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Automated fluorescence lifetime imaging high-content analysis of forster resonance energy transfer between endogenously labeled kinetochore proteins in live budding yeast cells

Abstract

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Forster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by microManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime >/=250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein-protein interactions at spatially confined macromolecular complexes.

Type Journal
ISBN 2472-6311 (Electronic) 2472-6303 (Linking)
Authors Guo, W.; Kumar, S.; Gorlitz, F.; Garcia, E.; Alexandrov, Y.; Munro, I.; Kelly, D. J.; Warren, S.; Thorpe, P.; Dunsby, C.; French, P.
Responsible Garvan Author Dr Sean Warren
Publisher Name SLAS Technology
Published Date 2019-06-30
Published Volume 24
Published Issue 3
Published Pages 308-320
Status Published in-print
DOI 10.1177/2472630318819240
URL link to publisher's version https://www.ncbi.nlm.nih.gov/pubmed/30629461