Biology subjects

Simpson, M. L.

Publications and source records attributed to Simpson, M. L..

6 recordsLinked to original sources

bioRxiv2021

Adsorption of semiflexible polymers in crowded environments

Macromolecular crowding is a feature of cellular and cell-free systems that, through depletion effects, can impact the interactions of semiflexible biopolymers with surfaces. In this work, we use computer simulations to study crowding-induced adsorption of semiflexible polymers on otherwise repulsive surfaces. Crowding particles are modeled explicitly, and we investigate the interplay between the bending stiffness of the polymer and the volume fraction and size of crowding particles. Adsorption is promoted by stiffer polymers, smaller crowding particles, and larger volume fractions of crowders. We characterize transitions from non-adsorbed to partially and strongly adsorbed states as a function of the bending stiffness. The crowding-induced transitions occur at smaller values of the bending stiffness as the volume fraction of crowders increases. Concomitant effects on the size and shape of the polymer are reflected by crowding- and stiffness-dependent changes to the radius of gyration. We also demonstrate that curvature of the confining surface can induce desorption when the bending stiffness is sufficiently large. The results of our simulations shed light on the interplay of crowding and bending stiffness on the spatial organization of biopolymers in encapsulated cellular and cell-free systems.

biophysics↗

bioRxiv2019

Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

Essential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

biophysics↗

bioRxiv2019

Controlling Cell-Free Gene Expression Behavior by Tuning Membrane Transport Properties

Controlled transport of molecules across boundaries for energy exchange, sensing, and communication is an essential step toward cell-like synthetic systems. This communication between the gene expression compartment and the external environment requires reaction chambers that are permeable to molecular species that influence expression. In lipid vesicle reaction chambers, species that support expression - from small ions to amino acids - may diffuse across membranes and amplify protein production. However, vesicle-to-vesicle variation in membrane permeability may lead to low total expression and high variability in this expression. We demonstrate a simple optical treatment method that greatly reduces the variability in membrane permeability. When transport across the membrane was essential for expression, this optical treatment increased mean expression level by ~6-fold and reduced expression variability by nearly two orders of magnitude. These results demonstrate membrane engineering may enable essential steps toward cell-like synthetic systems. The experimental platform described here provides a means of understanding controlled transport motifs in individual cells and groups of cells working cooperatively through cell-to-cell molecular signaling.

biophysics↗

bioRxiv2018

Synergistic interactions between confinement and macromolecular crowding spatially order transcription and translation in cell-free expression

Synergistic interactions between macromolecular crowding and confinement spatially organize transcription and translation in cells. Yet, reproducing such spatial ordering in cell-free expression platforms has proven to be elusive. Here we report crowding- and confinement-driven spatial self-organization of cell-free expression that mimics expression behavior within and around the nucleoid of prokaryotes. These experiments use Ficoll-70 to approximate cellular macromolecular crowding conditions within cell-size lipid vesicles. Intriguingly, there was an abrupt change in transcriptional dynamics when crowding reached physiologically relevant levels. Imaging experiments revealed that this change in transcriptional dynamics was coincident with localization of plasmid DNA and mRNA at the vesicle wall. Computer simulations demonstrated that crowding leads to an entropically induced attraction between plasmid DNA and the wall, causing localization of DNA near the wall at sufficiently high crowding levels. The experiments demonstrate cell-like spatial organization of translation, where translational activity is controlled by chromosomally-templated positioning of mRNA. This cell-free system provides a flexible experimental platform to probe the underlying mechanisms of self-organization of membrane-less structures in cells and the spatial control of gene expression.

synthetic biology↗

bioRxiv2017

Cytoplasmic import and processing of mRNA amplify transcriptional bursts accounting for the majority of cellular noise

Transcription is an episodic process characterized by probabilistic bursts; but how these bursts are modulated by cellular physiology remains unclear and has implications for how selection may act on these fluctuations. Using simulations and single-molecule RNA counting, we examined how cellular processes influence cell-to-cell variability (noise). The results show that RNA noise is amplified in the cytoplasm compared to the nucleus in ~85% of genes across diverse promoters, genomic loci, and cell types (human and mouse). Surprisingly, measurements show further amplification of RNA noise in the cytoplasm, fitting a model of biphasic mRNA conversion between translation- and degradation-competent states. The multi-state translation-degradation of mRNA also causes substantial noise amplification in protein levels, ultimately accounting for ~74% of intrinsic protein variability in cell populations. Overall, the results demonstrate how transcriptional bursts are intrinsically amplified by mRNA processing and indicate mechanisms through which noise could act as a substrate for evolutionary selection.

cell biology↗

bioRxiv2017

Non-latching positive feedback enables robust bimodality by de-coupling expression noise from the mean

Fundamental to biological decision-making is the ability to generate bimodal expression patterns where two alternate expression states simultaneously exist. Here, we use a combination of single-cell analysis and mathematical modeling to examine the sources of bimodality in the transcriptional program controlling HIVs fate decision between active replication and viral latency. We find that the HIV Tat protein manipulates the intrinsic toggling of HIVs promoter, the LTR, to generate bimodal ON-OFF expression, and that transcriptional positive feedback from Tat shifts and expands the regime of LTR bimodality. This result holds for both minimal synthetic viral circuits and full-length virus. Strikingly, computational analysis indicates that the Tat circuits non-cooperative non-latching feedback architecture is optimized to slow the promoters toggling and generate bimodality by stochastic extinction of Tat. In contrast to the standard Poisson model, theory and experiment show that non-latching positive feedback substantially dampens the inverse noise-mean relationship to maintain stochastic bimodality despite increasing mean-expression levels. Given the rapid evolution of HIV, the presence of a circuit optimized to robustly generate bimodal expression appears consistent with the hypothesis that HIVs decision between active replication and latency provides a viral fitness advantage. More broadly, the results suggest that positive-feedback circuits may have evolved not only for signal amplification but also for robustly generating bimodality by decoupling expression fluctuations (noise) from mean expression levels.

systems biology↗