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. 2024 Mar 4;35(4):ar51. doi: 10.1091/mbc.E23-09-0379

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics

Zhen Dou a,†, Ran Liu a,†, Ping Gui a,b,c,†, Chuanhai Fu a, Jennifer Lippincott-Schwartz d, Xuebiao Yao a,*, Xing Liu a,b,*
Editor: Guangshuo Oue
PMCID: PMC11064673  PMID: 38381564

Abstract

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

  • Visualization of specific molecules and their assembly in real time is essential to delineate how cellular dynamics and signaling circuit are orchestrated during the cell cycle.

  • The authors introduced a method for optically imaging tetrameric protein interactions at nanometer resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). The approach was used to visualize centromere CENP-SXTW tetramer assembly dynamics, and dimeric interactions between CENP-TW dimer and Spc24/25 dimer in live cells.

  • FC-FRET allows the delineation of spatiotemporal dynamics of trimerized or tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric complexes in live cells.

INTRODUCTION

Accurate chromosome segregation relies on the centromere, a specialized chromatin domain present throughout the cell cycle that acts as a platform on which transient assembly of the kinetochore occurs during mitosis (Cleveland et al., 2003; McKinley and Cheeseman, 2016). In eukaryotic cells, kinetochores, which are sophisticated molecular machines for cell division fate decisions, constitute a dynamic link between kinetochore-microtubule attachment and spindle checkpoint, a signaling cascade that prevents chromosome segregation before completion of biorientation during cell division. In fact, early electron microscopic (EM) analyses have indicated that the morphology of the outer kinetochore structure changes dynamically during mitotic progression (Yao et al., 1997; McIntosh et al., 2002), suggesting that the kinetochore is dynamically assembled and functionally matured during mitosis. Recent cryo-EM studies have shown the molecular architecture of the human constitutive centromere associated network (CCAN; Pesenti et al., 2022; Tian et al., 2022; Yatskevich et al., 2022). However, the molecular dynamics underlying CCAN assembly and its functional coupling to the kinetochore microtubule network and chromosome movements have not been fully characterized.

Two major CCAN subunits, CENP-C and CENP-T, provide a platform for assembly of the outer layer of kinetochores (Hori et al., 2008; Schleiffer et al., 2012; Hara et al., 2018; Walstein et al., 2021). The outer kinetochore comprises three subcomplexes: KNL1, MIS12, and NDC80, which are collectively referred to as the KMN network (Cheeseman and Desai, 2008). In addition to its regulatory functions, the KMN network, through its NDC80 complex, provides a site for accurate segregation of chromosomes to daughter cells (Musacchio and Desai, 2017; Zhao et al., 2019; Liu et al., 2020; Ismail et al., 2021). However, the spatiotemporal order of CCAN molecular assembly and the dynamic integration of the NDC80-CCAN network for spindle microtubule interactions during mitosis remain unclear.

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells (Chu et al., 2012; Rodriguez et al., 2017). We aimed to develop a new optical reporter to visualize the spatiotemporal dynamics of centromere super-complex assembly via the combination of fluorescence complementation with FRET (FC-FRET) measurements. Here, we generated complementary mTurquoise2 (abbreviated as mTQ2 hereafter) and enhanced yellow fluorescence protein (EYFP), which indicate protein dimerization and tetramerization in space and time, respectively. The fluorescence complementary mTQ2 and EYFP have the same spectral characteristics as their parents, which was confirmed by fusing FC to the CENP-T/W and Spc24/25 heterodimers. We also performed FC-FRET imaging in fixed and live cells expressing mTQ2n/c (the N-terminal or C-terminal fragment of the mTQ2 protein) tagged CENP-T/W and EYFPn/c (the N-terminal or C-terminal fragment of the EYFP protein) tagged Spc24/25 proteins, and revealed the spatiotemporal dynamics of the CENP-T/W and Spc24/25 dimers and their tetrameric interactions. Importantly, our FC-FRET measurements of the interactions of CENP-T with Spc24/25 in the presence of CDK1 chemical inhibitor Ro3306 highlighted the importance of CDK1 kinase activity in the recruitment of Spc24/25 via CENP-T at entry into mitosis. Intriguingly, the interaction between Spc24/25 and CENP-T was found to be independent of CDK1 kinase activity after prometaphase. Our results provide an effective new method on detecting dimeric and tetrameric protein-protein interactions at the nanoscale in space and time during cell division and further our understanding of the molecular dynamics of centromere assembly and reorganization during chromosome segregation in mitosis.

RESULTS

Engineering fluorescence complementation of the mTQ2 protein

The specificity of biological regulatory mechanisms relies on selective interactions between cognate partners in the right place at the right time in response to different extracellular signals. To visualize the spatiotemporal dynamics of protein interactions in centromere assembly, we sought to establish an approach to visualize dimeric and tetrameric protein-protein interactions using fluorescence complementation followed by FRET (Patterson and Lippincott-Schwartz, 2002).

The standard genetically encoded FRET pair consists of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), but many CFP-YFP reporters suffer from a low FRET dynamic range, phototoxicity from CFP excitation light and complex photokinetic events such as irreversible photobleaching. To this end, mTQ2n/c was used to replace classic CFPs and was designed to fuse with two proteins to generate FC-mTQ2 while EYFPn/c was designed to fuse with another pair of dimeric proteins to engineer FC-EYFP (Figure 1A). In principle, FC-FRET technology can be used to visualize the dynamic interactions of 4 proteins, namely, CENP-T, CENP-W, Spc24, and Spc25 (Supplemental Figure S1A). The interaction between proteins A and B provides an opportunity for FC-mTQ2 protein maturation. To test the complementation property of FC-mTQ2, we engineered an important centromere scaffold, the CENP-T/W heterodimer (Figure 1B). To avoid interference from the CENP-T unstructured N-terminal region via fluorescence complementation, we engineered a truncated CENP-T construct in which the histone fold domain (HFD) and Spc24/25 binding region were retained (Figure 1B; CENP-TBonsai). The fusion proteins were expressed at comparable levels as determined by Western blot analysis (Figure 1C). We then examined the mTQ2n-CENP-TBonsai/mTQ2c-CENP-W fusion proteins in HeLa cells using fluorescence microscopy. As shown in Figure 1D, the localization of the engineered mTQ2n-CENP-TBonsai/mTQ2c-CENP-W dimer was readily apparent in the centromere, which was superimposed onto that of the centromere marker ACA, as judged by immunofluorescence.

FIGURE 1:

FIGURE 1:

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Design and characterization of a tetrameric optical sensor. (A) Proteins A and B were conjugated with mTQ2n (aa. 1-155) and mTQ2c (aa. 156-239), respectively. Upon interaction of protein A with protein B, mTQ2n and mTQ2c were complemented to form a functional FC- mTQ2 protein. Proteins C and D were conjugated with EYFPn (aa. 1-155) and EYFPc (aa. 156-239), respectively. Upon interaction of protein C with protein D, EYFPn and EYFPc were complemented to form a functional FC-EYFP protein. When protein A and protein B interact, mTQ2 fluorescence can be excited; when protein C and protein D interact, EYFP fluorescence can be excited. When there is further binding between AB and CD, and the distance between mTQ2 and EYFP is less than 10 nM, which can generate FRET signals. (B) Schematic representation of the mTQ2n-CENP-TBonsai and mTQ2c-CENP-W fusion proteins. (C) Western blot showing the comparable expression of mTQ2n-CENP-TBonsai and mTQ2c-CENP-W. 24 h after transfection into HeLa cells, cell lysates were prepared. After separation by SDS–PAGE, the samples were probed with the indicated antibodies. (D) Representative images of HeLa cells coexpressing mTQ2n-CENP-TBonsai and mTQ2c-CENP-W. Cells were fixed and stained for ACA (red) and DAPI (blue). Bar: 10 μm. (E) Representative live-cell imaging stills illustrating mitotic progression in cells coexpressing mTQ2n-CENP-TBonsai, mTQ2c-CENP-W, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. The scale bar represents 10 μm.

To determine whether the engineered heterodimer CENP-T/W assembles during the cell division cycle, HeLa cells were transiently transfected with mTQ2n-CENP-TBonsai, mTQ2c-CENP-W, and mCherry-H2B for expression followed by thymidine synchronization. As shown in Figure 1E, the mTQ2n-CENP-TBonsai/mTQ2c-CENP-W dimer localizes to the centromere in interphase cells and persists at the centromere as chromosome aligns at the metaphase equator during mitosis. The dimeric interaction between CENP-T and CENP-W was also visualized and quantified by inter-molecular FRET using the mTQ2-CENP-TBonsai and EYFP-CENP-W pair of fluorescent proteins. As shown in Supplemental Figure S1B, the YFP:CFP emission ratio increased at the interphase nuclear centromere, indicating that the CENP-T/W dimer formed in interphase.

Engineering a fluorescence complementation-based FRET sensor to visualize tetramer dynamics

Various lines of structural analyses have indicated the functional importance of the CENP-S/X dimer in centromere function and chromosome segregation. However, the functional interactions between the CENP-S/X dimer and the CENP-T/W dimer during centromere assembly remains elusive (Tao et al., 2012; Tian et al., 2022). To visualize the spatiotemporal dynamics of CENP-S/X-T/W tetramer assembly and interactions at the centromere during cell division, we constructed plasmids expressing complementary EYFP fragments fused with CENP-S or CENP-X (Figure 2A). Western blot confirmed that the fusion proteins were expressed at comparable levels with the correct expected molecular weight (Supplemental Figure S2A). We then examined both the mTQ2-CENP-T/W and EYFP-CENP-S/X fusion proteins in HeLa cells using fluorescence microscopy. As shown in Figure 2B, centromere localization of engineered mTQ2-CENP-T/W and EYFP-CENP-S/X is ready in interphase nuclei judged by immunofluorescence, indicating a successful complementation.

FIGURE 2:

FIGURE 2:

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Visualizing the CENP-TWSX complex using FC-FRET imaging of live cells. (A) Schematic representation of the mTQ2n-CENP-TBonsai, mTQ2c-CENP-W, EYFPn-CENP-S, and EYFPc-CENP-X fusion proteins. (B) Representative immunofluorescence images of HeLa cells coexpressing mTQ2n-CENP-TBonsai, mTQ2c-CENP-W and mCherry-H2B (upper panel) and images of HeLa cells coexpressing EYFPn-CENP-S, EYFPc-CENP-X and mCherry-H2B (lower panel). Bar: 10 μm. (C) Time-lapse images of HeLa cells coexpressing mTQ2n-CENP-TBonsai, mTQ2c-CENP-W, EYFPn-CENP-S, EYFPc-CENP-X, and mCherry-H2B. The mTQ2 signal on the kinetochore shows that CENP-TBonsai directly binds to CENP-W, and the EYFP signal shows that CENP-S directly binds to CENP-X. The warm-colored FRET ratio indicates that CENP-TBonsai/W subcomplex interacts with CENP-S/X subcomplex. Bar: 10 μm. (D) Bar graph showing the curve of the FRET ratio of 15 kinetochores over time in the cell described in C. There was no obvious trend of change overall. The error bars represent SDs. (E) Dot plot showing the FRET ratio at the indicated time points (15 cells from three independent experiments) as in C. The error bars represent SDs. Student’s t test was used to calculate the P value. ns, no significance.

We next sought to monitor the interaction of CENP-T/W with CENP-S/X in the centromere using a FRET-based sensor that reports quantitative changes in the distance between the CENP-T/W dimer and the CENP-S/X dimer in living cells. We adapted a sensor design (Chu et al., 2011), in which changes in inter-molecular FRET between mTQ2 and EYFP depend on changes in the distance between the aforementioned CENP-T/W and CENP-S/X dimers.

To validate the sensor response to physical interactions between CENP-T/W and CENP-S/X dimers in living cells, we first imaged their centromere interactions in interphase cells. For the sensor the EYFP:mTQ2 emission ratio is almost constant during cell cycle progression (Figure 2C; Supplemental Figure S2C), indicating that the CENP-T/W-S/X tetramer was assembled before entry into mitosis (Dornblut et al., 2014). The quantification of the EYFP:mTQ2 emission ratio indicated that interaction between the CENP-T/W dimer and the CENP-S/X dimer was persistent (Figure 2D; Supplemental Figure S2D). Scatter plot of FRET values of 15 cells from three independent experiments (Figure 2E) indicates the rigor and reproducibility of our FRET analysis. Thus, we conclude that FC-FRET analyses provide an optical sensor for reporting tetrameric interactions in space and time.

As shown in Supplemental Figure S2E, cells coexpressing mTQ2n-CENP-TBonsai and mTQ2c-CENP-W displayed clear kinetochore signals in CFP channel under live-cell imaging. Whereas cells expressing mTQ2n-CENP-TBonsai or mTQ2c-CENP-W individually displayed no specific signal. Thus, due to half of the donor partner can’t produce emission signal, there is no FRET signal when expressing only half of the donor partner.

Probing dynamic interactions between the inner and outer kinetochore using FC-FRET

Stable kinetochore-microtubule attachment is essential for cell division and requires CENP-T-dependent recruitment of outer kinetochore microtubule binders such as the Ndc80 complex (Nishino et al., 2013; Rago et al., 2015; Huis In ‘t Veld et al., 2016). To visualize the mechanism of outer kinetochore assembly mediated by CENP-T (Figure 3A), we engineered FC-EYFP with kinetochore microtubule binders Spc24 and Spc25 (Spc24/25) dimer (Figure 3B). As judged by Western blot, Spc24-EYFPn and Spc25-EYFPc were expressed at nearly equivalent levels (Supplemental Figure S2B). As shown in Figure 3C, the Spc24/25-FC-EYFP displayed a bright EYFP signal and was correctly localized at the kinetochore upon prophase, and the chromosomes were faithfully segregated, suggesting that Spc24/25 forms a stable dimer during chromosome segregation in mitosis. As shown in Supplemental Figure S3A, cells coexpressing Spc24-EYFPn and Spc25-EYFPc displayed clear kinetochore signals (mitotic cells) or cytoplasmic signal (interphase cells) in YFP channel under live-cell imaging. However, cells expressing Spc24-EYFPn or Spc25-EYFPc individually displayed no specific signal. Thus, the signals observed in cells coexpressing Spc24-EYFPn and Spc25-EYFPc are specific. Interestingly, cells coexpressing EYFPn-Spc24 and EYFPc-Spc25 retained EYFP signals in the cytoplasm but failed to localize to kinetochores. In addition, in contrast to untransfected cells (Supplemental Figure S3B, arrow), cells expressing EYFPn-Spc24 and EYFPc-Spc25 had severe chromosomal alignment defects (Supplemental Figure S3B). These observations suggest that placing FC-EYFP at the N-terminus of Spc24/25 interferes with the correct kinetochore localization of Spc24/25 and chromosomal alignment, echoing the importance of Ndc80 complex in microtubule attachment (Cheeseman et al., 2006; DeLuca et al., 2006; Dou et al., 2015).

FIGURE 3:

FIGURE 3:

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Probing dynamic interactions between the inner and outer kinetochore using FC-FRET. (A) Schematic representation of how CDK1 phosphorylations towards CENP-T recruit the Ndc80 complex and Mis12 complex. (B) Schematic representation of the Spc24-EYFPn and Spc25-EYFPc fusion protein. (C) Representative live-cell imaging stills illustrating mitotic progression in cells coexpressing Spc24-EYFPn, Spc25-EYFPc, mTQ2-Tubulin, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. The scale bar represents 10 μm. To facilitate the display of merge images, the EYFP and mTQ2 signals were converted into green and blue representations (pseudocolor), respectively. (D) Time-lapse images of HeLa cells coexpressing mTQ2n-CENP-TBonsai, mTQ2c-CENP-W, Spc24-EYFPn, Spc25-EYFPc, and mCherry-H2B. The mTQ2 signal on the kinetochore shows that CENP-TBonsai directly binds to CENP-W, the EYFP signal shows that Spc24 directly binds to Spc25. In addition, both mTQ2n-CENP-TBonsai/mTQ2c-CENP-W and Spc24-EYFPn/Spc25-EYFPc displayed correct kinetochore localization. The warm-colored FRET ratio indicates that the CENP-TBonsai/W subcomplex interacts with the Spc24/Spc25 subcomplex. Bar: 10 μm. (E) Bar graph showing the curve of the FRET ratio value of 20 kinetochores over time in the cells described in D. Error bars represent SDs. (F) Dot plot showing the FRET ratio value of indicated time points (15 cells from three independent experiments) as in D. Error bars represent SDs. The student’s t test was used to calculate the P value. ****, P < 0.0001.

To visualize the dynamic interactions between the Spc24/25 dimer and the CENP-T/W dimer, synchronized HeLa cells were transiently transfected to express Spc24/25-FC-EYFP, FC-mTQ2-CENP-TBonsai/W, and mCherry-H2B. As shown in Figure 3D, mTQ2-CENP-T/W appeared at the interphase centromere while EYFP-Spc24/25 located to the kinetochore after cells entered mitosis when the nuclear envelope was broken down. Quantitative analyses revealed that the interaction between CENP-T/W and Spc24/25 dramatically strengthened as the cells entered mitosis as the EYFP:mTQ2 emission ratio increased (Figure 3E). A scatter plot of FRET values of the indicated time points of 15 cells from three independent experiments revealed that the FRET ratio increased significantly 30 min after nuclear envelope breakdown (NEBD, Figure 3F). Additional representative cell images for the FRET assay are shown in Supplemental Figure S4, A–D. These findings echo an elegant high-resolution kinetochore protein architecture study showing the CENP-T moved 16-nm closer to the position of Spc24/25 in taxol treated cells (Wan et al., 2009). As a negative control, HeLa cells were transiently transfected to express Spc24/25-FC-EYFP, FC-mTQ2-CENP-THFD/W, and mCherry-H2B. Although the FC-EYFP and FC-mTQ2 signals localized to the kinetochore, no FRET signal was observed, indicating that there was no interaction between CENP-THFD and Spc24/25 (Supplemental Figures S5, A and B). Thus, we conclude that FC-FRET analyses provide an optical sensor to report tetrameric interactions bridging the centromere core complex and kinetochore microtubule binders.

Delineating the temporal regulation of CENP-T interactions with Spc24/25 by CDK1 using FC-FRET

Several lines of evidence demonstrate that CENP-T recruits the KMN network to kinetochores and that CENP-T-KMN interactions are essential in chicken DT40 cells which is regulated by CDK1 phosphorylation (Hara et al., 2018; Takenoshita et al., 2022). To delineate the temporal regulation of CENP-T function by CDK1-elicited phosphorylation, synchronized HeLa cells were transiently transfected to express Spc24/25-FC-EYFP, mTQ2-CENP-T, and mCherry-H2B. As shown in Figure 4A and Supplemental Figure S6A, the localization of mTQ2-CENP-T is superimposition onto that of Spc24/25-FC-EYFP and the EYFP:mTQ2 emission ratio increased as the cells entered mitosis. However, the addition of 10 µM Ro3306 and 20 µM MG132 into G2/M cells resulted in a perturbation of the EYFP:mTQ2 emission ratio, indicating that CDK1 activity is critical for CENP-T binding to Spc24/25 (Figure 4B; Supplemental Figure S6B). Quantitative analyses of the EYFP:mTQ2 emission ratio from 20 kinetochores over time in cells in Figure 4, A and B indicated that CDK1 activity is required for accurate interactions between CENP-T and Spc24/25 (Figure 4C).

FIGURE 4:

FIGURE 4:

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Delineating the temporal regulation of CENP-T interactions with Spc24/25 by CDK1 using FC-FRET. (A and B) Time-lapse images illustrating mitotic progression of DMSO-treated HeLa cell (A) and Ro3306-treated HeLa cell (B) coexpressing mTQ2-CENP-T, Spc24-EYFPn, Spc25-EYFPc, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. Ten minutes before imaging, the cells were treated with DMSO and Ro3306, respectively. Bars: 10 μm. (C) Bar graph showing the curve of the FRET ratio value of 20 kinetochores over time in the cells described in A and B. (D) Time-lapse images illustrating the mitotic progression of HeLa cells coexpressing mTQ2-CENP-T, Spc24-EYFPn, Spc25-EYFPc, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. At the indicated time point, the cells were treated with Ro3306. Bar: 10 μm. (E) Bar graph showing the curve of the FRET ratio value of 20 kinetochores over time in the cells described in D. Error bars represent SDs. (F) Dot plot showing the FRET ratio value of cells with indicated drug treatment for 30 min (20 cells from three independent experiments) as in A, B, and D. Error bars represent SDs. The student’s t test was used to calculate the P value. ns, no significance. ****, P < 0.0001.

If CDK1 activity is required for CENP-T to recruit Spc24/25, the addition of CDK1 inhibitor into cells that have already entered mitosis would not interfere CENP-T interactions with Spc24/25. Consistent with our hypothesis, the addition of 10 µM Ro3306 and 20 µM MG132 into G2/M cells resulted in no detectable change of the EYFP:mTQ2 emission ratio but caused chromosome segregation paused (Figure 4, D and E; Supplemental Figure S7, A–D), suggesting that CDK1 kinase activity is not required for maintaining CENP-T interaction with Spc24/25 after cells enter mitosis. Thus, we conclude that phosphorylation of CENP-T by CDK1 is required for recruiting but not sustaining Spc24/25 to the centromere. Quantification of the FRET value of 20 cells at the 30 min (after the NEBD) time point revealed that the FRET ratio of cells treated with Ro3306 at the NEBD was significantly lower than that of cells treated with DMSO. However, the FRET ratio did not significantly differ between cells treated with Ro3306 at prometaphase and cells treated with DMSO (Figure 4F).

Probing the role of kinetochore microtubules in CENP-T interactions with Spc24/25 using FC-FRET

We next sought to examine whether kinetochore microtubule attachment regulates Spc24/25 interaction with CENP-T, as the Spc24/25 dimer is an important kinetochore microtubule binder. To this end, synchronized HeLa cells were transiently transfected to express Spc24/25-FC-EYFP, mTQ2-CENP-T, and mCherry-H2B. As shown in Figure 5A and Supplemental Figure S8, A–D, the localization of mTQ2-CENP-T is superimposition onto that of Spc24/25-FC-EYFP and the EYFP:mTQ2 emission ratio increased as the cells entered mitosis. We then added 0.33 µM nocodazole to depolymerize kinetochore microtubules at the indicated time (46 min after the cell entered mitosis). Despite an apparent chromosome clustering phenotype due to nocodazole-elicited microtubule depolymerization, the FRET ratio of EYFP:mTQ2 emission did not exhibit any significant changes after nocodazole treatment (Figure 5B), indicating that kinetochore microtubules are not necessary for maintaining dynamic interactions between CENP-T and Spc24/25.

FIGURE 5:

FIGURE 5:

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Probing the role of kinetochore microtubules in CENP-T interactions with Spc24/25 using FC-FRET. (A) Time-lapse images illustrating the mitotic progression of HeLa cells coexpressing mTQ2-CENP-T, Spc24-EYFPn, Spc25-EYFPc, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. Cells were treated with nocodazole at the indicated time point. Bar: 10 μm. (C) Time-lapse images illustrating the mitotic progression of HeLa cells coexpressing mTQ2-CENP-T, Spc24-EYFPn, Spc25-EYFPc, and mCherry-H2B. Images were acquired at the indicated time points after NEBD. The cells were treated with syntelin and imaged immediately. Bar: 10 μm. (B and D) Bar graph showing the curve of the FRET ratio value of 20 kinetochores over time in the cells described in A (B) and C (D). Error bars represent SDs. (E) Dot plot showing the FRET ratio value of cells with indicated drug treatment for 30 min (20 cells from three independent experiments) as in A and C. Error bars represent SDs. The student’s t test was used to calculate the P value. ns, no significance.

CENP-E is a key protein mediating correct kinetochore microtubule attachment. Inhibiting the activity of CENP-E induces a large proportion of chromosomes with incorrect kinetochore microtubule attachment. However, whether the binding of CENP-T and Spc24/25 was affected by incorrect kinetochore microtubule attachment remains unclear. To this end, we employed chemical inhibitor of CENP-E syntelin to create syntelic chromosomes. Consistent with the findings of previous report (Ding et al., 2010), syntelin did not inhibit progression through the S and G2 phases of the cell cycle but caused mitotic arrest with lagging chromosomes (Figure 5C). Syntelin treatment did not perturb bipolar spindles but produced misaligned chromosomes near the spindle poles (Figure 5C; arrows). The kinetochore position relative to the pole is an accurate reporter for judging chromosome misalignment. However, our quantitative analysis of the FRET ratio of the EYFP:mTQ2 emission indicated that the kinetochore microtubule attachment state has no effect on the dynamic interactions between CENP-T and Spc24/25 (Figure 5D; additional representative images are shown in Supplemental Figure S9, A–D). Thus, we conclude that CENP-T is required to recruit Spc24/25 to the kinetochore but that the interaction of Spc24/25 with CENP-T is independent of the microtubule attachment state during chromosome segregation. Quantification of the FRET value of 20 cells at 30 min time point after drug treatment revealed that the FRET ratio of cells treated with nocodazole or syntelin has no significant difference with cells treated with DMSO (Figure 5E).

DISCUSSION

The FC-FRET is based on a combination of complementary fluorescent proteins with FRET (Yang et al., 1996; Hu et al., 2002). Upon their maturation, FC molecules are excited to emit distinct spectra of fluorescence. An important consideration is the FRET efficiency achievable by the fluorescent protein pair. mTQ2 is recently described CFPs with dramatically increased quantum yield compared with those of previous CFPs (Goedhart et al., 2012). We used mTQ2 containing monomerizing mutation A206K as a FRET donor to generate functional FC-mTQ2. This feature of the FC-FRET pair provides benefits for super-resolution microscopic analyses applying for visualizing and quantifying spatiotemporally matched molecular interactions. Thus, FC-FRET provides an excellent tool kit for delineating physiochemical determinants underlying dimeric and tetrameric protein–protein interactions in space and time at a resolution of 10 nm.

This work provides a new method for observing dimeric, trimeric, and tetrameric protein–protein interactions in live cells at the single-molecule level so that their structure-functional relationship can be visualized at high precision. For proof-of-principle purposes, in the current study, we successfully demonstrated the efficiency of the FC-FRET system and observed the precise localization of the dimerized centromere proteins CENP-T/W, CENP-S/X and kinetochore proteins Spc24/25 on the mitotic chromosome with a resolution less than 10 nm. We further showed that CENP-T/W assembled onto centromeres in interphase cells, while the Spc24/25 dimer was recruited to kinetochores by CENP-T after the nuclear envelop is broken down. The localization of Spc24/25 to the kinetochore was dependent on CDK1 kinase activity, as phosphorylation of CENP-T at its N-terminus is essential for Spc24/25 binding (Gascoigne and Cheeseman, 2013; Huis In ‘t Veld et al., 2016; Zhao et al., 2019). Interestingly, CDK1 kinase activity is no longer needed upon Spc24/25 localization to the kinetochore. A recent study on fission yeast CENP-T reported that the CENP-T N-terminus has a Ccp1 (the yeast CENP-A homologue) interacting motif adjacent to the Spc24/25 binding motif (Dong et al., 2021). This study suggested that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T by CDK1 phosphorylation ensures faithful kinetochore assembly. Thus, it would be great excitements and challenges clarifying the temporal regulatory mechanisms of underlying the hierarchical interactions and posttranslational modifications in CENP-T pathway.

In the future, FC-FRET technology should benefit from improvements of the palette of available FRET pairs including newly developed dual FRET systems (Laviv et al., 2016), as well as from the discovery of means to improve photostability. This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity (Laviv et al., 2016). The combination of FC-FRET with functional protein interactome data will enable us to visualize their interactions in space and time during cell division, and build a high-resolution atlas of protein society with precise localization in eukaryotic cells in various contexts (Mo et al., 2016; Song et al., 2021). The successful demonstration of CENP-T/W interactions with Spc24/25 in centromere-kinetochore dynamics present here provides proof of concept.

It is worth noting that complementary fluorescent proteins exhibit some artificial activities when they are applied for those proteins with unknown ability of dimerization or less characterized dimerization activity (Honnappa et al., 2009). In addition, fluorescence complementation would perturb native dimerization dynamics and we noticed that FC-mTQ2-CENP-T/W caused delays in mitotic progression. Nevertheless, the use of both N- and C-terminal orientations for the FC together with classic biochemical characterization of protein dimerization will alleviate artifacts if trace amount exists in cells.

In sum, FC-FRET provides an excellent tool kit for delineating structural determinants underlying dimeric, trimeric, and tetrameric protein–protein interactions in space and time at a resolution of 10 nm. The FC-FRET approach established here will enable the simultaneous visualization of interactions between one copy of the wild-type protein and another copy of the protein with disease-prone mutations and/or single nucleotide polymorphisms. In this case, high-resolution correlation maps between genotype and phenotype can be established with ease. It is also expected to enable a new generation of single molecule-based screening approaches aiming modulating dimeric protein interactions.

MATERIALS AND METHODS

DNA Constructs

To generate plasmids expressing N-terminal mTQ2n/c-tagged CENP-THFD (aa. 450-561), CENP-W, PCR-amplified CENP-THFD or CENP-W cDNAs were cloned and inserted into pcDNA3.1 backbone-base vectors expressing mTQ2n/c via the BamHI and XhoI sites, respectively. To generate plasmids expressing N-terminal EYFPn/c-tagged CENP-S, CENP-X, PCR-amplified CENP-S or -X cDNAs were cloned and inserted into pcDNA3.1 backbone-base vectors expressing EYFPn/c via the BamHI and XhoI sites, respectively. To generate plasmids expressing C-terminal EYFPn/c-tagged Spc24, Spc25, PCR-amplified Spc24/25 cDNAs were cloned and inserted into pcDNA3.1 backbone-base vectors expressing EYFPn/c via the BamHI and XhoI sites, respectively. To generate plasmids expressing N-terminal mTQ2n-tagged CENP-TBonsai, PCR was used to amplify the Spc24/25 binding region of CENP-T (aa. 76-107), and PCR-amplified CENP-THFD cDNAs were inserted into the pcDNA3.1 backbone-base vector expressing mTQ2n using the ClonExpress Entry One Step Cloning Kit (Vazyme C112). PrimeSTAR DNA polymerase (Takara R045) was used to amplify different cDNAs. All the constructs were verified by sequencing.

Cell Culture and Transfection

HeLa cells were grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 units/ml penicillin (Life Technologies) plus 100 μg/ml streptomycin (Life Technologies). Cells were grown on cover glasses and were transfected 24 h before the experiments. Live cell experiments were performed in CO2 independent culture medium (Invitrogen/Life Technologies 18045088) containing 10% FBS. All the plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen 11668019) according to user’s manual.

Immunofluorescence Microscopy

HeLa cells grown on coverslips were fixed and permeabilized simultaneously with PTEMF buffer (50 mM PIPES pH 6.8, 0.2% Triton X-100, 10 mM EGTA, 1 mM MgCl2, 4% formaldehyde) at room temperature and were processed for indirect immunofluorescence microscopy. The samples were examined on a DeltaVision microscope (Applied Precision) with a 60 × objective lens (NA = 1.42), with optical sections acquired 0.25 μm apart in the z axis. Deconvoluted images from each focal plane were projected into a single picture using softWoRx (Applied Precision). Images were taken at identical exposure times within each experiment and were acquired as 16-bit gray-scale images. After deconvolution, the images were exported as 24-bit RGB images and processed in Adobe Photoshop. The images shown in the same panel have been identically scaled.

Antibodies and Drugs

Myc-tag antibody (Cell Signaling Technology, 9B11, #2276, 1:1,000), mouse anti-α-tubulin (Cell Signaling Technology, DM1A, #3873, 1:5,000) and human anticentromere autoantibody (ACA, Immunovision, HCT-0100, 1:5000 for immunofluorescence) were obtained from commercial suppliers. Thymidine was used at 2 mM, nocodazole at 0.33 μM, Ro3306 at 10 μM, syntelin at 1 μM (Ding et al., 2010; Mullen et al., 2022), and MG132 at 20 μM.

Western Blot

The samples were separated via standard SDS–PAGE electrophoresis and subsequently transferred to nitrocellulose membranes. Western blotting was performed using standard protocols. For all the Western blots, signals were detected using HRP-conjugated antimouse or antirabbit antibodies (Jackson ImmunoResearch) through an GE ImageQuant LAS 4000 machine.

Time-lapse Imaging

For time-lapse imaging, HeLa cells were cultured in glass-bottom culture dishes (MatTek) and maintained in CO2-independent medium (Life Technologies) supplemented with 10% FBS and 2 mM glutamine. During imaging, the dishes were placed in a sealed chamber at 37°C. Images of living cells were taken with a DeltaVision microscope. For presentation of the details of the real-time imaging, projection images were constructed from 0.5 μm/section for three sections within a cell.

Live Cell Imaging and FC-FRET Assay

HeLa cells expressing FC-FRET plasmids and mCherry-H2B plasmid were grown on glass-bottomed dishes. When most cells entered the late G2 phase of mitosis (∼7 h after thymidine washout), the cells were supplemented with CO2-independent medium (Life Technologies) and observed at 37°C using the DeltaVision RT system (Applied Precision). Images of mCherry were captured every 5 min with an exposure time of 0.1 s. In live cell imaging of FC-FRET sensors, four channels were typically used: CFPex/CFPem, CFPex/YFPem, YFPex/YFPem, and mCherryex/mCherryem. CFP was excited at 470 nm, and a dual-view beam splitter (Optical Insights) was used to collect emission light from both CFP and YFP. Fluorescence images were acquired using an inverted microscope (Olympus IX-70) with a 60 × NA 1.42 PlanApo objective lens, capturing three z-stack images with a slice thickness of 0.5 μm. The acquisition parameters such as exposure, focus, and illumination, were controlled by softWoRx software (Applied Precision).

Z-stack projection and subsequent image analysis and processing were performed using softWoRx software. Image deconvolution was applied to the acquired images for kinetochore localization analysis using DeltaVision software (Applied Precision). FRET ratio analysis was performed on unprocessed images. The same exposure settings and scaling were used for all images in a specific experiment. The image data after z-stack projection were analyzed using softWoRx software to calculate the FRET ratio using formula 1, resulting in RTH format files. Additionally, the average FRET ratio of kinetochores at each time point was calculated.

graphic file with name mbc-35-ar51-e001.jpg 1

Finally, to convert RTH files to a cool-to-warm color scheme using the softWoRx Explorer, the following steps were used: 1) Open softWoRx Explorer and select the Color Tool; 2) Check the Palette option and choose “Cold to Hot no Background” under the Type section; 3) Use the Display Scaling Tool to adjust the appropriate numerical range for the FRET Ratio values; 4) The images of the FRET ratio were saved in the cool-to-warm color scheme according to the respective time points.

To calculate the FRET value of the aforementioned sensor from individual cells from various experiments, softWoRx Explorer automatically selects 15 to 20 kinetochores in a given cell and gives the FRET average for all kinetochores in the examined cell. To streamline data presentation, we selected FRET values at two time points from more than 15 cells across three experimental sets, depicted them in specific bar graphs, performed statistical analysis, and illustrated scatter plots. For all scatter plots, the means represent the average FRET values of 15 to 20 cells. Error bars represent standard deviations (SDs).

Supplementary Material

mbc-35-ar51-s001.pdf (4.7MB, pdf)

Acknowledgments

This work was supported by the MOST-NSFC grants (2022YFA1303100, 32090040, 92254302, 2022YFA0806800, 92153302, 31621002, 2017YFA0503600 to X. L.; and 32170733 to Z.D.); Plans for Major Provincial Science & Technology Projects of Anhui Province (202303a0702003 to X. L.); the Ministry of Education (IRT_17R102 to X. L.); the Fundamental Research Funds for the Central Universities (KB9100000007, KB9100000013 to X. L.).

Abbreviations used:

ACA

anti-centromere antibody

CCAN

constitutive centromere associated network

CENP

centromere protein

CFP

cyan fluorescent protein

EYFP

enhanced yellow fluorescence protein

EYFPn/c

the N-terminal or C-terminal fragment of the EYFP protein

FC-FRET

fluorescence complementation-based Förster resonance energy transfer

FRET

Förster resonance energy transfer

HFD

histone fold domain

HRP

horseradish peroxidase

NEBD

nuclear envelope breakdown

SDS–PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Footnotes

This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E23-09-0379) on February 21, 2024.

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Supplementary Materials

mbc-35-ar51-s001.pdf (4.7MB, pdf)

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