Alice Ting
Professor of Genetics, of Biology and, by courtesy, of Chemistry
Academic Appointments
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Professor, Genetics
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Professor, Biology
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Professor (By courtesy), Chemistry
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Member, Bio-X
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Faculty Fellow, Sarafan ChEM-H
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Member, Stanford Cancer Institute
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Member, Wu Tsai Neurosciences Institute
Administrative Appointments
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Professor of Genetics, Biology, and by courtesy, Chemistry, Stanford University (2016 - Present)
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Investigator, Chan Zuckerberg Biohub - San Francisco (2017 - Present)
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Professor of Chemistry, MIT (2014 - 2016)
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Associate Professor of Chemistry, MIT (2007 - 2014)
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Assistant Professor of Chemistry, MIT (2002 - 2007)
Honors & Awards
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Elected Member, National Academy of Sciences (2023)
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John Kendrew Lecture, LMB-MRC (2022)
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Priestley Lecture, Penn State (2021)
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Paul D. Gottlieb Distinguished Lecture, UT Austin (2020)
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Bruce Merrifield Distinguished Lecture, Rockefeller (2020)
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NIH Director’s Transformative Research Award, NIH (2018)
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Chan Zuckerberg Biohub Investigator, Chan Zuckerberg Biohub (2017)
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Arthur C. Cope Scholar Award, American Chemical Society (2010)
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NIH Director’s Pioneer Award, NIH (2008)
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McKnight Technological Innovations in Neuroscience Award, McKnight Foundation (2005)
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HHMI Collaborative Innovation Award, HHMI (2012)
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NIH Director’s Transformative Research Award, NIH (2013)
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Alexander M. Cruickshank Award Lecture, . (2018)
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Prize for Creative Promise in Biomedical Science, Vilcek Foundation (2012)
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Ellen Swallow Richards Chair, MIT (2011)
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Eli Lilly Award in Biological Chemistry, American Chemical Society (2010)
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E. Bright Wilson Prize Lecture, Harvard University (2008)
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Buck-Whitney Award, American Chemical Society (2007)
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Camille Dreyfus Teacher-Scholar Award, Dreyfus Foundation (2006)
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Technology Review TR35 Award, MIT (2006)
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Alfred P. Sloan Foundation Research Fellowship, Sloan Foundation (2005)
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EJLB Foundation Scholar Research Program Award, EJLB Foundation (2003)
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NIH K22 Career Development Award, NIH (2003)
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Young Investigator Award, Office of Naval Research (2003)
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Camille and Henry Dreyfus New Faculty Award, Dreyfus Foundation (2002)
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Pfizer-Laubach Career Development Chair, MIT (2002)
Professional Education
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Post-doc, University of California, San Diego, Biochemistry (with Roger Tsien) (2002)
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Ph.D., University of California, Berkeley, Chemistry (with Peter Schultz) (2000)
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A.B., Harvard University, Chemistry (research advisor E. J. Corey) (1996)
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High school, Texas Academy of Math and Science (1992)
Current Research and Scholarly Interests
The goal of our laboratory is to develop, scale up, and broadly disseminate molecular technologies for mapping cells and functional circuits. At the sub-cellular scale, maps document the spatial organization of proteins, RNA, DNA, and metabolites with nanometer precision and temporal acuity on the order of seconds. Maps also chart the connectivity between these molecules, elucidating the circuits and signaling processes that give rise to function.
Beyond the single cell, we also strive to map cellular ensembles, such as brain tissue. We develop tools that can be used to construct cell and tissue atlases, and map the cellular circuits that give rise to function and behavior. To achieve these goals, our laboratory has focused on the development of scalable technologies to detect, measure, and manipulate molecules and circuits, both at the sub-cellular level, and at the level of cell populations.
2024-25 Courses
- Synthetic proteins and genetic circuits
BIO 246, BIOE 266, GENE 246 (Aut) -
Independent Studies (16)
- Advanced Undergraduate Research
CHEM 190 (Aut, Win, Spr, Sum) - Directed Instruction/Reading
CHEM 90 (Aut, Win, Spr, Sum) - Directed Reading in Biochemistry
BIOC 299 (Aut, Win, Spr, Sum) - Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum) - Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum) - Directed Study
BIOE 391 (Aut, Win, Spr, Sum) - Graduate Research
BIO 300 (Aut, Win, Spr, Sum) - Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum) - Graduate Research
CBIO 399 (Aut, Win, Spr, Sum) - Graduate Research
GENE 399 (Aut, Win, Spr, Sum) - Graduate Research
NBIO 399 (Aut, Win, Spr, Sum) - Research and Special Advanced Work
CHEM 200 (Aut, Win, Spr, Sum) - Research in Chemistry
CHEM 301 (Aut, Win, Spr, Sum) - Supervised Study
GENE 260 (Aut, Win, Spr, Sum) - Undergraduate Research
BIO 199 (Aut, Win, Spr, Sum) - Undergraduate Research
GENE 199 (Aut, Win, Spr, Sum)
- Advanced Undergraduate Research
Stanford Advisees
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Doctoral Dissertation Reader (AC)
Harrison Besser, Xinyi Chen, Yixin Hu, Jeewoo Kang, Jingjia Liu, Sophia Shi, Chuanyun Xu -
Postdoctoral Faculty Sponsor
Bo Cai, Shizhong Dai, Gabriele Kockelkoren, Sangsin Lee, Chang Lin, Ana Novacic, Reika Tei, Shang Jui Tsai -
Doctoral Dissertation Advisor (AC)
Peter Cavanagh, Andrew Xue -
Doctoral Dissertation Co-Advisor (AC)
Sifei Yin
All Publications
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Dynamic mapping of proteome trafficking within and between living cells by TransitID.
Cell
2023
Abstract
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
View details for DOI 10.1016/j.cell.2023.05.044
View details for PubMedID 37385249
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Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.
Nature methods
2023
Abstract
The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.
View details for DOI 10.1038/s41592-023-01880-5
View details for PubMedID 37188954
View details for PubMedCentralID 2766670
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Directed evolution of genetically encoded LYTACs for cell-mediated delivery.
Proceedings of the National Academy of Sciences of the United States of America
2024; 121 (13): e2320053121
Abstract
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.
View details for DOI 10.1073/pnas.2320053121
View details for PubMedID 38513100
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Cell-Type-Specific Intracellular Protein Delivery with Inactivated Botulinum Neurotoxin.
Journal of the American Chemical Society
2023
Abstract
The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells.
View details for DOI 10.1021/jacs.3c01145
View details for PubMedID 37098770
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A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein.
Journal of the American Chemical Society
2022
Abstract
Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of Escherichia coli and Saccharomyces cerevisiae. This biosensor is deployed in multiple homogeneous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an unaugmented cell phone camera. Binding-activated tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.
View details for DOI 10.1021/jacs.2c04192
View details for PubMedID 35876794
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A light-gated transcriptional recorder for detecting cell-cell contacts.
eLife
2022; 11
Abstract
Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here, we report TRACC (Transcriptional Readout Activated by Cell-cell Contacts), a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019), reported in this journal. TRACC incorporates light gating via the light-oxygen-voltage-sensing (LOV) domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.
View details for DOI 10.7554/eLife.70881
View details for PubMedID 35311648
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A Dual-Purpose Real-Time Indicator and Transcriptional Integrator for Calcium Detection in Living Cells.
ACS synthetic biology
2022
Abstract
Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator that combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides a real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts calcium-generated bioluminescence into stable gene expression. We also show that LuCID's modular design enables it to read out other cellular events such as protein-protein interactions. LuCID adds to the arsenal of tools for studying cells and cell populations that utilize calcium for signaling.
View details for DOI 10.1021/acssynbio.1c00597
View details for PubMedID 35254056
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An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens.
eLife
2021; 10
Abstract
The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.
View details for DOI 10.7554/eLife.69142
View details for PubMedID 34414886
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Deciphering molecular interactions by proximity labeling.
Nature methods
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
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Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery.
Nature communications
2021; 12 (1): 4980
Abstract
Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.
View details for DOI 10.1038/s41467-021-25259-2
View details for PubMedID 34404792
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A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion.
Cell
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
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RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.
Proceedings of the National Academy of Sciences of the United States of America
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
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Split-TurboID enables contact-dependent proximity labeling in cells.
Proceedings of the National Academy of Sciences of the United States of America
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
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Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.
Cell
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
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Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease.
Proceedings of the National Academy of Sciences of the United States of America
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
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Directed evolution improves the catalytic efficiency of TEV protease.
Nature methods
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
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Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions
ELIFE
2019; 8
View details for DOI 10.7554/eLife.43826
View details for Web of Science ID 000463337200001
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Directed Evolution of Split APEX2 Peroxidase.
ACS chemical biology
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
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Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID.
eLife
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
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Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
Cell
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
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Efficient proximity labeling in living cells and organisms with TurboID
NATURE BIOTECHNOLOGY
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
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Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation
ELIFE
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
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An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells
CELL
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
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Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells.
Cell chemical biology
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
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A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.
Nature biotechnology
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
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RNA targeting with CRISPR-Cas13.
Nature
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
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Time-gated detection of protein-protein interactions with transcriptional readout.
eLife
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
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Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking.
eLife
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
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Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.
Cell
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
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A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses.
Nature biotechnology
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 DOI 10.1038/nbt.3563
View details for PubMedID 27240195
View details for PubMedCentralID PMC4942342
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Directed evolution of APEX2 for electron microscopy and proximity labeling
NATURE METHODS
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
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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
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
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Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging
MOLECULAR CELL
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
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Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging
SCIENCE
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
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Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation
PLOS ONE
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
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Quantum Dot Targeting with Lipoic Acid Ligase and Halo Tag for Single-Molecule Imaging on Living Cells
ACS NANO
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
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Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
NATURE BIOTECHNOLOGY
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
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Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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Diels-Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
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
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Imaging Protein-Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells
BIOCHEMISTRY
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
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Imaging LDL Receptor Oligonnerization during Endocytosis Using a Co-internalization Assay
ACS CHEMICAL BIOLOGY
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
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Synthesis of 7-Aminocoumarin by Buchwald-Hartwig Cross Coupling for Specific Protein Labeling in Living Cells
CHEMBIOCHEM
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
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A fluorophore ligase for site-specific protein labeling inside living cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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
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Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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Fluorescent probes for super-resolution imaging in living cells
NATURE REVIEWS MOLECULAR CELL BIOLOGY
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
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Protein-protein interaction detection in vitro and in cells by proximity biotinylation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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Monovalent, reduced-size quantum dots for imaging receptors on living cells
NATURE METHODS
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
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Expanding the substrate tolerance of biotin ligase through exploration of enzymes from diverse species
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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An engineered aryl azide ligase for site-specific mapping of protein-protein interactions through photo-cross-linking
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
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
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Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin
NATURE PROTOCOLS
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
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Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes
NATURE BIOTECHNOLOGY
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
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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
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
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Synthesis of a ketone analogue of biotin via the intramolecular Pauson-Khand reaction
ORGANIC LETTERS
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
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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
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
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A monovalent streptavidin with a single femtomolar biotin binding site
NATURE METHODS
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
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Giving cells a new sugar-coating
NATURE CHEMICAL BIOLOGY
2006; 2 (3): 127-128
View details for Web of Science ID 000235424100007
View details for PubMedID 16485002
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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
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
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Site-specific labeling of proteins with small molecules in live cells
CURRENT OPINION IN BIOTECHNOLOGY
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
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Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
NATURE METHODS
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
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Genetically encoded fluorescent reporters of histone methylation in living cells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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
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A genetically encoded fluorescent reporter of histone phosphorylation in living cells
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
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