347 12.7 * 1.24mm Bakin Karfe nada tubing, Molecular inji na synchronous electrostatic condensation da coaggregation na α-synuclein da tau

Na gode da ziyartar Nature.com.Kuna amfani da sigar burauza tare da iyakancewar tallafin CSS.Don ƙwarewa mafi kyau, muna ba da shawarar ku yi amfani da sabuntar burauza (ko kuma musaki Yanayin dacewa a cikin Internet Explorer).Bugu da ƙari, don tabbatar da goyon baya mai gudana, muna nuna shafin ba tare da salo da JavaScript ba.
Sliders suna nuna labarai uku a kowane faifai.Yi amfani da maɓallan baya da na gaba don motsawa ta cikin nunin faifai, ko maɓallan masu sarrafa nunin faifai a ƙarshen don matsawa ta kowane faifan.

347 Bakin Karfe Musamman Bututu

347 12.7 * 1.24mm Bakin Karfe nada bututu

Diamita na Waje: 6.00 mm OD har zuwa 914.4 mm OD, Girman har zuwa 24 "NB akwai Ex-stock, OD Size Steel Tubes akwai Ex-stock

SS 347 Bututu Range: 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, da sauransu (0.5-12mm) Ko girman da ba na yau da kullun ba don daidaitawa kamar yadda ake buƙata.

Nau'in: SS 347 Bututu mara kyau |SS 347 ERW Bututu |SS 347 Welded Bututu |SS 347 Fabricated Bututu |SS 347 CDW Tubes, LSAW Bututu / Kabu-welded / Redrawn

Form: SS 347 Zagaye Bututu / Bututu, SS 347 Square Bututu / tubes, SS 347 Rectangular bututu / tubes, SS 347 Coiled tubes, SS 347 "U" siffar, SS 347 Pan Cake Coils, SS 347 na'ura mai aiki da karfin ruwa tubes.

Tsawon: Bazuwar Guda Guda, Bazuwar Biyu & Ƙarshen Tsawon Da ake Bukata: Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshen Ƙarshe, Taka

Ƙarshen Kariya: Filastik |Ƙarshen Waje: 2B, No.4, No.1, No.8 Mirror Gama ga Bakin Karfe Bututu, Gama kamar yadda abokin ciniki Bukatun

Yanayin Bayarwa: Gurasa da tsinke, goge, Mai haske, Janyewar sanyi

Dubawa, Rahoton Gwaji: Takaddun Gwajin Mill, EN 10204 3.1, Rahoton Sinadarai, Rahoton Injini, Rahoton Gwajin PMI, Rahoton Duban gani, Rahoton Dubawa na ɓangare na uku, Rahoton Lab da aka Amince na NABL, Rahoton Gwajin lalata, Rahoton Gwaji mara lalacewa.

Shiryawa: Cushe a cikin Akwatunan katako, Jakunkuna na Filastik, Tushen Karfe, ko kamar yadda Buƙatun Abokan ciniki

Musamman: Girma da ƙayyadaddun bayanai ban da sama ana iya kera su akan buƙata

SS 347 Girman Girman Bututu: 1/2 inch NB, OD zuwa 24 inch

ASTM A312 347: Bututun austenitic mara nauyi da madaidaiciyar kabu wanda aka yi niyya don babban zafin jiki da sabis na lalata gabaɗaya.Ba a yarda da ƙarfe mai cikawa yayin walda.

ASTM A358 347 Haɗaɗɗen wutar lantarki mai walƙiya bututu don lalata da/ko sabis na zafin jiki.Yawanci kawai bututu har zuwa 8 inch ana samar da wannan ƙayyadaddun.An ba da izinin ƙara ƙarfe mai filler yayin walda.

ASTM A790 347 mara kyau kuma madaidaiciya-kabu mai waldadden bututun ferritic/austenitic (duplex) wanda aka yi niyya don sabis na lalata na gabaɗaya, tare da fifiko na musamman kan juriya ga lalata lalata.

ASTM A409 347 Daidaitaccen kabu ko karkace-kabu-kabu na lantarki welded babban diamita austenitic haske-bangon bututu a cikin masu girma dabam 14 "zuwa 30" tare da bango Sch5S da Sch 10S don lalata da / ko babba

ASTM A376 347: Bututu austenitic mara kyau don aikace-aikacen zafin jiki

ASTM A813 347: Kabu guda ɗaya, guda- ko biyu-welded austenitic bututu don babban zafin jiki da aikace-aikacen lalata gabaɗaya.

ASTM A814 347 mai aikin sanyi mai waldadden bututun austenitic don babban zafin jiki da sabis na lalata gabaɗaya.

347H Bakin Karfe Bututun Sinadari

Bakin Karfe 347H Bututu Mechanical Properties

Bakin Karfe 347H Bututun Jiki

Daidai Maki na 347H Bakin Karfe Bututu

Amyloid alpha-synuclein (αS) aggregation alama ce ta cutar Parkinson da sauran synucleinopathies.Kwanan nan, furotin tau da ke da alaƙa da cutar Alzheimer yana da alaƙa da ilimin cututtuka na αS kuma an gano shi tare da haɗin gwiwa a cikin abubuwan da ke da wadatar αS, ko da yake tsarin kwayoyin halitta na haɗuwa da sunadaran biyu ya kasance ba a sani ba.Mun bayar da rahoto a nan cewa lokaci na αS ya rabu zuwa cikin condensates na ruwa ta hanyar hadaddun hadaddun electrostatic tare da ingantaccen cajin polypeptides kamar tau.Dangane da kusancin αS don polycations da ƙimar valence depletion na cibiyar sadarwar coagulation, ƙwanƙwasa suna yin saurin gelation ko haɗin gwiwa tare da jinkirin tarawar amyloid.Ta hanyar haɗa rukunin dabarun fasahar halittu na ci gaba, mun sami damar siffata rabewar lokaci na ruwa-ruwa αS/Tau da kuma gano mahimman abubuwan da ke haifar da samuwar tararraki iri-iri masu ɗauke da sunadaran duka biyu a cikin haɗin furotin na ruwa.
Baya ga ɓangarorin membrane, ana iya samun rabuwar sararin samaniya a cikin sel ta hanyar samar da wadataccen furotin, jikuna masu kama da ruwa da ake kira biomolecular condensates ko droplets, ta hanyar da aka sani da rabuwar ruwa-ruwa (LLPS).Waɗannan ɗigon ruwa suna samuwa ta hanyar hulɗar ɗan lokaci mai yawa, yawanci tsakanin sunadarai ko sunadaran da RNA, kuma suna yin ayyuka iri-iri a kusan dukkanin tsarin rayuwa.Babban adadin sunadaran da ke da ikon LLP suna nuna jerin ƙananan ƙananan abubuwa waɗanda ke da rikicewa sosai a cikin yanayi da kuma samar da condensates na biomolecular3,4,5.Yawancin gwaje-gwajen gwaje-gwaje sun bayyana sassauƙa, sau da yawa rikice-rikice, da nau'ikan sunadaran da ke haɗa waɗannan nau'ikan ruwa-kamar condensates, kodayake an san kaɗan game da ƙayyadaddun ƙayyadaddun kwayoyin halitta waɗanda ke sarrafa girma da maturation na waɗannan condensates zuwa mafi ƙarfi-kamar. jihar.
Sabbin bayanan suna goyan bayan hasashen cewa LLPS mai haifar da furotin mai ɓarna da canza ɗigon ruwa zuwa tsayayyen sifofi na iya zama hanyoyin salon salula masu dacewa da ke haifar da samuwar tara masu guba waɗanda galibi alamomi ne na cututtukan lalacewa.Yawancin sunadaran da ke da alaƙa da LLPS (IDPs), galibi ana caje su da sassauƙa, an daɗe suna haɗuwa da neurodegeneration ta hanyar haɓakar amyloid.Musamman ma, ƙwayoyin IDP na biomolecular kamar FUS7 ko TDP-438 ko sunadaran da ke da manyan ƙananan ƙananan yankuna kamar hnRNPA19 an nuna su zuwa shekaru zuwa gel-kamar ko ma da karfi ta hanyar tsarin da ake kira ruwa.fili.zuwa m lokaci mika mulki (LSPT) a matsayin aiki na lokaci ko a mayar da martani ga wasu bayan-fassara gyare-gyare ko pathologically maye gurbi1,7.
Wani IDP da ke da alaƙa da LLPS a cikin vivo shine Tau, furotin da ke da alaƙa da microtubule wanda haɗin amyloid ya kasance cikin cutar Alzheimer10 amma kuma kwanan nan an haɗa shi da cutar Parkinson (PD) da sauran synaptic proteinopathies 11, 12, 13 suna da alaƙa.An nuna Tau ya rabu ba da daɗewa ba daga mafita/cytoplasm saboda ingantacciyar hulɗar electrostatic14, wanda ya haifar da samuwar ɗigon ruwa masu wadatar tau da aka sani da electrostatic coacervates.An kuma lura cewa irin wannan nau'in hulɗar da ba ta dace ba ita ce motsa jiki a bayan yawancin condensates na biomolecular a cikin yanayi15.Game da furotin tau, za a iya samar da tarawar electrostatic ta hanyar tarawa mai sauƙi, wanda a cikin saɓanin cajin yankuna na furotin ke haifar da tsarin tsagewa, ko ta hanyar haɗaɗɗiyar haɗaɗɗiyar ta hanyar mu'amala da polymers da ba su da caji kamar RNA.
Kwanan nan, α-synuclein (αS), wani amyloid IDP da ke cikin PD da sauran cututtuka na neurodegenerative da aka sani da suna synucleinopathy17,18, an nuna su a cikin salon salula da kuma nau'in dabba19,20 da aka mayar da hankali a cikin ƙwayoyin furotin tare da hali mai kama da ruwa.Nazarin in vitro ya nuna cewa αS yana jure wa LLPS ta hanyar tarawa mai sauƙi ta hanyar hulɗar hydrophobic galibi, kodayake wannan tsari yana buƙatar babban adadin furotin na musamman da kuma lokaci mai tsawo na incubation19,21.Ko condensates mai ɗauke da αS da aka lura a cikin vivo an samo su ta wannan ko wasu hanyoyin LLPS ya kasance babban batu da ba a warware shi ba.Hakazalika, kodayake αS amyloid aggregation an lura da su a cikin neurons a cikin PD da sauran synucleinopathies, ainihin hanyar da αS ke shiga cikin ƙwayar amyloid na ciki ya kasance ba a sani ba, kamar yadda overexpression na wannan furotin ba ya bayyana don haifar da wannan tsari da kanta.Ana buƙatar ƙarin lalacewar salula sau da yawa, yana ba da shawarar cewa ana buƙatar wasu wurare na salon salula ko microenvironments don sake sakewa na intracellular αS amyloid majalisai.Ɗayan yanayin salon salula wanda ke da mahimmanci ga haɗuwa zai iya zama ciki na condensates na gina jiki 23.
Abin sha'awa, αS da tau an samo su tare da haɗin gwiwa a cikin halayen halayen cututtuka a cikin mutane tare da cutar ta Parkinson da sauran synucleinopathies 24,25 da gwaje-gwajen sun ba da rahoton dangantakar da ke tsakanin kwayoyin halitta guda biyu 26,27 suna ba da shawarar yiwuwar dangantaka tsakanin tarawa αS da tau a cikin cututtukan neurodegenerative.rashin lafiya.αS da tau an samo su don yin hulɗar juna da inganta haɓakar juna a cikin vitro da kuma a cikin vivo 28,29 da kuma nau'i-nau'i daban-daban da suka hada da waɗannan sunadarai guda biyu a cikin kwakwalwar marasa lafiya tare da synucleinopathies 30.Duk da haka, an san kadan game da tushen kwayoyin halitta na hulɗar tsakanin αS da tau da tsarin haɗin gwiwarsa.An ba da rahoton αS don yin hulɗa tare da tau ta hanyar jan hankali na lantarki tsakanin yankin C-terminal na αS da ake cajin da ba daidai ba da kuma yankin tsakiyar proline mai arzikin tau, wanda kuma ya wadatar a cikin abubuwan da aka caje.
A cikin wannan binciken, mun nuna cewa αS na iya ɓata cikin ɗigon ruwa ta hanyar haɗaɗɗen haɗaɗɗiyar electrostatic a gaban furotin tau, sabanin hulɗar sa tare da sauran ingantaccen cajin polypeptides kamar poly-L-lysine (pLK), kuma a cikin wannan tsari.αS yana aiki azaman ƙwayar cuta don cibiyar sadarwar droplet.Mun gano bambance-bambance masu ban sha'awa a cikin tsarin maturation na electrostatic αS coacervates, waɗanda ke da alaƙa da bambance-bambance a cikin valency da ƙarfin hulɗar sunadaran da ke cikin cibiyar sadarwar coacervate.Abin sha'awa, mun lura da haɗin gwiwar αS da furotin tau amyloid a cikin abubuwan haɗin gwiwar ruwa na dogon lokaci kuma mun gano wasu mahimman abubuwan da ke haifar da haɗakar waɗannan sunadaran guda biyu a cikin irin wannan coacervates.Anan mun bayyana wannan dalla-dalla dalla-dalla wannan tsari, wanda shine yuwuwar tsarin kwayoyin halitta wanda ke haifar da hadewar sunadaran sunadarai guda biyu a cikin takamaiman cututtukan cututtuka.
αS yana da wutsiya mai tsananin anionic C-terminal a tsaka tsaki pH (Fig. 1a), kuma mun yi hasashe cewa zai iya jurewa LLPS ta hanyar haɗaɗɗen ɗakunan lantarki tare da ƙwayoyin polypeptide marasa lalacewa.Mun yi amfani da 100-residue poly-L-lysine (pLK) a matsayin samfurin samfurin farawa saboda yanayin da yake da kyau da kuma rashin daidaituwa na polymeric a tsaka tsaki pH 32. Da farko, mun tabbatar da cewa pLK yana hulɗa tare da yankin Ct na αS ta hanyar Magani NMR spectroscopy. (Hoto na 1b) ta amfani da 13C/15N-labeled αS a gaban haɓaka αS: pLK molar rabo.Ma'amala na pLK tare da Ct-domain na αS yana bayyana kansa a cikin rikice-rikice na canjin sinadarai da raguwa a cikin ƙarfin kololuwa a cikin wannan yanki na furotin.Abin sha'awa, lokacin da muka haxa αS tare da pLK a αS taro na kusan.5-25 µM a gaban polyethylene glycol (5-15% PEG-8) (na al'ada LLPS buffer: 10 mM HEPES pH 7.4, 100 mM NaCl, 15% PEG-8) nan da nan muka shiga cikin fage na gina jiki mai yawa. .An lura da ɗigon ruwa ta hanyar amfani da haske (WF) da haske-filin (BF) microscopy (Fig. 1c).1-5 µm droplets dauke da maida hankali αS (ƙara 1 µM AlexaFluor488-labeled αS, AF488-αS), su electrostatic kaddarorin za a iya samu daga juriya zuwa 10% 1,6-hexanediol (1,6-HD) da hankalinsa zuwa. karuwa a cikin NaCl maida hankali (Fig. 1c).Halin ruwa-kamar yanayin coacervates na αS/pLK electrostatic hadaddun ana nuna su ta hanyar iya haɗawa tsakanin milliseconds (Fig. 1d).Yin amfani da turbidimetry, mun ƙididdige samuwar digo a ƙarƙashin waɗannan yanayi, mun tabbatar da yanayin electrostatic na babban hulɗar da ke da alaƙa da kwanciyar hankali (Fig. 1e), kuma mun kimanta tasirin nau'o'in polymer daban-daban akan tsarin LLPS (Fig. 1f).Ko da yake ana lura da samuwar droplet sama da kewayon ma'auni na polymer, tsarin yana da kyau sosai lokacin da pLK ya wuce αS.Hakanan an lura da LLPs ta hanyar amfani da dextran-70 (70 kDa) na kemikal daban-daban ko amfani da nau'ikan samfura iri-iri, gami da faɗuwar gilashin gilashi, rijiyoyin microplate na abubuwa daban-daban, Eppendorf ko ma'adini capillaries.
wakilcin tsari na yankuna sunadaran daban-daban a cikin bambance-bambancen WT-αS da ΔCt-αS da aka yi amfani da su a cikin wannan binciken.Yankin amphipathic N-terminal, yankin hydrophobic amyloid-forming (NAC), da yankin C-terminal maras kyau ana nuna su cikin shuɗi, orange, da ja, bi da bi.Ana nuna taswirar net Charge Per Residual (NCPR) na WT-αS.b NMR nazarin hulɗar αS / pLK a cikin rashin macromolecular clumps.Yayin da haɓakar pLK ke ƙaruwa (αS: pLK molar rabo na 1:0.5, 1:1.5, da 1:10 ana nuna su cikin haske kore, kore, da duhu kore, bi da bi).c Coacervate αS/pLK (molar rabo 1:10) a 25 µM (1 µM AF488-labeled αS ko Atto647N-labeled pLK don WF imaging) a cikin LLPS buffer (saman) ko ƙari tare da 500 mM NaCl (bayan hagu 1) ko % 1,6-hexanediol (1,6-HD; kasa dama).Ma'aunin Sikeli = 20 µm.d Hotunan Hotuna masu mahimmanci na BF droplet fusion na αS / pLK (molar rabo 1: 10) a maida hankali na 25 μM;Kibiyoyi suna nuna haɗuwar digo ɗaya ɗaya (kibiyoyi masu ja da rawaya) zuwa sabon digo (kiban orange) tsakanin 200 ms) .Ma'aunin Sikeli = 20 µm.e Watsawa mai haske (a 350 nm) αS / pLK tarawa a cikin buffer LLPS kafin da bayan ƙari na 500 mM NaCl ko 10% 1,6-HD a 25 µM αS (N = 3 samfurin kwafi, ma'ana da daidaitattun daidaituwa kuma an nuna).f Hoton BF (saman) da bincike na watsawa mai haske (a 350 nm, kasa) na αS / pLK aggregation a 25 μM αS tare da ƙara αS: pLK molar rabo (N = 3 samfurin kwafi, ma'ana da daidaitattun daidaituwa kuma ya nuna).Ma'aunin Sikeli = 10 µm.Ma'aunin ma'auni a kan hoto ɗaya yana nuna ma'aunin duk hotuna a cikin panel ɗaya.Ana ba da bayanan ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
Dangane da abubuwan da muka lura na αS/pLK electrostatic condensation condensation da kuma abubuwan da suka gabata na αS a matsayin kwayar abokin ciniki na tau/RNA condensate ta hanyar hulɗar kai tsaye tare da tau31, mun ɗauka cewa αS da tau na iya haɗawa tare da sauran ƙarfi idan babu RNA. kumburi.ta hanyar hadaddun wutar lantarki, kuma αS shine furotin na scaffold a cikin αS/Tau coacervates (duba rarraba cajin tau a Hoto 2e).Mun lura cewa lokacin da aka haɗu da 10 μM αS da 10 μM Tau441 (wanda ya ƙunshi 1 μM AF488-αS da 1 μM Atto647N-Tau, bi da bi) a cikin buffer na LLPS, cikin sauƙi suna samar da tarin furotin da ke ɗauke da sunadarai biyu, kamar yadda WF microscopy ya gani.(Hoto na 2 a).An tabbatar da ƙaddamar da ƙwayoyin sunadaran sunadaran guda biyu a cikin ɗigon ruwa ta hanyar microscopy (CF) (Ƙarin Hoto 1a).An lura da irin wannan hali lokacin da aka yi amfani da dextran-70 a matsayin wakili na tarawa (Ƙarin Hoton 1c).Yin amfani da ko dai FITC-labeled PEG ko dextran, mun gano cewa duka masu cunkoson jama'a an rarraba su a ko'ina cikin samfurori, suna nuna ba rabuwa ko ƙungiya (Ƙarin Hoton 1d).Maimakon haka, yana ba da shawarar cewa a cikin wannan tsarin suna haɓaka rarrabuwar lokaci ta hanyar tasirin cunkoson jama'a, tunda PEG wakili ne mai tsayayye na cunkoson jama'a, kamar yadda aka gani a cikin sauran tsarin LLP33,34.Waɗannan ɗigon furotin masu wadatar furotin sun kasance masu kula da NaCl (1 M) amma ba zuwa 1,6-HD (10% v / v), suna tabbatar da kaddarorinsu na lantarki (Ƙarin Hoton 2a, b).An tabbatar da halayensu na ruwa ta hanyar lura da abubuwan da suka faru na haɗe-haɗe na millisecond ta amfani da microscopy BF (Fig. 2b).
Hotunan microscopy na Confocal (CF) na αS/Tau441 yana haɓakawa a cikin buffer LLPS (10 μM na kowane furotin, 0.5 μM na AF488-labeled αS da Atto647N-labeled Tau441).b Wakilin bambancin tsangwama (DIC) hotuna na abubuwan αS/Tau441 droplet fusion events (10 μM ga kowane furotin).c Tsarin tsari dangane da watsawar haske (a 350 nm) na Tau441 LLPS (0-15 µM) a cikin rashi (hagu) ko kasancewar (dama) na 50 µM αS.Launuka masu zafi suna nuna ƙarin watsawa.d Hasken haske na samfuran αS / Tau441 LLPS tare da haɓaka haɓakar αS (Tau441 a 5 µM, N = 2-3 samfurin maimaitawa kamar yadda aka nuna).e wakilcin tsari na wasu bambance-bambancen furotin tau da yankuna daban-daban na furotin da aka yi amfani da su a cikin wannan binciken: yankin N-terminal mara kyau (ja), yanki mai wadatar albarkatu (blue), yankin microtubule-binding (MTBD, wanda aka haskaka a cikin orange), da kuma amyloid-kamar karkace.Yankunan filament (PHF) dake cikin MTBD (launin toka).Ana nuna taswirar Tau441 na Taswirar Net Charge Per Residue (NCPR).Yin amfani da 1 µM AF488-labeled αS da Atto647N-labeled ΔNt-, ta amfani da 1 µM AF488-labeled αS ko ΔCt-αS a gaban ΔNt-Tau (saman, 10 µM da furotin) ko K18 (kasa, 5M da furotin). ))) micrographs na WF wanda aka tattara a cikin LLPS ko K18 buffer.Sandunan ma'auni a cikin hoto ɗaya suna wakiltar ma'aunin duk hotuna a cikin panel ɗaya (20 µm don bangarori a, b da f).Raw data ga bangarori c da d ana bayar da su azaman fayilolin bayanai danye.
Don gwada rawar αS a cikin wannan tsari na LLPS, mun fara bincikar tasirin αS akan kwanciyar hankali na droplet ta hanyar nephelometry ta amfani da ƙara yawan NaCl (Fig. 2c).Mafi girman adadin gishiri a cikin samfuran da ke ɗauke da αS, mafi girman ƙimar watsawar haske (a 350 nm), wanda ke nuna rawar da αS ke da shi a cikin wannan tsarin LLPS.Ana iya lura da irin wannan tasiri ta hanyar ƙara haɓakar αS (saboda haka αS:Tau441 rabo) zuwa kusan.10-ninka karuwa dangane da tau maida hankali (5 µM) (Fig. 2d).Don nuna cewa αS shine furotin mai lalacewa a cikin coacervates, mun yanke shawarar bincika halin LLPS-rushe Tau mutant, wanda ba shi da wani mummunan cajin yankin N-terminal (sauran 1-150, duba Fig. 2e) da ake kira ΔNt-Tau.WF microscopy da nephelometry sun tabbatar da cewa ΔNt-Tau kanta bai sha LLPS ba (Fig. 2f da Ƙarin Hoto 2d), kamar yadda aka ruwaito a baya 14. Duk da haka, lokacin da aka ƙara αS zuwa hanyoyin watsawa na wannan bambance-bambancen Tau, tsarin LLPS ya kasance gaba daya. maidowa tare da ɗigon ɗigon ruwa kusa da ɗigon digo na cikakken girman mafita na Tau da αS a ƙarƙashin yanayi iri ɗaya da ƙididdigar furotin.Hakanan ana iya lura da wannan tsari a ƙarƙashin yanayin ƙarancin cunkoson macromolecular (Ƙarin Hoto 2c).Matsayin yankin C-terminal αS, amma ba duka tsawonsa ba, a cikin tsarin LLPS an nuna shi ta hanyar hana samuwar droplet ta amfani da bambance-bambancen C-terminal truncated αS wanda ba shi da ragowar 104-140 (Fig. 1a) na (ΔCt- αS) furotin (Fig. 2f da Ƙarin Hoto 2d).An tabbatar da haɗin gwiwar αS da ΔNt-Tau ta hanyar microscopy na fluorescence (Ƙarin Hoton 1b).
Don ƙara gwada tsarin LLPS tsakanin Tau441 da αS, an yi amfani da ƙarin bambance-bambancen Tau, wato guntu mai haɗaɗɗiyar helical filament core (PHF) a cikin yankin microtubule-binding (MTBD), wanda idan ya ƙunshi yankuna maimaita halaye huɗu, wanda aka fi sani da shi. kamar guntun K18 (duba siffa 2e).Kwanan nan an ba da rahoton cewa αS ya fi dacewa yana ɗaure da furotin tau da ke cikin yanki mai wadatar proline a cikin jerin da ke gaba da yankin dauri na microtubule.Duk da haka, yankin PHF kuma yana da wadata a cikin ragowar abubuwan da aka caje (duba Hoto 2e), musamman ma lysine (15% residues), wanda ya sa mu gwada ko wannan yanki yana ba da gudummawa ga ƙaddamar da hadaddun αS / Tau.Mun lura cewa K18 kadai ba zai iya haifar da LLPS a ƙididdigewa har zuwa 100 μM a ƙarƙashin yanayin da aka gwada (LLPS buffer tare da 15% PEG ko 20% dextran) (Hoto 2f).Duk da haka, lokacin da muka ƙara 50 µM αS zuwa 50 µM K18, saurin samuwar furotin da ke dauke da K18 da αS an lura da su ta hanyar nephelometry (Ƙarin Hoton 2d) da WF microscopy (Fig. 2f).Kamar yadda aka zata, ΔCt-αS ya kasa dawo da halin LLPS na K18 (Fig. 2f).Mun lura cewa tarawar αS / K18 yana buƙatar ƙananan ƙwayoyin sunadaran sunadaran don haifar da LLPS idan aka kwatanta da αS / ΔNt-Tau ko αS / Tau441, wasu abubuwa daidai suke.Wannan ya dace da haɗin gwiwa mai ƙarfi na yankin αS C-terminal tare da yankin Tau mai arzikin proline idan aka kwatanta da yankin microtubule-binding, kamar yadda aka bayyana a baya 31.
Ganin cewa ΔNt-Tau ba zai iya yin LLPS ba idan babu αS, mun zaɓi wannan bambance-bambancen Tau azaman samfuri don siffanta αS/Tau LLPS da aka ba da sauƙi a cikin tsarin LLPS tare da cikakken Tau (isotype, Tau441/Tau441).tare da hadaddun (heterotypic, αS/Tau441) matakan tarawa.Mun kwatanta matakin haɗin αS (a matsayin wani ɓangare na furotin na zamani, fαS, c) a cikin tsarin αS / Tau da αS / ΔNt-Tau ta hanyar centrifugation da tarwatsa lokaci SDS-PAGE bincike (duba 2e), sami kamancen dabi'u. ga duk sunadaran a daidai taro.Musamman, mun sami fαS, c 84 ± 2% da 79 ± 7% don αS / Tau da αS / ΔNt-Tau, bi da bi, yana ba da shawarar cewa hulɗar heterotypic tsakanin αS da tau ya fi dacewa da hulɗar tsakanin kwayoyin tau.tsakanin.
An fara nazarin hulɗar tare da polycations daban-daban da kuma tasirin tsarin daɗaɗɗa a kan αS motsin rai ta hanyar farfadowa da haske bayan hanyar photobleaching (FRAP).Mun gwada αS/Tau441, αS/ΔNt-Tau da αS/pLK coacervates (100 μM αS da 2 μM αS AF488-αS da 100 μM Tau441 ko ΔNt-Tau ko 1 mM pLK).An samo bayanai a cikin mintuna 30 na farko bayan haxa samfuran samfuran.Daga Hotunan FRAP masu wakilci (Fig. 3a, αS / Tau441 condensation) da kuma daidaitattun lokutan lokaci (Fig. 3b, Ƙarin Hoto 3), ana iya ganin cewa αS kinetics suna kama da na Tau441 coacervates.da ΔNt-Tau, wanda ya fi sauri tare da pLK.Ƙididdigar ƙididdigar ƙididdiga don αS a cikin coacervate bisa ga FRAP (kamar yadda Kang et al. 35 ya bayyana) sune D = 0.013 ± 0.009 µm2 / s da D = 0.026 ± 0.008 µm2 / s don αS / Tau441 da αS / Tau441 αS/ tsarin.pLK, Tau, da D = 0.18 ± 0.04 µm2/s, bi da bi (Fig. 3c).Koyaya, αS mai rarrabawa a cikin lokacin da aka watse yana da umarni da yawa na girma sama da duk matakan da aka haɗa, kamar yadda Fluorescence Correlation Spectroscopy ya ƙaddara (FCS, duba ƙarin siffa 3) ƙarƙashin yanayi iri ɗaya (LLPS buffer) amma idan babu polycations. (D = 8 ± 4 µm2/s).Saboda haka, motsin motsi na fassarar αS yana raguwa sosai a cikin coacervates idan aka kwatanta da sunadaran a cikin tarwatsewar lokaci saboda fa'idodin cunkoson kwayoyin halitta, kodayake duk coacervates suna riƙe da kaddarorin masu kama da ruwa a farkon rabin sa'a bayan samuwar su, sabanin lokacin tau.sauri motsi a cikin pLK condensate.
a-c FRAP bincike na αS kuzari (2% AF488-labeled αS) a cikin coacervates electrostatic.Hotunan wakilci na ƙididdigar αS/Tau441 FRAP a cikin nau'i uku ana nuna su a cikin (a), inda jajayen da'irar ke nuna wuraren da aka canza launin.Tsawon sikelin shine 5 µm.b Matsakaicin FRAP masu lanƙwasa da (c) ƙididdiga ƙididdiga masu rarrabawa (D) don 5-6 (N) ɗigogi daban-daban daga gwaje-gwaje uku ta amfani da 100 µM αS da ƙididdigar daidaitattun Tau441 (ja) ko ΔNt-Tau (blue) ko pLK (kore) a sau goma maida hankali na LLPS.Madaidaicin karkata na FRAP yana nunawa a cikin launi mai inuwa.Don kwatankwacin, ƙimar αS na watsawa a cikin lokacin da aka tarwatse an ƙaddara ta sau uku ta amfani da yanayin daidaita yanayin haske (FCS) (duba Ƙarin Hoto 3 da hanyoyin don ƙarin bayani).d Ci gaba na X-band EPR spectra na 100 μM TEMPOL-122-αS a cikin buffer LLPS ba tare da wani polycation (baƙar fata) ko a gaban 100 μM Tau441 (ja) ko ΔNt-Tau (blue) ko 1 mM pLK (kore).Saitin yana nuna ƙaƙƙarfan ra'ayi na layukan filin filin inda mafi girman sauye-sauye ke faruwa.e Ƙwaƙwalwar ɗaure na 50 μM TEMPOL-122-αS tare da polycations daban-daban in babu LLPS (babu PEG).Rage girman girman band III idan aka kwatanta da band II (IIII/III) na daidaitaccen bakan EPR an nuna shi don ƙara ƙimar molar tau441 (ja), ΔNt-Tau (blue) da pLK (kore).Launuka masu launi suna nuna dacewa da bayanai ta amfani da ƙirar ɗauri mai ƙaƙƙarfan tsari tare da n daidai da wuraren ɗaurin kai akan kowane lanƙwasa.Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
A matsayin ƙarin, mun bincika haɓakar αS a cikin coacervates daban-daban ta yin amfani da lakabin jujjuyawar kai tsaye (SDSL) da ci gaba da resonance paramagnetic electron (CW-EPR).Wannan hanya ta tabbatar da cewa tana da fa'ida sosai wajen bayar da rahoton sassauƙa da sauye-sauye na IDP tare da tabbataccen saura ƙuduri36,37,38.Don wannan karshen, mun gina ragowar cysteine ​​​​a cikin ƙwayoyin Cys guda ɗaya kuma mun yi amfani da bincike na 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL).Abubuwan Maleimide suna lakafta su.Musamman ma, mun saka TEMPOL bincike a matsayi na 122 ko 24 αS (TEMPOL-122-αS da TEMPOL-24-αS).A cikin shari'ar farko, mun yi niyya ga yankin C-terminal na furotin, wanda ke cikin hulɗa tare da polycations.Madadin haka, matsayi na 24 zai iya ba mu bayani game da gabaɗayan haɓakar sunadaran da ke cikin condensate.A cikin duka biyun, siginar EPR da aka samu don sunadaran da aka tarwatsa sun yi daidai da radicals nitroxide a cikin yanayin motsi mai sauri.Bayan rabuwa na lokaci a gaban tau ko pLK (100 μM TEMPOL-αS, Tau441 ko ΔNt-Tau a wani rabo na 1: 1 ko pLK a cikin rabo na 1: 10), an sami karuwa a cikin girman girman dangi. Farashin EPR na αS.Layin asara ya faɗaɗa, yana nuna raguwar αS reorientation kinetics a cikin ɗigon ruwa idan aka kwatanta da furotin a cikin tsawan lokaci (Fig. 3d, Ƙarin Hoto. 4a).Wadannan canje-canje sun fi bayyana a matsayi na 122. Yayin da yake a matsayi na 24 kasancewar pLK bai shafi kinetics na bincike ba, a matsayi na 122 siffar layin layi ya canza sosai (Ƙarin Hoto 4a).Lokacin da muka yi ƙoƙarin yin ƙirar ƙirar a matsayi na 122 na tsarin αS / polycation guda biyu ta amfani da samfurin isotropic (Ƙarin Hoto 5a) da aka saba amfani da shi don kwatanta ƙarfin hali na IDP38,39 mai lakabin layi, ba mu sami ikon sake gina fasalin gwaji ba..Simulation Spectral na matsayi na 24 spins ya bambanta (Ƙarin Hoto 5a).Wannan yana nuna cewa akwai fifikon matsayi a cikin sarari na daidaitawar juzu'i na yankin C-terminal na αS a gaban polycations.Lokacin la'akari da juzu'in αS a cikin lokaci mai ƙarfi a ƙarƙashin yanayin EPR na gwaji (84 ± 2%, 79 ± 7%, da 47 ± 4% don αS/Tau441, αS/ΔNt-Tau, da αS/pLK, bi da bi-duba Ƙarin Hoton 2e na nazarin bayanan c), ana iya ganin cewa fadadawa da aka gano ta hanyar EPR ya fi nuna ma'amalar yankin C-terminal na αS tare da polycations daban-daban a cikin lokaci mai mahimmanci (babban canji lokacin amfani da TEMPOL-122- αS), kuma ba gurɓataccen furotin ba.Ana lura da haɓaka microviscosity a cikin binciken.Kamar yadda aka zata, an sake dawo da nau'in EPR na furotin a ƙarƙashin yanayi ban da LLPS gaba ɗaya lokacin da aka ƙara 1 M NaCl zuwa gaurayawan (Ƙarin Hoton 4b).Gabaɗaya, bayananmu suna ba da shawarar cewa canje-canjen da CW-EPR ta gano sun fi nuna ma'amalar yankin C-terminal na αS tare da polycations daban-daban a cikin lokaci mai ƙarfi, kuma wannan hulɗar ta bayyana tana da ƙarfi tare da pLK fiye da Tau.
Domin samun ƙarin bayanan tsarin game da sunadaran da ke cikin coacervate, mun yanke shawarar yin nazarin tsarin LLPS ta amfani da NMR a cikin bayani.Koyaya, zamu iya gano juzu'in αS ɗin da ya rage a cikin lokacin tarwatsewa, wanda zai iya kasancewa saboda rage ƙarfin furotin a cikin coacervate da wani lokaci mai yawa a ƙasan mafita a cikin binciken NMR.Lokacin da muka bincika tsari da haɓakar furotin da suka ragu a cikin tarwatsa lokaci na samfurin LLPS ta amfani da NMR (Ƙarin Hoton 5c, d), mun lura cewa furotin ya yi kusan daidai a gaban pLK da ΔNt-Tau, duka biyu na waɗanda ke cikin tsari na biyu da haɓakar kashin bayan furotin, wanda aka bayyana ta hanyar gwaje-gwaje akan canjin sinadarai na biyu da shakatawa R1ρ.Bayanan NMR sun nuna cewa C-terminus na αS yana fama da hasara mai yawa na sassaucin ra'ayi yayin da yake riƙe da yanayin rashin lafiyarsa, kamar sauran jerin furotin, saboda mu'amalarsa da polycations.
Tun da faɗaɗa siginar CW-EPR da aka gani a cikin TEMPOL-122-αS ɗin da aka haɗe yana nuna ma'amalar furotin tare da polycations, mun yi titration na EPR don kimanta alaƙar alaƙar αS zuwa polycations daban-daban in babu LLPS (ba tarawa ba. Buffer LLPS), yana ba da shawarar cewa hulɗar iri ɗaya ce a cikin matakai masu nisa da tattarawa (wanda aka tabbatar da bayananmu, Ƙarin Hoto 4a da Ƙarin Hoto 6).Manufar ita ce a ga ko duk suna ba da haɗin kai, duk da kaddarorinsu na gama gari irin na ruwa, suna nuna kowane hali na bambance-bambance a matakin ƙwayoyin cuta.Kamar yadda aka zata, bakan EPR ya faɗaɗa tare da ƙara yawan maida hankali na polycation, yana nuna raguwa a cikin sassaucin kwayoyin halitta saboda hulɗar kwayoyin halitta na duk abokan hulɗar kusan zuwa jikewa (Fig. 3e, Ƙarin Hoto 6).pLK ya sami wannan jikewa a ƙaramin rabo na molar (polycation: αS) idan aka kwatanta da ΔNt-Tau da Tau441.A zahiri, kwatanta bayanan tare da ƙirar ɗaurin ƙima wanda aka ɗauka n daidai da wuraren ɗauri masu zaman kansu sun nuna cewa rarrabuwar kai tsaye na pLK (~ 5 μM) tsari ne na girma ƙasa da na Tau441 ko ΔNt-Tau (~ 50 μM). ).µM).Ko da yake wannan ƙididdigewa ne, wannan yana nuna cewa αS yana da alaƙa mafi girma don mafi sauƙi polycations tare da ci gaba da ingantaccen cajin yankuna.Ganin wannan bambance-bambance na kusanci tsakanin αS da polycations daban-daban, mun ɗauka cewa kayan ruwan su na iya canzawa daban-daban akan lokaci kuma don haka suna fama da matakai daban-daban na LSPT.
Ganin yanayin cunkoson jama'a a cikin furotin coacervate da yanayin amyloid na furotin, mun lura da halayen coacervate na tsawon lokaci don gano yiwuwar hanyoyin LSPT.Yin amfani da BF da CF microscopy (Hoto 4), mun lura cewa αS / Tau441 yana haɓakawa da yawa a cikin bayani, yana samar da manyan ɗigon ruwa waɗanda ke tuntuɓar da rigar saman ƙasa a ƙasan rijiyar / zamewa azaman cikakkun ɗigon ruwa, kamar yadda ake tsammani (Ƙarin Fig. ku 7d);muna kiran waɗannan sifofin da aka kafa a ƙasa "protein rafts".Waɗannan sifofin sun kasance masu ruwa yayin da suke riƙe da ikon haɗawa (Ƙarin Hoton 7b) kuma ana iya ganin su na sa'o'i da yawa bayan an kunna LLPS (Fig. 4 da Ƙarin Hoto 7c).Mun lura da cewa wetting tsari da aka yi falala a kan surface na hydrophilic maimakon hydrophobic kayan (Karin siffa. 7a), kamar yadda sa ran ga electrostatic coacervates tare da m zargin da haka high electrostatic surface m.Musamman ma, αS / ΔNt-Tau coalescence da rafting sun ragu sosai, yayin da αS / pLK condensates sun ragu sosai (Fig. 4).A cikin ɗan gajeren lokacin shiryawa, ɗigon αS / pLK sun sami damar haɗuwa da jika saman hydrophilic, amma wannan tsari ya tsaya da sauri kuma bayan sa'o'i 5 na incubation, ƙayyadaddun abubuwan haɗin gwiwa kawai kuma ba a lura da wetting ba.- canjin gel-drip.
Wakilin BF (bankunan greyscale) da CF (bankunan dama, AF488-labeled αS a cikin kore) na samfuran coacervate dauke da 100 µM αS (lakabin kyalli na 1%) a cikin buffer LLPS a gaban 100 µM Tau441 (ΔN) hotuna masu kyan gani. -Tau (tsakiya) ko 1 mM pLK (kasa) a lokuta daban-daban na shiryawa da tsayi mai tsayi (z, nisa daga kasan farantin rijiyar).An maimaita gwaje-gwajen sau 4-6 ba tare da juna ba tare da sakamako iri ɗaya.αS/Tau441 coacervates ana jika bayan sa'o'i 24, suna samar da rafts girma fiye da hoton.Matsakaicin ma'auni don duk hotuna shine 20µm.
Sai muka tambayi ko manyan wuraren tafkunan sunadaran sunadaran ruwa da aka kafa a cikin αS/Tau441 LLPS zasu haifar da amyloid aggregation na kowane sunadaran da aka yi nazari.Mun bi maturation na αS / Tau441 droplets a kan lokaci tare da WF microscopy a karkashin yanayi guda kamar yadda na sama, amma ta amfani da 1 μM AF488-labeled αS da Atto647N-labeled Tau441 (Fig. 5a).Kamar yadda aka sa ran, mun lura da cikakken kasancewar sunadaran sunadaran a cikin tsarin balaga.Abin sha'awa, daga ca.Bayan sa'o'i 5, an lura da tsarin da ba na madauwari ba a cikin rafts, wanda muka kira "maki", wasu daga cikinsu sun kasance tare da αS, kuma wasu sun wadata a Tau441 (Fig. 5a, fararen kiban).Ana lura da waɗannan tabo koyaushe a cikin rafts zuwa mafi girma ga αS/ΔNt-Tau fiye da na αS/ΔNt-Tau.Babu tabo daban-daban a cikin ɗigon ɗigon pLK da tsarin Tau waɗanda ba su iya haɗawa da jikewa ba.Don gwada ko waɗannan tabo masu ɗauke da αS da Tau441 sune amyloid-kamar aggregates, mun yi irin wannan gwaji ta amfani da CF microscopy wanda aka yiwa Tau441 lakabi da Atto647N da 12.5 μM amyloid-specific thioflavin-T (ThT) an ƙara daga farkon.rini.Ko da yake ThT-tabon αS / Tau441 droplets ko rafts ba a lura ba ko da bayan 24 h na shiryawa (Fig. 5b, saman jere-sauran droplets a kan furotin rafts), ThT-tabbatacce Tsarin da ke dauke da Atto647N-Tau441 a cikin rafts suna da rauni sosai.wannan yana maimaita girman, siffar, da wuri na wuraren da aka bayyana a baya (Fig. 5b, layuka na tsakiya da na kasa), yana nuna cewa spots na iya dacewa da amyloid-kamar aggregates da aka kafa a cikin tsufa ruwa coacervates.
WF 25 μM αS a lokuta daban-daban na shiryawa da tsayi mai tsayi (z, nisa daga ƙasa mara tushe) a gaban 25 μM Tau441 (1 μM AF488-labeled αS da Atto647N-labeled Tau441) a cikin rijiyar farantin microscope tare da buffer LLPS) .An maimaita gwaje-gwaje shida da kansa tare da sakamako iri ɗaya.b CF hoton microscopic na 25 μM αS a gaban 25 μM Tau441 (1 μM Atto647N-labeled Tau441) da 12.5 μM thioflavin-T (ThT).Ana nuna ɗigon furotin da aka auna da rafts ɗin furotin da aka ajiye a cikin layuka na sama da na tsakiya, bi da bi.Layin ƙasa yana nuna hotunan rafts da faɗuwa daga kwafi masu zaman kansu 3.Fararen kibau suna nuna ɗigo masu inganci na ThT a cikin bangarorin biyu.Matsakaicin ma'auni don duk hotuna shine 20µm.
Don bincika dalla-dalla sauye-sauye a cikin hanyar sadarwar furotin na coacervate yayin sauyawa daga ruwa zuwa ƙarfi, mun yi amfani da hoton rayuwa mai haske (FLIM) da Förster resonance energy transfer microscopy (FRET) (Hoto na 6 da Ƙarin Figures 8 da 9).Mun yi hasashe cewa coacervate maturation na Layer zuwa mafi natsuwa ko ma mai ƙarfi-kamar tsarin gina jiki mai ƙarfi yana haifar da kusanci tsakanin furotin da bincike mai kyalli da ke haɗe da shi, mai yuwuwar samar da tasirin kashewa wanda ke bayyana a cikin gajeriyar binciken rayuwa (τ) , kamar yadda aka bayyana a baya 40.,41,42.Bugu da ƙari, don samfurori masu alamar sau biyu (AF488 da Atto647N a matsayin mai ba da gudummawar FRET da dyes masu karɓa, bi da bi), wannan raguwa a cikin τ kuma yana iya kasancewa tare da coacervate condensation da karuwa a cikin FRET (E) dacewa a lokacin LSPT.Mun lura da raft da tabo samuwar lokaci a cikin LLPS αS/Tau441 da αS/ΔNt-Tau samfurori (25 µM na kowane furotin a cikin buffer LLPS dauke da 1 µM AF488 mai suna αS da / ko Atto647N mai lakabi Tau441 ko ΔNt-Tau).Mun lura da yanayin gaba ɗaya a cikin cewa hasken rayuwa na AF488 (τ488) da Atto647N (τ647N) binciken ya ragu kaɗan yayin da coacervates balagagge (Fig. 6 da Ƙarin siffa 8c).Abin sha'awa, an inganta wannan canji sosai don ɗigogi a cikin rafi (Fig. 6c), yana nuna cewa ƙarin haɓakar furotin ya faru a ɗigogi.Don goyan bayan wannan, ba a sami wani muhimmin canji a cikin rayuwa mai haske ba don αS / ΔNt-Tau droplets shekaru 24 h (Ƙarin Hoto 8d), yana nuna cewa droplet gelation wani tsari ne da ya bambanta da tabo kuma wannan ba ya tare da gagarumin sake fasalin kwayoyin halitta. cikin coacervates.Ya kamata a lura cewa ɗigon suna da nau'i daban-daban da kuma abun ciki mai mahimmanci a cikin αS, musamman ga tsarin αS / Tau441 (Ƙarin Hoton 8e).Ragewar a cikin rayuwa mai haske yana tare da haɓaka mai ƙarfi, musamman ga Atto647N mai suna Tau441 (Ƙarin Hoto 8a), da mafi girman ingancin FRET don duka tsarin αS/Tau441 da αS/ΔNt-Tau, yana nuna ƙarin haɓakawa a cikin LLPS Five hours. bayan kunnawa, sunadaran da ke cikin wutar lantarki a tsaye sun takure.Idan aka kwatanta da αS/ΔNt-Tau, mun lura da ƙananan τ647N da ɗan ƙaramin darajar τ488 a cikin αS/Tau441, tare da ƙananan ƙimar FRET marasa daidaituwa.Yiwuwa, wannan yana iya kasancewa da alaƙa da gaskiyar cewa a cikin tsarin αS / Tau441, abubuwan da aka lura da kuma tsammanin αS yawa a cikin tarawa sun fi yawa, sau da yawa substoichiometric idan aka kwatanta da Tau, tunda Tau441 da kanta na iya jurewa LLPS da tarawa (Ƙarin Fig. 8e) .Koyaya, matakin coalescence na droplet, haɓakar raft, kuma, mahimmanci, haɓakar furotin a cikin abubuwan haɗin ruwa-kamar coacervates yana da girma lokacin da duka Tau441 da αS suke.
Hotunan αS/Tau441 da αS/ΔNt-Tau a 25 μM na kowane furotin (1 μM AF488-labeled αS da 1 μM Atto647N-labeled Tau441 ko ΔNt-Tau) a cikin LLPS.ginshiƙan suna nuna hotunan wakilci na samfuran LLPS a lokuta daban-daban na girma (minti 30, 5 h da 24 h).Firam ɗin ja yana nuna yankin mai ɗauke da tabo αS/Tau441.Ana nuna tsawon rayuwa azaman sanduna masu launi.Ma'aunin Sikeli = 20 µm ga duk hotuna.b Hoton FLIM mai zuƙowa na yankin da aka zaɓa, wanda aka nuna a cikin akwatin ja a cikin panel a.Ana nuna kewayon rayuwa ta amfani da ma'aunin launi iri ɗaya kamar na panel a.Ma'aunin Sikeli = 5 µm.c Histograms da ke nuna AF488 (wanda aka haɗa da αS) ko Atto647N (wanda aka haɗa da Tau) don nau'ikan furotin daban-daban ( droplets-D-, raft-R- da speckle-P) waɗanda aka gano a cikin hotunan FLIM da aka yi rikodin don αS-) lokacin rarraba lokacin rayuwar Tau441 da αS/ΔNt-Tau samfuran coacervate (N = 17-32 ROI don D, 29-44 ROI don R, da 21-51 ROI don maki).Ana nuna ma'ana da matsakaiciyar ƙima azaman murabba'in rawaya da layukan baƙi a cikin kwalaye, bi da bi.Iyakoki na ƙasa da na sama na akwatin suna wakiltar jigon farko da na uku, bi da bi, kuma mafi ƙanƙanta da matsakaicin ƙima a cikin kewayon tsaka-tsaki mai ninki 1.5 (IQR) ana nuna su azaman wutsiya.Ana nuna abubuwan waje azaman baƙar fata lu'u-lu'u.An ƙayyade mahimmancin ƙididdiga tsakanin nau'i-nau'i na rarrabawa ta amfani da samfurin t-gwajin guda biyu, yana ɗaukan bambance-bambance marasa daidaituwa.Ana nuna ƙimar p-gwaji biyu tare da asterisks ga kowane nau'in bayanan da aka kwatanta (* p-darajar> 0.01, ** p-darajar> 0.001, *** p-darajar> 0.0001, **** p-darajar> 0.00001), ns Yana Nuna sakaci (p-darajar> 0.05).Ana ba da madaidaicin ƙimar p a cikin ƙarin Teburin 1, kuma ana gabatar da ainihin bayanan azaman fayilolin ɗanyen bayanai.
Don ci gaba da nuna yanayin amyloid-kamar speckles / aggregates, mun bi da samfurori na coacervate maras kyau na tsawon sa'o'i 24 tare da babban taro na (1 M) NaCl, wanda ya haifar da rabuwa da tarawa daga furotin coacervates.Lokacin da aka lura da abubuwan da aka keɓe (watau bayani mai tarwatsewa na aggregates) ta amfani da microscopy mai ƙarfi na atomic (AFM), mun lura da yanayin yanayin yanayin yanayin yanayin yanayin yanayi mai tsayi na yau da kullun na kusan 15 nm, wanda ke da alaƙa da haɗuwa a ƙarƙashin yanayin babban taro na gishiri, kama da halayen fibrils na amyloid na yau da kullun saboda tasirin hydrophobic mai ƙarfi akan farfajiya (lura cewa fibrils yawanci suna da tsayin ~ 10 nm) (Ƙarin Hoto 10a).Abin sha'awa shine, lokacin da aka keɓance haɗin kai tare da ThT a cikin ma'auni na ThT fluorescence assay, mun lura da karuwa mai girma a cikin ThT fluorescence quantum yawan amfanin ƙasa, kwatankwacin abin da aka lura lokacin da aka sanya rini tare da fibrils αS amyloid na al'ada (Ƙarin Hoton 10b), yana ba da shawarar cewa. tarin coacervate sun ƙunshi sifofi masu kama da amyloid..A gaskiya ma, abubuwan da aka tara sun kasance masu jurewa ga yawan gishiri mai yawa amma suna kula da 4 M guanidine chloride (GdnHCl), kamar fibrils amyloid na al'ada (Ƙarin Fig. 10c).
Bayan haka, mun yi nazarin abubuwan da ke tattare da tarawa ta hanyar amfani da kyalli guda ɗaya, ƙayyadaddun yanayin daidaitawar haske / giciye-daidaitawar spectroscopy (FCS/FCCS), da fashe bincike na gano daidaituwar launi biyu (TCCD).Don wannan karshen, mun keɓe tarin abubuwan da aka kafa bayan sa'o'i 24 na shiryawa a cikin samfuran 100 μl LLPS da ke ɗauke da αS da Tau441 (duka 25 μM) tare da 1 μM AF488-labeled αS da 1 μM Atto647N-labeled Tau441.Rarraba sakamakon tarwatsewar jimlar bayani zuwa yanayin monomolecular ta amfani da buffer mara amfani da PEG iri ɗaya da 1 M NaCl (makullin buffer iri ɗaya da ake amfani da shi don raba tarawa daga coacervate) don hana duk wata hulɗar lantarki tsakanin LLPS da furotin.Ana iya ganin misalin yanayin lokaci na kwayoyin halitta guda a cikin siffa 7a.FCCS/FCS bincike (giciye-daidaitacce, CC da autocorrelation, AC) ya nuna cewa aggregates dauke da αS da tau sun kasance mai yawa a cikin samfurori (duba CC lankwasa a cikin siffa 7b, hagu panel), da kuma wuce haddi na saura monomeric furotin ya tashi a matsayin sakamakon tsarin dilution (duba AC masu lanƙwasa a hoto na 7b, ɓangaren hagu).Gwaje-gwajen sarrafawa da aka yi a ƙarƙashin yanayin bayani guda ɗaya ta amfani da samfurori da ke dauke da sunadaran monomeric kawai ba su nuna alamun CC ba, kuma AC masu lankwasa sun dace da kyau tare da nau'i na nau'i na nau'i guda ɗaya (Eq. 4), inda sunadaran monomeric suna da matakan da ake sa ran watsawa (Fig. 7b). ), panel na dama).Matsakaicin adadin abubuwan da aka tara bai wuce 1 µm2/s ba, kuma na furotin monomeric kusan 1 µm2/s.50-100 µm/s;dabi'u sunyi kama da dabi'un da aka buga a baya don sonicated αS amyloid fibrils da monomeric αS daban a ƙarƙashin yanayin bayani iri ɗaya44.Lokacin da muka yi nazarin abubuwan da aka tattara tare da ƙididdigar fashewar TCCD (Fig. 7c, babban panel), mun gano cewa a cikin kowane yanki mai mahimmanci (αS / Tau heteroaggregate), game da 60% na abubuwan da aka gano sun ƙunshi duka αS da tau, game da 30% ya ƙunshi kawai. tau, kusan 10% αS kawai.Binciken Stoichiometric na αS / Tau heteroaggregates ya nuna cewa yawancin heteroaggregates an wadatar da su a cikin tau (stoichiometry da ke ƙasa 0.5, matsakaicin adadin ƙwayoyin tau a kowace tarawa shine sau 4 fiye da ƙwayoyin αS), wanda ya dace da aikinmu da aka lura a cikin FLIM a cikin wurin. gwaje-gwaje..Binciken FRET ya nuna cewa waɗannan tarin sun ƙunshi sunadarai biyu, kodayake ainihin ƙimar FRET a cikin wannan yanayin ba su da mahimmanci, tun da rarraba fluorophores a cikin kowane tarawa ya kasance bazuwar saboda wuce haddi na furotin da ba a yi amfani da su ba a cikin gwajin.Abin sha'awa, lokacin da muka yi bincike iri ɗaya ta amfani da 45,46 balagagge amyloid aggregation-rauni Tau bambance-bambancen (duba Ƙarin Fig. 11a,b), mun lura cewa ko da yake αS electrostatic aggregation ya kasance iri ɗaya (Ƙarin Fig. 11c, d), ikon samar da tara a cikin coacervate ya ragu sosai kuma FLIM ta gano tabo da yawa a cikin gwaje-gwajen wurin, kuma an lura da raƙuman haɗin giciye don keɓantaccen samfuran samfuran.Koyaya, don ƙaramin adadin abubuwan da aka gano (kashi ɗaya cikin goma na Tau441), mun lura cewa kowane jimlar an wadatar da shi a cikin αS fiye da wannan bambance-bambancen Tau, tare da kusan 50% na abubuwan da aka gano waɗanda ke ɗauke da ƙwayoyin αS kawai, kuma αS ya kasance iri-iri fiye da kima. .aggregates (duba Ƙarin Hoto 11e), ya bambanta da nau'i-nau'i daban-daban da Tau441 ya haifar (Fig. 6f).Sakamakon waɗannan gwaje-gwajen ya nuna cewa ko da yake αS kanta yana iya tarawa tare da tau a cikin coacervate, tau nucleation ya fi dacewa a karkashin waɗannan yanayi, kuma sakamakon amyloid-like aggregates suna iya yin aiki a matsayin nau'i na αS da tau.Duk da haka, da zarar an samar da ma'aunin tau-rich core, ana fifita mu'amala tsakanin αS da tau a cikin jimillar mu'amalar homotypic tsakanin kwayoyin tau;muna kuma lura da hanyoyin sadarwar furotin a cikin ruwa αS/tau coacervates.
Alamun haske mai wakilci na ɗan lokaci na kwayoyin halitta guda ɗaya na keɓantattun tarin da aka kafa a cikin αS/Tau441 electrostatic coacervates.Fashe daidai da αS/Tau441 coaggregates (fashe sama da alamar kofa) an lura a cikin uku gano tashoshi (AF488 da Atto647N watsi bayan kai tsaye tashin hankali, blue da ja Lines, Atto647N watsi bayan kai tsaye tashin hankali), FRET, violet line).b FCS/FCCS bincike na samfurin αS/Tau441 keɓe da aka samu daga LLPS (fashin hagu).Ana nuna masu lanƙwasa ta atomatik (AC) don AF488 da Atto647N da shuɗi da ja, bi da bi, kuma masu lanƙwasa-daidaitacce (CC) masu alaƙa da tarawa masu ɗauke da rinayen biyu ana nuna su da shunayya.Wuraren AC suna nuna kasancewar nau'in nau'in furotin da aka yiwa lakabi da monomeric, yayin da CC masu lankwasa suna nuna kawai yaduwa na tara masu lakabi biyu.Binciken iri ɗaya, amma a ƙarƙashin yanayin mafita iri ɗaya kamar a cikin wuraren da aka keɓe, samfuran da ke ɗauke da monomeric αS da Tau441 kawai ana nuna su azaman masu sarrafawa a cikin madaidaicin panel.c Fluorescence flash bincike na kwayoyin halitta guda ɗaya na keɓaɓɓen aggregates da aka kafa a cikin αS/Tau441 electrostatic coacervates.Bayani ga kowane jimlar da aka samu a cikin maimaitawa daban-daban guda huɗu (N = 152) an ƙirƙira su ne a kan stoichiometry, ƙimar S, da ingancin FRET (samfurin saman, mashaya launi yana nuna abin da ya faru).Za'a iya rarrabe nau'ikan tarin yawa: -αs-kawai tara tare da s ~ 1 da kuma fret na uku tare da s da kuma heterogeneous ta matsakaici s da kuma kimanin kimanin adadin na dukkanin sunadaran alamomin da aka gano a cikin kowane nau'i mai nau'i (N = 100) ana nuna su a cikin ƙananan panel (ma'auni na launi yana nuna abin da ya faru).Ana ba da albarkatun ɗanyen bayanai a cikin nau'in fayilolin ɗanyen bayanai.
Balagagge ko tsufa na furotin na ruwa yana haɗuwa a cikin gel-kamar ko tsattsauran sifofi na tsawon lokaci an ba da rahoton cewa yana shiga cikin ayyuka da yawa na physiological na condensate47 da kuma a cikin cututtuka, a matsayin wani tsari mara kyau wanda ya rigaya amyloid aggregation 7, 48, 49. Anan muna nazarin rabuwa da hali daki-daki.LSPT αS a gaban polycations na bazuwar a cikin yanayi mai sarrafawa a ƙananan ƙananan micromolar da kuma yanayin da ya dace da ilimin lissafi (lura cewa ƙididdige ƙididdigar ilimin lissafin jiki na αS shine> 1 µM50), bin dabi'un halayen yanayin zafi na LPS.Mun gano cewa αS, wanda ya ƙunshi wani babban cajin C-terminal yankin a pH na ilimin lissafi, yana iya samar da ɗigon furotin mai wadatar furotin a cikin maganin ruwa ta hanyar LLPS a gaban manyan peptides marasa ƙarfi kamar pLK ko Tau ta hanyar hanyar lantarki. hadaddun condensation a gaban aggregation macromolecules.Wannan tsari na iya samun tasirin da ya dace a cikin yanayin salon salula inda αS ta ci karo da ƙwayoyin polycationic daban-daban waɗanda ke da alaƙa da haɗakar cutar ta duka a cikin vitro da vivo51,52,53,54.
A cikin binciken da yawa, an yi la'akari da ƙarfin furotin a cikin ɗigon ruwa a matsayin ɗayan mahimman abubuwan da ke ƙayyade tsarin balaga55,56.A cikin electrostatic αS coacervates tare da polycations, tsarin balaga a fili ya dogara da ƙarfin hulɗa tare da polycations, valence, da yawa na waɗannan hulɗar.Ka'idar ma'auni ta nuna cewa ma'auni mai faɗi na jihohin ruwa biyu zai kasance kasancewar babban ɗigon ruwa mai wadata a cikin biopolymers wanda ke motsa LLPS57,58.Za a iya samun ci gaban digo ta hanyar Ostwald maturation59, coalescence60 ko amfani da monomer kyauta a cikin watsewar lokaci61.Don αS da Tau441, ΔNt-Tau ko pLK, yawancin furotin an tattara su a cikin condensate a ƙarƙashin yanayin da aka yi amfani da su a cikin wannan binciken.Duk da haka, yayin da cikakken girman tau droplets coalesced da sauri a kan saman jika, droplet coalescence da wetting sun kasance da wahala ga ΔNt-Tau da pLK, yana nuna saurin asarar kayan ruwa a cikin waɗannan tsarin guda biyu.Dangane da binciken mu na FLIM-FRET, tsofaffin pLK da ΔNt-Tau droplets sun nuna nau'in nau'in haɓakar furotin (irin wannan lokacin rayuwa mai haske) azaman ɗigon asali na asali, yana ba da shawarar cewa cibiyar sadarwar furotin ta asali ta kasance, kodayake ta fi tsayi.
Muna daidaita sakamakon gwajin mu a cikin samfurin mai zuwa (Hoto 8).Abubuwan da aka fara na ɗan lokaci na ɗan lokaci galibi su ne cibiyoyin sadarwa na furotin ba tare da ramuwa ta lantarki ba, don haka akwai wuraren rashin daidaituwar caji, musamman a wurin ɗigon ruwa, yana haifar da ɗigon ruwa tare da yuwuwar yuwuwar wutar lantarki.Don rama cajin (wani al'amari da aka fi sani da valence depletion) da kuma rage girman yuwuwar ɗigon ruwa, ɗigon ruwan na iya haɗawa da sabbin polypeptides daga lokacin dilution, sake tsara hanyoyin sadarwar furotin don haɓaka hulɗar caji, da yin hulɗa tare da sauran ɗigon ruwa.tare da saman (wetting).Ƙwayoyin αS / pLK, saboda hanyar sadarwar sunadaran sunadaran su (kawai hulɗar heterotypic tsakanin αS da pLK) da kuma mafi girma ga haɗin gwiwar furotin-protein, suna da alama suna iya daidaita cajin condensate da sauri;lalle ne, mun lura da saurin haɓakar furotin a farkon αS/pLK coacervates fiye da na αS/Tau.Bayan valence depletion, da huldar zama m ephemeral da droplets rasa su ruwa kaddarorin da kuma juya zuwa gel-kamar, non-flammable droplets tare da low electrostatic surface m (sabili da haka kasa jika saman).Sabanin haka, αS/Tau droplets ba su da inganci wajen inganta ma'auni na cajin droplet saboda ƙarin hadaddun hanyoyin sadarwar furotin (tare da haɗin gwiwar homotypic da heterotypic) da kuma raunin yanayin hulɗar furotin.Wannan yana haifar da ɗigon ruwa waɗanda ke riƙe halayen ruwa na tsawan lokaci kuma suna nuna yuwuwar yuwuwar yanayin lantarki wanda ke ƙoƙarin ragewa ta hanyar haɗawa da girma (don haka rage girman yanki / girman rabo na ɗigon ruwa) da kuma ta hanyar jika chem na hydrophilic.Wannan yana haifar da manyan ɗakunan karatu na sunadaran gina jiki waɗanda ke riƙe kaddarorin ruwa yayin da hulɗar ta kasance mai saurin wucewa saboda ci gaba da neman haɓaka caji a cikin hanyar sadarwar furotin.Abin sha'awa, N-terminally truncated nau'i na Tau, ciki har da wasu isoforms62 da ke faruwa ta halitta, suna nuna halin tsaka-tsaki, tare da wasu suna ƙarfafa tsufa tare da αS zuwa ɗigon gel-kamar ɗigon ruwa mai tsayi, yayin da wasu ke canzawa zuwa manyan ruwa.Wannan duality a cikin maturation na αS electrostatic coacervates ya yi daidai da kwanan nan LLPS ka'idar da binciken gwaji waɗanda suka gano alaƙa tsakanin valence depletion da electrostatic sieving a cikin condensates a matsayin mabuɗin don sarrafa girman condensate da kaddarorin ruwa.Injiniyanci 58.61.
Wannan makirci yana nuna hanyar tara amyloid putative don αS da Tau441 ta LLPS da LSPT.Tare da ƙarin yankuna masu wadatar anion (ja) da cation-rich (blue), αS da tau electrostatic coacervates tare da valence mai gamsarwa suna da ƙarancin kuzarin ƙasa don haka ƙasan coalescence, yana haifar da saurin tsufa.An sami kwanciyar hankali mara-agglomerated gel jihar..Wannan yanayin yana da kyau sosai a cikin yanayin tsarin αS / pLK saboda kusancinsa mafi girma da kuma hanyar sadarwa mai sauƙi na furotin-biyu, wanda ke ba da damar saurin gel-kamar sauyawa.Akasin haka, ɗigon ruwa tare da valence mara gamsarwa kuma, saboda haka, yankuna masu cajin furotin da ke akwai don hulɗar, suna sauƙaƙa wa coacervate don haɗawa da jika saman hydrophilic don rage ƙarfin samansa.Wannan yanayin ya fi dacewa don αS/Tau441 coacervates, waɗanda ke da cibiyar sadarwa mai mahimmanci mai mahimmanci wanda ya ƙunshi raƙuman hulɗar Tau-Tau da αS-Tau.Bi da bi, manyan coacervates za su fi sauri rike kaddarorinsu kamar ruwa, kyale sauran furotin-zuwa furotin mu'amala faruwa.Daga ƙarshe, amyloid heterogeneous aggregates dauke da duka biyu αS da tau form a cikin coacervate ruwa, wanda zai iya zama alaka da waɗanda aka samu a cikin hadawa jikin, waxanda suke da alamomi na neurodegenerative cututtuka.
Manyan sifofi masu kama da ruwa da aka kirkira a lokacin balaga na αS/Tau441 tare da cunkoso sosai amma yanayin furotin mai ƙarfi kuma, zuwa ƙarami, αS/ΔNt-Tau coacervates sune madaidaitan tafki don ƙaddamar da haɓakar furotin.Lallai mun lura da samuwar ɗumbin furotin mai ƙarfi a cikin irin wannan nau'in furotin na coacervates, galibi yana ɗauke da duka αS da tau.Mun nuna cewa waɗannan heteroaggregates an daidaita su ta hanyar hulɗar da ba ta lantarki ba, suna iya ɗaure dyes na musamman na amyloid kamar yadda fibrils na amyloid na al'ada, kuma hakika suna da irin wannan juriya ga tasiri daban-daban.Abubuwan αS/tau da LLPS suka kafa an nuna suna da kaddarorin amyloid.Lallai, bambance-bambancen da balagagge na Tau ya gaza a cikin tarawar amyloid yana da matukar rauni a cikin samuwar waɗannan tarukan αS iri-iri a cikin ruwan electrostatic coacervate.An lura da samuwar αS/Tau441 aggregates kawai a cikin coacervates, wanda ya riƙe kaddarorin masu kama da ruwa, kuma ba, idan coacervates / droplets ba su kai ga gel jihar.A cikin akwati na ƙarshe, ƙara ƙarfin hulɗar electrostatic kuma, a sakamakon haka, tsattsauran ra'ayi na sunadaran suna hana gyare-gyaren da suka dace na sunadaran don kafa sababbin hulɗar furotin da ake bukata don amyloid nucleation.Duk da haka, ana iya samun wannan a cikin mafi sassauƙa, nau'ikan ruwa-kamar coacervates, wanda hakanan zai iya zama ruwa yayin da suke haɓaka girma.
Gaskiyar cewa samuwar tarawa a cikin lokaci mai mahimmanci ya fi dacewa a cikin manyan αS / Tau condensates fiye da a cikin ƙananan ɗigon ruwa wanda ke da sauri gel, yana nuna mahimmancin gano abubuwan da ke sarrafa coalescence droplet.Don haka, ba wai kawai akwai yanayi don rabuwar lokaci ba, amma girman condensate dole ne a sarrafa shi don aiki mai kyau da kuma rigakafin cututtuka58,61.Sakamakonmu kuma yana nuna mahimmancin ma'auni tsakanin LLPS da LSPT don tsarin αS/Tau.Yayin da samuwar digo na iya karewa daga haɗuwar amyloid ta hanyar rage adadin furotin monomers da ake samu a ƙarƙashin yanayin jikewa, kamar yadda aka tsara a cikin wasu tsarin63,64, haɗuwar droplet a manyan matakan ɗigon ruwa na iya haifar da haɗuwar furotin na ciki ta hanyar sannu a hankali sake tsarawa.hanyoyin sadarwa na gina jiki..
Gabaɗaya, bayananmu suna jaddada mahimmancin haɗin kai da kuma gamsuwa/rashin gamsuwa a cikin hanyoyin sadarwa a cikin mahallin LSPT.Musamman ma, muna nuna cewa cikakken tsawon αS / Tau441 condensates suna iya yin amfani da su da kyau da kuma samar da amyloid-kamar heteroaggregates wanda ya haɗa da sunadarai biyu da kuma ba da shawarar tsarin kwayoyin halitta bisa ga sakamakon gwajin mu.Haɗin haɗin sunadaran guda biyu a cikin αS/Tau fluid coacervate da muka bayar da rahoto a nan na iya kasancewa da alaƙa da haɗin gwiwar sunadaran guda biyu a cikin abubuwan da aka haɗa, waɗanda alamun cutar ne, kuma suna iya ba da gudummawa ga fahimtar alaƙar da ke tsakanin LLPS da amyloid aggregation, yana buɗe hanya don IDP mai caji sosai a cikin neurodegeneration.
Monomeric WT-αS, cysteine ​​mutants (Q24C-αS, N122C-αS) da bambance-bambancen ΔCt-αS (Δ101-140) an bayyana su a cikin E. coli kuma an tsarkake su kamar yadda aka bayyana a baya.5 mM DTT an haɗa shi a cikin duk matakan tsarkakewa na αS cysteine ​​​​mutant don hana haɓakar haɗin disulfide.Tau441 isoform (plasmid samu daga Addgene #16316), ΔNt-Tau bambance-bambancen (Δ1-150, samu ta hanyar cloning IVA tare da premers CTTTAAGAAGGAGATACATATGATCGCCACACACCGG, CATATGTATATCCTCTCTTCTTAAAGTTAAAC) da AggDef-Tau755 bambance-bambancen, da kuma AggDef-Tau75-Premier. al'adu sun kasance girma zuwa OD600 = 0.6-0.7 a 37 ° C da 180 rpm, kuma an jawo magana tare da IPTG na 3 hours a 37 ° C.Kwayoyin girbi a 11,500 xg na 15 min a 4 ° C kuma a wanke tare da buffer saline mai dauke da 150 mM NaCl.Dakatar da pellet a cikin buffer lysis (20 ml da 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).An yi matakin sonication akan kankara tare da amplitude na 80% don bugun jini na 10 (minti 1 akan, kashe min 1).Kada ku wuce 60 ml a cikin duban dan tayi daya.E. coli lysates an yi zafi a 95 ° C. na minti 20, sa'an nan kuma sanyaya a kan kankara da kuma centrifuged a 127,000 × g na 40 minutes.An yi amfani da madaidaicin da aka bayyana akan membrane 3.5 kDa (Spectrum ™ Thermo Fisher Scientific, UK) kuma an yi masa maganin 4 L na buffer dialysis (20 mM MES, pH 6.8, NaCl 50 mM, EDTA 1 mM, MgCl2 2 mM, DTT 2 mM , PMSF 0.1 mM) na 10 hours.An daidaita ginshiƙin musayar cation na 5 ml (HiTrap SPFF, Cytiva, MA, Amurka) tare da buffer daidaitawa (20 mM MES, pH 6.8, 50 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).An tace tau lysate ta 0.22 μm PVDF tace kuma an allura a cikin ginshiƙi a yawan gudu na 1 ml/min.An gudanar da hasashe a hankali, tau ya ƙare tare da 15-30% buffer elution (20 mM MES, pH 6.8, 1 M NaCl, 1 mM EDTA, 2 mM MgCl2, 2 mM DTT, 0.1 mM PMSF).SDS-PAGE ne yayi nazarin ɓangarorin, kuma duk wani ɓangaren da ke ɗauke da bandeji ɗaya tare da nauyin kwayoyin da ake tsammani na tau an tattara su ta amfani da matatar centrifuge 10 kDa kuma an maye gurbinsu da buffer mai ɗauke da 10 mM HEPES, pH 7.4, NaCl 500 mM da DTT 2 mM don Matsakaicin furotin na ƙarshe shine 100 μM.An wuce maganin furotin ta hanyar tace PVDF 0.22 μm, daskarewa da sauri kuma an adana shi a -80 ° C.Protein K18 Farfesa Alberto Boffi ya ba da kyauta.Tsaftataccen shiri shine> 95% kamar yadda SDS-PAGE da MALDI-TOF/TOF suka tabbatar.An yi wa nau'in nau'in cysteine ​​​​mai lakabi tare da AlexaFluor488-maleimide (AF488, ThermoFisher Scientific, Waltham, MA, Amurka) ko TEMPOL-maleimide (Toronto Research Chemicals, Toronto, Canada).An tabbatar da su ta hanyar sha da MALDI-TOF/TOF.Tau441, ΔNt-Tau, AggDef-Tau da K18 an lakafta su tare da ragowar cysteine ​​na asali a matsayi na 191 da 322 ta amfani da Atto647N-maleimide (ATTO-TEC GmbH, Siegen, Jamus) bin wannan hanya.Taswirorin da suka rage na αS da Tau441 an ƙirƙira su ta amfani da CIDER66.
M poly-L-lysine (pLK DP 90-110 bisa ga NMR daga maroki, Alamanda Polymers Inc, Huntsville, Alabama, Amurka) an narkar da a 10 mM HEPES, 100 mM NaCl, pH 7.4 zuwa 10 mM taro, tsari sonicated ga 5 minti a cikin wani ultrasonic ruwa wanka da kuma adana a -20 ° C.PEG-8, dextran-70, FITC-PEG-10 (Biochempeg, Watertown, MA, Amurka) da FITC-dextran-500 (Sigma -Aldrich, Sant Louis, MI, Amurka) ruwa ne mai narkewa kuma an rarraba su sosai a cikin buffer LLPS.Dialysis yana cire gurɓataccen gishiri.Daga nan an tace su ta hanyar tace sirinji mai girman ramuka na 0.22 μm, kuma an ƙididdige adadinsu ta hanyar amfani da refractometer (Mettler Toledo, Columbus, Ohio, USA).An shirya samfurori na LLPS a dakin da zafin jiki a cikin tsari mai zuwa: an haɗa buffer da extrusion da 1 mM tris (2-carboxyethyl) phosphine (TCEP, Carbosynth, Compton, UK), 1 mM 2,2,2,2-(Ethane- 1, 2-diyldinitrile) tetraacetic acid (EDTA, carboxynth) da kuma cakuda 1% protease inhibitor (PMSF 100 mM, benzamide 1 mM, leupeptin 5 μM).Sannan ana ƙara αS da fused polycations (options pLK ko Tau).Don gwaje-gwajen jerin lokaci na thioflavin-T (ThT, Carbosynth, Compton, UK), yi amfani da jimlar taro na ThT don zama rabin taro na αS.A hankali amma a haxa samfuran sosai don tabbatar da sun yi kama.Matsakaicin kowane bangare ya bambanta daga gwaji zuwa gwaji, kamar yadda aka bayyana a cikin sashin Sakamako.An yi amfani da Azide a matakin 0.02% (w/v) a duk lokacin da tsawon gwajin ya wuce sa'o'i 4.Don duk nazarin ta amfani da samfuran LLPS, ba da izinin cakuda don daidaitawa na mintuna 5 kafin bincike.Don nazarin watsawar haske, 150 µl na samfurori an ɗora su a kan 96 microplates mara kyau (µClear®, baki, F-Bottom / Chimney Well, Greiner bio-one, Kremsmünster, Austria) kuma an rufe shi da fim mai mannewa.Ana kula da LLPs ta hanyar auna ɗaukar nauyi a 350 nm a tsakiyar mafita a cikin mai karanta farantin CLARIOstar (BMG Labtech, Ortenberg, Jamus).An gudanar da gwaje-gwajen a cikin sau uku a 25 ° C, kuma an ƙididdige kurakurai a matsayin daidaitattun daidaituwa daga ma'ana.An ƙididdige lokaci na dilute ta hanyar samfurin centrifugation da SDS-PAGE gel bincike, kuma an ƙididdige juzu'in αS a cikin matakan tsarkewa da haɓaka a cikin hanyoyin LLPS daban-daban.An shirya samfurin 100 μl LLPS mai ɗauke da 1 μM AF488-labeled αS ta hanyar haɗuwa sosai sannan ta hanyar centrifugation a 9600 × g na mintuna 30, bayan haka ana iya ganin hazo.An yi amfani da 50 μl na sama na mai ƙarfi don ƙididdige furotin ta amfani da gel SDS-PAGE.An duba gels tare da masu tacewa na AF488 ta amfani da tsarin hoton gel na ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) ko kuma an lalata su da tabon Coomassie kuma an gani tare da tacewa masu dacewa.An yi nazarin ƙungiyoyin da aka samu ta amfani da sigar ImageJ 1.53i (Cibiyoyin Lafiya na Ƙasa, Amurka).An gudanar da gwaje-gwajen a cikin kwafi a cikin gwaje-gwaje daban-daban guda biyu tare da sakamako iri ɗaya.
Yawanci, 150 μl na samfurori an yi amfani da su zuwa ƙananan ƙananan rijiyoyin 96 da ba su ɗaure ba kuma an gani a cikin dakin da zafin jiki a kan Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Jamus).Don gwaje-gwajen tabo, an kuma yi amfani da faranti na µ-Slide Angiogenesis (Ibidi GmbH, Gräfelfing, Jamus) ko 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts).EL6000 halogen ko mercury karfe halide fitulun aka yi amfani da matsayin haske kafofin (ga BF/DIC da WF imaging, bi da bi).Don microscope na WF, an yi amfani da maƙasudin haɓakawa na 40x (Leica Microsystems, Jamus) don mai da hankali kan haske akan samfurin kuma tattara shi.Don samfuran AF488 da ThT masu lakabi, tace tashin hankali da fitarwa tare da daidaitattun saitin tacewar GFP, tashin hankali da matattarar bandpass, bi da bi, 460-500 nm da 512-542 nm matattarar bandpass, da 495nm dichroic madubi.Don samfuran da aka yi wa lakabi da Atto647N, an yi amfani da daidaitaccen saitin masu tacewa na Cy5 tare da tashin hankali da ficewar bandpass na 628-40 nm da 692-40 nm, bi da bi, da kuma madubin dichroic 660 nm.Don microscope na BF da DIC, yi amfani da manufar tattara haske iri ɗaya.An yi rikodin hasken da aka tattara akan kyamarar Leica DFC7000 CCD (Leica Microsystems, Jamus).Lokacin bayyanarwa shine 50 ms don hoton BF da DIC microscope da 20-100 ms don hoton microscope na WF.Don kwatantawa, lokacin fallasa ga duk gwaje-gwaje tare da ThT ya kasance 100 ms.An yi gwaje-gwajen da ba a wuce lokaci ba don ganin haɗin kai, tare da tattara hotuna kowane 100 ms na mintuna da yawa.An yi amfani da ImageJ (NIH, Amurka) don nazarin hoto.An gudanar da gwaje-gwajen sau uku tare da sakamako iri ɗaya.
Don gwaje-gwaje na colocalization, FRAP da 3D sake ginawa, an samo hotuna a kan Zeiss LSM 880 mai jujjuya microscope ta amfani da bugu na blue ZEN 2 (Carl Zeiss AG, Oberkochen, Jamus).An yi amfani da samfurori na 50 µl zuwa µ-Slide Angiogenesis Petri jita-jita (Ibidi GmbH, Gröfelfing, Jamus), wanda aka bi da shi tare da polymer hydrophilic (ibiTreat) kuma an saka shi a cikin maƙasudin nutsar mai na 63 × (Plan-Apochromat 63 × / NA 1.4 Oil) da DIC).An samo hotuna ta amfani da 458 nm, 488 nm, da 633 nm argon laser Lines tare da ƙuduri na 0.26 μm/pixel da lokacin bayyanarwa na 8 μs/pixel don haɓakawa da ganowa windows na 470-600 nm, 493-628 nm, kuma an yi amfani da 638-755 nm don ganin ThT, AF488 da Atto647N, bi da bi.Don gwaje-gwajen FRAP, an yi rikodin ɗaukar hoto na kowane samfurin a firam 1 a sakan daya.An gudanar da gwaje-gwajen a cikin sau uku a zazzabi na ɗaki tare da sakamako iri ɗaya.Anyi nazarin dukkan hotuna ta amfani da software na Zen 2 blue edition (Carl Zeiss AG, Oberkochen, Jamus).An daidaita ma'aunin FRAP, ƙirƙira kuma an daidaita su zuwa ƙarfi / bayanan lokaci da aka fitar daga hotuna ta amfani da Zen 2 ta amfani da OriginPro 9.1.An daidaita maƙallan dawo da su zuwa ƙirar ƙira-ɗaya don yin lissafin yaɗuwar kwayoyin halitta tare da ƙarin ƙayyadaddun lokaci don yin la'akari da tasirin bleaching na saye.Sannan mun ƙididdige D ta amfani da radius na bleaching mara kyau da rabin rayuwar da aka ƙaddara a baya kamar yadda yake cikin lissafin Kang et al.535 nuna.
An haɗa bambance-bambancen cysteine ​​guda ɗaya na αS tare da 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) a matsayi 24 (TEMPOL-24-αS) da 122 (TEMPOL-122-αS), bi da bi.Lakabin Spin Label Don gwaje-gwajen EPR, an saita ƙaddamarwar αS a 100 μM kuma ƙaddamarwar PEG shine 15% (w/v).Don yanayin tarawa daban-daban, rabon αS: pLK shine 1:10, yayin da αS: ΔNt-Tau da αS: Tau441 rabo aka kiyaye a 1:1.Don ɗaurin gwaje-gwajen titration a cikin rashin cunkoson jama'a, TEMPOL-122-αS an kiyaye shi a 50 μM kuma an ƙididdige polycations a haɓaka haɓakawa, shirya kowane yanayi daban.An gudanar da ma'aunin CW-EPR ta amfani da Bruker ELEXSYS E580 X-band spectrometer sanye take da resonator Bruker ER4118 SPT-N1 wanda ke aiki a mitar microwave (SHF) na ~9.7 GHz.An saita zafin jiki a 25 ° C kuma an sarrafa shi ta hanyar cryostat na ruwa na nitrogen.An samo siginar a ƙarƙashin yanayi mara kyau a ƙarfin MW na 4mW, girman juzu'i na 0.1 mT, da mitar daidaitawa na 100 kHz.An daidaita ƙarfin bakan gizo don guje wa bambance-bambance a cikin ƙima tsakanin samfuran da yuwuwar rage juzu'i saboda raguwar raguwar wakilai a cikin samfuran da ke ɗauke da Tau441 ko ΔNt-Tau (a halin yanzu a cikin mafitacin furotin na asali).Abubuwan da aka bayar na g an samo su ne sakamakon ƙirar ƙirar EPR da aka yi ta amfani da software na Easyspin (aya 6.0.0-dev.34) da aka aiwatar a cikin Matlab®67.An yi amfani da nau'ikan isotropic sassa ɗaya/biyu don ƙirar bayanan.Bayan daidaita duk sigina, an ƙididdige ragowar ta hanyar cire kowane siminti daga madaidaicin bakan gwaji.Don nazarin titration na ɗaure, an yi amfani da ƙarfin dangi na rukuni na uku zuwa rukuni na biyu na daidaitaccen bakan EPR (IIII/III) don saka idanu da ɗaurin polycations zuwa αS.Don ƙididdige yawan rarrabuwar kawuna (Kd), sakamakon lanƙwan an daidaita shi zuwa ƙimayar ƙira wanda aka ɗauka n wuraren ɗauri iri ɗaya da masu zaman kansu.
An gudanar da gwaje-gwajen duban gani na NMR ta amfani da na'urar sikeli ta Bruker Neo 800 MHz (1H) NMR sanye da na'urar cryoprobe da Z-gradient.An yi duk gwaje-gwajen ta amfani da 130-207 µM αS da αS / ΔNt-Tau da pLK daidai a cikin 10 mM HEPES, 100 mM NaCl, 10% DO, pH 7.4 kuma an yi su a 15 ° C.Don saka idanu LPS ta NMR, an ƙara 10% PEG zuwa samfuran da aka riga aka haɗa.Makircin rikicewar canjin sinadarai (Hoto 1b) yana nuna matsakaicin 1H da 15N canje-canjen sinadarai.An sanya nau'in αS 2D1H-15N HSQC bisa ga aikin da aka yi a baya (shigarwa BMRB #25227) kuma an tabbatar da shi ta hanyar yin rikodi da kuma nazarin yanayin 3D na HNCA, HNCO da CBCAcoNH.An ƙididdige 13Cα da 13Cβ canje-canjen sinadarai a gaban ΔNt-Tau ko pLK don auna yiwuwar sauye-sauye a cikin tsarin tsarin sakandare idan aka kwatanta da αS canjin sinadarai a cikin tsabtataccen coil conformation 68 (Ƙarin Hoto 5c).An auna ƙimar R1ρ ta hanyar yin rikodin gwaje-gwajen hsqctretf3gpsi (wanda aka samo daga ɗakin karatu na Bruker) tare da jinkiri na 8, 36, 76, 100, 156, 250, 400, da 800 ms, kuma an daidaita ayyukan ma'auni zuwa mafi girman jinkirin jinkiri daban-daban. lokuta don tantance R1ρ da rashin tabbas na gwaji.
An yi gwaje-gwajen ƙwanƙolin haske mai launi biyu-lokaci akan na'urar ƙirgawa mai ɗaukar hoto MT200 da aka warware lokacin kasuwanci (PicoQuant, Berlin, Jamus) tare da na'urar kirga photon guda ɗaya (TCSPC).Ana amfani da shugaban diode na Laser don pulsed interleaved excitation (PIE), katako yana wucewa ta hanyar jagorar yanayin guda ɗaya kuma ana kunna shi zuwa ikon laser na 10 zuwa 100 nW don 481 nm da 637 nm layin laser da aka auna bayan madubi dichroic.Wannan yana tabbatar da mafi kyawun ƙimar ƙidayar photon, guje wa tasirin photon aliasing, photobleaching da jikewa.μ-Slide angiogenesis murfin ko faranti (Ibidi GmbH, Gräfelfing, Jamus) an sanya su kai tsaye a cikin ruwa mai nutsewa akan ruwan tabarau na Super Apochromat 60x NA 1.2 tare da abin wuya mai gyara (Olympus Life Sciences, Waltham, Amurka).An yi amfani da madubi dichroic 488/640 nm (Semrock, Lake Forest, IL, Amurka) azaman babban mai raba katako.Radiyoyin da ba a mayar da hankali ba suna toshewa ta hanyar rami mai diamita na microns 50, sannan an raba hasken da aka mayar da hankali zuwa hanyoyin ganowa guda 2 ta hanyar mai raba katako mai 50/50.An yi amfani da matatar fitar da iska (Semrock, Lake Forest, IL, Amurka) 520/35 don launin kore (AF488) da 690/70 don rini ja (Atto647N) a gaban mai ganowa.Avalanche diodes (SPAD) (Micro Photon Devices, Bolzano, Italiya) an yi amfani da su azaman masu ganowa.Duka tattara bayanai da bincike an yi su ta amfani da software na SymphoTime64 na kasuwanci (PicoQuant GmbH, Berlin, Jamus).
An yi amfani da microliters hamsin na samfuran LLPS zuwa rijiyoyin angiogenesis na μ-Slide (Ibidi GmbH, Gräfelfing, Jamus).Hotunan da aka samo suna mayar da hankali ga 20 µm sama da rijiyar ƙasa don kyakkyawan nisa na aiki don ɗigogi da aka dakatar da ~ 1 µm don rafts da dige tare da ƙudurin axial na aƙalla 0.25 µm/pixel da lokacin jinkiri na 400 µs/pixel.Zaɓi bayanai ta hanyar amfani da madaidaicin madaidaicin bisa matsakaicin matsakaicin ƙarfin siginar baya (PBG, ma'ana + 2σ) ga kowane tashoshi don kawai ana zaɓar ɗigon furotin na ruwa, rafts, ko aibobi, tace duk wata yuwuwar asali daga lokacin tarwatsewa.Don bincika tsawon rayuwar kowane nau'in (τ) na kowane tashar (kore, "g" don AF488 da ja, "r" don Atto647N), mun zaɓi yankuna masu sha'awa (ROIs) waɗanda ke ɗauke da ɗigon ruwa, rafts, ko spots (Ƙarin Hoto 1). ).8b) kuma ya samo su ta hanyar dacewa da lalatawar rayuwarsu (τD, τR da τP don ɗigon ruwa, rafts ko spots, bi da bi, duba Ƙarin Hoto 8c) a cikin kowane tashoshi ta amfani da bincike mai dacewa da wutsiya da samfurin lalata mai sassa biyu.Matsakaicin τ daga τ .ROIs waɗanda suka samar da ƴan hotuna kaɗan don dacewa da ma'auni da yawa an cire su daga bincike.Yanke da aka yi amfani da shi shine <104 photons don rafts da dige da 103 don digo.Digo-digon suna da ƙananan ƙofa saboda yana da wahala a sami ɓangarorin ɓarna tare da ƙima mafi girma, tunda ɗigon ruwa a cikin filin hoton yawanci ƙanana ne kuma ƙasa da yawa.ROI tare da ƙididdigar photon sama da iyakar tarawar photon (saita zuwa> 500 ƙididdiga/pixel) an kuma jefar da su don bincike.Daidaita madaidaicin ɓarna mai ƙarfi da aka samu daga yankin sha'awa tare da ƙarfi a 90% na matsakaicin (dan kadan bayan matsakaicin girman lalacewa) daga farkon rayuwar sabis don tabbatar da ƙaramin tsangwama na IRF yayin kiyaye iri ɗaya don duk ɓarna mai ƙarfi. saituna An bincika taga lokacin dangi 25 zuwa 50 ROI don rafts da spots da 15-25 ROI don faɗuwar, hotunan da aka zaɓa daga fiye da 4 kwafi da aka yi rikodin daga aƙalla gwaje-gwaje masu zaman kansu 3.An yi amfani da gwaje-gwaje masu wutsiya biyu don kimanta bambance-bambancen kididdiga tsakanin nau'in ko tsakanin tsarin coacervate.Don nazarin pixel-by-pixel na tsawon rayuwa (τ), an ƙididdige jimillar attenuation na tsawon rayuwa a kan filin don kowane tashoshi kuma an aiwatar da kimanin nau'i na 2/3-bangaren ƙira mai ƙima.An daidaita ƙimar rayuwar kowane pixel ta amfani da ƙididdige ƙimar τ a baya, wanda ya haifar da hoton da ya dace da pseudocolor FLIM.Tsawon rayuwar da ya dace da wutsiya ya kasance iri ɗaya a duk hotunan tashar guda ɗaya, kuma kowane ruɓe yana samar da isassun hotuna don samar da ingantaccen abin dogaro.Don bincike na FRET, an zaɓi pixels ta hanyar amfani da ƙaramin ƙarfi na photon 100, wanda ya kai matsakaicin siginar bango (FBG) na photon 11.An gyara ƙarfin haske na kowane tashoshi ta hanyar gyare-gyare na gwaji da aka ƙayyade: 69 spectral crosstalk α shine 0.004, ƙaddamarwa kai tsaye β shine 0.0305, ingantaccen ganowa γ shine 0.517.Ana ƙididdige ingancin FRET a matakin pixel ta amfani da ma'auni mai zuwa:
inda FDD shine ƙarfin haske da aka gani a cikin tashar mai bayarwa (kore), FDA ita ce hasken haske da aka gani a cikin tashar mai karɓa (ja) a ƙarƙashin tashin hankali kai tsaye, kuma FAA ita ce hasken haske da aka gani a cikin tashar mai karɓa (ja) a ƙarƙashin tashin hankali kai tsaye ( PIE).Ana lura da bugun jini mai ƙarfi a cikin tashar).
Sanya 100 µl na maganin amsawar LLPS mai ɗauke da 25 µM monomeric Tau441 mara lakabi (tare da ko ba tare da 25 µM αS) a cikin buffer na LLPS (wanda aka ƙara kamar yadda yake sama) akan ƙananan microplates na rijiyoyin 96 da ba na ɗaure ba tare da murfin foil mai mannewa da haɓakar digo bayan WF microscopy an duba shi. daidaitawa.cikin 10 min.Bayan sa'o'i 48 na shiryawa a cikin zafin jiki, an tabbatar da kasancewar rafts na furotin da tabo.Sa'an nan kuma a hankali cire ruwa a kan rafts daga rijiyoyin, sa'an nan kuma ƙara 50 L na dissociation buffer (10 mM HEPES, pH 7.4, 1 M NaCl, 1 mM DTT) da kuma incubate na 10 min.Babban yawan gishirin gishiri yana tabbatar da cewa LLPS ba zai maimaita ba saboda ragowar PEG, kuma yuwuwar taron furotin da aka kafa ta hanyar hulɗar lantarki kawai za a tarwatsa.Bayan haka an goge kasan rijiyar a hankali tare da tip micropipette kuma an mayar da sakamakon da aka samu zuwa rijiyar kallo mara komai.Bayan shigar da samfurori tare da 50 μM ThT na 1 h, an duba kasancewar wuraren da aka ware ta hanyar microscopy WF.Shirya sonicated αS fibrils ta incubating 300 µl na wani 70-µM αS bayani a PBS tare da pH 7.4, sodium azide 0.01% a 37 °C da 200 rpm a kan orbital shaker for 7 kwanaki.Bayan haka, an ba da maganin a 9600 × g don 30 min, an sake dakatar da pellet a cikin PBS pH 7.4 kuma ana sonicated (1 min, 50% sake zagayowar, 80% amplitude a cikin Vibra-Cell VC130 sonicator, Sonics, Newton, Amurka) samfuran fibril tare da in mun gwada da uniform size rarraba kananan fibrils.
Binciken FCS/FCCS da gano daidaituwar daidaituwa mai launi biyu (TCCD) an yi su akan MT200-lokaci-lokaci-ƙuduri mai ɗaukar hoto mai kyalli (Pico-Quant, Berlin, Jamus) da aka yi amfani da shi don gwaje-gwajen microscopy na FLIM-FRET ta amfani da yanayin PIE.An ƙara ƙarfin laser don waɗannan gwaje-gwajen zuwa 6.0 µW (481 nm) da 6.2 µW (637 nm).An zaɓi haɗin waɗannan ƙarfin laser don samar da irin wannan haske don nau'ikan nau'ikan fluorophores da aka yi amfani da su yayin samun ƙimar ƙididdiga mafi kyau da kuma guje wa ɗaukar hoto da jikewa.Duka tattara bayanai da bincike an yi su ta amfani da software na SymphoTime64 na kasuwanci da ake samu na 2.3 (PicoQuant, Berlin, Jamus).
Samfurori na keɓaɓɓen tarawar αS/Tau da aka samu ta amfani da LLPS ana diluted a cikin keɓewar keɓewa zuwa madaidaicin maida hankali na monomolecular (yawanci 1: 500 dilution, tunda tarin sun riga sun kasance a cikin ƙananan ƙima idan aka ware su daga samfuran coacervate).An yi amfani da samfurori kai tsaye zuwa murfin rufewa (Corning, Amurka) da aka riga aka yi da maganin BSA a maida hankali na 1 MG / ml.
Don nazarin PIE-smFRET a cikin tashoshi masu kore da ja, an yi amfani da ƙaramin ƙarami na photons 25 don tace ƙananan sigina masu ƙarfi da ke haifar da abubuwan da suka faru na monomeric (lura cewa monomers sun fi tara tarin samfurori idan aka kwatanta da keɓaɓɓen tara).An ƙididdige wannan bakin kofa a matsayin matsakaicin ƙarfin monomeric αS sau biyar da aka samu daga nazarin samfuran monomer tsarkakakku domin a zaɓa musamman tari don bincike.Da'irar tuƙi na PIE, tare da siyan bayanan TSCPC, sun ba da damar aikace-aikacen tace mai nauyi na rayuwa wanda ke taimakawa kawar da bangon bango da magana mai ban mamaki.An gyara ƙarfin wutan da aka zaɓa ta amfani da ƙofofin da ke sama ta amfani da matsakaicin siginar baya da aka ƙaddara daga lissafin abubuwan da suka faru tare da ƙarfi/bin samfuran buffer-kawai.Fashewar da ke da alaƙa da manyan tarawa yawanci suna mamaye kwanoni da yawa a jere a cikin saƙon lokaci ( saita zuwa 1 ms).A cikin waɗannan lokuta, an zaɓi kwandon mafi girman ƙarfi.Don FRET da bincike na stoichiometric, an yi amfani da ƙayyadaddun ƙayyadaddun gamma factor γ (0.517).Spectral crosstalk da gudunmawar tashin hankali kai tsaye ba a cika yin komai ba (an ƙayyadaddun gwaji) a ƙarfin Laser mai ban sha'awa da aka yi amfani da shi.Ana ƙididdige inganci da stoichiometry na FRET a cikin fashewa kamar haka.