Biology subjects

Belliveau, N. M.

Publications and source records attributed to Belliveau, N. M..

13 recordsLinked to original sources

bioRxiv2026

Sterilization and material compatibility of 3D-printed devices for cell culture

Three-dimensional (3D) printing is increasingly used in cell biology to prototype custom cell culture devices, microscopy chambers, and other experimental hardware, but practical guidance on sterilization and material compatibility remains limited. Here, we develop and validate a low-temperature paraformaldehyde vapor sterilization method for fused filament fabrication (FFF) components that avoids heat-induced deformation. Using bacterial challenge assays with Escherichia coli and Geobacillus stearothermophilus, we confirm effective sterilization of 3D printed components. We also assess the effects of common filaments and adhesives on HL-60 human cell growth to identify materials suitable for cell culture workflows. Most untreated plastics were well tolerated over 48 hours, whereas some formaldehyde-treated materials required post-treatment with ammonia vapor to restore compatibility. Together, these results provide a practical framework for sterilizing and deploying 3D-printed materials in cell culture and biological research. Multidisciplinary Abstract3D printers allow laboratories to quickly make custom experimental equipment, but many printed materials cannot be sterilized using standard methods. We developed a low-temperature sterilization procedure using paraformaldehyde vapor and tested whether common printing materials and adhesives are compatible with cultured human cells. We identified several plastics, silicone-based adhesives and acrylic tapes that can be safely used after sterilization, while others require additional ammonia treatment. These results provide guidance for researchers using 3D printing to build custom laboratory and cell culture devices. Methods SummaryA low-temperature paraformaldehyde vapor sterilization method for 3D-printed FFF components was developed and validated using bacterial challenge assays. Compatibility of common printing filaments, adhesives, and tapes with cell culture workflows was assessed by exposing HL-60 cells to untreated, sterilized, and ammonia- neutralized materials and quantifying cell growth by flow cytometry.

cell biology↗

bioRxiv2025

Protein diffusion controls how single cells respond to electric fields

Cells sense and respond to electric fields, using these fields as a guidance cue in wound healing and development. This sensing is done by redistribution of charged membrane proteins on the cells surface ("sensors") via electrophoresis and electroosmotic flow. If membrane proteins have to physically rearrange on the cells surface, how quickly can a cell respond to an applied signal? What limits the cells ability to respond? Are galvanotaxing cells, like chemotaxing cells, limited by stochasticity from the finite number of molecules? Here, we develop a model for the response dynamics of galvanotaxing cells and show that, for weak enough field strengths, two relevant timescales emerge: the time for the cells sensors to rearrange, which depends on their diffusion across the cell, and the time for the cells orientation to respond to an applied field, which may be very different. We fit this model to experimental measurements on the recently-identified sensor galvanin (TMEM154) in neutrophil-like HL-60 cells, finding that given the dynamics of a cell responding to an applied field, we can predict the dynamics of the cell after the field is turned off. This fit constrains the noise of the galvanotaxis process, demonstrating that HL-60 is not limited by the stochasticity of finite sensor number. Our model also allows us to explain the effect of media viscosity on cell dynamics, and predict how cells respond to pulsed DC fields. These results place constraints on the ability to guide cells with pulsed fields, predicting that a field on half of the time is no better than a field that is always on with half the magnitude.

cell biology↗

bioRxiv2024

Galvanin is an electric-field sensor for directed cell migration

Directed cell migration of immune and epithelial cells is critical for their rapid response to tissue injury or infection. Endogenous electric fields generated by disruption of the transepithelial potential across the skin have been postulated to play an important role in guiding cells to wound sites, though how individual cells sense these tissue-scale physical cues remains largely unknown. We have identified Galvanin (TMEM154), a previously uncharacterized single-pass transmembrane protein, as being required for electric-field-guided migration of individual rapidly moving cells. Galvanin functions in both immune and epithelial cell types. Upon exposure of cells to an electric field, Galvanin rapidly relocalizes to the anodal side of a cell, and the net charge on its extracellular domain is necessary and sufficient to drive this spatial relocalization. Furthermore, expression of Galvanin is sufficient to confer electric field-guided migration on otherwise non-responsive epithelial cells. In human neutrophils, we show that Galvanin relocalization is immediately followed by changes in the spatial pattern of cellular protrusion and retraction. The strong directional response of these cells is lost upon truncation of Galvanins intracellular domain, suggesting that Galvanin acts as a direct sensor of the electric field, transducing spatial information about a cells electrical environment to the intracellular migratory apparatus. This sensor relocalization mechanism of cell steering defines a new paradigm for directed cell migration.

cell biology↗

bioRxiv2024

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

molecular biology↗

bioRxiv2022

Mapping variation in the morphological landscape of human cells with optical pooled CRISPRi screening

The contributions of individual genes to cell-scale morphology and cytoskeletal organization are challenging to define due to the wide intercellular variation of these complex phenotypes. We leveraged the controlled nature of image-based pooled screening to assess the impact of CRISPRi knockdown of 366 genes on cell and nuclear morphology in human U2OS osteosarcoma cells. Screen scale-up was facilitated by a new, efficient barcode readout method that successfully genotyped 85% of cells. Phenotype analysis using a deep learning algorithm, the {beta}-variational autoencoder, produced a feature embedding space distinct from one derived from conventional morphological profiling, but detected similar gene hits while requiring minimal design decisions. We found 45 gene hits and visualized their effect by rationally constrained sampling of cells along the direction of phenotypic shift. By relating these phenotypic shifts to each other, we construct a quantitative and interpretable space of morphological variation in human cells.

cell biology↗

bioRxiv2022

Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. Their exquisite sensitivity to chemical gradients and ability to rapidly migrate make them especially suited to protect against infection. However, their terminal differentiation status and short lifetime (on the order of days) have hindered their study. Furthermore, while modern CRISPR-based gene perturbation strategies now allow comprehensive, genome-scale screens in human cells, their application to complex and dynamic processes like cell migration remain limited. Using HL-60 cells, a leukemia cell line that can be differentiated into neutrophil-like cells, we have developed multiple cell migration screen strategies that provide comprehensive, genome-wide discovery of molecular factors that are critical for directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. Combining these assays with additional, pooled, genome-wide CRISPR interference dropout screens of cell proliferation and neutrophil differentiation, we have identified a comprehensive set of genes that are important across the processes of cellular growth, differentiation, and migration. This combined dataset highlights a particular reliance upon mTORC1 signaling that alters neutrophil lifetime, migration phenotype, and sensitivity to chemotactic cues. Across our cell migration screens, we identified several hundred genes important for migration including those with specific roles only in particular migratory contexts. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

cell biology↗

bioRxiv2020

Fundamental limits on the rate of bacterial growth

Recent years have seen an experimental deluge interrogating the relationship between bacterial growth rate, cell size, and protein content, quantifying the abundance of proteins across growth conditions with unprecedented resolution. However, we still lack a rigorous understanding of what sets the scale of these quantities and when protein abundances should (or should not) depend on growth rate. Here, we seek to quantitatively understand this relationship across a collection of Escherichia coli proteomic data covering {approx} 4000 proteins and 36 growth rates. We estimate the basic requirements for steady-state growth by considering key processes in nutrient transport, cell envelope biogenesis, energy generation, and the central dogma. From these estimates, ribosome biogenesis emerges as a primary determinant of growth rate. We expand on this assessment by exploring a model of proteomic regulation as a function of the nutrient supply, revealing a mechanism that ties cell size and growth rate to ribosomal content.

systems biology↗

bioRxiv2020

Deciphering the regulatory genome of Escherichiacoli, one hundred promoters at a time

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for {approx} 65% of the promoters we remain completely ignorant of their regulation. Until we have cracked this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method (Reg-Seq) linking a massively-parallel reporter assay and mass spectrometry to produce a base pair resolution dissection of more than 100 promoters in E. coli in 12 different growth conditions. First, we show that our method recapitulates regulatory information from known sequences. Then, we examine the regulatory architectures for more than 80 promoters in the E. coli genome which previously had no known regulation. In many cases, we also identify which transcription factors mediate their regulation. The method introduced here clears a path for fully characterizing the regulatory genome of model organisms, with the potential of moving on to an array of other microbes of ecological and medical relevance.

biophysics↗

bioRxiv2019

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively.\n\nSummaryV(D)J recombination is a genomic cut-and-paste process for generating diverse antigen-receptor repertoires. The RAG enzyme brings separate gene segments together by binding the neighboring sequences called RSSs, forming a paired complex (PC) before cutting the DNA. There are limited quantitative studies of the sequence-dependent dynamics of the crucial inter-mediate steps of PC formation and cleavage. Here, we quantify individual RAG-DNA dynamics for various RSSs. While RSSs of frequently-used segments do not comparatively enhance PC formation or cleavage, the rare use of some segments can be explained by their neighboring RSSs crippling PC formation and/or cleavage. Furthermore, PC lifetimes reveal DNA-nicking is not required for forming the PC, but PCs with nicks are more stable.

immunology↗

bioRxiv2019

The Energetics of Molecular Adaptation in Transcriptional Regulation

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.\n\nSummaryWe present a biophysical model of allosteric transcriptional regulation that directly links the location of a mutation within a repressor to the biophysical parameters that describe its behavior. We explore the phenotypic space of a repressor with mutations in either the inducer binding or DNA binding domains. Using the LacI repressor in E. coli, we make sharp, falsifiable predictions and use this framework to generate a null hypothesis for how double mutants behave given knowledge of the single mutants. Linking mutations to the parameters which govern the system allows for quantitative predictions of how the free energy of the system changes as a result, permitting coarse graining of high-dimensional data into a single-parameter description of the mutational consequences.

biophysics↗

bioRxiv2018

Mapping DNA sequence to transcription factor binding energy in vivo

Despite the central importance of transcriptional regulation in systems biology, it has proven difficult to determine the regulatory mechanisms of individual genes, let alone entire gene networks. It is particularly difficult to analyze a promoter sequence and identify the locations, regulatory roles, and energetic properties of binding sites for transcription factors and RNA polymerase. In this work, we present a strategy for interpreting transcriptional regulatory sequences using in vivo methods (i.e. the massively parallel reporter assay Sort-Seq) to formulate quantitative models that map a transcription factor binding sites DNA sequence to transcription factor-DNA binding energy. We use these models to predict the binding energies of transcription factor binding sites to within 1 kBT of their measured values. We further explore how such a sequence-energy mapping relates to the mechanisms of trancriptional regulation in various promoter contexts. Specifically, we show that our models can be used to design specific induction responses, analyze the effects of amino acid mutations on DNA sequence preference, and determine how regulatory context affects a transcription factors sequence specificity.

biophysics↗

bioRxiv2017

A systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

Gene regulation is one of the most ubiquitous processes in biology. But while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here we show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect bacterial promoters in a systematic and scalable way. We demonstrate this method on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input-output relationships. This method opens up the possibility of exhaustively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

biophysics↗

bioRxiv2017

An allosteric theory of transcription factor induction

Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC50]. Finally, we derive an expression for the free energy of allosteric repressors which enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.

biophysics↗