PAbFold: Linear Antibody Epitope Prediction using AlphaFold2.

Defining the binding epitopes of antibodies is essential for understanding how they bind to their antigens and perform their molecular functions. However, while determining linear epitopes of monoclonal antibodies can be accomplished utilizing well-established empirical procedures, these approaches are generally labor- and time-intensive and costly. To take advantage of the recent advances in protein structure prediction algorithms available to the scientific community, we developed a calculation pipeline based on the localColabFold implementation of AlphaFold2 that can predict linear antibody epitopes by predicting the structure of the complex between antibody heavy and light chains and target peptide sequences derived from antigens. We found that this AlphaFold2 pipeline, which we call PAbFold, was able to accurately flag known epitope sequences for several well-known antibody targets (HA / Myc) when the target sequence was broken into small overlapping linear peptides and antibody complementarity determining regions (CDRs) were grafted onto several different antibody framework regions in the single-chain antibody fragment (scFv) format. To determine if this pipeline was able to identify the epitope of a novel antibody with no structural information publicly available, we determined the epitope of a novel anti-SARS-CoV-2 nucleocapsid targeted antibody using our method and then experimentally validated our computational results using peptide competition ELISA assays. These results indicate that the AlphaFold2-based PAbFold pipeline we developed is capable of accurately identifying linear antibody epitopes in a short time using just antibody and target protein sequences. This emergent capability of the method is sensitive to methodological details such as peptide length, AlphaFold2 neural network versions, and multiple-sequence alignment database. PAbFold is available at https://github.com/jbderoo/PAbFold.

Single molecule imaging of the central dogma reveals myosin-2A gene expression is regulated by contextual translational buffering.

While protein homeostasis is a hallmark of gene regulation, unraveling the hidden regulatory mechanisms that maintain homeostasis is difficult using traditional methods. To confront this problem, we CRISPR engineered a human cell line with multiple tags in the endogenous MYH9 gene, which encodes the essential and ubiquitous myosin-2A cytoskeletal motor. Using these cells, we imaged MYH9 transcription, translation, and mature mRNA and protein in distinct colors, enabling a full dissection of the central dogma. Our data show that MYH9 transcription is upregulated in an SRF-dependent manner in response to cytoskeletal cues and that MYH9 translation can either buffer or match the transcriptional response depending on context. Upon knockdown of actin-depolymerizing proteins like cofilin, translation efficiency drops by a factor of two to buffer strong transcriptional upregulation, likely to help prevent excessive myosin activity. In contrast, following serum stimulation, translation matches the transcriptional response to readily reestablish steady state. Our results identify contextual translational buffering as an important regulatory mechanism driving stable MYH9 expression. They also demonstrate the power and broad applicability of our cell line, which can now be used to accurately quantify central dogma dynamics in response to diverse forms of cellular perturbations.

Synonymous codon usage regulates translation initiation.

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

Translation Dynamics of Single mRNAs in Live Cells.

The translation of messenger RNA (mRNA) into proteins represents the culmination of gene expression. Recent technological advances have revolutionized our ability to investigate this process with unprecedented precision, enabling the study of translation at the single-molecule level in real time within live cells. In this review, we provide an overview of single-mRNA translation reporters. We focus on the core technology, as well as the rapid development of complementary probes, tags, and accessories that enable the visualization and quantification of a wide array of translation dynamics. We then highlight notable studies that have utilized these reporters in model systems to address key biological questions. The high spatiotemporal resolution of these studies is shedding light on previously unseen phenomena, uncovering the full heterogeneity and complexity of translational regulation. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Live-cell imaging uncovers the relationship between histone acetylation, transcription initiation, and nucleosome mobility.

Histone acetylation and RNA polymerase II phosphorylation are associated with transcriptionally active chromatin, but their spatiotemporal relationship in live cells remains poorly understood. To address this problem, we combine Fab-based labeling of endogenous protein modifications with single-molecule tracking to quantify the dynamics of chromatin enriched with histone H3 lysine-27 acetylation (H3K27ac) and RNA polymerase II serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals that chromatin enriched with these two modifications is generally separate. In these separated sites, we show that the two modifications are inversely correlated with one another on the minutes time scale and that single nucleosomes within each region display distinct and opposing dynamics on the subsecond time scale. While nucleosomes diffuse ~15% faster in chromatin enriched with H3K27ac, they diffuse ~15% slower in chromatin enriched with RNAP2-Ser5ph. These results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each marks distinct transcriptionally poised or active sites, respectively.

Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD.

C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.

An expanded view of transcription.

A new method expands chromatin to provide detailed images of transcription sites.

Using mechanistic models and machine learning to design single-color multiplexed nascent chain tracking experiments.

mRNA translation is the ubiquitous cellular process of reading messenger-RNA strands into functional proteins. Over the past decade, large strides in microscopy techniques have allowed observation of mRNA translation at a single-molecule resolution for self-consistent time-series measurements in live cells. Dubbed Nascent chain tracking (NCT), these methods have explored many temporal dynamics in mRNA translation uncaptured by other experimental methods such as ribosomal profiling, smFISH, pSILAC, BONCAT, or FUNCAT-PLA. However, NCT is currently restricted to the observation of one or two mRNA species at a time due to limits in the number of resolvable fluorescent tags. In this work, we propose a hybrid computational pipeline, where detailed mechanistic simulations produce realistic NCT videos, and machine learning is used to assess potential experimental designs for their ability to resolve multiple mRNA species using a single fluorescent color for all species. Our simulation results show that with careful application this hybrid design strategy could in principle be used to extend the number of mRNA species that could be watched simultaneously within the same cell. We present a simulated example NCT experiment with seven different mRNA species within the same simulated cell and use our ML labeling to identify these spots with 90% accuracy using only two distinct fluorescent tags. We conclude that the proposed extension to the NCT color palette should allow experimentalists to access a plethora of new experimental design possibilities, especially for cell Signaling applications requiring simultaneous study of multiple mRNAs.

Using Mechanistic Models and Machine Learning to Design Single-Color Multiplexed Nascent Chain Tracking Experiments.

mRNA translation is the ubiquitous cellular process of reading messenger-RNA strands into functional proteins. Over the past decade, large strides in microscopy techniques have allowed observation of mRNA translation at a single-molecule resolution for self-consistent time-series measurements in live cells. Dubbed Nascent chain tracking (NCT), these methods have explored many temporal dynamics in mRNA translation uncaptured by other experimental methods such as ribosomal profiling, smFISH, pSILAC, BONCAT, or FUNCAT-PLA. However, NCT is currently restricted to the observation of one or two mRNA species at a time due to limits in the number of resolvable fluorescent tags. In this work, we propose a hybrid computational pipeline, where detailed mechanistic simulations produce realistic NCT videos, and machine learning is used to assess potential experimental designs for their ability to resolve multiple mRNA species using a single fluorescent color for all species. Through simulation, we show that with careful application, this hybrid design strategy could in principle be used to extend the number of mRNA species that could be watched simultaneously within the same cell. We present a simulated example NCT experiment with seven different mRNA species within the same simulated cell and use our ML labeling to identify these spots with 90% accuracy using only two distinct fluorescent tags. The proposed extension to the NCT color palette should allow experimentalists to access a plethora of new experimental design possibilities, especially for cell signalling applications requiring simultaneous study of multiple mRNAs.

Visualization, Quantification, and Modeling of Endogenous RNA Polymerase II Phosphorylation at a Single-copy Gene in Living Cells.

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle. Graphical abstract: Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.

Imaging translational control by Argonaute with single-molecule resolution in live cells.

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering sensor reflects endogenous miRNA-mediated gene silencing. Using the sensor, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. Silencing was occasionally interrupted by brief bursts of translational activity and took 3-4 times longer than a single round of translation, consistent with a gradual increase in the inhibition of translation initiation. At later time points, Ago2-tethered mRNAs cluster and coalesce with P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying RNA-protein interactions.

A Drosophila toolkit for HA-tagged proteins unveils a block in autophagy flux in the last instar larval fat body.

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we have developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: 'HA Frankenbody'. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

Single-Molecule Imaging of mRNA Interactions with Stress Granules.

Single-molecule imaging in living cells enables the investigation of molecular dynamics and interactions underlying the physiology of a cell. We recently developed a method to visualize translation events at single-mRNA resolution in living cells. Here we describe how we apply this method to visualize mRNA interactions with stress granules in the context of translational activity during cell stress.

Generation and diversification of recombinant monoclonal antibodies.

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells.

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells.

Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind complementary epitopes in the two separate ORFs of the bicistronic biosensor. This causes the biosensor to fluoresce in different colors depending on which ORF/epitopes are translated. Using the biosensor together with high-resolution fluorescence microscopy and single-molecule tracking analysis allows for the quantitative comparison of translation dynamics between the two ORFs at a resolution of tens-of-nanometers in space and sub-seconds in time, which is not possible with more traditional GFP or luciferase reporters. Since both ORFs are on the same biosensor, they experience the same microenvironment, allowing a fair comparison of their relative translational activities. In this protocol, we describe how to get this assay up and running in cultured human cells so that translation dynamics can be studied under both normal and stressful cellular conditions. We also provide a number of useful tips and notes to help express components at appropriate levels inside cells for optimal live cell imaging. Graphical abstract: Steps required for 3-color single-molecule translation imaging and analysis.

Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene.

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster.

Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

Author Correction: Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

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

Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells.

Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5' cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.