Single-cell protein analysis reveals metastable states during the transition to a sensory organ fate

Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make to undergo sensory organ differentiation has been sucessfully described as such. We have extended these studies by focusing on the Senseless protein, which orchestrates the sensory fate transition. Wing cells contain intermediate Senseless numbers prior to their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are not consistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the four states are stable over time, the number of molecules in each state vary with time. Remarkably, the fold-change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.

Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling

Urinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

Variance Reducing and Noise Correction in Protein Quantification by Measuring Fluctuations in Fluorescence due to Photobleaching

Quantifying protein number using the ratio between the variance and the mean of the protein distribution is a straightforward calibration method in the experimental conditions for microscopy imaging. Recently the model has been expanded to decaying processes with binomial distribution. In this paper, we examine the model proposed, and show how the algorithm can be adapted to the case of variance in the initial number of proteins between cells. We propose improving the algorithm so that the information processing of each frame is done independently from other frames. By doing so, the variance in the process of determining the protein number can be reduced. In addition, we examine the handling of unwanted noises in the measurement, offer a solution for shot noise and background noise, and examine the expected error caused in calculating the decay constant. We also analyze the expected difficulties in conducting a practical experiment, which includes non-exponential decay, and variance in the decay constants of the cells. These methods can be applied to any superposition of n_0 discrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions, and for the evaluation of the error.

TiFoSi: an efficient tool for mechanobiology simulations of epithelia

Motivation Emerging phenomena in developmental biology and tissue engineering are the result of feedbacks between gene expression and cell biomechanics. In that context, in silico experiments are a powerful tool to understand fundamental mechanisms and to formulate and test hypotheses. Results Here, we present TiFoSi, a computational tool to simulate the cellular dynamics of planar epithelia. TiFoSi allows to model feedbacks between cellular mechanics and gene expression (either in a deterministic or a stochastic way), the interaction between different cell populations, the custom design of the cell cycle and cleavage properties, the protein number partitioning upon cell division, and the modeling of cell communication (juxtacrine and paracrine signaling). Availability and implementation http://tifosi.thesimbiosys.com. Supplementary information Supplementary data are available at Bioinformatics online.

TiFoSi: an Efficient Tool for Mechanobiology Simulations of Epithelia

AboutThis document is an extended version of the main text where some details and results are fleshed out. Further details can be also found in the manual of the code and at TiFoSi’s website: http://tifosi.thesimbiosys.com.MotivationEmerging phenomena in developmental biology and tissue engineering are the result of feedbacks between gene expression and cell biomechanics. In that context, in silico experiments are a powerful tool to understand fundamental mechanisms and to formulate and test hypotheses.ResultsHere we present TiFoSi, a computational tool to simulate the cellular dynamics of planar epithelia. TiFoSi allows to model feedbacks between cellular mechanics and gene expression (either in a deterministic or a stochastic way), the interaction between different cell populations, the custom design of the cell cycle and cleavage properties, the protein number partitioning upon cell division, and the modeling of cell communication (juxtacrine and paracrine signalling). TiFoSi fills a niche in the field of software solutions to simulate the mechanobiology of epithelia because of its functionalities, computational efficiency, and its user-friendly approach to design in silico experiments using XML configuration files.Availabilityhttp://[email protected]

Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

Background The order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag -1, encompassing ~ 50 genes. A discrete locus, flag -2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized. Results and Discussion Comparative genomic analyses showed that orthologous flag -2 loci are present in 592/4,028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of only the outermost flag- 2 genes in many taxa suggests that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag -2 loci range in size from ~3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag -2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria. Conclusions The flag -2 flagellar system is a fairly common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.

Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

Background The order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag -1, encompassing ~ 50 genes. A discrete locus, flag -2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized.Results and Discussion Comparative genomic analyses showed that orthologous flag -2 loci are present in 592/4,028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of only the outermost flag- 2 genes in many taxa suggests that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag -2 loci range in size from ~3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag -2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria.Conclusions The flag -2 flagellar system is a fairly common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.

Stochasticity in bacterial division control: Preliminary consequences for protein concentration

ABSTRACTThe stochastic nature of protein concentration inside cells can have important consequences in their physiology and population fitness. Classical models of gene expression consider these processes as first-order reactions with little dependence with the cell size. However, the concentrations of the relevant molecules depend directly on the cellular volume. Here we model the cell size dynamics as exponential growth followed by division with occurrence rate proportional to the size. This framework, together with known models of chromosome replication and both protein and mRNA synthesis, lets us predict relationships between cell size and both protein number and concentration. As a main result, we find that protein production strategies (constant rate or rate proportional to either chromosome number, cell size or chromosome number times cell size) can be experimentally distinguished from the correlation between protein concentration and cell size.

Comparative genomic analysis of the secondary flagellar (flag-2) system in the order Enterobacterales

Background The order Enterobacterales encompasses a broad range of metabolically and ecologically versatile bacterial taxa, most of which are motile by means of peritrichous flagella. Flagellar biosynthesis has been linked to a primary flagella locus, flag -1, encompassing ~ 50 genes. A discrete locus, flag -2, encoding a distinct flagellar system, has been observed in a limited number of enterobacterial taxa, but its function remains largely uncharacterized.Results and Discussion Comparative genomic analyses showed that orthologous flag -2 loci are present in 592/4,028 taxa belonging to 5/8 and 31/76 families and genera, respectively, in the order Enterobacterales. Furthermore, the presence of the outermost flag- 2 genes only in many taxa suggest that this locus was far more prevalent and has subsequently been lost through gene deletion events. The flag -2 loci range in size from ~3.4 to 81.1 kilobases and code for between five and 102 distinct proteins. The discrepancy in size and protein number can be attributed to the presence of cargo gene islands within the loci. Evolutionary analyses revealed a complex evolutionary history for the flag -2 loci, representing ancestral elements in some taxa, while showing evidence of recent horizontal acquisition in other enterobacteria.Conclusions The flag -2 flagellar system is a relatively common, but highly variable feature among members of the Enterobacterales. Given the energetic burden of flagellar biosynthesis and functioning, the prevalence of a second flagellar system suggests it plays important biological roles in the enterobacteria and we postulate on its potential role as locomotory organ or as secretion system.