HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

A guide to genetically-encoded redox biosensors: State of the art and opportunities.

Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.

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.

Halo-RPD: searching for RNA-binding protein targets in plants.

Study of RNA-protein interactions and identification of RNA targets are among the key aspects of understanding RNA biology. Currently, various methods are available to investigate these interactions with, RNA immunoprecipitation (RIP) being the most common. The search for RNA targets has largely been conducted using antibodies to an endogenous protein or to GFP-tag directly. Having to be dependent on the expression level of the target protein and having to spend time selecting highly specific antibodies make immunoprecipitation complicated. Expression of the GFP-fused protein can lead to cytotoxicity and, consequently, to improper recognition or degradation of the chimeric protein. Over the past few years, multifunctional tags have been developed. SNAP-tag and HaloTag allow the target protein to be studied from different perspectives. Labeling of the fusion protein with custom-made fluorescent dyes makes it possible to study protein expression and to localize it in the cell or the whole organism. A high-affinity substrate has been created to allow covalent binding by chimeric proteins, minimizing protein loss during protein isolation. In this paper, a HaloTag-based method, which we called Halo-RPD (HaloTag RNA PullDown), is presented. The proposed protocol uses plants with stable fusion protein expression and Magne® HaloTag® magnetic beads to capture RNA-protein complexes directly from the cytoplasmic lysate of transgenic Arabidopsis thaliana plants. The key stages described in the paper are as follows: (1) preparation of the magnetic beads; (2) tissue homogenization and collection of control samples; (3) precipitation and wash of RNA-protein complexes; (4) evaluation of protein binding efficiency; (5) RNA isolation; (6) analysis of the RNA obtained. Recommendations for better NGS assay designs are provided.

Application of bioluminescence resonance energy transfer to quantitate cell-surface expression of membrane proteins.

We report a bioluminescence resonance energy transfer (BRET) assay to quantitate the fraction of an engineered membrane protein at the cell surface versus inside the cell. As test cases, we engineered two different G protein-coupled receptors (GPCRs) in which a NanoLuc luciferase (NLuc) and a HaloTag are fused to the extracellular amino-terminal tail of the receptors. We then employed a pulse-chase labeling approach relying on two different fluorescent dyes with distinctive cell permeability properties. The dyes are efficiently excited by luminescence from NLuc, but are spectrally distinct. Measuring BRET from the chemiluminescence of the NLuc to the fluorophores bound to the HaloTag minimizes the limitations of in-cell fluorescence resonance energy transfer (FRET)-based approaches such as photobleaching and autofluorescence. The BRET surface expression assay can quantitatively differentiate between the labeling of receptors at the cell surface and receptors inside of the cell. The assay is shown to be quantitative and robust compared with other approaches to measure cell surface expression of membrane proteins such as enzyme-linked immunosorbent assay or immunoblotting, and significantly increases the throughput because the assay is designed to be carried out in microtiter plate format.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

mSphere of Influence: Hooray for HaloTag! A novel tool to study autophagy in mammalian cells.

Laura Cheney works at the crossroads of HIV and autophagy, a critical biological process for cellular homeostasis, to understand more completely the pathogenesis of HIV-associated neurocognitive impairment. In this mSphere of Influence article, she reflects on how "A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag" by Willa Wen-You Yim, Hayashi Yamamoto, and Noboru Mizushima (eLife 11:e78923, 2022, https://doi.org/10.7554/eLife.78923) expands the tools for studying autophagy and inspired her to use this technology to develop a reporter to study autophagy of mitochondria, termed mitophagy, to further her own research goals.

Construction of Coumarin-Based Bioorthogonal Macromolecular Probes for Photoactivation.

Photoactivatable fluorescence imaging is one of the most valuable methods for visualizing protein localization, trafficking, and interactions. Here, we designed four bioorthogonal fluorescent probes K1-K4 by installing photoactive cages and HaloTag ligands onto the different positions of the coumarin fluorophore. Although K1-K4 all exhibited rapid photostimulated responses in aqueous solution, only K3 was found to have an obvious aggregation-induced emission (AIE). Next, macromolecular fluorescent probes Kn=1/2/3/4_POIs were obtained by covalently attaching K1-K4 to HaloTag-fused proteins of interest (POIs). Kn=3/4_POIs exhibited a higher fluorescence increase than that of Kn=1/2_POIs upon photoactivation in both liquid and solid phases. Moreover, K3_GFP_Halo and K4_GFP_Halo presented the fluorescence resonance energy transfer (FRET) from photocleaved K3 and K4 to GFP in the protein complex. We further examined the fluorescence labeling ability of K1-K4 to intracellular IRE1_Halo protein and found that K3 and K4 containing the HaloTag ligand on the C4 position of coumarin could be retained in cells for long-term tracking of the IRE1_Halo protein. Hence, we established a platform of novel bioorthogonal fluorescent probes conjugating onto Halo-tagged POIs for rapid photoactivation in vitro and in cells.

HaloTag display enables quantitative single-particle characterization and functionalization of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells, and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity, and other properties. Measuring how incorporation varies across a population of EVs is important for characterizing such materials and understanding their function, yet it remains challenging to quantitatively characterize the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterization platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labeling to antibody-labeling of EVs using single vesicle flow cytometry, enabling us to quantify the substantial degree to which antibody labeling can underestimate the absolute number of proteins present on an EV. Finally, we demonstrate use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterization tool which complements and expands existing methods.

Synthesis and Evaluation of Cereblon-Recruiting HaloPROTACs.

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

Live cell single-molecule imaging to study DNA repair in human cells.

The genetic material in human cells is continuously exposed to a wide variety of insults that can induce different DNA lesions. To maintain genomic stability and prevent potentially deleterious genetic changes caused by DNA damage, mammalian cells have evolved a number of pathways that repair specific types of DNA damage. These DNA repair pathways vary in their accuracy, some providing high-fidelity repair while others are error-prone and are only activated as a last resort. Adding additional complexity to cellular mechanisms of DNA repair is the DNA damage response which is a sophisticated a signaling network that coordinates repair outcomes, cell-cycle checkpoint activation, and cell fate decisions. As a result of the sheer complexity of the various DNA repair pathways and the DNA damage response there are large gaps in our understanding of the molecular mechanisms underlying DNA damage repair in human cells. A key unaddressed question is how the dynamic recruitment of DNA repair factors contributes to repair kinetics and repair pathway choice in human cells. Methodological advances in live cell single-molecule imaging over the last decade now allow researchers to directly observe and analyze the dynamics of DNA repair proteins in living cells with high spatiotemporal resolution. Live cell single-molecule imaging combined with single-particle tracking can provide direct insight into the biochemical reactions that control DNA repair and has the power to identify previously unobservable processes in living cells. This review summarizes the main considerations for experimental design and execution for live cell single-molecule imaging experiments and describes how they can be used to define the molecular mechanisms of DNA damage repair in mammalian cells.

TCAB1 prevents nucleolar accumulation of the telomerase RNA to facilitate telomerase assembly.

Localization of a variety of RNAs to non-membrane-bound cellular compartments such as nucleoli and Cajal bodies is critical for their stability and function. The molecular mechanisms that underly the recruitment and exclusion of RNAs from these phase-separated organelles is incompletely understood. Telomerase is a ribonucleoprotein composed of the reverse transcriptase protein telomerase reverse transcriptase (TERT), the telomerase RNA (TR), and several auxiliary proteins, including TCAB1. Here we show that in the absence of TCAB1, a large fraction of TR is tightly bound to the nucleolus, while TERT is largely excluded from the nucleolus, reducing telomerase assembly. This suggests that nuclear compartmentalization by the non-membrane-bound nucleolus counteracts telomerase assembly, and TCAB1 is required to retain TR in the nucleoplasm. Our work provides insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase separation and demonstrates that TCAB1 plays an important role in telomerase assembly.

Functional Expression of the Ectodomain of Plant Receptor Kinases in Plant Suspension Culture.

Extracellular signals are usually perceived by membrane-localized receptors that transduce intercellular signals to activate various pathways. In plants, single transmembrane receptor kinases act as receptors for extracellular signals. Endogenous secreted peptide hormones have been recognized as novel signaling molecules, functioning through the formation of ligand-receptor pairs in plants. Recently, research on plant peptide hormone-receptor interactions based on the structural biology approach has greatly improved; however, the dissociation constant of recombinant receptor molecules expressed in insect cells using the baculovirus expression system is relatively low. We introduce here a method for creating a stable and functional homogeneous expression system for plant receptor kinases using tobacco BY-2 cells while maintaining conventional ligand-binding activity. This strategy will help improve our understanding of plant endogenous peptide ligand-receptor interactions.

Analysis of Wild Type and Variant B Cystatin C Interactome in Retinal Pigment Epithelium Cells Reveals Variant B Interacting Mitochondrial Proteins.

Cystatin C, a secreted cysteine protease inhibitor, is abundantly expressed in retinal pigment epithelium (RPE) cells. A mutation in the protein's leader sequence, corresponding to formation of an alternate variant B protein, has been linked with an increased risk for both age-related macular degeneration (AMD) and Alzheimer's disease (AD). Variant B cystatin C displays intracellular mistrafficking with partial mitochondrial association. We hypothesized that variant B cystatin C interacts with mitochondrial proteins and impacts mitochondrial function. We sought to determine how the interactome of the disease-related variant B cystatin C differs from that of the wild-type (WT) form. For this purpose, we expressed cystatin C Halo-tag fusion constructs in RPE cells to pull down proteins interacting with either the WT or variant B form, followed by identification and quantification by mass spectrometry. We identified a total of 28 interacting proteins, of which 8 were exclusively pulled down by variant B cystatin C. These included 18 kDa translocator protein (TSPO) and cytochrome B5 type B, both of which are localized to the mitochondrial outer membrane. Variant B cystatin C expression also affected RPE mitochondrial function with increased membrane potential and susceptibility to damage-induced ROS production. The findings help us to understand how variant B cystatin C differs functionally from the WT form and provide leads to RPE processes adversely affected by the variant B genotype.

Investigation of Sequence-Penetration Relationships of Antisense Oligonucleotides.

A major limitation for the development of more effective oligonucleotide therapeutics has been a lack of understanding of their penetration into the cytosol. While prior work has shown how backbone modifications affect cytosolic penetration, it is unclear how cytosolic penetration is affected by other features including base composition, base sequence, length, and degree of secondary structure. We have applied the chloroalkane penetration assay, which exclusively reports on material that reaches the cytosol, to investigate the effects of these characteristics on the cytosolic uptake of druglike oligonucleotides. We found that base composition and base sequence had moderate effects, while length did not correlate directly with the degree of cytosolic penetration. Investigating further, we found that the degree of secondary structure had the largest and most predictable correlations with cytosolic penetration. These methods and observations add a layer of design for maximizing the efficacy of new oligonucleotide therapeutics.

Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

HaloTag-Based Reporters for Fluorescence Imaging and Biosensing.

Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self-labelling proteins, which combine a genetically encoded tag with a small-molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell-permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.

Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional ß1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image ß1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous ß1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal ß1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.

Targeted Protein Unfolding at the Golgi Apparatus.

Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in the cell, manages misfolded proteins to maintain proteostasis is still poorly understood. Here we present a strategy for targeted protein unfolding at the Golgi that enables studying Golgi-related protein quality control and stress-signaling pathways. Targeted protein unfolding is induced by small molecule-based chemical biology approaches-hydrophobic tagging and the use of a destabilization domain. Imaging studies allow visualizing quality control (QC) phenotypes, such as the formation of QC carriers and Golgi-to-endoplasmic reticulum trafficking, and correlating these phenotypes with other trafficking processes.

A HaloTag-based reporter processing assay to monitor autophagic flux.

Monitoring mammalian macroautophagic/autophagic flux is necessary in most autophagy studies but has generally been difficult to do. Here, we discuss our recent report of a HaloTag-based processing method that offers a straightforward readout for autophagic flux. We found that the self-labeling protein HaloTag becomes resistant to proteolysis when labeled with its ligand. Fusing HaloTag to an autophagy protein such as LC3 results in a reporter that is completely degraded when delivered into lysosomes but, when pulse-labeled with HaloTag ligand, releases free HaloTagligand when processed by lysosomal enzymes. The quantifiable amount of free HaloTagligand, observed by immunoblotting or in-gel fluorescence detection, reflects autophagic flux. Besides being compatible with fluorescence microscopy and flow cytometry applications, this quantitative assay can be readily adapted to monitor most autophagy pathways or the autophagic degradation of a protein of interest.