Biology subjects

Spoelstra, W. K.

Publications and source records attributed to Spoelstra, W. K..

3 recordsLinked to original sources

bioRxiv2025

BEST4/CA7+ and goblet cells are interdependent regulators of intestinal mucus homeostasis

The intestinal mucus layer is essential for the integrity of the intestinal barrier. It is produced by goblet cells, whose depletion is common in colonic inflammation but remains poorly understood. Here, we show that goblet cell survival relies on a reciprocal dependence with newly discovered BEST4/CA7+ cells. We developed a method to follow BEST4/CA7+ and goblet cells in time from birth to death in human colon organoids. Notably, goblet cells induce BEST4/CA7+ fates in sister cells and other neighbors, using DLL1-mediated lateral activation of Notch-signaling. BEST4/CA7+ cells in turn promote goblet survival, with the latter depleting rapidly after differentiation in absence of BEST4/CA7+ cells. This apoptosis inhibition does not require direct cell-cell contact and instead depends on their shared lumen. Such differentiation and survival interdependencies may be relevant beyond the maintenance of mucosal homeostasis.

cell biology↗

bioRxiv2019

Enzymatic activity induces transient non-spherical coacervate shapes

Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient non-spherical shapes, and we systematically study how PNPase concentration, UDP concentration and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of non-spherical coacervate shapes may have broader implications for the relationship between (bio-)chemical activity and coacervate biology.

biophysics↗

bioRxiv2018

CRISPR-based DNA and RNA detection with liquid phase separation

The ability to detect specific nucleic acid sequences allows for a wide range of applications including identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence both enzymes activate a unique, indiscriminate nucleic acid cleavage activity, which has been exploited for detection of sequence specific nucleotides using labelled reporter molecules. We here present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Turbidity arises from coacervates of positively charged polyelectrolytes with long poly(dT) or poly(U) oligonucleotides. In the presence of a target sequence, long oligonucleotides are progressively shortened, changing the solution from turbid to transparent. We explain how oligonucleotide cleavage resolves LLPS by using a mathematical model which we validate with poly(dT) phase separation experiments. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

biophysics↗