Simultaneous Stabilization ntawm Actin Cytoskeleton nyob rau hauv ntau yam Nephron-specific Cells tiv thaiv lub raum los ntawm ntau yam mob.
Sep 26, 2023
Mob raum mobthiabmob raum raug mobyog mechanicallytxawv kab mob raum. Thaum mob raum kab mob yog txuam nrogkev raug mob podocyte, mob raum raug mobcuam tshuam rau lub raum tubular epithelial hlwb. Txawm tias muaj qhov sib txawv no,ib tug cardinal featurentawm ob qho tib simob raum thiab mob raumyog dysregulated actin cytoskeleton. Peb tau qhia tiasPharmaological activation ntawm GTPase dynaminameliorates podocyte raug mob hauvmurine qauv ntawm cov kab mob raum moblos ntawmtxhawb nqa actin polymerization. Ntawm no peb tsim dynamin lub luag haujlwm hauv kev hloov kho qhov tawv thiab polarity ntawmlub raum tubular epithelial hlwblos ntawm crosslinking actin filaments rau hauv tes hauj lwm. Ua kom muaj peev xwm ntawm dynamin's crosslinking muaj peev xwm los ntawm ib qho me me molecule agonist stabilizes actomyosin cortex ntawm apical membrane tiv thaiv kev raug mob, uas nyob rau hauv lemkhaws cia lub raum ua haujlwmnyob rau hauv ntau yam murine qauv ntawm mob raum raug mob. Qhov tseem ceeb, dynamin agonist ib txhij attenuates podocyte thiabtubular raug mobnyob rau hauv genetic murine qauv ntawm Alport syndrome. Peb txoj kev tshawb fawb muab pov thawj rau qhov ua tau thiab hais txog qhovCov txiaj ntsig ntawm qhov tshiab holistic nephron-tiv thaiv kev kho mob.
Covua rau mob raum raug mob(AKI) yogischemia, hypoxia, los yognephrotoxicity 1. Thaum nws tuaj yeem thim rov qab, AKI sawv cev rau qhov teeb meem kev noj qab haus huv tseem ceeb nrog kev tuag siab thiab tsis muaj kev kho mob meej. Tsis hais txog ntawm nws cov etiology, AKI feem ntau raug mob polarized epithelial hlwb ntawm lub raum tubules uas nws apical microvilli tsim cov tubular txhuam ciam teb uas koom nrog kev sib koom tes tseem ceeb electrolyte thiab dej thauj. Ib qho tshwj xeeb morphological thaum ntxov ntawm AKI yog qhov poob ntawm txhuam ciam teb thiab cell polarity vim yog kev tawg ntawm actomyosin cortex ntawm apical membrane1.
Kev tsim thiab kev saib xyuas ntawm cell polarity koom nrog cov teeb liab cascades, kev lag luam membrane, thiab cytoskeletal dynamics, tag nrho cov uas muaj kev sib koom tes zoo heev3. Lub koom haum ntawm apical daim nyias nyias yog qhov loj heev txiav txim siab los ntawm cov qauv ntawm actomyosin networks4, uas tsim kom muaj qhov tawv nqaij cortex, yog li ua kom yooj yim rau pawg ntawm cov polarity proteins. Thaum myosin II motors raug suav tias yog thawj lub tshuab hluav taws xob ntawm cortical txhav 5,6, cov qauv ntawm actomyosin cortex yog tsim los ntawm ntau yam ntawm actin-binding proteins (ABPs)7.
Ntxiv nrog rau cov paub ABPs, txhuam ciam teb ntawm lub raum tubules yog nplua nuj nyob rau hauv dynamin8, GTPase paub zoo tshaj plaws rau nws lub luag haujlwm hauv endocytosis9. Dynamin muaj lub tswv yim zoo los sib sau ua ke rau hauv ntau lub xeev oligomerization xws li dimers, tetramers, rings, thiab spirals9. Peb tau txheeb xyuas thawj zaug ncaj qha dynamin-actin interactions10 thiab pom tias dynamin's oligomerization tswj actin polymerization hauv podocytes11,12, cov hlwb tshwj xeeb tseem ceeb rau kev xaiv ntawm lub raum lim. Siv cov qauv murine ntawm CKD, peb tau pom tias kev ua kom muaj zog ntawm dynamin-dependent actin polymerization thim rov qab kev raug mob podocyte los ntawm kev kho lawv cov qauv tshwj xeeb thiab kev ua haujlwm12.
Ntawm no peb qhia tias nyob rau hauvlub raum tubular epithelial hlwb, dynamin cross-links filamentous actin (F-actin) rau hauv cov tes hauj lwm sib txuas. Dynamin txoj kev sib txuas ntawm kev muaj peev xwm yog txhais los ntawm nws lub xeev oligomerization thiab qhov ntev ntawm F-actin. Pharmacological activation ntawm dynamin oligomerization counteracts AKI los ntawm stabilizing cov actin tes hauj lwm thiab yog li kev ncaj ncees ntawm tes, uas ib feem tiv thaiv lub raum epithelial hlwb los ntawm oxidative kev nyuaj siab vim raug mob. Peb txoj kev tshawb fawb txheeb xyuas cov actomyosin cortex ntawm apical membrane ntawm lub raum tubular cell raws li lub hom phiaj tshuaj hauv AKI ntawm dynamin ua tus neeg sawv cev.
Cov txiaj ntsig Dynamin oligomerization tsim kom muaj qhov tawv thiab morphology ntawm apical membrane.
Txhawm rau tshuaj xyuas lub luag haujlwm ntawm dynamin-actin kev sib cuam tshuam hauv polarized lub raum tubular epithelial hlwb, peb siv Bis-T-23, allosteric activator ntawm actin-dependent dynamin oligomerization nyob rau hauv lub reconstituted system13, nyob rau hauv lub cell11,13, thiab nyob rau hauv lub tag nrho lub cev 12. Cellular phenotypes raug soj ntsuam nyob rau hauv Madin-Darby Canine raum (MDCK) hlwb los ntawm kev ua raws li cov xwm txheej ntawm F-actin thiab staining qauv ntawm ib tug nruj junction protein zonula occludens-1 (ZO-1), uas yog suav tias yog biomarker ntawm cell polarity. Cytochalasin D (CytoD) thiab latrunculin A (LatA), paub inhibitors ntawm actin polymerization, txo F-actin qib, thiab induced discontinuous ZO-1 staining (Ntxiv daim duab 1a). Hauv qhov sib piv, Bis-T-23 induced me ntsis nce qib F-actin yam tsis muaj kev cuam tshuam rau ZO-1 staining. Ntxiv ntawm Bis-T-23 ua ntej tab sis tsis yog tom qab LatA, qee qhov khaws cia F-actin qib thiab cell polarity. Tsis yog DMSO lub tsheb lossis dynamin inhibitor dynole14 tsis pom muaj txiaj ntsig (Ntxiv Fig. 1a).
Scanning electron microscopy (SEM) tso cai rau peb pom cov tshuaj cuam tshuam kev hloov pauv ntawm cov cell morphology tsom mus rau apical membrane (Fig. 1a). Qhov nruab nrab MDCK cell qhov siab yog 11 ± 2 µm, thiab qhov nruab nrab ntev ntawm lub microvilli yog 0.63 ± 0.2 µm (Table 1), uas yog nyob rau hauv qhov ntau pom nyob rau hauv lub raum15. LatA txo qis ntawm tes qhov siab, thiab qhov ntev ntawm microvilli thiab hloov cov microvilli sib npaug sib npaug rau hauv pawg, whereas Bis-T-23 ua rau muaj qhov cuam tshuam tsis zoo (Table 1, Fig. 1a). Thaum ntxiv ua ntej LatA, Bis-T-23 ib nrab khaws cia ntawm tes qhov siab thiab microvilli ntev. Txij li thaum microvilli nthuav qhia qhov ntev ntawm kev tswj hwm tau hais los ntawm cortical actin ntawm lawv lub hauv paus 16, cov ntaub ntawv no muab pov thawj tias Bis-T-23 hloov kho actomyosin cortex ntawm apical membrane.
Txhawm rau txiav txim siab qhov tshwm sim tiag tiag uas Bis-T-23 muaj rau ntawm cortical actin, peb pom cov actomyosin cortex hauv lamellipodia siv platinum replica electron microscopy (PR-EM). LatA txo qhov ceev ntawm cov actin networks, thiab cov nyhuv no tau raug tshem tawm ib nrab los ntawm kev ntxiv ntawm Bis-T-23 ua ntej LatA (Fig. 1b). Raws li LatA accelerates actin filament depolymerization los ntawm sequestering actin monomers, tom ntej no peb tau soj ntsuam seb qhov kev saib xyuas ntawm actomyosin cortex los ntawm Bis-T -23 yog vim nws cov txiaj ntsig zoo ntawm actin polymerization. Nyob rau hauv sib piv rau lub zog stimulation ntawm actin polymerization pom nyob rau hauv podocyte cell extracts10,11, Bis-T-23 tsuas yog marginally nce actin polymerization nyob rau hauv MDCK cell extract (Supplementary Fig. 1b). Ib yam li ntawd, immunodepletion ntawm endogenous dynamin -2 (Dyn2) los ntawm cov extract los yog inhibition ntawm nws cov kev ua GTPase los ntawm dynode ua rau marginal impairment ntawm actin polymerization (Supplementary Fig. 1c). Thaum LatA thiab CytoD cuam tshuam loj heev rau actin polymerization, qhov sib ntxiv ntawm Bis-T-23 ua ntej LatA lossis CytoD tsis tuaj yeem kov yeej lawv cov teebmeem inhibitory (Ntxiv Fig. 1d, e). Jasplakinolide, ib yam tshuaj uas induces actin polymerization los ntawm stimulating actin filament nucleation17, tsis tau nce tag nrho cov theem ntawm polymerization (Supplementary Fig. 1d), qhia tias MDCK cell lysate nthuav tawm ze-qhov siab tshaj plaws ntawm polymerized actin. Ua ke, cov ntaub ntawv no tau qhia tias qhov cuam tshuam ntawm Bis-T-23 ntawm morphology ntawm apical membrane nyob rau hauv MDCK hlwb tau tsav los ntawm ib tug mechanism uas tsis yog actin polymerization.
Muab qhov kev paub dav dav ntawm kev ua kom muaj txiaj ntsig zoo ntawm Dyn2 thiab F-actin ntawm txhuam ciam teb ntawm lub raum epithelial cells8, thiab dynamin lub luag haujlwm hauv endocytosis, peb txuas ntxiv mus tshawb xyuas yog tias Bis-T-23 cuam tshuam rau actin tsis ncaj los ntawm kev hloov pauv hauv endocytosis. Raws li kev cia siab, Dyn2 thiab F-actin co-localized ntawm actomyosin cortex hauv qab lub apical membrane, hauv microvilli, thiab ntawm clathrin-coated pits (CCPs), txhais los ntawm lawv cov duab sib txawv thiab qhov loj me (Ntxiv daim duab 1f). Peb tau tshuaj xyuas qhov dynamics ntawm CCPs siv tag nrho cov kev xav hauv nruab nrog fluorescence (TIRF) microscopy18,19. Bis-T-23, txawm tias nws qhov siab tshaj plaws, tsis muaj kev cuam tshuam rau kev faib tawm ntawm CCPs lub neej, thaum dynole txo cov naj npawb ntawm cov khoom tsim tau CCPs (Cov Lus Qhia Ntxiv 1g). Qhov no tsis muaj kev sib raug zoo ntawm qib ntawm endocytosis thiab kev hloov pauv hauv cell morphology hais tias Bis-T- 23 tsom rau cortical actin yam tsis muaj kev cuam tshuam dynamin lub luag haujlwm hauv endocytosis.
Txij li thaum lub raum cell polarity yog tswj los ntawm cov architecture thiab kev sib cog lus ntawm cov actomyosin tes hauj lwm, uas tsim kom muaj cell tawv ntawm lub apical membrane20, peb tom ntej no ntsuas cell txhav siv atomic force microscopy (AFM). Nanowizard IV system thiab JPK tsom xam software tau raug siv los txiav txim siab hloov pauv hauv Young's Modulus21 nyob rau hauv qhov chaw sib txawv ntawm qhov kev sim (Ntxiv Fig. 2a). Kev kho mob nrog LatA ua rau muaj qhov txo qis hauv cell-cell contact stiffness thiab apical cell stiffness hauv MDCK cells (Fig. 1c–e). Hauv qhov sib piv, Bis-T-23 ua rau muaj zog ntawm tes ntau dua piv rau DMSO lub tsheb (Daim duab 1c–e), zoo ib yam nrog nws cov txiaj ntsig zoo ntawm qhov siab ntawm tes, microvilli tooj, thiab qhov ceev ntawm actin networks (Table 1 thiab Ib. 1b) 22. Qhov sib ntxiv ntawm Bis-T-23 ua ntej LatA tau txo qis cov txiaj ntsig tsis zoo ntawm LatA ntawm kev txhav ntawm tes (Fig. 1c–e), raws li Bis-T-23 cov txiaj ntsig zoo ntawm actin networks thiab apical cell morphology (Table 1, Fig. 1b).
Fig. 1 Dynamin oligomerization txhais cov tawv nqaij ntawm tes los ntawm kev cuam tshuam cov actin architecture hauv lub raum epithelial hlwb. Tus Neeg Sawv Cev SEM cov duab ntawm MDCK hlwb kho nrog DMSO (0.1%) lossis Bis-T-23 (30 µM, 0.1% DMSO) rau 1 0 min ua ntej qhov sib ntxiv ntawm DMSO (0.1%) lossis LatA (0.2 µM, 0.1% DMSO) rau 2{{23} } min. Cov duab loj ntawm cov insets (cov cheeb tsam txiv kab ntxwv-boxed) qhia txog kev teeb tsa, kev faib tawm, thiab qhov ntom ntom ntawm microvilli ntawm apical membrane. b Tus Neeg Sawv Cev PR-EM cov duab ntawm MDCK hlwb raug kho raws li tau piav qhia hauv (a). Cov duab qhia kev hloov pauv hauv lub koom haum ntawm actomyosin cortex hauv MDCK hlwb raws li cov xwm txheej qhia. c Cov duab sawv cev ntawm Young's Modulus maps ntawm MDCK hlwb raug kho raws li tau piav qhia hauv (a). d, e Bar graphs sawv cev rau Young's Modulus piav qhia txog kev tawv nqaij ntawm tes ntsuas ntawm lub cell-cell junction (d) lossis ntawm apical membrane (e). Txhua lub cim sawv cev rau qhov nruab nrab nruj ntawm ib leeg. Cov txiaj ntsig tau pom hauv d, e tau tsim los ntawm tsawg kawg 10 hlwb los ntawm tsawg kawg peb kab lis kev cai tais diav. Kev ua yuam kev, txhais tau tias ± SD (* P Tsawg dua lossis sib npaug rau 0.05, ** P Tsawg dua lossis sib npaug rau 0.01, *** P Tsawg dua lossis sib npaug rau 0.001, **** P Tsawg dua lossis sib npaug rau 0.0001, tsis muaj khub ob-tailed t-test). ns, tsis tseem ceeb.
Peb kuj tau txiav txim siab nruj ntawm tes siv BioScope II system ua lwm txoj hauv kev sim rau AFM. Hauv qhov piv txwv no, kev quab yuam indentation curves tau ua raws li tus qauv ntawm Discher thiab cov neeg ua haujlwm nrog suav nrog Matlab software23. Cov xwm txheej zoo sib xws hais txog kev tawv nqaij ntawm tes tau raug kaw rau kev sib cuam tshuam ntawm LatA thiab Bis-T-23 (Cov duab ntxiv. 2b, 2c). Tsis tas li ntawd, dynode tsis pom muaj kev cuam tshuam rau ntawm tes tawv, qhov CytoD txo qis ntawm tes tawv nqaij (Supplementary Fig. 2d, e), raws li lawv cov actin phenotypes (Ntxiv daim duab 1a). Ua ke, cov ntaub ntawv no tsim kom muaj kev sib raug zoo ntawm cov xwm txheej ntawm actomyosin cortex, cell stiffness, morphology ntawm apical membrane, thiab cell polarity. Cov kev tshawb pom no kuj ua kom pom tseeb lub luag haujlwm ntawm dynamin oligomerization hauv kev txheeb xyuas cov khoom tsis sib xws ntawm epithelial cell polarity ntawm nws cov nyhuv ntawm actomyosin cortex.
Dynamin cross-links actin filaments rau hauv cov tes hauj lwm sib txuas uas underlie cell polarity. Txhawm rau kom paub meej txog cov txheej txheem molecular uas dynamin oligomerization cuam tshuam rau cov qauv ntawm actomyosin cortex, peb txuas ntxiv mus tshuaj xyuas cov nyhuv ntawm dynamin ntawm actin filaments hauv ib qho kev rov tsim dua tshiab. Raws li qhov kev xav tam sim no, qhov ntev ntawm actin filaments txhais lawv hom kev sib txuas 6. Raws li qhov nruab nrab ntev ntawm cortical actin filaments nyob rau hauv ib lub network ntawm cov ntug kev yog nyob nruab nrab ntawm 100 thiab 150 nm24, peb tau tshuaj xyuas cov teebmeem ntawm dynamin ntawm lub koom haum ntawm luv filaments generated los ntawm capping F-actin nrog gelsolin (Gsn-actin) (Fig. 2 a). Qhov sib ntxiv ntawm Dyn2 ua rau muaj kev loj hlob ntawm cov tes hauj lwm, ceg (Fig. 2b, c). Raws li qhov ntau thiab tsawg thiab cov duab ntawm recombinant Dyn2 (Fig. 2d), cov tes hauj lwm tau tsim feem ntau los ntawm Dyn2 dimers (Dyn2DIMER) thiab tetramers (Dyn2TETRA) uas cuam tshuam nrog ob peb actin filaments (Fig. 2e): Dyn2DIMER khi mus txog ob lub filaments, Dyn2TETRA khi mus txog plaub filaments, thiab Dyn2RING khi mus txog rau rau filaments. Tsawg magnification ntawm cov dluab qhia tau hais tias dynamin-dependent networks tsim ib tug qauv ntawm me me thiab loj lub nplhaib zoo li cov duab (Fig. 2c).
Txhawm rau txheeb xyuas cov kev soj ntsuam los ntawm cov txheej txheem rov tsim dua nrog dynamin lub luag haujlwm hauv cov hlwb, peb tom ntej tau txiav txim siab qhov chaw ntawm endogenous Dyn2 ntawm cortical actin tes hauj lwm siv monoclonal anti-Dyn2 antibody ua raws li kub-conjugated secondary antibody (Ntxiv daim duab 3a–c). Raws li pom nyob rau hauv lub reconstituted system, dynamin yog txuam nrog ib tug txawv tus naj npawb ntawm F-actin nyob rau hauv branched tes hauj lwm (Fig. 2f). Ua ke, cov ntaub ntawv no txheeb xyuas cov haujlwm tshiab ntawm dynamin, uas yog kev sib txuas ntawm F-actin rau hauv cov tes hauj lwm sib txuas.
Txhawm rau txheeb xyuas dynamin txoj kev sib txuas sib txuas thiab kev tiv thaiv ntawm Bis-T-23 ntawm actomyosin cortex thiab morphology ntawm apical membrane, peb txuas ntxiv mus tshuaj xyuas cov teebmeem ntawm Bis-T-23 ntawm dynamin-mediated networks nyob rau hauv reconstituted systems (Fig. 3a). Raws li contour plots, uas muab topographical sawv cev ntawm txawv filament densities, Bis T-23 nce tag nrho cov network ceev (Fig. 3a), uas yuav piav qhia los ntawm kev nce nyob rau hauv tus naj npawb ntawm F-actin khi rau dynamin vim nce nyob rau hauv nws oligomerization. Tsis tas li ntawd, dynamin muaj peev xwm hla cov kab txuas luv luv dua li ntev F-actin (Daim duab 3a-c), qhia tias dynamin lub peev xwm los tsim cov tes hauj lwm sib txuas yog txhais los ntawm nws cov xwm txheej oligomerization thiab qhov ntev ntawm actin filaments. Lub peev xwm los hla-txuas actin filaments rau hauv tes hauj lwm tau muab faib los ntawm ob dynamin isoforms, ubiquitously qhia Dyn2 thiab neuron-specific dynamin-1 (Dyn1) (Fig. 3b, c).
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