Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster 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 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.

Keywords: FRET; budding yeast; fluorescence lifetime imaging; high-content analysis; kinetochore protein interactions.

Figure 1.

Budding yeast kinetochore. (a) The kinetochore is a multiprotein complex comprising various types of KPs that belong to several subcomplexes, together with some microtubule-associated proteins (MAPs) and motor proteins. The architecture of the kinetochore can be divided into three domains: the inner kinetochore is in direct contact with the centromeric DNA; the outer kinetochore interacts with the microtubule; the central kinetochore serves as a bridge between the other two domains. During the metaphase of mitosis, the KPs at each kinetochore span a length of ~70 nm along the kinetochore–microtubule axis. (b) Under conventional fluorescence microscopy, the multiple kinetochores (16, 32, or 64, depending on the cell cycle stage and the ploidy) cluster into one or two diffraction-limited foci. For example, in the metaphase/anaphase cell shown on the right-hand side of the micrograph, the fluorescently tagged kinetochores containing Mtw1p-mTq2FP appeared as two foci, each of which contained multiple kinetochores linked to microtubules nucleated from a single spindle-pole body (SPB). Scale bar: 5 µm. (c) We chose six KPs (Ndc10p, Mtw1p, Spc24p, Ndc80p, Nuf2p, Ask1p) to be tagged by the FRET donor mTq2FP, respectively, for the FLIM experiments in this study; see proteins with blue circles, which are representative of KPs along the kinetochore–microtubule axis.

Figure 2.

Schematic of the automated multiwell plate FLIM microscope system for FLIM HCA. This includes the electronically controlled micropump for maintaining the immersion water for the microscope objective during automated unsupervised imaging. Refer to the main text and Görlitz et al. for a detailed description of the system.

Figure 3.

Strategy for segmenting FLIM images. (a) Exemplar image showing the fluorescence intensity integrated over the 12 time gates of a FLIM image of donor-only cells expressing Ask1p-mTq2FP. Note the appearance of the spot-like kinetochore clusters, the cellular autofluorescence (indicating the profiles of the cells), and the large bright dead cell. Scale bar: 20 µm. (b) Exemplar image showing the kinetochore segmentation mask (blue) and the dead cell exclusion mask (green) overlaid on the original image. We segmented the kinetochore clusters using a modified version of the NTH algorithm (see main text for details), whereas the dead cell was picked out by both its brightness and size. Scale bar: 20 µm. (c) Enlarged view of a segmented kinetochore region. Each kinetochore was segmented as a 3 × 3 pixel region (blue) centered on a pixel selected by the modified NTH algorithm. To remove the local cellular background for each kinetochore region, the median value from the pixels in a 7 × 7 hollow square (red) was subtracted off the pixel values in the kinetochore region in every temporal frame.

Figure 4.

FLIM results of the strains in the Spc24p-mTq2FP and Ndc80p-mTq2FP groups. (a–c) Schematics of the positions of the fluorophores in the three strains in the Spc24p-mTq2FP group: (a) the positive control containing a plasmid expressing Spc24p-mTq2FP-YFP; (b) the donor-only negative control; and (c) the query strain where YFP was tagged to Spc25p, which was known to form a dimer with Spc24p in the NDC80 complex. (d) The query strain expressing Spc24p-mTq2FP and Spc25p-YFP exhibited a significantly reduced donor fluorescence lifetime compared with the negative control. (e) The query strain expressing Ndc80p-mTq2FP and Nuf2p-YFP exhibited a significantly reduced donor fluorescence lifetime compared with the negative control (note that this query strain is heterozygous for both FP-tagged KP genes). Each box plot shows the median value (bar) and the quartiles (box) of the image-wise lifetime values. The notches indicate the 95% confidence interval of the median. The whiskers correspond to the minimum of the data range or 1.5× the interquartile range, whereas extreme data points are marked as outliers. The number of FOVs analyzed for each strain is shown in parentheses above the corresponding box. The statistical test performed was the two-sided Wilcoxon rank-sum test. A Bonferroni correction was performed to correct the α value for multiple comparisons. p values smaller than the corrected α values are shown in the box plots.

Figure 5.

FLIM results of the strains in the (a) Ndc10p-mTq2FP, (b) Mtw1p-mTq2FP, (c) Nuf2p-mTq2FP, and (d) Ask1p-mTq2FP groups. None of the query strains in these groups exhibited a significantly changed lifetime compared with the donor-only negative controls. The statistical test performed was the two-sided Wilcoxon rank-sum test. A Bonferroni correction was performed to correct the α value for multiple comparisons. p values smaller than the corrected α values are shown in the box plots. n.s. = not significant.