The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24h rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.
Single cell data is available from https://gitlab.com/slcu/teamJL/Gould_etal_2018
- James CW Locke
- James CW Locke
- Peter D Gould
- Mirela Domijan
- Anthony JW Hall
- James CW Locke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Richard Amasino, University of Wisconsin, United States
© 2018, Gould et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that obtained by the pre-optimized culture in terms of the pigmentation scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.
Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor mRNA (pre-mRNA) structure and downstream function are particularly challenging. Here, we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on pre-mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the Bact complex with pre-mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a β-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and 6 newly discovered mutations on inclusion of exon 10 of MAPT. This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.