Biology subjects

Gerber, A. P.

Publications and source records attributed to Gerber, A. P..

4 recordsLinked to original sources

bioRxiv2025

QuantGUV: Quantifying Encapsulation Efficiency of Small Molecules in GUVs

1Synthetic cells, constructed through the self-assembly of small molecules, are designed to mimic life-like behaviours by encapsulating functional molecules. For such synthetic cells to accurately replicate cellular reactions, it is critical that the concentrations of encapsulated molecules mirror those in living systems, as reaction kinetics and cellular network states are highly sensitive to these concentrations. However, methods for precisely determining encapsulation efficiency in synthetic cells at single cell resolution have been limited. To address this challenge, we developed QuantGUV, a software-driven, image-based analysis method that determines the concentrations of fluorescent molecules encapsulated within giant unilamellar vesicles (GUVs). We used Quant-GUV to measure the encapsulation efficiencies of fluorescent molecules, ranging in size from 0.5 nm to 20 nm. These measurements were conducted on GUVs formed via the water-in-oil emulsion transfer method under various experimental conditions. Using QuantGUV, we have measured the encapsulation efficiencies of three fluorescence molecules, sulforhodamine B, mEGFP and polystyrene bead, in GUVs formed via the water-in-oil emulsion transfer method. The encapsulation efficiencies for polystyrene bead was close to 100% in most of the conditions while sulforhodamine B and mEGFPs encapsulation efficiencies depended on the parameters during the GUV formation such as concentrations of lipids and oil-water ratio during the GUV formation. By providing crucial insights into encapsulation efficiencies, QuantGUV offers a valuable tool to monitor the building quantitative synthetic cell systems with accurately controlled internal environments which is a critical step towards the creation of synthetic cells.

synthetic biology↗

bioRxiv2025

The yeast phosphofructokinase β-subunit has ATP-dependent RNA unwinding activity and modulates cell cycle progression

Phosphofructokinase (PFK) is a rate-limiting glycolytic enzyme that also possesses an unexplored RNA binding activity. Here, we show that the - and {beta}-subunits of yeast PFK, encoded by PFK1 and PFK2, respectively, bind hundreds of mRNAs in cells, including ones coding for proteins involved in regulation of mitotic cell cycle. Pkf2p directly binds in a Mg-ATP-dependent manner to short GA-, UC-, AU- and U-rich motifs overrepresented in its mRNA targets. Strikingly, Pfk2p displays directional 5 - 3 double-stranded RNA unwinding activity not seen with Pfk1p. Furthermore, Pfk2p dynamically associates with ribosomes and promotes translation of cell cycle genes. Consequently, pfk2{Delta}, but not pfk1{Delta}, mutant cells show severely delayed G1/S phase transition which is independent of the enzymes glycolytic activity. Our results uncovered a hidden function for the Pfk2 subunit as a translational activator of mitotic cell cycle gene transcripts possibly through energy-dependent RNA unwinding activity.

molecular biology↗

bioRxiv2024

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient "RNA world" PUN-like motifs may have supported the correct folding of early ribozymes.

biochemistry↗

bioRxiv2021

The mechanical regulation of RNA binding protein hnRNPC in the failing heart

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

pathology↗