Yoichiroh Hosokawa

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.

Nondestructive micropatterning of living animal cells using focused femtosecond laser-induced impulsive force

Micropatterning of mouse NIH3T3 fibroblast cells was performed using focused femtosecond laser-induced impulsive force in a culture medium. The cells were detached from an upper substrate by the force and transferred to an underlying substrate with less than spatial resolution of 80μm full width at half maximum. About 80% of the cells were confirmed to be alive at 3h after the patterning. The force exerted to the cell was investigated by high-speed imaging and estimated to be an order of micronewtons. The force origin was not only due to cavitation bubble but also due to shockwave and jet flow.

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.

Trapping and manipulation of a single micro-object in solution with femtosecond laser-induced mechanical force

A nondestructive and noncontact method to trap and manipulate a microparticle in solution is proposed by utilizing femtosecond laser-induced nonlinear phenomena. A 90μm diameter polystyrene bead in solution was trapped and manipulated by scanning femtosecond laser pulses around it, which was ascribed to shockwave, cavitation bubble, and jet flow. The maximum mechanical force exerted by laser irradiation was estimated to be over 1μN. In comparison with conventional optical tweezers, this method provides not only a much larger trapping force but also a noninvasive advantage.

Actin Migration Driven by Directional Assembly and Disassembly of Membrane-Anchored Actin Filaments

Actin and actin-associated proteins migrate within various cell types. To uncover the mechanism of their migration, we analyzed actin waves, which translocate actin and actin-associated proteins along neuronal axons toward the growth cones. We found that arrays of actin filaments constituting waves undergo directional assembly and disassembly, with their polymerizing ends oriented toward the axonal tip, and that the lateral side of the filaments is mechanically anchored to the adhesive substrate. A combination of live-cell imaging, molecular manipulation, force measurement, and mathematical modeling revealed that wave migration is driven by directional assembly and disassembly of actin filaments and their anchorage to the substrate. Actin-associated proteins co-migrate with actin filaments by interacting with them. Furthermore, blocking this migration, by creating an adhesion-free gap along the axon, disrupts axonal protrusion. Our findings identify a molecular mechanism that translocates actin and associated proteins toward the cells leading edge, thereby promoting directional cell motility.

Laser microfabrication and rotation of ship-in-a-bottle optical rotators

We have fabricated optical rotators inside a silica substrate and rotated them by a laser trapping technique. The fabrication method used was femtosecond laser-assisted etching, i.e., modification of the host material by irradiation with femtosecond laser pulses along a predesigned pattern, followed by selective chemical etching. The rotators, which consist of the same material as the substrate, can move inside the microcavity but cannot get out. The rotation speed was proportional to the trapping laser power, and the maximum achieved was about 100rpm. Such rotators will be applicable to micro-total-analysis systems and microfluidics.

Explosive Crystallization of Urea Triggered by Focused Femtosecond Laser Irradiation

The crystallization of urea was triggered using an intense 800 nm femtosecond laser that was focused to a supersaturated solution through an objective lens. An explosive crystallization proceeded in the entire sample glass tube for a few seconds at a concentration that no spontaneous nucleation occurred even after a few days. The crystallization was precisely monitored using a high-speed complementary metal oxide semiconductor (CMOS) camera attached to a microscope with a time resolution of 100 µs. On the basis of the results, the dynamic process of crystallization triggered by femtosecond laser ablation was discussed.

Femtosecond laser ablation dynamics of amorphous film of a substituted Cu-phthalocyanine

Abstract Femtosecond laser ablation dynamics of an amorphous thin film of a substituted Cu–phthalocyanine was studied using time-resolved absorption spectroscopic and scattering imaging methods. Above the ablation threshold of 45 mJ/cm 2 , the etch-depth is about 400 nm and almost constant up to a laser fluence of 300 mJ/cm 2 . Transient absorption spectra confirmed that the electronic excitation energy is converted to heat through exciton–exciton annihilation within 100 ps after excitation. The etching profile and ablation threshold are discussed in connection with the heating rate.

Statistical organelle dissection of Arabidopsis guard cells using image database LIPS

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.