Micrografting device for testing systemic signaling in Arabidopsis.

Grafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting, and showed that a temperature of 27°C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of iron accumulation in shoots was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot- and root-ward translocation of nicotianamine-iron complexes and/or nicotianamine is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants.

Intracellular localization of histone deacetylase HDA6 in plants.

Histone modification is an important epigenetic mechanism in eukaryotes. Histone acetyltransferase and deacetylase regulate histone acetylation levels antagonistically, leading to dynamic control of chromatin structure. One of the histone deacetylases, HDA6, is involved in gene silencing in the heterochromatin regions, chromocenter formation, and metabolic adaptation under drought stress. Although HDA6 plays an important role in chromatin control and response to drought stress, its intracellular localization has not been observed in detail. In this paper, we generated transformants expressing HDA6-GFP in the model plant, Arabidopsis thaliana, and the crops, rice, and cassava. We observed the localization of the fusion protein and showed that HDA6-GFP was expressed in the whole root and localized at the nucleus in Arabidopsis, rice, and cassava. Remarkably, HDA6-GFP clearly formed speckles that were actively colocalized with chromocenters in Arabidopsis root meristem. In contrast, such speckles were unlikely to be formed in rice or cassava. Because AtHDA6 directly binds to the acetate synthesis genes, which function in drought tolerance, we performed live imaging analyses to examine the cellular dynamics of pH in roots and the subnuclear dynamics of AtHDA6 responding to acetic acid treatment. The number of HDA6 speckles increased during drought stress, suggesting a role in contributing to drought stress tolerance.

Capability of tip-growing plant cells to penetrate into extremely narrow gaps.

Plant cells are covered with rigid cell walls, yet tip-growing cells can elongate by providing new cell wall material to their apical regions. Studies of the mechanical properties of tip-growing plant cells typically involve measurement of the turgor pressure and stiffness of the cells' apical regions. These experiments, however, do not address how living tip-growing cells react when they encounter physical obstacles that are not substantially altered by turgor pressure. To investigate this issue, we constructed microfabricated platforms with a series of artificial gaps as small as 1 µm, and examined the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to penetrate into these gaps. The cells were grown inside microfluidic chambers and guided towards the gaps using microdevices customized for each cell type. All types of tip-growing cells could grow through the microgaps with their organelles intact, even though the gaps were much smaller than the cylindrical cell diameter. Our findings reveal the dramatic physiological and developmental flexibility of tip-growing plant cells. The microfluidic platforms designed in this study provide novel tools for the elucidation of the mechanical properties of tip-growing plant cells in extremely small spaces.

Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis.

Intercellular communications are essential for cell proliferation and differentiation during plant embryogenesis. However, analysis of intercellular communications in living material in real time is difficult owing to the restricted accessibility of the embryo within the flower. We established a live-embryo imaging system to visualize cell division and cell fate specification in Arabidopsis thaliana from zygote division in real time. We generated a cell-division lineage tree for early embryogenesis in Arabidopsis. Lineage analysis showed that both the direction and time course of cell division between sister cells differed along the apical-basal or radial axes. Using the Arabidopsis kpl mutant, in which single-fertilization events are frequent, we showed that endosperm development is not required for pattern formation during early embryogenesis. Optical manipulation demonstrated that damage to the embryo initial cell induces cell fate conversion of the suspensor cell to compensate for the disrupted embryo initial cell even after cell fate is specified.

Quantification of pollen tube attraction in response to guidance by female gametophyte tissue using artificial microscale pathway.

We developed two types of artificial platforms, T-junction and crossroad microchannel devices, and obtained guidance response ratio of pollen tubes to the female tissue as 56-57%. The crossroad device was also able to collect the attracted pollen tubes with high purity, which is useful for future omics analysis.

Poly(dimethylsiloxane)-based microdevices for studying plant reproduction.

Long-term holding and precise handling of growing plant tissues during in vitro cultivation has been a major hurdle for experimental studies related to plant development and reproduction. In the present review, we introduce two of our newly developed poly(dimethylsiloxane)-based microdevices: a T-shaped microchannel device for pollen tube chemoattraction and a microcage array for long-term live imaging of ovules. Their design, usage and advantages are described, and future prospects of experimental approaches to plant reproduction using such microdevices are discussed.

Rapid sub-attomole microRNA detection on a portable microfluidic chip.

Microfluidic devices are an attractive choice for meeting the requirements of point-of-care microRNA detection. A method using a microfluidic device can drastically shorten the incubation time because the device conveys sample molecules right straight to the surface-immobilized probe DNAs by hydrodynamic force. In this review, we present an overview of a new method for rapid and sensitive microRNA detection from a small sample volume using a power-free microfluidic device driven by degassed poly-dimethylsiloxane (PDMS). Two key technologies for this detection method are summarized. One of the methods relies on the coaxial stacking effect of nucleic acids during sandwich hybridization. This effect is also efficient for stabilizing sandwich hybridization consisting of small DNA and microRNA. The other is the laminar flow-assisted dendritic amplification, which increases the fluorescent signal by supplying two amplification reagents from laminar streams to surface-bound molecules. Utilizing both technologies, microRNA detection is possible with a 0.5 pM detection limit from a 0.5 µL sample corresponding to 0.25 attomoles, with a detection time of 20 min. Since microRNAs are associated with various human diseases, future studies of these technologies might contribute to improved healthcare and may have both industrial and societal impacts.

Rapid and sensitive microRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip.

Detection of microRNAs, small noncoding single-stranded RNAs, is one of the key topics in the new generation of cancer research because cancer in the human body can be detected or even classified by microRNA detection. This report shows rapid and sensitive microRNA detection using a power-free microfluidic device, which is driven by degassed poly(dimethylsiloxane), thus eliminating the need for an external power supply. MicroRNA is detected by sandwich hybridization, and the signal is amplified by laminar flow-assisted dendritic amplification. This method allows us to detect microRNA of specific sequences at a limit of detection of 0.5 pM from a 0.5 µL sample solution with a detection time of 20 min. Together with the advantages of self-reliance of this device, this method might contribute substantially to future point-of-care early-stage cancer diagnosis.

Rapid microRNA detection using power-free microfluidic chip: coaxial stacking effect enhances the sandwich hybridization.

We present a new method for rapid microRNA detection with a small volume of sample using the power-free microfluidic device driven by degassed PDMS. Target microRNA was detected by sandwich hybridization taking advantage of the coaxial stacking effect. This method allows us to detect miR-21 in 20 min with a 0.5 µL sample volume at a limit of detection of 0.62 nM. Since microRNAs can act as cancer markers, this method might substantially contribute to future point-of-care cancer diagnosis.

Miniaturized thermocontrol devices enable analysis of biomolecular behavior on their timescales, second to millisecond.

To establish general-purpose methods and tools for biological experiments on a short time scale is an essential requirement for future research in molecular biology because most of the functions of living organisms at the molecular level take place on a time scale from 1-second to millisecond. Thermal control with on-chip micro-thermodevices is one of the strongest and most useful ways to realize biological experiments at molecular level on these time scales. Novel biological phenomena revealed by the experiments using micro-thermodevices on a 1-second and millisecond time scale will be shown for the proof. Finally, the advantages and impact of this methodology in molecular biology will be discussed.

Direct observation of twisting steps during Rad51 polymerization on DNA.

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 A per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.

Millisecond analysis of double stranded DNA with fluorescent intercalator by micro-thermocontrol-device.

Study of interaction between DNA and intercalator at molecular level is important to understand the mechanisms of DNA replication and repair. A micro-fabricated local heating thermodevice was adapted to perform denaturation experiments of DNA with fluorescent intercalator on millisecond time scale. Response time of complete unzipping of double stranded DNA, 16 microm in length, was measured to be around 5 min by commercial thermocycler. Response time of quenching of double stranded DNA with fluorescent intercalator SYBR Green was measured to be 10 ms. Thus, quenching properties owing to strand unzipping and denaturation at base pair level were distinguished. This method has provided easy access to measure this parameter and may be a powerful methodology in analyzing biomolecules on millisecond time scale.

Millisecond denaturation dynamics of fluorescent proteins revealed by femtoliter container on micro-thermodevice.

Real-time observation of biomolecular behavior focusing on high speed temperature response is an essential endeavor for further biological study at the molecular level. This is because most of the important biological functions at the molecular level happen at the sub-second time scale. We used our own on-chip microheaters and microcontainers to observe the denaturation dynamics of fluorescent proteins at the millisecond time scale. The microheater controls the temperature in 1 ms under the microscope. Fluorescent proteins were contained in 28 fL PDMS microcontainers to prevent them from diffusing into the solution. The proteins were denatured by high temperatures and observed by a high speed CCD camera with 5 ms per frame. Hence, denaturation speeds of red fluorescent proteins (rDsRed and rHcRed) were measured to be 5-10 ms. Green fluorescent proteins (rAcGFP and rGFPuv) denatured with bi-exponential decay. rAcGFP denatured with time constants of 5 ms and 75 ms while rGFPuv denatured with 10 ms and 130 ms. This may be the reverse process of a two step renaturation of GFP observed in a previous report. This micro-thermodevice is applicable to other biomaterials such as nucleic acids or other proteins. It does not require any chemical treatment nor mutation to the biomaterial itself. Therefore, the methodology using this general purpose device gives access to biomolecular studies in short time scales and acts as a powerful tool in molecular biology.

Towards single biomolecule handling and characterization by MEMS.

Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation, a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology.

Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber.

Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application. Hence here, we developed a novel technique for fluorescent imaging of LAMP at a single molecule level. The experiment was conducted in a polyacrylamide (PAA) gel-based microchamber where a single DNA template, freely suspended in a solution containing primers and polymerase was initially encapsulated. In order to activate the amplification reaction, a microheater regulated by an automatic computerized feedback system was used for localized heating. This microchamber-based approach for LAMP demonstrated the effective exploitation of minute amount of templates and primers, and the overall reduction in LAMP detection time. An average efficiency of 80% was evaluated for conducting DNA amplification after 50 min of incubation at 65 degrees C. As the total time for reaction including detection can be completed in less than 1 h, this one-step, direct observation method displays the potential as a simple alternative to conventional techniques for genetic analysis and diagnosis in the clinical laboratory.

Thermally responsive supramolecular nanomeshes for on/off switching of the rotary motion of F1-ATPase at the single-molecule level.

The artificial regulation of protein functions is essential for the realization of protein-based soft devices, because of their unique functions conducted within a nano-sized molecular space. We report that self-assembled nanomeshes comprising heat-responsive supramolecular hydrogel fibers can control the rotary motion of an enzyme-based biomotor (F(1)-ATPase) in an on/off manner at the single-molecule level. Direct observation of the interaction of the supramolecular fibers with a microbead unit tethered to the F(1)-ATPase and the clear threshold in the size of the bead required to stop ATPase rotation indicates that the bead was physically blocked so as to stop the rotary motion of ATPase. The temperature-induced formation and collapse of the supramolecular nanomesh can produce or destroy, respectively, the physical obstacle for ATPase so as to control the ATPase motion in an off/on manner. Furthermore, this switching of the F(1)-ATPase motion could be spatially restricted by using a microheating device. The integration of biomolecules and hard materials, interfaced with intelligent soft materials such as supramolecular hydrogels, is promising for the development of novel semi-synthetic nano-biodevices.

On-chip thermal calibration with 8 CB liquid crystal of micro-thermal device.

A temperature sensor integrated on a micro-device for biological experiments requires affordable, rapid and easy thermal calibration. However, such calibration cannot usually be done directly under the microscope, a fact that impedes biological experiments. We present in this paper an inexpensive and rapid method to achieve thermal calibration directly under the microscope. It is based on the use of a thermotropic liquid crystal: the 4-n-octyl-4-cyanobiphenyl (8 CB) exhibiting an isothermal phase change at 313 K that can be monitored optically. We demonstrate the advantages of this method by calibrating the temperature sensor integrated onto a micro-device.

Temperature alternation by an on-chip microheater to reveal enzymatic activity of beta-galactosidase at high temperatures.

A method to measure enzymatic activity at high temperatures by rapid temperature alternation of a microreactor with a microheater is proposed. On-chip microreactor and microheater were integrated on a glass plate by MEMS technology; this microheater can control the temperature of the microreactor with a response speed of 34.2 and 31.5 K/s for temperature rise and fall, respectively, with an accuracy of 3 degrees C. The enzyme, beta-galactosidase, was revealed to survive short exposure (4-s pulses) to temperatures above that which would "normally" denature them. Its activity at 60 degrees C was revealed to be approximately 4 times greater than that at room temperature. This method not only gives new kinetic information in biochemistry but also enables application in highly sensitive biosensors.

Microfabricated arrays of femtoliter chambers allow single molecule enzymology.

Precise understanding of biological functions requires tools comparable in size to the basic components of life. Single molecule studies have revealed molecular behaviors usually hidden in the ensemble- and time-averaging of bulk experiments. Although most such approaches rely on sophisticated optical strategies to limit the detection volume, another attractive approach is to perform the assay inside very small containers. We have developed a silicone device presenting a large array of micrometer-sized cavities. We used it to tightly enclose volumes of solution, as low as femtoliters, over long periods of time. The microchip insures that the chambers are uniform and precisely positioned. We demonstrated the feasibility of our approach by measuring the activity of single molecules of beta-galactosidase and horseradish peroxidase. The approach should be of interest for many ultrasensitive bioassays at the single-molecule level.