FRAP Assay to Trace Lipid Mixing of the Inner and Outer Leaflet of Yeast Vacuoles: Assessing the Fusion State in Live Cells

Ana Catarina Alves; Christin Bissig; Andreas Mayer

doi:10.1007/978-1-0716-4314-3_14

FRAP Assay to Trace Lipid Mixing of the Inner and Outer Leaflet of Yeast Vacuoles: Assessing the Fusion State in Live Cells

Methods Mol Biol. 2025:2887:197-206. doi: 10.1007/978-1-0716-4314-3_14.

Authors

Ana Catarina Alves¹, Christin Bissig¹, Andreas Mayer²

Affiliations

¹ Department of Immunobiology, University of Lausanne, Epalinges, Switzerland.
² Department of Immunobiology, University of Lausanne, Epalinges, Switzerland. [email protected].

PMID: 39806156
DOI: 10.1007/978-1-0716-4314-3_14

Abstract

Fluorescence recovery after photobleaching (FRAP) can be employed to investigate membrane lipid mixing of vacuoles in live budding yeast cells and distinguish the fused, hemi-fused or non-fused states of these organelles under physiological conditions. Here, we describe a protocol for labeling the outer and inner leaflets of vacuoles in live cells that allow to detect hemifusion intermediates and, thus, identify components necessary for fusion pore opening.

Keywords: FRAP; Fusion pores; Hemifusion; Lipid probe; SNAREs; Vacuole; Yeast.

MeSH terms

Fluorescence Recovery After Photobleaching* / methods
Membrane Fusion*
Membrane Lipids / metabolism
Saccharomyces cerevisiae* / cytology
Saccharomyces cerevisiae* / metabolism
Vacuoles* / metabolism

Substances

Membrane Lipids