Showing 1 - 25 of 758 results
A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica.
Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.
Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine.
In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.
Engineered Allosteric Regulation of Protein Function.
Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.
Engineered Cas9 extracellular vesicles as a novel gene editing tool.
Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.
Synthetic cells with self-activating optogenetic proteins communicate with natural cells.
Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.
Design and engineering of light-sensitive protein switches.
Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.
Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies.
Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, proces-sivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.
LILAC: Enhanced actin imaging with an optogenetic Lifeact.
We have designed an improved Lifeact variant that binds to actin under the control of light using the LOV2 protein. Light control enables one to subtract the pre-illumination signal of the unbound label, yielding an enhanced view of F-actin dynamics in cells. Furthermore, the tool eliminates actin network perturbations and cell sickness caused by Lifeact overexpression.
Optogenetic tools for microbial synthetic biology.
Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.
Light-Sensitive Lactococcus lactis for Microbe-Gut-Brain Axis Regulating via Upconversion Optogenetic Micro-Nano System.
The discovery of the gut-brain axis has proven that brain functions can be affected by the gut microbiota's metabolites, so there are significant opportunities to explore new tools to regulate gut microbiota and thus work on the brain functions. Meanwhile, engineered bacteria as oral live biotherapeutic agents to regulate the host's healthy homeostasis have attracted much attention in microbial therapy. However, whether this strategy is able to remotely regulate the host's brain function in vivo has not been investigated. Here, we engineered three blue-light-responsive probiotics as oral live biotherapeutic agents. They are spatiotemporally delivered and controlled by the upconversion optogenetic micro-nano system. This micro-nano system promotes the small intestine targeting and production of the exogenous L. lactis in the intestines, which realizes precise manipulation of brain functions including anxiety behavior, Parkinson's disease, and vagal afferent. The noninvasive and real-time probiotic intervention strategy makes the communiation from the gut to the host more controllable, which will enable the potential for engineered microbes accurately and effectively regulating a host's health.
A guide to designing photocontrol in proteins: methods, strategies and applications.
Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.
mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration.
By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanoresponsive mTORC2 program in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the mTORC2 kinase arm is essential for regulation of Rho activity and Myosin II-based contraction at the trailing edge. Cells required mTORC2 for spatial and temporal coordination between the front and back polarity programs and persistent migration under confinement. mTORC2 is in a mechanosensory cascade, but membrane stretch did not suffice to stimulate mTORC2 unless the co-input PIP3 was also present. Our work suggests that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs allowing efficient coordination of neutrophil shape and movement.
Optical control of protein delivery and partitioning in the nucleolus.
The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline-arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.
Local temporal Rac1-GTP nadirs and peaks restrict cell protrusions and retractions.
Cells probe their microenvironment using membrane protrusion-retraction cycles. Spatiotemporal coordination of Rac1 and RhoA GTP-binding activities initiates and reinforces protrusions and retractions, but the control of their finite lifetime remains unclear. We examined the relations of Rac1 and RhoA GTP-binding levels to key protrusion and retraction events, as well as to cell-ECM traction forces at physiologically relevant ECM stiffness. High RhoA-GTP preceded retractions and Rac1-GTP elevation before protrusions. Notable temporal Rac1-GTP nadirs and peaks occurred at the maximal edge velocity of local membrane protrusions and retractions, respectively, followed by declined edge velocity. Moreover, altered local Rac1-GTP consistently preceded similarly altered traction force. Local optogenetic Rac1-GTP perturbations defined a function of Rac1 in restricting protrusions and retractions and in promoting local traction force. Together, we show that Rac1 plays a fundamental role in restricting the size and durability of protrusions and retractions, plausibly in part through controlling traction forces.
Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation.
Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.
Optogenetic actuator/ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics.
Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedbacks operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs and its perturbation can enhance the efficiency of MAPK inhibitors.
Persistent cell migration emerges from a coupling between protrusion dynamics and polarized trafficking.
Migrating cells present a variety of paths, from random to highly directional ones. While random movement can be explained by basal intrinsic activity, persistent movement requires stable polarization. Here, we quantitatively address emergence of persistent migration in (hTERT)-immortalizedRPE1 (retinal pigment epithelial) cells over long timescales. By live cell imaging and dynamic micropatterning, we demonstrate that the Nucleus-Golgi axis aligns with direction of migration leading to efficient cell movement. We show that polarized trafficking is directed toward protrusions with a 20-min delay, and that migration becomes random after disrupting internal cell organization. Eventually, we prove that localized optogenetic Cdc42 activation orients the Nucleus-Golgi axis. Our work suggests that polarized trafficking stabilizes the protrusive activity of the cell, while protrusive activity orients this polarity axis, leading to persistent cell migration. Using a minimal physical model, we show that this feedback is sufficient to recapitulate the quantitative properties of cell migration in the timescale of hours.
Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression.
Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling edge pulse detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.
Using single-cell models to predict the functionality of synthetic circuits at the population scale.
SignificanceAt the single-cell level, biochemical processes are inherently stochastic. For many natural systems, the resulting cell-to-cell variability is exploited by microbial populations. In synthetic biology, however, the interplay of cell-to-cell variability and population processes such as selection or growth often leads to circuits not functioning as predicted by simple models. Here we show how multiscale stochastic kinetic models that simultaneously track single-cell and population processes can be obtained based on an augmentation of the chemical master equation. These models enable us to quantitatively predict complex population dynamics of a yeast optogenetic differentiation system from a specification of the circuit's components and to demonstrate how cell-to-cell variability can be exploited to purposefully create unintuitive circuit functionality.
A rich get richer effect governs intracellular condensate size distributions.
Phase separation of biomolecules into condensates has emerged as a ubiquitous mechanism for intracellular organization and impacts many intracellular processes, including reaction pathways through clustering of enzymes and their intermediates. Precise and rapid spatiotemporal control of reactions by condensates requires tuning of their sizes. However, the physical processes that govern the distribution of condensate sizes remain unclear. Here, we utilize a combination of synthetic and native condensates to probe the underlying physical mechanisms determining condensate size. We find that both native nuclear speckles and FUS condensates formed with the synthetic Corelet system obey an exponential size distribution, which can be recapitulated in Monte Carlo simulations of fast nucleation followed by coalescence. By contrast, pathological aggregation of cytoplasmic Huntingtin polyQ protein exhibits a power-law size distribution, with an exponent of −1.41 ± 0.02. These distinct behaviors reflect the relative importance of nucleation and coalescence kinetics: introducing continuous condensate nucleation into the Monte Carlo coarsening simulations gives rise to polyQ-like power-law behavior. We demonstrate that the emergence of power-law distributions under continuous nucleation reflects a “rich get richer” effect, whose extent may play a general role in the determination of condensate size distributions.
NIR-Responsive Photodynamic Nanosystem Combined with Antitumor Immune Optogenetics Bacteria for Precise Synergetic Therapy.
Photodynamic therapy (PDT) and immunotherapy are considered promising methods for the treatment of tumors. However, these treatment systems are still suffering from shortcomings such as hypoxia, easy metastasis, and delayed immune response during PDT. Therefore, it is still challenging to establish a programmed and rapid response immune combination therapy platform. Here, we construct a two-step synergetic therapy platform for the treatment of primary tumors and distant tumors using upconversion nanoparticles (UCNPs) and engineered bacteria as therapeutic media. In the first step, erbium ion (Er3+)-doped UCNPs act as a photoswitcher to activate the photosensitizer ZnPc to produce 1O2 for primary tumor therapy. In the second step, thulium ion (Tm3+)-doped UCNPs can emit blue-violet light under the excitation of near-infrared (NIR) light to activate the engineered bacteria to produce interferon (INF-γ) and release them in the intestine, which can not only treat tumors directly but also act with PDT to regulate immune pathways to activate the immune system, resulting in a joint immunotherapy effect to inhibit the growth of distant tumors. As a new type of programmatic combination therapy, we have proved that this platform can jointly activate the body's immune system during PDT and immunization treatment and can effectively inhibit tumor metastasis.
Optogenetics Illuminates Applications in Microbial Engineering.
Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Optogenetic Application to Investigating Cell Behavior and Neurological Disease.
Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field.
Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.
Fluorescent protein (FP) maturation can limit the accuracy with which dynamic intracellular processes are captured and reduce the in vivo brightness of a given FP in fast-dividing cells. The knowledge of maturation timescales can therefore help users determine the appropriate FP for each application. However, in vivo maturation rates can greatly deviate from in vitro estimates that are mostly available. In this work, we present the first systematic study of in vivo maturation for 12 FPs in budding yeast. To overcome the technical limitations of translation inhibitors commonly used to study FP maturation, we implemented a new approach based on the optogenetic stimulations of FP expression in cells grown under constant nutrient conditions. Combining the rapid and orthogonal induction of FP transcription with a mathematical model of expression and maturation allowed us to accurately estimate maturation rates from microscopy data in a minimally invasive manner. Besides providing a useful resource for the budding yeast community, we present a new joint experimental and computational approach for characterizing FP maturation, which is applicable to a wide range of organisms.
Optical Sensors and Actuators for Probing Proximity-Dependent Biotinylation in Living Cells.
Proximity-dependent biotinylation techniques have been gaining wide applications in the systematic analysis of protein-protein interactions (PPIs) on a proteome-wide scale in living cells. The engineered biotin ligase TurboID is among the most widely adopted given its enhanced biotinylation efficiency, but it faces the background biotinylation complication that might confound proteomic data interpretation. To address this issue, we report herein a set of split TurboID variants that can be reversibly assembled by using light (designated "OptoID"), which enable optogenetic control of biotinylation based proximity labeling in living cells. OptoID could be further coupled with an engineered monomeric streptavidin that permits real-time monitoring of biotinylation with high temporal precision. These optical actuators and sensors will likely find broad applications in precise proximity proteomics and rapid detection of biotinylation in living cells.