Jonathan W. Jarvik

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.

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.

Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate.

The Src tyrosine kinase associates with the focal adhesion adaptor protein Cas (Crk‐associated substrate) to suppress the expression of potential tumor suppressor genes. For example, Src utilizes Cas to suppress the expression of the LIM‐only protein Fhl1 (four and a half LIM domains 1), in order to promote non‐anchored tumor‐cell growth and migration. Here, we report that the promoter region of the Fhl1 gene was methylated more in Src‐transformed cells than non‐transformed cells. In addition, global expression analysis indicates that Fhl1 induced expression of serum deprivation response factor (Sdpr) in Src‐transformed cells. Moreover, Fhl1 and Sdpr was expressed in approximately 87% and 40% of samples obtained from non‐transformed breast, 100% of samples obtained from non‐transformed kidney, and over 60% of samples obtained from non‐transformed prostate. In contrast, Fhl1 and Sdpr was detected in approximately 40% and 7% of matched samples from mammary carcinoma, less than 11% of matched samples from kidney carcinoma, and in less than 22% of matched samples from prostate carcinoma. These data indicate that Fhl1 and Sdpr expression was significantly reduced in tumors of the breast (P < 0.02 and P < 0.001), kidney (P < 0.01), and prostate (P < 0.05). In addition, although Src can activate mitogen‐activated protein kinase (MAPK) to promote tumor‐cell growth, our data indicate that Src did not rely on MAPK activity to suppress the expression of Fhl1 and Sdpr in transformed cells. Thus, Src induced methylation of the promoter region of the Fhl1 gene; Src suppressed Fhl1 and Sdpr expression independent of mitogen‐activated protein kinase (MAPK) activity; Fhl1 induced the expression of Sdpr in Src‐transformed cells; and Fhl1 and Sdpr expression was suppressed in tumors of the breast, kidney, and prostate. (Cancer Sci 2008; 99: 1326–1333)

Location proteomics: building subcellular location trees from high-resolution 3D fluorescence microscope images of randomly tagged proteins

The overall object of proteomics is to characterize all of the proteins expressed in a given cell type. With the rapid development of random gene tagging technology and high resolution fluorescence microscopy, it has become possible to generate libraries of digital images depicting the location patterns of most proteins in any given cell type. While the subcellular location of a protein is important to its function, no established methods exist for the systematic description, comparison or organization of protein location patterns. We have previously described classification methods that accurately recognize all major subcellular location patterns in both 2D and 3D images, as well as methods for rigorous statistical comparison of such patterns. We describe here the application of the numerical features from the previous work to images obtained by random tagging of proteins. Spinning disk confocal microscopy was used to collect images depicting the location patterns of 46 NIH 3T3 cell clones expressing proteins randomly tagged with a fluorescent protein. A set of 42 numerical features describing both image texture and object morphology were calculated and used to build subcellular location trees that group the tagged proteins by similarity of location pattern.

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).

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform.

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

Determining the subcellular location of new proteins from microscope images using local features

MOTIVATION Evaluation of previous systems for automated determination of subcellular location from microscope images has been done using datasets in which each location class consisted of multiple images of the same representative protein. Here, we frame a more challenging and useful problem where previously unseen proteins are to be classified. RESULTS Using CD-tagging, we generated two new image datasets for evaluation of this problem, which contain several different proteins for each location class. Evaluation of previous methods on these new datasets showed that it is much harder to train a classifier that generalizes across different proteins than one that simply recognizes a protein it was trained on. We therefore developed and evaluated additional approaches, incorporating novel modifications of local features techniques. These extended the notion of local features to exploit both the protein image and any reference markers that were imaged in parallel. With these, we obtained a large accuracy improvement in our new datasets over existing methods. Additionally, these features help achieve classification improvements for other previously studied datasets. AVAILABILITY The datasets are available for download at http://murphylab.web.cmu.edu/data/. The software was written in Python and C++ and is available under an open-source license at http://murphylab.web.cmu.edu/software/. The code is split into a library, which can be easily reused for other data and a small driver script for reproducing all results presented here. A step-by-step tutorial on applying the methods to new datasets is also available at that address. CONTACT murphy@cmu.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

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.

ISOLATION AND PRELIMINARY CHARACTERIZATION OF THREE CHLAMYDOMONAS STRAINS INTERFERTILE WITH CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Three new strains of the unicellular green alga Chlamydomonas reinhardtii Dangeard were isolated from soil. The isolates differed from one another and from standard laboratory strains of C. reinhardtii in a number of traits, including heavy metal resistance, protein composition, and mitochondrial DNA length. The new isolates also exhibited distinctive restriction fragment length polymorphisms in their nuclear, chloroplast, and mitochondrial genomes. The new isolates were interfertile with the standard laboratory strains and appeared to transfer chloroplast and mitochondrial genomes in a similar manner, that is, predominantly from the material (mt+) and paternal (mt−) parents, respectively.