Provider: - Institution: - Data provided by Europeana Collections- Cilj istraživanja: Prvenstveni cilj ovoga rada bila je uspostava modela za praćenje putovanja MHC molekula I. razreda, ali i ostalih površinskih molekula, u neinficiranim stanicama i u stanicama inficiranim MCMV divljim-tipom ili različitim mutantama virusa MCMV kojima nedostaje/ju geni čija je funkcija u literaturi opiasana. Poznato je da se MHC-I molekule puno brže uklanjaju sa površine inficirane stanice nego sa površine neinficirane stanice, upravo je stoga mjerena kinetika uklanjanja površinskih MHC-I molekula. Također su istražena načela ulaska MHC-I molekula u put recikliranja te njihov ulazak u degradacijski put, odnosno u kasne endosome. Materijal i metode: U pokusima smo koristili staničnu liniju mišjih embrionalnih fibroblasta (MEF-a). U istraživanjima smo koristili neinficirane i inficirane stanice te određivali možebitne razlike. Slijedeće rekombinante virusa MCMV su korištene: Δm138-MCMV (ΔMC95.15) s delecijom fcr1 (m138) gena , Δie1-MCMV s delecijom egzona 4 ie1 gena, Δie2-MCMV s delecijom ie2 (m128) gena, Δie3-MCMV s delecijom ie3 gena, Δm4m6m152-MCMV s delecijom tri poznata imunoinvaziva koji utječu na sazrijevanje MHC-I molekula te MCMV divlji-tip, koji je kao i gotovo sve MCMV-delecijske mutante proizvedene i umnažan na kulturi MEF porijeklom iz BALB/c miševa. Δie3-MCMV uzgajan je na komplementarnoj NIH3T3 BAM25 staničnoj liniji. U svrhu praćenja MHC-I molekula koristili smo monoklonska protutijela (mPt): MA-215 za konformirane Kd i 34-5-8S za konformirane Dd. Transferinski receptor (TfR) smo pratili pomoću mPt R17, a molekule GM1 pomoću obilježene podjedinice B kolera toksina. Kako bismo utvrdili koji su putovi uključeni u ulazak molekula u stanicu, endosomalno putovanje, recikliranje i degradaciju, koristili smo različite kemijske inhibitore. Kinetiku internalizacije pratili smo protočnom citometrijom, a unutarstanično putovanje konfokalnom mikroskopijom koristeći princip površinskog vezivanja odgovarajućeg mPt i njegovog praćenja nakon određene kinetike internalizacije. Internalizirane molekule kolokalizirali smo međusobno, te s određenim endosomalnim markerima. Recikliranje molekula pratili smo pomoću dva različita protokola metodom protočne citometrije i konfokalne mikroskopije. U cilju identifikacije sudbine površinskih proteina, koristili smo metode površinske biotinilacije, imunoprecipitacije, elektroforeze na poliakrilamidnim gelovima (SDS-PAGE), Western-blota i kemiluminiscencije. Rezultati: Obrazac izražaja MHC-I molekula kako na površini tako i u unutrašnjosti stanice mijenja se u uvjetima MCMV-infekcije. MHC-I molekule nishodno se reguliraju s površine inficirane stanice procesom koji nije isključivo za njih specifičan, potom se nakupljaju u hibridnom odjeljku koji je pozitivan na mnogobrojne biljege unutarstaničnih endosomalnih putova ili odjeljaka (EEA-1+, TfR, GM130). MHC-I molekule internaliziraju se brže u inficiranim nego u neinficiranim stanicama putem koji je neovisan o dinaminu. Niti jedan od ispitivanih kemijskih inhibitora endocitoze (klorpromazin, imipramin, filipin i AlF4-) nije značajnije blokirao internalizaciju MHC-I molekula. Nishodna regulacija MHC-I molekula s površine inficirane stanice je proces 5-6 puta brži od konstitutivne internalizacije te je najvjerojatnije induciran virusnim proteinima. MCMV usmjerava MHC-I molekule nakon internalizacije sa površine stanice, u odjeljak u kojem ih i nakuplja. Internalizirane MHC-I molekule nalaze se u mjehurićima koji ne mogu otpustiti EEA-1 odnosno rane endosomalne membrane, što bi bio jedan od razloga njihovog odgođenog ulaska u endolizosomalni put u proces degradacije. Iz odjeljka koji se formira u MCMV-inficiranim stanicama u kojem se nakupljaju mnogobrojne internalizirane molekule moguće je recikliranje, ali je kapacitet recikliranja smanjen. Retencijski odjeljak sadrži većinom membrane ranih endosoma s obzirom da samo mali dio molekula može reciklirati pri 16 °C i uz prisustvo LY294002, što su karakteristike jukstanuklearnog reciklirajućeg odjeljka. Najvjerojatniji uzrok promjene recikliranja u MCMV-inficiranim stanicama je smanjenje ukupne količine Rab11 molekule te smanjenje mogućnosti stvaranja reciklirajućh membranskih domena. Ulazak u put degradacije za mnoge je molekule odgođen u MCMV-inficiranim stanicama neovisno o njihovu putu ulaska u stanicu te je otežano formiranje endolizosomalnih mebrana. U MCMV-inficiranim stanicama dolazi do promjena u izražavanju pojedinih molekula Rab. Razina Rab5 u inficiranim stanicama ne smanjuje se poput ostalih molekula Rab, te dolazi do otežane pretvorbe Rab5 u Rab7, kao i do smanjenja razine Rab7 u inficiranim stanicama, što za posljedicu ima odgođeno odvođenje molekula u put degradacije. Za nishodnu regulaciju MHC-I molekula s površine inficirane stanice odgovoran je ili ie3 gen ili oni geni koji su pod kontrolom ie3 gena. m06/gp48 protein odgovoran je za usmjeravanje kako novo-sintetiziranih tako i internaliziranih MHC-I molekula u proces degradacije. Zaključak: Endosomalno remodeliranje je mehanizam evoluirao u svrhu MCMV morfogeneze u inficiranih stanica, jer ne utječe samo na MHC-I molekule, nego i na druge endocitirane molekule koje koriste različite endosomalne putove. Ovo remodeliranje se nadopunjuje blokiranjem izlaza MHC-I molekula iz sekretnog puta što dovodi do ubrzanog gubitka MHC-I molekula s površine stanice u ranim razdobljima infekcije.- Objectives: The aim of this study was to establish experimental model that will enable us to follow constitutive internalization of MHC class I. molecules and other cell surface expressed molecules in uninfected or in the cell infected with either MCMV wild-type or with different MCMV deletion mutant. It is well known that MHC-I molecules are downregulated with higher rate from infected cell than from uninfected cell, so we measured the kinetics of MHC-I molecules downregulation. Additionally, we investigated the principles of MHC-I molecules endocytosis, their recycling pathway and also MHC-I molecules entrance in degradation pathway respectively, in late endosomes. Material and methods: Experiments were performed on murine embrional fibroblasts (MEFs). In our experiment we used uninfected and cell infected with numerous MCMV: Δm138-MCMV (ΔMC95.15) with the deletion of the fcr1 (m138) gene, Δie1-MCMV with the deletion of exon 4 ie1 gene, Δie2-MCMV with the deletion of the ie2 (m128) gene, Δie3-MCMV with the deletion of the ie3 gene, Δm4m6m152-MCMV with deletion of three well known immunoevasins that affect MHC-I molecules maturation. The MCMV wild-type and MCMV deletion mutants were propagated on BALB/c MEFs. The Δie3-MCMV was grown on the complementing cell line NIH3T3 BAM25. In order to follow MHC-I molecules, we have used a monoclonal antibodies (mAbs): MA-215 for conformed Kd molecules and 34-5-8S for conformed Dd molecules. Transferrin receptor (TfR) was followed by mAb R17, and GM1 molecules with labeled cholera toxin B subunits. In order to determine which pathways are involved in endocytosis, endosomal trafficking, recycling and degradation of these molecules, we used different chemical inhibitors of endocytosis. The kinetics of surface molecules was followed by flow citometry and their intracellular trafficking by confocal microscopy after binding of appropriate mAbs. Internalized molecules were colocalized with each other as well as with specific endosomal markers. After rearranging two different protocols from the literature we followed molecules recycling by flow citometry and confocal microscopy. In order to monitor the kinetics of degradation, we used the method of surface biotinilation and imunoprecipitation, electrophoresis on polyacrylamide gels (SDS-PAGE), Western-blot and chemiluminiscence. Results: The pattern of MHC-I molecules expression on the cell surface and intracellular was changing during the infection. MHC-I molecules were downregulated from the surface of infected cell with process that is not specific for them since the other cell surface expressed molecules have also been downregulated. After their downregulation MHC-I molecules were accumulated in hybrid compartment which is positive on various markers of endosomal pathways and compartments (EEA-1+, TfR, GM130). Kinetics of MHC-I molecules internalization were significantly faster in infected cells than in uninfected cells following the pathway that is dynamin independent. Neither of the tested chemical inhibitors (chlorpromazine, imipramin, filipin and AlF4-) did not block internalization of MHC-I molecules. Downregulation of cell surface expressed MHC-I molecules is process that is 5-6 times faster than constitutive internalization and it is probably induced with viral proteins. MCMV directs after internalization from the cell surface MHC-I molecules in the retention compartment. Internalized MHC-I+ vesicles are unable to release EEA-1 respectively early endosomal membrane what could clarify their delayed entry into endolysosomal route in the process of degradation. In MCMV-infected cells many of the internalized molecules have been accumulated in the retention compartment from which recycling was possible although the capacity of the recycling was reduced. Retention compartment contains most of the membrane of early endosomes since only small amount of the molecules was capable to recycle on 16 °C even in the presence of LY294002, which are the characteristics juxtanuclear recycling compartment. The most likely cause of changes in the recycling of MCMV-infected cells was reduction of the total amount of Rab11 molecules that decreased the possibility of creating recycling membrane domains. Degradation pathway has been affected in MCMV-infected cells for numerous molecules regardless to their pathway of entry into the cell. Besides the amount of the Rab11 molecules in MCV-infected cells the level of other Rab molecules was altered. The level of Rab5 in infected cells did