Opazovnaje mobilnosti stresnih granul s pomočjo optične pincete v stabilni celični liniji FlpIn SHSY-5Y-Halo-G3BP

Nograšek, Teo (author), Rogelj, Boris (mentor)

Stresne granule se tvorijo v celicah za omilitev posledic zunanjih stresnih dejavnikov. Nastanejo iz komponent zaustavljene translacije, vsebujejo RNA vezavne proteine (RBP), RNA in ribosome. G3BP1 je eden od ključnih proteinov stresnih granul in ima ključno vlogo pri njihovi tvorbi. Kondenzacija je tako intenzivna, da pride do fazne ločitve dveh tekočin (LLPS). Strukture, nastale prek LLPS, imajo lastnosti tekočinskih kapljic: so sferične, imajo ločeno sestavo ter se manjše kapljice lahko zlivajo in združujejo v večje kapljice. Visoka koncentracija molekul in sferična oblika predstavljata odlično tarčo za manipulacijo z optično pinceto. Optična pinceta temelji na intenzivnem snopu svetlobe (laser), ki lahko ujame sferične strukture z visokim relativnim lomnim količnikom. Podobne lastnosti imajo stresne granule, saj imajo višjo koncentracijo od citoplazme. Na podlagi tega dejstva smo začrtali temelje tega dela. Izbrali smo celično linijo SH-SY5Y, v katero smo prek transfekcije vnesli konstrukt za izražanje rekombinantnega proteina HaloTag-G3BP1. Oznaka HaloTag nam omogoči kovalentno vezavo stabilnega sintetičnega fluorfora na protein G3BP1. Z dodatkom natrijevega arzenita (NaAsO2) smo v celicah sprožili oksidativni stres, ki je induciral tvorbo stresnih granul. Rekombinantni protein je pokazal podobno kondenzacijo in lokalizacijo kot endogeni G3BP1 pod stresnimi pogoji. Izbrani HaloTag ligand Oregon Green je uspešno prešel v sestavljene stresne granule, kar nam je omogočilo nadaljnje spremljanje granul pod optično pinceto. S pinceto smo testirali 2928 granul. Okoli 5 % (156) granul se je ob približanju laserskega žarka optične pincete premaknilo. Večina uspešno premaknjenih granul se ob približanju laserskega žarka premaknilo proti žarku. Približno 31 % uspešno premaknjenih granul pa se je od laserskega žarka odmaknilo. 10 granul se je popolnoma ujelo v optično past laserskega žarka, kar nam je omogočilo popoln nadzor nad premikanjem granule. Opazili smo, da večja kot je bila granula, bolj dovzetna je bila na manipulacijo z optično pinceto. Večje granule so bolj dozorene in koncentrirane, kar pomeni, da imajo višji relativni lomni količnik. Skozi delo smo uspešno uporabili optično pinceto za manipulacijo in ujetje stresnih granul, kar ponuja pot za nadaljnje eksperimente, ki se fokusirajo na bolj fizikalne lastnosti stresnih granul pod različnimi pogoji.

• Language: Slovenian
• Keywords: stresne granule, optična pinceta, mobilnost, G3BP1
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-188848
• Publication date in RUL: 29.09.2026

Secondary language

• Language: English
• Title: Obesrving the mobility of stress granules using optical tweezers in a stable Flp In SHSY-5Y-Halo-G3BP cell line

Abstract

Stress granules represent one of the many way cells can mitigate external stressors. Formed out of stalled translation machinery, they are primarily made up of RNA binding proteins (RBPs), RNA and ribosomes. G3BP1 is a crucial protein in stress granule assembly. Stress granules are condensed to the point in which a liquid-liquid phase separation occurs (LLPS). Structures formed through LLPS have the properties of droplets: smaller droplets can combine into bigger ones, they have an autonomous composition and are spherical. With them having a high relative concentration of molecules and a spherical form they represent a potentially useful target for manipulation with optical tweezers. Optical tweezers consist of a high intensity beam of light (laser) that can trap spherical objects with a high relative refractive index, such as a highly concentrated stress granule has compared to cellular cytoplasm. Based on these facts, we designed this study. We chose to transfect SH-SY5Y cells with a construct that expresses a recombinant HaloTag-G3BP1 protein. The HaloTag tag enabled us to covalently bond a stable synthetic fluorophore to the G3BP1 protein. We induced a cellular oxidative stress response by adding sodium arsenite (NaAsO2). The recombinant protein showed similar condensation and assembly into stress granules as the endogenic G3BP1. The successful binding of HaloTag ligand Oregon Green into already assembled stress granules enabled us to move on to manipulation test using optical tweezers. During which we tested 2928 stress granules with the optical tweezers. Around 5 % (156) of the tested granules exhibited movement as the optical tweezer laser moved closer. Most of the movement we saw was the result of the laser attracting stress granules. Around 31 % of the moved granules moved in the opposite direction of the laser as if they were repelled by it. 10 granules were successfully trapped with the optical trap of the laser enabling us to completely control the movement of the granule. In the end we concluded that bigger granules were more likely to be manipulated with optical tweezer, which could be because bigger granules are more concentrated meaning they have a higher refraction ratio. All in all, we successfully used optical tweezers to manipulate and trap stress granules paving the way for future experiments focusing on measuring physical properties of granules under different conditions.

• Keywords: stress granules, optical tweezers, mobility, G3BP1