Advances in Use of Capsule-Based Fluorescent Sensors for Measuring Acidification of Endocytic Compartments in Cells with Altered Expression of V-ATPase Subunit V₁G₁

Abstract

Acidification of eukaryotic cell compartments is accomplished by vacuolar H+-ATPases (V-ATPases), large multisubunit complexes able to pump protons into the lumen of organelles or in the extracellular medium. V-ATPases are involved in a number of physiological cellular processes, and thus regulation of V-ATPase activity is of crucial importance for the cell. Indeed, dysfunction of V-ATPase or alterations of acidification have been recently recognized as key factors in a variety of human diseases. In this study, we applied capsule-based pH sensors and a real-time tracking method for investigating the role of the V1G1 subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V1G1. Second, we used fluorescent capsule-based pH sensors to monitor acidification before and during internalization by modified and control living cells. By using a simple real-time method for tracking capsule internalization, we were able to identify different capsule acidification levels with respect to each analyzed cell and to establish the kinetics for each. The intracellular pH measurements indicate a delay in acidification in either V1G1-overexpressing or V1G1-silenced cells compared to controls. Finally, in an independent set of experiments, we applied transmission electron microscopy and confocal fluorescence microscopy to further investigate the internalization of the capsules. Both analyses confirm that capsules are engulfed in acidic vesicular structures in modified and control cell lines. The use of capsule-based pH sensors allowed demonstration of the importance of the V1G1 subunit in V-ATPase activity concerning intravesicular acidification. We believe that the combined use of these pH-sensor system and such a real-time method for tracking their internalization path would contribute to systematically measure the proton concentration changes inside the endocytic compartments in various cell systems. This approach would provide fundamental information regarding molecular mechanisms and factors that regulate intracellular acidification, vesicular trafficking, and cytoskeletal reorganizations.

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive‐Manufactured Scaffolds

“…These data are in agreement with our previous results, in which we studied the fluorescence response of SNARF–dextran conjugates loaded in the cavities of polyelectrolyte capsules inside living cells. [ 52 ] The pKa calculated for SNARF‐1‐dextran conjugate was 7.09 ± 0.04, which is lower compared to the pKa of SNARF‐1 (pKa ≈7.5). The possible reason for lowering of pKa could be the result of interaction between SNARF‐1 and surrounding microparticle matrix.…”

“…[ 59 ] Namely, this deformation does not impair the sensing properties of the capsules that retain both the pH‐sensitive and the reference fluorophores within their cavities, but additionally confirm their intracellular localization. [ 52 ] Figure S4, Supporting Information shows the normal lysosome quantity and distribution in hMSCs growing in 3D AM scaffolds, where cells were separately stained with calcein and lysotracker (control). The internalization of capsules at day 7 was further assessed via TEM analysis (Figure 2c,d).…”

Ratiometric Organic Fibers for Localized and Reversible Ion Sensing with Micrometer‐Scale Spatial Resolution

“…Capsule‐based sensors have previously been developed by loading various ion‐sensitive probes, and related barcoded sensors have been reported for the simultaneous measurement of H + , Na + , and K + concentration in test tubes . Most studies validated the use of these sensors for the analysis of intracellular pH (pHi) changes . In contrast, here we focus on designing a mechanically robust and flexible architecture suited for pHe analyses.…”

Design and characterization of microcapsules-integrated collagen matrixes as multifunctional three-dimensional scaffolds for soft tissue engineering

“…In these approaches, the protein-loaded carriers can be easily added onto the prefabricated scaffolds or be added to the polymer phase while processing the scaffolds (Tessmar and Göpferich, 2007). Polymer capsules fabricated by the layer-by-layer (LbL) method are considered highly tunable delivery vehicles and easy to combine with other materials (De Luca et al, 2015;del Mercato et al, 2016del Mercato et al, , 2015del Mercato et al, , 2014Delcea et al, 2011;Kolbe et al, 2011;Wohl and Engbersen, 2012;Xiong et al, 2013). Biodegradable multilayer capsules integrated within 3D scaffolds would serve as containers of bioactive molecules that would allow for controlling the release of the payload and for protecting it from the surgical procedures (washing, debridement, bleeding, fluid aspiration) applied during the implantation of the scaffold which challenge the integrity of the material (De Cock et al, 2012;Facca et al, 2010;She et al, 2012).…”