Vpliv diferenciacijske stopnje celic urotelija na izražanje mehanoreceptorjev Piezo

Deutsch, Maja (author), Romih, Rok (mentor)

Dokončno diferenciran urotelij sečnega mehurja med mikcijskimi cikli vzdržuje krvno- urinsko pregrado in ima pomembno mehanosenzorično vlogo. Ključni mediatorji tega zaznavanja so mehanosenzorični ionski kanali, med njimi tudi kanala Piezo1 in Piezo2. Namen magistrske naloge je bil proučiti izražanje in lokalizacijo proteinov Piezo v različno diferenciranih urotelijskih celicah in vivo ter in vitro ter pokazati njuno povezavo s proteini medceličnih stikov in citoskeleta. Ugotovili smo, da je bil mišji urotelij sestavljen iz 3 skladov, medtem ko je imel podganji 1-2 sklada. V dežnikastih celicah so bili prisotni zreli fuziformni vezikli, imunooznačevanje uroplakinov pa je pokazalo njihovo močno izražanje. Pri podgani so bili prisotni urotelijski plaki in tesni stiki. Izražanje Piezo1 in Piezo2 smo pri miših in podganah potrdili v uroteliju in tkivih pod urotelijem (lamina propria, mišica) s prenosom western, pri čemer so bile lise šibkejše pri uroteliju. Izražanje proteinov Piezo smo potrdili tudi v celičnih linijah SV-HUC-1, RT4 in T24. Imunofluorescenčna analiza je pokazala Piezo1 na bazolateralni plazmalemi ter citosolu dežnikastih celic ter plazmalemi vmesnih in bazalnih celic. Označevanje obeh Piezo z imunoelektronsko mikroskopijo na ultratankih rezinah je bilo negativno, medtem ko je postopek ločevanja celic urotelija z dodatkom EDTA pokazal prisotnost Piezo1 na plazmalemi. V in vitro modelih se je pokazalo, da je bil signal za Piezo1 pri normalnih SV-HUC-1 prisoten v citoplazmi, pri Piezo2 tudi na plazmalemi, pri RT4 (neinvazivni karcinom) v citoplazmi, pri T24 (invazivni karcinom) pa je bilo izražanje proteinov Piezo zelo nizko. V uroteliju sta bila E-kadherin in β-katenin prisotna na vseh plazmalemah, razen apikalne, β-aktin pa ob adherentnih stikih. Reakcija za vinkulin je bila negativna. Kolokalizacije med proteini Piezo in E-kadherinom ali β-aktinom nismo zaznali, zaznali pa smo potencialno kolokalizacijo med Piezo1 in β-kateninom. Rezultati kažejo, da je urotelij sečnega mehurja miši in podgan dokončno diferenciran. Proteinov Piezo je v urotelijskih celicah malo v primerjavi s tkivi pod urotelijem. Dokazali smo, da je v modelu in vitro izražanje proteinov Piezo odvisno od diferenciacijske stopnje, medtem ko v normalnem uroteliju miši tega nismo mogli dokazati. Pokazali smo razporeditev proteinov medceličnih stikov in citoskeleta v uroteliju, vendar nismo potrdili povezave s Piezo1 ali Piezo2, ki bi kazala na aktivacijo s prenosom mehanske sile preko nitastih proteinov.

• Language: Slovenian
• Keywords: urotelij, proteini Piezo, diferenciacija, prenos western, imunofluorescenca, elektronska mikroskopija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-173242
• COBISS.SI-ID: 257172995
• Publication date in RUL: 15.09.2025

Secondary language

• Language: English
• Title: Effect of the differentiation stage of urothelial cells on the expression of Piezo mechanoreceptors

Abstract

The terminally differentiated urothelium of the urinary bladder maintains the blood-urine permeability barrier and plays an important mechanosensory role. Key mediators of this sensing are ion channels Piezo1 and Piezo2. We wanted to study the expression and localization of Piezo proteins in differently differentiated urothelial cells in vivo and in vitro, and to show a connection with cell adhesion and cytoskeletal proteins. The mouse urothelium consisted of 3 layers, while the rat had 1-2 layers. Mature fusiform vesicles and uroplakins were present in umbrella cells. In rats, urothelial plaques and tight junctions were present. We confirmed the expression of Piezo proteins in mice and rats in the urothelium and tissues beneath the urothelium (lamina propria, muscle) by Western blotting, with weaker bands on the urothelium. We also confirmed the expression of Piezo proteins in SV-HUC-1, RT4, and T24 cell lines. Immunofluorescence showed Piezo1 on the basolateral plasma membrane and cytosol of umbrella cells and on the plasma membrane of intermediate and basal cells. iEM staining of Piezo proteins on ultrathin sections was negative, while the process of separating urothelium cells with the addition of EDTA showed Piezo1 on the plasma membrane. In vitro models showed that the signal was present in the cytoplasm (and plasma membrane for Piezo2) in normal SV-HUC-1 cells and RT4 (non-invasive carcinoma) cells, and that the expression of Piezo proteins was very low in T24 (invasive carcinoma) cells. In urothelium, E-cadherin and β-catenin were present on all plasma membranes except the apical plasma membrane, while β-actin was present at adherent junctions. The reaction for vinculin was negative. We did not detect any colocalization between Piezo proteins and E-cadherin or β-actin, while we did detect potential colocalization between Piezo1 and β-catenin. The results show that the urothelium in mice and rats is fully differentiated. Both Piezo proteins are present in small amounts in urothelial cells compared to the tissues beneath the urothelium. We demonstrated that in an in vitro model, the expression of Piezo proteins depends on the differentiation stage, whereas in mouse urothelium, we were unable to demonstrate this. We showed the distribution of cell adhesion and cytoskeletal proteins in the urothelium, but we did not confirm a connection with Piezo proteins, which would indicate their activation by force from filament model.

• Keywords: urothelium, Piezo proteins, differentiation, immunofluorescence, western blot, electron microscopy