Kazunori Matsumura

Role of timer and sizer in regulation of Chlamydomonas cell cycle

To estimate the role that time and size had in controlling the Chlamydomonas cell cycle, we used a new on-chip single-cell microcultivation system, which involved the direct observation of single cells captured in microchambers made on a thin glass slide. The dependence of the pattern of energy supply for cells on its cell cycle was examined through a series of different intensities of continuous illumination in a minimal medium, and we found that cell division occurred when cells reached the critical size, which was 2.2 times larger than that of the newly created cells. When illumination stopped before cells reached the critical size, even though growth had stopped, they continued dividing during the delay time, which was shorter when cells were larger. With re-illumination after darkness, cells began to grow again and the timing of cell division was again controlled by the critical size. This indicates that the co-existence of two cell cycle regulation mechanisms and the sizer mechanism had a stronger influence than the timer.

On-chip microcultivation chamber for swimming cells using visualized poly(dimethylsiloxane) valves

We have developed a new type of on-chip microcultivation chamber made of poly(dimethylsiloxane) (PDMS) for long-term cultivation of swimming cells. The advantages of this chamber are that (1) the microfluidic channel width of the valve can be controlled according to air pressure while monitoring the microscopic image of the channel width, and that (2) a simple single-step procedure is required for the fabrication of the valve structure and microfluidic pathway. Using this chamber, we can control the passage of swimming Chlamydomonas cells, through the channel while visualizing the opening and closing of the valve as the medium buffer passes any time. Thus the long-term observation of the behavior of a particular single cell is accomplished.

Differential Analysis of Cell Cycle Stability in Chlamydomonas Using On-Chip Single-Cell Cultivation System

We have developed an on-chip microcultivation system for differential analysis of photosynthesizing cells of Chlamydomonas as a means of comparing the difference in interdivision time between sister cells and cells of adjacent generations having the same DNA and environment. The system enabled us to compare the change in the interdivision time of four sister cells simultaneously for several days, we found that the interdivision times of the four sister cells synchronize well, while, those of cells of subsequent generations do not. The result showed the potential of the system for measuring how much epigenetic information of eukaryotic cells can be conserved between sister cells and cells of subsequent generations.

On-chip single-cell-based gene expression analysis using gold nano-particles

The authors have developed a novel method to measure the quantitative amount of mRNA expression in individual cells keeping their spatial distributions in the cell without any amplification process like PCR. In this method, a set of different sizes of gold nanoparticles attached with different probe-DNA respectively were used as a set of probes to detect different mRNAs existing in a cell. At first, the optimum condition of the immobilization of probe-DNA onto the gold nanoparticle surface was examined. Next, the selectivity of the probe-DNA immobilized onto the nanoparticle was tested using complementary oligonucleotide molecules. We confirmed the several different kinds of gold nanoparticle probes were hybridized with target oligonucleotides having complementary sequences with almost 100% selectively. For the counting and distinguishing each of the gold nanoparticles, we used two different methods and compared: one is atomic force microscopy and the other is scanning electron microscopy. Quantitative detection of mRNAs in individual cells keeping their spatial distributions was then examined using the gold nanoparticle-based detection system. In this meeting, we will present the results and will discuss about the potential and problems of this method for the single-cell-based quantitative expression analysis.