Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles

• Type: Article in International Scientific Journal
• Date: 2013

Authors

Rodrigues, J (Author) Other; Silva, RD (Author) Other; Noronha, H (Author) Other; Pedras, A (Author) Other; Geros, H (Author) Other; Corte Real, M (Author) Other

Journal

Title: Microbiology

Vol. 159

Pages: 848-856

ISSN: 1350-0872

Publisher: Microbiological Society

Indexing

ISI Web of Knowledge - 7 Citations

Scopus - 10 Citations

Other information

Authenticus ID: P-005-0TX

DOI: 10.1099/mic.0.062570-0

Abstract (EN): 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 9