Gregory W. Fisher

Fluorogen-activating single-chain antibodies for imaging cell surface proteins

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, κ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG2a, κ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Non-invasive image acquisition and advanced processing in optical bioimaging

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Tumor Detection and Visualization Using Cyanine Fluorochrome‐Labeled Antibodies

Tumor localization using fluorescence has been made practical by current improvements in tumor targeting molecules, especially monoclonal antibodies and their derivatives, by the development of convenient near‐infrared emitting fluorochromes and by the availability of digital cameras having high sensitivity in this spectral region. Recent studies in animals have demonstrated that fluorochrome labeling of monoclonal antibodies confers adequate sensitivity and improved resolution. Distribution and catabolism of fluorochrome‐labeled and radiolabeled antibodies are similar. Simultaneous localization of multiple reagents is made possible by labeling with several different near‐infrared emitting fluorochromes; thus background subtraction and differential labeling of multiple tumor‐associated components can be performed. Difficulties in using the fluorochrome labels are mainly related to light scattering and absorption in tissues, but detection of small tumors at depths of several millimeters is feasible. The major medical use of this new technology is likely to be endoscopic location of tumors. Scientific uses include studies of tumor metastasis, uptake and distribution of drugs and tumor‐targeting molecules by tumors, and migration patterns of near‐infrared labeled cells in vivo.

Signal Amplification in the Detection of Single-copy DNA and RNA by Enzyme-catalyzed Deposition (CARD) of the Novel Fluorescent Reporter Substrate Cy3.29-Tyramide

We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent In Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of S-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa S3 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads. (J Histochem Cytochem 45:365–373, 1997)

Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

Life in the Atacama: Searching for life with rovers (science overview)

[1] The Life in the Atacama project investigated the regional distribution of life and habitats in the Atacama Desert of Chile. We sought to create biogeologic maps through survey traverses across the desert using a rover carrying biologic and geologic instruments. Elements of our science approach were to: Perform ecological transects from the relatively wet coastal range to the arid core of the desert; use converging evidence from science instruments to reach conclusions about microbial abundance; and develop and test exploration strategies adapted to the search of scattered surface and shallow subsurface microbial oases. Understanding the ability of science teams to detect and characterize microbial life signatures remotely using a rover became central to the project. Traverses were accomplished using an autonomous rover in a method that is technologically relevant to Mars exploration. We present an overview of the results of the 2003, 2004, and 2005 field investigations. They include: The confirmed identification of microbial habitats in daylight by detecting fluorescence signals from chlorophyll and dye probes; the characterization of geology by imaging and spectral measurement; the mapping of life along transects; the characterization of environmental conditions; the development of mapping techniques including homogeneous biological scoring and predictive models of habitat location; the development of exploration strategies adapted to the search for life with an autonomous rover capable of up to 10 km of daily traverse; and the autonomous detection of life by the rover as it interprets observations on-the-fly and decides which targets to pursue with further analysis.

Fluorogen-Activating Proteins as Biosensors of Cell-Surface Proteins in Living Cells

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen‐activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent‐Gyorgyi et al., Nat Biotechnol 2010;00:000–000).

Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β2-adrenergic receptor (β2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2ARs, all 33 known β2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization

G protein–coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology—receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.