Biology subjects

Brown, A. I.

Publications and source records attributed to Brown, A. I..

10 recordsLinked to original sources

bioRxiv2026

Kinetic Control of Nuclear-encoded Mitochondrial mRNA Localization and Local Translation

Most biological processes are dynamic, yet experimental methods predominantly rely on steady-state measurements to investigate their underlying mechanisms. RNA localization is a fundamental aspect of eukaryotic cell organization and is dynamically regulated by cells. While extensively studied in specialized cell types for a limited number of candidate RNAs, the general principles governing dynamic RNA localization at a transcriptome-wide scale remain largely unexplored. Existing transcriptome-wide studies provide only a static snapshot of RNAs residing in specific cellular locales, in part due to the limited availability of tools for probing cellular spatial organization at biologically relevant scales. Here, we leverage the high spatial (tens of nanometers) and temporal (minute) resolution of APEX-seq to quantitatively measure the dependence of RNA transport on molecular motors at a transcriptome-wide scale in living cells. We conducted these experiments in the context of the localization of mRNAs to the mitochondria, which are essential for cellular function. Our findings indicate that the majority of nuclear-encoded RNAs encoding mitochondrial proteins localize to the outer mitochondrial membrane (OMM) for local translation. We reveal a crucial role of retrograde dynein-based motor transport in RNA localization, demonstrating that its disruption severely impairs RNA targeting to the OMM. Time-resolved profiling of RNAs at the OMM revealed that localization is an active process, and even a brief disruption of transport for a few minutes results in a dramatic loss of localization. Moreover, we demonstrate that the translation efficiency (TE) of localized RNAs is a critical determinant of RNA localization in the context of motor-driven transport, as RNAs that delocalize following motor-transport perturbations exhibit lower TE. Using our temporal perturbation data, we also developed a spatiotemporal model that utilizes translation kinetics to capture key features of RNA localization dynamics at the OMM. Together, experiments and modeling suggest that the process of local translation at the OMM is kinetically controlled by the cell, and reveal an unappreciated mechanism by which active transport of RNAs enables cells to modulate their translation within minutes through RNA localization control. Our study demonstrates how simultaneously capturing the kinetics of hundreds of transcripts with minute resolution can uncover general principles of cellular and organelle organization. Together, these experiments and modeling reveal how active transport and translation jointly maintain the OMM-localized transcriptome. More broadly, they identify RNA localization to cellular membranes as a rapidly tunable mechanism for controlling local translation, even in non-polarized cells.

cell biology↗

bioRxiv2026

Spatially localized ligand binding to receptors affects magnitude and timing of signaling response

Cell surface receptors are activated by ligand binding and transmit signals into the cell. Epidermal growth factor (EGF) receptor (EGFR) signaling regulates cell growth, differentiation, and survival, and its dysregulation is linked to cancer. Recent experiments show that ligand binding to EGFR is enhanced for receptors in tetraspanin nanodomains on the cell surface. We use kinetic modeling of receptor confinement, ligand binding, and internalization to compare confinement and signaling behavior for EGFR with spatially localized ligand binding to a hypothetical receptor that has uniform ligand binding anywhere on the cell surface. We find that introducing a membrane domain that confines and enhances ligand binding to receptors leads to more consistent confinement across ligand levels, raises necessary ligand levels for steady-state signaling, and flattens and extends the signaling response to sudden ligand concentration increases. This confining domain that enhances ligand binding provides the cell with a distinct regulatory mechanism to tune its signaling response. We also find that the concentration of receptors in signaling states and the fraction of receptors in signaling states respond to ligand at different ligand concentrations, with substantial increase of the concentration of receptors in signaling states occurring at a much lower ligand concentration than a substantial increase of the fraction of surface receptors in signaling states. This quantitative modeling of spatially restricted receptor activation applies to other receptors with similar characteristics and builds towards physical principles of receptor signaling.

biophysics↗

bioRxiv2025

Mitochondrial pull-out controls mitochondrial structure in response to mitochondrial activity

Mitochondrial networks undergo continuous remodeling through fusion and fission, processes that are essential for maintaining energy production and cellular homeostasis. Rapid elongation of the tip of mitochondrial tubules, a process termed dynamic tubulation, also participates in mitochondrial network formation. However, these mechanisms alone cannot fully account for the formation of highly interconnected mitochondrial networks that are required for rapid distribution of mitochondria material. Here, we identify a distinct type of dynamic tubulation, mitochondrial pull-outs characterized by the lateral extrusion from pre-existing mitochondrial tubules, as metabolically regulated determinants of mitochondrial network formation. Pull-outs are distinct from the tip elongation form of dynamic tubulation as they are modulated by the mitochondrial dynamins MFN1 and DRP1 and are stimulated by conditions favoring oxidative phosphorylation. Pull-outs also depend on mitochondrial actin polymerization and are required to increase mitochondrial connectivity and respiratory activity. Together, these findings establish mitochondrial pull-out as a metabolically sensitive mechanism that promotes mitochondrial network connectivity and links organelle architecture to cellular energy demands.

cell biology↗

bioRxiv2024

Mitochondrial network branching enables rapid protein spread with slower mitochondrial dynamics

Mitochondrial network structure is controlled by the dynamical processes of fusion and fission, which merge and split mitochondrial tubes into structures including branches and loops. To investigate the impact of mitochondrial network dynamics and structure on the spread of proteins and other molecules through mitochondrial networks, we used stochastic simulations of two distinct quantitative models that each included mitochondrial fusion and fission, and particle diffusion via the network. Better-connected mitochondrial networks and networks with faster dynamics exhibit more rapid particle spread on the network, with little further improvement once a network has become well-connected. As fragmented networks gradually become better-connected, particle spread either steadily improves until the networks become well-connected for slow-diffusing particles or plateaus for fast-diffusing particles. We compared model mitochondrial networks with both end-to-end and end-to-side fusion, which form branches, to non-branching model networks that lack end-to-side fusion. To achieve the optimum (most rapid) spread that occurs on well-connected branching networks, non-branching networks require much faster fusion and fission dynamics. Thus the process of end-to-side fusion, which creates branches in mitochondrial networks, enables rapid spread of particles on the network with relatively slow fusion and fission dynamics. This modeling of protein spread on mitochondrial networks builds towards mechanistic understanding of how mitochondrial structure and dynamics regulate mitochondrial function.

biophysics↗

bioRxiv2023

Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence

A decline in mitochondrial function is a hallmark of aging and neurodegenerative diseases. It has been proposed that changes in mitochondrial morphology, including fragmentation of the tubular mitochondrial network, can lead to mitochondrial dysfunction, yet the mechanism of this loss of function is unclear. Most proteins contained within mitochondria are nuclear-encoded and must be properly targeted to the mitochondria. Here, we report that sustained mRNA localization and co-translational protein delivery leads to a heterogeneous protein distribution across fragmented mitochondria. We find that age-induced mitochondrial fragmentation drives a substantial increase in protein expression noise across fragments. Using a translational kinetic and molecular diffusion model, we find that protein expression noise is explained by the nature of stochastic compartmentalization and that co-translational protein delivery is the main contributor to increased heterogeneity. We observed that cells primarily reduce the variability in protein distribution by utilizing mitochondrial fission-fusion processes rather than relying on the mitophagy pathway. Furthermore, we are able to reduce the heterogeneity of the protein distribution by inhibiting co-translational protein targeting. This research lays the framework for a better understanding of the detrimental impact of mitochondrial fragmentation on the physiology of cells in aging and disease.

cell biology↗

bioRxiv2023

Quantitative modeling of EGF receptor ligand discrimination via internalization proofreading

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology that is stimulated by multiple distinct ligands. Although ligands bind to EGFR while the receptor is exposed on the plasma membrane, EGFR incorporation into endosomes following receptor internalization is an important aspect of EGFR signaling, with EGFR internalization behavior dependent upon the type of ligand bound. We develop quantitative modeling, both kinetic and with spatial details, for EGFR recruitment to and internalization from clathrin domains and competition with ligand unbinding from EGFR. We find that a combination of spatial and kinetic proofreading leads to enhanced EGFR internalization ratios in comparison to unbinding differences between ligand types. Various stages of the EGFR internalization process, including recruitment to and internalization from clathrin domains, modulate the internalization differences between receptors bound to different ligands. Our results indicate that following ligand binding, EGFR may encounter multiple clathrin domains before successful recruitment and internalization. The quantitative modeling we have developed describes competition between EGFR internalization and ligand unbinding and the resulting proofreading.

biophysics↗

bioRxiv2022

Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR was more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurred preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impaired EGFR ligand binding and altered the conformation of the receptors ectodomain. We thus reveal a novel mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.

cell biology↗

bioRxiv2022

Mitochondrial mRNA localization is governed by translation kinetics and spatial transport

For many nuclear-encoded mitochondrial genes, mRNA localizes to the mitochondrial surface co-translationally, aided by the association of a mitochondrial targeting sequence (MTS) on the nascent peptide with the mitochondrial import complex. For a subset of these co-translationally localized mRNAs, their localization is dependent on the metabolic state of the cell, while others are constitutively localized. To explore the differences between these two mRNA types we developed a stochastic, quantitative model for MTS-mediated mRNA localization to mitochondria in yeast cells. This model includes translation, applying gene-specific kinetics derived from experimental data; and diffusion in the cytosol. Even though both mRNA types are co-translationally localized we found that the steady state number, or density, of ribosomes along an mRNA was insufficient to differentiate the two mRNA types. Instead, conditionally-localized mRNAs have faster translation kinetics which modulate localization in combination with changes to diffusive search kinetics across metabolic states. Our model also suggests that the MTS requires a maturation time to become competent to bind mitochondria. Our work indicates that yeast cells can regulate mRNA localization to mitochondria by controlling mitochondrial volume fraction (influencing diffusive search times) and gene translation kinetics (adjusting mRNA binding competence) without the need for mRNA-specifc binding proteins. These results shed light on both global and gene-specific mechanisms that enable cells to alter mRNA localization in response to changing metabolic conditions.

biophysics↗

bioRxiv2021

The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

genetics↗

bioRxiv2019

High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.\n\nHighlightsO_LIA single-molecule unzipping assay mimics DNA unwinding by Pol II and maps the topography of human canonical, H2A.Z and uH2B nucleosome barriers at high resolution\nC_LIO_LIReal-time dynamics and full molecular trajectories of Pol II crossing the nucleosomal barrier reveal the transcriptional landscape of the barrier at high accuracy\nC_LIO_LIH2A.Z enhances the width and uH2B the height of the barrier\nC_LIO_LIA unified mechanical model links position-dependent dwell times of Pol II on the nucleosome with energetics of the barrier\nC_LI

biophysics↗