Superločljivostna mikroskopija kanalov akvaporina 4, Kir4.1 in ORF3a v plazemski membrani podganjih astrocitov

Rupnik, Marko (author), Jorgačevski, Jernej (mentor)

Delovanje astrocitov je ključnega pomena za uravnavanje vodne in ionske homeostaze v centralnega živčnega sistema (CŽS). V astrocitih igrajo pri teh procesih vodilno vlogo kalijevi kanalčki iz družine Kir (Kir4.1) in akvaporini tipa 4 (AQP4). Domneva se, da se kanala v plazemski membrani astrocitov izražata na podobnih mestih, zaradi česa se sklepa na njuno povezano funkcijo. Pri okužbah z nevtrotropnimi virusi kot so SARS-CoV-2 lahko pride do kliničnih znakov in simptomov, ki so posledica motenega delovanja CŽS. Mednje spadajo npr. ageusia, anosmia, težave s koncentracijo in utrujenost med in po prebolevanju COVID-19. Eden možnih krivcev za neravnovesje v CŽS ob okužbi je verjetno protein ORF3a, ki ga kodira genom SARS-CoV-2. Protein ORF3a je viroporin, ki lahko opravlja funkcijo ionskega kanala, poleg tega bi naj v celicah motil tudi zlivanje avtofagosomov z lizosomi. V raziskavi smo s pomočjo visokoločljivostne metode fluorescenčne mikroskopije (mikroskopija s strukturirano osvetlitvijo – SIM) preučili lokalizacijo Kir4.1 in AQP4 v celični kulturi podganjih astrocitov. V nadaljevanju smo raziskali še vpliv virusnega proteina ORF3a na izražanje AQP4 in Kir4.1. Pokazali smo, da se signala AQP4 in Kir4.1 ne prekrivata, kljub temu se v astrocitih izražata v neposredni bližini. Prav tako smo pokazali, da izražanje ORF3a v celični kulturi podganjih astrocitov vpliva na gostoto izražanja AQP4, čeprav se ORF3a in AQP4 v astrocitih prostorsko izražata povsem ločeno.

• Language: Slovenian
• Keywords: AQP4, Kir4.1, ORF3a, SARS-CoV-2, celična kultura, podganji astrociti, visokoločljivostna mikroskopija, SIM, kolokalizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [M. Rupnik]
• Year: 2022
• PID: 20.500.12556/RUL-140438
• UDC: 576
• COBISS.SI-ID: 122106883
• Publication date in RUL: 15.09.2022

Secondary language

• Language: English
• Title: Super-resolution imaging of aquaporin-4, Kir4.1 and ORF3a in the plasma membrane of rat astrocytes

Abstract

Astrocytes play a crucial role in the regulation of the ion and water homeostasis in the central nervous system (CNS). It is thought that inward rectifying potassium channels (Kir4.1) alongside aquaporin 4 (AQP4) play an important role in maintaining this homeostasis. Furthermore, it is also believed that Kir4.1 and AQP4 are expressed in close vicinity to each other, which gives additional support to the claims of a causal link in function between the two channels. Viral infections, such as SARS-CoV-2 infection, can lead to the development of neural symptoms caused by the dysfunction of water and ion homeostasis. These symptoms include ageusia, anosmia, lack of focus and persistent tiredness while and after COVID-19. One of the possible culprits is the protein ORF3a in the genome of the virus SARS-CoV-2. ORF3a is a viroporin and can facilitate passage of ions across cell membranes. ORF3a is also known to stunt the autophagosomes ability to fuse with lysosomes. In this work we used super resolution fluorescent microscopy (structured illumination microscopy – SIM) to analyze the distance between AQP4 and Kir4.1. We also studied the influence expression of ORF3a has on Kir4.1 and AQP4 expression in astrocytes. We showed that AQP4 and Kir4.1 do not directly colocalize in rat astrocyte cell culture, however they are expressed in close proximity to each other. We also showed that the presence of ORF3a in rat astrocytes influenced the expression of AQP4 even though ORF3a and AQP4 are expressed in different parts of astrocytes.

• Keywords: AQP4, Kir4.1, ORF3a SARS-CoV-2, cell culture, rat astrocytes, super resolution microscopy, SIM, colocalization