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

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Title: Automated fluorescence lifetime imaging high content analysis of Förster resonance energy transfer between endogenously-labeled kinetochore proteins in live budding yeast cells
Authors: Guo, W
Kumar, S
Gorlitz, F
Garcia, EC
Alexandrov, Y
Munro, I
Kelly, D
Warren, S
Thorpe, P
Dunsby, C
French, P
Item Type: Journal Article
Abstract: We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster 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 µManager 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.
Issue Date: 1-Jun-2019
Date of Acceptance: 23-Nov-2018
URI: http://hdl.handle.net/10044/1/66504
DOI: https://doi.org/10.1177/2472630318819240
ISSN: 2472-6303
Publisher: SAGE Publications
Start Page: 308
End Page: 320
Journal / Book Title: Slas Technology
Volume: 24
Issue: 3
Copyright Statement: © 2019 Society for LaboratoryAutomation and Screening. This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Biotechnology and Biological Sciences Research Council (BBSRC)
Funder's Grant Number: BB/E003621/1
BB/M006786/1
Keywords: Science & Technology
Life Sciences & Biomedicine
Physical Sciences
Biochemical Research Methods
Chemistry, Analytical
Biochemistry & Molecular Biology
Chemistry
fluorescence lifetime imaging
high-content analysis
budding yeast
kinetochore protein interactions
FRET
MICROTUBULE ATTACHMENT
NDC80 COMPLEX
LIVING CELLS
PLATE READER
FLIM
MICROSCOPY
ARCHITECTURE
REVEALS
IDENTIFICATION
FRET
budding yeast
fluorescence lifetime imaging
high-content analysis
kinetochore protein interactions
Publication Status: Published
Online Publication Date: 2019-01-10
Appears in Collections:Physics
Photonics
Faculty of Natural Sciences



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