Biology subjects

Mrowka, R.

Publications and source records attributed to Mrowka, R..

2 recordsLinked to original sources

bioRxiv2023

Monitoring oligomerization dynamics of individual human neurotensin receptors 1 in living cells and in SMALP nanodiscs

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based structured illumination microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.

biophysics↗

bioRxiv2021

Toll-like receptor 5 as a novel receptor for fungal zymosan

Microbial pathogens carry specific structural patterns which were termed pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) as key elements for the recognition of microbial pathogens are necessary for the activation of innate immune pathways. TLRs are activated by binding PAMPs of bacteria, viruses and fungi and initiate a signaling pathway resulting in the activation of transcription factors which modulate the production of various proinflammatory cytokines. It is not fully clear in detail which microbial pattern is recognized by which TLR. Here we show for the first time that TLR5 is a strong receptor for the yeast particle zymosan. We have generated stable human cell lines with combinations of TLR2 and TLR5 knock in/knock out together with stable nuclear factor kappaB (NF-{kappa}B) luciferase reporters. We found that both receptors TLR5 and TLR2 lead to an independent activation of the NF-{kappa}B pathway when simulated with zymosan. Our results demonstrate that TLR5 is a receptor for the fungal particle zymosan in addition to bacterial fragments like flagellin. Distinct cytokine patterns might suggest that TLR5 is potentially important for the differentiation in the recognition of the specific type of the foreign microorganisms and in the specific host defense response.

immunology↗