Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that small interfering RNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA-deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/serum response factor-regulated genes such as that encoding myosin-IIA (MYH9). Our studies utilizing live cell imaging and pharmacological inhibition of myosin-II support a mechanism of deregulated myosin-II self-organizing activity at the nexus of divergent actin cytoskeletal aberrations resulting from LMNA loss. In light of our results, we propose a model of how the nucleus, via linkage to the cytoplasmic actomyosin network, may act to control myosin-II contractile behavior through both mechanical and transcriptional feedback mechanisms.

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.

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.

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.

Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation.

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.

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

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.

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.

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated proteins (MAPs) that have the capacity to affect various cellular functions, including motor-mediated transport. One such MAP is She1, a dynein effector that polarizes dynein-mediated spindle movements in budding yeast. Here, we characterize the molecular basis by which She1 affects dynein, providing the first such insight into which a MAP can modulate motor motility. We find that She1 affects the ATPase rate, microtubule-binding affinity, and stepping behavior of dynein, and that microtubule binding by She1 is required for its effects on dynein motility. Moreover, we find that She1 directly contacts the microtubule-binding domain of dynein, and that their interaction is sensitive to the nucleotide-bound state of the motor. Our data support a model in which simultaneous interactions between the microtubule and dynein enables She1 to directly affect dynein motility.

The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.

Advances in fluorescence microscopy have introduced new assays to quantify live-cell translation dynamics at single-RNA resolution. We introduce a detailed, yet efficient sequence-based stochastic model that generates realistic synthetic data for several such assays, including Fluorescence Correlation Spectroscopy (FCS), ribosome Run-Off Assays (ROA) after Harringtonine application, and Fluorescence Recovery After Photobleaching (FRAP). We simulate these experiments under multiple imaging conditions and for thousands of human genes, and we evaluate through simulations which experiments are most likely to provide accurate estimates of elongation kinetics. Finding that FCS analyses are optimal for both short and long length genes, we integrate our model with experimental FCS data to capture the nascent protein statistics and temporal dynamics for three human genes: KDM5B, β-actin, and H2B. Finally, we introduce a new open-source software package, RNA Sequence to NAscent Protein Simulator (rSNAPsim), to easily simulate the single-molecule translation dynamics of any gene sequence for any of these assays and for different assumptions regarding synonymous codon usage, tRNA level modifications, or ribosome pauses. rSNAPsim is implemented in Python and is available at: https://github.com/MunskyGroup/rSNAPsim.git.

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.

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (∼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (∼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process 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.

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.