TIRF microscopy enables the precise measurement of the lifetime of endocytic orthologs individually tagged with EGFP in three fungal species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis.

The lifetimes in each species are registered together using the appearance of an actin marker tagged with mCherry, which is imaged simultaneously to the EGFP-tagged orthologs and acts as a fiducial. The alignment of the different lifetimes defines a fingerprint of the endocytic process in each species.

Differential evanescent TIRF precisely identifies the beginning of the invagination movement with respect to the actin marker and, thus, within the endocytic fingerprint of each species. The timing of the start of the invagination movement allows species-to-species comparison. Time 0 marks the beginning of the invagination movement globally.

References:

image thumbnail
Bar graph showing the time of appearance and disappearance of each endocytic ortholog in respect to the beginning of the invagination movement, in each species. Error bars represent the SD for each measurement. Time 0 s marks the estimation of the beginning of the invagination movement. The dashed vertical lines represent its SD. (Picco, Toret, Rivier, and Kaksonen, Biorxiv, 2024)