Three-color spectral FRET microscopy localizes three interacting proteins in living cells.

FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough. The approach quantifies FRET signals and calculates the apparent energy transfer efficiencies (Es). The method was validated by measurement of a genetic (FRET standard) construct consisting of three different fluorescent proteins (FPs), mTFP, mVenus, and tdTomato, linked sequentially to one another. In addition, three 2-FP reference constructs, tethered in the same way as the 3-FP construct, were used to characterize the energy transfer pathways. Fluorescence lifetime measurements were employed to compare the relative relationships between the FPs in cells producing the 3-FP and 2-FP fusion proteins. The 3sFRET microscopy method was then applied to study the interactions of the dimeric transcription factor C/EBPalpha (expressing mTFP or mVenus) with the heterochromatin protein 1alpha (HP1alpha, expressing tdTomato) in live-mouse pituitary cells. We show how the 3sFRET microscopy method represents a promising live-cell imaging technique to monitor the interactions between three labeled cellular components.

Dynamic interactions between Pit-1 and C/EBPalpha in the pituitary cell nucleus.

The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPalpha) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPalpha in different ways. The results showed that the first alpha-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPalpha, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.

Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy.

The genetically encoded fluorescent proteins (FP), used in combination with Forster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells. The results demonstrate that when compared to Cerulean, mTFP has increased brightness, optimal excitation using the standard 458-nm laser line, increased photostability, and improved spectral overlap with Venus. In addition, the two-photon excitation and fluorescence lifetime characteristics are determined for mTFP. Together, these measurements indicate that mTFP is an excellent donor fluorophore for FRET studies, and that its use may improve the detection of interactions involving proteins that are difficult to express, or that need to be produced at low levels in cells.

Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain.

The restriction of transcription factors to certain domains within the cell nucleus must serve an important regulatory function. The silencing mediator of retinoic acid and thyroid hormone (SMRT) and other members of the corepressor complex are enriched in spherical intranuclear foci, and repress estrogen receptor alpha (ERalpha)-dependent transcriptional activity. When fluorescent protein (FP)-labeled SMRT and ERalpha were co-expressed, the proteins co-localized. The subnuclear organization and positioning of the complexes, however, depended on the ligand state of the receptor. Automated image analysis was used to quantify the ERalpha-dependent change in SMRT organization in randomly selected living cell populations. The results demonstrate that the subnuclear positioning of SMRT is influenced by the ligand-bound ERalpha, and this activity is dependent on the ratio of the co-expressed ERalpha and SMRT. A deletion mutant of ERalpha showed that the receptor DNA-binding domain was necessary for the ligand-dependent positioning of SMRT. These results define important organizational mechanisms that underlie nuclear receptor regulation of gene expression.

Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression.

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus.

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPalpha are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

Computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell populations.

Many nuclear proteins, including the nuclear receptor co-repressor (NCoR) protein are localized to specific regions of the cell nucleus, and this subnuclear positioning is preserved when NCoR is expressed in cells as a fusion to a fluorescent protein (FP). To determine how specific factors may influence the subnuclear organization of NCoR requires an unbiased approach to the selection of cells for image analysis. Here, we use the co-expression of the monomeric red FP (mRFP) to select cells that also express NCoR labeled with yellow FP (YFP). The transfected cells are selected for imaging based on the diffuse cellular mRFP signal without prior knowledge of the subnuclear organization of the co-expressed YFP-NCoR. The images acquired of the expressed FPs are then analyzed using an automated image analysis protocol that identifies regions of interest (ROIs) using a set of empirically determined rules. The relative expression levels of both fluorescent proteins are estimated, and YFP-NCoR subnuclear organization is quantified based on the mean focal body size and relative intensity. The selected ROIs are tagged with an identifier and annotated with the acquired data. This integrated image analysis protocol is an unbiased method for the precise and consistent measurement of thousands of ROIs from hundreds of individual cells in the population.

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser.

Orange fluorescent proteins (FPs) are attractive candidates as Forster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.

Monitoring dynamic protein interactions with photoquenching FRET.

The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.

Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus.

The co-repressor proteins SMRT and NCoR concentrate in specific subnuclear compartments and function with DNA-binding factors to inhibit transcription. To provide detailed mechanistic understanding of these activities, this study tested the hypothesis that functional interactions with transcription factors, such as the pituitary-gland-specific Pit-1 homeodomain protein, direct the subnuclear organization and activity of co-repressor complexes. Both SMRT and NCoR repressed Pit-1-dependent transcription, and NCoR was co-immunoprecipitated with Pit-1. Immunofluorescence experiments confirmed that endogenous NCoR is concentrated in small focal bodies and that incremental increases in fluorescent-protein-tagged NCoR expression lead to progressive increases in the size of these structures. In pituitary cells, the endogenous NCoR localized with endogenous Pit-1 and the co-expression of a fluorescent-protein-labeled Pit-1 redistributed both NCoR and SMRT into diffuse nucleoplasmic compartments that also contained histone deacetylase and chromatin. Automated image-analysis methods were applied to cell populations to characterize the reorganization of co-repressor proteins by Pit-1 and mutation analysis showed that Pit-1 DNA-binding activity was necessary for the reorganization of co-repressor proteins. These data support the hypothesis that spherical foci serve as co-repressor storage compartments, whereas Pit-1/co-repressor complexes interact with target genes in more widely dispersed subnuclear domains. The redistribution of co-repressor complexes by Pit-1 might represent an important mechanism by which transcription factors direct changes in cell-specific gene expression.