Rice dwarf disease is caused by rice dwarf virus (RDV) transmitted to rice plants by leafhoppers (Recilia dorsalis and Nephotettix cincticeps). We proposed the mechanism how RDV, which can multiply in both plants and insect vectors, transmit to adjacent cells in insect vector cells in previous report. That idea was conceived from the tomogram of the RDV-infected cells reconstructed from dual axis tilt series. We also reported the role of outer capsid of RDV composed of double shells by investigating the tubular crystals of P8, major outer capsid protein. This review summarizes these two topics.
Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. It has been suggested by electrophysiological studies that gap junctions possess multiple gating mechanisms while only one structural model, a subunit rotation model, was proposed. Here we report the electron crystallographic structure of a connexin 26 mutant (Cx26M34A) and a projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2-7 (Cx26M34Adel2-7). The three-dimensional map of Cx26M34A shows a prominent density in the pore of each hemichannel, suggesting that physical blockage may play an important role that underlies gap junction channel regulation. The projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full length Cx26 channel. Our structures allow us to suggest that the two docked hemichannels can independently function and may regulate their activity autonomously with a plug that most likely consists of the connexin N-terminus in the vestibule. This gating model offers insights into an alternate mechanism on how gap junction channels gate in response to cellular stimuli.
Dynein is a molecular motor that moves along a microtubule. Dynein interacts with a microtubule using the microtubule-binding domain at the tip of its stalk, and a predominant model for the movement involved a rotation of the stalk relative to microtubules. However, the stalk, which is a single coiled-coil, was difficult to observe in the microtubule-bound state, either by negative staining or by cryo EM. Here, using a new method “cryo-positive stain EM”, which is a combination of uranyl acetate staining and cryo EM, we succeeded in observing microtubule-bound stalks. Contrary to the above model, the stalks did not change angles relative to microtubules between the two nucleotide states we observed.
Single particle image analysis by electron cryomicroscopy (cryoEM) is a powerful tool for structural analysis of macromolecular complexes such as ribosomes and viruses. However, because of the extremely low signal/noise ratio and low contrast of cryoEM images, molecules smaller than a molecular weight of 100 kDa are generally difficult to visualize and analyze their structures. We solved the structure of a very small DNA nanostructure whose molecular weight is only 78 kDa by cryoEM and single particle image analysis. Here, we demonstrate that this technique allows us to deduce structural information of sufficient resolution to reveal the absolute 3D configuration of a designed DNA nanostructure. We successfully visualized the DNA helix of a self-assembled DNA tetrahedron, each edge of which consists only of a 7-nm, 20-basepair duplex. Structural analysis at such high resolution by cryoEM image analysis is unprecedented for any biological molecule of such a small size.
The laser irradiated GaAs-cathode with NEA surface can produce an electron beam with spin polarization and other attractive performances. Present status of best performances, such as polarization, quantum efficiency, current density, brightness, energy resolution, and continuous operation time etc. are explained using our experimental data. Finally, a recent work to enable the real-time observation of the magnetic domain formation process using a new polarized electron source is reported.
Aquaporins are membrane water channel proteins through which water as well as some small solutes permeates the lipid bilayer. So far thirteen isoforms (AQP0-AQP12) have been identified in mammals. They are widely distributed in most water-handling organs. In the kidney, segment-specific expression of AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11 in the renal tubular epithelium enables water reabsorption to produce concentrated urine. AQP2 in collecting duct cells translocates between intracellular vesicles and the cell surface via a membrane trafficking mechanism. In salivary glands, AQP5 is present in the apical membrane of the acinar cells and plays an important role in saliva secretion. There are no histochemical data showing the change of the intracellular distribution of AQP5 according to the regulation of water secretion. On the other hand, exocytotic release of secretory granules and following endocytic membrane retrieval cause some change of AQP5-distribution in the membrane. This is a very interesting result to understand the membrane dynamics according to exocytosis and endocytosis.
Spermatozoa are specialized for transport of genome and egg-activating factors. It is important to understand the structural molecular basis of sperm flagella to comprehend sperm movement. In addition to axoneme, flagella of mammalian spermatozoa have a peri-axonemal structure composed of accessory structures, such as outer dense fibers, fibrous sheath, satellite fibrils, mitochondrial sheath, which is connected to axoneme in flagella. Its structure is more complex than that of lower vertebrates that lacks the accessory structures. Recent progress in identification and analyses of the molecules consisting of sperm flagella provides important information for understanding the mechanisms of sperm flagella movement. We here outline advances of recent researches concerning the structures and functions of the accessory structures in mammalian sperm flagella.
In this lecture, a concept of three-dimensional imaging characteristics of optical imaging systems, applicable to both optical and electron microscopes, are described with a demonstration of image observations using a transmission optical microscope. These experimental results are, then, interpreted and discussed on the basis of the three-dimensional optical transfer function (3D-OTF) of the transmission microscopes. Finally, new imaging techniques, using active control for the illumination/imaging systems followed by successive image processing, are proposed to improve the tree-dimensional imaging characteristics of the original microscope.
Mammals have two cell types for melanin-producing cells based on their distinct embryonic origins. Retinal pigment epithelium (RPE) cells originate from the dorsal portion of the optic vesicle. RPE is essential for the visual acuity. Melanocytes are derived from the neural crest uniquely formed in vertebrate embryos. They are indispensable not only for protecting organisms from UV damage but also for the hearing acuity. One of the long-term goals of our research group is to elucidate the molecular mechanisms by which pigment cells develop and differentiate in multicellular organisms in order to infer the evolution of those mechanisms and to predict the roles of these cells. To this end, morphological analyses of these pigment cells are a central and indispensable line of research. Here we introduce our recent progress from our related researches.
During cytokinesis, the final step of cell cycle, actin and myosin fibers assemble at the equatorial cell cortex and form the contractile ring. The motor activity of myosin II is believed to generate constriction force that cleaves a parent cell into two daughter cells. Anaphase microtubules provide the specification signal for the contractile ring, yet the nature of the signal remains unclear. In this review, I discuss how microtubules specify the contractile ring. I show that the small GTPase Rho and a complex of Rho regulators (ECT2 RhoGEF, MgcRacGAP, MKLP1 kinesin) act as a cleavage signal that specifies the contractile ring. I also discuss the visualization of the cleavage signal generated by anaphase microtubule, using live cell imaging techniques.
Analytical electron microscopy (AEM) with an X-ray energy-dispersive spectrometry (XEDS) method is one of the most powerful approaches for materials characterization in the nanometer scale. This approach is further improved with the recent developments of aberration-correction technique, which can provide sub-nm incident probes with sufficient currents to generate efficient X-ray signals. In addition, new approaches such as spectrum imaging are available. In this manuscript, we describe (1) improvement of quantitative X-ray analysis, (2) applications of multivariate statistical analysis to spectrum image datasets to improve analytical sensitivities of limited signals and (3) atomic-level X-ray analysis using aberration-corrected instruments, mainly based on our recent research projects. Furthermore, the future trends on X-ray analysis in AEM are also discussed.
A novel characterization technique based on atomic force microscopy (AFM) is introduced, where AFM is utilized as nano-scale palpation technique. The technique maps surface mechanical properties of polymeric materials as well as their artifact-free true topographic images. Several analytical models those can be used in this technique and their applicable limits are also discussed. Finally, a example to exhibit the advantage of this technique is shown.
Aiming at controlling self-assembly of nanostructures using surface atomic steps as templates, we observed various surface phenomena on Si(111) dynamically using a low-energy electron microscope. Motion of steps during annealing, crystal growth, and phase transition enabled us to clarify the mechanism of the surface mass diffusion and ways of controlling the step shapes dynamically. We also showed from dynamical observations of nucleation and growth of nanostructures that the steps are useful in controlling their arrangements.