Biology subjects

Bohrer, C. H.

Publications and source records attributed to Bohrer, C. H..

13 recordsLinked to original sources

bioRxiv2026

Highly Constrained Kinetic Models for Single-Cell Gene Expression Analysis

Advances in single-cell RNA sequencing (scRNA-seq) and high-resolution imaging techniques, such as single-molecule tracking (SMT) of RNA and transcription factors, allow researchers to quantitatively explore dynamics and variation but have never been integrated into a single coherent model. In this study, we propose a kinetic model that intakes multiple data types, including steady-state and time-resolved datasets, to simulate and fit stochastic models of gene transcription to experimental data. We find that 3-state models provide an essential improvement over the widely used 2-state model for most genes and have the property of kinetic proofreading, which we argue is advantageous in the cellular context. We further identify two dimensionless quantities derived from the rate equations which are broadly conserved across genes. Finally, we extend this model to scRNA-seq datasets to infer kinetic rates under defined perturbations and reveal biochemical insight into the mechanism of action of transcription factors.

bioinformatics↗

bioRxiv2025

Chromosomal Topological Domain Formation Modulates Transcription and the Coupling of Neighboring Genes in Escherichia coli

Chromosomal topology and transcription are tightly coupled, yet the quantitative impact of topological constraints on transcription, supercoiling, and the potential coupling between neighboring genes in vivo remains unclear. In this work, we constructed synthetic chromosomal domains in Escherichia coli that contained two genes inside a topology-controllable domain and a third gene outside. Using three-color single-molecule fluorescence in situ hybridization (smFISH), we measured transcription output from the three genes in individual cells under conditions in which gene orientation, domain formation state, and global chromosomal supercoiling density were varied. We found that topological domain formation repressed transcription, diminished gene orientation-dependent differences in transcription, and modulated the supercoiling sensitivity of genes located both within and near the domain. Relaxing global negative supercoiling through gyrase inhibition broadly repressed transcription; increasing global negative supercoiling level through topoisomerase I inhibition repressed highly expressed genes, while activating lowly expressed ones. Besides single-gene effects, we also observed an intrinsic coupling between neighboring genes with a non-monotonic dependence on the underlying supercoiling state, which shifted with domain topology and gene syntax. Our results establish chromosome topology as a major regulator of both transcription levels and the coupling between adjacent genes.

biophysics↗

bioRxiv2025

Cell size-dependent mRNA transcription drives proteome remodeling

Increasing cell size drives proteomic changes that impact cell physiology. However, the molecular basis of size-dependent proteome remodeling has remained unclear. Here, we develop an inducible Cyclin D1 expression system in human cells to generate populations of proliferating cells spanning over a two-fold size range. We use this genetic system to make comprehensive genome-wide measurements of mRNA and protein concentrations and stability. We find that protein and mRNA turnover rates are weakly related to cell size, but that mRNA concentrations are strongly size-dependent. This establishes that transcriptional regulation is the basis of proteome remodeling. Live-cell imaging of endogenous mRNAs using MS2 fluorescent protein binding motifs is used to measure how transcriptional dynamics change with cell size. Larger cells prolong transcriptional bursts and shorten inactive periods between bursts but maintain similar burst amplitudes to achieve transcriptional scaling. Taken together, our results show how transcription is modulated by cell size to remodel the proteome and alter cell physiology.

cell biology↗

bioRxiv2025

A methodology to reduce the localization error in multi-loci microscopy provides new insights into enhancer biology

Numerous functions hinge on the spatial dynamics of different genomic loci. Hence, microscopy techniques, such as chromatin tracing, have been developed to localize multiple loci in living and fixed cells. Depending on the throughput and specifics of the experiment, localization errors can still obscure the true spatial locations. We have developed a post-processing methodology to address this challenge without the need for additional experimentation: Loci Enabled Advanced Resolution (LEAR). By leveraging the fact that localization errors increase the variability of the displacements between loci, and given an approximation of the localization error, we can determine the ground truth spatial variation for each pair of loci to guide an iterative error correction process. After validating our approach with simulation and experiment, we then applied our approach to existing chromatin tracing data that probed the relation between chromatin organization and Sox2 regulation, where previous work found no correlation between enhancer-promoter proximity and transcription bursts in individual cells. We discovered a correlation previously obscured by localization error, clearly demonstrating the need for the methodology. We then investigated the influence of loop-extrusion on higher order multi-way contact frequencies, which dramatically increased with the application of the LEAR method, finding that certain multi-way contacts were only present with loop-extrusion.

biophysics↗

bioRxiv2023

HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.

bioinformatics↗

bioRxiv2022

Synthetic analysis of chromatin tracing and live-cell imaging indicates pervasive spatial coupling between genes

The role of the spatial organization of chromosomes in directing transcription remains an outstanding question in gene regulation. Here, we analyze two recent single-cell imaging methodologies applied across hundreds of genes to systematically analyze the contribution of chromosome conformation to transcriptional regulation. Those methodologies are: 1) single-cell chromatin tracing with super-resolution imaging in fixed cells; 2) high throughput labeling and imaging of nascent RNA in living cells. Specifically, we determine the contribution of physical distance to the coordination of transcriptional bursts. We find that individual genes adopt a constrained conformation and reposition toward the centroid of the surrounding chromatin upon activation. Leveraging the variability in distance inherent in single-cell imaging, we show that physical distance - but not genomic distance - between genes on individual chromosomes is the major factor driving co-bursting. By combining this analysis with live-cell imaging, we arrive at a corrected transcriptional correlation of{phi} {approx} 0.3 for genes separated by < 400 nm. We propose that this surprisingly large correlation represents a physical property of human chromosomes and establishes a benchmark for future experimental studies.

biophysics↗

bioRxiv2022

Light-dependent modulation of protein localization and function in living bacteria cells

Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a proteins subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.

synthetic biology↗

bioRxiv2021

A spatial stochastic model reveals the role of supercoiling in transcription regulation

In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we develop a spatially resolved supercoiling model of transcription, describing RNAP-supercoiling interactions, topoisomerase activities, stochastic topological domain formation, and supercoiling diffusion in all transcription stages. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes. It reveals that supercoiling transmits RNAP-accessible information through DNA and enables different RNAP molecules to communicate within and between genes. It thus predicts that a topological domain could serve as a transcription regulator, generating substantial transcription bursting and coordinating communications between adjacent genes in the domain. The model provides a quantitative platform for further theoretical and experimental investigations of how genome organization impacts transcription. Author SummaryDNA mechanics and transcription dynamics are intimately coupled. During transcription, the translocation of RNA polymerase overwinds the DNA ahead and underwinds the DNA behind, rendering the DNA supercoiled. The supercoiled DNA could, in return, influences the behavior of the RNA polymerase, and consequently the amount of mRNA product it makes. Furthermore, supercoils could propagate on the DNA over thousands of base pairs, impacting RNA polymerase molecules at faraway sites. These complicated interplays between supercoiling and RNA polymerase makes supercoiling an important transcription regulator. To quantitatively investigate the role of supercoiling in transcription, we build a spatially resolved model that links transcription with the generation, propagation, and dissipation of supercoiling. Our model reveals that supercoiling mediates transcription at multiple length scales. At a single-gene scale, we show that supercoiling gives rise to the collective motion of co-transcribing RNA polymerase molecules, supporting recent experimental observations. Additionally, large variations in mRNA production of a gene can arise from the constraints of supercoiling diffusion in a topological domain. At a multi-gene scale, we show that supercoiling dynamics allow two adjacent genes influence each others transcription kinetics, thus serving as a transcription regulator.

biophysics↗

bioRxiv2020

Tau forms oligomeric complexes on microtubules that are distinct from pathological oligomers in disease

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single molecule localization microscopy (SMLM), we show that, in vivo, tau forms small oligomers on microtubules under physiological conditions. These physiological oligomers are distinct from those found in cells exhibiting tau aggregation and could be pre-cursors of aggregated tau in pathology. Further, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates taus nanoscale composition under physiological and pathological conditions in vivo.

cell biology↗

bioRxiv2020

Spatially compartmentalized phase regulation of a Ca2+-cAMP-PKA oscillatory circuit

Signaling networks are spatiotemporally organized in order to sense diverse inputs, process information, and carry out specific cellular tasks. In pancreatic {beta} cells, Ca2+, cyclic adenosine monophosphate (cAMP), and Protein Kinase A (PKA) exist in an oscillatory circuit characterized by a high degree of feedback, which allows for specific signaling controls based on the oscillation frequencies. Here, we describe a novel mode of regulation within this circuit involving a spatial dependence of the relative phase between cAMP, PKA, and Ca2+. We show that nanodomain clustering of Ca2+-sensitive adenylyl cyclases drives oscillations of local cAMP levels to be precisely in-phase with Ca2+ oscillations, whereas Ca2+-sensitive phosphodiesterases maintain out-of-phase oscillations outside of the nanodomain, representing a striking example and novel mechanism of cAMP compartmentation. Disruption of this precise in-phase relationship perturbs Ca2+ oscillations, suggesting that the relative phase within an oscillatory circuit can encode specific functional information. This example of a signaling nanodomain utilized for localized tuning of an oscillatory circuit has broad implications for the spatiotemporal regulation of signaling networks.

cell biology↗

bioRxiv2019

A Pairwise Distance Distribution Correction (DDC) algorithm for blinking-free super-resolution microscopy

In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations void of blinking-artifacts, generating an accurate reconstruction of the underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking-artifact correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications. The simplicity and robustness of DDC will allow it to become the field standard in SMLM imaging, enabling the most accurate reconstruction and quantification of SMLM images to date.

biophysics↗

bioRxiv2018

RNA polymerase organizes into distinct spatial clusters independent of ribosomal RNA transcription in E. coli

Recent studies have shown that RNA polymerase (RNAP) is spatially organized into distinct clusters in E. coli and B. subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer new insights into pertinent functions and regulations. However, the function of RNAP clusters and whether its formation is driven by active ribosomal RNA (rRNA) transcription remain elusive. In this work, we investigated the spatial organization of RNAP in E. coli cells using quantitative superresolution imaging. We observed that RNAP formed large, distinct clusters under a rich medium growth condition and preferentially located in the center of the nucleoid. Two-color superresolution colocalization imaging showed that under the rich medium growth condition, nearly all RNAP clusters were active in synthesizing rRNA, suggesting that rRNA synthesis may be spatially separated from mRNA synthesis that most likely occurs at the nucleoid periphery. Surprisingly, a large fraction of RNAP clusters persisted under conditions in which rRNA synthesis was reduced or abolished, or when only one out of the seven rRNA operons (rrn) remained. Furthermore, when gyrase activity was inhibited, we observed a similar rRNA synthesis level, but multiple dispersed, smaller rRNA and RNAP clusters occupying not only the center but also the periphery of the nucleoid, comparable to an expanded nucleoid. These results suggested that RNAP was organized into active transcription centers for rRNA synthesis under the rich medium growth condition; their presence and spatial organization, however, were independent of rRNA synthesis activity under the conditions used but were instead influenced by the structure and characteristics of the underlying nucleoid. Our work opens the door for further investigations of the function and molecular nature of RNAP clusters and points to a potentially new mechanism of transcription regulation by the spatial organization of individual molecular components.

microbiology↗

bioRxiv2018

Improved single-molecule localization precision in astigmatism-based 3D superresolution imaging using weighted likelihood estimation

Astigmatism-based superresolution microscopy is widely used to determine the position of individual fluorescent emitters in three-dimensions (3D) with subdiffraction-limited resolutions. This point spread function (PSF) engineering technique utilizes a cylindrical lens to modify the shape of the PSF and break its symmetry above and below the focal plane. The resulting asymmetric PSFs at different z-positions for single emitters are fit with an elliptical 2D-Gaussian function to extract the widths along two principle x- and y-axes, which are then compared with a pre-measured calibration function to determine its z-position. While conceptually simple and easy to implement, in practice, distorted PSFs due to an imperfect optical system often compromise the localization precision; and it is laborious to optimize a multi-purpose optical system. Here we present a methodology that is independent of obtaining a perfect PSF and enhances the localization precision along the z-axis. By utilizing multiple calibration images of fluorescent beads at varying z-planes and characterizing experimentally measured background distributions, we numerically approximated the probability of observing a certain signal in a given pixel from a single emitter at a particular z-plane. We then used a weighted maximum likelihood estimator (WLE) to determine the 3D-position of the emitter. We demonstrate that this approach enhances z-axis localization precision in all conditions we tested, in particular when the PSFs deviate from a standard 2D Gaussian model.

biophysics↗