Mehansko občutljiv ionski kanal TRPV6 in prenašalec CorA ter njuna umestitev v transficiranih celicah HEK293T

Šimunović, Tina (author), Benčina, Mojca (mentor), Gunčar, Gregor (comentor)

Prilagajanje na signale iz okolja je ključna lastnost organizmov, ki jim omogoča preživetje. Celice signale sprejemajo preko membranskih proteinov, ki informacijo v notranjost celice posredujejo preko sekundarnih sporočevalcev. Mehansko občutljivi ionski kanali so ključni posredniki pri pretvarjanju mehanskega signala v nihanje koncentracije ionov. Pomembno vlogo sekundarnega sporočevalca imajo kalcijevi ioni, ki sodelujejo v skoraj vseh celičnih procesih, zaradi česar so ti visoko ohranjeni skozi evolucijo. V sklopu magistrskega dela smo želeli preveriti izražanje, delovanje in umestitev mehansko občutljivega kalcijevega kanala znotraj celice. Delo je obsegalo metode molekulskega kloniranja v bakterijskih celicah, transfekcijo sesalskih celic HEK293T, spremljanje jakosti signalov po stimulaciji z ultrazvokom ali kalcijevim ionoforom z luminometrom in umestitev kanala znotraj celice s fluorescenčnim konfokalnim mikroskopom. Pripravili smo genetska konstrukta podganjega mehansko občutljivega ionskega kanala TRPV6 iz naddružine TRP in prenašalca primarno magnezijevih ionov CorA iz glive Aspergillus niger. Genetska konstrukta sestavlja zapis za ionski kanal ali prenašalec, ki smo mu na C-konec dodali hemaglutininsko oznako, samoizrezujoči se peptid T2A in fluorescenčni protein TagBFP. Kot mehanski signal za aktivacijo kanalov smo testirali ultrazvok. Stimulacija z ultrazvokom povzroči odprtje kanalov, kar omogoči prehod kalcijevih ionov v celico, kjer se vežejo na kalcij vezavne proteine in posredno sprožijo izražanje tarčnih genov. Povišanje znotrajcelične koncentracije kalcijevih ionov smo spremljali kot izražanje luciferaze pod od kalcija odvisnim promotorjem NFAT. S kalcijevim ionoforom smo dosegli povišan odziv celic na kemijsko stimulacijo in dokazali, da se zasnovani poročevalski sistem odziva na kalcijeve ione. Celice, ki so izražale TRPV6, so sintetizirale nekoliko več luciferaze kot celice s pcDNA3. Nasprotno je bil odziv znižan v celicah s CorA. Pri stimulaciji z ultrazvokom smo pokazali, da v zasnovanem sistemu TRPV6 ne prispeva k občutljivosti celice, saj je bil odziv celic primerljiv celicam s pcDNA3. Podobno kot prej je pri CorA odziv znižan. S fluorescenčno konfokalno mikroskopijo smo pokazali, da se TRPV6 umešča na celično membrano, zaznamo pa ga tudi v znotrajceličnih organelih. Večinoma je na endosomih in v okolici jedra. CorA je v znotrajceličnih organelih. Celice, ki so izražale TRPV6 ali CorA, niso bolj občutljive na ultrazvočno stimulacijo od nespremenjenih celic HEK293T. Zasnovani sistem ni občutljiv na ultrazvok, kar bi bilo smiselno podrobneje preučiti v nadaljnjih raziskavah, saj ultrazvok, kot neinvazivno orodje za aktivacijo celic in globokih tkiv, pridobiva na prepoznavnosti in možnosti uporabe v sintezni biologiji, diagnostiki in terapiji.

• Language: Slovenian
• Keywords: sonogenetika, ultrazvok, mehansko občutljivi kanali, kalcij
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-109237
• COBISS.SI-ID: 1538321603
• Publication date in RUL: 28.08.2019

ŠIMUNOVIĆ, Tina, 2019, Mehansko občutljiv ionski kanal TRPV6 in prenašalec CorA ter njuna umestitev v transficiranih celicah HEK293T [online]. Master’s thesis. [Accessed 8 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=109237

Secondary language

• Language: English
• Title: Mechanosensitive TRPV6 Ion Channel and CorA Transporter and Their Localization in Transfected HEK293T Cells

Abstract

Fundamental survival feature of organisms is an adequate response to the environmental signal. Cells sense extracellular signals via membrane proteins, which transfer signals inside the cell through second messengers. Mechanosensitive ion channels are the key transductors of mechanical signals to ion flux. Calcium ions are important second messengers involved in many different cellular processes, and are therefore highly conserved through evolution. As a part of this master’s thesis, we aimed to study expression, activity and localization of mechanosensitive calcium channel. Experimental methods included molecular cloning in bacterial cells, transfection of mammalian HEK293T cells, monitoring the intesity of signals after ultrasound or calcium ionophore stimulation with a luminometer, and fluorescence confocal microscopy for localization imaging. We prepared gene constructs expressing rat mechanosensitive TRPV6 ion channel of TRP superfamily and CorA magnesium ion transporter from fungus Aspergillus niger. Genes for TRPV6 or CorA were tagged at C-terminus with hemagglutinin tag, self-cleaving T2A peptide and fluorescent protein TagBFP. Ultrasound was tested as a mechanical signal for activation. Ultrasound stimulation opens channels and enables the influx of calcium ions. Calcium ions bind calcium-binding proteins inside the cell and trigger expression of genes under calcium-dependent promotor, such as reporter protein luciferase and NFAT promotor. With calcium ionophore, we proved the functionality of reporter system, as there was an elevated cell response. Cells expressing TRPV6 synthesized more luciferase than cells expressing pcDNA3. In contrast, cells expressing CorA attenuated the response. Ultrasound stimulation results showed that TRPV6 does not contribute to ultrasound cell sensitivity, as the response was comparable to cells transfected with the empty pcDNA3 plasmid. As before, CorA response to ultrasound was attenuated. Fluorescence confocal microscopy showed TRPV6 located in cell membrane, endosomes and around the nucleus. CorA is observed in cellular organelles. Cells transfected with TRPV6 ion channel or CorA transporter were as sensitive to ultrasound stimulation as untransfected, control cells. These should be the subject of further research because ultrasound as a non-invasive tool for cell and deep tissue activation is gaining more recognition and potential applications in synthetic biology, diagnostics and therapy.

• Keywords: sonogenetics, ultrasound, mechanosensitive channels, calcium