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1.
Cell ; 187(18): 5081-5101.e19, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38996528

RESUMO

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.


Assuntos
Axônios , Proteínas de Drosophila , Drosophila melanogaster , Proteínas do Tecido Nervoso , Neurônios Receptores Olfatórios , Transdução de Sinais , Sinapses , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Axônios/metabolismo , Sinapses/metabolismo , Actinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Tenascina , Proteínas rac de Ligação ao GTP
2.
Cell ; 186(15): 3307-3324.e30, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37385249

RESUMO

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.


Assuntos
Mitocôndrias , Proteoma , Proteoma/metabolismo , Mitocôndrias/metabolismo , Nucléolo Celular/metabolismo , Espectrometria de Massas/métodos , Regulação da Expressão Gênica
3.
Cell ; 183(7): 2003-2019.e16, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33308478

RESUMO

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.


Assuntos
Comportamento Animal , Cálcio/metabolismo , Corpo Estriado/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Optogenética , Ratos , Análise de Célula Única , Transcriptoma/genética
4.
Cell ; 180(2): 373-386.e15, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31955847

RESUMO

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 in vivo 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.


Assuntos
Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteômica/métodos , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Nervo Olfatório/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Receptores de Lipoproteínas/metabolismo , Olfato/fisiologia
5.
Cell ; 178(2): 473-490.e26, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31230715

RESUMO

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.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Corantes Fluorescentes/química , Células HEK293 , Humanos , Microscopia de Fluorescência , Mitocôndrias/genética , RNA/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transcriptoma
6.
Cell ; 169(2): 350-360.e12, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388416

RESUMO

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.


Assuntos
Ascorbato Peroxidases/química , Mapas de Interação de Proteínas , Coloração e Rotulagem/métodos , Animais , Humanos , Lisossomos/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/metabolismo , Ubiquitina/metabolismo
7.
Cell ; 166(5): 1295-1307.e21, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-27565350

RESUMO

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.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Neurônios GABAérgicos/metabolismo , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Membranas Sinápticas/metabolismo , Animais , Antígenos CD/metabolismo , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Moléculas de Adesão de Célula Nervosa/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteômica , Ratos , Receptores de GABA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tálamo/metabolismo
8.
Proc Natl Acad Sci U S A ; 121(13): e2320053121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38513100

RESUMO

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.


Assuntos
Lisossomos , Humanos , Células HEK293 , Proteólise
9.
Nat Methods ; 20(6): 908-917, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37188954

RESUMO

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.


Assuntos
Mitocôndrias , Proteômica , Retículo Endoplasmático , Biotina
10.
Plant Cell ; 35(3): 975-993, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36660928

RESUMO

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.


Assuntos
Proteínas Quinases , Proteômica , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biotina/química , Biotinilação , Brassinosteroides/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteômica/métodos , Transdução de Sinais/fisiologia
11.
Cell ; 143(3): 456-69, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-20933261

RESUMO

The functions of trans-synaptic adhesion molecules, such as neurexin and neuroligin, have been difficult to study due to the lack of methods to directly detect their binding in living neurons. Here, we use biotin labeling of intercellular contacts (BLINC), a method for imaging protein interactions based on interaction-dependent biotinylation of a peptide by E. coli biotin ligase, to visualize neurexin-neuroligin trans-interactions at synapses and study their role in synapse development. We found that both developmental maturation and acute synaptic activity stimulate the growth of neurexin-neuroligin adhesion complexes via a combination of neurexin and neuroligin surface insertion and internalization arrest. Both mechanisms require NMDA receptor activity. We also discovered that disruption of activity-induced neurexin-neuroligin complex growth prevents recruitment of the AMPA receptor, a hallmark of mature synapses. Our results provide support for neurexin-neuroligin function in synapse maturation and introduce a general method to study intercellular protein-protein interactions.


Assuntos
Biotinilação/métodos , Moléculas de Adesão Celular Neuronais/metabolismo , Microscopia de Fluorescência/métodos , Proteínas do Tecido Nervoso/metabolismo , Animais , Carbono-Nitrogênio Ligases , Proteínas de Escherichia coli , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras , Sinapses/metabolismo
12.
Proc Natl Acad Sci U S A ; 119(30): e2201208119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35858434

RESUMO

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.


Assuntos
Acetamidas , Antivirais , Isoindóis , Vírus Lassa , Fatores de Terminação de Peptídeos , Piperidonas , RNA Polimerase Dependente de RNA , Proteínas Virais , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Humanos , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Febre Lassa/tratamento farmacológico , Vírus Lassa/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Piperidonas/metabolismo , Piperidonas/farmacologia , Piperidonas/uso terapêutico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteoma , Proteômica , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo
13.
Nat Methods ; 18(2): 133-143, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432242

RESUMO

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.


Assuntos
Ácidos Nucleicos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Espectrometria de Massas , Ligação Proteica
14.
Immunity ; 43(2): 227-39, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26231119

RESUMO

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.


Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Complexos Multiproteicos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/genética , Humanos , Hibridomas , Mecanotransdução Celular , Camundongos , Complexos Multiproteicos/genética , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estrutura Terciária de Proteína/genética , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética
15.
J Am Chem Soc ; 145(18): 10220-10226, 2023 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-37098770

RESUMO

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.


Assuntos
Toxinas Botulínicas Tipo A , Animais , Toxinas Botulínicas Tipo A/metabolismo , Citosol/metabolismo , Peptídeos , Luciferases , Mamíferos/metabolismo
16.
Nat Methods ; 17(2): 167-174, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31819267

RESUMO

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 (uTEV1Δ), 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.


Assuntos
Evolução Molecular Direcionada , Endopeptidases/metabolismo , Catálise , Endopeptidases/genética , Células HEK293 , Humanos , Saccharomyces cerevisiae/genética
17.
Nat Methods ; 17(2): 242, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31907448

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

18.
Nature ; 550(7675): 280-284, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-28976959

RESUMO

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.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Silenciamento de Genes/métodos , Leptotrichia/enzimologia , RNA/genética , RNA/metabolismo , Biocatálise , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Escherichia coli/genética , Genes Reporter/genética , Células HEK293 , Humanos , Leptotrichia/genética , Células Vegetais/metabolismo , RNA/análise , Interferência de RNA , Estresse Fisiológico , Especificidade por Substrato
19.
Mol Cell Proteomics ; 20: 100128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34332124

RESUMO

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.


Assuntos
Proteômica/métodos , Animais , Animais Geneticamente Modificados , Biotinilação , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , RNA Mensageiro , Transgenes , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(52): 33186-33196, 2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33323488

RESUMO

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.

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