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  1. Abstract

    We determined the macroscopic limit for phase synchronization of cellular clocks in an artificial tissue created by a “big chamber” microfluidic device to be about 150,000 cells or less. The dimensions of the microfluidic chamber allowed us to calculate an upper limit on the radius of a hypothesized quorum sensing signal molecule of 13.05 nm using a diffusion approximation for signal travel within the device. The use of a second microwell microfluidic device allowed the refinement of the macroscopic limit to a cell density of 2166 cells per fixed area of the device for phase synchronization. The measurement of averages over single cell trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, suggesting support for quorum sensing. Further evidence came from showing phase synchronization was density-dependent.

  2. Abstract

    Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific genemore »con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

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  3. Millet, Oscar (Ed.)
    System biology relies on holistic biomolecule measurements, and untangling biochemical networks requires time-series metabolomics profiling. With current metabolomic approaches, time-series measurements can be taken for hundreds of metabolic features, which decode underlying metabolic regulation. Such a metabolomic dataset is untargeted with most features unannotated and inaccessible to statistical analysis and computational modeling. The high dimensionality of the metabolic space also causes mechanistic modeling to be rather cumbersome computationally. We implemented a faster exploratory workflow to visualize and extract chemical and biochemical dependencies. Time-series metabolic features (about 300 for each dataset) were extracted by Ridge Tracking-based Extract (RTExtract) on measurements from continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) in Neurospora crassa under different conditions. The metabolic profiles were then smoothed and projected into lower dimensions, enabling a comparison of metabolic trends in the cultures. Next, we expanded incomplete metabolite annotation using a correlation network. Lastly, we uncovered meaningful metabolic clusters by estimating dependencies between smoothed metabolic profiles. We thus sidestepped the processes of time-consuming mechanistic modeling, difficult global optimization, and labor-intensive annotation. Multiple clusters guided insights into central energy metabolism and membrane synthesis. Dense connections with glucose 1-phosphate indicated its central position in metabolism in N . crassa .more »Our approach was benchmarked on simulated random network dynamics and provides a novel exploratory approach to analyzing high-dimensional metabolic dynamics.« less
  4. Most eukaryotes and cyanobacterial species have a biological clock that allows adaptation to the daily light/dark cycle of the planet. A central problem in the study of the biological clock is understanding the synchro-nization of the stochastic oscillators in different cells and tissues, but this problem is largely unstudied, particularly in the context of circadian rhythms. We developed a novel microfluidic platform to make high-throughput and high-precision measurements of biological clocks on a controlled number of Neurospora crassa (N. crassa) cells. Single cell measurements in this platform enabled us to test whether clocks of individual cells are able to communicate.
  5. The use of a microwell microfluidic device allows separating single cells and tracking single cells data. The measurement of single cell fluorescent intensity trajectories in the microwell device supported a deterministic quorum sensing model identified by ensemble methods for clock phase synchronization. A strong inference framework was used to test the communication mechanism in phase synchronization of quorum sensing versus cell-to-cell contact, and the results lent support for quorum sensing.
  6. We report a microfluidic device that mimics an artificial tissue to test the theory of quorum sensing as a method for synchronization of a model fungal system, Neurospora crassa (N. crassa). High synchronicity between cells were observed by calculating the Kuramoto order parameter (K) between different fields of view.The dimensions of the microfluidic chamber allows us to also calculate an upper limit of the radius of a hypothesized quorum sensing signal by using the diffusion approximation for signal travelling within the device.