Yoshitaka Shirasaki

Single-Cell Imaging of Caspase-1 Dynamics Reveals an All-or-None Inflammasome Signaling Response

Inflammasome-mediated caspase-1 activation is involved in cell death and the secretion of the proinflammatory cytokine interleukin-1β (IL-1β). Although the dynamics of caspase-1 activation, IL-1β secretion, and cell death have been examined with bulk assays in population-level studies, they remain poorly understood at the single-cell level. In this study, we conducted single-cell imaging using a genetic fluorescence resonance energy transfer sensor that detects caspase-1 activation. We determined that caspase-1 exhibits all-or-none (digital) activation at the single-cell level, with similar activation kinetics irrespective of the type of inflammasome or the intensity of the stimulus. Real-time concurrent detection of caspase-1 activation and IL-1β release demonstrated that dead macrophages containing activated caspase-1 release a local burst of IL-1β in a digital manner, which identified these macrophages as the main source of IL-1β within cell populations. Our results highlight the value of single-cell analysis in enhancing understanding of the inflammasome system and chronic inflammatory diseases.

Real-time single-cell imaging of protein secretion

Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1β secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1β secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.

Use of Folded Micromachined Pillar Array Column with Low-Dispersion Turns for Pressure-Driven Liquid Chromatography

In this study, we show for the first time that the separation efficiency of a pillar array column under pressure-driven liquid chromatography (LC) conditions can be improved using a separation channel with low-dispersion turns. The pillar array column was fabricated by reactive ion etching of a silicon substrate. With the low-dispersion-turn geometry, a column with a length and width of 110 mm and 400 microm, respectively, could be fabricated on a 20 x 20 mm microchip. Under nonretained conditions, the solute bands obtained for fluorescent compounds remained almost unchanged even after passing through the low-dispersion turns; however, significant skewing of the solute bands was observed in the case of constant-radius turns. Two coumarin dyes were well resolved under reversed-phase conditions, and a maximum theoretical plate number of 8000 was obtained. Successful separation of the fluorescent derivatives of six amino acids was achieved in 140 s. These results indicated that the separation efficiency of microchip chromatography could be significantly improved using a long separation channel with low-dispersion turns.

On-chip microfluidic sorting with fluorescence spectrum detection and multiway separation

The microfluidic platform is an important tool for diagnosis and biomedical studies because it enables us to handle precious cells and infectious materials safely. We have developed an on-chip microfluidic sorter with fluorescence spectrum detection and multiway separation. The fluorescence spectrum of specimens (495-685 nm) in the microchannels was obtained every 2 ms using a 1 x 16 arrayed photomultiplier tube. The specimen was identified by its spectrum and collected into the corresponding channel based on our previously reported thermoreversible gelation polymer technique (Y. Shirasaki, J. Tanaka, H. Makazu, K. Tashiro, S. Shoji, S. Tsukita and T. Funatsu, Anal. Chem., 2006, 78, 695-701). Four kinds of fluorescence microspheres and three kinds of Escherichia coli cells, expressing different fluorescent proteins, were successfully separated with accuracy and purity better than 90% at a throughput of about one particle per second.

Single-molecule imaging of β-actin mRNAs in the cytoplasm of a living cell

Beta-actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most beta-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D(MACRO)) at the leading edge was 0.3 microm(2)/s. On the other hand, D(MACRO) in the perinuclear region was 0.02 microm(2)/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize beta-actin mRNAs, led to an increase in D(MACRO) to 0.2 microm(2)/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of beta-actin mRNA.

A Novel Biomolecule Sorter Using Thermosensitve Hydrogel in Micro Flow System

A novel biomolecule sorter using thermoreversible hydrogel was developed. Thermosensitive block copolymer changes its state from sol to gel when it is heated. The solution containing fluorescent biomolecules and thermosensitive polymer was introduced to a Y-shaped microchannel. The sol-gel transition was locally induced by site directed IR (infrared) laser irradiation in order to plug the microchannel and sort out the biomolecules. The sorting time of 120 ms was achieved. As an application of this sorter, fluorescently labeled λ-phage DNA molecules were separated and collected.

Integration in a multilayer microfluidic chip of 8 parallel cell sorters with flow control by sol–gel transition of thermoreversible gelation polymer

Microfluidic systems have significant implications in the field of cell separation since they could provide platforms with inexpensive, disposable and sterile structures. Here, we present a novel strategy to integrate microfluidic sorters into a single chip for high throughput sorting. Our parallel sorter consists of a microfluidic chip with a three-dimensional channel network that utilizes flow switching by a heat-induced sol-gel transition of thermoreversible gelation polymer. The 8 parallel sheathed sample flows were realized by injecting sample and buffer solutions into only 2 inlets. The sheathed flows enabled disposal of unwanted sample waste without laser irradiation, and collection of wanted sample upon irradiation. As an application of the sorter, two kinds of fluorescent microspheres were separated with recovery ratio and purity of 70% or 90% at throughputs of about 100 or 20 particles per second, respectively. Next, Escherichia coli cells expressing green fluorescent protein were separated from those expressing DsRed with recovery ratio and purity of 90% at a throughput of about 20 cells per second.

High-speed particles and biomolecules sorting microsystem using thermosensitive hydrogel

Two types of high-speed particle and biomolecule sheath flow sorters were realized using thermosensitive hydrogel. One is all hydrogel sheath flow and the other is two-phase sheath flow that consists of a water flow including samples and two hydrogel carrier flows; these have been utilized instead of the air and water droplet two-phase flow used in the conventional commercialized cell sorting system. Flow switching is performed by the sol–gel transfer of the thermosensitive hydrogel generated by focused infrared (IR) laser irradiation. High-speed sorting less than 5.0 ms and no error sorting (120 000 counts) in the all hydrogel sheath flow system while 20 ms in the two-phase sheath flow was realized. Since sorting was performed in simple PDMS-glass microchannels without any electric stimulation and mechanical valve structures, the proposed system is suitable for many biochemical applications.

Size-dependent accumulation of mRNA at the leading edge of chicken embryo fibroblasts

beta-actin mRNA localizes to the leading edge of a living chicken embryo fibroblast. Recently we proposed that the mRNA maintains its localization at the leading edge by utilizing the heterogeneity of cytoplasmic microstructure (Yamagishi et al., 2009 [10]). In this study, we observed the intracellular distribution of beta-actin mRNA variants to elucidate the mechanism of mRNA localization at the leading edge. We found that the degree of localization correlated positively with the molecular mass of the mRNA variants. We further demonstrated that the molecular mass-dependent localization was found even with dextrans, which have no biological function. The dependency of localization on molecular mass suggested that the barrier effect caused by the physical obstruction of the cytoplasmic microstructure is one of the major factors controlling mRNA localization in motile fibroblasts.

Single cell real time secretion assay using amorphous fluoropolymer microwell array

Real time assay of protein secretion from living single cells was realized by coupling total internal reflection fluorescence (TIRF) microscopy observation and an optically optimized microwell array device. This system was based on a sandwich immunosorbent assay. Individual cells were cultured on an inverted microscope in microwell arrays fabricated of an amorphous fluoropolymer, CYTOP, to be monitored by TIRF imaging. CYTOP have no interference for the TIRF imaging because the refractive index of CYTOP is almost same as that of water. Using this system, we could succeed in real time secretion assay of lipopolysaccharide stimulated mast cells and the secretion time course of IL-1β from a single MC/9 cell was obtained successfully.