Takanori Iino

Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

Enhanced Nerve–Mast Cell Interaction by a Neuronal Short Isoform of Cell Adhesion Molecule-1

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve–mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a–d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve–mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca2+ concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve–mast cell interaction, and they suggest that nerve–mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (Of) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between Of and the cantilever, the fsLP-IF generated at Of was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte–endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between Of and the adhesive interface, were on the order of 10-13 and 10-12 N-s, respectively. When the total impulse at Of is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

Photoporation of biomolecules into single cells in living vertebrate embryos induced by a femtosecond laser amplifier.

Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.

Direct Measurement of Femtosecond Laser Impulse in Water by Atomic Force Microscopy

An impulsive force generated by focusing a femtosecond laser into water through an objective lens was directly measured utilizing an atomic force microscope (AFM). We analyzed the transient bending movement of the AFM cantilever initiated by the impulsive force and suggested a procedure to quantify it. The order was 10-12 N s, corresponding to the average force of µN in µs timescale. The quantification creates a new way of estimating intercellular adhesion strength, because it is possible to dissociate cell–cell adhesion by the impulsive force.

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve-mast cell interaction in a hapten-induced mouse model of atopic dermatitis

Background  Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve–skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast‐cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves.

On fundamental cellular processes for emergence of collective epithelial movement

Summary In all animals, collective cell movement is an essential process in many events, including wound healing and embryonic development. However, our understanding of what characterizes the emergence of multicellular collective behavior is still far from complete. In this article we showed the fundamental cellular processes that drive collective cell movement by means of integrated approaches, including precise quantification measurements and mathematical modeling of measured data. First, we observed the dependence of the collective behaviors of cultured human skin cells on Ca2+ concentrations. When the culturing area confined by a PDMS sheet was suddenly expanded by removing the sheet, the group of cells moved to the expanded area with higher collectivity at higher Ca2+ concentrations. Next, we quantitatively measured cellular responses to the Ca2+ treatments, such as cell growth, cell division, and the strength of intercellular adhesion. Using a femtosecond-laser-based assay, an original method for estimating intercellular adhesion, we found that the strength of intercellular adhesion has an approximately 13-fold range in our treatments. Incorporating the quantitative data into a mathematical model, we then confirmed that the model well reproduced the multicellular behaviors we observed, demonstrating that the strength of intercellular adhesion sufficiently determines the generation of collective cell movement. Finally, we performed extensive numerical experiments, and the results suggested that the emergence of collective cell movement is derived by an optimal balance between the strength of intercellular adhesion and the intensity of cell migration.

Controllability of femtosecond laser-induced impulse in water evaluated by local force measurement system using atomic force microscopy

A micro-sized impulse is generated in water by tightly focusing a femtosecond laser through an objective lens. Pulse energy dependence of the impulse was investigated for application as an external force to analyze single biological cells. The impulse proportionally increased between threshold (Eth) and 2 × Eth, and then it was saturated above 2 × Eth. In the proportional region, control precision of the impulse was 2.5 × 10−14 Ns, which was smaller than the uncertainty of the cell-cell adhesion force previously evaluated. This result indicates the impulse can be applied as a promising tool to estimate cell adhesion force.

Pulsed-laser-activated impulse response encoder: Sensitive detection of surface elastic waves on biomimetic microsized gel spheres

A femtosecond-laser-induced impulsive force was applied to microsized calcium alginate (CaAlg) gel spheres as an external force to excite elastic waves. To evaluate elasticity, atomic force microscopy (AFM) was applied to detect vibration propagation. The sphere size dependence of the vibration was well reproduced by finite element method (FEM) simulation for pressure waves and surface acoustic waves. The obtained results indicate that the pulsed-laser-activated impulse response encoder (PLAIRE) enables the sensitive detection of elasticities, not only on inside but also on the surface.

Ultrathin glass filter fabricated by femtosecond laser processing for high-throughput microparticle filtering

In this study, we developed a method of fabricating totally glass-based filters having micrometer-scale through holes for high-throughput filtration using a femtosecond laser. Filtration using a membrane-type filter is an indispensable technique for biological, chemical, and physical analysis fields. A larger flow rate or stronger driving pressure will result in a faster filtration. However, conventional high-throughput filtering methods often use a relatively slow flow rate or low pressure owing to the fracture toughness of the filter material. In this study, we introduce a customizable 4-µm-thick glass filter that could be used for high-throughput microparticle filtering at a flow velocity of 4 m/s.