Title Endocitozni put i recikliranje MHC molekula I razreda : doktorska disertacija Title (english) Endocytic pathway and recycling of MHC class I molecules Author Gordana Blagojević Zagorac Mentor Pero Lučin (mentor)Committee member Blaženka Grahovac (predsjednik povjerenstva)Committee member Alenka Gagro (član povjerenstva)Committee member Sanja Balen (član povjerenstva)Committee member Pero Lučin (član povjerenstva)Granter University of Rijeka Defense date and country 2010-02-11, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 577 - Biochemistry. Molecular biology. Biophysics Abstract Cilj istraživanja: Prvenstveni cilj ovoga rada bila je karakterizacija konstitutivne internalizacije konformiranih i nekonformiranih MHC molekula I razreda, odnosno karakterizacija njihove endocitoze, recikliranja i degradacije. Poznato je da razlika u konformaciji MHC-I molekula uzrokuje njihovo razdvajanje u sekretornom putu. Stoga su u ovom radu istražena načela njihova razdvajanja tijekom endocitoze i putovanja kroz endosomalne odjeljke. Na temelju eksperimentalnih mjerenja cilj nam je bio i uspostavljanje matematičkog modela za kinetičku analizu endosomalnih putova.
Materijal i metode: Istraživanja smo proveli na mišjim fibroblastima J26 transficiranim s genom koji kodira molekule HLA-B7 (J26-B7), HLA-Cw6 (J26-Cw6) ili HLA-G1 (J26-G1), na humanoj tumorskoj staničnoj liniji HeLa koja konstitutivno izražava sve klasične HLA molekule I razreda, Balb 3T3 mišjim fibroblastima, te L-Ld stanicama (mišji fibroblasti transficirani genom za molekule H2-Ld). Za praćenje MHC molekula I razreda koristili smo monoklonska protutijela (mPt): mPt W6/32, koje prepoznaje sve klasične konformirane HLA molekule i konformirane HLA-G1 molekule, mPt HC-10, koje prepoznaje sve klasične nekonformirane HLA molekule, mPt MEM-G/1 koje prepoznaje nekonformirane molekule HLA-G1, mPt 30-5-7 koje prepoznaje konformirane H2-Ld molekule, te mPt 64-3-7 koje prepoznaje nekonformirane H2-Ld molekule. Transferinski receptor (TfR) smo pratili pomoću mPt R17, a molekule GM1 pomoću obilježene podjedinice B kolera toksina. Neionski detergent Triton X-100 nam je poslužio za ispitivanje nazočnosti molekula u lipidnim splavima. Oponašanjem uvjeta endosomalnog pH izvan stanice, utvrdili smo promjenu konformacije MHC molekula I razreda u zadanim uvjetima. Kako bismo utvrdili koji putovi su uključeni u endocitozu, endosomalno putovanje, recikliranje i degradaciju ovih molekula, koristili smo različite kemijske inhibitore. Kinetiku internalizacije pratili smo protočnom citometrijom, a unutarstanično putovanje konfokalnom mikroskopijom koristeći princip površinskog vezivanja odgovarajućeg monoklonskog protutijela i njegovog praćenja nakon određene kinetike internalizacije. Internalizirane molekule kolokalizirali smo međusobno, te s određenim endosomalnim markerima. Recikliranje molekula pratili smo modificirajući tri različita protokola iz literature i to protočnom citometrijom i konfokalnom mikroskopijom. U cilju praćenja kinetike degradacije koristili smo metodu površinske biotinilacije, te imunoprecipitacije, elektroforeze na poliakrilamidnim gelovima (SDS-PAGE), Western-blota i kemiluminiscencije. Za matematičko modeliranje i simulacije endocitoznih procesa koristili smo programski paket Mathematica (Wolfram Research Europe Ltd.). Rezultati: Konformirane MHC-I molekule dobro su izražene na staničnoj površini za razliku od nekonformiranih molekula. Djelovanjem kiselog pH (fiziološke i nefiziološke vrijednosti) izražaj konformiranih MHC-I molekula pada, dok izražaj nekonformiranih raste. Različite konformacije MHC-I molekula u ustaljenom stanju samo djelomično se kolokaliziraju unutar stanice i to prvenstveno u ranim endosomalnim odjeljcima. Niti jedan od ispitivanih kemijskih inhibitora (klorpromazin, Dynasore, filipin i AlF4-) nije blokirao internalizaciju MHC-I molekula. Nekonformirane MHC-I molekule se znatno brže internaliziraju i degradiraju od konformiranih MHC-I molekula što je najvjerojatnije posljedica činjenice da se konformirane MHC-I molekule usmjeravaju u reciklirajuće odjeljke, a nekonformirane MHC-I molekule u degradacijski put. Tome u prilog govori rezultat da se konformirane MHC-I molekule nakon internalizacije nalaze prvenstveno u jukstanuklearnom području zajedno s Tf/TfR i Rab11 GTPazom, dobro poznatim biljezima jukstanuklearnih reciklirajućih endosoma. Internalizirane konformirane MHC-I recikliraju s učinkovitošću od oko 30%, znatno manje od TfR/Tf koji recikliraju s učinkovitošću od 85-99%, što ukazuje da konformirane MHC-I molekule ulaze u zasebne reciklirajuće odjeljke. Ubrzo nakon internalizacije konformirane i nekonformirane MHC-I se kolokaliziraju s biljegom ranih endosoma (EEA-1), kao i međusobno, ali nedugo zatim stupanj kolokalizacije se smanjuje što znači da se razmjerno brzo razdvajaju. To razdvajanje blokira LY294002 (blokator PI3K), kao i konkanamicin A (blokator endosomalne H+ crpke). Djelovanjem Tx-ispiru se konformirane MHC-I molekula i TfR sa staničnih membrana, dok se nekonformirane MHC-I molekule i GM1 ne ispiru. Matematičkim modeliranjem potvrđeni su eksperimentalni rezultati, te izračunati slijedeći parametri: stopa endocitoze, stopa recikliranja i stopa degradacije.
Zaključak: Konformirane MHC-I molekule kao i TfR nalaze se u detergent-osjetljivim membranskim mikrodomenama za razliku od nekonformiranih MHC-I molekula i GM1, kako na staničnoj površini tako i u unutrašnjosti stanice. Internalizacija MHC-I molekula nije ovisna o klatrinu i dinaminu, već najvjerojatnije o funkciji malih GTPaza (Cdc42 i Arf6). Sortiranje, odnosno odvajanje endosomalnih putova konformiranih i nekonformiranih MHC-I molekula odvija se na razini ranih endosoma i ovisno je o pH. Nakon sortiranja konformirane MHC-I molekule se prvenstveno usmjeravaju u recikliranje, a nekonformirane u degradaciju.
Abstract (english) Objectives: The aim of this study was to characterize constitutive internalization of conformed and non-conformed MHC class I molecules, with respect to their endocytosis, recycling and degradation. Conformational difference is known to segregate MHC-I molecules in secretory pathway. Therefore, we investigated endocytic pathway and putative segregation principles of these molecules during endocytic trafficking. Additionally, based on experimental measurements our goal was to establish a mathematical model for kinetics analysis of endosomal transport.
Material and methods: Experiments were performed on murine fibroblasts J26 transfected with HLA-B7 (J26-B7), HLA-Cw6 (J26-Cw6) or HLA-G1 molecules (J26-G1), on human HeLa cell line that constitutively expresses all classical HLA class I molecules, Balb 3T3 murine fibroblasts, and murine L cells stably transfected with Ld molecules (L-Ld). In order to follow MHC class I molecules, we used the following monoclonal antibodies (mAb): mAb W6/32 for all classical conformed HLA-I molecules and for conformed HLA-G1 molecules, mAb HC-10 for all classical non-conformed HLA-I molecules, mAb MEM-G/1 for non-conformed HLA-G1 molecules, mAb 30-5-7 for conformed H2-Ld molecules, and mAb 64-3-7 for non-conformed H2-Ld molecules. Transferrin receptor (TfR) was followed by mAb R17, and GM1 molecules with labeled cholera toxin B subunits. Non-ionic detergent Triton X-100 was used for testing the presence of molecules in lipid rafts. Conditions of endosomal pH were simulated outside the cell and the conformation changes of MHC-I molecules were under given conditions. In order to determine which pathways are involved in endocytosis, endosomal trafficking, recycling and degradation of these molecules, we used different chemical inhibitors. 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 three 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. For mathematical modeling and simulation of endocytic processes we used a software package Mathematica (Wolfram Research Europe Ltd.). Results: Conformed MHC-I molecules are well expressed on the cell surface, unlike non-conformed molecules. Expression of conformed MHC-I molecules decreases due to treatment with acid pH (physiological and non- physiological values), while expression of non-conformed MHC-I molecules increases. Different conformations of MHC-I molecules in steady state conditions are only partially colocalized intracellular, primarily in the early endosomal compartments. Neither of the tested chemical inhibitors (chlorpromazine, Dynasore, filipin and AlF4-) did not block internalization of MHC-I molecules. Kinetics of internalization, as well as degradation of non-conformed MHC-I molecules are significantly faster than kinetics of conformed MHC-I molecules, due to fact that conformed MHC-I molecules recycle, unlike non-conformed molecules. These thesis are supported also by the finding that conformed MHC-I molecules after internalization are directed primarily in juxtanuclear region where they colocalize with Tf/TfR, and with Rab11. Internalized conformed MHC-I molecules recycle with 30% efficiency, while TfR/Tf recycling efficiency is 85-99%. Shortly after internalization conformed and non-conformed MHC-I molecules colocalize with early endosomal marker EEA-1, as well as to each other, but soon thereafter the level of colocalization decreases. This separation is blocked due to the influence of LY294002 (inhibitor of PI3K) or concanamycin A (inhibitor of endosomal H+ pump). Treatment with Tx-100 removes conformed MHC-I molecules and TfR from the cell membranes, while non-conformed MHC-I molecules and GM1 are resistant to the Tx-100 treatment. Experimental results are confirmed by mathematical modeling, by which the following parameters are also calculated: the rate of endocytosis, the rate of recycling and the rate of degradation.
Conclusion: Conformed MHC-I molecules, as well as transferrin receptor are localized in detergent-sensitive membrane micro domains, while non-conformed MHC-I molecules and GM1 are localized in detergent-resistant membrane micro domains, not only on the cell surface, but also intracellularly. Internalization of MHC-I molecules is clathrin- and dynamin-independent, but probably depends on the function of small GTPases (Cdc42 and Arf6). Sorting and separation of endosomal pathways of conformed and non-conformed MHC-I molecules occur at the level of early endosomes and is pH dependent. After sorting, conformed MHC-I molecules are primarily directed to recycling while non-conformed are directed to degradation.
Keywords
Endosomalno recikliranje
Internalizacija
endocitoza
Lipidne splavi
Matematičko modeliranje
MHC molekule I razreda
Sortiranje proteina
Vezikularni transport
Keywords (english)
Endosomal recycling
Internalization
endocytosis
Lipid rafts
Mathematical modeling
MHC class I molecules
Protein sorting
Vesicular transport
Language croatian URN:NBN urn:nbn:hr:188:855159 Project Number: 0062030 Project Number: 062-0620238-0223 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL http://libraries.uniri.hr/cgi-bin/ucat/unilib.cgi?form=D1120729055 Type of resource Text Extent 220 str. File origin Born digital Access conditions Open access Terms of use Created on 2017-01-19 18:03:03
