In cells, numerous membraneless organelles are formed through the process of liquid
liquid phase separation (LLPS). Among these are stress granules, which assemble in the
cytosol when the cell is exposed to various forms of stress. Chronic exposure to stress
and the resulting dysregulation of LLPS lead to the death of neurons and the development
of neurodegenerative diseases. One of the key components of stress granules is the Ras
GTPase-activating protein-binding protein 2 (G3BP2), which, compared to its better
known paralog G3BP1, exhibits higher expression levels in neurons. In this master’s
thesis, we studied the formation of G3BP2 condensates in an in vitro system. We focused
on how the composition of the condensates influences their physicochemical properties
and their interactions with lipid membranes. The condensates were formed in the presence
of either RNA molecules or polyethylene glycol (PEG). We found that condensates
formed in the presence of PEG were smaller in size and had a larger immobile fraction.
They were capable of wetting lipid membranes, but the extent of wetting was not
dependent on membrane charge. In contrast, RNA-condensates did not wet lipid
membranes. Since RNA-condensates are more similar in composition and formation
mechanism to stress granules, we further examined what enables their interaction with
lipid membranes in cellular systems. Specifically, we investigated whether the known
adaptor protein annexin A11 (ANXA11) is present in stress granules that interact with
damaged endolysosomal and lysosomal membranes. To this end, we used a FlpIn SH
SY5Y cell line expressing mScarlet I-G3BP1-myc. We confirmed that upon
endolysosomal and lysosomal membrane damage, there is an increased interaction
between damaged organelles and stress granules, and that nearly 90 % of stress granules
interacting with damaged membranes contain ANXA11.
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