Super-resolution imaging reveals the mechanism of endosomal acidification inhibitors against SARS-CoV-2 infection.

In this study, super-resolution structured illumination microscope (SIM) was used to analyze molecular mechanism of endocytic acidification inhibitors in the SARS-CoV-2 pandemic, such as Chloroquine (CQ), Hydroxychloroquine (HCQ) and Bafilomycin A1 (BafA1). We fluorescently labeled the SARS-CoV-2 RBD and its receptor ACE2 protein with small molecule dyes. Utilizing SIM imaging, the real-time impact of inhibitors (BafA1, CQ, HCQ, Dynasore) on the RBD-ACE2 endocytotic process was dynamically tracked in living cells. Initially, the protein activity of RBD and ACE2 was ensured after being labeled. And then our findings revealed that these inhibitors could inhibit the internalization and degradation of RBD-ACE2 to varying degrees. Among them, 100 nM BafA1 exhibited the most satisfactory endocytotic inhibition (~63.9%) and protein degradation inhibition (~97.7%). And it could inhibit the fusion between endocytic vesicles in the living cells. Additionally, Dynasore, a widely recognized dynein inhibitor, also demonstrated cell acidification inhibition effects. Together, these inhibitors collectively hinder SARS-CoV-2 infection by inhibiting both the viral internalization and RNA release. The comprehensive evaluation of pharmacological mechanisms through super-resolution fluorescence imaging has laid a crucial theoretical foundation for the development of potential drugs to treat COVID-19.

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.

Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation.

A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells.

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,ß-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1-5 µM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 µM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones.

Recent Advancements in Subcellular Proteomics: Growing Impact of Organellar Protein Niches on the Understanding of Cell Biology.

The mammalian cell is a complex entity, with membrane-bound and membrane-less organelles playing vital roles in regulating cellular homeostasis. Organellar protein niches drive discrete biological processes and cell functions, thus maintaining cell equilibrium. Cellular processes such as signaling, growth, proliferation, motility, and programmed cell death require dynamic protein movements between cell compartments. Aberrant protein localization is associated with a wide range of diseases. Therefore, analyzing the subcellular proteome of the cell can provide a comprehensive overview of cellular biology. With recent advancements in mass spectrometry, imaging technology, computational tools, and deep machine learning algorithms, studies pertaining to subcellular protein localization and their dynamic distributions are gaining momentum. These studies reveal changing interaction networks because of "moonlighting proteins" and serve as a discovery tool for disease network mechanisms. Consequently, this review aims to provide a comprehensive repository for recent advancements in subcellular proteomics subcontexting methods, challenges, and future perspectives for method developers. In summary, subcellular proteomics is crucial to the understanding of the fundamental cellular mechanisms and the associated diseases.

Cysteine-independent CRISPR-Associated Protein Labeling for Presentation and Co-delivery of Molecules Toward Genetic and Epigenetic Regulations.

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.

Visualizing the invisible: novel approaches to visualizing bacterial proteins and host-pathogen interactions.

Host-pathogen interactions play a critical role in infectious diseases, and understanding the underlying mechanisms is vital for developing effective therapeutic strategies. The visualization and characterization of bacterial proteins within host cells is key to unraveling the dynamics of these interactions. Various protein labeling strategies have emerged as powerful tools for studying host-pathogen interactions, enabling the tracking, localization, and functional analysis of bacterial proteins in real-time. However, the labeling and localization of Salmonella secreted type III secretion system (T3SS) effectors in host cells poses technical challenges. Conventional methods disrupt effector stoichiometry and often result in non-specific staining. Bulky fluorescent protein fusions interfere with effector secretion, while other tagging systems such as 4Cys-FLaSH/Split-GFP suffer from low labeling specificity and a poor signal-to-noise ratio. Recent advances in state-of-the-art techniques have augmented the existing toolkit for monitoring the translocation and dynamics of bacterial effectors. This comprehensive review delves into the bacterial protein labeling strategies and their application in imaging host-pathogen interactions. Lastly, we explore the obstacles faced and potential pathways forward in the realm of protein labeling strategies for visualizing interactions between hosts and pathogens.

Quantification of local matrix deposition during muscle stem cell activation using engineered hydrogels.

Adult stem cells occupy a niche that contributes to their function, but how stem cells remodel their microenvironment remains an open-ended question. Herein, biomaterials-based systems and metabolic labeling were utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts were observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, the increased nascent matrix deposition was associated with stem cell activation. Reducing the ability to deposit nascent matrix in muscle stem cells attenuated function and mimicked impairments observed from muscle stem cells isolated from old aged muscles, which could be rescued with therapeutic supplementation of insulin-like growth factors. These results highlight how nascent matrix production is critical for maintaining healthy stem cell function.

An Efficient Homologous Recombination-Based In Situ Protein-Labeling Method in Verticillium dahliae.

Accurate determination of protein localization, levels, or protein-protein interactions is pivotal for the study of their function, and in situ protein labeling via homologous recombination has emerged as a critical tool in many organisms. While this approach has been refined in various model fungi, the study of protein function in most plant pathogens has predominantly relied on ex situ or overexpression manipulations. To dissect the molecular mechanisms of development and infection for Verticillium dahliae, a formidable plant pathogen responsible for vascular wilt diseases, we have established a robust, homologous recombination-based in situ protein labeling strategy in this organism. Utilizing Agrobacterium tumefaciens-mediated transformation (ATMT), this methodology facilitates the precise tagging of specific proteins at their C-termini with epitopes, such as GFP and Flag, within the native context of V. dahliae. We demonstrate the efficacy of our approach through the in situ labeling of VdCf2 and VdDMM2, followed by subsequent confirmation via subcellular localization and protein-level analyses. Our findings confirm the applicability of homologous recombination for in situ protein labeling in V. dahliae and suggest its potential utility across a broad spectrum of filamentous fungi. This labeling method stands to significantly advance the field of functional genomics in plant pathogenic fungi, offering a versatile and powerful tool for the elucidation of protein function.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria.

Cell-wall-less (L-form) bacteria exhibit morphological complexity and heterogeneity, complicating quantitative analysis of them under internal and external stimuli. Stable and efficient labeling is needed for the fluorescence-based quantitative cell analysis of L-forms during growth and proliferation. Here, we evaluated the expression of multiple fluorescent proteins (FPs) under different promoters in the Bacillus subtilis L-form strain LR2 using confocal microscopy and imaging flow cytometry. Among others, Pylb-derived NBP3510 showed a superior performance for inducing several FPs including EGFP and mKO2 in both the wild-type and L-form strains. Moreover, NBP3510 was also active in Escherichia coli and its L-form strain NC-7. Employing these established FP-labeled strains, we demonstrated distinct morphologies in the L-form bacteria in a quantitative manner. Given cell-wall-deficient bacteria are considered protocell and synthetic cell models, the generated cell lines in our work could be valuable for L-form-based research.

Decaging-to-labeling: Development and investigation of quinone methide warhead for protein labeling.

Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

Split-GFP complementation at the bacterial cell surface for antibody-free labeling and quantification of heterologous protein display.

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1-10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.

Affinity-Directed Site-Specific Protein Labeling and Its Application to Antibody-Drug Conjugates.

Chemically modified proteins have diverse applications; however, conventional chemo-selective methods often yield heterogeneously labeled products. To address this limitation, site-specific protein labeling holds significant potential, driving extensive research in this area. Nevertheless, site-specific modification of native proteins remains challenging owing to the complexity of their functional groups. Therefore, a method for site-selective labeling of intact proteins is aimed to design. In this study, a novel approach to traceless affinity-directed intact protein labeling is established, which leverages small binding proteins and genetic code expansion technology. By applying this method, a site-specific antibody labeling with a drug, which leads to the production of highly effective antibody-drug conjugates specifically targeting breast cancer cell lines is achieved. This approach enables traceless conjugation of intact target proteins, which is a critical advantage in pharmaceutical applications. Furthermore, small helical binding proteins can be easily engineered for various target proteins, thereby expanding their potential applications in diverse fields. This innovative approach represents a significant advancement in site-specific modification of native proteins, including antibodies. It also bears immense potential for facilitating the development of therapeutic agents for various diseases.

Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate.

Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.

Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging.

This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed in vitro, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for in vitro and in vivo imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or in vivo biological activities of the POI.

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing.

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.

Triazinium Ligation: Bioorthogonal Reaction of N1-Alkyl 1,2,4-Triazinium Salts.

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1-alkylation of 1,2,4-triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4-triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1-alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5-tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.