20.500.12556/RUL-187076 Vpliv receptorja GPR27 na organizacijo citoskeleta in mehanske lastnosti astrocitov Regulatory role of GPR27 in cytoskeletal dynamics and mechanical behavior of astrocytes Astrociti, najštevilčnejše celice glije, so morfološko plastične celice centralnega živčnega sistema. Vse več dokazov kaže, da noradrenalin (NA) pomembno uravnava spremembe v njihovi morfologiji, kot je tvorba celičnih podaljškov, kar pa je neposredno povezano z reorganizacijo citoskeleta in posledično mehanskimi lastnostmi celic. GPR27 je receptor sirota iz družine z G-proteini sklopljenih receptorjev, katerega vloga v astrocitih še ni pojasnjena. V magistrskem delu smo raziskovali vpliv GPR27 in NA na organizacijo citoskeleta ter mehanske lastnosti mišjih astrocitov. Z visokoločljivostno mikroskopijo STED in mikroskopijo z detektorjem Airyscan smo analizirali organizacijo in prostorsko zasedenost aktinskih filamentov, mikrotubulov in intermediarnih filamentov fiksiranih celic, z mikroskopijo atomskih sil (AFM) pa smo določili Youngov modul živih celic, ki odraža togost oz. elastičnost celic. NA je povzročil reorganizacijo vseh treh citoskeletnih omrežij, vendar je bila odvisnost odziva od GPR27 različna. Najizrazitejši vpliv GPR27 smo ugotovili pri aktinskem citoskeletu. Pri astrocitih divjega tipa je NA povečal vzporednost in gostoto aktinskega omrežja ter njegovo prostorsko zasedenost na ravni celotne celice in periferne regije, medtem ko pri GPR27 KO primerljivega odziva ni bilo oz. je bila sprememba vzporednosti nasprotna. Gostota mikrotubulnega omrežja in omrežja intermediarnih filamentov se je po tretiranju z NA povečala neodvisno od genotipa. Pri mikrotubulih je NA povečal tudi prostorsko zasedenost celotne celice ter perinuklearne in periferne regije, medtem ko se je pri intermediarnih filamentih od genotipa odvisna sprememba pokazala predvsem perinuklearno. Rezultati kažejo, da GPR27 predvsem modulira aktinsko plastičnost astrocitov ob adrenergični stimulaciji. Razlike med genotipoma so bile pri nekaterih parametrih prisotne že pred stimulacijo. Kljub citoskeletnim spremembam vpliva GPR27 ali NA na mehanske lastnosti celic nismo potrdili. Youngov modul se ni razlikoval ne glede na genotip ne tretma, medtem ko je bil pri vseh celicah značilno višji na periferiji kot pa nad jedrom. Astrocytes, the most abundant glial cells, are morphologically plastic cells of the central nervous system. Growing evidence indicates that noradrenaline (NA) plays an important role in regulating changes in their morphology, such as the formation of cellular processes, which are directly associated with cytoskeletal reorganization and, consequently, with the mechanical properties of the cells. GPR27 is an orphan G protein-coupled receptor whose role in astrocytes remains unclear. In this master's thesis, we investigated the effects of GPR27 and NA on cytoskeletal organization and the mechanical properties of mouse astrocytes. Using high-resolution STED microscopy and Airyscan microscopy, we analysed organisation and spatial occupancy of actin filaments, microtubules, and intermediate filaments of fixed cells, while atomic force microscopy (AFM) was used to determine the Young’s modulus of living cells, reflecting their stiffness or elasticity. NA induced reorganization of all three cytoskeletal networks; however, the dependence of these responses on GPR27 differed among the networks. The most pronounced effect of GPR27 was observed in the actin cytoskeleton. In wild-type astrocytes, NA increased the parallelness and density of the actin network, as well as its spatial occupancy at the whole-cell level and in the peripheral region, whereas no comparable response was observed in GPR27 KO astrocytes, and the change in parallelness was in the opposite direction. The density of both the microtubule and intermediate filament networks increased following NA treatment independently of genotype. In the microtubule network, treatment with NA also increased spatial occupancy across the whole cell and in both the perinuclear and peripheral regions, whereas in the intermediate filament network, a genotype-dependent change was observed primarily in the perinuclear region. For some parameters, differences between the genotypes were already present before stimulation. These results suggest that GPR27 primarily modulates actin plasticity in astrocytes during adrenergic stimulation. Despite the cytoskeletal changes, we found no evidence that GPR27 or NA affected the mechanical properties of the cells. Young’s modulus did not differ with either genotype or treatment, while it was significantly higher at the cell periphery than above the nucleus in all cells. AFM detektor Airyscan astrociti citoskelet GPR27 STED Youngov modul AFM Airyscan detector astrocytes cytoskeleton GPR27 STED Young's modulus true false false [J. Gale] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-09 07:18:17 2026-09-09 07:18:21 2026-09-10 04:30:37 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 576.31:577.175.5(043.2) 287563 290480899 61680.pdf 61680.pdf 1 DB9ABCFD348842DACFF8DD20A2C6403A 2a1d15a62d623b053b82be39a3368e9f1b5ce94a95310362d80abac1695fcf66 dac6088b-ac0d-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246512 Biotehniška fakulteta 0 0 0