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A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion. Cell Kim, C. K., Sanchez, M. I., Hoerbelt, P., Fenno, L. E., Malenka, R. C., Deisseroth, K., Ting, A. Y. 2020
Abstract

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE's modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

View details for DOI 10.1016/j.cell.2020.11.015

View details for PubMedID 33308478
Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell Fazal, F. M., Han, S. n., Parker, K. R., Kaewsapsak, P. n., Xu, J. n., Boettiger, A. N., Chang, H. Y., Ting, A. Y. 2019
Abstract

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should�be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

View details for DOI 10.1016/j.cell.2019.05.027

View details for PubMedID 31230715
Efficient proximity labeling in living cells and organisms with TurboID NATURE BIOTECHNOLOGY Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., Ting, A. Y. 2018; 36 (9): 880-+
Abstract

Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 h of labeling time or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-min PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.

View details for PubMedID 30125270

View details for PubMedCentralID PMC6126969
A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nature biotechnology Wang, W. n., Wildes, C. P., Pattarabanjird, T. n., Sanchez, M. I., Glober, G. F., Matthews, G. A., Tye, K. M., Ting, A. Y. 2017
Abstract

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

View details for PubMedID 28650461
Deciphering molecular interactions by proximity labeling. Nature methods Qin, W., Cho, K. F., Cavanagh, P. E., Ting, A. Y. 2021
Abstract

Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.

View details for DOI 10.1038/s41592-020-01010-5

View details for PubMedID 33432242
RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting. Proceedings of the National Academy of Sciences of the United States of America Han, S., Zhao, B. S., Myers, S. A., Carr, S. A., He, C., Ting, A. Y. 2020
Abstract

RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N 6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.

View details for DOI 10.1073/pnas.2006617117

View details for PubMedID 32839320
Split-TurboID enables contact-dependent proximity labeling in cells. Proceedings of the National Academy of Sciences of the United States of America Cho, K. F., Branon, T. C., Rajeev, S., Svinkina, T., Udeshi, N. D., Thoudam, T., Kwak, C., Rhee, H., Lee, I., Carr, S. A., Ting, A. Y. 2020
Abstract

Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

View details for DOI 10.1073/pnas.1919528117

View details for PubMedID 32424107
Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators. Cell Li, J., Han, S., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Xu, C., Guajardo, R., Xie, Q., Li, T., Luginbuhl, D. J., Wu, B., McLaughlin, C. N., Xie, A., Kaewsapsak, P., Quake, S. R., Carr, S. A., Ting, A. Y., Luo, L. 2020
Abstract

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed invivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

View details for DOI 10.1016/j.cell.2019.12.029

View details for PubMedID 31955847
Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease. Proceedings of the National Academy of Sciences of the United States of America Sanchez, M. I., Nguyen, Q. A., Wang, W. n., Soltesz, I. n., Ting, A. Y. 2020
Abstract

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

View details for DOI 10.1073/pnas.2006521117

View details for PubMedID 33323488
Directed evolution improves the catalytic efficiency of TEV protease. Nature methods Sanchez, M. I., Ting, A. Y. 2019
Abstract

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Delta), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.

View details for DOI 10.1038/s41592-019-0665-7

View details for PubMedID 31819267
Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions ELIFE Kim, C. K., Cho, K. F., Kim, M., Ting, A. Y. 2019; 8
View details for DOI 10.7554/eLife.43826

View details for Web of Science ID 000463337200001
Directed Evolution of Split APEX2 Peroxidase. ACS chemical biology Han, Y. n., Branon, T. C., Martell, J. D., Boassa, D. n., Shechner, D. n., Ellisman, M. H., Ting, A. n. 2019; 14 (4): 619?35
Abstract

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). A total of 20 rounds of fluorescence activated cell sorting (FACS)-based selections from yeast-displayed fragment libraries, using 3 different surface display configurations, produced a 200-amino-acid N-terminal fragment (with 9 mutations relative to APEX2) called "AP" and a 50-amino-acid C-terminal fragment called "EX". AP and EX fragments were each inactive on their own but were reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within a non-coding RNA scaffold, and at mitochondria-endoplasmic reticulum contact sites.

View details for PubMedID 30848125
Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID. eLife Mair, A. n., Xu, S. L., Branon, T. C., Ting, A. Y., Bergmann, D. C. 2019; 8
Abstract

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and N. benthamiana and versatile vectors enable customization by plant researchers.

View details for DOI 10.7554/eLife.47864

View details for PubMedID 31535972
Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation ELIFE Hung, V., Lam, S. S., Udeshi, N. D., Svinkina, T., Guzman, G., Mootha, V. K., Carr, S. A., Ting, A. Y. 2017; 6
Abstract

The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP's ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.

View details for DOI 10.7554/eLife.24463

View details for Web of Science ID 000400017500001

View details for PubMedID 28441135

View details for PubMedCentralID PMC5404927
An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells CELL Lobingier, B. T., Huttenhain, R., Eichel, K., Miller, K. B., Ting, A. Y., von Zastrow, M., Krogan, N. J. 2017; 169 (2): 350-360
Abstract

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

View details for DOI 10.1016/j.cell.2017.03.022

View details for Web of Science ID 000398349500018

View details for PubMedID 28388416
Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells. Cell chemical biology Han, S., Udeshi, N. D., Deerinck, T. J., Svinkina, T., Ellisman, M. H., Carr, S. A., Ting, A. Y. 2017
Abstract

A recurring challenge in cell biology is to define the�molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.

View details for DOI 10.1016/j.chembiol.2017.02.002

View details for PubMedID 28238724
Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking. eLife Kaewsapsak, P. n., Shechner, D. M., Mallard, W. n., Rinn, J. L., Ting, A. Y. 2017; 6
Abstract

The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.

View details for PubMedID 29239719
RNA targeting with CRISPR-Cas13. Nature Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P. n., Han, S. n., Joung, J. n., Belanto, J. J., Verdine, V. n., Cox, D. B., Kellner, M. J., Regev, A. n., Lander, E. S., Voytas, D. F., Ting, A. Y., Zhang, F. n. 2017; 550 (7675): 280?84
Abstract

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

View details for PubMedID 28976959

View details for PubMedCentralID PMC5706658
Time-gated detection of protein-protein interactions with transcriptional readout. eLife Kim, M. W., Wang, W. n., Sanchez, M. I., Coukos, R. n., von Zastrow, M. n., Ting, A. Y. 2017; 6
Abstract

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

View details for PubMedID 29189201

View details for PubMedCentralID PMC5708895
Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts. Cell Loh, K. H., Stawski, P. S., Draycott, A. S., Udeshi, N. D., Lehrman, E. K., Wilton, D. K., Svinkina, T., Deerinck, T. J., Ellisman, M. H., Stevens, B., Carr, S. A., Ting, A. Y. 2016; 166 (5): 1295-1307 e21
Abstract

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

View details for DOI 10.1016/j.cell.2016.07.041

View details for PubMedID 27565350
A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nature biotechnology Martell, J. D., Yamagata, M. n., Deerinck, T. J., Phan, S. n., Kwa, C. G., Ellisman, M. H., Sanes, J. R., Ting, A. Y. 2016; 34 (7): 774?80
Abstract

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.

View details for PubMedID 27240195

View details for PubMedCentralID PMC4942342
Directed evolution of APEX2 for electron microscopy and proximity labeling NATURE METHODS Lam, S. S., Martell, J. D., Kamer, K. J., Deerinck, T. J., Ellisman, M. H., Mootha, V. K., Ting, A. Y. 2015; 12 (1): 51-54
Abstract

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

View details for Web of Science ID 000347668600017

View details for PubMedID 25419960
Computational design of a red fluorophore ligase for site-specific protein labeling in living cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, D. S., Nivon, L. G., Richter, F., Goldman, P. J., Deerinck, T. J., Yao, J. Z., Richardson, D., Phipps, W. S., Ye, A. Z., Ellisman, M. H., Drennan, C. L., Baker, D., Ting, A. Y. 2014; 111 (43): E4551-E4559
Abstract

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

View details for DOI 10.1073/pnas.1404736111

View details for Web of Science ID 000343729500005

View details for PubMedID 25313043

View details for PubMedCentralID PMC4217414
Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging MOLECULAR CELL Hung, V., Zou, P., Rhee, H., Udeshi, N. D., Cracan, V., Svinkina, T., Carr, S. A., Mootha, V. K., Ting, A. Y. 2014; 55 (2): 332-341
Abstract

Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.

View details for DOI 10.1016/j.molcel.2014.06.003

View details for Web of Science ID 000340641700016

View details for PubMedID 25002142

View details for PubMedCentralID PMC4743503
Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging SCIENCE Rhee, H., Zou, P., Udeshi, N. D., Martell, J. D., Mootha, V. K., Carr, S. A., Ting, A. Y. 2013; 339 (6125): 1328-1331
Abstract

Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

View details for DOI 10.1126/science.1230593

View details for Web of Science ID 000316053400040

View details for PubMedID 23371551

View details for PubMedCentralID PMC3916822
Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation PLOS ONE Liu, D. S., Loh, K. H., Lam, S. S., White, K. A., Ting, A. Y. 2013; 8 (2)
Abstract

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.

View details for DOI 10.1371/journal.pone.0052823

View details for Web of Science ID 000315602700002

View details for PubMedID 23457442

View details for PubMedCentralID PMC3573046
Quantum Dot Targeting with Lipoic Acid Ligase and Halo Tag for Single-Molecule Imaging on Living Cells ACS NANO Liu, D. S., Phipps, W. S., Loh, K. H., Howarth, M., Ting, A. Y. 2012; 6 (12): 11080-11087
Abstract

We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.

View details for DOI 10.1021/nn304793z

View details for Web of Science ID 000312563600073

View details for PubMedID 23181687

View details for PubMedCentralID PMC3528850
Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy NATURE BIOTECHNOLOGY Martell, J. D., Deerinck, T. J., Sancak, Y., Poulos, T. L., Mootha, V. K., Sosinsky, G. E., Ellisman, M. H., Ting, A. Y. 2012; 30 (11): 1143-?
Abstract

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments or require light and can be difficult to use. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.

View details for DOI 10.1038/nbt.2375

View details for Web of Science ID 000311087500035

View details for PubMedID 23086203

View details for PubMedCentralID PMC3699407
Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yao, J. Z., Uttamapinant, C., Poloukhtine, A., Baskin, J. M., Codelli, J. A., Sletten, E. M., Bertozzi, C. R., Popik, V. V., Ting, A. Y. 2012; 134 (8): 3720-3728
Abstract

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

View details for DOI 10.1021/ja208090p

View details for Web of Science ID 000301161600025

View details for PubMedID 22239252

View details for PubMedCentralID PMC3306817
Diels-Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Liu, D. S., Tangpeerachaikul, A., Selvaraj, R., Taylor, M. T., Fox, J. M., Ting, A. Y. 2012; 134 (2): 792-795
Abstract

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

View details for DOI 10.1021/ja209325n

View details for Web of Science ID 000301084300013

View details for PubMedID 22176354

View details for PubMedCentralID PMC3381951
Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Uttamapinant, C., Tangpeerachaikul, A., Grecian, S., Clarke, S., Singh, U., Slade, P., Gee, K. R., Ting, A. Y. 2012; 51 (24): 5852-5856
View details for DOI 10.1002/anie.201108181

View details for Web of Science ID 000304814400010

View details for PubMedID 22555882

View details for PubMedCentralID PMC3517120
Imaging Protein-Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Slavoff, S. A., Liu, D. S., Cohen, J. D., Ting, A. Y. 2011; 133 (49): 19769-19776
Abstract

We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein-protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA(W37V), which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA(W37V) labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein-protein interactions can be imaged (FRB-FKBP, Fos-Jun, and neuroligin-PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution.

View details for DOI 10.1021/ja206435e

View details for Web of Science ID 000298719800044

View details for PubMedID 22098454

View details for PubMedCentralID PMC3547671
Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells BIOCHEMISTRY Cohen, J. D., Thompson, S., Ting, A. Y. 2011; 50 (38): 8221-8225
Abstract

Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.

View details for DOI 10.1021/bi201037r

View details for Web of Science ID 000295058700013

View details for PubMedID 21859157

View details for PubMedCentralID PMC3181222
Imaging LDL Receptor Oligonnerization during Endocytosis Using a Co-internalization Assay ACS CHEMICAL BIOLOGY Zou, P., Ting, A. Y. 2011; 6 (4): 308-313
Abstract

Methods to probe receptor oligomerization are useful to understand the molecular mechanisms of receptor signaling. Here we report a fluorescence imaging method to determine receptor oligomerization state in living cells during endocytic internalization. The wild-type receptor is co-expressed with an internalization-defective mutant, and the internalization kinetics of each are independently monitored. If the receptor internalizes as an oligomer, then the wild-type and mutant isoforms will mutually influence each others' trafficking properties, causing co-internalization of the mutant or co-retention of the wild-type at the cell surface. Using this approach, we found that the low density lipoprotein (LDL) receptor internalizes as an oligomer into cells, both in the presence and absence of LDL ligand. The internalization kinetics of the wild-type receptor are not changed by LDL binding. We also found that the oligomerization domain of the LDL receptor is located in its cytoplasmic tail.

View details for DOI 10.1021/cb100361k

View details for Web of Science ID 000289455400003

View details for PubMedID 21194239
Synthesis of 7-Aminocoumarin by Buchwald-Hartwig Cross Coupling for Specific Protein Labeling in Living Cells CHEMBIOCHEM Jin, X., Uttamapinant, C., Ting, A. Y. 2011; 12 (1): 65-70
View details for DOI 10.1002/cbic.201000414

View details for Web of Science ID 000285771200009

View details for PubMedID 21154801

View details for PubMedCentralID PMC4857190
A fluorophore ligase for site-specific protein labeling inside living cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uttamapinant, C., White, K. A., Baruah, H., Thompson, S., Fernandez-Suarez, M., Puthenveetil, S., Ting, A. Y. 2010; 107 (24): 10914-10919
Abstract

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

View details for DOI 10.1073/pnas.0914067107

View details for Web of Science ID 000278807400027

View details for PubMedID 20534555

View details for PubMedCentralID PMC2890758
Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Puthenveetil, S., Liu, D. S., White, K. A., Thompson, S., Ting, A. Y. 2009; 131 (45): 16430-16438
Abstract

Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007 , 25 , 1483 - 1487 ) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008 , 47 , 7018 - 7021 ) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved beta-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A approximately 10(7) library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a k(cat)/K(m) of 0.99 muM(-1) min(-1), >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483-1487), and only 8-fold worse than the k(cat)/K(m) values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.

View details for DOI 10.1021/ja904596f

View details for Web of Science ID 000271723000036

View details for PubMedID 19863063

View details for PubMedCentralID PMC2799336
Fluorescent probes for super-resolution imaging in living cells NATURE REVIEWS MOLECULAR CELL BIOLOGY Fernandez-Suarez, M., Ting, A. Y. 2008; 9 (12): 929-943
Abstract

In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

View details for DOI 10.1038/nrm2531

View details for Web of Science ID 000261126800013

View details for PubMedID 19002208
Protein-protein interaction detection in vitro and in cells by proximity biotinylation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fernandez-Suarez, M., Chen, T. S., Ting, A. Y. 2008; 130 (29): 9251-?
Abstract

We report a new method for detection of protein-protein interactions in vitro and in cells. One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA's "acceptor peptide" (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3epsilon phosphoserine/threonine binding protein. Protein-protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.

View details for DOI 10.1021/ja801445p

View details for Web of Science ID 000257796500036

View details for PubMedID 18582056

View details for PubMedCentralID PMC2635094
Monovalent, reduced-size quantum dots for imaging receptors on living cells NATURE METHODS Howarth, M., Liu, W., Puthenveetil, S., Zheng, Y., Marshall, L. F., Schmidt, M. M., Wittrup, K. D., Bawendi, M. G., Ting, A. Y. 2008; 5 (5): 397-399
Abstract

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

View details for DOI 10.1038/NMETH.1206

View details for Web of Science ID 000255411700013

View details for PubMedID 18425138

View details for PubMedCentralID PMC2637151
Expanding the substrate tolerance of biotin ligase through exploration of enzymes from diverse species JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Slavoff, S. A., Chen, I., Choi, Y., Ting, A. Y. 2008; 130 (4): 1160-?
View details for DOI 10.1021/ja0766551

View details for Web of Science ID 000252634600024

View details for PubMedID 18171066

View details for PubMedCentralID PMC3501195
An engineered aryl azide ligase for site-specific mapping of protein-protein interactions through photo-cross-linking ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Baruah, H., Puthenveetil, S., Choi, Y., Shah, S., Ting, A. Y. 2008; 47 (37): 7018-7021
View details for DOI 10.1002/anie.200802088

View details for Web of Science ID 000259041000011

View details for PubMedID 18677791

View details for PubMedCentralID PMC2745654
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin NATURE PROTOCOLS Howarth, M., Ting, A. Y. 2008; 3 (3): 534-545
Abstract

This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.

View details for DOI 10.1038/nprot.2008.20

View details for Web of Science ID 000254137100019

View details for PubMedID 18323822

View details for PubMedCentralID PMC2671200
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes NATURE BIOTECHNOLOGY Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L., Xie, K. T., Baskin, J. M., Bertozzi, C. R., Ting, A. Y. 2007; 25 (12): 1483-1487
Abstract

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

View details for DOI 10.1038/nbt1355

View details for Web of Science ID 000251457800039

View details for PubMedID 18059260

View details for PubMedCentralID PMC2654346
Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, I., Choi, Y., Ting, A. Y. 2007; 129 (20): 6619-6625
Abstract

Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

View details for DOI 10.1021/ja071013g

View details for Web of Science ID 000246535300052

View details for PubMedID 17472384

View details for PubMedCentralID PMC2629800
Synthesis of a ketone analogue of biotin via the intramolecular Pauson-Khand reaction ORGANIC LETTERS McNeill, E., Chen, I., Ting, A. Y. 2006; 8 (20): 4593-4595
Abstract

We report an improved synthesis of 5-(5-oxohexahydrocyclopenta[c]thiophen-1-yl)pentanoic acid (ketone biotin, 1) based on the intramolecular Pauson-Khand cyclization. The synthesis proceeds in eight steps and in 2.7% overall yield from cyclohexene.

View details for DOI 10.1021/ol061862t

View details for Web of Science ID 000240654700053

View details for PubMedID 16986958

View details for PubMedCentralID PMC2531226
Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lin, C. W., Ting, A. Y. 2006; 128 (14): 4542-4543
Abstract

Site-specific protein labeling methods allow cell biologists to access the vast array of existing chemical probes for the study of specific proteins of interest in the live cell context. Here we describe the use of the transglutaminase enzyme from guinea pig liver (gpTGase), whose natural function is to cross-link glutamine and lysine side chains, to covalently conjugate various small-molecule probes to recombinant proteins fused to a 6- or 7-amino acid transglutaminase recognition sequence, called a Q-tag. We demonstrate labeling of Q-tag fusion proteins both in vitro and on the surface of living mammalian cells with biotin, fluorophores, and a benzophenone photoaffinity probe. To illustrate the utility of this labeling, we tagged the NF-kappaB p50 transcription factor with benzophenone, cross-linked with UV light, and observed increased levels of p50 homodimerization in the presence of DNA and the binding protein myotrophin.

View details for DOI 10.1021/ja0604111

View details for Web of Science ID 000236770300023

View details for PubMedID 16594669

View details for PubMedCentralID PMC2561265
A monovalent streptavidin with a single femtomolar biotin binding site NATURE METHODS Howarth, M., Chinnapen, D. J., Gerrow, K., Dorrestein, P. C., Grandy, M. R., Kelleher, N. L., El-Husseini, A., Ting, A. Y. 2006; 3 (4): 267-273
Abstract

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

View details for DOI 10.1038/NMETH861

View details for Web of Science ID 000236501400013

View details for PubMedID 16554831

View details for PubMedCentralID PMC2576293
Giving cells a new sugar-coating NATURE CHEMICAL BIOLOGY Howarth, M., Ting, A. Y. 2006; 2 (3): 127-128
View details for Web of Science ID 000235424100007

View details for PubMedID 16485002
Targeting quantum dots to surface proteins in living cells with biotin ligase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Howarth, M., Takao, K., Hayashi, Y., Ting, A. Y. 2005; 102 (21): 7583-7588
Abstract

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

View details for DOI 10.1073/pnas.0503125102

View details for Web of Science ID 000229417500034

View details for PubMedID 15897449

View details for PubMedCentralID PMC1129026
Site-specific labeling of proteins with small molecules in live cells CURRENT OPINION IN BIOTECHNOLOGY Chen, I., Ting, A. Y. 2005; 16 (1): 35-40
Abstract

The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest. Recently developed methods for labeling proteins with small-molecule probes in cells employ special protein or peptide handles that recruit small-molecule ligands, harness enzymes to catalyze small-molecule conjugation or hijack the cell's protein translation machinery.

View details for DOI 10.1016/j.copbio.2004.12.003

View details for Web of Science ID 000227383000006

View details for PubMedID 15722013
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase NATURE METHODS Chen, I., Howarth, M., Lin, W. Y., Ting, A. Y. 2005; 2 (2): 99-104
Abstract

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.

View details for DOI 10.1038/NMETH735

View details for Web of Science ID 000226754100010

View details for PubMedID 15782206
Genetically encoded fluorescent reporters of histone methylation in living cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lin, C. W., Jao, C. Y., Ting, A. Y. 2004; 126 (19): 5982-5983
Abstract

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.

View details for Web of Science ID 000221416700027

View details for PubMedID 15137760
A genetically encoded fluorescent reporter of histone phosphorylation in living cells ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Lin, C. W., Ting, A. Y. 2004; 43 (22): 2940-2943
View details for DOI 10.1002/anie.200353375

View details for Web of Science ID 000221829700013

View details for PubMedID 15170310

Alice Ting

Professor of Genetics, of Biology and, by courtesy, of Chemistry

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