Biology subjects

Tanenbaum, M. E.

Publications and source records attributed to Tanenbaum, M. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Linking live-cell imaging to single-cell sequencing using genetic barcoding

Linking cellular phenotype to genotype and gene expression is a central goal in biomedical research. Live-cell imaging captures dynamic cellular phenotypes, whereas single-cell sequencing measures genome-wide molecular states, but pairing these readouts for the same cell remains challenging. Here we present Waldo, a genetically encoded single-cell barcoding strategy that can be read out by both live-cell imaging and single-cell sequencing to connect single cells across both modalities. Waldo barcodes are generated by combinatorial expression of synthetic cell-surface receptors, each detected by a cognate peptide probe coupled either to a fluorescent dye or a DNA barcode. Iterative fluorescent probe staining achieves large barcode diversity, while preserving fluorescence channels for phenotypic reporters. As a proof-of-principle, we use Waldo to link cell division phenotypes after chromosome missegregation to the karyotype and transcriptome of the same cells. Waldo provides a general strategy for connecting imaging phenotypes to genome-wide sequencing readouts in single cells.

cell biology↗

bioRxiv2026

Decoding the Sequence Requirements for Translation Initiation

Accurate selection of start codons by ribosomes is a fundamental determinant of proteome composition. Although the Kozak sequence--an 8-nucleotide sequence flanking the start codon--has long been viewed as the primary determinant of initiation in eukaryotes, it fails to explain the large diversity of start codon usage across transcripts. Here we combine massively parallel reporter assays, bioinformatics, machine learning, single-molecule imaging and cryo-electron microscopy to define the extended translation initiation sequence (eTIS), an [~]80-nucleotide sequence surrounding the start codon that governs initiation efficiency. A deep-learning model trained on eTIS features accurately predicts translation initiation across transcripts. Unexpectedly, we find that the Kozak sequence is not optimal for initiation as is widely presumed, and we identify the origin of this discrepancy. eTIS nucleotides that promote efficient initiation are enriched in the human transcriptome and are evolutionarily conserved, underscoring their functional importance. Biophysical and structural analyses reveal that specific eTIS residues--including the key +6 position and residues in the mRNA entry and exit channel--engage ribosomal proteins, rRNA and initiation factors to promote start codon recognition by stabilizing the ribosome at the start codon and facilitating the structural transitions required for initiation. Finally, optimization of the eTIS markedly enhances translational fidelity and protein output from therapeutic mRNAs, highlighting its practical utility. Together, these findings redefine the sequence logic of translation initiation and establish a framework for precise control of protein expression.

molecular biology↗

bioRxiv2026

In vivo kinetics of protein degradation by individual proteasomes

Protein degradation by the proteasome is central to cellular homeostasis and has been studied extensively using biochemical and structural studies. Despite an in-depth understanding of core proteolytic activity, it has remained largely unresolved how individual proteasomes process substrates inside living cells where many substrate types and co-factors exist. Here, we establish a live-cell single-molecule imaging approach that enables direct visualization and quantification of protein degradation by individual proteasomes. Using this approach, we find that substrate identity, folding and protein-protein interaction have a surprisingly modest impact on processing efficiency, whereas the mode of substrate engagement greatly impacts substrate processing; degradation initiated from protein termini typically proceeds rapidly and with high processivity, whereas internal engagement constitutes a distinct processing mode that exhibits poor processivity and a specific requirement for the AAA+ family ATPase p97/VCP. Furthermore, degradation initiated from opposite termini proceeds with asymmetric rates in a sequence-dependent manner, demonstrating that directionality is an important feature of proteasomal processing in vivo. Notably, poly-glutamine substrates associated with neurodegenerative disease are efficiently degraded from one terminus but resist degradation when engaged from the opposite terminus, highlighting the importance of substrate engagement mode. Together, our results show that different modes of substrate engagement lead to different proteasomal processing outcomes in vivo and revise the prevailing view of the proteasome as a uniform degradation machine.

molecular biology↗

bioRxiv2025

MeCP2 binding and genome-lamina reorganization precede long gene activation during mouse corticogenesis

During corticogenesis, neural gene expression is tightly coordinated by chromatin and epigenetic changes, whose misregulation can lead to neurodevelopmental disorders1-4. The role of spatial genome organization--particularly interactions with the nuclear lamina--during these developmental programs remains poorly understood. Here, we combined in utero electroporation with scDam&T-seq to jointly profile genome-lamina contacts and transcriptomes in single cells of the mouse embryonic cortex. Interestingly, we find extensive genome-lamina reorganization during corticogenesis that is strongly biased towards long neuronal genes ([≥]100 kb), which are associated with neurodevelopmental disorders including autism spectrum disorder. Detachment of these genes frequently precedes transcriptional activation, positioning lamina disengagement as an early gene regulatory event. We identify the methyl CpG binding protein 2 (MeCP2)--mutated in Rett syndrome--as a candidate mediator of this process. MeCP2 binds lamina-associated, hydroxymethylated long genes before their repositioning, suggesting that MeCP2 may play a role in genome-lamina reorganization. These findings suggest a link between prevalent genome-lamina reorganization and MeCP2 regulation to ensure proper spatiotemporal activation of long neuronal genes during corticogenesis.

molecular biology↗

bioRxiv2025

Development of a live-cell imaging assay to elucidate spatiotemporal dynamics of extracellular vesicle fusion with target cells

Cells communicate via extracellular vesicles (EVs) containing functional RNAs, proteins and lipids. Knowledge on the fate of internalized EVs, especially their capacity to fuse with target cell membranes and deliver luminal cargo, is limited. Currently available EV-cargo delivery assays are indirect and thus unlikely to uncover molecular players and conditions that specifically control the EV-fusion step. Here, we present a novel live-cell imaging assay for detection of EV-binding, -uptake, and -fusion in time and space. We employed the SunTag system for exceptional signal amplification. EV-donor cells were engineered to tag the luminal EV-membrane with a fluorescent label coupled to SunTag peptides. Recipient cells express fluorescent single-chain anti-SunTag antibody (STAb), which binds EV-enclosed SunTag upon its cytosolic exposure. Using SunTagged EVs carrying fusogen VSV-G, we visualize the EV-fusion process, quantify fusion kinetics and efficiency, and determine subcellular localization of fusion events. We term this methodology EV-FUSIM (Extracellular Vesicle Fusion Spatiotemporal Imaging Method). In the future, this technology can support identification of fusogenic EV-subsets, as well as molecular players and drugs that modulate EV-fusion, without confounding effects of post-fusion processes. This will extend knowledge on EV-biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads.

cell biology↗

bioRxiv2025

Pre-assembly of biomolecular condensate seeds drives RSV replication

During infection many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, inclusion bodies (IBs), where viral transcription and replication occur1-4. Paradoxically, high protein concentrations are typically required for condensate nucleation5, yet attaining sufficient protein levels in infection is thought to require IBs for viral transcription and replication. To uncover how viruses solve this paradox to establish IBs, we visualized early infection of RSV in real-time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that IBs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. IB nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form pre-replication centers (PRCs). PRCs are potent condensate nucleation seeds due to their resistance to disassembly and efficient recruitment of newly-synthesized viral proteins. The high protein affinity of PRCs also results in increased polymerase complex association, allowing efficient viral transcription even in the absence of IBs. Together, these activities create a feed-forward loop that drives rapid IB formation. Intriguingly, PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as the origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry, and explains cell-to-cell heterogeneity in RSV infection.

microbiology↗

bioRxiv2025

WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Mechanistically, WEE1i treatment triggers a translational shut-down, which is lethal in combination with the reduced translation of EIF2AKO cells. A genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinases GCN1/2 rescued WEE1i-mediated cytotoxicity. WEE1i induced GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. Loss of the collided ribosome sensor ZNF598 conversely increased sensitivity to WEE1i. Notably, the ISR was not required for WEE1i to induce DNA damage, premature mitotic entry or sensitization to DNA-damaging chemotherapeutics. ISR activation was independent of CDK1/2 activation. Importantly, WEE1i-mediated ISR activation was independent of WEE1 presence, pointing at off-target effects, which are shared by multiple chemically distinct WEE1i. This response was also observed in peripheral blood mononuclear cells. Importantly, low-dose WEE1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i triggers toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.

cancer biology↗

bioRxiv2025

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

microbiology↗

bioRxiv2025

Mapping the complete influenza A virus infection cycle through single vRNP imaging

Cell-to-cell heterogeneity is a common feature of viral infection that can generate enormous complexity, complicating understanding of infection progression and interpretation of differences between viral variants. To overcome these challenges, we developed a technology to visualize the infection cycle of human influenza A virus (IAV) from start-to-finish in individual living cells with unprecedented spatial and temporal resolution, which identified numerous distinct pathways through which infection can progress. We show that heterogeneous viral gene expression drives infection cycle heterogeneity, and identified genome packaging, vRNP transcriptional activation and host cell division as major determinants of gene expression variability. Our work maps out the complete IAV infection cycle, identifies the origin of infection heterogeneity and provides a broadly-applicable technology for studying IAV and other viruses.

cell biology↗

bioRxiv2024

Ribosome cooperativity promotes fast and efficient translation

The genetic information stored in mRNA is decoded by ribosomes during mRNA translation. mRNAs are typically translated by multiple ribosomes simultaneously, but it is unclear if different ribosomes translating the same mRNA functionally interact with each other. Here, we combine single-ribosome imaging and simulations to compare translation dynamics of individual ribosomes in either monosomes or polysomes to determine how ribosomes interact during translation. We find that ribosomes frequently undergo transient collisions, but that transient collisions, unlike persistent collisions, escape detection by cellular quality control pathways. Instead, our results indicate that transient ribosome collisions promote productive translation by suppressing ribosome pausing when ribosomes encounter problems, a process we term ribosome cooperativity. Ribosome cooperativity also suppresses ribosome recycling on problematic sequences, likely by reducing pause duration, thus stimulating processive translation. Together, our results show that ribosomes cooperate during translation to ensure fast and efficient translation.

molecular biology↗

bioRxiv2024

Dissecting translation elongation dynamics through ultra-long tracking of single ribosomes

mRNA translation by ribosomes is a highly dynamic and heterogeneous process. However, current approaches cannot readily resolve individual ribosomes during translation, limiting our understanding of translation dynamics. Here, we develop an imaging approach based on Stopless-ORF circular RNAs (socRNAs) to monitor individual translating ribosomes for hours. Using the socRNA imaging technology we obtained accurate measurements of ribosome pausing on various problematic RNA sequences or induced by ribosome-targeting drugs. In addition, we identified a novel translation factor involved in translation elongation, and revealed that translocation rates of ribosomes vary, indicative of intracellular ribosomal heterogeneity. Finally, socRNAs allow very sensitive measurements of translation elongation fidelity, revealing widespread frameshifting during translation. In summary, our single-ribosome imaging approach provides a detailed view of ribosome translocation kinetics and a powerful new tool to study the translation elongation phase.

molecular biology↗

bioRxiv2024

N-glycosylated molecules act as a co-precipitant in RNA purification

A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA) 1. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, we likewise identified an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of co-purifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA, and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from co-purified glycoconjugates.

molecular biology↗

bioRxiv2023

Zero-mode waveguide nanowells for single-molecule detection in living cells

Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs - subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a five-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging, as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce cytoplasmic background fluorescence, enabling detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/546504v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@156ab8borg.highwire.dtl.DTLVardef@1185744org.highwire.dtl.DTLVardef@109370corg.highwire.dtl.DTLVardef@1fd2bba_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Heterogeneity in viral replication dynamics shapes the antiviral response

In response to virus infection, host cells can activate antiviral signaling to restrict virus replication and communicate viral infection to neighboring cells. For poorly understood reasons, antiviral response activation is highly heterogeneous among infected cells; both quantitatively (level of pathway activation) and qualitatively (transcribed antiviral gene set). Here, we used live-cell single-molecule imaging to simultaneously visualize viral infection and antiviral signaling, providing quantitative insights into antiviral response activation in single cells; first, the probability of activating an antiviral response varies throughout infection, with most efficient activation occurring several hours after the first viral replication. Second, cell-to-cell heterogeneity in viral replication rates early in infection determine the efficiency of antiviral response activation. Finally, variation in signaling strength of the viral sensing pathway result in qualitatively distinct antiviral responses. Together, this works identifies key parameters that shape the antiviral response and provides quantitative insights into the origin of heterogeneity in the antiviral response.

immunology↗

bioRxiv2021

Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit

Accurate control of the cell cycle is critical for development and tissue homeostasis and requires precisely-timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally-controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.

cell biology↗

bioRxiv2020

High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

CRISPR (clustered regularly interspaced short palindromic repeats) -based gene inactivation provides a powerful means of linking genes to particular cellular phenotypes. CRISPR-based screening has mostly relied upon using large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to computationally select cells displaying a particular CRISPR-induced phenotype, mark them by photo-conversion of an expressed photo-activatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software Manager to automate the phenotypic identification and photo-conversion of cells, allowing ~1.5 million individual cells to be screened in 8 hr. We used this approach to screen 6092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a highly scalable approach to facilitate imaging-based pooled CRISPR screens.

cell biology↗

bioRxiv2020

Pharmaceutical-grade Rigosertib is a Microtubule-destabilizing Agent

We recently used CRISPRi/a-based chemical-genetic screens and targeted cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. In a recent manuscript, Reddy and co-workers suggest that this microtubule-destabilizing activity of rigosertib is mediated not by rigosertib itself but by a contaminating degradation product of rigosertib, ON01500, present in formulations obtained from commercial vendors (Baker et al., 2019). Here, we demonstrate that treatment of cells with pharmaceutical-grade rigosertib (>99.9% purity) results in qualitatively indistinguishable phenotypes as treatment with commercially obtained rigosertib across multiple assays. The two compounds have indistinguishable chemical-genetic interactions with genes involved in modulating the microtubule network (KIF2C and TACC3), both destabilize microtubules in cells and in vitro, and both show substantially reduced toxicity in cell lines expressing a rationally-designed mutant of tubulin (L240F TUBB mutant), in which the rigosertib binding site in tubulin is mutated. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents, which is disputed by Reddy and co-workers, was recently confirmed by an independent research group (Patterson et al., 2019). We conclude that rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

cancer biology↗

bioRxiv2019

mRNA structural dynamics shape Argonaute-target interactions

Small RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

molecular biology↗