Self-labeling Enzymes Research Articles

Using Localization Microscopy to Quantify Calcium Channels at Presynaptic Boutons.

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes-expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons. Key features • Super-resolution imaging of live anesthetized C. elegans. • Three-color super-resolution reconstruction of synapses. • Nanodomains and the distribution of proteins. • Quantification of the number of proteins at synapses from single-molecule localization data.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging.

Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s-1 ) and reHaloTagF (≈0.055 s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.

Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

Quantitative Analysis of Protein Bioconjugation in Cells with an Expanded Genetic Code

Protein bioconjugation – specific covalent bond formation between a protein of interest (POI) and a label – has a major impact on protein imaging, protein function studies, and drug discovery. Ideal protein bioconjugation should be fast, quantitative, one step with no off‐target binding, and have minimal impact on protein function, no matter the complexity of the biological environment. The impressive selectivity and kinetics of bioorthogonal reactions, specifically the catalyst‐free inverse‐electron demand Diels‐Alder (IEDDA) cycloaddition of tetrazines and strained alkene dienophiles, make IEDDA‐reactive unnatural amino acids an exceptional tool for protein bioconjugation via genetic code expansion. Analyses of these bioconjugation reactions in live cells has been mostly limited to fluorophore labeling, and thus is rarely quantitative. Self‐labeling enzymes, like HaloTag, have been used for protein isolation and purification, studies of protein‐protein interactions, and in vivo and in vitro imaging. The excellent permeability and low reactivity of the chloroalkane substrate of HaloTag yields bioconjugation detection independent of ligand permeability. The objective of this research is to utilize the HaloTag technology in combination with genetic code expansion to develop a generalized method for quantification of intracellular bioconjugation to any POI at defined sites. We hypothesize that bioconjugation efficiency could be assessed through site‐specific installation of a reactive handle on a POI, followed by ligand‐mediated protein labeling via ternary complex formation after cell lysis and exposure to HaloTag. Here we describe the optimization of concentrations, reaction times, and reaction partners for bioconjugation and pulldown for POIs expressing different IEDDA reactive surface residues. Additionally, we describe the attempted application of this method for labeling protein targets, such as kinases and nuclear receptors. With this new methodology, we have demonstrated quantitative assessment of protein bioconjugation reactions in live cells.

Recent biomedical advances enabled by HaloTag technology

The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment. As a general approach, the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest (POI). However, the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags. Conversely, HaloTag, a novel self-labeling enzyme (SLE) tag, could quickly form a covalent bond with its ligand, enabling fast and specific labeling of POI. These desirable features greatly increase the accuracy and specificity, making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI. Notably, the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency. In this review, we summarize the development of HaloTag and recent advanced investigations associated with HaloTag, including in vitro imaging (e.g., POI imaging, cellular condition monitoring, microorganism imaging, system development), in vivo imaging, biomolecule immobilization (e.g., POI collection, protein/nuclear acid interaction and protein structure analysis), targeted degradation (e.g., L-AdPROM), and more. We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique.

A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles.

Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches for determining the orientation of a membrane-inserted protein are based on limited proteolytic digest, impermeable labeling reagents for specific amino acids, or membrane-impermeable quenchers for fluorescent proteins. Here, we describe a simple site-specific fluorescent assay based on self-labeling enzyme tags to determine the orientation of membrane proteins after reconstitution, exemplified on a reconstituted SNAP-tag plant H + -ATPase. This versatile method should benefit the optimization of reconstitution conditions and the analysis of many types of membrane proteins. Graphical abstract.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Harnessing self-labeling enzymes for selective and concurrent A-to-I and C-to-U RNA base editing

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.

Molecular Basis for Rep HUH‐endonuclease Mediated Protein‐DNA Bioconjugation

Self-labeling enzymes such as the SNAP-tag, CLIP-tag, and HALO-tag are popular systems for specific protein-DNA bioconjugation. While these fusion tags are useful and efficient, the limited number of orthogonal or non-cross reactive self-labeling enzymes pose a bottleneck for development of multiplexed applications reliant on protein-DNA linkages, such as protein barcoding. We are addressing this bottleneck by exploiting the single-stranded DNA (ssDNA) processing mechanism of HUH-endonucleases, so named for a histidine-hydrophobic residue-histidine (HUH) motif, to create site-specific and covalent linkages with unmodified DNA. Several recently developed applications in the fields of gene editing, cell targeting, live cell imaging, among others, are reliant on these so called HUH-tags. The most efficient and versatile HUH-tags are 11-16 kDa replication initiator proteins (Reps), which initiate rolling circle replication in a variety of organisms. We characterized the underlying molecular basis by which Reps recognize ssDNA sequences using structural analysis of co-crystal structures and by profiling the ssDNA specificity of a panel of Reps using a novel deep sequencing-based cleavage assay we developed termed HUH-seq. Combining the information from these results, we swapped residues between two different Reps to create a chimeric Rep with predictably altered specificity. These results are a first step in the direction of creating an HUH-tag toolkit that may spawn the development of new biotechnologies.

Fluorescent Labeling of Proteins of Interest in Live Cells: Beyond Fluorescent Proteins.

Live cell imaging brings us into a new era of direct visualization of biological processes and molecular dynamics in real time. To visualize dynamic cellular processes and virus-host interactions, fluorescent labeling of proteins of interest is often necessary. Fluorescent proteins are widely used for protein imaging, but they have some intrinsic deficiencies such as big size, photobleaching, and spectrum restriction. Thus, a variety of labeling strategies have been established and continuously developed. To protect the natural biological function(s) of the protein of interest, especially in viral life cycle, in vivo labeling requires smaller-sized tags, more specificity, and lower cytotoxicity. Here, we briefly summarized the principles, development, and their applications mainly in the virology field of three strategies for fluorescent labeling of proteins of interest including self-labeling enzyme derivatives, stainable peptide tags, and non-canonical amino acid incorporation. These labeling techniques greatly expand the fluorescent labeling toolbox and provide new opportunities for imaging biological processes.

Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue.

Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemically synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethylene glycol linker (RhoVR-Halo). When applied to cells, RhoVR-Halo dyes selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halo dyes maintain high voltage sensitivities-up to 34% ΔF/F per 100 mV-and fast response times typical of untargeted RhoVRs, while gaining the selectivity of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca2+ indicators like GCaMP to provide simultaneous voltage and Ca2+ imaging. In a brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read-out of action potentials from labeled cortical neurons in a rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically encodable systems, permitting imaging modalities inaccessible to either technique individually.

Translokation bakterieller Effektorproteine — live und in Farbe

Intracellular pathogens thrive by invading host cells, thereby creating protected replicative niches. The manipulation of host cells is accomplished by means of translocated bacterial effector proteins. The ability to track effector proteins during an infection is essential to understand host-pathogen interaction. Here, we describe a novel method to target bac terial effector proteins using Salmonella enterica as a model organism and deploying self-labeling enzyme tags.

Self-Labeling Enzyme Tags for Analyses of Translocation of Type III Secretion System Effector Proteins.

Type III secretion systems (T3SS) are molecular machines in Gram-negative pathogens that translocate effector proteins with central roles in virulence. The analyses of the translocation, subcellular localization, and mode of action of T3SS effector proteins are of central importance for the understanding of host-pathogen interaction and pathogenesis of bacterial infections. The analysis of translocation requires dedicated techniques to address the temporal and spatial dynamics of translocation. Here we describe a novel approach to deploy self-labeling enzymes (SLE) as universal tags for localization and tracking of translocated effector proteins. Effector-SLE fusion proteins allow live-cell imaging of translocation by T3SS, superresolution microscopy, and single-molecule tracking of effector motility in living host cells. We describe the application of the approach to T3SS effector proteins for invasion and intracellular lifestyle of Salmonella enterica serovar Typhimurium and to a T3SS effector of Yersinia enterocolitica The novel approach enables analyses of the role of T3SS in host-pathogen interaction at the highest temporal and spatial resolution, toward understanding the molecular mechanisms of their effector proteins.IMPORTANCE Type III secretion systems mediate translocation of effector proteins into mammalian cells. These proteins interfere with host cell functions, being main virulence factors of Gram-negative pathogens. Analyses of the process of translocation, the subcellular distribution, and the dynamics of effector proteins in host cells have been hampered by the lack of suitable tags and detection systems. Here we describe the use of self-labeling enzyme tags for generation of fusions with effector proteins that are translocated and functional in host cell manipulation. Self-labeling reactions with cell-permeable ligand dyes are possible prior to or after translocation. We applied the new approach to superresolution microscopy for effector protein translocation. For the first time, we show the dynamic properties of effector proteins in living host cells after translocation by intracellular bacteria. The new approach of self-labeling enzyme tags fusions will enable analyses of type III secretion system effector proteins with new dimensions of temporal and spatial resolution.

Covalent adaptor synNotch and chimeric antigen receptors (CARs) for programmable antigen targeting

Abstract Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically bind and kill tumor cells. Adoptive cell therapy with CAR T cells targeting CD19 has revolutionized treatment of refractory B cell acute lymphoblastic leukemia, and there is great interest in generating CAR T cells treating other cancers by targeting additional tumor antigens. Another promising class of engineered receptors are synthetic Notch (synNotch) receptors that can sense an antigen of interest on a neighboring cell and turn on expression of any transgene(s) of interest. To expand the targeting capabilities of these receptors, we have developed “universal” CAR and SynNotch receptors whose antigen-specificity can be re-directed by co-administered tumor-specific antibodies. Instead of directly targeting a tumor antigen, our universal receptors contain the SNAPtag self-labeling enzyme, which reacts with antibodies conjugated to benzylguanine (BG) to post-translationally assemble complete covalently associated antigen receptors. We demonstrate that the activation of SNAP CAR and SNAP-SynNotch receptors can be successfully re-targeted by several clinically relevant antibodies including: Rituximab, FMC63, Herceptin, and Cetuximab. SNAP-SynNotch cells demonstrate potent transgene activation, and SNAP-CAR T cells are capable of performing effector functions in a BG-antibody-directed antigen-specific manner. Additionally, the receptor response is titratable by BG-antibody dose. Finally, a continuous mathematical model was constructed to describe and optimize system activity. SNAP synNotch and SNAP CAR T cells provide a powerful new strategy to retarget engineered cells to multiple antigens.

On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates.

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.

Beyond O6-Benzylguanine: O6-(5-Pyridylmethyl)guanine as a Substrate for the Self-Labeling Enzyme SNAP-Tag.

SNAP-tag is a genetically encoded label for tracking proteins of interest that is known to utilize O6-benzylguanine derivatives as substrates. In this work, mass spectrometric analysis revealed that SNAP-tag also accepts O6-(5-pyridylmethyl)guanine derivatives as substrates. A fluorescently conjugated O6-(5-pyridylmethyl)guanine was synthesized and used to selectively label intracellular compartments. This compound also acts as a zinc(II) ion-sensitive fluorescent indicator and therefore can be used in organelle-specific zinc(II) imaging.

Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.

New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported “cell-penetrating peptides” (CPPs) transport a model protein cargo—the self-labeling enzyme SNAP-tag—beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo—the engineered peroxidase APEX2—and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales.

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.