title: Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles.
abstract: The yeast vacuole is functionally analogous to the mammalian lysosome. Both play important roles in fundamental cellular processes such as protein degradation, detoxification, osmoregulation, autophagy and apoptosis which, when deregulated in humans, can lead to several diseases. Some of these vacuolar roles are difficult to study in a cellular context, and therefore the use of a cell-free system is an important approach to gain further insight into the different molecular mechanisms required for vacuolar function. In the present study, the potentialities of flow cytometry for the structural and functional characterization of isolated yeast vacuoles were explored. The isolation protocol resulted in a yeast vacuolar fraction with a degree of purity suitable for cytometric analysis. Moreover, isolated vacuoles were structurally and functionally intact and able to generate and maintain electrochemical gradients of ions across the vacuolar membrane, as assessed by flow cytometry. Proton and calcium gradients were dissipated by NH4Cl and calcimycin, respectively. These results established flow cytometry as a powerful technique for the characterization of isolated vacuoles. The protocols developed in this study can also be used to enhance our understanding of several molecular mechanisms underlying the development of lysosome-related diseases, as well as provide tools to screen for new drugs that can modulate these processes, which have promising clinical relevance.
authors: Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.|Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Jorge
authors: J
authors: Rodrigues
authors: Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.|Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Rui D
authors: RD
authors: Silva
authors: Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB), Quinta de Prados, 5001-801 Vila Real, Portugal.|Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Henrique
authors: H
authors: Noronha
authors: Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.|Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Andreia
authors: A
authors: Pedras
authors: Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB), Quinta de Prados, 5001-801 Vila Real, Portugal.|Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Hernâni
authors: H
authors: Gerós
authors: Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.|Centro de Biologia Molecular e Ambiental (CBMA), Departamento de Biologia, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Manuela
authors: M
authors: Côrte-Real
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry* / Q000378:metabolism*