An analysis of semaphorin-mediated cellular interactions in the Caenorhabditis elegans epidermis using the IR-LEGO single-cell gene induction system.

One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.

Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism.

Involvement of the synaptotagmin/stonin2 system in vesicular transport regulated by semaphorins in Caenorhabditis elegans epidermal cells.

Vesicular transport serves as an important mechanism for cell shape regulation during development. Although the semaphorin signaling molecule, a well-known regulator of axon guidance, induces endocytosis in the growth cone and the axonal transport of vertebrate neurons, the underlying molecular mechanisms remain largely unclear. Here, we show that the Caenorhabditis elegans SNT-1/synaptotagmin-UNC-41/stonin2 system, whose role in synaptic vesicle recycling in neurons has been studied extensively, is involved in semaphorin-regulated vesicular transport in larval epidermal cells.

Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination.

Ion pumps and channels are responsible for a wide variety of biological functions. Ion pumps transport only one ion during each stimulus-dependent reaction cycle, whereas ion channels conduct a large number of ions during each cycle. Ion pumping rhodopsins such as archaerhodopsin-3 (Arch) are often utilized as light-dependent neural silencers in animals, but they require a high-density light illumination of around 1 mW/mm.

Optical silencing of body wall muscles induces pumping inhibition in Caenorhabditis elegans.

Feeding, a vital behavior in animals, is modulated depending on internal and external factors. In the nematode Caenorhabditis elegans, the feeding organ called the pharynx ingests food by pumping driven by the pharyngeal muscles. Here we report that optical silencing of the body wall muscles, which drive the locomotory movement of worms, affects pumping.

Characterizing Semaphorin Signaling In Vivo Using C. elegans.

A small model animal Caenorhabditis elegans is particularly suitable for genetic analysis, but cell-type-specific biochemistry is a formidable task in this organism. Here we describe techniques utilizing transgenic C. elegans strains expressing epitope-tagged proteins for analyzing biochemical events, such as protein phosphorylation and formation of protein complex, in a small number of a specific group of cells at a defined stage of development.

Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator.

Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations.

X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified.

Optical silencing of C. elegans cells with light-driven proton pumps.

Recent development of optogenetic techniques, which utilize light-driven ion channels or ion pumps for controlling the activity of excitable cells, has greatly facilitated the investigation of nervous systems in vivo. A new generation of optical silencers includes outward-directed proton pumps, such as Arch, which have several advantages over currently widely used halorhodopsin (NpHR). These advantages include the resistance to inactivation during prolonged illumination and the ability to generate a larger optical current from low intensity light.

Pulsed irradiation improves target selectivity of infrared laser-evoked gene operator for single-cell gene induction in the nematode C. elegans.

Methods for turning on/off gene expression at the experimenter's discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application.

Japanese studies on neural circuits and behavior of Caenorhabditis elegans.

The nematode Caenorhabditis elegans is an ideal organism for studying neural plasticity and animal behaviors. A total of 302 neurons of a C. elegans hermaphrodite have been classified into 118 neuronal groups.

Effect of geometry and microstructure of honeycomb TCP scaffolds on bone regeneration.

In recent years, artificial biological materials have been commonly used for the treatment of bone tissue defects caused by trauma, tumors, or surgical stress. Although tricalcium phosphate (TCP) is a promising absorbent bone tissue reconstruction biomaterial, it has been reported that its biocompatibility and osteoconductivity depend on its preparation method and sintering temperature. In addition, although it is thought that the microenvironment produced by the extracellular matrix plays an important role in cell growth and differentiation, there have been few studies on how the geometric structure of artificial biological materials affects cells.

A blue-shifted light-driven proton pump for neural silencing.

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing.

Infrared laser-induced gene expression in targeted single cells of Caenorhabditis elegans.

Since the dawn of transgenic technology some 40 years ago, biologists have sought ways to manipulate, at their discretion, the expression of particular genes of interest in living organisms. The infrared laser-evoked gene operator (IR-LEGO) is a recently developed system for inducing gene expression in living organisms in a targeted fashion. It exploits the highly efficient capacity of an infrared laser for heating cells, to provide a high level of gene expression driven by heat-inducible promoters.

The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model.

An optogenetic application of proton pump ArchT to C. elegans cells.

Application of novel light-driven ion channel/pumps would benefit optogenetic studies of Caenorhabditis elegans. A recent study showed that ArchT, a novel light-driven outward proton pump, is >3 times more light-sensitive than the Arch proton pump. Here we report the silencing effect of ArchT in C.

Optical silencing of C. elegans cells with arch proton pump.

Background: Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses.

A shift of the TOR adaptor from Rictor towards Raptor by semaphorin in C. elegans.

The target of rapamycin (TOR), a central regulator for cell growth and metabolism, resides in the two functionally distinct complexes TORC1 and TORC2, which are defined by their adaptors Raptor and Rictor, respectively. How the formation of the two TORCs is orchestrated remains unclear. Here we show the control of TOR partnering by semaphorin-plexin signalling in Caenorhabditis elegans.

Effect of fluoride-substituted apatite on in vivo bone formation.

Biological apatites are characterized by the presence of minor constituents such as magnesium (Mg), chloride (Cl), or fluoride (F) ions. These ions affect cell proliferation and osteoblastic differentiation during bone tissue formation. F-substituted apatites are being explored as potential bonegraft materials.

Localization of antimicrobial peptides human beta-defensins in minor salivary glands with Sjögren's syndrome.

Sjögren's syndrome is a common systemic autoimmune disease associated with inflammatory cells that infiltrate exocrine glands. The antimicrobial peptides human beta-defensin-1, human beta-defensin-2, and human beta-defensin-3 are expressed in various human epithelial cells and in normal salivary glands. Antimicrobial peptides provide local protection against infection and participate in inflammatory responses.

Effect of a new titanium coating material (CaTiO3-aC) prepared by thermal decomposition method on osteoblastic cell response.

Titanium and hydroxyapatite (HA) are widely used as biomaterials for dental and medical applications. HA-coated titanium implants have excellent biocompatibility and mechanical properties. However, the adherence of HA film formed on titanium substrate is weak because of the lack of chemical interaction between HA and titanium.

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser-evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock-mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Semaphorin controls epidermal morphogenesis by stimulating mRNA translation via eIF2alpha in Caenorhabditis elegans.

Conserved semaphorin-plexin signaling systems govern various aspects of animal development, including axonal guidance in vertebrates and epidermal morphogenesis in Caenorhabditis elegans. Here we provide in vivo evidence that stimulation of mRNA translation via eukaryotic initiation factor 2alpha (eIF2alpha) is an essential downstream event of semaphorin signaling in C. elegans.

The PLEXIN PLX-2 and the ephrin EFN-4 have distinct roles in MAB-20/Semaphorin 2A signaling in Caenorhabditis elegans morphogenesis.

Semaphorins are extracellular proteins that regulate axon guidance and morphogenesis by interacting with a variety of cell surface receptors. Most semaphorins interact with plexin-containing receptor complexes, although some interact with non-plexin receptors. Class 2 semaphorins are secreted molecules that control axon guidance and epidermal morphogenesis in Drosophila and Caenorhabditis elegans.

Improved method for visualizing cells revealed dynamic morphological changes of ventral neuroblasts during ventral cleft closure of Caenorhabditis elegans.

The formation of intricate and functional biological structures depends on the dynamic changes of cellular morphology. Confocal laser scanning microscopy (CLSM) is a widely used method to reveal the three-dimensional (3-D) structure of cells during the development of Caenorhabditis elegans (C. elegans) and other model organisms.