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Bacterial plasmids have evolved segregation machineries to partition replicated DNA to the daughter cells at cell division. P1 phage lysogenizes as a low-copy-number plasmid in Escherichia coli. Its partition system consists of three components, a centromere-like region, parS, an adaptor protein that binds to the centromere, ParB and a partition ATPase, ParA. In general, it is known that a ParB/parS partition complex is formed when ParB oligomerize onto the centromere. This large nucleoprotein complex interacts with ParA and is thought to couple ATP hydrolysis to drive the movement and segregation of plasmids to opposite cell-halves. To understand ATP-driven DNA segregation, we reconstituted the P1 plasmid partition system in a cell-free reaction and visualized the spatiotemporal dynamics using TIRF microscopy. We coated a flow cell surface with non-specific DNA to mimic the bacterial nucleoid surface and flowed in the three-component reaction system. We found that ParA coats the artificial nucleoid creating a reference scaffold for plasmid movement. ParA assembles onto the ParB/parS complexes and anchors them onto the ParA-coated nucleoid surface. ParB stimulates ParA disassembly leading to vigorous Brownian motion of the plasmid as the complex loses bridging interactions with the nucleoid. The plasmid detaches from the nucleoid surface leaving a hole devoid of ParA, which is refilled rapidly with ParA rebinding onto the nucleoid. FRAP experiments demonstrate the dynamic exchange of proteins on the nucleoid surface and the partition complex. We present a Par partition model of ParB-stimulated ParA assembly/disassembly triggering dynamic instability leading to plasmid segregation and movement.

Temperature is a fundamental physical quantity that governs every biological reaction within living cells, and temperature distribution reflects cellular thermodynamics and function. In medical studies, the cellular pathogenesis of diseases (e.g., cancer) is characterized by extraordinary heat production. Therefore, intracellular temperature imaging of living cells should promote better understanding of cellular events and the establishment of novel diagnoses and therapies. However, imaging of temperature distributions in living cells has never been achieved. Here we demonstrate the first intracellular temperature imaging based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy (FLIM). The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.2 °C, respectively. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These findings demonstrate an intrinsic connection between temperature and organelle function. Thus, our intracellular temperature imaging has a significant impact on the comprehension of cell function and will provide insights into the regulatory mechanisms of intracellular signaling.

DNA segregation is an essential process that ensures that every daughter cell inherits a copy of genomic DNA. Many bacterial chromosomes and low-copy-number plasmids such as P1 in Escherichia coli have partitioning systems to separate and transport genomes towards opposite cell-halves before cell division. Only three components are required to partition plasmids: a parS sequence on the plasmid and two proteins, ParA and ParB. It was widely believed that P1 plasmids segregate similar to a mitotic mechanism in eukaryotes. Here, we propose that the partition proteins pattern the bacterial nucleoid using it as a track for plasmid motion. We reconstituted the P1 partition system in a DNA-coated flowcell to mimic an artificial nucleoid, and visualized the dynamics with TIRF microscopy. ParA and ParB coated the DNA surface uniformly. ParB binds specifically to parS site on the plasmid and stimulated the disassembly of ParA, an ATP-dependent DNA-binding protein. This generated a transient depletion zone surrounding the plasmid prior to its motion on the DNA surface. FRAP measurements showed the partition proteins exchanging rapidly on DNA without forming any filamentous structures. Our results support a reaction-diffusion based mechanism, where ParB on the plasmid chases and redistributes ParA patterns on the nucleoid and in turn drives plasmid movement.

In eukaryotic cells mRNA plays a key role in gene regulation. However, the function of mRNA is not fully understood because direct analysis of endogenous mRNAs in living cells has been difficult. We developed a method for the observation of endogenous mRNA in living cells using two fluorescently labeled linear antisense 2′O-methyl RNA oligonucleotides. When those two antisense probes, each is labeled with different fluorescent dyes, are hybridized to an adjacent sequence of the target mRNA, the distance between two fluorophores becomes close and FRET occurs.Here we applied linear antisense probes to the real time monitoring of endogenous mRNA, which will be useful in understanding the function of mRNA as well as the intracellular localization. First, two kinds of linear antisense probes were microinjected into the cytoplasm of living COS7 cells and the FRET signal from cells was recorded over time to examine the kinetics of the hybridization reaction with c-fos mRNA. The hybridization reaction of linear antisense probes proceeded quickly and time constants of linear antisense probe was estimated to be less than one minute. When using Molecular Beacon, the conventional probe for endogenous mRNAs, it took more than one hour to complete the hybridization. Next, the induction of c-fos mRNA in the cytoplasm of COS7 cells was investigated in real time using linear antisense probes. As a result, the elevation of c-fos mRNA expressed in the cytoplasm was observed within one hour after the stimulation with PMA (phorbol 12-myristate 13-acetate). In conclusion, we showed the linear antisense probes are advantageous in monitoring of mRNAs due to their prominent kinetics in hybridizing with target mRNAs in living cells.

Eukaryotic gene expression is regulated by chromatin structures and/or DNA modification such as CpG methylation. The basic unit of eukaryotic chromatin structure is a nucleosome consisting of approximately 150 bp DNA wrapped in 1.7 superhelical turns around a histone octamer. The histone octamer consists of two copies each of H2A, H2B, H3 and H4. Posttranslational histone modifications such as acetylation, methylation, phosphorylation and ubiquitylation regulate chromatin structure, resulting in activation or repression of gene expression. On the other hand, CpG methylation represses gene expression and is essential for silencing of parasitic DNA, genomic imprinting and embryogenesis. During DNA replication, methylated CpGs are converted into hemi-methylated CpGs and newly replicated CpGs should be methylated to inherit methylation pattern. DNA methyltransferase 1 (Dnmt1) is the enzyme to methylate hemi-methylated CpG regions. Uhrf1 is methylated CpG binding protein and interacts with Dnmt1, followed by recruitment of Dnmt1 to hemi-methylated CpG regions. SRA domain of Uhrf1 is responsible for hemi-methylated CpG binding activity. We characterize the process of hemi-methylated CpG recognition by SRA domain using Single-Molecule technique, and in this symposium, we will show our present data.