Takako Ichinose

In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.

Sexual reproduction is widespread in eukaryotes; however, only asexual reproduction has been observed in unicellular red algae, including Galdieria , which branched early in Archaeplastida. Galdieria possesses a small genome; it is polyextremophile, grows either photoautotrophically, mixotrophically, or heterotrophically, and is being developed as an industrial source of vitamins and pigments because of its high biomass productivity. Here, we show that Galdieria exhibits a sexual life cycle, alternating between cell-walled diploid and cell wall–less haploid, and that both phases can proliferate asexually. The haploid can move over surfaces and undergo self-diploidization or generate heterozygous diploids through mating. Further, we prepared the whole genome and a comparative transcriptome dataset between the diploid and haploid and developed genetic tools for the stable gene expression, gene disruption, and selectable marker recycling system using the cell wall–less haploid. The BELL/KNOX and MADS-box transcription factors, which function in haploid-to-diploid transition and development in plants, are specifically expressed in the haploid and diploid, respectively, and are involved in the haploid-to-diploid transition in Galdieria , providing information on the missing link of the sexual life cycle evolution in Archaeplastida. Four actin genes are differently involved in motility of the haploid and cytokinesis in the diploid, both of which are myosin independent and likely reflect ancestral roles of actin. We have also generated photosynthesis-deficient mutants, such as blue-colored cells, which were depleted in chlorophyll and carotenoids, for industrial pigment production. These features of Galdieria facilitate the understanding of the evolution of algae and plants and the industrial use of microalgae.

Live cell imaging by fluorescence microscopy is a useful tool for elucidating the localization and function of proteins and organelles in single cells. Especially, time-lapse analysis observing the same field sequentially can be used to observe cells of many organisms and analyze the dynamics of intracellular molecules. By single-cell analysis, it is possible to elucidate the characteristics and fluctuations of individual cells, which cannot be elucidated from the data obtained by averaging the characteristics of an ensemble of cells. The primitive red alga Cyanidioschyzon merolae has a very simple structure and is considered a useful model organism for studying the mechanism of organelle division, since the division is performed synchronously with the cell cycle. However, C. merolae does not have a rigid cell wall, and environmental changes such as low temperature or high pH cause morphological change and disruption easily. Therefore, morphological studies of C. merolae typically use fixed cells. In this study, we constructed a long-term time-lapse observation system to analyze the dynamics of proteins in living C. merolae cells. From the results, we elucidate the cell division process of single living cells, including the function of intracellular components.