Spremljanje internalizacije fluorescentno označene α-D-manozilirane sonde v dendritične celice s superločljivostnim slikanjem

Purić, Edvin (author), Anderluh, Marko (mentor), Mravljak, Janez (co-mentor)

S superločljivostno mikroskopijo lahko opazujemo znotrajcelične procese pod magično mejo valovne dolžine vidne svetlobe 200 nm na nanometrskem nivoju. Za mikroskopijo z vzbujenim praznjenjem emisije, ki je ena izmed izvedbenih variant superločljivostne mikroskopije, so primerne le fluorescentne sonde, ki so kompatibilne z dodatnim laserskim žarkom, ki omogoča praznjenje emisije. Karakterizacija tovrstne sonde je bila osrednji cilj naših raziskav. V magistrski nalogi smo preučevali proces internalizacije fluorescentne sonde v različne tipe celic. Največ pozornosti smo namenili dendritičnim celicam in receptorju DC-SIGN (za dendritične celice specifični, intracelularno adhezijsko molekulo–3 vezoč neintegrin), ki igra izredno pomembno vlogo pri okužbah z mnogimi patogeni, kot je npr. okužba z virusom humane imunske pomanjkljivosti (HIV). Njegove glavne naloge so prepoznavanje patogenov oz. njihovih površinskih molekul in internalizacije ter prispevek k uničenju patogenov. Kljub vsem tem lastnostim pa lahko DC-SIGN s tem, ko zagotovi vstop patogenov v dendritične celice, tudi pospeši razvoj okužbe, kar je značilno ravno za virus HIV. DC-SIGN veže različne manozilirane proteine, oligomanozide in molekule, ki vsebujejo le eno enoto D-manoze. Preiskovali smo α-D-manozilirano fluorescentno sondo, ki je odporna na glikozidaze in tako zagotavlja stabilnost v celičnih testih. S fluorescenčno spektrofluorimetrijo smo ugotovili, da se njeni emisijski spektri ne spreminjajo v odvisnosti od pH okolice. Nadalje smo dokazali, da sonda prehaja v notranjost celic le, če te na svoji membrani izražajo DC-SIGN receptorje oz. druge lektine, ki vežejo manozo (angl. Mannose binding lectins, MBL). Dodana vrednost preiskovane sonde je primernost za mikroskopijo z vzbujenim praznjenjem emisije, saj je nabor sond, primernih za tovrstno mikroskopijo, precej omejen. Fluorescentna sonda predstavlja visoko učinkovito orodje za opazovanje bioloških procesov, kot je z lektini posredovan celični privzem v različnih bioloških okoljih.

• Language: Slovenian
• Keywords: Fluorescenčna mikroskopija, BODIPY, DC-SIGN, STED mikroskopija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-129805
• Publication date in RUL: 08.09.2021

Secondary language

• Language: English
• Title: Internalization monitoring of a fluorescently labelled α-D-mannosylated probe into dendritic cells by super-resolution imaging

Abstract

Super-resolution microscopy allows observation of cellular phenomena below the “magic” limit of the visible light wavelength of 200 nm to the nanometer level. For Stimulated Emission Depletion microscopy, only fluorescence probes compatible with an additional laser beam that allows emission depletion are suitable. Characterisation of such a probe was an overarching aim of our research work. In the master thesis we studied the process of internalisation of a fluorescent probe into different cell types. Most attention was paid to the dendritic cells and the DC-SIGN receptor (Dendritic Cell-Specific, Intracellular adhesion molecule-3-Grabbing Non-integrin), which plays an important role in infections with many pathogens, such as human immunodeficiency virus (HIV) infection. The main roles of the DC-SIGN are recognition of pathogens/their molecules and internalisation, as well as their contribution to pathogen destruction. Despite all these properties, the DC-SIGN receptor can also accelerate the development of infection, which is a hallmark of HIV virus, by ensuring pathogen entry into dendritic cells. DC-SIGN binds various manosylated proteins, oligomanosides, and molecules containing only one unit of D-mannose. We have investigated an α-D-mannosylated fluorescent probe that is resistant to glycosidases. Fluorescence spectrometry showed that its emission spectra did not change as a function of environmental pH. The behaviour of the probe in different cell types was observed using a fluorescence microscope. It was demonstrated that the probe passes the interior of cells only when they express DC-SIGN receptors or other Mannose binding lectins (MBL) on their membranes. The added value of the probe is its suitability for Stimulated Emission Depletion Microscopy, as the choice of probes suitable for this type of microscopy is quite limited. All these experiments demonstrated that the fluorescent probe is a highly effective tool for observing biological processes, such as lectin-mediated cell uptake in a variety of biological environments.

• Keywords: Fluorescence microscopy, BODIPY, DC-SIGN, STED microscopy