Biology subjects

Sears, R. M.

Publications and source records attributed to Sears, R. M..

7 recordsLinked to original sources

bioRxiv2026

Live-cell co-translational folding tracking reveals bidirectional coupling between translation and folding

Translation elongation and protein folding have long been proposed to coordinate during co-translational folding, yet the lack of technologies capable of simultaneously tracking both processes in live cells has hindered mechanistic understanding of this relationship. Here, we developed co-translational folding tracking (coTFT), a live-cell imaging platform that directly and simultaneously tracks translation and folding from individual mRNAs. Using reporters with distinct folding kinetics, we found that differences in folding kinetics were accompanied by corresponding changes in translation elongation rates. Conversely, altering translation elongation markedly affected protein folding outcomes. Combining coTFT with mathematical modeling enabled estimation of reporter folding times on translating ribosomes in live cells, confirming their distinct folding kinetics. Together, our results reveal that translation elongation and folding are bidirectionally coupled during co-translational folding.

biophysics↗

bioRxiv2026

UTag, a cysteine-free thermostable tagging system for tracking single mRNA translation live

Spatiotemporal regulation of mRNA translation is central to gene expression. Over the past decade, translation has become directly observable in live cells at single-mRNA resolution by tagging nascent chains with tandem arrays of short epitope tags recognized by genetically encodable fluorescent intracellular antibodies (intrabodies). While this technology has revolutionized our understanding of translation regulation, the current toolbox of tagging systems remains limited. Here, we developed a novel and tight-binding intrabody against a short (11-amino acid) HIV protease epitope (named UTag). To ensure robust intracellular folding of the anti-UTag intrabody, we further engineered a cysteine-free variant that folds and functions independently of disulfide-bond formation, as validated by X-ray crystallography. The cysteine-free anti-UTag intrabody retains high binding affinity comparable to the parental intrabody while exhibiting significantly improved thermostability ([~]80 {degrees}C). Importantly, the cysteine-free UTag system enables real-time tracking of single-mRNA translation in live cells with performance on par with the parental UTag system as well as the established SunTag and ALFA-tag. Collectively, these results demonstrate that the newly developed UTag system expands the toolbox for live-cell translation tracking and provides complementary tools for multiplexed applications.

biophysics↗

bioRxiv2025

MicroLive: An Image Processing Toolkit for Quantifying Live-cell Single-Molecule Microscopy

Advances in live-cell fluorescence microscopy have enabled us to visualize single molecules (such as mRNAs and nascent proteins) in real time with high spatiotemporal resolution. However, these experiments generate large datasets that require complex computational processing pipelines to derive meaningful and quantitative information, which is a technical barrier for many researchers. To address this barrier, here, we introduce MicroLive, an open-source Python-based application for quantifying live-cell microscopy images. MicroLive provides an interactive Graphical User Interface (GUI) to perform key tasks, including cell segmentation, photo-bleaching correction, single-particle detection/tracking, spot intensity quantification, inter-channel colocalization, and time-series correlation analysis. As a ground-truth testing dataset, we used synthetic live-cell imaging data generated with the rSNAPed toolkit, demonstrating accurate extraction of biologically relevant parameters. Microscopy images of U-2 OS cells expressing a gene construct smHA-KDM5B-BoxB-MS2 were used to demonstrate the use of this software. Availability and implementationMicroLive is distributed under a GPLv3 license and available on GitHub. https://github.com/ningzhaoAnschutz/microlive. Contactning.zhao@cuanschutz.edu.

biophysics↗

bioRxiv2024

Devaluation of response-produced safety signals reveals circuits for goal-directed versus habitual avoidance in dorsal striatum

Active avoidance responses (ARs) are instrumental behaviors that prevent harm. Adaptive ARs may contribute to active coping, whereas maladaptive avoidance habits are implicated in anxiety and obsessive-compulsive disorders. The AR learning mechanism has remained elusive, as successful avoidance trials produce no obvious reinforcer. We used a novel outcome-devaluation procedure in rats to show that ARs are positively reinforced by response-produced feedback (FB) cues that develop into safety signals during training. Males were sensitive to FB-devaluation after moderate training, but not overtraining, consistent with a transition from goal-directed to habitual avoidance. Using chemogenetics and FB-devaluation, we also show that goal-directed vs. habitual ARs depend on dorsomedial vs. dorsolateral striatum, suggesting a significant overlap between the mechanisms of avoidance and rewarded instrumental behavior. Females were insensitive to FB-devaluation due to a remarkable context-dependence of counterconditioning. However, degrading the AR-FB contingency suggests that both sexes rely on safety signals to perform goal-directed ARs.

neuroscience↗

bioRxiv2023

Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

The nuclear envelope (NE) creates a barrier between the cytosol and nucleus during interphase that is key for cellular compartmentalization and protecting genomic DNA. NE rupture can expose genomic DNA to the cytosol and allow admixture of the nuclear and cytosolic constituents, a proposed mechanism of cancer and NE-associated diseases. Barrier-to-autointegration factor (BAF) is a DNA-binding protein that localizes to NE ruptures where it recruits LEM-domain proteins, A-type lamins, and participates in rupture repair. To further reveal the mechanisms by which BAF responds to and aids in repairing NE ruptures, we investigated known properties of BAF including LEM domain binding, lamin binding, compartmentalization, phosphoregulation of DNA binding, and BAF dimerization. We demonstrate that it is the cytosolic population of BAF that functionally repairs NE ruptures, and phosphoregulation of BAFs DNA-binding that enables its ability to facilitate that repair. Interestingly, BAFs LEM or lamin binding activity appears dispensable for its role in functional repair. Furthermore, we demonstrate that BAF functions to reduce the extent of leakage though NE ruptures, suggesting that BAF effectively forms a diffusion barrier prior to NE repair. Collectively, these results enhances our knowledge of the mechanisms by which BAF responds to NE ruptures and facilitates their repair.

cell biology↗

bioRxiv2022

Mechanisms of A-type lamin targeting to nuclear ruptures are disrupted in LMNA- and BANF1- associated progerias

Mutations in the genes LMNA and BANF1 can lead to accelerated aging syndromes called progeria. The protein products of these genes, A-type lamins and BAF, respectively, are nuclear envelope (NE) proteins that interact and participate in various cellular processes, including nuclear envelope rupture and repair. BAF localizes to sites of nuclear rupture and recruits NE-repair machinery, including the LEM-domain proteins, ESCRT-III complex, A-type lamins, and membranes. Here, we show that it is a mobile, nucleoplasmic population of A-type lamins that is rapidly recruited to ruptures in a BAF-dependent manner via BAFs association with the Ig-like {beta} fold domain of A-type lamins. These initially mobile lamins become progressively stabilized at the site of rupture. Farnesylated prelamin A and lamin B1 fail to localize to nuclear ruptures, unless that farnesylation is inhibited. Progeria-associated LMNA mutations inhibit the recruitment affected A-type lamin to nuclear ruptures, due to either permanent farnesylation or inhibition of BAF binding. A progeria-associated BAF mutant targets to nuclear ruptures but is unable to recruit A-type lamins. Together, these data reveal the mechanisms that determine how lamins respond to nuclear ruptures and how progeric mutations of LMNA and BANF1 impair recruitment of A-type lamins to nuclear ruptures.

cell biology↗

bioRxiv2019

A brainstem-central amygdala circuit underlies defensive responses to learned threats

Norepinephrine (NE) plays a central role in the acquisition of aversive learning via actions in the lateral nucleus of the amygdala (LA)1,2. However, the function of NE in expression of aversively-conditioned responses has not been established. Given the role of the central nucleus of the amygdala (CeA) in the expression of such behaviors3, and the presence of NE projections in this brain nucleus, we assessed the effects of NE activity in the CeA on behavioral expression using receptor-specific pharmacology and cell-and projection-specific chemogenetic manipulations. We found that inhibition and activation of locus coeruleus (LC) neurons decreases and increases freezing to aversively conditioned cues, respectively. We then show that locally inhibiting or activating LC terminals in CeA is sufficient to achieve this bidirectional modulation of defensive reactions. These findings support the hypothesis that LC projections to CeA are required for the expression of defensive responses elicited by conditioned threats.

neuroscience↗