Hu rau: jerry.he@wecistanche.com

Yuto Mitsuhata1, Takaya Abe2, Kazuyo Misaki 3, Yuna Nakajima1, Keita Kiriya1,

Miwa Kawasaki4, Hiroshi Kiyonari2, Masatoshi Takeichi4*, Mika Toya1,4,5,6*&

Masamitsu Sato 1,6,7

Cistanche tuaj yeem txhim kho lub raum ua haujlwm

Epithelial hlwb teeb tsa ib qho kev txiav txim ntawm cov uas tsis yog-centrosomal microtubules, qhov rho tawm ntawm qhov kawg uas tau tswj los ntawm CAMSAP3. Lub luag haujlwm ntawm cov microtubules hauv cov haujlwm epithelial, txawm li cas los xij, tsis to taub. Ntawm no, peb qhia tias covlub raumntawm cov nas uas Camsap3 tau hloov pauvcystsnyob rau hauv proximal convoluted tubules (PCTs). PCTs tau nthuav dav heev hauv cov mutantlub raum, thiab lawv kuj tau nthuav tawm txhim kho cell proliferation. Hauv cov PCTs, cov hlwb epithelial tau dhau los ua fattened nrog rau kev cuam tshuam ntawm microtubule arrays nrog rau qee cov qauv subcellular xws li interdigitating basal txheej txheem. Tsis tas li ntawd, YAP thiab PIEZO1, uas yog hu ua mechanosensitive regulators rau cell shaping thiab proliferation, tau qhib rau hauv cov mutant PCT hlwb. Cov kev soj ntsuam no qhia tau hais tias CAMSAP3 - mediated microtubule tes hauj lwm yog ib qho tseem ceeb rau kev tswj kom zoo txhua yam khoom ntawm PCT hlwb, thiab nws poob ua rau cell deformation thiab proliferation ntawm activation ntawm mechanosensors, uas ua rau lub dilation ntawm PCTs.

Epithelial hlwb yog cov khoom tseem ceeb ntawm ntau yam kabmob. Tey qhia apical-rau-basal polarity uas txuas nrog lawv cov haujlwm, xws li nqus thiab tso tawm. Hauv cov hlwb sib txawv, feem ntau ntawm cov microtubules tsis yog centrosomal thiab ua raws li apical-rau-basal axis1-3. CAMSAP3 yog ib tug tswv cuab ntawm calmodulin-regulated spectrin-associated protein (CAMSAP)/Nezha/Patronin protein tsev neeg thiab yog ib qho tseem ceeb rau epithelial microtubule koom haum4-8. CAMSAP3 khi thiab stabilizes lub minus xaus ntawm microtubules4,6,9. Nyob rau hauv cov hlwb ntawm cov hnyuv me hauv nas, CAMSAP3 yog concentrated nyob rau hauv apical cortical cheeb tsam, anchoring lub minus xaus ntawm non-centrosomal microtubules rau cov chaw no 10,11, uas ua rau cov tsim ntawm microtubule arrays taw qhia rau apicobasal kev taw qhia nrog rau ntxiv xaus taw tes rau hauv paus. . Txawm li cas los xij, lub koom haum microtubule tus yam ntxwv no ploj hauv cov nas uas muaj cov noob caj noob ces encoding ib qho truncated Camsap3 (Camsap3dc/dc nas), uas C-terminal microtubule-binding domain raug tshem tawm. Lub plab hnyuv epithelial hlwb hauv cov nas no muaj ntau yam cytological tsis xws luag, suav nrog txo qis cell siab thiab mispositioned organelles xws li cov nucleus thiab Golgi10. Txawm hais tias muaj kev cuam tshuam loj heev hauv kev tsim vaj tsev hauv tsev, cov hlwb epithelial tuav ib txwm saib ob sab nplooj ntawv 10,12, thiab cov hnyuv mutated tsis muaj qhov pom tseeb ntawm lub cev tsis xws luag.

tubules (PCTs), lub voj ntawm Henle, distal convoluted tubules, thiab sau cov duct. Tese nephron substructures muaj ib txheej ntawm epithelium, uas ua haujlwm rau kev pom, reabsorption, thiab secretion.

A lub raum cystNws yog ib tug pathological lesion ntawm tus kab moblub raum. Te cystic lesion yog variable; focal los yog ntau cysts tsim nyob rau hauv ib sab los yog ob sab nyob rau hauv ob lub raum, qhia ib tug txawv degree ntawm cov tsos mob13-15. Muaj ntau congenital cysticlub raumkab mob, suav nrog polycysticraumkab mob (PKD), kab mob glomerulocystic raum (GCKD) thiab medullary cysticraumkab mob (MCKD), paub tias ua rau ntau yamlub raumcysts nyob rau hauv ob lub raum. Hauv PKD, cov hlwv tau muab faib ua ib feem ntawm tag nrho lub cev nrog rau PCTs, thaum GCKD thiab MCKD tsim cov hlwv hauv Bowman's capsules thiab khaws cov ducts (tsuas yog nyob rau thaj tsam medulla), feem ntau 16-19.

Raws li rau PKD, ob qho laj thawj tseem ceeb tau paub: 1) kev hloov pauv hauv cov noob Pkd1 lossis Pkd2, thiab 2) kev hloov pauv hauv cov noob xav tau rau thawj cilium biogenesis. Pkd1 thiab Pkd2 encode polycystin1 thiab polycystin2, ntsig txog, uas tsis yog xaiv cation channels20 uas nyob hauv ntau lub cellular compartments suav nrog thawj cilia21,22. Hauv cov nas uas nqa ib daim ntawv hloov pauv ntawm PKD1 lossis PKD2, polycystin proteins tsis nyob rau hauv cilia23. Cov noob uas xav tau rau biogenesis ntawm thawj cilia suav nrog If88 thiab Kif3a, uas koom nrog hauv kev thauj mus los hauv intrafagellar24-26. Kev hloov pauv hauv cov noob no kuj ua raulub raumkab mob cystic 24-27. Txawm hais tias txoj kev sib raug zoo ntawm ob txoj hauv kev no yog qhov nyuaj23, ib txoj hauv kev uas tau pom tias yuav ua rau PKD cuam tshuam nrog Hippo signaling effector Yap28 . Hauv Pkd1 mutant nas, cov ntawv sau ua ke ua haujlwm YAP / TAZ thiab nws cov nyhuv c-Myc tau qhib thiab txhawb nqa cystogenesis28. Txawm li cas los xij, cov txheej txheem hauv qab ntawm kev loj hlob ntawm polycysticlub raumnyob twj ywm tsis paub ntau.

Hauv qhov kev tshawb fawb tam sim no, peb tau tshawb xyuas lub luag haujlwm ntawm CAMSAP3 hauvraumqauv thiab kev ua haujlwm. Peb cov kev soj ntsuam pom tau hais tias apically localized CAMSAP3 pab txhawb rau kev taw qhia kom zoo ntawm non-centrosomal microtubules nrog rau kev tsim cov txheej txheem intracellular ntawm epithelial hlwb hauv PCTs, raws li tau pom hauv cov hnyuv me. Tsis zoo li lub plab hnyuv epithelia, txawm li cas los xij, CAMSAP3kev ua haujlwm tsis zooua rau lub cev-qib txawv txav hauv ob lub raum - uas yog, cyst tsim hauv PCTs. Kev poob ntawm CAMSAP3 muaj nuj nqi ua rau muaj kev ua haujlwm ntawm cov neeg tswj xyuas mechanosensitive uas ib txwm pab khaws cov ntaub so ntswg ncaj ncees. Peb txoj kev tshawb fawb qhia tias CAMSAP3- cov koom haum kho mob uas tsis yog-centrosomal microtubule yog qhov tseem ceeb rau kev tswj xyuas cov khoom mechanosensitive ntawm PCTs, thiab kev poob ntawm cov kab ke no ua rau thaj tsam cyst tsim hauv lub raum.

Cov txiaj ntsig

Cysttsim nyob rau hauvlub raumntawm Camsap3 mutant nas. Phenotypic tsom xam ntawm Camsap3dc/dc mutant nas qhia tiaslub raumnyob rau hnub nyoog (~ 2 xyoos) Camsap3dc/dc nas yog hypertrophic thiab discolored (Fig. 1a). Txhawm rau txheeb xyuas qhov txawv txav hauv Camsap3dc/dclub raum, peb tshuaj xyuas cov nas yau. Hnub tom qab hnub yug 21 (P21), Camsap3dc/dc ob lub raum tau zoo li qub. Txawm li cas los xij, ntu ntawm lub raum qhia txog cov qauv cystic ntawm thaj chaw cortical (Fig. 1b thiab Supplementary Fig. S1a), raws li tau lees paub los ntawm kev suav cov cystic Performance index, uas yog qhov piv ntawm cov cyst-muaj thaj chaw rau tag nrho lub raum cheeb tsam (Fig. 1c). Peb soj ntsuam mutant embryos los txiav txim seb thaum lub cysts pib tsim thaum lub sij hawm txoj kev loj hlob, uas qhia ib tug kos npe rau ntawm nce cyst tsim nyob rau hauv kwv yees li embryonic hnub 17.5 (E17.5) (Ntxiv daim duab S1a, xub). Ntawm P0, cortical cysts tau dhau los ua qhov pom tau yooj yim (Ntxiv Fig. S1a, xub), thiab lawv qhov loj thiab tus lej tau nce los ntawm P21 (Daim duab 1b, Ntxiv Fig. S1a, xub). Raws li cov nas loj hlob tuaj, cystic phenotypes tau tshwm sim ntau dua, txawm hais tias qib ntawm cov cyst tsim tau txawv ntawm cov tib neeg (Fig. 1c thiab Supplementary Fig. S1b). Tsis tas li ntawd, lub raum-rau-lub cev qhov hnyav piv tau siab dua rau cov nas Camsap3dc/dc dua li cov nas qus (WT) nas dhau lawv lub neej (Fig. 1d thiab Supplementary Fig. S1c, d). Tus, Camsap3dc/dc ob lub raum nthuav tawm cyst tsim, uas tau txhim kho nyob rau lub sijhawm, ua rau muaj mob hypertrophy hauv cov laus lub raum.

Camsap3dc/dc nas qhia C-terminal domain-truncated CAMSAP3 (CAMSAP3-dc)10, uas tuaj yeem koom nrog hauv kev soj ntsuam cystic phenotype ntawm nas lub raum. Txhawm rau tshawb xyuas qhov muaj peev xwm no, peb tau tsim lub Camsap3–/– (Camsap3-null) nas (Ntxiv Fig. S2a,b) (saib Cov Txheej Txheem hauv kev nthuav dav). Cov nas no kuj muajlub raumnrog ntau lub hlwv, thiab lawv cov phenotype yog qhov txawv ntawm qhov pom hauv Camsap3dc/dc nas; tshwj xeeb tshaj yog, cov nas heterozygous (Camsap3 ntxiv /–) cov nas tsis pom muaj cov tsos mob ntawm polycystic ob lub raum (Ntxiv Fig. S2c).

Cyst tsim hauv PCTs ntawm Camsap3dc/dclub raum. Txhawm rau txiav txim siab seb cov qauv twg los ua cystic hauv cov mutantlub raum, peb tau ua cov tshuaj tiv thaiv ntawm peb ntu nephron rau peb cov proteins, xws li Megalin (localizes rau PCTs), Na plus / Cl- cotransporter (distal convoluted tubules), thiab aquaporin -2 (sau ducts). Te cov txiaj ntsig tau pom tias qhov dilation tshwm sim tshwj xeeb ntawm Megalin-positive tubules ntawm P21 (Daim duab 1e thiab Supplementary Fig. S1e), qhia tias cystogenesis raug txwv rau PCTs. PCTs raug faib ua peb ntu, xws li S1, S2 thiab S3, thiab peb tau sib txawv ntawm lawv los ntawm kev tiv thaiv kab mob rau cov piam thaj thauj khoom SGLT1 thiab SGLT2, uas nyob hauv thaj tsam ntawm S3 thiab S1 / S2 ntu, raws li 29,30. Te dilated tubules tau cim ib leeg los ntawm SGLT2 (Fig. 1f), qhia tias cystogenesis tshwm sim ntawm S1 / S2 ntu. Hauv cov nas laus, txawm li cas los xij, cyst tsim kuj txuas ntxiv mus rau Bowman's capsule (Daim duab 1g).

Disorganized cellular architecture hauv cystic PCTs. Txhawm rau tshawb xyuas qhov tsis xws ntawm cellular-theem hauv cystic PCTs, peb tau kuaj xyuas lawv thawj zaug los ntawm kev tshuaj ntsuam xyuas electron microscopy, pom tias cov pej xeem ntawmlub raumtubules swelled nyob rau hauv Camsap3dc/dc ob lub raum (Fig. 2a), txawm hais tias qhov o ntawm qhov chaw ntawm qhov chaw ntawm lub tubule. Hla ntu tau qhia tias, ntawm qhov o ntawm ib lub tubule, nws cov lumen tau nthuav dav nrog rau thinning ntawm cov epithelial txheej uas ua rau cov tubules (Fig. 2a). Quantification siv hematoxylin thiab eosin-stained kuaj tau lees tias qhov siab ntawm tes tau txo qis hauv cov tubules dilated (Daim duab 2b), thaum lub apical perimeter ntawm txhua lub xovtooj tau nce (Fig. 2c), qhia tias cov hlwb mutant tau rog dua cov hauv WT.lub raum.

Peb tom ntej no tau tshuaj xyuas seb puas yog cell architecture thiab / lossis subcellular qauv ntawm PCTs tau cuam tshuam los ntawm Camsap3 kev hloov pauv. Kev txhaj tshuaj tiv thaiv kab mob rau DPP IV, ib qho apical membrane marker, thiab rau Na plus / K plus -ATPase, basolateral membrane marker, qhia tias tag nrho cov cell polarity yog ib txwm nyob rau hauv txhua txhua.lub raumtubule cell ntawm Camsap3dc/dc

Daim duab 1. Cyst tsim nyob rau hauv proximal convoluted tubules ntawm Camsap3dc/dclub raum. (a) Morphology ntawm ob lub raum ntawm WT (ntxiv / ntxiv) thiab Camsap3 mutant (Camsap3dc/dc) nas ntawm P666. (b) H&E staining ntawm WT thiab Camsap3dc/dc ob lub raum ntawm P21. Ntau lub hlwv tsim nyob rau hauv lub cortex ntawm Camsap3dc/dc ob lub raum. (c) Hloov cov cystic Performance index ntawm WT thiab Camsap3dc/dc nas dhau sijhawm. 9 nas tau kuaj xyuas WT lossis Camsap3dc/dc. Saib cov ntaub ntawv thiab cov txheej txheem rau defnition ntawm cystic index. (d) Qhov piv ntawm lub raum rau lub cev hnyav. 11 nas tau kuaj xyuas WT lossis Camsap3dc/dc. (e) Kev tiv thaiv kab mob rau cov cim ntawm cov kab sib txuas sib txuas (PCT) (Megalin), cov tubular distal convoluted (DCT), NaCl co-transporter, thiab sau cov ducts (CD) (aquaporin{12}}; AQP2) hauv WT thiab Camsap3dc/ dc ualub raumhauv p21. Cov duab ntawm qhov pom loj ntawm Megalin qhia delocalized Megalin nyob rau hauv cystic hlwb hauv Camsap3dc/dc ob lub raum, raws li qhia los ntawm tus xub xub. Cov kab tawg thiab txuas mus ntxiv qhia txog apical thiab basal margin ntawm lub xov tooj ntawm tes, feem. (f) Kev tiv thaiv kab mob rau cov cim ntawm S1 / S2 ntu (SGLT2, ntsuab) thiab S3 ntu (SGLT1, liab) hauv WT thiab Camsap3dc/dc raum ntawm P21. Cov duab ntawm qhov pom loj ntawm SGLT2 qhia tau tias txo qis SGLT2 teeb liab siv nyob rau hauv cystic hlwb hauv Camsap3dc/dc ob lub raum, raws li qhia los ntawm xub xub thawj. (g) Morphology ntawm glomerulus ntawm P21 thiab P487. Arrowheads qhia lub dilated glomerulus. Saib cov duab S1 thiab S2.

Daim duab 2. Disorganized cellular architecture nyob rau hauv cystic PCT hlwb. (a) Scanning electron microscopy dluab ▸

Cistanche tuaj yeem txhim kho lub raum ua haujlwm

ntawm WT thiab Camsap3dc/dclub raumhauv p21. Lub raum tubule hauv Camsap3dc/dclub raumyog ib nrab o. (b) Quantifcation ntawm cell siab nyob rau hauv lub cortex [ntxiv / ntxiv , n=121 hlwb; dc/dc (dilated), n=128 hlwb; dc/dc (tsis yog dilated), n=66 hlwb]. Cov kab yuam kev qhia SD ***p<0.001, t-test.="" see="" materials="" and="" methods="" for="" the="" defnition="" of="" dilated="" and="" non-dilated="" tubules.="" (c)="" quantifcation="" of="" cell="" width="" in="" the="" cortex="" [+="" +="" ,="" n="26" tubules;="" dc/="" dc="" (dilated),="" n="8" tubules;="" dc/dc="" (non-dilated),="" n="14" tubules].="" error="" bars="" indicate="" s.d.="" ***p=""><0.001, t-test.="" (d)="" cell="" polarity="" markers="" were="" visualized:="" dpp="" iv="" (apical="" marker)="" and="" na+/k+-atpase="" (basal="" marker).="" te="" localization="" of="" cell="" polarity="" markers="" in="" camsap3dc/dc="" kidney="" is="" overall="" normal.="" te="" arrowhead="" indicates="" basal="" plasma="" membranes,="" whose="" invagination="" is="" not="" normal.="" (e)="" apical="" structures="" (actin="" and="" ezrin)="" and="" the="" golgi="" were="" visualized.="" arrowheads="" indicate="" disrupted="" structures="" such="" as="" the="" lack="" of="" actin="" signal="" and="" misplacement="" of="" the="" golgi.="" broken="" and="" continuous="" lines="" indicate="" the="" apical="" and="" basal="" margin="" of="" the="" cell,="" respectively,="" in="" d="" and="" e.="" (f)="" transmission="" electron="" microscopy="" images="" of="" wt="" and="" camsap3dc/dc="" kidneys="" at="" p21.="" subcellular="" defects="" such="" as="" a="" fattened="" nucleus="" and="" misaligned="" mitochondria="" are="" observed="" in="" camsap3dc/dc="" kidneys.="" arrowheads="" indicate="" invaginated="" basal="" membranes.="" note="" that="" their="" extensive="" invagination="" is="" suppressed="" in="" camsap3dc/dc.="" asterisks="" denote="" mitochondria.="" drawings="" are="" the="" traced="" invagination="" in="" electron="" microscopy="" images.="" (g)="" quantifcation="" of="" the="" shape="" of="" the="" nucleus="" [+="" +="" ,="" n="72" cells;="" dc/dc="" (dilated),="" n="72" cells;="" dc/dc="" (non-dilated),="" n="71" cells].="" (h)="" immunostaining="" for="" acetylated="" tubulin,="" a="" marker="" for="" the="" primary="" cilium,="" in="" wt="" and="" camsap3dc/dc="" kidneys="" at="" p21.="" te="" primary="" cilium="" was="" present="" in="" both="" wt="" and="" camsap3dc/dc="">lub raum. (i) Tus naj npawb ntawm thawj cilia ntawm PCTs. Tus naj npawb ntawm thawj cilia tau suav nrog siv qhov ntev ntawm qhov ntev ntawm qhov hla ntawm PCT, nrog rau cov lej ntawm thawj cilia thiab nuclei. [ plus / plus , N=340 cells, 63 cross sections of PCT, No. of primary cilia=268; dc/dc, N=221 hlwb, 29 hla ntu ntawm PCT, No. ntawm thawj cilia=133].

nas (Fig. 2d). Hauv qhov sib piv, txawm hais tias Megalin tau khaws cia ntawm thaj chaw apical hauv WT hlwb, nws tau tawg mus rau hauv cytoplasm hauv qee lub cystic Camsap3dc/dc hlwb (Daim duab 1e magnifed saib, thiab daim duab 2d). Fluorescence staining rau F-actin, uas sib sau ua ke ntawm qhov ntom ntom hauv apical microvilli, qhia tias microvilli qee zaus cuam tshuam hauv cov hlwb fattened mutant.lub raum(Fig. 2e arrowhead), raws li confrmed los ntawm kis tau tus mob electron microscopy (Fig. 2f). Ezrin, ib feem ntawm ERM-complex uas koom nrog apical plasma membrane31, tau txo los yog poob hauv cystic hlwb ntawm Camsap3dc/dclub raum(Daim duab 2e), qhia tias cytoarchitecture yog txawv txav hauv cov hlwb mutant. Tsis tas li ntawd, qhov Giantin-zoo Golgi apparatus feem ntau yog faib raws li nyias strands juxtaposed rau lub nucleus hauv WT hlwb, lub Golgi qee zaus los ua condensed hauv Camsap3dc/dc hlwb (Fig. 2e). Kev xa tawm electron microscopy qhia ntxiv qhov tsis xws luag hauv subcellular hauv dilated PCTs ntawm Camsap3dc/dclub raum. Nyob rau hauv WT PCT hlwb, mitochondria tau elongated, qhia ib qho kev taw qhia raws lub apico-basal axis ntawm lub cell, whereas nyob rau hauv cystic PCT hlwb, mitochondria tended mus puag ncig - tsis pom ib qho kev taw qhia (Fig. 2f). Nyob rau hauv cov hlwb no, cov basal striations kuj tsis muaj teeb meem, poob lawv cov parallel thiab sib sib zog nqus invaginations uas muaj nyob rau hauv WT hlwb (Fig. 2f arrowheads thiab traced drawing), raws li tau qhia los ntawm immu- nostaining rau Na plus / K ntxiv -ATPase (Fig. . 2d arrowheads). Ntxiv mus, nuclei tau fattened nyob rau hauv lub cystic hlwb, piv nrog rau lawv puag ncig nyob rau hauv WT hlwb (Fig. 2f,g). Thawj cilia tau kuaj pom ntawm sab luminal ntawm PCT hlwb hauv WT thiab mutant ob lub raum (Daim duab 2h, i), tsis zoo li cov nas mutant uas tsis zoo rau ciliogenesis, uas ua rau lub raum polycystic 24-27, qhia tias kev puas tsuaj ntawm ciliogenesis yog. tsis yog vim li cas rau qhov

pom polycystic phenotypes hauv Camsap3dc/dc nas. Ua ke, cov kev soj ntsuam no qhia tau tiaskev ua haujlwm tsis zoontawm CAMSAP3 perturbs lub subcellular architecture ntawm PCT cystic hlwb.

Camsap3dc/dc nas qhia qee qhov tsis xws luag ntawm lub cev. Peb mam li tshuaj xyuas seb Camsap3dc/dc lub raum puas muaj qhov tsis xws luag ntawm lub cev. Te aforementioned immunocytological kev tshawb fawb pom tau hais tias Megalin, uas ua hauj lwm raws li ib tug multiligand endocytic receptor32,33, tended mus rau hauv lub cytoplasm ntawm Camsap3dc/dc PCT hlwb (Fig. 1e arrowhead, thiab Fig. 2d), nyob rau hauv sib piv rau nws meej apical localization nyob rau hauv WT PCT hlwb. Tsis tas li ntawd, txawm hais tias cov piam thaj thauj khoom SGLT2, uas yog lub luag haujlwm rau 90 feem pua ​​​​ntawm cov piam thaj reabsorption29, thaj chaw feem ntau ntawm cov cheeb tsam apical ntawm Camsap3dc / dc cells (raws li pom hauv WT hlwb), nws cov teeb liab siv tau txo qis hauv qee thaj tsam ntawm cystic tubules ( Fig. 1f arrowheads), qhia tias lub cev ntawm cov mutant tubules tuaj yeem raug cuam tshuam tsis zoo. Yog li ntawd, peb tau kuaj zis thiab ntshav, siv P396- rau P424- cov nas qub. Kev kuaj zis qhia tau tias muaj kev cuam tshuam ntau dhau ntawm K ntxiv, Mg2 ntxiv, thiab urea nitrogen hauv cov nas Camsap3dc/dc (Supplemen-tary Fig. S3a). Kev kuaj ntshav ntau pom tau hais tias Camsap3dc / dc nas tau txo qis qis ntawm K ntxiv thiab Mg2 ntxiv piv nrog WT lossis Camsap3 ntxiv / dc nas, txawm tias tsis muaj kev hloov pauv hauv cov qib ntawm urea nitrogen thiab qabzib (Ntxiv Fig. S3b) . Cov zis thiab ntshav creatinine qib tsis txawv ntawm WT thiab cov nas mutant (Ntxiv Fig. S3c). Yog li, peb pom tsuas yog ib feem ntawm lub cev tsis xws luag hauv Cam-sap3dc/dc nas.

Microtubule koom haum hauvlub raumtubules epithelial hlwb. Raws li Camsap3 kev hloov pauv ua rau muaj kev cuam tshuam ntawm microtubules hauv cov hlwb epithelial ntawm txoj hnyuv me 10,12, peb tau tshuaj xyuas seb qhov no puas tshwm sim hauvraumepithelial hlwb. Peb frst immunostained -tubulin ua ke nrog Megalin. Qhov tseem ceeb, ob qho tib si hauv WT thiab Camsap3dc / dc ob lub raum, Megalin-negative tubules, uas yog, ob qho tib si distal convoluted tubules los yog khaws cov ducts, pom muaj zog-tubulin immunostaining siv ntau dua li pom rau -tubulin hauv Megalin-zoo PCTs (Fig. 3a, b) . Raws li pom nyob rau hauv cov hnyuv me 10, CAMSAP3 nyob rau hauv apically nyob rau hauv tag nrho cov epithelial hlwb ntawm WTlub raum, overlapping lub api-cal kawg ntawm microtubules uas yog raws li lub apico-basal axis (Fig. 3c lef). Camsap3dc/dc ob lub raum epithelial hlwb kuj tau me ntsis immunopositive rau CAMSAP3 tab sis ntawm cov hauj lwm tsis xwm yeem (Daim duab 3c txoj cai, xub xub), qhia tias mutated CAMSAP3 yog khaws cia nyob rau hauv cov hlwb, anchored rau ib co unidentifed qauv. Hauv cystic hlwb ntawm lub raum mutant, cov array ntawm microtubules tau tangled thiab disorganized (Fig. 3c txoj cai), raws li pom nyob rau hauv cov hnyuv hlwb ntawm Camsap3dc/dc nas10 . Hauv cov hlwb uas tsis yog cystic, txawm li cas los xij, lub koom haum microtubule tsis cuam tshuam

Daim duab 3. Microtubules nyob rau hauv lub raum tubule epithelial hlwb. (a) Immunostaining rau -tubulin, Megalin, thiab nuclei hauv WT thiab Camsap3dc/dc ob lub raum ntawm P21. (b) Quantifcation ntawm -tubulin siv hauv PCT thiab DCT / CD ntawm WT ob lub raum (PCT, n=28 tubules; DCT/CD, n=18 tubules) thiab Camsap3dc/dc ob lub raum (PCT, n { {8}} tubules; DCT/CD, n=9 tubules). Cov kab yuam kev qhia SD ***p<0.001, t-test.="" (c)="" immunostaining="" for="" α-tubulin,="" camsap3,="" and="" dapi="" in="" wt="" and="" camsap3dc/dc="" kidneys="" at="" p21.="" arrowheads="" indicate="" camsap3-dc="" proteins="" anchoring="" to="" unidentifed="" structures.="" te="" apical="" localization="" of="" camsap3="" was="" absent="" in="" camsap3dc/dc="" cells.="" see="" also="" figure="">

ntau txawm tias tsis muaj kev ua haujlwm CAMSAP3, uas tuaj yeem piav qhia vim li cas cov hlwb no tsis pom qhov txawv txav ntawm cytological. Tsis tas li ntawd, Camsap3-null lub raum epithelial hlwb kuj pom zoo li microtubule tsis xws luag tab sis tsuas yog nyob rau hauv cystic hlwb (Supplementary Fig. S2d). Sib sau ua ke, cov txiaj ntsig no tau qhia tias CAMSAP3 ua lub luag haujlwm hauv kev tswj hwm lub koom haum apico-basal ntawm microtubules tsuas yog rau cov subpopulation ntawm lub raum epithelial hlwb.

Txhim kho cell proliferation hauv Camsap3dc/dc PCTs. Kev tsim cov cyst hauv ob lub raum muaj feem cuam tshuam nrog kev txhim kho lub raum cell proliferation34 . Txhawm rau kuaj seb puas muaj cell proliferation tau hloov pauv hauv Camsap3dc/dc ob lub raum, peb muab piv rau kev loj hlob ntawm PCT hlwb ntawm WT thiab mutant ob lub raum los ntawm kev sib koom ua ke rau Megalin thiab Ki-67, ib qho cim rau kev loj hlob ntawm tes. Ki-67– cov hlwb tsis tshua pom zoo hauv PCTs ntawm WT ob lub raum, thaum lawv tau pom meej meej hauv PCT hlwb ntawm Camsap3dc/dc ob lub raum. Txawm li cas los xij, Ki-67 tau kuaj pom nyob rau hauv ob qho tib si tsis yog dilated thiab dilated tubules (Fig. 4a,b), uas qhia tsis muaj kev sib raug zoo ntawm cell proliferation thiab cyst tsim. Tus, CAMSAP3kev ua haujlwm tsis zootau txhawb nqa cell proliferation, tab sis cov txheej txheem no tsis yog ib txwm txuam nrog cyst tsim.

Kev hloov pauv hauv zos ntawm YAP thiab cov leeg tsis yog myosin txuas nrog cell deformation. YAP yog ib tug transcriptional activator uas tswj cell proliferation35 raws li zoo raws li mechanical tension-dependent cell shaping36 . YAP tau qhib ua haujlwm nrog rau nws qhov chaw nyob hauv nuclear hauv ob lub raum ntawm Pkd1-cov nas tsis muaj zog, tus qauv ntawm PKD37, thiab Yap ua kom muaj zog txhawb nqa cell proliferation thiab tubular dilation hauv lub raum28 . Yog li ntawd, peb tau sim seb YAP puas tau qhib rau hauv PCTs ntawm Camsap3dc/dc ob lub raum. Immunostaining rau YAP qhia nws cov cytoplasmic localization hauv PCTs ntawm WT ob lub raum. Zoo kawg li, YAP tau ua kom muaj zog hauv cov nuclei ntawm Camsap3dc/dc PCTs, tab sis tsuas yog nyob rau hauv lawv qhov chaw dilated (Fig. 5a), raws li tau lees paub los ntawm quantifcation (Fig. 5b), qhia tias qhov pom ntawm YAP redistribution tshwm sim nyob rau hauv teb rau kev hloov hauv cell morphology tsis yog CAMSAP3kev ua haujlwm tsis zoorau se.

YAP yog ib tug downstream efector ntawm Hippo txoj kev, uas yog tswj los ntawm mechanical cues38 . Qhov tseeb, cystic hlwb hauv Camsap3dc / dc lub raum tau nthuav tawm, qhia tias lawv yuav raug cuam tshuam rau qhov nro.

Daim duab 4. Cell proliferation hauv Camsap3dc/dc PCTs. (a) Immunostaining rau Ki-67, Megalin, thiab DAPI hauv WT thiab Camsap3dc/dc ob lub raum ntawm P22. Arrowheads qhia Ki-67–cov hlwb zoo hauv cystic tubules. (b) Feem pua ​​ntawm Ki-67-cov hlwb zoo nyob rau hauv PCT epithelial hlwb [ntxiv / ntxiv , n=700 hlwb; dc/dc (dilated), n=224 hlwb; dc/dc (non- dilated), n=396 hlwb]. Cov kab yuam kev qhia SD *p<0.05,>

Yog li peb tau tshawb xyuas seb puas muaj kev hloov pauv hauv kev hais kwv txhiaj nrog cyst tsim. Vim hais tias tsis yog-muscle myosin II ua kom muaj zog-rhiab heev txheej txheem39, peb tau soj ntsuam qhov kev faib tawm ntawm myosin teeb saw 2 (MLC2), uas koom nrog hauv kev ua kom myosin II, hauv ob lub raum. Txawm hais tias MLC2 difused mus rau hauv cytoplasm hauv WT tubule hlwb, nws tau ua kom muaj zog nyob ze rau ntawm apical feem ntawm cystic cells hauv Camsap3dc/dc ob lub raum (Fig. 5c arrowhead), qhia tias cov hlwb no raug quab yuam tensile. Cells hauv cov PCTs uas tsis yog dilated tsis qhia cov MCL2 concentration. Tese fndings tau ua raws li lub tswv yim hais tias YAP nkag mus rau hauv lub nucleus thaum lub hlwb yog mechanically stretched40,41 .

Kev hloov pauv hauv PIEZO1 kev faib tawm hauv Camsap3dc/dc PCTs. Txhawm rau kuaj ntxiv seb puas muaj cov kab mob cystic raug cuam tshuam rau lub zog ncab, peb tau tshuaj xyuas cov subcellular faib ntawm PIEZO1, ib qho mechanosensitive ion channel42-45, vim qhov no yog ib qho cim rau cov neeg kho tshuab ncab; PIEZO1 localization shifs los ntawm plasma membrane / cytoplasm mus rau lub hnab ntawv nuclear thaum nws hnov ​​​​cov teeb liab stretch46 . Immunostaining rau PIEZO1 tau pom tias PIEZO1 feem ntau nyob hauv cytoplasm ntawm WT PCT hlwb, thaum nws qhov chaw nyob sib tshooj nrog cov nucleus hauv cystic hlwb ntawm Camsap3dc/dc PCTs (Fig. 6a). Kev txhaj tshuaj tiv thaiv kab mob rau YAP thiab PIEZO1 hauv ntu ntu kuj tau qhia tias lawv qhov chaw nyob hauv nuclear sib koom ua ke ntawm dilated PCTs (Supplementary Fig. S4, arrowheads). Ntawm qhov tod tes, hauv cov tsis-dilated Camsap3dc/dc PCTs, PIEZO1 faib ntsia cytoplasmic (Fig. 6a). Txawm li cas los xij, kev soj ntsuam ze ntawm cov duab immunofuorescence nrog rau kev ua kom muaj nuj nqis tau qhia tias nws qhov chaw nyob hauv nuclear tau nce mus rau qee qhov, piv nrog WT hlwb (Fig. 6a, b). Tus, tsis zoo li YAP, nws zoo nkaus li tias PIEZO1 pib ua haujlwm ua ntej kev kuaj pom ntawm cov hlwb hauv Camsap3dc/dc PCTs.

Kev sib tham

Hauv txoj kev tshawb no, peb pom tias CAMSAP3 kev hloov pauv lossis nws qhov poob tau ua rau muaj kev nthuav dav ntawm PCT hlwb, uas ua rau cov ntu ntu ntawm tubules coj mus rau qhov zoo li cystic. Ib qho cim ntawm cystogenesis twb tau pom thaum ntxov li E17.5, nrog rau kev maj mam nce ntawm dilation thaum lub hnub nyoog laus thiab nws qhov kev ncua mus rau Bowman's capsule. Epithelial hlwb nyob rau hauv cov cheeb tsam dilated ua thinner, nthuav tawm ntau yam cytological tsis xws luag, raws li pom nyob rau hauv cov hlwb ntawm cov hnyuv me ntawm Camsap3dc/dc nas10,12 . Qee lub raum tshwj xeeb tsis xws luag kuj tau pom, suav nrog kev cuam tshuam ntawm mitochondria alignment thiab basal plasma membrane patterning. Tese hloov pauv nrog kev cuam tshuam ntawm kev teeb tsa ntawm microtubules, tawm tswv yim tias CAMSAP3- cov xov tooj sib txuas nrog microtubule yog lub luag haujlwm rau lub koom haum ib txwm ntawm cov txheej txheem subcellular hauv PCT hlwb, raws li pom hauv lwm hom cell. Ntawm qhov tod tes, txawm hais tias CAMSAP3 tau tshwm sim los qhia hauv txhua lub raum tubule epithelial hlwb, qhov tsis zoo ntawm cov qauv phenotypes tau pom tsuas yog hauv PCTs, qhia tias muaj cov txheej txheem rov qab los tswj cov microtubule tes hauj lwm thaum tsis muaj CAMSAP3. Nws yog qhov ua tau tias cov molecules uas tsis yog CAMSAP3, xws li lwm yam CAMSAPs, tuaj yeem koom nrog kev tswj xyuas kev ncaj ncees ntawm microtubule tes hauj lwm hauv cov hlwb. PCT hlwb muaj qhov tsawg dua microtubule ntom dua li lwm cov tubule hlwb. Qhov no tej zaum yuav pab txhawb cell deformation, raws li cov microtubule tes hauj lwm uas yuav nyob twj ywm afer CAMSAP3 poob tej zaum yuav tsis txaus los tswj.

tsim nyog cell architecture. Txawm hais tias nyob hauv PCT hlwb, txawm li cas los xij, cov txheej txheem zoo li muaj nyob uas them rau qhov poob ntawm CAMSAP3, vim tias qhov kev nthuav dav ntawm PCTs tsis tshwm sim sib npaug.

Daim duab 5. Localization ntawm YAP thiab non-muscle myosin txuam nrog cell deformation. (a) Kev tiv thaiv kab mob rau YAP, Megalin, thiab DAPI hauv WT thiab Camsap3dc/dc ob lub raum ntawm P22. Fluorescence signals ntawm DAPI thiab YAP tau raug tshuaj xyuas raws kab kos hauv cov duab loj, thiab cov kev hloov pauv ntawm fuorescence siv tau pom hauv xiav rau DAPI thiab ntsuab rau YAP. Dashed kab qhia qhov kwv yees thaj tsam ntawm lub nucleus. Arrowheads qhia txoj hauj lwm ntawm nucleus hauv cov cell sawv cev. (b) Qhov ratio ntawm nuclear YAP siv rau cytoplasmic YAP siv [ntxiv / ntxiv , n=81 hlwb; dc/dc (dilated), n =43 hlwb; dc/dc (tsis yog dilated), n=53 hlwb]. Lub thawv ntawv qhia txog frst quartile, nruab nrab, thiab thib peb quartile qhov tseem ceeb. ***p<0.001, t-test.="" (b)="" immunostaining="" for="" mlc2,="" actin,="" and="" dapi="" in="" wt="" and="" camsap3dc/dc="" kidneys="" at="" p22.="" arrowheads="" indicate="" apical="" accumulation="" of="" mlc2="" in="" cystic="">

Daim duab 6. Hloov pauv hauv PIEZO1 kev faib tawm hauv Camsap3dc/dc PCTs. (a) Immunostaining rau PIEZO1, Megalin, thiab DAPI hauv WT thiab Camsap3dc/dc ob lub raum ntawm P22. Fluorescence signals ntawm DAPI thiab PIEZO1 tau luam tawm raws kab kos hauv cov duab loj, thiab cov kev hloov pauv ntawm fuorescence siv tau pom hauv xiav rau DAPI thiab ntsuab rau PIEZO1. Dashed kab qhia qhov kwv yees thaj tsam ntawm lub nucleus. Arrowheads qhia txoj hauj lwm ntawm cov nucleus hauv cov neeg sawv cev. (b) Qhov ratio ntawm nuclear piezo1 siv rau cytoplasmic piezo1 siv [ntxiv / ntxiv , n=66 hlwb; dc/dc (dilated), n =44 hlwb; dc/dc (tsis yog dilated), n=81 hlwb]. Lub thawv ntawv qhia txog frst quartile, nruab nrab, thiab thib peb quartile qhov tseem ceeb. ***p<0.001, **p=""><0.01, t-test.="" see="" also="" figure="">

Yog li ntawd, PCTs tau nthuav dav li cas hauv ib cheeb tsam, nrog rau cov epithelial-cell fattening? Qhov tseem ceeb, kev tsim cov cyst tuaj yeem kuaj pom ntawm E17.5, thiab lub sijhawm no cuam tshuam nrog qhov pib ntawm cov zis thaum ntxov hauv nas47. Yog li ntawd, peb tuaj yeem xav tias 'cov zis fow' yog qhov muaj peev xwm ua kom muaj peev xwm ua kom muaj zog ntawm lub raum tubules. Nws yog qhov tshwm sim tias PCT cov hlwb uas tau poob CAMSAP3-kho cov microtubule cytoskeleton tau kho tau yooj yim dua rau sab nraud quab yuam, uas yog, ntau deformable, piv nrog WT PCT hlwb. Yog li, lub zog tsim los ntawm cov zis fow nyob rau hauv lub raum tubules yuav ua rau tubule dilation, nqa txog lawv fattening. Cov rog zoo li no yuav tsis sib xws, ua rau muaj qhov sib txawv ntawm qhov sib txawv ntawm PCTs. Lub tswv yim no, tsis tas yuav hais, tsis suav nrog lwm cov txheej txheem ua tau rau PCT dilation.

Peb cov kev soj ntsuam pom tau hais tias PIEZO1 tau hloov chaw los ntawm cytoplasm mus rau thaj tsam nuclear hauv dilated PCT hlwb ntawm Camsap3dc / dc ob lub raum, qhia tias lub tshuab hluav taws xob no tau qhib los teb rau cov cell deformation, feem ntau yuav yog 'stretching' ntawm cov hlwb raws li tau tshaj tawm yav dhau los46. Te stretched xeev ntawm dilated PCT hlwb tau confrmed los ntawm kev soj ntsuam tias MLC2 tau nce nyob rau hauv lawv cov cheeb tsam cortical. Peb qhov kev soj ntsuam ntxiv tau qhia tias PIEZO1 twb tau qhib rau qee qhov hauv cov hlwb uas tsis yog-dilated PCT, qhia tias txawm tias cov hlwb no tau hloov pauv txhua yam txawm tias lawv zoo li qub. Lwm lub tshuab hluav taws xob, YAP, kuj tau qhib rau hauv PCT hlwb, tab sis nws qhov kev ua kom zoo li tshwm sim hauv cov hlwb uas ncab xwb.

Yuav ua li cas, yog li ntawd, cov kev tswj xyuas mechanosensitive pab txhawb rau cell deformation, nrog rau kev loj hlob ntawm tes uas tau txhawb nqa thaum tsis muaj CAMSAP3? Nws tau raug pom tias stretch-induced activation ntawm PIEZO1 txhawb kev loj hlob ntawm tes nyob rau hauv epithelial cells46, qhia tias cov txheej txheem no kuj ua haujlwm hauv PCT hlwb ntawm Camsap3dc/dc ob lub raum. Lub tswv yim no zoo ib yam nrog peb qhov kev soj ntsuam uas txhim kho cov cell proliferation thiab ib feem ntawm PIEZO1 ua kom tshwm sim ua ke, txawm tias nyob rau hauv cov hlwb uas tsis yog dilated ntawm Camsap3dc/dc PCTs. Hais txog YAP, nws txoj haujlwm tau ncua qeeb, tsis qhia meej txog kev sib raug zoo nrog qhov pib ntawm kev loj hlob ntawm tes. YAP paub tsis yog tsuas yog kev loj hlob ntawm tes tab sis kuj yog tus tswj hwm ntawm cov neeg kho tshuab homeostasis, raws li tau pom yav dhau los 36,48 . Yog li ntawd, nws yog qhov ua tau tias YAP, uas tau hloov mus rau hauv lub nucleus nrog rau qhov deformation ntawm PCT hlwb, tuaj yeem ua haujlwm los tswj lawv cov duab, uas yog, los kho cov duab puas ntawm cov hlwb. Txwv tsis pub, YAP tuaj yeem koom tes nrog PIEZO1 hauv kev txhim kho kev loj hlob ntawm cov hlwb dilated, raws li YAP paub tias muaj kev koom tes hauv kev tsim cov raum polycystic los ntawm kev txhim kho ntawm cov cell proliferation28.

Peb pom tias Camsap3dc / dc nas tau txo qis cov ntshav K ntxiv thiab Mg2 ntxiv, qhia tias lawv lub raum yuav raug cuam tshuam los ntawm qee qhov kev ua haujlwm tsis zoo. Cov teeb meem cytological hnyav uas peb tau pom nyob rau hauv apical thiab basal plasma daim nyias nyias ntawm PCT hlwb, qhov twg cov khoom siv thauj khoom nyob, yuav cuam tshuam rau qhov txawv txav no. Txawm li cas los xij, peb tsis tuaj yeem tshem tawm qhov ua tau tias lwm feem ntawm lub raum tubules lossis lwm yam kabmob tuaj yeem ua haujlwm tsis zoo, ua rau muaj qhov phenotype no. Tsis tas li ntawd, peb tsis tau txais ib qho pov thawj tias lub luag haujlwm ntawm glomerulus raug puas tsuaj, vim tias cov ntshav ntawm creatinine thiab urea nitrogen, uas tau tawm los ntawm glomerulus, yog qhov qub.

Kev cuam tshuam ntawm thawj cilium biogenesis ua rau PKD24-27 . Hauv qhov xwm txheej ntawm Camsap3 kev hloov pauv, tsim ntawm thawj cilia zoo li tsis muaj kev cuam tshuam. Ib txoj kev tshawb fawb tsis ntev los no tau pom tias poob ntawm CAMSAP3 hauv nasal multi-ciliated epithelial hlwb cuam tshuam rau kev tsim ntawm lub hauv paus khub ntawm microtubules nyob rau hauv axoneme, ua rau aberrant motility nyob rau hauv motile cilia49. Txawm hais tias thawj cilia ntawm cov hlwb epithelial yog nonmotile thiab tsis muaj lub hauv paus khub ntawm microtubules50, nws tseem tsis paub meej tias thawj cilium hauv Camsap3 mutant nas tseem ua haujlwm. Txawm hais tias qhov tsis paub tseeb no, qhov txawv txav ntawm PKD thiab Camsap3 mutants yog qhov sib txawv heev, vim tias cyst tsim tshwm sim tsis sib xws ntawm tag nrho lub raum nyob rau hauv lub qub, qhov uas nws yog ib qho kev tshwm sim hauv zos nyob rau tom kawg. Qhov sib txawv no feem ntau yog tshwm sim los ntawm qhov txawv ntawm cov keeb kwm ntawm cov cyst tsim ntawm lawv.

Txhawm rau kom ua tiav, peb txoj kev tshawb fawb qhia tias CAMSAP3 yog qhov tseem ceeb rau kev tswj hwm tus qauv thiab kev ua haujlwm ntawm lub raum tubules ntawm thaj tsam ze, yuav yog los ntawm kev tswj hwm ntawm non-centrosomal microtubule sib dhos. Cov kev tshawb fawb ntxiv yuav tsum tau, txawm li cas los xij, kom nkag siab meej txog qhov kev poob ntawm lub koom haum microtubule no cuam tshuam rau lub cev lub cev ntawm lub raum.

Cov ntaub ntawv thiab cov txheej txheem

Txhua qhov kev sim tau ua tiav raws li cov lus qhia, cov cai, thiab pom zoo los ntawm RIKEN Center rau Biosystems Dynamics Research thiab/lossis Waseda University's Institutional Biosafety Committee.

Cov nas. Camsap3 mutant (Camsap3dc/dc) nas siv nyob rau hauv txoj kev tshawb no tau tsim nyob rau hauv peb qhov kev tshawb fawb yav dhau los10 (acces- sion no. CDB0716K; http://www2.clst.riken.jp/arg/mutant feem pua ​​20mice feem pua ​​20list.html). Hauv cov nas no, thaj tsam C-terminal ntawm Camsap3, uas yog exons 14-17, raug tshem tawm, thiab yav dhau los peb tau lees paub tias cov nas hlwb qhia qhov tsis ua haujlwm C-terminally truncated CAMSAP3 (CAMSAP3-dc)10 . Hauv kev tshawb fawb tam sim no, N4 thiab 5 tiam nas tau siv hauv kev sim.

Camsap3 knockout (Camsap3–/–) nas (accession no. CDB0033E), uas tsis muaj tag nrho 22-kb Camsap3 cod- cheeb tsam, tau tsim nrog CRISPR/Cas9- kho genome kho ntawm zygote electroporation51. Luv luv, ob phau ntawv qhia RNAs thiab ib leeg-strand oligodeoxynucleotide (ssODN) tau tsim los tshem tawm thaj tsam 22-kb, thiab ib qho chaw EcoRI tau ntxiv rau lub hom phiaj ntawm kev lees paub rau kev ua tiav hauv cov nas mutant. Rau electroporation, cRNAs, tracrRNA, ssODN thiab Cas9 protein tau nkag rau hauv C57BL / 6 zygotes. Afer electroporation, cov zygotes tau pauv mus rau pseudopregnant ICR poj niam nas, thiab tus tsim nas tau kuaj xyuas los ntawm PCR thiab DNA sequencing. Tsob ntoo knockout nas (# 4, # 10, # 23) tau txais, thiab ib qho ntawm lawv (# 23) tau tsim EcoRI knock-in allele. Qhia RNA cov kab ke tau tsim los ntawm CRISPRdirect (https://crispr.dbcls.jp). Te crRNAs, tracrRNA, thiab ssODN sequences used for genome editing was synthesized as follows: 5′-cRNA, 5′-UGA UCC CAC ACA UAU GCA GGg uuu uag agc uau gcu guu uug-3′, 3′-cRNA , 5′-CAGCCACCUCUGAUC UGACCguuuuagagcuaugcuguuuug-3′; tracrRNA, 5′-AAACAGCAAGCAAGUAAAAAGGCUAGUCC

GUUAUCAACUUGAAAGUGGCACCGAGUCGGUGCU-3'; ssODN, 5′-TAGAACTTACTGTATAGTAAT

AGCCCACAACCATICGCATCCATICGTGAATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAGAAGATCAGACCAGCAG

CTCTCCTCTTCTGGGCCTCCTCCCCCCCCCCACAT-3′ . Cov tsiaj ntawv loj thiab cov tsiaj ntawv me qhia cov coding thiab pab ua ntu zus, raws li. Te nas #23 kab tau hla nrog C57BL / 6 peb zaug, thiab N3 tiam nas tau siv hauv kev sim.

Cov kev sim siv cov nas tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Pawg ntawm RIKEN Center rau Biosystems Dynamics Research lossis Waseda University, thiab tau ua raws li cov txheej txheem muab los ntawm cov pawg neeg ntawd. Te kawm tau ua raws li ARRIVE cov lus qhia: https://arriveguidelines.org.

Genotyping. Genotyping rau nas tau ua los ntawm PCR. Rau Camsap3dc/dc nas, peb siv primers piav nyob rau hauv Toya li al. (2016). Rau Camsap3–/– nas, peb siv cov primers hauv qab no txhawm rau kuaj cov tsiaj qus (WT) (P1/P2, 411 bp) lossis Camsap3–/– (P1/P3, 614 bp) allele (Ntxiv Fig. S2a,b) : P1, 5′- CATGTACCTACCACC ATCC-3′; P2, 5′- GGACCCTGGAGAAGGGCTTAG-3′; P3, 5′- GCAAGGCTGTAGTGAGCC-3′ . Te P1/P3 cheeb tsam hauv WT tsis yog amplifed raws li PCR tej yam kev mob siv.

Histology: Kev npaj cov ntaub so ntswg khov. Dissected raum cov ntaub so ntswg yog fxed nyob rau hauv 2 feem pua ​​paraform- aldehyde (Termo Fisher) / 0.1 M sorbitol (Nacalai Tesque) / PEM bufer rau 1 h ntawm chav tsev kub. Daim ntawv qhia rau PEM yog: 100 mM PIPES (Nacalai Tesque), 5 mM EGTA (Nacalai Tesque), thiab 5 mM MgCl2 (Wako), kho pH 6.9 nrog 2 M KOH. Afer fxation, lub raum cov ntaub so ntswg tau yaug peb zaug nrog PEM rau 10 feeb. Kaum, cryoprotection tau ua nrog sucrose / PEM: 15 feem pua ​​sucrose hauv PEM tov rau 2 h ntawm 4 degree, ua raws li 20 feem pua ​​sucrose hauv PEM daws thaum hmo ntuj ntawm 4 degree, thiab hloov nrog 30 feem pua ​​sucrose hauv PEM tov rau ~ 2 h ntawm 4 degree. Te raum cov ntaub so ntswg tau khov nyob rau hauv OCT compound nyob rau hauv ib tug embedding pwm (Polysciences, Inc.) nyob rau hauv kua nitrogen thiab khaws cia ntawm −80 degree. Cov qauv khov tau muab hlais ntawm 5 µm tuab nrog CM1850 Cryostat (Leica). Cov ntu ntawm cov iav slides tau khaws cia ntawm -80 degree kom txog thaum muaj xim.

Histology: Kev npaj ntawm parafn embedded cov ntaub so ntswg. Dissected raum cov ntaub so ntswg yog fxed nyob rau hauv Super Fix tov (Kurabo) rau 2 h ntawm chav tsev kub, ces pauv mus rau 4 degree thaum hmo ntuj. Txhua tus qauv raug nthuav tawm rau peb qhov kev daws teeb meem aqueous ethanol ntawm kev nce siab ntawm chav tsev kub: 30 feem pua ​​​​ethanol rau 3{{20}} min ob zaug, 50 feem pua ​​ethanol rau 3{ {35}} min, thiab fnally 7 0 feem pua ​​​​ethanol rau 30 min. Txhua tus qauv tau ua tiav nrog ASP300 Tissue Processor (Leica). Kaum, cov qauv ntawm lub raum tau muab tso rau hauv parafn wax (Sakura Fine- tek) nrog EG1160 (Leica) thiab khaws cia ntawm 4 degree. Parafn-embedded kuaj tau hlais ntawm 6 µm thickness nrog RV240 Rotary Microtome (Yamato Kohki). Cov seem ntawm cov iav slides tau khaws cia ntawm 4 degree kom txog thaum muaj xim. Immunofuorescent staining. Frozen raum seem tau muab tso rau hauv 0.1 feem pua ​​Triton X-100 (Nacalai Tesque) / PEM rau 10 min ntawm chav tsev kub. Kaum, kuaj tau permeabilized los ntawm kev thov 0.2 feem pua ​​Triton X-100 / PEM rau 10 min ntawm chav tsev kub. Ib zaug ntxiv, cov qauv tau muab tso rau hauv 0.1 feem pua ​​​​Triton X-100 / PEM rau 10 min ntawm chav tsev kub. Tom ntej no, cov qauv raug thaiv nrog 3 feem pua ​​​​bovine serum albumin (BSA; Roche) / 0.1 feem pua ​​Triton X-100 / PEM rau 30 min ntawm chav tsev kub, ua raws li cov tshuaj tiv thaiv thawj zaug rau hmo ntuj ntawm 4 degree. Thawj cov tshuaj tiv thaiv tau diluted hauv 3 feem pua ​​BSA / 0.1 feem pua ​​Triton X-100 / PEM. Rau thawj cov tshuaj tiv thaiv uas tau tsa hauv nas, ntxiv kev thaiv nrog MOM Blocking Reagent (Vector Laboratories) tau ua ua ntej thaiv hauv 3 feem pua ​​BSA / 0.1 feem pua ​​Triton X-100 / PEM. MOM Blocking Reagent tau diluted hauv 0.1 feem pua ​​​​Triton X-100 / PEM thiab siv rau cov qauv rau 30 min ntawm chav tsev kub. Afer MOM kev kho mob, cov qauv raug ntxuav nrog 0.1 feem pua ​​​​Triton X-100 / PEM peb zaug rau 5, 10 thiab 15 min ntawm chav tsev kub. Afer thawj cov tshuaj tiv thaiv kab mob, cov qauv raug ntxuav rau 15 feeb ntawm chav tsev kub nrog 0.1 feem pua ​​Triton X-100 / PEM peb zaug. Cov tshuaj tiv thaiv kab mob thib ob tau ua tiav rau 2 teev ntawm chav tsev kub hauv qhov tsaus ntuj. Cov tshuaj tiv thaiv thib ob tau diluted hauv 3 feem pua ​​BSA / 0.1 feem pua ​​Triton X-100 / PEM. Afer cov tshuaj tiv thaiv, cov qauv raug ntxuav nrog 0.1 feem pua ​​​​Triton X-100 / PEM peb zaug rau 5, 10 thiab 15 min ntawm chav tsev kub thiab kaw nrog VECTASHIELD Mounting Medium (Vec-tor Laboratories). Antibodies thiab reagents. DNA tau stained nrog DAPI tov (Dojindo, D523, 1:1000). F-actin tau stained nrog Alexa Fluor 594 phalloidin (Termo Fisher, A12381, 1:400). Microtubules tau stained nrog nas anti- -tubulin-FITC (DM1A; Sigma Aldrich, F2168, 1:200). Siv cov tshuaj tiv thaiv hauv qab no: tshis anti-Megalin (P-20; Santa Cruz Biotechnology, sc-16478, 1:200), luav anti-NaCl co-transporter (MERCK, AB3533, 1:500 ), luav anti-aquaporin-2 (abcam, AB3274, 1:400), tshis anti-SGLT1 (M-19; Santa Cruz Biotech-nology, sc-20582, 1:200) , luav anti-SGLT2 (Novus Biologicals, NBP1-92384, 1:200), nas anti-ezrin (3C12; abcam, ab4069, 1:500), luav anti-giantin (Covance, PRB-114 C, 1:700), luav anti-aPKC (Santa Cruz Biotech- nology, sc-216, 1:400), tshis anti-DPP IV (R&D Systems, AF954, 1:100), nas anti-Na plus /K plus -ATPase (abcam, ab7671, 1:100), nas anti-acetylated tubulin (Sigma Aldrich, T6793, 1:200), luav anti-CAMSAP3 (Tanaka et al., 2012, 1:400), nas anti-tyrosinated tubulin (Sigma Aldrich, T9028, 1:200), luav anti-Ki-67 (SP6; abcam, ab16667, 1:250), luav anti-YAP (Cell Signaling, #4912, 1:100) ), luav anti-myosin teeb saw 2 (Cell Signaling, #8505, 1:100), luav anti-piezo1 (Novus Biologicals, N BP1-78446, 1:100), nas anti-E-cadherin (52, 1:400), thiab luav anti-merlin (Sigma Aldrich, HPA003097, 1:100). Te nram qab no cov tshuaj tiv thaiv kab mob thib ob tau siv: don-key Dylight 594-conjugated anti-tshis IgG (Jackson, 1:500), tshis Alexa Fluor 488/594-conjugated anti-luv IgG (Termo Fisher, A-11034, A -11037, 1:500), tshis Alexa Fluor 488/594–conjugated anti-nas IgG (Termo Fisher, A-11029, A-11032, 1:500), thiab tshis Alexa Fluor 488-conjugated anti-rat IgG (Termo Fisher, A{196}},

1:400). Te binding specifcity ntawm anti-YAP (Cell Signaling, #4912) thiab anti-PIEZO1 (Novus Biologicals, NBP1-78446) cov tshuaj tiv thaiv siv hauv txoj kev tshawb fawb tau txheeb xyuas los ntawm kev ua haujlwm dhau los46,53. Hematoxylin thiab eosin (H&E) staining. Cov iav slides nrog raum seem tau muab tso rau ntawm 55 degree phaj rau 30 min kom ncab cov parafn. Tom qab ntawd cov qauv tau muab tso rau hauv xylene rau 15 feeb ob zaug tom qab los ntawm kev muab tso rau hauv 70-100 feem pua ​​​​ethanol: 100 feem pua ​​​ethanol rau 5 feeb ob zaug, 90 feem pua ​​ethanol rau 5 feeb, 80 feem pua ​​ethanol rau 5 feeb, thiab 70 feem pua. ethanol rau 5 feeb. Afer yaug hauv dej rau 20 feeb thiab hauv distilled / deionized H2O (ddH2O) rau 1 min, cov qauv tau stained hauv Mayer's hematoxylin tov (Wako) rau 5 min. Cov qauv tau dipped hauv ddH2O ob zaug, tom qab ntawd yaug nrog ddH2O rau 10 feeb. Cov qauv tau rov dipped hauv ddH2O thiab tom qab ntawd stained hauv eosin tov (Merck) rau 2 min. Rau kev ntxuav tawm, cov qauv tau muab tso rau hauv ddH2O rau 30 s, tom qab ntawd immersion hauv series ntawm 70-100 feem pua ​​​​ethanol thiab xylene: 70 feem pua ​​​​ethanol rau 5 s, 80 feem pua ​​​​ethanol rau 5 s, 90 feem pua ​​​ethanol rau 5 s, 100 feem pua. rau 5 s ob zaug, thiab xylene rau 10 feeb. Thaum kawg, cov qauv raug kaw hauv glycerol. Microscopy. Cov duab tau txais nrog BZ-X710 All-in-one Fluorescence Microscope (Keyence) lossis LSM880 laser scanning confocal microscope (Zeiss). Rau BZ-X710, lub hom phiaj lo ntsiab muag siv yog Nikon CFI Plan Apo λ 2 × , 10 × , 40 × , 100 × thiab Nikon CFI Plan Fluor 4 × . Cov duab tau raug ua tiav nrog BZ-X Analyzer sofware. Te LSM880 tau nruab nrog Axio Observer Z1 inverted microscope nrog Plan- Apochromat 63 × / 1.40 NA roj immersion hom phiaj lens (Zeiss). Cov duab tau ua tiav thiab tshuaj xyuas siv ImageJ thiab Adobe Photoshop CC2019. Cystic index tsom xam. Cystic index tau txheeb xyuas los ntawm ImageJ siv H&E-stained dluab ntawm kab rov tav ntawm lub raum. Raws li kev ntsuas ntawm thaj chaw muaj cyst, thaj tsam ntawm lub dilated tubular lumen tau taug qab siv "Freehand selections" muaj nuj nqi, thiab nws cheeb tsam tau ntsuas los ntawm "Kev Ntsuas" muaj nuj nqi. Qhov kev ua haujlwm no tau ua rau txhua lub hlwv nyob rau hauv lub raum seem, thiab cov cheeb tsam ntawm tag nrho cov hlwv tau ntxiv ua ke los txiav txim qhov chaw muaj cyst. Te tag nrho lub raum cheeb tsam tau ntsuas tib yam. Lub sab nraud ntawm lub raum tau taug qab siv qhov "Freehand selections", thiab nws cheeb tsam tau soj ntsuam. Thaum kawg, peb suav cov cystic Performance index los ntawm kev faib cov cyst-muaj cheeb tsam los ntawm tag nrho lub raum cheeb tsam.

Txhais ntawm dilated tubules thiab non-dilated tubules. Hauv kev tshawb fawb no, tubules nrog thaj tsam lumen ntawm Ntau dua lossis sib npaug ntawm 500 µm2 tau raug tiv thaiv raws li "dilated", hos cov uas muaj thaj tsam lumen ntawm<500 µm2="" have="" been="" defned="" as"non-dilated".="" when="" selecting="" a="" "dilated"="" tubule="" in="" camsap3dc/dc="" samples,="" we="" frst="" visually="" picked="" up="" a="" cyst="" that="" was="" not="" recognized="" in="" wt="" samples="" and="" then="" roughly="" measured="" its="" lumen="" area="" using="" imagej.="" if="" the="" value="" was="" ≧500="" µm2,="" it="" was="" regarded="" as="">

Electron microscopy. Rau electron microscopy, P21 txiv neej nas tau kuaj. Cov qauv rau kev tshuaj ntsuam xyuas hluav taws xob microscopy tau npaj raws li Takahashi-Iwanaga54 nrog qee qhov kev hloov kho, raws li hauv qab no. Cov nas raug tshuaj loog nrog isofurane thiab perfused nrog 2 feem pua ​​(w/vol) formaldehyde thiab 2.5 feem pua ​​(vol/vol) glutaralde-hyde hauv 0.1 M cacodylate bufer, pH 7.4. Txhua lub raum raug excised thiab txiav mus rau hauv cubes uas yog immersed nyob rau hauv tib fxative hmo ntuj, thiab ces yaug nrog 0.1 M cacodylate bufer. Te fxed cov qauv raug xa mus los ntawm 30 feem pua ​​aqueous tov ntawm dimethyl sulfoxide rau hauv 60 feem pua ​​dimethyl sulfoxide, thiab tom qab ntawd tawg hauv cov kua N2. Te fractured cov qauv tau muab yaug nrog 0.1 M cacodylate bufer thiab muab tso rau hauv 6 N NaOH rau 1{{30}}-20 min ntawm 60 degree . Afer maceration hauv cov tshuaj NaOH, txhua tus qauv tau yaug nrog 0.1 M cacodylate bufer uas muaj 0.05 feem pua ​​​​Tween 20 thiab postfxed nrog 1 feem pua ​​OsO4 hauv 0.1 M cacodylate bufer ntawm dej khov rau 2 h. Cov qauv postfxed yog: (1) yaug nrog ddH2O thiab muab tso rau hauv 1 feem pua ​​​​aqueous thiocarbohydrazide rau 20 min ntawm chav tsev kub, thiab (2) yaug nrog ddH2O thiab postfxed dua hauv 1 feem pua ​​OsO4 hauv 0.1 M cacodylate bufer rau 1 h ntawm chav tsev kub. . Afer rov ua cov kauj ruam (1) thiab (2), cov qauv tau muab yaug nrog ddH2O, thiab stained nrog 0.5 feem pua ​​​​uranyl acetate daws thaum hmo ntuj ntawm chav tsev kub. Te cov ntaub so ntswg tau rinsed nrog ddH2O thiab lub cev qhuav dej los ntawm ib tug graded series ntawm ethanol, pauv mus rau isopentyl acetate, thiab tseem ceeb point-dried siv kua CO2. Te qhuav cov qauv tau evaporation-coated nrog osmium thiab tshuaj xyuas nrog JSM-5600LV scanning electron microscope ntawm qhov nrawm nrawm ntawm 10 kV. Cov duab tau ua tiav nrog Adobe Photoshop.

Rau kev sib kis electron microscopy, lub qhov cub ntawm lub raum dissected tau fxed rau yam tsawg 2 h hauv 2 feem pua ​​(w/vol) formaldehyde thiab 2.5 feem pua ​​(vol/vol) glutaraldehyde hauv 0.1 M cacodylate bufer ntawm chav tsev kub. Te cov qauv raug ntxuav nrog 0.1 M cacodylate bufer thiab postfxed nrog 1 feem pua ​​OsO4 hauv {{10}}.1 M cacodylate bufer rau dej khov rau 2 teev. Tom qab ntawd cov qauv raug ntxuav nrog ddH2O thiab stained ib hmos nrog 0.5 feem pua ​​aqueous uranyl acetate ntawm chav tsev kub. Te stained samples dehydrated with a graded series of ethanol, transfer to propylene oxide, and embedded in Polybed 812 (Polysciences). Ultrathin ntu raug txiav nrog pob zeb diamond riam, muab ob npaug rau-stained nrog uranyl acetate thiab lead citrate, thiab pom ntawm 100 kV accelerating voltage siv ib tug kis tau tus mob electron microscope (JEM{20}}; JEOL) nruab nrog ib tug charge-coupled ntaus ntawv lub koob yees duab ( BioScan qauv 792; Gatan). Cov duab tau ua tiav nrog Adobe Photoshop.

kuaj zis thiab kuaj ntshav. Rau kev sib piv nrog Camsap3dc / dc nas, WT (ntxiv / ntxiv) thiab heterozygous (ntxiv / dc) nas tau siv los tswj hauv cov kev ntsuas no. Cov nas Heterozygous tsis pom qhov tshwm sim ntawm Camsap3kev ua haujlwm tsis zoo 10thiab tau teeb tsa apico-basal arrays ntawm microtubules. Cov nas muaj hnub nyoog 56-60 lub lis piam.

Cov nas tau nyob hauv lub tawb me me (TECNIPLAST). Cov zis kuaj tau raug sau txhua hnub rau tsib hnub sib law liag, thiab cov qauv sau tau khaws cia ntawm -80 degree. Thaum kawg, tag nrho cov qauv fve rau txhua tus nas tau muab sib xyaw kom cov fnal sib xyaw ua kom pom qhov nruab nrab ntawm txhua tus nas nyob rau lub sijhawm ntawd.

Rau kev sau cov ntshav, cov nas tau tswj hwm {{{0}}.02 mL tshuaj loog ib gram lub cev hnyav. Cov tshuaj loog muaj xws li Domitor (ZENOAQ) 30 µL, Midazolam (Sandoz) 80 µL, Vetorphale (Meiji Seika Pharma) 100 µL, thiab 0.9 feem pua ​​aqueous NaCl rau 1 mL. Ntshav tau sau los ntawm kev mob plawv. Txhawm rau kom tsis txhob hemolysis, lub koob txhaj tshuaj tau maj mam muab tso thiab rub tawm. Txhua tus qauv ntshav tau khaws cia hauv chav sov li 1 teev kom coagulate. Tom qab ntawd txhua tus qauv raug centrifuged (10,{10}}×g, 25 degree , 15 min), thiab cov ntshav tau sau thiab khaws cia ntawm −80 degree . Tag nrho cov zis thiab ntshav kuaj tau soj ntsuam los ntawm Oriental Yeast Co.