Biology subjects

Bar-Ziv, R.

Publications and source records attributed to Bar-Ziv, R..

9 recordsLinked to original sources

bioRxiv2024

Transcription swells chromosomes in vitro

Transplanting chromosomes from living to artificial cells would impact our understanding of chromosome organization and DNA transactions, with implications for autonomous biological systems. Here, we transplanted Escherichia coli chromosomes into artificial cells, enabling real-time labeling, manipulation, and steady-state gene expression down to the single-molecule limit. Chromosomes stripped of native proteins transitioned from a swollen to compacted state induced by transcription inhibition, in contrast to protein-bound chromosomes retaining an organization with blobs. In a cell-free expression reaction, RNA polymerases were uniformly distributed along the entire chromosome and rapidly detached, consistent with a global transcriptional activity. We used tailored surfaces to capture and count 20 nascent proteins per hour from a single gene on the chromosome. We mapped stably bound condensins to the blobs, supporting a model where swelling by transcription is counterbalanced by condensin-mediated compaction. Our data suggest transplanted chromosomes as active gels organized by molecular machines.

biophysics↗

bioRxiv2024

On-chip large-scale-integration and 2D collective modes of genetically programmed artificial cells

The on-chip large-scale-integration of genetically programmed artificial cells capable of exhibiting collective modes is an important goal for fundamental research and technology. Here, we report assembly of a 2D layout of 1024 monolithic DNA compartments as artificial cells on a 5-millimeter square silicon chip. Homeostatic cell-free protein synthesis reactions driven by genetic circuits occur inside the compartments. We created a reaction-diffusion system with a 30x30 square lattice of artificial cells interconnected by thin capillaries for diffusion of products. Driving the system by a genetic oscillator revealed emergent collective modes of synchrony and propagating phase waves in 2D, with dynamics controlled by geometry. This demonstrates a class of nonequilibrium autonomous systems, where chemical energy consumed to make proteins induces 2D collective patterns of gene expression on multicellular scales, with applications in biological computing, sensing, and materials synthesis.

biophysics↗

bioRxiv2023

A genetic circuit on a single DNA molecule as autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcriptiontranslation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we used a genetically encoded fluorescent reporter system with improved spatiotemporal resolution and observed the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally designed a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibited enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that coexpressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

synthetic biology↗

bioRxiv2023

Glial-derived mitochondrial signals impact neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored non-neuronal cells of the nervous system. Here, we found that UPRMT activation in four, astrocyte-like glial cells in the nematode, C. elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Surprisingly, we find that glial cells utilize small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then impact neuron-mediated effects in organismal homeostasis and longevity. One-Sentence SummaryGlial cells sense mitochondrial stress and signal a beneficial stress signal to promote neuronal health and longevity.

genetics↗

bioRxiv2022

Large-scale genetic screens identify BET-1 as a cytoskeleton regulator promoting actin health and lifespan.

The actin cytoskeleton is a three-dimensional scaffold of proteins that is a regulatory, energy-consuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well understood. Here, we performed a multi-pronged, cross-organismal screen combining a whole-genome CRISPR-Cas9 screen in human fibroblasts with in vivo C. elegans synthetic lethality screening. We identified the bromodomain protein, BET-1, as a key regulator of actin health and longevity. Overexpression of bet-1 preserves actin health at late age and promotes lifespan and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET-1. Together, our discovery assigns a key role for BET-1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.

cell biology↗

bioRxiv2022

SKN-1 is a metabolic surveillance factor that monitors amino acid catabolism to control stress resistance

The deleterious potential to generate oxidative stress and damage is a fundamental challenge to metabolism. The oxidative stress response transcription factor, SKN-1/NRF2, can sense and respond to changes in metabolic state, although the mechanism and physiological consequences of this remain unknown. To explore this connection, we performed a genetic screen in C. elegans targeting amino acid catabolism and identified multiple metabolic pathways as regulators of SKN-1 activity. We found that genetic perturbation of the conserved amidohydrolase T12A2.1/amdh-1 activates a unique subset of SKN-1 regulated detoxification genes. Interestingly, this transcriptional program is independent of canonical P38-MAPK signaling components but requires the GATA transcription factor ELT-3, nuclear hormone receptor NHR-49, and mediator complex subunit MDT-15. This activation of SKN-1 is dependent on upstream histidine catabolism genes HALY-1 and Y51H4A.7/UROC-1 and may occur through accumulation of a catabolite, 4-imidazolone-5-propanoate (IP). Triggering SKN-1 activation results in a physiological trade off of increased oxidative stress resistance but decreased survival to heat stress. Together, our data suggest that SKN-1 is a key surveillance factor which senses and responds to metabolic perturbations to influence physiology and stress resistance.

genetics↗

bioRxiv2020

Decision-making in a synthetic cell: the limits of biological computation

We measured the dynamics of decision-making by a minimal bistable gene network integrated in a synthetic cell model, free of external perturbations. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow computation to a fuzzy and rapid regime dominated by singleprotein fluctuations. Fuzzy computation appeared at DNA and protein concentrations 100-fold lower than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. Whereas the high-copy regime was characterized by a sharp transition, hysteresis and robust memory, the low-copy limit showed incipient strong fluctuations, switching between states, and a signature of cellular individuality across the decision-making point. Our work establishes synthetic cells operating rapidly at the single molecule level to integrate gene regulatory networks with metabolic pathways for sustained survival with low energetic cost.One Sentence Summary Decision-making in a synthetic cell can be slow and precise or rapid and probabilistic by reducing the number of computing molecules by five decades down to single-molecule fluctuations.Competing Interest StatementThe Noireaux laboratory receives research funds from Arbor Biosciences, a distributor of the myTXTL cell-free protein synthesis kit.View Full Text

synthetic biology↗

bioRxiv2019

Transcription-factor binding to replicated DNA

Genome replication perturbs the DNA regulatory environment by displacing DNA-bound proteins, replacing nucleosomes, and introducing dosage-imbalance between regions replicating at different S phase stages. Recently, we showed that these effects are integrated to maintain transcription homeostasis: replicated genes increase in dosage, but their expression remains stable due to replication-dependent epigenetic changes that suppress transcription. Here, we examined whether reduced transcription from replicated DNA results from limited accessibility to regulatory factors, by measuring the time-resolved binding of RNA polymerase II (RNAPII) and specific transcription factors (TFs) to DNA during S phase in budding yeast. We show that RNAPII binding-pattern is largely insensitive to DNA dosage, indicating limited binding to replicated DNA. By contrast, binding of three TFs (Reb1, Abf1 and Rap1) to DNA increased with the increasing DNA dosage. We conclude that the replication-specific chromatin environment remains accessible to regulatory factors, but suppresses RNA polymerase recruitment.

systems biology↗

bioRxiv2017

Optimizing gene expression by adapting splicing

Can splicing be used by cells to adapt to new environmental challenges? While various adaptation mechanisms for regulating gene expression have been revealed for transcription and translation, the role of splicing and how it evolves to optimize gene-expression patterns has not been thoroughly investigated. To tackle this question, we employed a lab-evolution experimental approach that challenged yeast cells to increase expression levels of a gene that carries an inefficiently-spliced intron. We followed the evolution of multiple lines and found independent routes by which cells adapted. Surprisingly, we did not observe an intron loss event, a mechanism believed to be common in intron evolution. Instead, we identified mutations in cis that improved the introns splicing efficiency and increased the overall expression level of the entire gene. One of these cis-acting mutations occurred in an adjacent exon and hampered the functionality of the gene that was not under selection - demonstrating that adaptation of splicing efficiency may sometimes come at the expense of protein activity. Additionally, we observed adaptations in trans, which increased the cellular availability of the splicing machinery. These adaptations were achieved either by elevated expression levels of the splicing apparatus or, unexpectedly, by reduced expression levels of other intron-containing genes that are the natural consumers of this process. Ultimately, our work reveals novel molecular means by which the splicing machinery is changed by natural selection to optimize gene-expression patterns of cells.

evolutionary biology↗