Advances in Use of Capsule-Based Fluorescent Sensors for Measuring Acidification of Endocytic Compartments in Cells with Altered Expression of V‑ATPase Subunit V 1 G 1 Maria De Luca, †,§ Marzia M. Ferraro, †,‡,⬡ Raimo Hartmann, †,⊥ Pilar Rivera-Gil, ⊥,○ Andreas Klingl, #,△ Moritz Nazarenus, ⊥ Agnese Ramirez, ⊥ Wolfgang J. Parak, ⊥,|| Cecilia Bucci,* ,§ Rosaria Rinaldi, ‡,□ and Loretta L. del Mercato* ,‡,⬡ § Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Universita ̀ del Salento, Via Monteroni, 73100, Lecce, Italy ‡ Istituto Nanoscienze - CNR and □ Dipartimento di Matematica e Fisica “Ennio De Giorgi”, via per Arnesano, 73100, Lecce, Italy ⊥ Fachbereich Physik, Philipps-Universitä t Marburg, Renthof 7, 35037, Marburg, Germany # LOEWE Centre for synthetic Microbiology (Synmikro) and Department of Cell Biology, Philipps-Universitä t Marburg, Karl-von-Frisch-Strasse 8, 35043, Marburg, Germany || CIC biomaGUNE, Parque Tecnoló gico de San Sebastia ́ n, Ed. P° Miramó n 182, 20009, San Sebastian, Spain * S Supporting Information 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 V 1 G 1 subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V 1 G 1 . 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 V 1 G 1 -overexpressing or V 1 G 1 -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 V 1 G 1 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. KEYWORDS: V-ATPase, layer-by-layer microcapsules, biosensors, cell uptake, pH measurements ■ INTRODUCTION To process multiple reactions efficiently and timely, eukaryotic cells are compartmentalized into distinct membrane-bound organelles. Intracellular pH plays a pivotal role in cellular processes and is highly regulated in every organelle. 1 Indeed, proper function of a number of organelles requires acidic pH. Acidification has multiple roles: for instance, it is fundamental for ligand-receptor dissociation in endosomes, for activation of hydrolases in lysosomes, for antigen processing in immune cells, for sorting of molecules in the trans golgi network, and for neurosecretion. 2,3 Acidification of compartments is accom- plished by vacuolar H + -ATPases (V-ATPases), large multi- subunit complexes that are able to pump protons into the lumen of the organelles or in the extracellular medium. 4,5 Also, the structural stability and function of proteins are tightly associated with pH. 6 For example, it has been shown that Received: May 20, 2015 Accepted: June 18, 2015 Published: June 18, 2015 Research Article www.acsami.org © 2015 American Chemical Society 15052 DOI: 10.1021/acsami.5b04375 ACS Appl. Mater. Interfaces 2015, 7, 15052-15060