Biology subjects

Aguilera, L. U.

Publications and source records attributed to Aguilera, L. U..

5 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

Integrating Single-Cell Experiments and Stochastic Models to Understand and Predict Glucocorticoid Receptor Transport and DUSP1 mRNA Expression Dynamics

Glucocorticoids activate the glucocorticoid receptor (GR) to suppress inflammation, yet it remains unclear how GR transport dynamics and downstream gene regulation are coordinated within single cells. We combine immunocytochemistry (ICC) and single-molecule fluorescence in situ hybridization (smFISH) to quantify endogenous GR transport and DUSP1 transcription dynamics across thousands of individual cells following dexamethasone (Dex) stimulation. Performing multiple rounds of statistical inference based on Chemical Master Equations (CME), we determine the most likely mechanisms and reaction rates for Dex-driven GR nuclear import; compartment-specific GR degradation; GR-dependent control of the DUSP1 promoter; and DUSP1 transcription, elongation, transport, and degradation. Our inferred model suggests that nuclear GR degradation is the dominant mechanism of receptor clearance, that GR primarily regulates promoter activation, and that time-dependent AU-rich element (ARE)-mediated mRNA degradation contributes heavily to DUSP1 clearance. With these mechanisms, the fully-parameterized model quantitatively predicts joint distributions of GR translocation and decay dynamics, DUSP1 transcription site activity, and nuclear and cytoplasmic DUSP1 mRNA heterogeneity among clonal cells as functions of time and across seven orders of magnitude for Dex induction concentrations. Our results establish an integrated quantitative framework to link receptor dynamics to gene expression heterogeneity and predict single-cell hormone-responsive transcription programs.

cell biology↗

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↗

bioRxiv2018

Live-cell single RNA imaging reveals bursts of translational frameshifting

Ribosomal frameshifting during the translation of RNA is implicated in both human disease and viral infection. While previous work has uncovered many mechanistic details about single RNA frameshifting kinetics in vitro, very 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 in living cells. Frameshifting RNA are preferentially in multi-RNA \"translation factories,\" are translated at about the same rate as non-frameshifting RNA (~2 aa/sec), 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 novel insight into retroviral frameshifting and could lead to new strategies to perturb the process in living cells.

biochemistry↗