20.500.12556/RUL-172588 Vpliv plektina na mobilnost avtofagosomov in avtolizosomov ter določanje deleža laktilacije citoskeletnih proteinov v mišjih astrocitih Exploring Plectin's Role in Autophagic Compartment Dynamics and Cytoskeletal Lactylation in Mouse Astrocytes. Astrociti so najštevilčnejše celice glije v možganih. Nemoten potek avtofagije v astrocitih je ključnega pomena za razgrajevanje okvarjenih organelov in preprečuje nabiranje proteinskih agregatov. Pri uspešnem poteku avtofagne poti ima pomembno vlogo citoskelet, ki omogoča učinkovito potovanje avtofagnih predelkov in zorenje avtofagosomov v avtolizosome. V diplomskem delu smo raziskovali vpliv, ki ga ima plektin, pomemben povezovalec proteinov citoskeleta, na mobilnost avtofagosomov in avtolizosomov v mišjih astrocitih. Analizo smo izvedli s programom ImageJ, kjer smo z vtičnikom TrackMate analizirali premikanje avtofagnih predelkov na fluorescenčnih mikrografijah mišjih astrocitov divjega tipa in astrocitov, ki plektina niso izražali. V realnem času smo spremljali premikanje avtofagosomov in avtolizosomov, ki so bili fluorescenčno označeni s proteinom mRFP-EGFP-LC3. Astrocite smo predhodno transficirali z vektorjem ptfLC3. Pokazali smo, da so v mišjih astrocitih, ki niso izražali plektina, avtofagni predelki potovali hitreje in bolj usmerjeno v primerjavi z astrociti, ki so izražali plektin. V drugem delu diplomske naloge nas je zanimal vpliv, ki ga ima receptor GPR27 na delež laktilacije proteinov citoskeleta in plektina. L-laktilacija je posttranslacijska modifikacija, ki so jo odkrili leta 2019 in o kateri ostaja še veliko odprtih vprašanj. Gre za vezavo laktil CoA oz. laktoilglutationa na lizinski aminokislinski ostanek v proteinih in je povezana s koncentracijo znotrajceličnega L-laktata. GPR27 je GPCR, ki se izraža v možganih. Vloga GPR27 v celici ni točno določena, vemo pa, da vpliva na koncentracijo znotrajceličnega L-laktata. V mišjih astrocitih, v katerih smo fluorescenčno označili proteine citoskeleta (vimentin, α-tubulin in F-aktin) in plektin, smo z drugim fluorescenčnim označevalcem označili tudi L-laktiliran lizin. Pod konfokalnim mikroskopom smo opazovali astrocite divjega tipa in astrocite, ki niso izražali receptorja GPR27, nakar smo izvedli računalniško analizo s programom ImageJ. Izkazalo se je, da so proteini citoskeleta imeli višji delež laktilacije pri astrocitih divjega tipa v primerjavi z astrociti, ki receptorja GPR27 niso izražali. Delež laktilacije je naraščal z naraščajočim deležem lizina v aminokislinskem zaporedju proteina. Pri plektinu je bil pojav ravno obraten, predvidoma zaradi globularne oblike proteina in nizke izpostavljenosti aminokislinskih ostankov laktilaciji. Astrocytes are the most abundant glial cells in the brain and play a crucial role in the central nervous system. Normal progression of the autophagic pathway in astrocytes is important for the removal of defective organelles and to prevent the accumulation of protein aggregates. The cytoskeleton acts as a stabilizing network for the mobility of autophagic vesicles and the maturation of autophagosomes into autolysosomes. The first part of the thesis focuses on the analysis of autophagic compartment mobility. We compared the mobility of autophagic compartments between wild-type cells expressing plectin and plectin-knockout cells. Plectin is a cytolinker protein that is essential for maintaining cellular integrity by anchoring the cytoskeleton to various cellular structures. Specifically, we analysed confocal micrographs of mouse astrocytes using the plugin TrackMate in ImageJ. We monitored the trafficking of autophagosomes and autolysosomes that were fluorescently labeled with the mRFP-EGFP-LC3 protein. Astrocytes were previously transfected with the ptfLC3 vector. The analysis showed that the autophagic compartments in plectin-knockout mouse astrocytes had higher mean speed and a higher ratio of directional movement compared to wild-type astrocytes. In the second part of the thesis, we investigated the effect of the GPR27 receptor on the degree of lactylation of cytoskeletal proteins and plectin. L-lactylation is a post translational modification that was discovered in 2019. It consists of the addition of a molecule of lactyl-CoA or lactoylglutathione on the lysine residue and is related to the intracellular L-lactate concentration. GPR27 is a GPCR expressed in the brain. Its role in the cell has not been elucidated yet, however it is known to affect intracellular L-lactate concentration. Mouse astrocytes that were then observed under the confocal microscope were immunolabeled with two different fluorescent markers: the first binds to a cytoskeletal protein (vimentin, α-tubulin or F-actin) or to plectin, while the second binds to the L-lactylated amino acid lysine. We analyzed wild-type astrocytes and astrocytes lacking the GPR27 receptor by using ImageJ software. The analysis showed that wild type astrocytes had a higher degree of lactylation of the cytoskeletal proteins compared to GPR27-knockout astrocytes. The degree of lactylation was higher for proteins with a higher proportion of lysine in the amino acid sequence. Interestingly, plectin showed the opposite trend, presumably due to the protein’s globular structure compared to the filamentous structure of the cytoskeletal proteins, where lysine amino acids have a much higher exposure to lactylation. astrociti avtofagni predelek plektin GPR27 laktilacija astrocytes autophagic compartment plectin GPR27 lactylation true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-09-09 12:05:00 2025-09-09 12:05:09 2025-10-30 12:16:09 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25723 255417091 Vogric_Vanja_-_Vpliv_plektina_na_mobilnost_avtofagosomov_in_avtolizosomov_ter_dolocanje_deleza_l.pdf Vogric_Vanja_-_Vpliv_plektina_na_mobilnost_avtofagosomov_in_avtolizosomov_ter_dolocanje_deleza_l.pdf 1 87CEE8981447EF656B44203B88B31268 87d2486fa22d1829e80a43177c2c3b3548417708f6f8475455452f782344726d 4f65a5c8-8d64-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=216494 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0