347 12.7 * 1.24mm ʻO ke kila kila i hoʻopaʻa ʻia i nā paipu wili, ʻo ka mīkini molekala o ka condensation electrostatic synchronous a me ka coaggregation o α-synuclein a me tau.

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347 Kūlana Kūlana Pipe

347 12.7*1.24mm paipu wiliwili kila kila

Ka Anawaena Mawaho: 6.00 mm OD a hiki i 914.4 mm OD, Nui a hiki i 24” NB loaʻa Ex-stock, OD Size Steel Tubes loaʻa Ex-stock

SS 347 Paipu Mānoanoa: 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, etc. (0.5-12mm) A i ʻole ka nui maʻamau ʻole e hoʻopili ʻia e like me ka makemake.

ʻAno: SS 347 Paipu ʻole |SS 347 ERW Paipu |SS 347 Paipu Weld |SS 347 Paipu Hana |SS 347 CDW Paipu, LSAW Paipu / Seam-welded / Hoʻohou

ʻAno: SS 347 Puʻupuʻu Puʻupuʻu/ Paipu, SS 347 Paipu Hui Pū ʻIa/ Paipu, SS 347 Pahu Puʻupuʻu ʻAiʻa/ Paipu, SS 347 Puʻupuʻu Coiled, SS 347 "U" Kino, SS 347 Pan Cake Coils, SS 347 Hydraulic Tubes

Length: Hoʻokahi Random, ʻAlua Random & Pono Loaʻa Hopena: Plain End, Beveled End, Treaded

Hoʻopaʻa Hoʻopau: Nā Paʻi Paʻa |Hoʻopau i waho: 2B, No.4, No.1, No.8 Mirror Finish no nā paipu kila kila, hoʻopau e like me nā mea kūʻai aku.

Kūlana Hoʻouna: Annealed and Pickled, Polished, Bright Annealed, Cold Drawn

Nānā, Hōʻike Hōʻike: Nā Palapala Hoʻāʻo Mill, EN 10204 3.1, Hōʻike Kemika, Hōʻike Mechanical, Hōʻike hoʻāʻo PMI, Hōʻike Nānā Kiʻi, Hōʻike Nānā ʻekolu ʻaoʻao, NABL Approved Lab Reports, Destructive Test Report, Non Destructive Test Reports

Hoʻopili ʻia: Hoʻopili ʻia i loko o nā pahu lāʻau, nā ʻeke palaki, nā ʻāpana kila i hui ʻia, a i ʻole e like me nā noi a nā mea kūʻai aku.

Nā mea kūikawā: Hiki ke hanaʻia nā nui a me nā kiko'ī'ē aʻe ma luna o ke noi

SS 347 Paipu Nui Launa:1/2 iniha NB, OD i 24 iniha

ASTM A312 347: ʻO ka paipu austenitic i hoʻopaʻa ʻia a pololei-seam i manaʻo ʻia no ka wela wela a me ka lawelawe corrosive maʻamau.ʻAʻole ʻae ʻia ka metala hoʻopiha i ka wā wili.

ASTM A358 347: Hoʻohui uila wili austenitic paipu no ka lawelawe corrosive a / a i ʻole ka wela wela.ʻO ka maʻamau wale nō ka paipu a hiki i 8 iniha ke hana ʻia i kēia kikoʻī.ʻAe ʻia ka hoʻohui ʻana i ka metala hoʻopiha i ka wā wili.

ASTM A790 347: ʻO ka paipu ferritic/austenitic (duplex) i hoʻopaʻa ʻia no ka lawelawe corrosive maʻamau, me ka manaʻo nui i ke kūʻē ʻana i ka pohā ʻino.

ASTM A409 347: ʻO ka hui ʻana o ka uila pololei a i ʻole ka spiral-seam i hoʻohui ʻia i ke anawaena nui austenitic light-wall paipu i ka nui 14 "a 30" me nā paia Sch5S a me Sch 10S no ka corrosive a / a kiʻekiʻe.

ASTM A376 347: Paipu austenitic seamless no nā noi wela kiʻekiʻe.

ASTM A813 347: Hoʻokahi-seam, hoʻokahi- a pālua-welded austenitic paipu no ka wela kiʻekiʻe a me nā noi corrosive maʻamau.

ASTM A814 347: ʻO ka paipu austenitic welded i hana anu no ka wela kiʻekiʻe a me ka lawelawe corrosive maʻamau.

347H Nā Paipu Kinohi Kinohi

Na Mea Mechanical Pipe 347H

Pilikino Pilikino 347H Paipu Pono Kino

Nā Papa Kūlike no 347H Pipe kila ʻiliahi

ʻO Amyloid alpha-synuclein (αS) aggregation kahi hōʻailona o ka maʻi o Parkinson a me nā synucleinopathies ʻē aʻe.I kēia mau lā, ua pili pū ka protein tau i pili pū me ka maʻi o Alzheimer me ka αS pathology a ua ʻike ʻia e hui pū i loko o nā hoʻohui waiwai αS, ʻoiai ʻaʻole maopopo ke ʻano molecular o ka coaggregation o nā protein ʻelua.Hōʻike mākou ma aneʻi ua hoʻokaʻawale ka māhele αS i nā condensates wai ma o ka condensation complex electrostatic me nā polypeptides i hoʻopiʻi maikaʻi ʻia e like me tau.Ma muli o ka pili o αS no nā polycations a me ka helu o ka valence depletion o ka coagulation network, nā clots e hana i ka gelation wikiwiki a i ʻole coalescence i ukali ʻia e ka lohi amyloid aggregation.Ma ka hoʻohui ʻana i kahi hui o nā ʻenehana biophysical kiʻekiʻe, ua hiki iā mākou ke ʻike i ka hoʻokaʻawale ʻana o ka wai-wai αS / Tau a ʻike i nā kumu koʻikoʻi e alakaʻi ai i ka hoʻokumu ʻana o nā heterogeneous aggregates i loaʻa nā protein ʻelua i loko o kahi condensate protein wai.
Ma waho aʻe o nā keʻena membrane, hiki ke hoʻokō ʻia ka hoʻokaʻawale spatial i loko o nā cell ma o ka hoʻokumu ʻana i nā kino momona e like me ka wai i kapa ʻia ʻo biomolecular condensates a i ʻole droplets, ma o ke kaʻina hana i kapa ʻia ʻo ka wai-wai-wai (LLPS).Hoʻokumu ʻia kēia mau droplets e nā pilina kino multivalent, maʻamau ma waena o nā protein a me nā protein a me RNA, a lawelawe i nā ʻano hana like ʻole i nā ʻōnaehana ola āpau.Hōʻike ka nui o nā protein hiki i ka LLP i nā kaʻina o ka paʻakikī haʻahaʻa i hoʻopilikia nui ʻia i ke ʻano a me ka hoʻokumu ʻana o nā condensates biomolecular3,4,5.Ua hōʻike ʻia nā haʻawina hoʻokolohua he nui i ke ʻano maʻalahi, pinepine ʻole, a me nā ʻano like ʻole o nā protein i hana i kēia mau condensates e like me ka wai, ʻoiai ʻaʻole i ʻike nui ʻia e pili ana i nā mea hoʻoholo molekala kikoʻī e hoʻomalu i ka ulu a me ka oʻo ʻana o kēia mau condensates i ʻoi aku ka paʻa. mokuʻāina..
Ke kākoʻo nei ka ʻikepili hou i ke kuhiakau e pili ana i ka LLPS i alakaʻi ʻia i ka protein a me ka hoʻololi ʻana o nā kulu i loko o nā hale paʻa e pili ana i nā ala cellular e alakaʻi ai i ka hoʻokumu ʻana i nā aggregates insoluble i hōʻike pinepine ʻia i nā maʻi degenerative.ʻO ka nui o nā protein intrinsically disordered (IDPs) e pili ana i ka LLPS, i hoʻopiʻi pinepine ʻia a maʻalahi, ua pili lōʻihi me ka neurodegeneration ma o ke kaʻina o ka amyloid aggregation.ʻO ka mea nui, biomolecular IDP condensates e like me FUS7 a i ʻole TDP-438 a i ʻole nā ​​protein me nā kikowaena haʻahaʻa haʻahaʻa haʻahaʻa nui e like me hnRNPA19 ua hōʻike ʻia i ka makahiki i loko o ka gel-like a i ʻole nā ​​ʻano paʻa ma o ke kaʻina i kapa ʻia ʻo fluidization.pūhui.i ka hoʻololi pae paʻa (LSPT) ma ke ʻano he hana o ka manawa a i ʻole ma ka pane ʻana i kekahi mau hoʻololi ma hope o ka unuhi ʻana a i ʻole nā ​​hoʻololi koʻikoʻi pathologically1,7.
ʻO kekahi IDP e pili ana me LLPS i loko o vivo ʻo Tau, kahi pūmua maʻi e pili ana i ka microtubule nona ka amyloid aggregation i hoʻopili ʻia i ka maʻi o Alzheimer10 akā ua pili pū kekahi i ka maʻi o Parkinson (PD) a me nā synaptic nuclear proteinopathies 11, 12, 13 pili.Ua hōʻike ʻia ʻo Tau i ka wehe wale ʻana mai ka solution/cytoplasm ma muli o nā pilina electrostatic maikaʻi14, ka hopena i ka hoʻokumu ʻia ʻana o nā droplets i hoʻonui ʻia i ka tau i ʻike ʻia he electrostatic coacervates.Ua ʻike ʻia hoʻi ʻo kēia ʻano o ka launa pū ʻole kikoʻī ʻo ia ka ikaika hoʻokele ma hope o nā condensates biomolecular ma ke ʻano15.I ka hihia o ka protein tau, hiki ke hoʻohui ʻia ka electrostatic aggregation e ka hoʻohui maʻalahi, kahi i hoʻopiʻi kūʻē ʻia ai nā ʻāpana o ka protein i ke kaʻina hana, a i ʻole ma ka hoʻohui paʻakikī ma o ka launa pū ʻana me nā polymers i hoʻopiʻi ʻia e like me RNA.
I kēia mau lā, ua hōʻike ʻia ʻo α-synuclein (αS), kahi amyloid IDP i hoʻopili ʻia i ka PD a me nā maʻi neurodegenerative ʻē aʻe i ʻike ʻia ʻo synucleinopathy17,18, i hōʻike ʻia i loko o nā cellular a me nā holoholona holoholona19,20 i hoʻopaʻa ʻia i nā condensates protein me ka ʻano like me ka wai.Ua hōʻike ʻia nā haʻawina in vitro e loaʻa ana ʻo αS i ka LLPS ma ka hoʻohui maʻalahi ma o nā pilina hydrophobic nui, ʻoiai ʻo kēia kaʻina e koi ai i nā ʻano protein kiʻekiʻe kiʻekiʻe a me nā manawa incubation lōʻihi loa19,21.Inā hoʻokumu ʻia nā condensates i loko o ka αS i loko o vivo e kēia a i ʻole nā ​​kaʻina hana LLPS ʻē aʻe, he pilikia nui i hoʻoholo ʻole ʻia.Pēlā nō, ʻoiai ua ʻike ʻia ka αS amyloid aggregation i loko o nā neurons ma PD a me nā synucleinopathies ʻē aʻe, ʻaʻole maopopo ke ʻano o ka αS e komo ai i ka intracellular amyloid aggregation, no ka mea, ʻaʻole ʻike ʻia ka overexpression o kēia protein e hoʻomaka i kēia kaʻina.Pono pinepine ʻia nā pōʻino kelepona hou, e manaʻo ana e koi ʻia kekahi mau wahi kelepona a i ʻole microenvironment no ka hoʻihoʻi ʻana i nā hui amyloid intracellular αS.ʻO kekahi kaiapuni pūnaewele e pili pono ana i ka hōʻuluʻulu ʻana, ʻo ia paha ka loko o nā condensates protein 23 .
ʻO ka mea e mahalo ai, ua ʻike ʻia ka αS a me ka tau e hui pū i nā maʻi maʻi i loko o nā kānaka me ka maʻi o Parkinson a me nā synucleinopathies ʻē aʻe 24,25 a ua hōʻike ʻia nā hoʻokolohua i kahi pilina pathological synergistic ma waena o nā protein ʻelua 26,27 e hōʻike ana i kahi pilina ma waena o ka aggregation αS a me tau i nā maʻi neurodegenerative.mai.Ua ʻike ʻia ʻo αS a me tau e launa pū a hoʻoikaika i ka hoʻohui ʻana o kēlā me kēia i loko o ka vitro a me ka vivo 28,29 a me nā heterogeneous aggregates i haku ʻia i kēia mau protein ʻelua ua ʻike ʻia i loko o ka lolo o nā maʻi me ka synucleinopathies 30.Eia naʻe, liʻiliʻi ka ʻike e pili ana i ke kumu molekala o ka pilina ma waena o αS a me tau a me ke ʻano o kona hui pū ʻana.Ua hōʻike ʻia ʻo αS e launa pū me ka tau ma o ka huki ʻana i ka electrostatic ma waena o ka ʻāpana C-terminal i hoʻopiʻi maikaʻi ʻia o αS a me ka ʻāpana waiwai nui o ka proline o tau, i hoʻonui ʻia i nā koena i hoʻopiʻi maikaʻi ʻia.
Ma kēia haʻawina, hōʻike mākou hiki iā αS ke hoʻokaʻawale i loko o nā droplets ma o ka condensation paʻakikī electrostatic i mua o ka protein tau, ʻokoʻa i kāna pilina me nā polypeptides i hoʻopiʻi maikaʻi ʻia e like me ka poly-L-lysine (pLK), a ma kēia hana.Hana ʻo αS ma ke ʻano he molekele scaffold no ka pūnaewele droplet.Ua ʻike mākou i nā ʻokoʻa i ʻike ʻia i ke kaʻina o ka maturation o nā coacervates electrostatic αS, e pili ana me nā ʻokoʻa i ka valency a me ka ikaika o ka pilina o nā protein i komo i loko o ka pūnaewele coacervate.ʻO ka mea mahalo, ua ʻike mākou i ka hui pū ʻana o nā protein αS a me tau amyloid i loko o nā coacervates wai lōʻihi a ʻike i kekahi mau kumu nui e alakaʻi ai i ka hui pū ʻana o kēia mau protein ʻelua i loko o ia mau coacervates.Maʻaneʻi mākou e wehewehe kikoʻī i kēia kaʻina hana, ʻo ia kahi ʻano molecular hiki ke hoʻokumu i ka colocalization o ʻelua mau protein i loko o nā inclusions kikoʻī maʻi.
Loaʻa iā αS kahi huelo C-terminal anionic i ka pH kūʻokoʻa (Fig. 1a), a ua manaʻo mākou e hiki ke hana i ka LLPS ma o ka condensation o nā paʻakikī electrostatic me nā molekele polypeptide polycationic disordered.Ua hoʻohana mākou i ka 100-residue poly-L-lysine (pLK) ma ke ʻano he moleke kumu hoʻomaka ma muli o kona ʻano polymeric i hoʻopiʻi maikaʻi ʻia a hoʻopilikia ʻia i ka pH neutral 32. ʻO ka mua, ua hōʻoia mākou e pili ana ka pLK me ka Ct domain o αS ma o Solution NMR spectroscopy. (Figure 1b) me ka 13C/15N-labeled αS i mua o ka hoʻonui ʻana i ka αS:pLK molar ratios.ʻO ka pilina o pLK me ka Ct-domain o αS e hōʻike ana iā ia iho i nā pilikia o ka hoʻololi kemika a me ka emi ʻana o ka ikaika kiʻekiʻe ma kēia māhele o ka protein.ʻO ka mea hoihoi, i ka wā i hui pū ai mākou i ka αS me ka pLK ma kahi ʻano αS o ka approx.5–25 µM i mua o ka polyethylene glycol (5–15% PEG-8) (nā LLPS buffer maʻamau: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) ua hele koke mākou ma kahi ākea ākea o ka hoʻokumu ʻana i ka protein. .Ua ʻike ʻia nā droplets me ka microscopy fluorescence (WF) a me bright-field (BF) (Fig. 1c).1-5 µm droplets i loaʻa i ka αS concentrated (hoʻohui ʻia 1 µM AlexaFluor488-labeled αS, AF488-αS), hiki ke loaʻa i ko lākou mau waiwai electrostatic mai ko lākou kū ʻana i ka 10% 1,6-hexanediol (1,6-HD) a me kona ʻike ka piʻi ʻana o ka ʻike NaCl (Fig. 1c).Hōʻike ʻia ke ʻano like me ka wai o nā coacervates o ka αS/pLK electrostatic complex e ko lākou hiki ke hoʻohui i loko o nā milliseconds (Fig. 1d).Ke hoʻohana nei i ka turbidimetry, ua helu mākou i ka hoʻokumu ʻana o nā droplets ma lalo o kēia mau kūlana, ua hōʻoia i ke ʻano electrostatic o ka pilina nui e pili ana i kona kūpaʻa (Fig. 1e), a loiloi i ka hopena o nā ʻano like ʻole polymer ma ke kaʻina LLPS (Fig. 1f).ʻOiai ʻike ʻia ka hoʻokumu ʻana o ka droplet ma luna o kahi ākea o nā ratio polymer, maikaʻi loa ke kaʻina hana inā ʻoi aku ka pLK ma mua o αS.Ua ʻike ʻia nā LLP me ka hoʻohana ʻana i ka mea hoʻoneʻe kemika ʻokoʻa dextran-70 (70 kDa) a i ʻole ka hoʻohana ʻana i nā ʻano laʻana like ʻole, me ka hāʻule ʻana o ke aniani, nā luawai microplate o nā mea like ʻole, Eppendorf a i ʻole quartz capillaries.
he hōʻike Schematic o nā ʻāpana protein ʻokoʻa i nā ʻano like ʻole WT-αS a me ΔCt-αS i hoʻohana ʻia i kēia haʻawina.Hōʻike ʻia ka domain amphipathic N-terminal, ka hydrophobic amyloid-forming (NAC), a me ka domain C-terminal i hoʻopiʻi maikaʻi ʻia i ka uliuli, ʻalani, a me ka ʻulaʻula.Hōʻike ʻia ka palapala ʻāina Net Charge Per Residual (NCPR) o WT-αS.b NMR ka hoʻopili ʻana o ka αS / pLK pili i ka nele o ka macromolecular clumps.Ke piʻi aʻe nei ka manaʻo pLK (αS:pLK molar ratios o 1:0.5, 1:1.5, a me 1:10 i hōʻike ʻia i ka ʻōmaʻomaʻo māmā, ka ʻōmaʻomaʻo, a me ka ʻōmaʻomaʻo ʻeleʻele, kēlā me kēia).c Coacervate αS/pLK (molar ratio 1:10) ma 25 µM (1 µM AF488-labeled αS a i ʻole Atto647N-labeled pLK no WF kiʻi) ma LLPS buffer (luna) a i hoʻohui ʻia me 500 mM NaCl (lalo hema) a i ʻole ma hope o 10 % 1,6-hexanediol (1,6-HD; lalo ʻākau).ʻO ka pā unahi = 20 µm.d Nā kiʻi microscopic o ka BF droplet fusion o αS/pLK (molar ratio 1:10) ma kahi o 25 μM;hōʻike nā pua i ka hui ʻana o nā kulu pākahi (nā pua ʻulaʻula a me nā pua melemele) i kahi hāʻule hou (pua ʻalani) i loko o 200 ms).ʻO ka pā unahi = 20 µm.e Hoʻopuehu māmā (ma 350 nm) αS/pLK aggregation ma LLPS buffer ma mua a ma hope o ka hoʻohui ʻana o 500 mM NaCl a i ʻole 10% 1,6-HD ma 25 µM αS (N = 3 sample replicates, mean and standard deviation).f BF kiʻi (luna) a me ka malamalama hoʻopuehu ana (ma 350 nm, lalo) o αS/pLK aggregation ma 25 μM αS me ka hoonui αS:pLK molar ratio (N = 3 sample replicates, mean and standard deviation also shown).ʻO ka pā unahi = 10 µm.Hōʻike ka pā unahi ma hoʻokahi kiʻi i ka unahi o nā kiʻi a pau ma ka papa hoʻokahi.Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila data raw.
Ma muli o kā mākou ʻike ʻana i ka αS/pLK electrostatic complex condensation a me ka ʻike mua ʻana o αS ma ke ʻano he mea kūʻai aku o ka condensate tau / RNA ma o ka launa pū ʻana me tau31, ua manaʻo mākou e hiki ke hoʻokaʻawale ʻia ʻo αS a me tau me ka solvent me ka ʻole o RNA. condensation.ma o ka electrostatic complexes, a ʻo αS ka protein scaffold ma αS/Tau coacervates (e ʻike i ka puʻunaue tau ma ke Kiʻi 2e).Ua ʻike mākou i ka wā i hui pū ʻia ai ka 10 μM αS a me 10 μM Tau441 (ʻo 1 μM AF488-αS a me 1 μM Atto647N-Tau, kēlā me kēia) i loko o ka buffer LLPS, ua hana maʻalahi lākou i nā ʻāpana protein i loaʻa nā protein ʻelua, e like me ka WF microscopy.(Fig. 2a).Ua hōʻoia ʻia ka colocalization o nā protein ʻelua i nā droplets e ka microscopy confocal (CF) (Supplementary Fig. 1a).Ua ʻike ʻia ke ʻano like i ka wā i hoʻohana ʻia ai ʻo dextran-70 ma ke ʻano he mea hoʻohui (Supplementary Fig. 1c).Ma ka hoʻohana ʻana i ka PEG a i ʻole dextran i kapa inoa ʻia ʻo FITC, ua ʻike mākou ua puʻunaue like ʻia nā mea hoʻopili ʻelua ma nā laʻana, ʻaʻole hōʻike i ka hoʻokaʻawale a i ʻole ka hui ʻana (Supplementary Fig. 1d).Akā, manaʻo ia i loko o kēia ʻōnaehana hoʻolaha lākou i ka hoʻokaʻawale ʻana ma o nā hopena macromolecular crowding, ʻoiai ʻo PEG kahi mea hoʻokūkū paʻa paʻa, e like me ka ʻike ʻia ma nā ʻōnaehana LLP ʻē aʻe33,34.Ua maʻalahi kēia mau kulu waiwai protein i ka NaCl (1 M) akā ʻaʻole i ka 1,6-HD (10% v / v), e hōʻoia ana i kā lākou mau waiwai electrostatic (Supplementary Fig. 2a, b).Ua hōʻoia ʻia ko lākou ʻano wai e ka nānā ʻana i nā hanana droplet millisecond me ka hoʻohana ʻana i ka microscopy BF (Fig. 2b).
a Confocal (CF) microscopy kiʻi o αS/Tau441 coacervates i LLPS buffer (10 μM o kēlā me kēia protein, 0.5 μM o AF488-labeled αS a me Atto647N-labeled Tau441).b Nā kiʻi hoʻohālikelike ʻokoʻa ʻokoʻa (DIC) o αS/Tau441 droplet fusion hanana (10 μM no kēlā me kēia protein).c Hōʻuluʻulu manaʻo kiʻi ma muli o ka hoʻopuehu māmā ʻana (ma 350 nm) o Tau441 LLPS (0–15 µM) i ka ʻole (hema) a i ʻole ke alo (akau) o 50 µM αS.Hōʻike nā kala mehana i ka hoʻopuehu ʻana.d Ka hoʻopuehu māmā ʻana o nā laʻana αS/Tau441 LLPS me ka hoʻonui ʻana i ka manaʻo o ka αS (Tau441 ma 5 µM, N = 2-3 hōʻano hou e like me ka mea i hōʻike ʻia).ʻO ka hōʻike schematic o kekahi mau ʻano protein tau a me nā ʻāpana like ʻole o ka protein i hoʻohana ʻia i kēia noiʻi: N-terminal domain (ʻulaʻula), proline-rich region (blue), microtubule-binding domain (MTBD, hōʻike ʻia i ka ʻalani), a amyloid-forming pair spiral.nā ʻāpana filament (PHF) i loko o ka MTBD (hina).Hōʻike ʻia ka palapala ʻāina Net Charge Per Residue (NCPR) o Tau441.f Ke hoʻohana nei i ka 1 µM AF488-labeled αS a me Atto647N-labeled ΔNt-, me ka hoʻohana ʻana i 1 µM AF488-labeled αS a i ʻole ΔCt-αS i mua o ΔNt-Tau (luna, 10 µM no ka protein) a i ʻole K18 (lalo, 50 µM ) ) ) nā micrographs o WF i hoʻopaʻa ʻia ma LLPS a i ʻole K18 buffer.Hōʻike nā kaola unahi i hoʻokahi kiʻi i ka pālākiō o nā kiʻi āpau i hoʻokahi panel (20 µm no nā panela a, b a me f).Hāʻawi ʻia nā ʻikepili maka no nā panela c a me d ma ke ʻano he faila ʻikepili maka.
No ka hoʻāʻo ʻana i ke kuleana o ka αS i kēia kaʻina LLPS, ua noiʻi mua mākou i ka hopena o ka αS i ka paʻa droplet e ka nephelometry me ka hoʻohana ʻana i ka hoʻonui ʻana o ka NaCl (Fig. 2c).ʻOi aku ka kiʻekiʻe o ka paʻakai paʻakai i loko o nā laʻana i loaʻa iā αS, ʻoi aku ka kiʻekiʻe o nā waiwai hoʻopuehu māmā (ma 350 nm), e hōʻike ana i ka hana hoʻokū o αS i kēia ʻōnaehana LLPS.Hiki ke ʻike ʻia kahi hopena like ma ka hoʻonui ʻana i ka ʻike αS (a no laila ka αS:Tau441 ratio) a kokoke.Hoʻonui 10-fold e pili ana i ka ʻike tau (5 µM) (Fig. 2d).No ka hōʻike ʻana he scaffold protein ka αS i loko o nā coacervates, ua hoʻoholo mākou e noiʻi i ka ʻano o ka LLPS-disrupted Tau mutant, ka mea i nele i kahi ʻāpana N-terminal i hoʻopiʻi maikaʻi ʻia (koe 1-150, ʻike Fig. 2e) i kapa ʻia ʻo ΔNt-Tau.Ua hōʻoia ʻo WF microscopy a me nephelometry ʻaʻole i loaʻa ʻo ΔNt-Tau iā ia iho i ka LLPS (Fig. 2f a me ka Fig. ho'iho'i 'ia me ka mānoanoa kulu kokoke i ka mānoanoa kulu o nā hā'ina piha piha o Tau a me αS ma lalo o nā kūlana like a me ka ho'opalekana pūmua.Hiki ke ʻike ʻia kēia kaʻina hana ma lalo o nā kūlana haʻahaʻa haʻahaʻa macromolecular crowding (Supplementary Fig. 2c).Ua hōʻike ʻia ke kuleana o ka ʻāpana C-terminal αS, akā ʻaʻole i kona lōʻihi holoʻokoʻa, i ke kaʻina hana LLPS ma ke kāohi ʻana i ka hoʻokumu ʻana o ka droplet me ka hoʻohana ʻana i kahi ʻano ʻokoʻa C-terminal ʻoki ʻia αS nele i nā koena 104-140 (Fig. 1a) o ka (ΔCt- αS) protein (Fig. 2f a me ka Fig. 2d Hoʻohui).Ua hōʻoia ʻia ka colocalization o αS a me ΔNt-Tau e ka microscopy fluorescence confocal (Supplementary Fig. 1b).
No ka hoʻāʻo hou ʻana i ka mīkini LLPS ma waena o Tau441 a me αS, ua hoʻohana ʻia kahi ʻano Tau hou, ʻo ia hoʻi ka ʻāpana helical filament core (PHF) i hui pū ʻia i loko o ka microtubule-binding domain (MTBD), inā he ʻehā mau ʻano kikowaena hou, i ʻike ʻia hoʻi. e like me ka ʻāpana K18 (e nānā i ke kiʻi 2e).Ua hōʻike ʻia i kēia manawa ua hoʻopaʻa ʻia ʻo αS i kahi protein tau i loaʻa i loko o kahi waihona waiwai proline i kahi kaʻina ma mua o ka microtubule-binding domain.Eia nō naʻe, ua waiwai pū ka ʻāina PHF i nā koena i hoʻopiʻi maikaʻi ʻia (e nānā i ke kiʻi 2e), ʻoi aku ka lysine (15% koena), ka mea i koi iā mākou e hoʻāʻo inā hāʻawi pū kēia māhele i ka condensation o ka paʻakikī αS/Tau.Ua ʻike mākou ʻaʻole hiki iā K18 wale ke hoʻoulu i ka LLPS ma nā ʻano a hiki i 100 μM ma lalo o nā kūlana i hoʻāʻo ʻia (LLPS buffer me 15% PEG a i ʻole 20% dextran) (Figure 2f).Eia naʻe, i ka wā i hoʻohui ai mākou i ka 50 µM αS i ka 50 µM K18, ua ʻike ʻia ka hoʻokumu wikiwiki ʻana o nā kulu protein i loaʻa iā K18 a me αS e nephelometry (Supplementary Fig. 2d) a me WF microscopy (Fig. 2f).E like me ka mea i manaʻo ʻia, ʻaʻole hiki iā ΔCt-αS ke hoʻihoʻi i ka ʻano LLPS o K18 (Fig. 2f).Hoʻomaopopo mākou ʻo ka hui ʻana o αS/K18 e koi aku i nā ʻāpana protein kiʻekiʻe aʻe e hoʻoulu ai i ka LLPS i hoʻohālikelike ʻia me αS/ΔNt-Tau a i ʻole αS/Tau441, ua like nā mea ʻē aʻe.Kūlike kēia me ka pilina ikaika o ka αS C-terminal māhele me ka proline-rich Tau domain i hoʻohālikelikeʻia me ka microtubule-binding domain, e like me ka mea i hōʻike muaʻia 31.
Ma muli o ka hiki ʻole iā ΔNt-Tau ke hana i ka LLPS me ka ʻole o αS, ua koho mākou i kēia ʻano ʻano Tau ma ke ʻano he kumu hoʻohālike no ka hōʻike ʻana i ka αS / Tau LLPS i hāʻawi ʻia i kona maʻalahi i nā ʻōnaehana LLPS me Tau piha piha (isotype, Tau441/Tau441).me nā kaʻina hana hoʻohui paʻakikī (heterotypic, αS/Tau441).Ua hoʻohālikelike mākou i ke kiʻekiʻe o ka hōʻuluʻulu αS (ma ke ʻano o ka protein condensed phase, fαS, c) i nā ʻōnaehana αS / Tau a me αS / ΔNt-Tau ma o ka centrifugation a me ka dispersed phase SDS-PAGE analysis (e nānā i ka 2e), loaʻa nā waiwai like loa. no nā protein a pau i ka hoʻohālikelike like.Ma keʻano kūikawā, ua loaʻa iā mākou ka fαS, c 84 ± 2% a me 79 ± 7% no ka αS / Tau a me ka αS / ΔNt-Tau, e hōʻike ana i ka pilina heterotypic ma waena o ka αS a me ka tau ma mua o ka pilina ma waena o nā mole tau.ma waena.
ʻO ka pilina me nā polycations like ʻole a me ka hopena o ke kaʻina condensation ma nā kinetics αS i aʻo mua ʻia e ka fluorescence recovery after photobleaching (FRAP).Ua hoʻāʻo mākou i ka αS / Tau441, αS / ΔNt-Tau a me αS / pLK coacervates (100 μM αS i hoʻohui ʻia me 2 μM αS AF488-αS a me 100 μM Tau441 a i ʻole ΔNt-Tau a i ʻole 1 mM pLK).Loaʻa ka ʻikepili i loko o nā minuke 30 mua ma hope o ka hui ʻana i nā ʻāpana hāpana.Mai nā kiʻi FRAP ʻelele (Fig. 3a, condensation αS/Tau441) a me kā lākou mau pihi papa manawa kūpono (Fig. 3b, Hoʻohui Fig. 3), hiki ke ʻike ʻia he ʻano like loa nā kinetics αS me nā coacervates Tau441.a me ΔNt-Tau, ʻoi aku ka wikiwiki me ka pLK.ʻO nā coefficients diffusion i helu ʻia no αS i loko o ka coacervate e like me FRAP (e like me ka wehewehe ʻana e Kang et al. 35) ʻo D = 0.013 ± 0.009 µm2/s a me D = 0.026 ± 0.008 µm2/s no αS/TauS441 ka αS/ ʻōnaehana.pLK, Tau, a me D = 0.18 ± 0.04 µm2/s, pakahi (Fig. 3c).Eia nō naʻe, ʻo ka diffusion coefficient αS i loko o ka māhele dispersed he mau kauoha o ka nui ma mua o nā ʻāpana condensed a pau, e like me ka mea i hoʻoholo ʻia e Fluorescence Correlation Spectroscopy (FCS, e ʻike i ka Fig. ( D = 8 ± 4 µm2/s).No laila, ua ho'emi nui 'ia nā kinetics o ka unuhi αS i nā coacervates i ho'ohālikelike 'ia me nā protein i loko o ka māhele dispersed ma muli o ka ha'i 'ia 'ana o nā hopena molecular crowding, 'oiai na'e e pa'a ana nā coacervates a pau i nā waiwai e like me ka wai i ka hapalua hola ma hope o ko lākou ho'okumu 'ana, e like me ka māhele tau.ʻoi aku ka wikiwiki o nā kinetics i ka condensate pLK.
a-c FRAP ka nānā ʻana o ka dynamics αS (2% AF488-labeled αS) i nā coacervates electrostatic.Hōʻike ʻia nā kiʻi ʻelele o αS/Tau441 FRAP assays ma triplicate ma (a), kahi e hōʻike ai nā pōʻai ʻulaʻula i nā wahi decolorized.ʻO 5 µm ka pae pālākiō.b Awelika FRAP piʻi a me (c) helu diffusion coefficients (D) no 5-6 (N) ʻokoʻa droplets mai ʻekolu hoʻokolohua me ka hoʻohana ʻana i 100 µM αS a me nā equimolar o Tau441 (ʻulaʻula) a i ʻole ΔNt-Tau (uliuli) a i ʻole pLK ('ōmaʻomaʻo) i ʻumi manawa i ka hoʻopaʻa ʻana o LLPS.Hōʻike ʻia ka ʻokoʻa maʻamau o ka pihi FRAP ma ka waihoʻoluʻu.No ka hoʻohālikelike ʻana, ua hoʻoholo ʻia ka diffusion coefficient αS i loko o ka dispersed phase i triplicate me ka hoʻohana ʻana i ka fluorescence correlation spectroscopy (FCS) (e nānā i ke Kiʻi Hoʻohui 3 a me nā ala no ka ʻike hou aku).d Hoʻomau X-band EPR spectra o 100 μM TEMPOL-122-αS i ka LLPS buffer me ka ʻole o ka polycation (ʻeleʻele) a i ʻole i mua o 100 μM Tau441 (ʻulaʻula) a i ʻole ΔNt-Tau (uliuli) a i ʻole 1 mM pLK ('ōmaʻomaʻo).Hōʻike ka inset i kahi ʻike nui o nā laina kahua ikaika kahi i loaʻa ai nā loli nui loa.e Hoʻopaʻa i nā pihi o 50 μM TEMPOL-122-αS me nā polycations like ʻole me ka loaʻa ʻole o LLPS (ʻaʻohe PEG).Hōʻike ʻia ka hōʻemi o ka amplitude o ka band III i hoʻohālikelike ʻia me ka band II (IIII/III) o ka spectrum EPR maʻamau e hoʻonui i nā ratio molar o Tau441 (ʻulaʻula), ΔNt-Tau (uliuli) a me ka pLK ('ōmaʻomaʻo).Hōʻike nā laina kala i kūpono i ka ʻikepili me ka hoʻohana ʻana i ke kumu hoʻohālike paʻa paʻa me n kahua paʻa like a kūʻokoʻa i kēlā me kēia pihi.Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.
Ma ke ʻano he hoʻohui, ua noiʻi mākou i ka dynamics o αS i nā coacervates like ʻole me ka hoʻohana ʻana i ka lepili spin kuhikuhi (SDSL) a me ka hoʻomau electron paramagnetic resonance (CW-EPR).Ua hōʻike ʻia kēia ʻano hana i mea pono loa i ka hōʻike ʻana i ka maʻalahi a me ka dynamics o ka IDP me kahi hoʻonā ʻokoʻa i koe36,37,38.I kēia hopena, kūkulu mākou i nā koena cysteine ​​​​i loko o nā mutants Cys hoʻokahi a hoʻohana i ka 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) spin probe.Hoʻopaʻa inoa nā derivatives maleimide iā lākou.ʻOi aku ka kikoʻī, ua hoʻokomo mākou i nā ʻimi TEMPOL ma ke kūlana 122 a i ʻole 24 αS (TEMPOL-122-αS a me TEMPOL-24-αS).I ka hihia mua, ke kuhikuhi nei mākou i ka ʻāpana C-terminal o ka protein, kahi e pili ana i ka pilina me nā polycations.Akā, hiki i ke kūlana 24 ke hāʻawi iā mākou i ka ʻike e pili ana i ka dynamics holoʻokoʻa o nā protein i loko o ka condensate.Ma nā hihia ʻelua, ʻo nā hōʻailona EPR i loaʻa no nā protein o ka māhele i hoʻopuehu ʻia e pili ana i nā radical nitroxide i ke kūlana neʻe wikiwiki.Ma hope o ka hoʻokaʻawale ʻana o ka pae ma ke alo o ka tau a i ʻole pLK (100 μM TEMPOL-αS, Tau441 a i ʻole ΔNt-Tau ma kahi ratio o 1: 1 a i ʻole pLK ma kahi ratio o 1:10), ua ʻike ʻia ka piʻi ʻana o ka ikaika kiʻekiʻe pili i loko. ka EPR spectrum o αS.Hoʻonui ʻia ka laina pohō, e hōʻike ana i nā kinetics reorientation αS i hoʻemi ʻia i nā droplets i hoʻohālikelike ʻia me ka protein i ka dilute phase (Fig. 3d, Supplementary Fig. 4a).ʻOi aku ka nui o kēia mau hoʻololi i ke kūlana 122. ʻOiai ma ke kūlana 24 ʻaʻole i hoʻopilikia ka hele ʻana o ka pLK i nā kinetics o ka ʻimi, ma ke kūlana 122 ua loli nui ke ʻano o ka laina spectral (Supplementary Fig. 4a).Ke ho'āʻo nei mākou e hoʻohālike i ka spectra ma ke kūlana 122 o nā ʻōnaehana αS / polycation ʻelua me ka hoʻohana ʻana i ke ʻano isotropic (Supplementary Figure 5a) i hoʻohana mau ʻia e wehewehe i ka dynamics o ka spin-labeled IDP38,39, ʻaʻole hiki iā mākou ke kūkulu hou i ka spectra hoʻokolohua..Spectral simulation o ke kūlana o 24 milo like 'ole (Hoʻohui Fig. 5a).Hōʻike kēia aia nā kūlana makemake i ka manawa o nā hoʻonohonoho wili o ka ʻāpana C-terminal o αS i mua o nā polycations.I ka noʻonoʻo ʻana i ka hakina o αS i loko o ka ʻāpana condensed ma lalo o nā kūlana EPR hoʻokolohua (84 ± 2%, 79 ± 7%, a me 47 ± 4% no αS/Tau441, αS/ΔNt-Tau, a me αS/pLK, i kēlā me kēia—e nānā i ka Pākuʻi. Fig. 2e o ka ʻikepili ʻikepili c), hiki ke ʻike ʻia ʻo ka hoʻonui ʻana i ʻike ʻia e ke ʻano EPR e hōʻike nui ana i ka pilina o ka C-terminal māhele o αS me nā polycations like ʻole i ka manawa condensed (ka hoʻololi nui i ka hoʻohana ʻana iā TEMPOL-122- αS), ʻaʻole ka condensation protein.ʻIke ʻia ka piʻi ʻana o ka microviscosity i ka probe.E like me ka mea i manaʻo ʻia, ua hoʻihoʻi hou ʻia ka spectrum EPR o ka protein ma lalo o nā kūlana ʻē aʻe ma mua o LLPS i ka wā i hoʻohui ʻia ai ka 1 M NaCl i ka hui ʻana (Supplementary Fig. 4b).Ma keʻano holoʻokoʻa, hōʻike kā mākou ʻikepili i nā loli i ʻike ʻia e CW-EPR e hōʻike nui i ka pilina o ka C-terminal māhele o αS me nā polycations like ʻole i ka manawa condensed, a ʻike ʻia kēia pilina me ka pLK ma mua o Tau.
I mea e loaʻa ai ka ʻike hou aʻe e pili ana i nā protein i loko o ka coacervate, ua hoʻoholo mākou e aʻo i ka ʻōnaehana LLPS me ka hoʻohana ʻana i ka NMR i ka hopena.Eia nō naʻe, hiki iā mākou ke ʻike i ka hapa αS i koe i ka māhele i hoʻopuehu ʻia, ʻo ia paha ma muli o ka hoʻohaʻahaʻa ʻana i ka protein dynamics i loko o ka coacervate a me kahi ʻāpana paʻa ma lalo o ka hopena i ka nānā ʻana NMR.I ko mākou nānā ʻana i ke ʻano a me ka dynamics o ka protein e koe ana i ka māhele dispersed o ka laʻana LLPS me ka hoʻohana ʻana i ka NMR (Supplementary Fig. 5c, d), ua ʻike mākou ua ʻano like ke ʻano o ka protein i mua o ka pLK a me ΔNt-Tau, ʻelua aia i loko o ka papa hana lua a me ka ikaika o ka iwi kuamoʻo, i hōʻike ʻia e nā hoʻokolohua ma ka hoʻololi kemika lua a me ka hoʻomaha R1ρ.Hōʻike ka ʻikepili NMR i ka hopena o ka C-terminus o αS i ka nalowale nui o ka conformational flexibility ʻoiai e mālama ana i kona ʻano ʻino, e like me ke koena o ke kaʻina protein, ma muli o kāna mau pilina me nā polycations.
Ma muli o ka hoʻonui ʻia ʻana o ka hōʻailona CW-EPR i ʻike ʻia ma ka TEMPOL-122-αS condensed phase e hōʻike ana i ka pilina o ka protein me nā polycations, ua hana mākou i kahi titration EPR e loiloi i ka pilina paʻa o αS i nā polycations like ʻole me ka loaʻa ʻole o LLPS (ʻaʻohe hōʻuluʻulu o Buffer LLPS), e hōʻike ana he like nā pilina ma nā māhele dilute a concentrated (i hōʻoiaʻiʻoʻia e kā mākouʻikepili, Supplementary Fig. 4a a me Supplementary Fig. 6).ʻO ka pahuhopu ka ʻike inā ʻike ʻia nā coacervates a pau, ʻoiai ko lākou mau waiwai like me ka wai, e hōʻike i kekahi ʻano ʻokoʻa ma lalo o ka pae molekala.E like me ka mea i manaʻo ʻia, ua hoʻonui ʻia ka spectrum EPR me ka hoʻonui ʻana i ka polycation concentrate, e hōʻike ana i ka emi ʻana o ka molecular flexibility ma muli o ka pilina molekala o nā hoa pili āpau kokoke i ka saturation (Fig. 3e, Supplementary Fig. 6).Ua loaʻa i ka pLK kēia saturation ma kahi ratio molar haʻahaʻa (polycation:αS) i hoʻohālikelike ʻia me ΔNt-Tau a me Tau441.ʻO ka ʻoiaʻiʻo, ʻo ka hoʻohālikelike ʻana i ka ʻikepili me kahi kumu hoʻohālike pili i manaʻo ʻia n nā kahua paʻa like a kūʻokoʻa i hōʻike ʻia ʻo ka dissociation mau o ka pLK (~ 5 μM) he kauoha o ka nui ma lalo o ka Tau441 a i ʻole ΔNt-Tau (~ 50 μM ).µM).ʻOiai he manaʻo koʻikoʻi kēia, hōʻike kēia he ʻoi aku ka pili o ka αS no nā polycations maʻalahi me nā wahi hoʻopiʻi maikaʻi mau.Ma muli o kēia ʻokoʻa i ka pilina ma waena o αS a me nā polycations like ʻole, ua kuhi mākou e loli ʻokoʻa ko lākou mau waiwai wai i ka manawa a no laila e loaʻa i nā kaʻina LSPT like ʻole.
Hāʻawi ʻia i ka puni nui o ka coacervate protein a me ke ʻano amyloid o ka protein, nānā mākou i ke ʻano o ka coacervate i ka manawa e ʻike ai i nā kaʻina hana LSPT.Ke hoʻohana nei i ka microscopy BF a me CF (Figure 4), ua ʻike mākou ua hui pū ʻo αS / Tau441 i kahi nui i ka hopena, e hana ana i nā droplets nui e hoʻopili a pulu i ka ʻili ma lalo o ka pūnāwai / paheʻe e like me nā kulu piha, e like me ka mea i manaʻo ʻia (Supplementary Fig .7d);kapa mākou i kēia mau hale i hana ʻia i lalo he "protein rafts".Ua mau kēia mau hale i ka wai i ka mālama ʻana i ka hiki ke fuse (Supplementary Fig. 7b) a hiki ke ʻike ʻia no kekahi mau hola ma hope o ka hoʻomaka ʻana o LLPS (Fig. 4 a me Supplementary Fig. 7c).Ua ʻike mākou ua makemake ʻia ke kaʻina hana pulu ma ka ʻili o ka hydrophilic ma mua o nā mea hydrophobic (Supplementary Fig. 7a), e like me ka mea i manaʻo ʻia no nā coacervates electrostatic me nā uku kaulike ʻole a no laila ke kiʻekiʻe electrostatic surface potentials.ʻO ka mea nui, ua hoʻemi nui ʻia ka coalescence αS/ΔNt-Tau a me ka rafting, ʻoiai ua hoʻemi nui ʻia nā condensates αS/pLK (Fig. 4).I loko o ka manawa incubation pōkole, hiki i nā kulu αS/pLK ke hui a pulu i ka ʻili hydrophilic, akā ua hoʻōki koke kēia kaʻina a ma hope o 5 mau hola o ka incubation, he mau hanana coalescence liʻiliʻi wale nō a ʻaʻohe pulu i ʻike ʻia.- ka hoʻololi gel-drip.
ʻO BF Lunamakaʻāinana (nā ʻāpana hina) a me CF (nā ʻaoʻao ʻākau, AF488-labeled αS ma ka ʻōmaʻomaʻo) o nā laʻana coacervate i loaʻa ka 100 µM αS (1% lepili fluorescent) i loko o ka pahu LLPS ma ke alo o 100 µM Tau441 (luna) fluorescence kiʻi microscopic. -Tau (waena) a i ʻole 1 mM pLK (lalo) i nā manawa hoʻoulu ʻokoʻa a me nā kiʻekiʻe kiko (z, mamao mai ka lalo o ka luawai).Ua hana hou ʻia nā hoʻokolohua 4-6 mau manawa kūʻokoʻa o kekahi me nā hopena like.Hoʻomaʻu ʻia nā coacervates αS/Tau441 ma hope o 24 mau hola, e hana ana i nā lāʻau ʻoi aku ka nui ma mua o ke kiʻi.ʻO 20 µm ka pae pālākiō no nā kiʻi a pau.
A laila ua nīnau mākou inā e alakaʻi ʻia nā loko protein e like me ka wai nui i αS/Tau441 LLPS i ka hōʻuluʻulu amyloid o kekahi o nā protein i aʻo ʻia.Ua hahai mākou i ka maturation o αS/Tau441 droplets i ka manawa me WF microscopy ma lalo o nā kūlana like i luna, akā hoʻohana i ka 1 μM AF488-labeled αS a me Atto647N-labeled Tau441 (Fig. 5a).E like me ka mea i manaʻo ʻia, ʻike mākou i ka localization protein piha i ke kaʻina o ka maturation.ʻO ka mea hoihoi, mai ca.Ma hope o 5 mau hola, ʻoi aku ka ikaika o nā hale ʻaʻohe pōʻai i ʻike ʻia i loko o nā lāʻau, a mākou i kapa ai he "points", ua hoʻopili ʻia kekahi o ia mau mea me ka αS, a ua hoʻonui ʻia kekahi ma Tau441 (Fig. 5a, nā pua keʻokeʻo).Ua ʻike mau ʻia kēia mau kiko i loko o nā lāʻau no ka αS/ΔNt-Tau ma mua o ka αS/ΔNt-Tau.ʻAʻohe wahi ʻokoʻa o nā kulu o nā ʻōnaehana pLK a me Tau i kūpono ʻole no ka hui ʻana.No ka hoʻāʻo ʻana inā he amyloid-like aggregates kēia mau stains i loko o ka αS a me Tau441, ua hana mākou i kahi hoʻokolohua like me ka microscopy CF kahi i hōʻailona ʻia ai ʻo Tau441 me Atto647N a me 12.5 μM amyloid-specific thioflavin-T (ThT) i hoʻohui ʻia mai ka hoʻomaka.kalai.ʻOiai ʻaʻole i ʻike ʻia ka ThT-staining o αS/Tau441 droplets a i ʻole nā ​​lāʻau ma hope o 24 mau hola o ka incubation (Fig. 5b, lālani luna-koe nā droplets ma luna o nā raft protein), ʻo nā hale ThT-positive i loaʻa iā Atto647N-Tau441 i loko o nā rafts ua nāwaliwali loa.Hoʻopili kēia i ka nui, ke ʻano, a me kahi o nā kiko i hōʻike mua ʻia (Fig. 5b, waena a me nā lālani lalo), e hōʻike ana e pili ana nā kiko me nā aggregates like me amyloid i hana ʻia i loko o nā coacervates wai kahiko.
WF 25 μM αS i nā manawa incubation like ʻole a me nā kiʻekiʻe kiko (z, mamao mai lalo i hoʻopaʻa ʻole ʻia) i mua o 25 μM Tau441 (1 μM AF488-labeled αS a me Atto647N-labeled Tau441) i loko o ka luawai o ka microscope plate me LLPS buffer) .ʻEono mau hoʻokolohua i hana hou ʻia me nā hopena like.b CF kiʻi microscopic o 25 μM αS i mua o 25 μM Tau441 (1 μM Atto647N-labeled Tau441) a me 12.5 μM thioflavin-T (ThT).Hōʻike ʻia nā kulu protein paona a me nā raft protein i waiho ʻia ma nā lālani luna a me waena.Hōʻike ka lālani lalo i nā kiʻi o nā lāʻau a me nā hāʻule mai 3 mau kope kūʻokoʻa.Hōʻike nā pua keʻokeʻo i nā kiko ThT-positive ma nā ʻaoʻao ʻelua.ʻO 20 µm ka pae pālākiō no nā kiʻi a pau.
No ka nānā ʻana i nā kikoʻī hou aku i nā loli i ka pūnaewele coacervate protein i ka wā o ka hoʻololi ʻana mai ka wai a paʻa, ua hoʻohana mākou i ka fluorescence lifetime imaging (FLIM) a me Förster resonance energy transfer microscopy (FRET) (Figure 6 a me nā Kiʻi Hoʻohui 8 a me 9).Manaʻo mākou ʻo ka hoʻomaʻamaʻa coacervate o ka papa i loko o kahi ʻano paʻa paʻa a ʻoi aku ka paʻa ʻana i hoʻohui ʻia e alakaʻi i ka pilina pili kokoke ma waena o ka protein a me ka ʻimi fluorescent i hoʻopili ʻia me ia, hiki ke hana i kahi hopena kinai i hōʻike ʻia i loko o ke ola pōkole pōkole (τ) , e like me ka mea i hoike mua ia40.,41 ,42.Eia hou, no nā la'ana lepili pālua (AF488 a me Atto647N ma ke ʻano he mea hāʻawi FRET a me nā mea hoʻonaninani ʻae, ʻo ia hoʻi), hiki ke hoʻopili ʻia kēia emi ʻana o τ me ka condensation coacervate a me ka piʻi ʻana o ka pono FRET(E) i ka wā LSPT.Ua nānā mākou i ka hoʻokumu ʻana o ka lāʻau a me ke kiko i ka manawa ma LLPS αS/Tau441 a me αS/ΔNt-Tau samples (25 µM o kēlā me kēia protein ma LLPS buffer i loaʻa ka 1 µM AF488 i kapa ʻia ʻo αS a me / a i ʻole Atto647N i kapa ʻia ʻo Tau441 a i ʻole ΔNt-Tau).Ua ʻike mākou i kahi ʻano maʻamau i ka emi iki ʻana o ke ola fluorescence o ka AF488 (τ488) a me Atto647N (τ647N) i ka wā i oʻo ai nā coacervates (Fig. 6 a me Supplementary Fig. 8c).ʻO ka mea e mahalo ai, ua hoʻonui nui ʻia kēia hoʻololi no nā kiko i loko o nā lāʻau (Fig. 6c), e hōʻike ana ua hiki mai ka condensation protein hou ma nā kiko.I ke kākoʻo ʻana i kēia, ʻaʻole i ʻike ʻia ka loli nui o ke ola fluorescence no nā droplets αS / ΔNt-Tau kahiko no 24 h (Supplementary Fig. 8d), e hōʻike ana he hana ʻokoʻa ka gelation droplet mai ka ʻike ʻana a ʻaʻole i hui pū ʻia me ka hoʻonohonoho hou ʻana o ka molekala. i loko o ka coacervates.Pono e hoʻomaopopo ʻia he ʻokoʻa ka nui o nā kiko a me nā ʻikepili hoʻololi i ka αS, ʻoi loa no ka ʻōnaehana αS / Tau441 (Supplementary Fig. 8e).ʻO ka emi ʻana o ke ola fluorescence wahi i hele pū ʻia me ka piʻi ʻana o ka ikaika, ʻoi aku no ka Atto647N i kapa ʻia ʻo Tau441 (Supplementary Fig. 8a), a me nā pono FRET kiʻekiʻe no nā ʻōnaehana αS / Tau441 a me αS / ΔNt-Tau, e hōʻike ana i ka condensation hou ma LLPS ʻElima hola. ma hope o ka hoʻoulu ʻia ʻana, hoʻopaʻa ʻia nā protein i loko o ka uila static.Ke hoʻohālikelike ʻia me αS/ΔNt-Tau, ua ʻike mākou i ka τ647N haʻahaʻa a ʻoi aku ka kiʻekiʻe o nā koina τ488 i nā kiko αS/Tau441, i hui pū ʻia me nā koina FRET haʻahaʻa a ʻoi aku ka like ʻole.Malia paha, pili paha kēia i ka ʻoiaʻiʻo i loko o ka ʻōnaehana αS / Tau441, ʻoi aku ka heterogeneous ka nui o ka αS i ʻike ʻia a manaʻo ʻia, ʻoi aku ka nui o ka substoichiometric i hoʻohālikelike ʻia me Tau, ʻoiai ʻo Tau441 ponoʻī e loaʻa i ka LLPS a me ka hōʻuluʻulu (Supplementary Fig. 8e) .Eia nō naʻe, ʻoi aku ka nui o ka degere o ka coalescence droplet, ka hoʻokumu ʻana i ka lāʻau, a, ʻo ka mea nui, ʻoi aku ka nui o ka hui pūmua i loko o nā coacervates e like me ka wai ke loaʻa ʻo Tau441 a me αS.
a Lifetime fluorescence microscopy (FLIM) nā kiʻi o αS/Tau441 a me αS/ΔNt-Tau ma 25 μM o kēlā me kēia protein (1 μM AF488-labeled αS a me 1 μM Atto647N-labeled Tau441 a i ʻole ΔNt-Tau) i LLPS buffer.Hōʻike nā kolamu i nā kiʻi kikoʻī o nā laʻana LLPS i nā manawa ʻokoʻa (30 min, 5 h a me 24 h).Hōʻike ke kiʻi ʻulaʻula i ka ʻāina i loaʻa nā kiko αS/Tau441.Hōʻike ʻia ke ola ma ke ʻano he mau kala kala.ʻO ka pā unahi = 20 µm no nā kiʻi āpau.b Kiʻi FLIM i hoʻonui ʻia o ka wahi i koho ʻia, i hōʻike ʻia ma ka pahu ʻulaʻula ma ka panel a.Hōʻike ʻia nā pae ola me ka hoʻohana ʻana i ka unahi kala like me ka panel a.ʻO ka pā unahi = 5 µm.c Nā hōʻike moʻolelo e hōʻike ana i ka AF488 (pili ʻia me αS) a i ʻole Atto647N (pili ʻia me Tau) no nā ʻano protein like ʻole (droplets-D-, raft-R- a me speckle-P) i ʻike ʻia ma nā kiʻi FLIM i hoʻopaʻa ʻia no αS-) ka māhele manawa manawa o Tau441 a Nā laʻana coacervate αS/ΔNt-Tau (N = 17-32 ROI no D, 29-44 ROI no R, a me 21-51 ROI no nā helu).Hōʻike ʻia nā waiwai median a me nā ʻāpana melemele a me nā laina ʻeleʻele i loko o nā pahu.ʻO nā palena haʻahaʻa a me luna o ka pahu e hōʻike ana i nā quartile mua a me ke kolu, ʻo ia hoʻi, a ʻo nā helu haʻahaʻa a me ka nui i loko o ka 1.5-fold interquartile range (IQR) e hōʻike ʻia ma ke ʻano he ʻumi.Hōʻike ʻia nā outliers e like me nā daimana ʻeleʻele.Ua hoʻoholo ʻia ke koʻikoʻi o ka helu helu ma waena o nā pālua o ka puʻunaue me ka hoʻohana ʻana i ka hoʻāʻo t-lua-lua, me ka manaʻo ʻana i nā ʻokoʻa like ʻole.Hōʻike ʻia nā waiwai p-hōʻike ʻelua-huelo me nā asterisk no kēlā me kēia pālua o ka ʻikepili i hoʻohālikelike ʻia (* p-value > 0.01, ** p-value > 0.001, *** p-value > 0.0001, **** p-waiwai > 0.00001), ns Hōʻike i ka negligibility (p-waiwai > 0.05).Hāʻawi ʻia nā koina p pololei ma ka Pākuʻi Pākuʻi 1, a hōʻike ʻia ka ʻikepili kumu e like me nā faila ʻikepili maka.
No ka hōʻike hou ʻana i ke ʻano amyloid-like o nā speckles/aggregates, ua mālama mākou i nā laʻana coacervate ʻole no 24 mau hola me nā kiʻekiʻe kiʻekiʻe o (1 M) NaCl, i hopena i ka hoʻokaʻawale ʻana o nā aggregates mai nā coacervates protein.I ka wā i ʻike ʻia ai nā hōʻiliʻili kaʻawale (ʻo ia hoʻi, kahi hopena i hoʻopuehu ʻia o nā aggregates) me ka hoʻohana ʻana i ka microscopy atomic force (AFM), ua ʻike mākou i kahi morphology spherical nui me ke kiʻekiʻe maʻamau ma kahi o 15 nm, e pili ana i nā kūlana o ka paʻakai kiʻekiʻe. ka hana o nā fibrils amyloid maʻamau ma muli o ka hopena hydrophobic ikaika ma luna o ka ʻili (e hoʻomaopopo i ke kiʻekiʻe o nā fibrils he ~ 10 nm) (Supplementary Fig. 10a).ʻO ka mea e mahalo ai, i ka wā i hoʻokomo ʻia ai nā aggregates kaʻawale me ThT i kahi hōʻike fluorescence ThT maʻamau, ʻike mākou i ka piʻi nui ʻana o ka ThT fluorescence quantum yield, e hoʻohālikelike ʻia me ka mea i ʻike ʻia i ka wā i hoʻomoʻa ʻia ai ka wai me nā fibrils amyloid αS maʻamau (Supplementary Fig. 10b), e hōʻike ana i kēlā. ʻO nā hui coacervate i loaʻa nā hale like-amyloid..ʻO ka ʻoiaʻiʻo, ua ʻae nā aggregates i ka paʻakai kiʻekiʻe akā pili i ka 4 M guanidine chloride (GdnHCl), e like me nā fibrils amyloid maʻamau (Supplementary Fig. 10c).
Ma hope aʻe, ua kālailai mākou i ka haku mele ʻana o nā aggregates me ka hoʻohana ʻana i ka fluorescence mole hoʻokahi, ka hoʻopili ʻana i ka fluorescence correlation/cross-correlation spectroscopy (FCS/FCCS), a me ka nānā ʻana o ka ʻike ʻana i nā kala ʻelua (TCCD).I kēia hopena, ua hoʻokaʻawale mākou i nā aggregates i hoʻokumu ʻia ma hope o 24 mau hola o ka incubation i 100 μl LLPS laʻana i loaʻa iā αS a me Tau441 (ʻelua 25 μM) me 1 μM AF488-labeled αS a me 1 μM Atto647N-labeled Tau441.E hoʻoheheʻe i ka hopena i hoʻoheheʻe ʻia i loko o kahi mokuʻāina monomolecular me ka hoʻohana ʻana i ka pahu PEG-noa like a me 1 M NaCl (ʻo ia ka pahu hoʻokahi i hoʻohana ʻia e hoʻokaʻawale i nā aggregates mai ka coacervate) no ka pale ʻana i nā pilina electrostatic ma waena o LLPS a me ka protein.Hiki ke ʻike ʻia kahi hiʻohiʻona o ke alahele manawa o kahi mole hoʻokahi ma Fig. 7a.ʻO ka hōʻike FCCS/FCS (cross-correlation, CC a me autocorrelation, AC) ua hōʻike i ka nui o nā aggregates i loaʻa i ka αS a me ka tau i loko o nā laʻana (e nānā i ka pihi CC ma Fig. 7b, panel hema), a ʻoi aku ka nui o ke koena monomeric protein i ala mai ma ke ʻano he ka hopena o ke kaʻina hana hoʻoheheʻe (e ʻike i nā pihi AC ma ke Kiʻi 7b, panel hema).ʻO nā hoʻokolohua hoʻomalu i hana ʻia ma lalo o nā kūlana hoʻonā like me ka hoʻohana ʻana i nā laʻana i loaʻa i nā protein monomeric wale nō i hōʻike ʻole i nā pihi CC, a ua kūpono nā pihi AC me ke kumu hoʻohālike hoʻokahi-hui (Eq. 4), kahi i loaʻa i nā protein monomeric nā coefficients diffusion i manaʻo ʻia (Fig. 7b ), ʻaoʻao ʻākau).ʻO ka helu diffusion o nā ʻāpana i hoʻohui ʻia ʻaʻole ia ma mua o 1 µm2/s, a ʻo nā protein monomeric ma kahi o 1 µm2/s.50–100 µm/s;Ua like nā waiwai me nā waiwai i hoʻopuka mua ʻia no nā sonicated αS amyloid fibrils a me ka monomeric αS ma lalo o nā kūlana hoʻonā like44.I ko mākou ʻimi ʻana i nā aggregates me ka TCCD explosion analysis (Fig. 7c, luna panel), ua ʻike mākou i kēlā me kēia hui kaʻawale (αS/Tau heteroaggregate), ma kahi o 60% o nā aggregates i ʻike ʻia i loko o ka αS a me ka tau, ma kahi o 30% wale nō. tau, ma kahi o 10% αS wale nō.Ua hōʻike ʻia ka loiloi Stoichiometric o αS/Tau heteroaggregates ua hoʻonui ʻia ka hapa nui o nā heteroaggregates i tau (stoichiometry ma lalo o 0.5, ʻo ka awelika helu o nā moleki tau i kēlā me kēia hui he 4 mau manawa ʻoi aku ma mua o nā molela αS), i kūlike me kā mākou hana i ʻike ʻia ma FLIM in situ. nā hoʻokolohua..Ua hōʻike ʻia ka hōʻike FRET i loko o kēia mau aggregates nā protein ʻelua, ʻoiai ʻaʻole koʻikoʻi nui nā waiwai FRET maoli i kēia hihia, ʻoiai ʻo ka hāʻawi ʻana i nā fluorophores i kēlā me kēia aggregate ma muli o ka nui o ka protein unlabeled i hoʻohana ʻia i ka hoʻokolohua.ʻO ka mea e mahalo ai, i ka wā i hana ai mākou i ka loiloi like me ka hoʻohana ʻana i ka 45,46 mature amyloid aggregation-deficient Tau variant (e nānā i ka Fig. ua hoemi loa ka hiki ke hana i na aggregates i loko o ka coacervate a ua ike ka FLIM i kekahi mau kiko ma na hoao in situ, a ua ikeia na auwaha hookui kea nawaliwali no na laana houluulu kaawale.Eia nō naʻe, no kahi helu liʻiliʻi o nā hōʻuluʻulu i ʻike ʻia (hoʻokahi hapaʻumi wale nō o Tau441), ua ʻike mākou ua hoʻonui ʻia kēlā me kēia hui i ka αS ma mua o kēia ʻano Tau, me kahi o 50% o nā aggregates i ʻike ʻia i loko o nā molekala αS wale nō, a he heterogeneous ka αS i keu. .nā hōʻuluʻulu (e nānā i ka Fig.Ua hōʻike ʻia nā hopena o kēia mau hoʻokolohua ʻoiai hiki iā αS ponoʻī ke hōʻiliʻili me ka tau i loko o ka coacervate, ua ʻoi aku ka maikaʻi o ka nucleation tau ma lalo o kēia mau kūlana, a hiki i nā ʻāpana like ʻole amyloid ke hana e like me ke ʻano o ka αS a me ka tau.Eia naʻe, i ka manawa e hoʻokumu ʻia ai kahi kumu waiwai tau, makemake ʻia nā pilina heterotypic ma waena o αS a me tau i nā aggregates ma luna o nā pilina homotypic ma waena o nā molekala tau;ʻIke pū mākou i nā pūnaewele protein i loko o ka wai αS/tau coacervates.
a Lunamaka'āinana fluorescence kaʻina kino o nā molekele hoʻokahi o nā aggregates kaʻawale i hana ʻia ma αS/Tau441 electrostatic coacervates.Ua ʻike ʻia nā ʻūhā e pili ana i nā coaggregates αS/Tau441 (nā ʻūhā ma luna o ka paepae i hōʻike ʻia) i ʻekolu kaila ʻike (AF488 a me Atto647N hoʻokuʻu ma hope o ka hoʻoulu pololei ʻana, nā laina uliuli a me ka ʻulaʻula, Atto647N ma hope o ka hoʻoulu ʻana ʻole), FRET, laina violet).b Ka nānā 'ana o FCS/FCCS o kahi la'ana o nā hui kaawale αS/Tau441 i loa'a mai LLPS (pane hema).Hōʻike ʻia nā pihi Autocorrelation (AC) no AF488 a me Atto647N i ka polū a me ka ʻulaʻula, a ʻo nā ʻōkuhi cross-correlation (CC) e pili ana i nā aggregates i loaʻa nā mea ʻelua e hōʻike ʻia i ka poni.Hōʻike nā ʻōkuhi AC i ka hiki ʻana mai o nā mea monomeric i kapa ʻia a me nā ʻano protein aggregated, aʻo nā curves CC e hōʻike wale i ka laha ʻana o nā hōʻuluʻulu inoa pālua.ʻO ka loiloi like, akā ma lalo o nā kūlana hoʻonā like e like me nā wahi kaʻawale, hōʻike ʻia nā laʻana i loko o ka monomeric αS a me Tau441 wale nō e like me nā mana ma ka ʻaoʻao ʻākau.c Ka nānā 'ana i ka uila uila o nā molekalaka ho'okahi o nā hui ka'awale i hana 'ia ma nā coacervates electrostatic αS/Tau441.Hoʻolālā ʻia ka ʻike no kēlā me kēia aggregate i ʻehā mau ʻano hou like ʻole (N = 152) e kūʻē i kā lākou stoichiometry, nā waiwai S, a me ka pono FRET (ka papa luna, ʻike ʻia ka pahu kala i ka hanana).Hiki ke ʻike ʻia ʻekolu mau ʻano hui: -αS-wale nō me S~1 a me FRET~0, Tau-wale nō me S~0 a me FRET~1, a me heterogeneous Tau/αS aggregates me waena S a me FRET Manaʻo o ka nui. ʻO nā protein marker ʻelua i ʻike ʻia i kēlā me kēia hui heterogeneous (N = 100) e hōʻike ʻia ma ka ʻaoʻao haʻahaʻa (e hōʻike ana ke kala kala i ka hanana).Hāʻawi ʻia ka ʻikepili maka ma ke ʻano o nā faila ʻike maka.
Ua hōʻike ʻia ka oʻo ʻana a i ʻole ke kahiko ʻana o ka pūmua wai i loko o ka gel-like a i ʻole nā ​​hale paʻa i ka wā lōʻihi e pili ana i kekahi mau hana physiological o ka condensate47 a me ka maʻi, ma ke ʻano he kaʻina hana ma mua o ka hoʻohui amyloid 7, 48, 49. aʻo mākou i ka hoʻokaʻawale ʻana o ka pae a me ke ʻano.LSPT αS i mua o nā polycations random i loko o kahi kaiapuni i hoʻopaʻa ʻia ma nā ʻano micromolar haʻahaʻa a me nā kūlana pili pili kino (e hoʻomaopopo i ka helu ʻana o ka physiological o ka αS he> 1 µM50), ma muli o ke ʻano maʻamau o ka thermodynamically driven o LPS.Ua ʻike mākou ʻo αS, aia kahi ʻāpana C-terminal i hoʻopiʻi maikaʻi ʻia i ka pH physiological, hiki ke hana i nā kulu waiwai waiwai i ka protein i loko o ka wai wai ma o LLPS i mua o nā peptides disordered cationic nui e like me pLK a i ʻole Tau ma o ke kaʻina hana electrostatic. paʻakikī condensation i mua o ka aggregation macromolecules.Hiki i kēia kaʻina hana ke loaʻa i nā hopena kūpono i loko o ke kaiapuni pūnaewele kahi e hālāwai ai ʻo αS i nā molekole polycationic like ʻole e pili ana i kāna hui ʻana i ka maʻi i loko o vitro a i vivo51,52,53,54.
I loko o nā haʻawina he nui, ua manaʻo ʻia ka dynamics protein i loko o nā droplets ʻo ia kekahi o nā kumu nui e hoʻoholo ai i ke kaʻina o ka maturation55,56.I ka electrostatic αS coacervates me polycations, ke kaʻina maturation apparently ma muli o ka ikaika o ka pilina me polycations, ka valence, a me ka multiplicity o keia mau pilina.Hōʻike ka manaʻo kaulike i kahi ʻāina kaulike o nā mokuʻāina wai ʻelua e loaʻa i kahi droplet nui i waiwai i nā biopolymers e hoʻokele LLPS57,58.Hiki ke hoʻokō ʻia ka ulu ʻana o Droplet ma o Ostwald maturation59, coalescence60 a i ʻole ka hoʻohana ʻana i ka monomer manuahi i ka māhele hoʻopuehu61.No ka αS a me Tau441, ΔNt-Tau a i ʻole pLK, ʻo ka hapa nui o ka protein i hoʻopaʻa ʻia i ka condensate ma lalo o nā kūlana i hoʻohana ʻia i kēia haʻawina.Eia nō naʻe, ʻoiai ua hui koke nā kulu piha piha i ka pulu ʻana o ka ʻili, ua paʻakikī ka hui ʻana o ka kulu a me ka pulu no ΔNt-Tau a me pLK, e hōʻike ana i ka nalowale wikiwiki o nā waiwai wai i kēia mau ʻōnaehana ʻelua.Wahi a kā mākou loiloi FLIM-FRET, ua hōʻike ʻia nā droplets pLK kahiko a me ΔNt-Tau i kahi pae like o ka hōʻuluʻulu protein (like fluorescence lifetime) e like me nā droplets kumu, e hōʻike ana ua mālama ʻia ka pūnaewele protein kumu, ʻoiai ʻoi aku ka paʻakikī.
Hoʻoponopono mākou i kā mākou hopena hoʻokolohua ma ke kumu hoʻohālike (Figure 8).ʻO nā droplets i hoʻokumu mua ʻia no ka manawa pōkole he mau pūnaewele protein me ka uku ʻole electrostatic, a no laila aia nā wahi o ka hoʻopiʻi ʻole ʻana, ʻoi aku ka nui ma ka interface droplet, ka hopena i nā droplets me kahi kiʻekiʻe electrostatic surface potential.No ka uku ʻana i ka uku (kahi ʻano i kapa ʻia ʻo valence depletion) a hōʻemi i ka hiki o ka ʻili o nā droplets, hiki i nā droplets ke hoʻokomo i nā polypeptides hou mai ka dilute phase, hoʻonohonoho hou i nā ʻupena protein e hoʻomaikaʻi i ka pili ʻana i ka uku-uku, a me ka launa pū me nā droplet ʻē aʻe.me nā ʻili (wetting).ʻO nā droplets αS/pLK, ma muli o kā lākou pūnaewele protein maʻalahi (nā pilina heterotypic wale nō ma waena o αS a me pLK) a me ka pili nui ʻana no nā pilina protein-protein, me he mea lā e hiki ke kaulike i ka uku o ka condensate me ka wikiwiki;ʻoiaʻiʻo, ua ʻike mākou i nā kinetics protein wikiwiki i nā coacervates αS/pLK i hana mua ʻia ma mua o ka αS/Tau.Ma hope o ka pau ʻana o ka valence, e emi iho ka ephemeral nā pilina a lilo nā kulu i ko lākou mau waiwai wai a lilo i ʻano gel-like, non-flammable droplets me kahi haʻahaʻa electrostatic ʻili (a no laila hiki ʻole ke pulu i ka ʻili).ʻO ka hoʻohālikelike ʻana, ʻoi aku ka maikaʻi o nā droplets αS/Tau i ka hoʻonui ʻana i ke koena o ka droplet charge ma muli o nā ʻoihana protein paʻakikī (me nā pilina homotypic a me heterotypic) a me ke ʻano nāwaliwali o nā pilina protein.ʻO kēia ka hopena i nā droplets e hoʻopaʻa i ka ʻano wai no ka manawa lōʻihi a hōʻike i kahi kiʻekiʻe electrostatic hiki ke hoʻemi ʻia e ka hui ʻana a me ka ulu ʻana (pēlā e hōʻemi ai i ka ʻili o ka ʻili o nā kulu) a me ka pulu ʻana i ka hydrophilic surface chem.Hoʻokumu kēia i nā hale waihona puke protein concentrated e paʻa i nā waiwai wai no ka mea e mau ana nā pilina ma muli o ka ʻimi mau ʻana no ka hoʻonui ʻana i ka uku ma ka pūnaewele protein.ʻO ka mea e mahalo ai, ʻo nā ʻano N-terminally truncated o Tau, me kekahi mau isoforms62 kūlohelohe, hōʻike i ke ʻano waena, me kekahi mau coacervates ʻelemakule me αS i loko o nā kulu gel-like lōʻihi, aʻo nā mea ʻē aʻe e hoʻololi i nā condensates wai nui.ʻO kēia duality i ka maturation o αS electrostatic coacervates ua kūlike me ka LLPS theoretical a me nā haʻawina hoʻokolohua hou i ʻike i ka pilina ma waena o ka pau ʻana o ka valence a me ka sieving electrostatic i nā condensates ma ke ʻano he kī i ka hoʻomalu ʻana i ka nui condensate a me nā waiwai wai.Mechanism 58.61.
Hōʻike kēia papahana i ke ala hoʻohui amyloid putative no αS a me Tau441 ma o LLPS a me LSPT.Me nā ʻāpana anion-waiwai (ʻulaʻula) a me ka cation-rich (uliuli), ʻo αS a me tau electrostatic coacervates me ka valence ʻoluʻolu he haʻahaʻa haʻahaʻa ka ikehu o ka ʻili a no laila ʻoi aku ka liʻiliʻi o ka coalescence, e hopena i ka ʻelemakule kulu wikiwiki.Loaʻa kahi kūlana gel non-agglomerated paʻa..He mea maikaʻi loa kēia kūlana ma ke ʻano o ka ʻōnaehana αS/pLK ma muli o kona kiʻekiʻe kiʻekiʻe a me ka maʻalahi o ka pilina o ka protein-pair, e hiki ai ke hoʻololi wikiwiki e like me ka gel.ʻO ka mea ʻē aʻe, ʻo nā droplets me ka valence maikaʻi ʻole a, no laila, loaʻa nā wahi i hoʻopaʻa ʻia i ka protein no ka launa pū ʻana, e maʻalahi i ka coacervate e fuse a pulu i ka ʻili hydrophilic i mea e hōʻemi ai i kona ikehu kiʻekiʻe.ʻOi aku ka maikaʻi o kēia kūlana no nā coacervates αS/Tau441, nona kahi pūnaewele paʻakikī multivalent i loko o nā pilina nāwaliwali Tau-Tau a me αS-Tau.Ma ka huli ʻana, ʻoi aku ka maʻalahi o nā coacervates nui i ko lākou mau waiwai e like me ka wai, e ʻae ana i nā pilina protein-a-protein e hana.ʻO ka hopena, ʻo ka amyloid heterogeneous aggregates i loaʻa ka αS a me ka tau i loko o ka wai coacervate, i pili paha i nā mea i loaʻa i loko o nā kino hoʻohui, ʻo ia nā hiʻohiʻona o nā maʻi neurodegenerative.
ʻO nā ʻano like ʻole o ka wai nui i hoʻokumu ʻia i ka wā o ka oʻo ʻana o ka αS/Tau441 me kahi kaiapuni protein ikaika loa a, i kahi liʻiliʻi, αS / ΔNt-Tau coacervates he mau punawai kūpono no ka nucleation o ka hui pūmua.Ua ʻike maoli mākou i ka hoʻokumu ʻia ʻana o nā hui pūmua paʻa i kēia ʻano coacervates protein, i loaʻa pinepine ʻia nā αS a me ka tau.Ua hōʻike mākou ua hoʻokūpaʻa ʻia kēia mau heteroaggregates e nā pilina non-electrostatic, hiki ke hoʻopaʻa i nā dyes ThT kikoʻī amyloid ma ke ʻano like me nā fibrils amyloid maʻamau, a loaʻa maoli ke kūʻē like ʻana i nā mana like ʻole.Ua hōʻike ʻia nā ʻāpana αS/tau i hoʻokumu ʻia e LLPS i nā waiwai like-amyloid.ʻOiaʻiʻo, ʻo ka ʻokoʻa oʻo o Tau hemahema i ka amyloid aggregation ua pilikia nui i ka hana ʻana o kēia mau heterogeneous αS aggregates i loko o ka coacervate electrostatic wai.Ua ʻike ʻia ka hoʻokumu ʻana o nā aggregates αS/Tau441 i loko wale nō o nā coacervates, ka mea i mālama i nā waiwai like me ka wai, ʻaʻole loa, inā ʻaʻole hiki i nā coacervates/droplets i ka mokuʻāina gel.I ka hihia hope, ʻo ka hoʻonui ʻana i ka ikaika o nā pilina electrostatic a, ma muli o ka hopena, ʻo ka rigidity o ka pūnaewele protein e pale i ka hoʻoponopono hou ʻana o nā protein e hoʻokumu i nā pilina protein hou e pono ai no ka nucleation amyloid.Eia nō naʻe, hiki ke hoʻokō ʻia i loko o nā coacervates ʻoi aku ka maʻalahi, e like me ka wai, a ʻoi aku ka nui o ka noho ʻana i ka wai ke piʻi lākou i ka nui.
ʻO ka mea ʻoi aku ka maikaʻi o ka hoʻokumu ʻana i nā aggregates i loko o ka māhele condensed i loko o nā condensates nui αS/Tau ma mua o nā kulu liʻiliʻi e wikiwiki ana i ka gel, e hōʻike ana i ka pili o ka ʻike ʻana i nā mea e hoʻomalu ai i ka coalescence droplet.No laila, ʻaʻole wale ka manaʻo no ka hoʻokaʻawale ʻana, akā pono e mālama ʻia ka nui o ka condensate no ka hana kūpono a me ka pale ʻana i ka maʻi58,61.Hōʻike pū kā mākou hopena i ke koʻikoʻi o ke kaulike ma waena o LLPS a me LSPT no ka ʻōnaehana αS/Tau.ʻOiai ʻo ka hoʻokumu ʻana o ka droplet e pale aku i ka amyloid aggregation ma ka hōʻemi ʻana i ka nui o nā monomer protein i loaʻa ma lalo o nā kūlana saturation, e like me ka mea i manaʻo ʻia ma nā ʻōnaehana ʻē aʻe63,64, hiki i ka hui pū ʻana i nā kiʻekiʻe droplet kiʻekiʻe ke alakaʻi i ka hōʻuluʻulu protein i loko ma o ka hoʻonohonoho hou ʻana o ka conformational.nā pūnaewele protein..
Ma ke ʻano holoʻokoʻa, koʻikoʻi kā mākou ʻikepili i ka pili o ka valence cohesive a me nā pilina ʻoluʻolu / ʻoluʻolu ʻole i nā pūnaewele hāʻule i ka pōʻaiapili o LSPT.Ma keʻano kūikawā, hōʻike mākou e hiki i nā condensates αS / Tau441 lōʻihi ke hoʻohui maikaʻi a me ka nucleate e hana i nā heteroaggregates like me amyloid e komo pū ana i nā protein ʻelua a hoʻolālā i kahi mīkini mole e pili ana i kā mākou hopena hoʻokolohua.ʻO ka hui pū ʻana o ʻelua mau protein i loko o ka αS/Tau fluid coacervate a mākou e hōʻike nei ma aneʻi e pili maoli paha i ka co-localization o ʻelua mau protein i loko o nā inclusions, he mau hōʻailona o ka maʻi, a hiki ke kōkua i ka hoʻomaopopo ʻana i ka pilina ma waena o LLPS a amyloid aggregation, e wehe ana i ke ala no ka IDP i uku nui ʻia i ka neurodegeneration.
Monomeric WT-αS, cysteine ​​​​mutants (Q24C-αS, N122C-αS) a me ΔCt-αS nāʻano (Δ101-140) i hōʻikeʻia ma E. coli a hoʻomaʻemaʻeʻia e like me ka mea i ho'ākāka muaʻia.Ua hoʻokomo ʻia ʻo 5 mM DTT i nā ʻanuʻu āpau i ka hoʻomaʻemaʻe ʻana i ka αS cysteine ​​​​mutants e pale ai i ka hoʻokumu ʻana o ka disulfide bond.Tau441 isoform (plasmid i loaʻa mai Addgene #16316), ΔNt-Tau ʻano like ʻole (Δ1–150, loaʻa ma ka cloning IVA me nā primers CTTTAAGAAGGAGATACATATGATCGCCACACCGCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) a me AggDef-Tau ʻano like ʻole (1ΔTC5-Tau kumu mua o ka moʻomeheu collar (1ΔTC5-Tau). ulu i OD600 = 0.6-0.7 ma 37 ° C a me 180 rpm, a ua hoʻokomo ʻia ka ʻōlelo me IPTG no 3 mau hola ma 37 ° C.E hōʻiliʻili i nā pūnaewele ma 11,500 xg no 15 min ma 4 °C a holoi me ka paʻakai paʻakai i loaʻa ka 150 mM NaCl.Hoʻopau hou i ka pellet i loko o ka lysis buffer (20 ml no 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 100).Ua hana 'ia ka sonication kapuai ma ka hau me ka amplitude o 80% no 10 pulses (1 min ma, 1 min aku).Mai ʻoi aku ma mua o 60 ml i hoʻokahi ultrasound.Ua wela ka E. coli lysates ma 95 ° C. no 20 mau minuke, a laila hoʻomaha ʻia ma ka hau a centrifuged ma 127,000 × g no 40 mau minuke.Hoʻopili ʻia ka supernatant i hoʻomaʻamaʻa ʻia i kahi membrane 3.5 kDa (Spectrum ™ Thermo Fisher Scientific, UK) a hoʻopaʻa ʻia i ka 4 L o ka dialysis buffer (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) no 10 mau hola.Ua hoʻohālikelike ʻia kahi kolamu hoʻololi cation 5 ml (HiTrap SPFF, Cytiva, MA, USA) me ka equilibration buffer (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).Ua kānana ʻia ka tau lysate ma o kahi kānana 0.22 μm PVDF a hoʻokomo ʻia i loko o ke kolamu ma kahi kahe kahe o 1 ml / min.Ua hoʻokō mālie ʻia ka elution, ua hoʻoheheʻe ʻia ka tau me 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).Ua kālailai ʻia nā hakina e SDS-PAGE, a ua hoʻopaʻa ʻia nā hakina i loaʻa i hoʻokahi pahu me ke kaumaha molekala i manaʻo ʻia o ka tau me kahi kānana centrifuge 10 kDa a pani ʻia me kahi paʻa i loaʻa he 10 mM HEPES, pH 7.4, NaCl 500 mM a me DTT 2 mM no ʻo 100 μM ka hopena o ka protein.A laila ua hoʻoholo ʻia ka hopena protein i loko o kahi kānana 0.22 μm PVDF, wikiwiki i ka hau a mālama ʻia ma -80°C.Ua hāʻawi lokomaikaʻi ʻia ʻo Protein K18 e Prof. Alberto Boffi.ʻO ka maʻemaʻe o ka hoʻomākaukau ʻana he> 95% i hōʻoia ʻia e SDS-PAGE a me MALDI-TOF/TOF.Ua hoʻopaʻa inoa ʻia nā cysteine ​​like ʻole me AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, USA) a i ʻole TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).ua hōʻoia ʻia e ka absorbance a me MALDI-TOF/TOF.Ua hōʻailona ʻia ʻo Tau441, ΔNt-Tau, AggDef-Tau a me K18 me nā koena cysteine ​​maoli ma nā kūlana 191 a me 322 me ka hoʻohana ʻana iā Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Kelemānia) ma hope o ke kaʻina hana like.Ua hana ʻia ka uku ʻupena no kēlā me kēia koena palapala ʻāina no αS a me Tau441 me ka hoʻohana ʻana iā CIDER66.
ʻO ka poly-L-lysine paʻa (pLK DP 90-110 e like me ka NMR mai ka mea hoʻolako, Alamanda Polymers Inc, Huntsville, Alabama, USA) ua hoʻoheheʻe ʻia ma 10 mM HEPES, 100 mM NaCl, pH 7.4 a 10 mM ka hoʻopaʻa ʻana, ka hana sonicated no 5 mau minuke i loko o kahi ʻauʻau wai ultrasonic a mālama i -20 ° C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, USA) a me FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, USA) he wai hoʻoheheʻe ʻia a hoʻolaha nui ʻia i loko o ka buffer LLPS.Hoʻopau ka dialysis i nā paʻakai hoʻohaumia.A laila ua kānana ʻia lākou ma o kahi kānana syringe me ka nui o ka pore o 0.22 μm, a ua helu ʻia kā lākou ʻike me ka refractometer (Mettler Toledo, Columbus, Ohio, USA).Ua hoʻomākaukau ʻia nā laʻana LLPS ma ka lumi wela ma ke ʻano penei: ua hui pū ʻia ka buffer a me ka extrusion a me 1 mM tris (2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) a me kahi hui o 1% protease inhibitor (PMSF 100 mM, benzimide 1 mM, leupeptin 5 μM).A laila hoʻohui ʻia ka αS a me nā polycations fused (koho pLK a i ʻole Tau).No nā hoʻokolohua thioflavin-T moʻo manawa (ThT, Carbosynth, Compton, UK), e hoʻohana i ka nui o ka ʻike ThT i ka hapalua o ka ʻike αS.Hoʻohui mālie akā hoʻohui pono i nā laʻana e hōʻoia i ka homogeneous.Ua ʻokoʻa ka manaʻo o kēlā me kēia ʻāpana mai ka hoʻokolohua a i ka hoʻokolohua, e like me ka mea i wehewehe ʻia ma ka ʻāpana hopena.Ua hoʻohana ʻia ʻo Azide ma kahi ʻano o 0.02% (w/v) i kēlā me kēia manawa i ʻoi aku ka lōʻihi o ka hoʻokolohua ma mua o 4 mau hola.No nā kānana a pau e hoʻohana ana i nā laʻana LLPS, e ʻae i ka hui ʻana e kaulike no 5 mau minuke ma mua o ka nānā ʻana.No ka nānā ʻana i ka hoʻopuehu māmā, ua hoʻouka ʻia he 150 µl o nā laʻana ma luna o nā microplates 96-well paʻa ʻole (µClear®, ʻeleʻele, F-Bottom/Chimney Well, Greiner bio-one, Kremsmünster, Austria) a uhi ʻia me ke kiʻi ʻoniʻoni.Ua nānā ʻia nā LLP ma ke ana ʻana i ka absorbance ma 350 nm ma ke kikowaena o ka hopena i kahi mea heluhelu papa CLARIOstar (BMG Labtech, Ortenberg, Kelemānia).Ua hoʻokō ʻia nā hoʻokolohua ʻekolu ma 25 ° C, a ua helu ʻia nā hewa ma ke ʻano he ʻokoʻa maʻamau mai ka mean.Ua helu ʻia ka māhele dilute e ka laʻana centrifugation a me ka SDS-PAGE gel analysis, a ua helu ʻia ka hakina αS i loko o ka dilute a me ka concentrated phase ma nā ʻano hoʻonā LLPS like ʻole.Ua hoʻomākaukau ʻia kahi laʻana 100 μl LLPS i loaʻa iā 1 μM AF488-labeled αS e ka hui pū ʻana i ukali ʻia e ka centrifugation ma 9600×g no 30 mau minuke, a laila ʻike mau ʻia ka precipitate.Ua hoʻohana ʻia ka 50 μl kiʻekiʻe o ka supernatant no ka helu ʻana i ka protein me ka hoʻohana ʻana i ka gel SDS-PAGE.Ua nānā ʻia nā gels me nā kānana AF488 me ka hoʻohana ʻana i kahi ʻōnaehana kiʻi kiʻi ChemiDoc gel (Bio-Rad Laboratories, Hercules, CA, USA) a i ʻole ʻia me ka ʻili Coomassie a ʻike ʻia me nā kānana kūpono.Ua kālailai ʻia nā hui hopena me ka hoʻohana ʻana i ImageJ version 1.53i (National Institutes of Health, USA).Ua hoʻokō ʻia nā hoʻokolohua ʻelua ma nā hoʻokolohua like ʻole me nā hopena like.
ʻO ka maʻamau, ua hoʻohana ʻia ka 150 μl o nā laʻana i nā microplates 96-well paʻa ʻole a ʻike ʻia ma ka lumi wela ma kahi microscope inverted Leica DMI6000B (Leica Microsystems, Wetzlar, Kelemānia).No nā hoʻokolohua ʻokoʻa, ua hoʻohana pū ʻia nā papa µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Kelemānia) a i ʻole 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts).Ua hoʻohana ʻia nā kukui halogen EL6000 a i ʻole mercury metal halide ma ke ʻano he kumu hoʻomālamalama (no ke kiʻi BF/DIC a me WF, kēlā me kēia).No ka microscopy WF, ua hoʻohana ʻia kahi pahuhopu ea hoʻonui 40x (Leica Microsystems, Kelemānia) e kālele i ke kukui i ka hāpana a hōʻiliʻili iā ia.No ka AF488 a me ka ThT hōʻailona hōʻailona, ​​kānana excitation a me ka hoʻokuʻu ʻana me nā papa kānana GFP maʻamau, nā kānana bandpass exitation a me ka hoʻokuʻu ʻana, kēlā me kēia, nā kānana bandpass 460-500 nm a me 512-542 nm, a me kahi aniani dichroic 495 nm.No nā laʻana i hōʻailona ʻia me Atto647N, kahi hoʻonohonoho maʻamau o nā kānana Cy5 me ka excitation a me nā kānana bandpass emission 628-40 nm a me 692-40 nm, kēlā me kēia, a ua hoʻohana ʻia kahi aniani dichroic 660 nm.No ka microscopy BF a me DIC, e hoʻohana i ka pahuhopu ohi kukui i ʻike ʻia.Ua hoʻopaʻa ʻia ke kukui i hōʻiliʻili ʻia ma kahi kāmela Leica DFC7000 CCD (Leica Microsystems, Kelemānia).ʻO ka manawa hoʻolaha he 50 ms no BF a me DIC microscopy imaging a me 20-100 ms no WF microscopy imaging.No ka hoʻohālikelike, ʻo ka manawa hōʻike no nā hoʻokolohua āpau me ThT he 100 ms.Ua hana ʻia nā hoʻokolohua manawa lōʻihi e ʻike i ka coalescence droplet, me nā kiʻi e hōʻiliʻili ʻia i kēlā me kēia 100 ms no kekahi mau minuke.Ua hoʻohana ʻia ʻo ImageJ (NIH, USA) no ka nānā ʻana i nā kiʻi.Ua hana ʻia nā hoʻokolohua ʻekolu me nā hopena like.
No nā hoʻokolohua colocalization, FRAP a me 3D hana hou, ua loaʻa nā kiʻi ma kahi Zeiss LSM 880 inverted confocal microscope me ka hoʻohana ʻana i kahi paʻi polū ZEN 2 (Carl Zeiss AG, Oberkochen, Kelemānia).Ua hoʻohana ʻia nā laʻana o 50 µl i nā kīʻaha µ-Slide Angiogenesis Petri (Ibidi GmbH, Gröfelfing, Kelemānia), mālama ʻia me kahi polymer hydrophilic (ibiTreat) a kau ʻia i kahi pahuhopu 63 × ʻaila immersion (Plan-Apochromat 63 × / NA 1.4 Oil) ma DIC).Ua kiʻi ʻia nā kiʻi me ka hoʻohana ʻana i 458 nm, 488 nm, a me 633 nm argon laser laina me ka hoʻonā ʻana o 0.26 µm / pixel a me kahi manawa hoʻolaha o 8 µs / pixel no ka excitation a me nā puka makani ʻike emission o 470-600 nm, 493-628 nm, a ua hoʻohana ʻia ʻo 638–755 nm e nānā i ka ThT, AF488 a me Atto647N.No nā hoʻokolohua FRAP, ua hoʻopaʻa ʻia ke kiʻi paʻi manawa o kēlā me kēia laʻana ma 1 kiʻi i kēlā me kēia kekona.Ua hoʻokō ʻia nā hoʻokolohua ʻekolu i ka lumi wela me nā hopena like.Hoʻopili ʻia nā kiʻi āpau me ka hoʻohana ʻana i ka polokalamu Zen 2 blue edition (Carl Zeiss AG, Oberkochen, Kelemānia).Hoʻopili ʻia nā ʻōkuhi FRAP, hoʻolālā ʻia a hoʻopili ʻia i ka ʻikepili ikaika/manawa i unuhi ʻia mai nā kiʻi me ka hoʻohana ʻana iā Zen 2 me ka hoʻohana ʻana iā OriginPro 9.1.Ua hoʻokomo ʻia nā ʻōkuhi hoʻihoʻi i kahi ʻano hoʻohālike mono-exponential e helu no ka laha ʻana o ka molekala me kahi huaʻōlelo exponential hou e helu ai i ka hopena bleaching loaʻa.A laila helu mākou iā D me ka hoʻohana ʻana i ka radius bleaching nominal a me ka hoʻihoʻi hou ʻana i ka hapalua o ke ola e like me ka hoʻohālikelike o Kang et al.5 35 i hoikeia.
Ua hoʻohui ʻia nā ʻano ʻano cysteine ​​hoʻokahi o αS me 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) ma nā kūlana 24 (TEMPOL-24-αS) a me 122 (TEMPOL-122-αS), pakahi.Spin Labeling No nā hoʻokolohua EPR, ua hoʻonohonoho ʻia ka manaʻo αS ma 100 μM a ʻo 15% ka manaʻo PEG (w / v).No nā kūlana hōʻuluʻulu like ʻole, ʻo ka ratio αS:pLK he 1:10, aʻo ka αS:ΔNt-Tau a me αS:Tau441 i mālama ʻia ma 1:1.No ka hoʻopaʻa ʻana i nā hoʻokolohua titration me ka ʻole o ka lehulehu, mālama ʻia ʻo TEMPOL-122-αS ma 50 μM a ua hoʻopaʻa ʻia nā polycations i ka hoʻonui ʻana i ka nui, e hoʻomākaukau ana i kēlā me kēia kūlana.Ua hana ʻia nā ana CW-EPR me ka Bruker ELEXSYS E580 X-band spectrometer i lako me kahi Bruker ER4118 SPT-N1 resonator e hana ana ma ka microwave (SHF) alapine o ~9.7 GHz.Hoʻonohonoho ʻia ka mahana ma 25 ° C a mālama ʻia e kahi cryostat nitrogen wai.Ua loaʻa ka spectra ma lalo o nā kūlana unsaturated ma ka mana MW o 4 mW, kahi amplitude modulation o 0.1 mT, a me kahi alapine modulation o 100 kHz.Ua hoʻomaʻamaʻa ʻia nā ʻano kikoʻī e pale i nā ʻokoʻa o ka wili ʻana ma waena o nā laʻana a me ka hōʻemi ʻana o ka milo ma muli o ke koena o nā mea hoʻohaʻahaʻa i loko o nā laʻana i loaʻa ʻo Tau441 a i ʻole ΔNt-Tau (i kēia manawa i loko o nā ʻōnaehana protein kumu).Ua loaʻa nā waiwai i hāʻawi ʻia o g ma muli o ke ʻano hoʻohālike EPR spectral i hana ʻia me ka polokalamu Easyspin (v. 6.0.0-dev.34) i hoʻokō ʻia ma Matlab®67.Hoʻokahi/ʻelua mau hiʻohiʻona isotropic i hoʻohana ʻia e hoʻohālike i ka ʻikepili.Ma hope o ka hoʻoponopono ʻana i nā hōʻailona a pau, ua helu ʻia nā koena ma ka unuhi ʻana i kēlā me kēia simulation mai ka spectrum hoʻokolohua kūpono.No ka hoʻopaʻa ʻana i ka hoʻopaʻa ʻana i ka titration, ua hoʻohana ʻia ka ikaika pili o ka pūʻulu ʻekolu i ka pūʻulu lua o ka spectrum EPR maʻamau (IIII/III) e nānā i ka hoʻopaʻa ʻana o nā polycations i ka αS.No ka hoʻohālikelike ʻana i ka hoʻokaʻawale ʻana (Kd), ua hoʻokomo ʻia ka pihi hopena i kahi kumu hoʻohālike e manaʻo ʻia ana nā kahua paʻa like a kūʻokoʻa.
Ua lawe ʻia nā hoʻokolohua NMR spectroscopy me ka Bruker Neo 800 MHz (1H) NMR spectrometer i lako me kahi cryoprobe a me Z-gradient.Hana ʻia nā hoʻokolohua āpau me ka hoʻohana ʻana i ka 130-207 µM αS a me nā mea like αS/ΔNt-Tau a me nā pLK ma 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 a ua hana ʻia ma 15 ° C.No ka nānā ʻana iā LPS e NMR, ua hoʻohui ʻia ʻo 10% PEG i nā laʻana pre-mixed.Hōʻike ka awelika 1H a me 15N i ka awelika o ka hoʻololi kemika (Fig. 1b).Hoʻonohonoho ʻia ka spectra αS 2D1H-15N HSQC ma muli o kahi hana mua (BMRB entry #25227) a hōʻoia ʻia ma ka hoʻopaʻa ʻana a me ka nānā ʻana i ka spectra 3D o HNCA, HNCO a me CBCAcoNH.Ua helu ʻia nā hoʻololi kemika 13Cα a me 13Cβ ma ke alo o ΔNt-Tau a i ʻole pLK e ana i nā loli hiki ke hoʻololi ʻia i nā ʻano hana kiʻekiʻe e hoʻohālikelike ʻia me nā hoʻololi kemika αS i ka conformation coil maʻemaʻe maʻemaʻe 68 (Supplementary Figure 5c).Ua ana ʻia nā kumukūʻai R1ρ ma ka hoʻopaʻa ʻana i nā hoʻokolohua hsqctretf3gpsi (loaʻa mai ka hale waihona puke Bruker) me nā lohi o 8, 36, 76, 100, 156, 250, 400, a me 800 ms, a ua hoʻoponopono ʻia nā hana exponential i nā lohi like ʻole. manawa e hoʻoholo ai i ka R1ρ a me kāna ʻike maopopo ʻole.
Ua hana ʻia nā hoʻokolohua microscopy fluorescence ʻelua kala ma kahi microscope confocal fluorescence MT200 i hoʻoholo ʻia i ka manawa kūʻai (PicoQuant, Berlin, Kelemānia) me kahi hāmeʻa helu photon hoʻokahi (TCSPC).Hoʻohana ʻia ke poʻo diode laser no ka pulsed interleaved excitation (PIE), hele ka beam i kahi alakaʻi nalu mode a hoʻopaʻa ʻia i ka mana laser o 10 a 100 nW no nā laina laser 481 nm a me 637 nm i ana ʻia ma hope o ke aniani dichroic.Mālama kēia i ka helu helu photon maikaʻi loa, e pale ana i nā hopena o ka inoa inoa photon, photobleaching a me ka saturation.μ-Slide angiogenesis coverslips or plates (Ibidi GmbH, Gräfelfing, Germany) ua hoʻokomo pololei ʻia i loko o ka wai hoʻolulu ma luna o ka Super Apochromat 60x NA 1.2 lens me kahi kola hoʻoponopono (Olympus Life Sciences, Waltham, USA).Ua hoʻohana ʻia kahi aniani dichroic 488/640 nm (Semrock, Lake Forest, IL, USA) ma ke ʻano he mea hoʻokaʻawale beam nui.Hoʻopaʻa ʻia ka radiation unfocused e kahi lua me ke anawaena o 50 microns, a laila hoʻokaʻawale ʻia ka radiation i ʻike ʻia i 2 ala ʻike e ka 50/50 beam splitter.Ua hoʻohana ʻia nā kānana emission Bandpass (Semrock, Lake Forest, IL, USA) 520/35 no ka wai ʻōmaʻomaʻo (AF488) a me 690/70 no ka ʻulaʻula (Atto647N) i mua o ka mea ʻike.Ua hoʻohana ʻia nā diodes avalanche hoʻokahi-photon (SPAD) (Nā Pūnaehana Micro Photon, Bolzano, Italia) i mea ʻike.Ua hana ʻia ka hōʻiliʻili ʻikepili a me ka nānā ʻana me ka lako polokalamu SymphoTime64 i kūʻai ʻia (PicoQuant GmbH, Berlin, Kelemānia).
Hoʻohana ʻia he kanalima microliters o nā laʻana LLPS i nā pūnāwai μ-Slide angiogenesis (Ibidi GmbH, Gräfelfing, Kelemānia).Hoʻopili ʻia nā kiʻi i loaʻa i ka 20 µm ma luna o ka luawai no ka mamao hana kūpono no nā droplets i hoʻokuʻu ʻia a i ~ 1 µm no nā lāʻau a me nā kiko me ka hoʻonā axial o ka liʻiliʻi he 0.25 µm/pixel a me ka manawa lohi o 400 µs/pixel.E koho i ka ʻikepili ma ka hoʻohana ʻana i ka paepae ikaika e pili ana i ka awelika o ka hōʻailona hōʻailona hope (PBG, mean + 2σ) no kēlā me kēia kahawai i koho ʻia nā kulu wai protein wai wale nō, nā lāʻau, a i ʻole nā ​​kikoʻī, e kānana ʻana i nā kumu kumu mai ka māhele hoʻopuehu.No ke kālailai ʻana i ke ola o kēlā me kēia ʻano (τ) o kēlā me kēia kahawai ('ōmaʻomaʻo, "g" no AF488 a me ka ʻulaʻula, "r" no Atto647N), ua koho mākou i nā wahi hoihoi (ROI) i loaʻa nā kulu, nā lāʻau, a i ʻole nā ​​kikoʻī. ).8b) a loaʻa iā lākou ma ka hoʻokomo ʻana i ko lākou pohō o ke ola ʻana (τD, τR a me τP no nā kulu, nā lāʻau a i ʻole nā ​​kikoʻī, e ʻike i ka Fig. 8c Hoʻohui) i kēlā me kēia kahawai me ka hoʻohana ʻana i kahi kānana tail-fit a me kahi kumu hoʻohālike palaho ʻelua.ʻAwelika τ mai τ .ʻO nā ROI i hoʻopuka i nā kiʻi liʻiliʻi loa no kahi kūpono multi-exponential ua kāpae ʻia mai ka nānā ʻana.ʻO ka ʻoki ʻoki i hoʻohana ʻia he <104 photons no nā lāʻau a me nā kiko a me 103 no nā kulu.He paepae haʻahaʻa ko nā droplets no ka mea paʻakikī ke kiʻi ʻana i nā pihi palaho me nā waiwai ʻoi aku ka ikaika, no ka mea, ʻoi aku ka liʻiliʻi o nā droplets ma ke kahua kiʻi.ʻO nā ROI me nā helu kiʻi kiʻi ma luna o ka palena hōʻiliʻili kiʻi (hoʻonoho ʻia i>500 helu/pixel) i hoʻolei ʻia no ka nānā ʻana.E hoʻohālikelike i ka pihi palaho ikaika i loaʻa mai ka ʻāina hoihoi me ka ikaika ma 90% o ka nui (ma hope iki o ka ikaika o ka palaho) mai ka hoʻomaka ʻana o ke ola lawelawe e hōʻoia i ka liʻiliʻi o ka hoʻopilikia ʻana o ka IRF me ka mālama ʻana i ka mea like no nā pohō ikaika a pau. Nā hoʻonohonoho puka makani pili Ua ʻike ʻia ʻo 25 a i 50 ROI no nā lāʻau a me nā kiko a me 15-25 ROI no nā hāʻule, nā kiʻi i koho ʻia mai nā mea hou aʻe ma mua o 4 i hoʻopaʻa ʻia mai ka liʻiliʻi o 3 mau hoʻokolohua kūʻokoʻa.Ua hoʻohana ʻia nā hoʻāʻo t-huelo ʻelua e loiloi i nā ʻokoʻa helu ma waena o nā ʻano a i ʻole ma waena o nā ʻōnaehana coacervate.No ka hoʻopili pixel-by-pixel o ke ola (τ), ua helu ʻia ka nui o ka attenuation o ke ola ma luna o ke kahua no kēlā me kēia kanal a ua hoʻokō ʻia kahi hoʻohālikelike o kahi kumu hoʻohālike hoʻonui exponential attenuation 2/3.A laila hoʻokomo ʻia ka attenuation ola no kēlā me kēia pika me ka hoʻohana ʻana i nā waiwai τ i helu mua ʻia, e loaʻa ana i kahi kiʻi kūpono FLIM pseudocolor.Ua like ka laulā o ke ola holoʻokoʻa o ka huelo ma nā kiʻi a pau o ke kahawai hoʻokahi, a ʻo kēlā me kēia palaho i hoʻopuka i nā kiʻi kiʻi lawa e hāʻawi i kahi kūpono kūpono.No ka loiloi FRET, ua koho ʻia nā pika ma ka hoʻohana ʻana i ka paepae haʻahaʻa haʻahaʻa o 100 photons, kahi i hoʻohālikelike ʻia i kahi hōʻailona hope (FBG) o 11 photons.Ua hoʻoponopono ʻia ka ikaika o ka fluorescence o kēlā me kēia kahawai e nā kumu hoʻoponopono i hoʻoholo ʻia: 69 spectral crosstalk α he 0.004, he 0.0305 ka excitation pololei, he 0.517 ka ʻike ʻana i ka pono.A laila helu ʻia ka pono FRET ma ka pae pika me ka hoʻohana ʻana i ka hoohalike penei:
ʻo FDD ka ikaika o ka fluorescence i ʻike ʻia i loko o ke kahawai hāʻawi ('ōmaʻomaʻo), ʻo FDA ka ikaika o ka fluorescence i ʻike ʻia i ke kahawai ʻae (ʻulaʻula) ma lalo o ka hoʻoulu ʻole ʻana, a ʻo FAA ka ikaika o ka fluorescence i ʻike ʻia ma ke kahawai ʻae (ulaʻula) ma lalo o ka hauʻoli pololei ( PIE).ʻIke ʻia nā pulupulu ikaika o ka fluorescence i ke kahawai).
E kau i 100 µl o LLPS reaction solutions me 25 µM unlabeled monomeric Tau441 (me 25 µM αS a i ole ole 25 µM αS) i loko o ka LLPS buffer (hoʻohui ʻia e like me luna) ma luna o nā microplates 96-well paʻa ʻole me ka hoʻopili ʻia ʻana o ka foil a me ka hoʻokumu ʻia ʻana o ka droplet i nānā ʻia e WF micros. kaulike.iloko o 10 min.Ma hope o 48 mau hola o ka incubation ma ka lumi wela, ua hōʻoia ʻia ka hele ʻana o nā raft protein a me nā kiko.A laila e wehe pono i ka wai ma luna o nā lāʻau mai nā pūnāwai, a laila e hoʻohui i 50 L o ka dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) a hoʻomoʻa no 10 min.ʻO ka paʻakai kiʻekiʻe e hōʻoiaʻiʻo ʻaʻole e hana hou ʻia ka LLPS ma muli o ke koena PEG, a hiki ke wehe ʻia nā hui pūmua i hana ʻia e nā pilina electrostatic wale nō.ʻO ka lalo o ka pūnāwai e ʻoki pono ʻia me kahi ʻaoʻao micropipette a ua hoʻoneʻe ʻia ka hopena i kahi lua nānā ʻole.Ma hope o ka incubation o nā laʻana me 50 μM ThT no 1 h, ua nānā ʻia ka hele ʻana o nā wahi kaʻawale e WF microscopy.E hoomakaukau i sonicated αS fibrils ma incubating 300 µl o ka 70-μM αS solution ma PBS me pH 7.4, sodium azide 0.01% ma 37 °C a me 200 rpm ma ka orbital shaker no 7 la.A laila ua centrifuged ka hopena ma 9600 × g no 30 min, ua hoʻokuʻu ʻia ka pellet ma PBS pH 7.4 a sonicated (1 min, 50% cycle, 80% amplitude i kahi Vibra-Cell VC130 sonicator, Sonics, Newton, USA) fibril samples. me ka puunaue like like o na fibrils liilii.
Ua hana ʻia ka nānā ʻana o FCS/FCCS a me ka ʻike ʻana i nā kala ʻelua (TCCD) ma ka MT200 manawa-hoʻoholo ʻia ʻo fluorescent confocal microscope (Pico-Quant, Berlin, Kelemānia) i hoʻohana ʻia no nā hoʻokolohua microscopy FLIM-FRET me ka hoʻohana ʻana i ke ʻano PIE.Hoʻohui ʻia ka mana laser no kēia mau hoʻokolohua i 6.0 µW (481 nm) a me 6.2 µW (637 nm).Ua koho ʻia ka hui pū ʻana o kēia mau mana laser e hana i ka ʻōlinolino like no nā pālua o nā fluorophores i hoʻohana ʻia ʻoiai e loaʻa ana nā helu helu maikaʻi loa a pale aku i ka photobleaching a me ka saturation.Ua hana ʻia ka hōʻiliʻili ʻikepili a me ka nānā ʻana me ka polokalamu SymphoTime64 version 2.3 (PicoQuant, Berlin, Kelemānia).
Hoʻoheheʻe ʻia nā laʻana o nā aggregates αS/Tau i hoʻokaʻawale ʻia me ka hoʻohana ʻana i ka LLPS i loko o ka paʻa kaʻawale i ka neʻe ʻana o ka monomolecular kūpono (maʻamau he 1:500 dilution, ʻoiai aia nā aggregates i nā haʻahaʻa haʻahaʻa ke hoʻokaʻawale ʻia mai nā laʻana coacervate).Ua hoʻopili pololei ʻia nā laʻana i nā uhi uhi (Corning, USA) i uhi mua ʻia me kahi hoʻonā BSA ma ke ʻano o 1 mg/mL.
No ka nānā 'ana o PIE-smFRET ma nā kaha 'ōma'oma'o a me ka 'ula'ula, ua ho'ohana 'ia kahi paepae ha'aha'a ha'aha'a o 25 photons no ka kānana 'ana i nā hō'ailona ha'aha'a i hana 'ia e nā hanana monomeric (e ho'omaopopo i ka nui o nā monomers i nā la'ana i ho'ohui 'ia i ka ho'ohālikelike 'ia me nā aggregates kaawale).Ua helu ʻia kēia paepae ma ke ʻano he ʻelima mau manawa o ka awelika ikaika o ka monomeric αS i loaʻa mai ka nānā ʻana i nā laʻana monomer maʻemaʻe i mea e koho pono ai i nā aggregates no ka nānā ʻana.ʻO ke kaapuni kaʻa PIE, a me ka loaʻa ʻana o ka ʻikepili TSCPC, ua hiki ke hoʻohana i kahi kānana paona ola e kōkua i ka hoʻopau ʻana i ke kua a me ka spectral crosstalk.Ua hoʻoponopono ʻia ka ikaika o ka lapalapa i koho ʻia me ka hoʻohana ʻana i nā paepae ma luna me ka hoʻohana ʻana i ka hōʻailona hope maʻamau i hoʻoholo ʻia mai nā histograms o ka hanana ʻana me ka ikaika/bin o nā mea hoʻohālike buffer-wale nō.ʻO nā pahu i pili me nā hōʻuluʻulu nui e noho maʻamau i kekahi mau pahu ʻokoʻa i ka ʻimi manawa (hoʻonoho ʻia i 1 ms).I kēia mau hihia, ua koho ʻia kahi bin o ka ikaika loa.No ka FRET a me ka stoichiometric analysis, ua hoʻohana ʻia ke kumu hoʻoholo gamma γ (0.517).ʻAʻole i hoʻopaʻa ʻia nā hāʻawi kikoʻī kikoʻī a me nā haʻawina hoʻohiwahiwa (i hoʻoholo ʻia ma ka hoʻokolohua) ma ka mana laser excitation i hoʻohana ʻia.Ua helu ʻia ka maikaʻi a me ka stoichiometry o FRET i kahi pahū.