Biology subjects

Wieneke, R.

Publications and source records attributed to Wieneke, R..

3 recordsLinked to original sources

bioRxiv2021

Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and mesoscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a chelator nanotool. Due to the multivalent interaction, we observed a dynamic exchange in the microscale confined regions. Fast Y2R enrichment in clustered areas triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading, and migration. We revealed that the cell response to ligand-induced activation was amplified when cells were pre-clustered by the nanotool. Ligand-independent signaling by clustering differed from ligand-induced activation in the binding of arrestin-3 as downstream effector, which was recruited to the confined regions only in the presence of the ligand. This approach enables in situ clustering of membrane receptors and raises the possibility to explore different modalities of receptor activation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/472742v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@9c7823org.highwire.dtl.DTLVardef@93660corg.highwire.dtl.DTLVardef@110c414org.highwire.dtl.DTLVardef@1b906d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Manipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

synthetic biology↗

bioRxiv2017

Multiplexed Live-Cell Visualization Of Endogenous Proteins With Nanometer Precision By Fluorobodies

The visualization of endogenous proteins in living cells is a major challenge. A fundamental requirement for spatiotemporally precise imaging is a minimal disturbance of protein function at high signal-to-background ratio. Current approaches for visualization of native proteins in living cells are limited by dark emitting, bulky fluorescent proteins and uncontrollable expression levels. Here, we demonstrate the labeling of endogenous proteins using nanobodies with site-specifically engineered bright organic fluorophores, named fluorobodies. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing allowed for low background, low toxicity, and high-throughput. Multiplexed imaging of distinct cellular structures was facilitated by specific protein targeting, culminating in live-cell super-resolution imaging of protein networks. The high-throughput delivery of engineered nanobodies will open new avenues in visualizing native cellular structures with unprecedented accuracy in cell-based screens.

cell biology↗