Vpliv mutacij E475G in P25L v keratinu 5 na fenotip keratinocitov gojenih v celični kulturi

Cvetkovska, Janina (author), Liović, Mirjana (mentor)

Keratini (K) so največja skupina intermediarnih filamentov. Zaradi svoje strukture se organizirajo v navite dimere. K5 tvori heterodimerne strukture s K14 in v veliki meri se izražata v bazalnih keratinocitih v epidermisu kože. Njuna glavna vloga je fizična stabilnost epidermisa. Določene mutacije v K5 ali K14 povzročajo bolezen, imenovano bulozna epidermoliza simpleks (EBS), katere značilnost je povišana krhkost kože in tvorba mehurjev. V magistrskem delu nas je zanimalo, kako na značilnosti keratinocitov, gojenih v celični kulturi, vpliva mutacija E475G v K5, ki povzroča hudo obliko EBS, in P25L v K5, ki povzroča blažjo obliko EBS z lisasto pigmentacijo. Uporabili smo celično linijo NEB-1, v katero je bil vstavljen konstrukt EGFP-K5, bodisi divji tip (WT), E475G ali P25L. Nameravali smo s klonsko selekcijo priti do klonov, kjer se bi omenjen konstrukt izražal v določenem deležu glede ne endogeni K5 in takšne klone medsebojno primerjati. S pomočjo NaDS-PAGE in prenosa po Westernu smo ugotovili, da smo imeli na voljo le klone, ki so izražali konstrukt v približno enaki, nizki količini. Z optično pinceto smo izmerili togost celičnih membran in ugotovili, da noben izmed konstruktov ne vpliva na togost membran. Ugotovili smo, da konstrukt EGFP-K5 E475G, tudi če je prisoten v majhni količini, povzroča povišano spontano tvorbo agregatov in ne le na periferiji celic. To je v nasprotju z našimi predvidevanji, saj v predhodnih študijah ni bilo potrjeno, da celice z mutacijo E475G zmotijo dinamiko keratinskega citoskeleta na ta način, da se poviša spontana tvorba agregatov. Pri teh celicah smo zaznali povišano izražanje K16 in K19. Oblikovali smo tkivne modele kože s keratinociti različnih celičnih linij. Izstopal je tkivni model s celicami EGFP-K5 E475G, kjer smo zaznali številne prazne prostore tako med dermisom in epidermisom, kot tudi med celicami epidermisa, kar je značilno za hudo obliko EBS in nakazuje na pomembno vlogo keratinskih filamentov pri adheziji.

• Language: Slovenian
• Keywords: Keratin 5, keratinociti, koža, bulozna epidermoliza simpleks, togost celične membrane, agregati, tkivni model kože
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [J. Cvetkovska]
• Year: 2024
• PID: 20.500.12556/RUL-154093
• UDC: 547.962.9:616.529.1(043.2)
• COBISS.SI-ID: 182556163
• Publication date in RUL: 25.01.2024

Secondary language

• Language: English
• Title: The effect of keratin 5 E475G and P25L mutations on the fenotype of keratinocytes grown in cell culture

Abstract

Keratins (K) are the largest group of intermediate filaments. They have a characteristic structure that causes them to organize into coiled dimers. K5 forms heterodimers with K14. They are mostly present in basal keratinocytes in the epidermis and their primary role is the physical stability of the epidermis. Mutations in K5 or K14 can cause epidermolysis bullosa simplex (EBS), which is characterized by skin fragility that results in blisters and erosions by minor mechanical trauma. In this master's thesis, we were interested in how the mutations E475G in K5, which causes severe EBS, and P25L in K5, which causes EBS with mottled pigmentation, effect the phenotype of keratinocytes grown in cell culture. We used the cell line NEB-1 with previously inserted construct EGFP-K5 wild type, E475G or P25L. Our goal was to select clones that express the construct in specific percentage in comparison with endogenous K5 and compare them. We used SDS-PAGE and Western blot to compare the quantity of EGFP-K5 and endogenous K5. We only got clones with the same, low, quantity of the construct. We used optical tweezers to measure the cortical stiffness of the cells and concluded that neither of the constructs causes important changes in the cortical stiffness and there are no bigger differences between the cell lines. The construct EGFP-K5 E475G, even in small amounts, increases spontaneous aggregate formation, where the aggregates are not only in the cell periphery. This is contrary to our predictions. Previous studies on cells with E475G mutation did not confirm that the mutation causes such disturbance of keratin cytoskeleton, where the spontaneous aggregate formation would be increased. We also noticed a higher expression of K16 and K19 in cells EGFP-K5 E475G. We created skin tissue models with keratinocytes expressing different constructs, where EGFP-K5 E475G stood out the most. We noticed several voids between the dermis and epidermis as well as between cells in epidermis, which is typical for severe EBS and shows the important role of keratin filaments in adhesion.

• Keywords: Keratin 5, keratinocytes, skin, epidermolysis bullosa simplex, cortical stiffness, aggregates, skin tissue model