kuongorora kwakafanana, asi pasi pemamiriro akafanana ekugadzirisa senge munzvimbo dzakasarudzika, sampuli dzine chete monomeric αS uye Tau441 inoratidzwa sezvinodzora mupaneru yekurudyi.c Fluorescence flash kuongororwa kwemamorekuru ega ega akaunganidzwa akaumbwa mu αS/Tau441 electrostatic coacervates.Ruzivo rwemuunganidzwa wega wega unowanikwa mune ina dzakasiyana kudzokorora (N = 152) yakarongerwa stoichiometry yavo, S values, uye FRET kunyatsoshanda (yepamusoro pani, bhara reruvara rinoratidza kuitika).Mhando nhatu dzeaggregates dzinogona kusiyaniswa: -αS-chete aggregates ane S ~ 1 uye FRET ~ 0, Tau-chete aggregates neS ~ 0 uye FRET ~ 1, uye heterogeneous Tau/αS aggregates ane yepakati S uye FRET Estimate yehuwandu. eese mapuroteni emucherechedzo akaonekwa mune yega yega heterogeneous aggregate (N = 100) inoratidzwa mupazasi pepaneru (chiyero chemavara chinoratidza chiitiko).Raw data inopiwa nenzira yemafaira e data.
Kukura kana kuchembera kweprotein yemvura inonamira kuita gel-yakafanana kana yakasimba zvimiro nekufamba kwenguva yakanzi inobatanidzwa mune akati wandei emuviri mabasa eiyo condensate47 pamwe nechirwere, senzira isina kujairika inotangira amyloid aggregation 7, 48, 49. Pano tinodzidza kuparadzaniswa kwechikamu uye maitiro mune zvakadzama.LSPT αS muhupo hwemapolycations asina kurongeka munzvimbo inodzorwa pane yakaderera micromolar concentrations uye physiologically akakodzera mamiriro (ona kuti yakaverengerwa physiological concentration ye αS ndeye> 1 µM50), ichitevera yakajairika thermodynamically inofambiswa maitiro eLPS.Takaona kuti αS, iyo ine yakanyanya kuchemerwa C-terminal dunhu pa physiological pH, inokwanisa kugadzira madonhwe ane mapuroteni ane aqueous mhinduro kuburikidza neLLPS pamberi peiyo yakanyanya cationic disordered peptides senge pLK kana Tau kuburikidza nemaitiro emagetsi. yakaoma condensation muhupo hwekuunganidza macromolecules.Iyi nzira inogona kuva nemigumisiro yakakodzera munharaunda yemasero apo αS inosangana nemhando dzakasiyana-siyana dzepolycationic molecules dzakabatana nehosha-yakabatana kuunganidza zvose mu vitro uye mu vivo51,52,53,54.
Mune zvidzidzo zvakawanda, mapuroteni emagetsi mukati memadonhwe akaonekwa sechimwe chezvinhu zvakakosha zvinogadzirisa maturation process55,56.Mu electrostatic αS coacervates with polycations, maturation process sezviri pachena inoenderana nesimba rekudyidzana nepolycations, valence, uye kuwanda kwekudyidzana uku.Equilibrium theory inoratidza kuti kuenzana kwenzvimbo mbiri dzemvura kungave kuvepo kwedonhwe rakakura rakapfuma mumabiopolymers anotyaira LLPS57,58.Droplet kukura kunogona kuwanikwa neOstwald maturation59, coalescence60 kana kushandiswa kwemahara monomer muchikamu chakapararira61.Kune αS uye Tau441, ΔNt-Tau kana pLK, yakawanda yeprotein yakaiswa mu condensate pasi pemamiriro akashandiswa muchidzidzo ichi.Nekudaro, nepo madonhwe akazara etau akabatana nekukurumidza pakunyorova kwepasi, kudonhedza kubatana uye kunyorova kwaive kwakaomera ΔNt-Tau uye pLK, zvichiratidza kukurumidza kurasikirwa kwezvinhu zvemvura mumasisitimu maviri aya.Maererano nekuongorora kwedu kweFLIM-FRET, madonhwe akwegura pLK uye ΔNt-Tau akaratidza dhigirii yakafanana yeprotein aggregation (yakafanana fluorescence lifetime) semadonhwe ekutanga, zvichiratidza kuti yekutanga protein network yakachengetwa, kunyange yakaoma.
Isu tinoruramisa mhedzisiro yedu yekuyedza mune inotevera modhi (Mufananidzo 8).Madonhwe akaumbwa kwenguva pfupi anowanzova mapuroteni network pasina muripo we electrostatic, uye nekudaro kune nzvimbo dzekuchajisa kusaenzana, kunyanya painodonhedza, zvichikonzera madonhwe ane simba repamusoro remagetsi.Kutsiva kubhadhariswa (chiitiko chinowanzonzi valence depletion) uye kudzikisa kugona kwepamusoro kwemadonhwe, madonhwe anogona kusanganisira mapolypeptides matsva kubva pachikamu chedilute, kurongazve maprotein network kuti akwidzise kushaja-chaji kupindirana, uye kupindirana nemamwe madonhwe.nepamusoro (wetting).Iyo αS / pLK madonhwe, nekuda kwekureruka kwavo kweprotein network (chete heterotypic interactions pakati pe αS uye pLK) uye hukuru hukuru hwekubatana kweprotein-protein, inoratidzika kunge inokwanisa kuenzanisa mutero we condensate nekukurumidza;zvechokwadi, takacherechedza kukurumidza mapuroteni kinetics mukutanga kuumbwa αS / pLK coacervates kupfuura mu αS / Tau.Mushure mekupera kwevalence, kupindirana kunova kushoma ephemeral uye madonhwe anorasikirwa neayo emvura uye anoshanduka kuita senge-se, madonhwe asingabatike anopisa ane yakaderera electrostatic surface inogona (uye nekudaro haikwanise kunyorova pamusoro).Kusiyana neizvi, madonhwe e αS/Tau haanyanyi kushanda zvakanaka pakugadzirisa chiyero chedonhwe rekuchaja nekuda kwekunyanya kuomarara mapuroteni network (nezvese homotypic uye heterotypic kudyidzana) uye husina kusimba hwekuita kweprotein.Izvi zvinoguma nemadonhwe anochengeta hunhu hwemvura kwenguva yakarebesa uye anoratidza yakakwirira electrostatic pamusoro inogona kuita shoma nekubatana uye kukura (nekudaro kuderedza nzvimbo yepasi/huwandu hwehuwandu hwemadonhwe) uye nekunyorovesa iyo hydrophilic surface chem.Izvi zvinogadzira mahombe akaiswa mapuroteni maraibhurari anochengeta zvimiro zvemvura sezvo kupindirana kunoramba kuri kwenguva pfupi nekuda kwekugara uchitsvaga kubhadharisa optimization muprotein network.Sezvineiwo, N-terminally truncated mafomu eTau, anosanganisira mamwe anongoitika isoforms62, anoratidza maitiro epakati, aine mamwe macoacervates kuchembera ne αS kuita madonhwe akareba-akararama segel, nepo mamwe achichinja kuita mahombe emvura emvura.Hunyambiri uhwu mukukura kwe αS electrostatic coacervates inopindirana nezvinoitwa LLPS zvedzidziso uye zvidzidzo zvekuyedza zvakaratidza kuwirirana pakati pekuderera kwevalence uye electrostatic sieving mumakondensa sekiyi yekudzora saizi yecondensate uye zvimiro zvemvura.Mechanism 58.61.
Ichi chirongwa chinoratidza iyo putative amyloid aggregation nzira ye αS uye Tau441 kuburikidza neLLPS uye LSPT.Nekuwedzera kweanion-rich (tsvuku) uye cation-rich (blue) matunhu, αS uye tau electrostatic coacervates ine inogutsa valence ine yakaderera pamusoro pesimba uye nokudaro kushoma coalescence, zvichikonzera kukurumidza kudonha kuchembera.Iyo yakagadzikana isina-agglomerated gel state inowanikwa..Mamiriro ezvinhu aya anonyanya kufadza munyaya ye αS/pLK system nekuda kwekubatana kwayo kwepamusoro uye nyore protein-pair interaction network, iyo inobvumira kukurumidza gel-kufanana nekuchinja.Pane zvinopesana, madonhwe ane valence isingafadzi uye, naizvozvo, mapuroteni-anopomerwa matunhu anowanikwa kuti awirirane, anoita kuti zvive nyore kuti coacervate isanganise uye inyorove iyo hydrophilic pamusoro kuitira kuderedza simba rayo repamusoro.Iyi mamiriro akanakira αS/Tau441 coacervates, ine multivalent yakaoma network inosanganisira isina simba Tau-Tau uye αS-Tau kusangana.Zvakare, macoacervates akakura anozochengetedza zviri nyore midziyo yavo-senge zvivakwa, zvichibvumira kumwe kupindirana kweprotein-kune-protein kuitika.Pakupedzisira, amyloid heterogeneous aggregates ane ese ari maviri αS uye tau fomu mukati mecoacervate fluid, iyo inogona kunge ine hukama neiyo inowanikwa mumiviri inosanganisirwa, inova chiratidzo chezvirwere zveurourodegenerative.
Iyo huru yemvura-yakafanana zvivako zvakaumbwa panguva yekukura kwe αS/Tau441 ine mhepo yakanyanyisa asi ine simba reprotein uye, kusvika pamwero muduku, αS / ΔNt-Tau coacervates inzvimbo dzakanaka dzekuchengetedza nucleation yeprotein aggregation.Takanyatsocherechedza kuumbwa kwemapuroteni akasimba mumhando iyi yeprotein coacervates, kazhinji ine zvose αS uye tau.Takaratidza kuti idzi heteroaggregates dzakadzikamiswa neasiri-electrostatic interactions, dzinokwanisa kusunga amyloid-specific ThT dyes nenzira yakafanana neyakajairika amyloid fibrils, uye zvechokwadi ine kupesana kwakafanana kune zvakasiyana-siyana.Iwo αS/tau akaunganidzwa akaumbwa neLLPS akaratidzwa kuve ane amyloid-senge zvivakwa.Chokwadi, musiyano wakakura weTau kushomeka muamyloid aggregation wakakanganisika zvakanyanya mukuumbwa kweizvi heterogeneous αS aggregates mukati memvura electrostatic coacervate.Kuumbwa kwe αS/Tau441 aggregates kwakaonekwa chete mukati me coacervates, iyo yakachengetedza mvura-yakafanana nezvinhu, uye kwete, kana coacervates / madonhwe asina kusvika kune gel state.Muchiitiko chekupedzisira, kuwedzera kwesimba rekusangana kwe electrostatic uye, semhedzisiro, kusimba kweprotein network kunodzivirira kurongeka kwekugadzirisazve mapuroteni kuti atange kusangana kutsva kweprotein inodiwa kune amyloid nucleation.Nekudaro, izvi zvinogona kuwanikwa mune inoshanduka-shanduka, mvura-yakafanana necoacervates, iyo inozoita ingangoramba iri mvura sezvavanowedzera muhukuru.
Icho chokwadi chekuti kuumbwa kweaggregates mukati mechikamu chakakomberedzwa kunodiwa muhombe αS/Tau condensates kupfuura mumadonhwe madiki anokurumidza gel, inosimbisa kukosha kwekuziva izvo zvinodzora madonhwe coalescence.Nokudaro, kwete chete kune tsika yekuparadzanisa kwechikamu, asi ukuru hwekondensate hunofanira kudzorwa nekushanda kwakakodzera pamwe nekudzivirira zvirwere58,61.Mhedzisiro yedu inoratidzawo kukosha kwechiyero pakati peLLPS neLSPT yeiyo αS/Tau system.Kunyange zvazvo kuumbwa kwemadonhwe kunogona kudzivirira kubva kuamyloid aggregation nekuderedza huwandu hweprotein monomers inowanikwa pasi pemamiriro ekuzara, sezvakarongwa mune mamwe maitiro63,64, droplet fusion pamadonhwe akakwirira anogona kutungamirira mukati meprotein aggregation kuburikidza nekugadzirisa zvishoma nezvishoma kugadzirisa.protein network..
Pakazere, data redu rinosimbisa zvakanyanya kukosha kwekubatana valence uye kugutsikana / kusagutsikana kupindirana mukudonha network mumamiriro eLSPT.Kunyanya, isu tinoratidza kuti yakazara-kureba αS/Tau441 condensates inokwanisa kunyatso fuse uye nucleate kuti iite amyloid-senge heteroaggregates inosanganisira ese mapuroteni uye inokurudzira mamorekuru maitiro anoenderana nemhedzisiro yedu yekuedza.Iko kubatanidzwa kwemapuroteni maviri mu αS/Tau fluid coacervate yatinoshuma pano inogona kunge ine hukama nekubatana kwemapuroteni maviri mune inclusions, ayo ari zviratidzo zvechirwere, uye anogona kubatsira mukunzwisisa hukama pakati peLLPS uye. amyloid aggregation, ichigadzira nzira yeIDP yakachajiswa zvakanyanya muurodegeneration.
Monomeric WT-αS, cysteine mutants (Q24C-αS, N122C-αS) uye ΔCt-αS kusiyana (Δ101-140) yakaratidzwa muE. coli uye yakacheneswa sezvakatsanangurwa kare.5 mM DTT yakaverengerwa mumatanho ese mukucheneswa kwe αS cysteine mutants kudzivirira disulfide bond kuumbwa.Tau441 Ovesomid Yakawanikwa Kubva kuScushgee # 16316), δnn-tau VariartTatTatTatTatTCTCGGN (δ275-311, Yakacheneswa neGGCC5 Primer) E. CLELY ures yaive yakakura kusvika kuOD600 = 0.6–0.7 pa37°C uye 180 rpm, uye kutaura kwakakonzerwa neIPTG kwemaawa matatu pa37°C.Kohwa maseru pa11,500 xg kwemaminetsi gumi nemashanu pa4 °C uye geza nesaline buffer ine 150 mM NaCl.Dzorerazve pellet mu lysis buffer (20 ml pa 1 L LB: MES 20 mM, pH 6.8, NaCl 500 mM, EDTA 1 mM, MgCl2 0.2 mM, DTT 5 mM, PMSF 1 mM, benzamidine 50 μM, copeptin μM1).Nhanho ye sonication yakaitwa pachando ine amplitude ye80% ye 10 pulses (1 min on, 1 min off).Usapfuure 60 ml mune imwe ultrasound.E. coli lysates yakapiswa pa 95 ° C. kwemaminitsi makumi maviri, ndokubva yatonhodzwa pachando uye centrifuged pa 127,000 × g kwemaminitsi makumi mana.Iyo yakajekeswa supernatant yakaiswa kune 3.5 kDa membrane (Spectrum™ Thermo Fisher Scientific, UK) uye dialyzed ichipesana ne4 L ye dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM. , PMSF 0.1 mM) kwemaawa gumi.A 5 ml cation exchange column (HiTrap SPFF, Cytiva, MA, USA) yakaenzaniswa nebhafa yekuenzanisa (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Iyo tau lysate yakasefa kuburikidza ne 0.22 μm PVDF sefa uye yakabaiwa mukoramu pakuyerera kwe1 ml/min.Elution yakaitwa zvishoma nezvishoma, tau yakaburitswa ne15–30% elution buffer (20 mM MES, pH 6.8, 1 M NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Zvidimbu zvakaongororwa neSDS-PAGE, uye chero zvikamu zvine bhendi rimwechete rine huremu hunotarisirwa hwema molecular etau zvakanangidzirwa pachishandiswa 10 kDa centrifuge sefa ndokutsiviwa nebuffer ine 10 mM HEPES, pH 7.4, NaCl 500 mM uye DTT 2 mM ye mapuroteni ekupedzisira aive 100 μM.Mushonga weprotein wakabva wapfuudzwa nepa 0.22 μm PVDF sefa, nekukasira kuomeswa nechando uye kuchengetwa pa -80°C.Protein K18 yakapihwa nemutsa naProf Alberto Boffi.Kuchena kwegadziriro yaive> 95% sekusimbiswa neSDS-PAGE uye MALDI-TOF/TOF.Ma cysteine akasiyana-siyana akanyorwa nemakemikari ane AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) kana TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).zvakasimbiswa nekunyura uye MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau uye K18 zvakanyorwa neanowanikwa cysteine masara panzvimbo 191 uye 322 achishandisa Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Germany) ichitevera maitiro akafanana.Mambure ekubhadhara pamepu dzakasara dze αS neTau441 dzakagadzirwa pachishandiswa CIDER66.
Solid poly-L-lysine (pLK DP 90-110 maererano neNMR kubva kumutengesi, Alamanda Polymers Inc, Huntsville, Alabama, USA) yakanyungudika mu10 mM HEPES, 100 mM NaCl, pH 7.4 kusvika 10 mM concentration, process sonicated for 5 maminetsi mune ultrasonic mvura yekugezera uye chengetedza pa -20 ° C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) uye FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) inonyungudika nemvura uye inoparadzirwa zvakanyanya muLLPS buffer.Dialysis inobvisa munyu unosvibisa.Ivo vakabva vasefa kuburikidza nesirinji sefa ine saizi yepore ye 0.22 μm, uye kutarisisa kwavo kwakaverengerwa pachishandiswa refractometer (Mettler Toledo, Columbus, Ohio, USA).Sampuli dzeLLPS dzakagadzirirwa pakamuri tembiricha nenzira inotevera: buffer uye extrusion zvakasanganiswa uye 1 mM tris(2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2- (Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) uye musanganiswa we1% protease inhibitor (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).Zvadaro αS uye fused polycations (sarudzo pLK kana Tau) inowedzerwa.Kune thioflavin-T nguva yakatevedzana kuyedza (ThT, Carbosynth, Compton, UK), shandisa iyo yakazara ThT yekumisikidza kuve hafu yeiyo αS concentration.Zvinyoro asi zvakanyatsosanganisa masampuli kuti ave nechokwadi chekuti ane homogeneous.Iko kusungirirwa kwechikamu chimwe nechimwe kwakasiyana kubva pakuedza kusvika pakuedza, sezvinotsanangurwa muchikamu cheMitsva.Azide yakashandiswa pamusangano we 0.02% (w / v) pose apo nguva yekuedza yakapfuura maawa mana.Pakuongorora kwese uchishandisa LLPS samples, tendera musanganiswa kuti uenzane kwemaminetsi mashanu isati yaongororwa.Pakuongorora kupararira kwechiedza, 150 µl yemasampuli akaiswa pane asina-kusunga 96-tsime microplates (µClear®, dema, F-Bottom/Chimney Zvakanaka, Greiner bio-one, Kremsmünster, Austria) uye yakafukidzwa nefirimu rinonamira.LLPs dzakatariswa nekuyera kunyura pa350 nm pakati pemhinduro muCLARIOstar plate reader (BMG Labtech, Ortenberg, Germany).Iwo maedzo akaitwa ari katatu pa25 ° C, uye zvikanganiso zvakaverengerwa seyakajairwa kutsauka kubva pazvinoreva.Iyo dilute phase yakaverengerwa nesample centrifugation uye SDS-PAGE gel kuongororwa, uye chikamu che αS muchikamu chekudzikisa uye chakamisikidzwa chakaverengerwa mumhando dzakasiyana dzeLLPS mhinduro.Sampuli ye100 μl LLPS ine 1 μM AF488-yakanyorwa αS yakagadzirwa nekunyatso kusanganiswa ichiteverwa necentrifugation pa9600 × g kwemaminetsi makumi matatu, mushure mezvo mvura yaiwanzoonekwa.Iyo yepamusoro 50 μl yeiyo supernatant yakashandiswa kuverengera mapuroteni uchishandisa SDS-PAGE gel.Gels akaongororwa neAF488 mafirita achishandisa ChemiDoc gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA) kana akasvibiswa neCoomassie tsvina uye akaonekwa aine mafirita akakodzera.Mabhendi akabuda akaongororwa achishandisa ImageJ version 1.53i (National Institutes of Health, USA).Iwo maedzo akaitwa muduplicate mukuedza kuviri kwakasiyana kwakafanana.
Kazhinji, 150 μl yemasampuli akaiswa kune asina-kusunga 96-tsime microplates uye akaonekwa pakamuri tembiricha paLeica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany).Pakuedza kwemavara, mahwendefa eµ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Germany) kana 96-tsime polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) akashandiswawo.EL6000 halogen kana mercury simbi halide marambi akashandiswa sezvivheneko masosi (yeBF/DIC uye WF imaging, zvichiteerana).KuWF microscopy, chinangwa chemhepo che40x magnification (Leica Microsystems, Germany) yakashandiswa kutarisa chiedza pamuenzaniso uye kuitora.Kune AF488 uye ThT yakanyorwa samples, kusefa mafambiro uye kuburitsa ine yakajairwa GFP mafirita seti, excitation uye emission bandpass mafirita, zvichiteerana, 460-500 nm uye 512-542 nm bandpass mafirita, uye 495 nm dichroic girazi.Kumasampuli akanyorwa neAtto647N, yakajairwa seti yeCy5 mafirita ane mafaro uye emission bandpass mafirita 628–40 nm uye 692–40 nm, zvichiteerana, uye 660 nm dichroic girazi rakashandiswa.KuBF neDIC maikorosikopu, shandisa chinangwa chekuunganidza chiedza chakafanana.Chiedza chakaunganidzwa chakanyorwa paLeica DFC7000 CCD kamera (Leica Microsystems, Germany).Nguva yekuratidzwa yaive 50 ms yeBF neDIC microscopy imaging uye 20-100 ms yeWF microscopy imaging.Kuenzanisa, nguva yekuratidzwa yezvese zviedzo neThT yaive 100 ms.Kuedza-kupfuura-nguva kwakaitwa kuti vaone kubatanidzwa kwedonhwe, nemifananidzo ichiunganidzwa yega 100 ms kwemaminitsi akati wandei.ImageJ (NIH, USA) yakashandiswa kuongorora mufananidzo.Iwo maedzo akaitwa ari katatu aine mhedzisiro yakafanana.
Pakuedza kwecolocalization, FRAP uye 3D kuvaka patsva, mifananidzo yakawanikwa paZeiss LSM 880 inverted confocal microscope vachishandisa ZEN 2 blue edition (Carl Zeiss AG, Oberkochen, Germany).Mienzaniso ye50 µl yakashandiswa kune µ-Slide Angiogenesis Petri ndiro (Ibidi GmbH, Gröfelfing, Germany), yakabatwa ne hydrophilic polymer (ibiTreat) uye yakaiswa mu 63 × chinangwa chekunyudza mafuta (Plan-Apochromat 63 ×/NA 1.4 Oil) paDIC).Mifananidzo yakatorwa pachishandiswa 458 nm, 488 nm, uye 633 nm argon laser mitsara ine resolution ye0.26 µm/pixel uye nguva yekuratidzwa ye8 µs/pixel yekunakidzwa uye kuona emission windows ye470-600 nm, 493-628 nm, uye 638-755 nm yakashandiswa kuona ThT, AF488 uye Atto647N, zvichiteerana.Kuyedzo yeFRAP, kutora-nguva-kupfuura mafoto emuenzaniso wega wega wakarekodhwa pa 1 furemu pasekondi.Iwo maedzo akaitwa ari katatu pakamuri tembiricha aine mhedzisiro yakafanana.Mifananidzo yese yakaongororwa pachishandiswa Zen 2 blue edition software (Carl Zeiss AG, Oberkochen, Germany).Iyo FRAP macurves akajairwa, akarongwa uye akaiswa kusimba / nguva data yakatorwa kubva pamifananidzo uchishandisa Zen 2 uchishandisa OriginPro 9.1.Iwo ekudzoreredza macurves akaiswa kune mono-exponential modhi kuti azvidavirire pakupararira kwema molecular pamwe neimwe exponential term yekuzvidavirira kune yekuwana bleaching maitiro.Isu takazoverenga D tichishandisa iyo inonzi bleaching radius uye yakambotemerwa kudzoreredza hafu yeupenyu semuequation yeKang et al.5 35 yakaratidzwa.
Single cysteine variants ye αS yakagadzirwa ne4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) panzvimbo 24 (TEMPOL-24-αS) uye 122 (TEMPOL-122-αS), zvichiteerana.Spin Labeling Yezviyedzo zveEPR, iyo αS concentration yakaiswa pa 100 μM uye iyo PEG concentration yaive 15% (w/v).Pamamiriro akasiyana-siyana ekubatanidza, chiyero che αS:pLK chaive 1:10, ukuwo αS:ΔNt-Tau uye αS:Tau441 zviyero zvakachengetwa pa1:1.Zvekusunga zviedzo zvetitration pakashaikwa kutsvikinyidzana, TEMPOL-122-αS yakachengetwa pa50 μM uye mapolycations akaiswa pakuwedzera, achigadzirira mamiriro ega ega.CW-EPR kuyerwa kwakaitwa pachishandiswa Bruker ELEXSYS E580 X-bhendi spectrometer ine Bruker ER4118 SPT-N1 resonator inoshanda pa microwave (SHF) frequency ~ 9.7 GHz.Tembiricha yakaiswa pa25°C uye inodzorwa ne-liquid nitrogen cryostat.Iyo spectra yakawanikwa pasi peunsaturated mamiriro paMW simba re4 mW, modulation amplitude ye0.1 mT, uye modulation frequency ye100 kHz.Spectral intensities yakajairwa kudzivirira misiyano yekutenderera kutenderera pakati pemasamples uye zvichibvira spin kuderedzwa nekuda kwekusara kwekutarisa kweanoderedza vamiririri mumasampuli ane Tau441 kana ΔNt-Tau (iripo mune yekutanga mapuroteni mhinduro).Hwanhu hwakapihwa hweg hwakawanikwa nekuda kweEPR spectral modelling yakaitwa pachishandiswa Easyspin software (v. 6.0.0-dev.34) yakaitwa muMatlab®67.Imwe/mbiri chikamu isotropic modhi yakashandiswa kuenzanisa data.Mushure mekugadzirisa zviratidzo zvose, zvakasara zvakaverengerwa nekubvisa kuenzanisa kwega kwega kubva kune inoenderana yekuedza spectrum.Pakusunga ongororo yetitration, kusimba kwebhandi rechitatu kune yechipiri bhendi yeyakajairwa EPR spectrum (IIII/III) yakashandiswa kutarisa kusungwa kwepolycations kune αS.Kufungidzira iyo dissociation yenguva dzose (Kd), iyo yakaguma curve yakashongedzerwa kune inofungidzira modhi inofungidzira n yakafanana uye yakazvimirira inosunga nzvimbo.
Kuedza kweNMR spectroscopy kwakaitwa pachishandiswa Bruker Neo 800 MHz (1H) NMR spectrometer ine cryoprobe uye Z-gradient.Zvose zviedzo zvakaitwa uchishandisa 130-207 µM αS uye inowirirana αS / ΔNt-Tau uye pLK yakaenzana mu10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 uye yakaitwa pa15 ° C.Kutarisa LPS neNMR, 10% PEG yakawedzerwa kune pre-yakasanganiswa samples.Mushonga wekemikari wekuchinja kukanganisa (Fig. 1b) inoratidza mavhareji 1H uye 15N kushandiswa kwemakemikari.Iyo αS 2D1H-15N HSQC spectra yakagoverwa zvichienderana nebasa rekare (BMRB yekupinda #25227) uye yakasimbiswa nekurekodha uye kuongorora iyo 3D spectra yeHNCA, HNCO uye CBCAcoNH.13Cα uye 13Cβ kushandiswa kwemakemikari kwakaverengwa pamberi peΔNt-Tau kana pLK kuti ienzanise kuchinja kunogona kuitika mumagadzirirwo echipiri ekugadzirisa maitiro akaenzaniswa neAS kemikari kuchinja mune yakachena random coil conformation 68 (Supplementary Figure 5c).Iwo maR1ρ akayerwa nekurekodha hsqctretf3gpsi kuyedza (yakawanikwa kubva kuBruker raibhurari) nekunonoka kwe8, 36, 76, 100, 156, 250, 400, uye 800 ms, uye mabasa eexponential akagadziridzwa kusvika pakakwirira pakunonoka kwakasiyana. nguva dzekuona iyo R1ρ uye kusavimbika kwayo kwekuyedza.
Kuedza-mavara maviri-akagadziriswa fluorescence microscopy akaitwa pane yekutengeserana nguva-yakagadziriswa MT200 fluorescence confocal microscope (PicoQuant, Berlin, Germany) ine nguva-yakabatana single photon counting (TCSPC) mudziyo.Musoro welaser diode unoshandiswa pulsed interleaved excitation (PIE), danda rinopfuura nepakati peimwe modhi waveguide uye rakagadziridzwa kune laser simba re10 kusvika 100 nW ye481 nm uye 637 nm laser mitsetse yakayerwa mushure me dichroic girazi.Izvi zvinogonesa chiyero chekuverenga fotoni, kudzivirira mhedzisiro yephoton aliasing, photobleaching uye saturation.μ-Slide angiogenesis inovhara kana mahwendefa (Ibidi GmbH, Gräfelfing, Germany) yakaiswa zvakananga mumvura yakanyudzwa pamusoro peSuper Apochromat 60x NA 1.2 lens ine collar inogadzirisa (Olympus Life Sciences, Waltham, USA).A 488/640 nm dichroic mirror (Semrock, Lake Forest, IL, USA) yakashandiswa seyakanyanya danda rinoparadzanisa.Mwaranzi isina kutariswa inovharwa negomba rine dhayamita ye50 microns, ipapo mwaranzi yakatariswa inokamurwa kuita 2 nzira dzekuona ne50/50 danda kupatsanura.Bandpass emission filters (Semrock, Lake Forest, IL, USA) 520/35 yegirinhi dhayi (AF488) uye 690/70 yedhayi tsvuku (Atto647N) yakashandiswa pamberi pe detector.Single-photon avalanche diodes (SPAD) (Micro Photon Devices, Bolzano, Italy) yakashandiswa sema detectors.Zvese zviri zviviri kuunganidzwa kwedata uye kuongororwa kwakaitwa pachishandiswa inotengeswa SymphoTime64 software (PicoQuant GmbH, Berlin, Germany).
Makumi mashanu microliters eLLPS samples akaiswa kune μ-Slide angiogenesis matsime (Ibidi GmbH, Gräfelfing, Germany).Mifananidzo inenge yabuda yakanangidzirwa ku20 µm pamusoro pezasi kwetsime kuitira chinhambwe chekushanda chakanyatsonangana pamadonhwe akamiswa uye kusvika ~1 µm yemarafti nemadots ane axial resolution inosvika 0.25 µm/pixel uye nguva yekunonoka ye400 µs/pixel.Sarudza data nekushandisa intensity pachikumbaridzo zvichienderana neavhareji yekumashure chiratidzo chesimba (PBG, zvinoreva + 2σ) yechiteshi chega chega kuitira kuti madonhwe eprotein emvura chete, marefu, kana makwapa asarudzwa, kusefa chero bviro kubva pachikamu chakapararira.Kuongorora hupenyu hwemhando yega yega (τ) yegwara rega rega (girini, "g" yeAF488 uye tsvuku, "r" yeAtto647N), takasarudza matunhu anofarira (ROIs) ane madonhwe, raft, kana mavara (Supplementary Figure 1 )8b) uye akazvitora nekukodzera kuora kwehupenyu hwavo ( τD, τR uye τP yemadonhwe, rafts kana mavara, maererano, ona Supplementary Fig. 8c) mugero rega rega uchishandisa tail-fit analysis uye maviri-component decay model.Avhareji τ kubva ku τ .ROIs iyo yakagadzira mashoma mafotoni e-multi-exponential fit akaiswa kunze kwekuongorora.Iyo cutoff yakashandiswa yaive <104 photons yemarafts nemadots uye 103 yemadonhwe.Madonhwe ane chikumbaridzo chepasi nekuti zvakaoma kuwana ma curves ekuora ane hutsika hwepamusoro, sezvo madonhwe ari mundima yemufananidzo anowanzo diki uye mashoma kuwanda.ROIs ine photon kuverenga pamusoro peiyo photon accumulation limit (yakagadzirirwa ku> 500 kuverenga / pixel) yakaraswawo kuti iongororwe.Batanidza iyo intensite decay curve inowanikwa kubva mudunhu rekufarira nekusimba pa90% yehupamhi (zvishoma mushure mekunyanya kusimba kwekuora) kubva pakutanga kwehupenyu hwesevhisi kuona kushoma kweIRF kupindira uku uchichengeta zvakafanana pakuora kwese kusimba. zvigadziriso Relative time window Yakaongororwa 25 kusvika 50 ROI ye rafts uye mavara uye 15-25 ROI yemadonhwe, mifananidzo yakasarudzwa kubva kune inodarika 4 replicates yakanyorwa kubva kunenge 3 yakazvimirira kuedza.Maviri-muswe t-bvunzo akashandiswa kuongorora nhamba mutsauko pakati pemarudzi kana pakati coacervate masisitimu.Kuongororwa kwepixel-by-pixel yehupenyu hwese (τ), kuderedzwa kwehupenyu hwese pamusoro pemunda kune imwe neimwe chiteshi kwakaverengerwa uye fungidziro ye2/3-chikamu exponential attenuation modhi yakaitwa.Hupenyu hwese hwepikisiki imwe neimwe hwakazoiswa pachishandiswa zvakaverengerwa kare τ values, zvichikonzera pseudocolor FLIM fit mufananidzo.Muswe-unokodzera hupenyu hwehupenyu hwese hwakafanana pamifananidzo yese yemugero mumwechete, uye kuora kwega kwega kwakaburitsa mafotoni akakwana kuti ape kukwana kwakavimbika.Pakuongorora FRET, mapixels akasarudzwa nekushandisa yakaderera intensity pachikumbaridzo che100 photons, iyo yakaenzana neyekumashure chiratidzo (FBG) yegumi nerimwe mafoto.Iyo fluorescence intensity yemugero wega wega yakagadziriswa nekuyedza yakagadziriswa zvinhu: 69 spectral crosstalk α yaive 0.004, yakananga excitation β yaive 0.0305, yekuona kugona γ yaive 0.517.Iko kushanda kweFRET padanho repixel inozoverengerwa uchishandisa inotevera equation:
uko FDD ndiyo fluorescence intensity inoonekwa mumupi (yegirini) chiteshi, FDA ndiyo fluorescence intensity inocherechedzwa munogamuchira (mutsvuku) chiteshi pasi pekunakidzwa kusiri kwakananga, uye FAA ndiyo fluorescence intensity inocherechedzwa mukugamuchira (tsvuku) chiteshi pasi pekunakidzwa kwakananga ( PIE).Fluorescence intensity pulses inoonekwa muchiteshi).
Isa 100 µl yeLLPS reaction solutions ine 25 µM isina kunyorwa monomeric Tau441 (ine kana isina 25 µM αS) muLLPS buffer (yakawedzerwa sepamusoro) pane isina-kusunga 96-tsime microplates ane adhesive foil coating uye droplet kugadzirwa kwakaongororwa neWF. equilibration.mukati me10 min.Mushure memaawa makumi mana nemasere ekuvharirwa pamhepo yekupisa, kuvapo kweprotein rafts uye mavara kwakasimbiswa.Wobva wanyatso bvisa mvura iri pamusoro pema rafts kubva mumatsime, wobva wawedzera 50 L ye dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) uye incubate kwemaminitsi gumi.Kuiswa kwemunyu kwakakwira kunovimbisa kuti LLPS haidzokorore nekuda kwePEG yasara, uye mapuroteni anogoneka akaumbwa chete nemagetsi emagetsi anoparadzaniswa.Pazasi pegomba rakabva rakweshwa zvakanyanyisa nemuromo wemicropipette uye mhinduro yacho yakaendeswa kune isina chinhu chekutarisa tsime.Mushure mekuiswa kwemasamples ane 50 μM ThT kwe1 h, kuvepo kwenzvimbo dzakasarudzika kwakatariswa neWF microscopy.Gadzirira sonicated αS fibrils nekupinza 300 µl ye 70-µM αS mhinduro muPBS ine pH 7.4, sodium azide 0.01% pa 37 ° C uye 200 rpm pane orbital shaker kwemazuva manomwe.Mhinduro yacho yakabva yaiswa centrifuged pa9600 × g ye30 min, pellet yakamiswa zvakare muPBS pH 7.4 uye sonicated (1 min, 50% cycle, 80% amplitude muVibra-Cell VC130 sonicator, Sonics, Newton, USA) fibril samples. ine yunifomu saizi kugovera kwediki fibrils.
Ongororo yeFCS/FCCS uye kuona kusawirirana kwemavara maviri-mavara (TCCD) kwakaitwa paMT200 imwe nguva-yakagadziriswa fluorescent confocal microscope (Pico-Quant, Berlin, Germany) yakashandiswa paFLIM-FRET maikorosikopu kuyedza uchishandisa iyo PIE modhi.Simba relaser rezviyedzo izvi rakawedzerwa ku6.0 µW (481 nm) uye 6.2 µW (637 nm).Iko kusanganiswa kweaya masimba e-laser kwakasarudzwa kuti ibudise kupenya kwakafanana kune mapairi efluorophores anoshandiswa uku uchiwana yakakwana mareti ekuverenga uye kudzivirira mapikicha uye saturation.Zvese zviri zviviri kuunganidzwa kwedata uye kuongorora kwakaitwa pachishandiswa inotengeswa SymphoTime64 vhezheni 2.3 software (PicoQuant, Berlin, Germany).
Masamples eakasarudzika αS/Tau aggregates akawanikwa achishandisa LLPS anodzikiswa mugaba buffer kune akakodzera monomolecular concentration (kazhinji 1:500 dilution, sezvo akaunganidzwa atove pakadzika kudzika kana akaparadzaniswa kubva kune coacervate samples).Masamples akaiswa zvakananga kune anovhara (Corning, USA) precoated neBSA mhinduro pakusangana kwe1 mg/mL.
Pakuongorora kwePIE-smFRET mumatanho akasvibira uye matsvuku, chikumbaridzo chakaderera chemafotoni makumi maviri neshanu chakaiswa kusefa masaini akaderera anokonzerwa nezviitiko zvemonomeric (ona kuti mamonomers anodarika masampuli akaunganidzwa kana achienzaniswa neakaunganidzwa ega).Ichi chikumbaridzo chakaverengerwa kakapetwa kashanu paavhareji kusimba kwemonomeric αS yakawanikwa kubva mukuongororwa kwemasamples akachena emonomeri kuitira kuti anyatso kusarudza akaunganidzwa ekuongorora.Iyo PIE drive yedunhu, pamwe neTSCPC yekutora data, yakagonesa kushandiswa kwehupenyu hwese huremu sefa inobatsira kubvisa kumashure uye spectral crosstalk.Iyo flare intensity yakasarudzwa uchishandisa zvikumbaridzo zviri pamusoro yakagadziriswa pachishandiswa avhareji yechiratidzo chekumashure chakatarwa kubva kuhistograms yechiitiko maringe nekusimba/bhini yemasampuli-chete.Kuputika kwakabatana neaggregates hombe kunowanzo gara akati wandei anoteedzana mabhini munguva yekutevera (yakaiswa ku1 ms).Muzviitiko izvi, bhini resimba guru rakasarudzwa.Kuongorora kweFRET uye stoichiometric, iyo theoretically yakatarwa gamma factor γ (0.517) yakashandiswa.Spectral crosstalk uye yakananga excitation mipiro haina basa (yakatemerwa kuyedza) pane excitation laser simba rinoshandiswa.Kubudirira uye stoichiometry yeFRET mukuputika inoverengwa sezvinotevera.
Nguva yekutumira: Mar-08-2023