Periodic Reporting for period 1 - QuantiCl (Deconstructing the role of trans-cellular ion transport in organ formation and function)
Reporting period: 2023-10-01 to 2026-03-31
Many organs have of a complex structure with lumina and ducts, for example, the pancreas, liver, intestine, etc. Such organs rely on the transport of nutrients and other chemicals from the lumens to the tissue or vice versa for their function and development. These lumens and ducts are lined with epithelial cells, a polarized cell type that faces the lumen on one side (apical side) and the tissue on the other (basolateral side). The transport of nutrients is carried out by transport proteins embedded in both the basolateral membrane and the apical membrane.
The interplay of individual transport proteins in transport processes cells is what ultimately leads to the net transport events, but a solid molecular understanding of these biochemical processes is lacking. Solute transport across epithelia is a molecular process, but leading edge studies until now mainly revolve around bulk measurements, where all transporters are considered but not their unique interplay. This implies that mathematical models based on such measurements have limited predictive value when it comes to the single transport proteins. Changes in single proteins can however easily lead to disruptions in function and to diseases.
QuantiCl aims to bridge this knowledge gap by studying transcellular transport using quantitative biochemistry in epithelial cells and organoids, with specifically Chloride as the target. Chloride is an important anion in the function of many organds, for example, it is exchanged for bicabonate throughout the pancreatic duct, which is then used to neutralize stomach acid, but it is also believed to play a role in lumen formation.
Using a combination of fluorescent chloride sensors in organoids, microscopy and an image and analysis pipeline QuantiCl aims to provide quantitative insights into transcellular chloride transport, within the full complexity of the native cellular environment. These insights will provide key fundamental knowledge on how (membrane) proteins work together and take part in forming complex biological systems.
The interplay of individual transport proteins in transport processes cells is what ultimately leads to the net transport events, but a solid molecular understanding of these biochemical processes is lacking. Solute transport across epithelia is a molecular process, but leading edge studies until now mainly revolve around bulk measurements, where all transporters are considered but not their unique interplay. This implies that mathematical models based on such measurements have limited predictive value when it comes to the single transport proteins. Changes in single proteins can however easily lead to disruptions in function and to diseases.
QuantiCl aims to bridge this knowledge gap by studying transcellular transport using quantitative biochemistry in epithelial cells and organoids, with specifically Chloride as the target. Chloride is an important anion in the function of many organds, for example, it is exchanged for bicabonate throughout the pancreatic duct, which is then used to neutralize stomach acid, but it is also believed to play a role in lumen formation.
Using a combination of fluorescent chloride sensors in organoids, microscopy and an image and analysis pipeline QuantiCl aims to provide quantitative insights into transcellular chloride transport, within the full complexity of the native cellular environment. These insights will provide key fundamental knowledge on how (membrane) proteins work together and take part in forming complex biological systems.
QuantiCl has paved the way to enable quantitative chloride measurements inside epithelial cells and organoids.
To do so, we have introduced a fluorescence based chloride sensor into epithelium-forming cells which can either grow in 2D on a support, or into 3D organoids that form a lumen. The 2D system allows for manipulation of fluids on either side of the epithelium, so cellular conditions can be mimicked.
The 3D system allows us to, next to chloride measurements, also look at organoid development in parallel.
We developed a microscopy pipeline and image analysis pipeline that can be used to study the effect of inhibitors on chloride transport and lumen formation. Using this pipeline, we can deconvolute the role of individual transport proteins in transepithelial chloride transport, under various physiological conditions.
To do so, we have introduced a fluorescence based chloride sensor into epithelium-forming cells which can either grow in 2D on a support, or into 3D organoids that form a lumen. The 2D system allows for manipulation of fluids on either side of the epithelium, so cellular conditions can be mimicked.
The 3D system allows us to, next to chloride measurements, also look at organoid development in parallel.
We developed a microscopy pipeline and image analysis pipeline that can be used to study the effect of inhibitors on chloride transport and lumen formation. Using this pipeline, we can deconvolute the role of individual transport proteins in transepithelial chloride transport, under various physiological conditions.
Up until this point the experimental system and analysis pipelines are in place, but took more time than anticipated due to biological challenges. Further research is currently going on to ensure further the success of quantitative chloride measurements over epithelial cells.