The last 20 years of biological research have brought countless advances in the technologies used and, by consequence, the invaluable, life-enriching information revealed through their use. Advances in our ability to label proteins fluorescently have revolutionized the study of protein function in all areas of research, from academia to drug development. Simply put, fluorescent labeling provides the means to monitor the precise localization and dynamics of proteins within living cells. Importantly, evolution of this technology is defined by advances in both the number of spectrally distinct fluorescent labels available and the instruments used to detect them. One great advance in instrument technology is the development of high-content analysis (HCA), an automated platform for performing fluorescence microscopy and quantitative image analysis on whole cells. Introduced in the mid 1990s, HCA was first used as a fixed-cell assay system employed only in the more advanced steps of preclinical research and primarily focused on simple intracellular changes indicative of cell toxicity. In time, and in parallel with the continued emergence of fluorescent markers, both the instrumentation and accompanying software analyses have become more high-throughput, versatile and affordable, allowing sophisticated live-cell analyses. Today’s HCA provides a powerful means by which multiple markers, indicative of two or more discrete cellular responses, can be measured in large-scale quantitative assays
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. When combined with quantitative analysis of cellular morphology and textures, the information content of the numerous available assays has dramatically increased.
In the spirit of pursuing further technical advancement in this field, two leading providers of biological research tools, Promega Corporation and PerkinElmer, Inc., have started collaboration by merging cutting-edge technologies that are exemplary in their versatility and ease of use: Promega Corporation’s HaloTag® protein reporter technology and the high-content capabilities of PerkinElmer Opera® High Content Screening System and Operetta® High Content Imaging System. While the HaloTag® platform offers fast and flexible ways of fluorescently tagging proteins, the Opera® and Operetta® systems enable high-content analyses of large numbers of cells at subcellular resolution.
The HaloTag® reporter technology can be used for imaging by specifically and efficiently labeling an expressed protein fusion with any one of the available spectrally distinct fluorescent tags
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(Figure 1). These small tags, called ligands, are comprised of a linker that covalently binds to the HaloTag® fusion protein and a fluorescent moiety. Thus, unlike standard protein reporters, a separate construct does not need to be made for each “color” of fluorescence. More importantly, unlike other available modular labeling technologies, the HaloTag® protein itself has no endogenous equivalent and does not interfere with proper cellular function of fusion partners. Furthermore, labeled HaloTag® proteins can withstand denaturation, making this system inclusive of fixed-cell analyses, unlike many conventional fluorescent labeling technologies. Moreover, if a fluorescent ligand is needed but not currently commercially available, it is possible to make novel HaloTag® ligands using simple chemistry. Reactive linkers that covalently attach to the HaloTag® protein and are easily conjugated to any fluorescent moiety of choice are available for this purpose. Ultimately, by combining color choice and differences in ligand cell permeability, the user is empowered with spatial and temporal control of cell labeling. This makes imaging experiments aimed at directly uncovering protein turnover or translocation possible
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Figure 1. HaloTag® technology. Panel A. A HaloTag® fusion protein displaying the linker to scale. Also shown are the TEV protease recognition site and binding pocket for covalent interaction with HaloTag® ligands. Panel B. Commercially available HaloTag® fluorescent ligands include cell-permeant and impermeant options in a variety of “colors”. Ligands suited to no-wash and rapid labeling protocols are available to accommodate individual workflows.
With more than a decade of leadership in high-content screening (HCS) and many millions of compounds screened on its platforms, PerkinElmer offers a portfolio of solutions for high-content screening and high-content imaging and analysis. Most importantly, these solutions address the diverse needs of throughput, flexibility and ease of use for scientists worldwide. The high-content Opera® and Operetta® systems are automated microscopes capable of acquiring fluorescence and brightfield images from samples in microplates or on slides. The Opera® system combines laser-based excitation with a microlens-enhanced Nipkow spinning disk, water objectives and multichannel simultaneous acquistion. The result is outstanding confocal image quality combined with high throughput and high flexibility for large-scale screening applications. The Operetta® system provides great ease of use and a unique combination of enabling hardware and software features. Both systems are equipped with spinning disk confocal scanners, which cause significantly less photodamage compared to other confocal systems
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and are therefore especially well suited for live-cell experiments.
Image acquisition on the Operetta® system can be unsupervised by using a reliable autofocus system and different objectives that range from low to high magnifications (2–100X). This, combined with a large field of view, allows imaging of the whole well with the option of choosing an area of interest for more detailed analysis. For exceptional image quality, high numerical aperture objectives are available, and objectives with adjustable working distances ensure compatibility with a range of different microplates. The ability to switch freely between widefield and confocal modes enables the user to choose the best imaging technology in a flexible way, depending on the study’s scope and biological samples. By combining a large number of different excitation and emission filters, researchers can image innumerable fluorophores by both methods, and brightfield imaging even enables label-free visualization of samples. For live-cell experiments, the live-cell chamber option on the Operetta® system ensures maintenance of optimal environmental conditions (temperature, CO2). Driving image acquisition as well as image analysis and data management, the Harmony® High Content Imaging and Analysis Software provides an intuitive user interface that allows a step-by-step approach to image analysis. This approach can be achieved by combining building blocks that are easy-to-understand. In this way, it is possible for every lab member to generate statistical unbiased data sets from imaging experiments without the need for an imaging expert.
The available assays are numerous and assorted, ranging from cell toxicity and cell cycle assays to different analyses of protein translocation. One of the most frequent high-content screening assays is monitoring G-protein coupled receptor (GPCR) internalization upon agonist stimulation. The first experimental step in this collaborative endeavor was, therefore, to evaluate the subcellular protein translocation of a labeled, membrane-bound HaloTag® fusion using the Operetta® system. The HaloTag® fusion protein model chosen for this evaluation is one in which HaloTag® protein is displayed on the cell surface and anchored to the membrane via a short transmembrane domain (HT-extracellular surface; HT-ECS). Prior to imaging, U2OS cells stably expressing this fusion protein were plated in 96-well format and labeled with the cell-impermeant HaloTag® Alexa Fluor® 488 Ligand. In this way, only the plasma-membrane-surface-displayed pool of HaloTag® fusion protein is labeled, leaving the internal endoplasmic reticulum, Golgi and vesicle-based pools unlabeled. Given that HT-ECS is brought into the cytoplasm via vesicle compartments that bud off from the plasma membrane as a result of normal membrane turnover, this model enables direct imaging of protein internalization over time (Figure 2). To record this phenomenon, images were acquired every 30 minutes over an 18-hour time period (Figure 2, Panel A). These images then were analyzed using a texture parameter analysis, “SER Spot” (Figure 2, Panel B), revealing a wide signal window for the quantitative increase of labeled vesicles in cells over time. The corresponding Z factor (0.51), which is a statistical measure of sensitivity and accuracy, indicates a successful and robust assay. As this research moves forward, more complex biologically relevant assays will be implemented such that the inherently powerful potential offered by the HaloTag® technology and Operetta® system is successfully exploited.
Figure 2. Monitoring internalization of a membrane-bound HaloTag® protein (HT-ECS) using the Operetta® system. Panel A. Internalization of a membrane-bound HaloTag® protein (HT-ECS) into endosomes. The extracellular pool of HaloTag® protein was labeled with the membrane-impermeant HaloTag® Alexa Fluor® 488 Ligand, and cells were imaged on the Operetta® system at 37°C and 5% CO2 for 18 hours using a measurement interval of 30 minutes. Panel B. Quantification of membrane protein internalization using the “SER Spot” texture feature (scale 2px) of the Harmony® software.
The HaloTag® technology also was recently used for a large screening project by Evotec AG, where over 250,000 unique compounds were screened for their ability to inhibit internalization of a novel membrane receptor using live cells. The HaloTag® platform allowed Evotec scientists to specifically label receptors with low background levels and increased the sensitivity compared to other technologies. Using a stably transfected cell line, the screen was conducted with a throughput of over 10,000 compounds per day using the Opera® system The results identified compounds that have the potential to be first in class for the therapeutic area.
Figure 3. Screening for inhibitors of ligand-induced cell-surface receptor internalization. Cells stably expressed an orphan receptor fused with HaloTag® protein on the C-terminus. Panel A. In the absence of ligand, the receptor is located mainly on the cell surface. Panel B. The presence of ligand leads to translocation of the receptor to the cytosol. Panel C. Treatment with Compound A inhibits receptor internalization to some extent. Panel D. Compound B leads to internalization of the receptor into endosomes. This exemplifies how high-content screening allows us to distinguish between different modes of action of compounds. Panel E. Exemplary compound showing dose-dependent inhibition of receptor internalization. Data courtesy of M. Slack, Evotec AG, Hamburg.Further facilitating research using the HaloTag® technology for high-content analysis is an ever-growing collection of available, validated human clones generated in partnership with the Kazusa DNA Research Institute in Japan. This collection includes many relevant gene families such as GPCRs, kinases, oncogenes and epigenetics-related genes (Table 1). Provided with these clones are sequence, insert and expression validation data and even subcellular localization data for over 80% of them. Overall, given their respective attributes, combining these technologies enables the generation of numerous powerful assay designs.
Conclusion
The HaloTag® technology is a flexible labeling technology for visualizing living cells in various experimental contexts. The interchangeable labeling simplifies multiplexing with green fluorescent protein (GFP) or other labeling reagents, and the high sensitivity and low background levels make the HaloTag® platform an ideal tool for high-content assays. Furthermore, using HaloTag® fusion proteins with the Opera® and Operetta® systems allows high-quality live-cell imaging with exceptionally low phototoxicity on a large scale, enabling the generation of unbiased statistically relevant data sets.