Ryuta Mizutani

X-ray microtomography in biology

Progress in high-resolution X-ray microtomography has provided us with a practical approach to determining three-dimensional (3D) structures of opaque samples at micrometer to submicrometer resolution. In this review, we give an introduction to hard X-ray microtomography and its application to the visualization of 3D structures of biological soft tissues. Practical aspects of sample preparation, handling, data collection, 3D reconstruction, and structure analysis are described. Furthermore, different sample contrasting methods are approached in detail. Examples of microtomographic studies are overviewed to present an outline of biological applications of X-ray microtomography. We also provide perspectives of biological microtomography as the convergence of sciences in X-ray optics, biology, and structural analysis.

Protein splicing: its discovery and structural insight into novel chemical mechanisms.

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self‐catalytically excised out from a nascent protein precursor and the two flanking sequences (N‐ and C‐exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70‐kDa subunit of the vacuolar H + ‐ATPase) and VDE (a 50‐kDa DNA endonuclease) as an in‐frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure‐directed chemistry, a series of crystal structural studies has been carried out with the use of splicing‐inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N‐terminal junction of the introduced extein polypeptide forms an intermediate containing a five‐membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N‐ and C‐extein segments. This article summarizes biochemical and structural studies on a self‐catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. IUBMB Life, 57: 563‐574, 2005

Computed tomography imaging of the neuronal structure of Drosophila brain

The neural circuit of the central nervous system (CNS) primarily determines brain functions and, as a consequence, controls animal behavior. This paper describes an X-ray microtomographic analysis of the Drosophila larvae CNS, visualizing the neural network embedded in the three-dimensional structure of the nerve tissue. In fluorescence confocal microscopy, absorbance at emission or excitation wavelengths interferes with the fluorescence detection. In contrast, transparency of the nerve tissue to hard X-rays enables tomographic analysis of the intact CNS without sectioning. Yet the nerve tissue is composed of light elements that give little contrast in a hard X-ray transmission image. The contrast was enhanced by staining neuropils in the CNS with metal elements. The obtained structure revealed the internal architecture of the CNS. This metal-staining microtomographic analysis can be applied to any nerve tissues, thereby shedding light on the underlying structural basis of neural functions.

X-Ray Microtomographic Imaging of Three-Dimensional Structure of Soft Tissues

We report the x-ray microtomographic imaging of three-dimensional (3D) structure of soft tissues. The transparency of biological tissue to hard x-rays enables radiographic analysis of tissue entrails. However, biological tissues are mainly composed of light elements, which produce little contrast in a hard x-ray transmission image. Tissue structures were visualized by contrasting biological constituents with heavy elements. Efficient x-ray absorption by heavy-element dyes allowed the radiographic visualization of microstructures of soft tissues. The high-resolution computed tomography analysis provided the 3D microstructure of these microcontrasted tissues. Element-selective visualization of the stained tissue using x-ray absorption edges revealed the specific architecture of internal components. The structures obtained were used for rapid prototyping, giving 3D copies of human capillary vessels and fruit fly body.

Microtomographic Analysis of Neuronal Circuits of Human Brain

We report a 3D analysis of the neuronal circuits of human cerebral cortex. Neuronal circuits, which are essential for brain functions, are built up by neurons as a 3D network, so tracing the 3D neuronal network of human cerebral cortex is the first step to understanding the mechanism of human brain functions. The cortical microstructures were visualized by X-ray microtomographic imaging of adult frontal cortex tissue stained with metal impregnation. Skeletonized wire models were built by tracing the 3D distribution of X-ray absorption coefficients. The obtained neuronal models were composed of 240 pyramidal neurons and 131 interneurons. Capillary vessel structures along with blood cells in the capillary lumen were also visualized and traced to build capillary network models. Possible neuronal circuits were analytically resolved from the skeletonized wire models. The operating mechanism of the resolved circuits is discussed on the basis of neurotransmission in the circuits. The results also indicate that X-ray microtomography is a potential method of visualizing the neuronal circuits of the brain.

Protein splicing of yeast VMA1-derived endonuclease via thiazolidine intermediates.

Protein splicing precisely excises out an internal intein segment from a protein precursor, and concomitantly ligates the N- and C-terminal extein polypeptides flanking the intein. A recombinant X10SNS bearing N- and C-extein polypeptides has been prepared for the intein endonuclease derived from the Saccharomyces cerevisiae VMA1 gene. X10SNS has replacements of C284S, H362N and C738S, and forms the intein and extein segments in the crystal lattice. The crystal structure of X10SNS revealed a linkage between the N- and C-extein segments, and showed that the C284 amino group of the resultant intein segment is in interaction with the G283 O atom of the N-extein segment. A mechanism for the final S --> N acyl shift step proposes that a tetrahedral intermediate involves a five-membered thiazolidine ring at G283-C738 junction. An oxyanion of the thiazolidine intermediate is to be stabilized by the C284 N atom.

Homing at an extragenic locus mediated by VDE (PI‐SceI) in Saccharomyces cerevisiae

PI‐SceI (VDE), a homing endonuclease with protein splicing activity, is a genomic parasite in the VMA1 gene of Saccharomyces cerevisiae. In a heterozygous diploid of the VDE‐less VMA1 allele and a VDE‐containing VMA1 allele, VDE specifically cleaves its recognition sequence (VRS) in the VDE‐less VMA1 allele at meiosis, followed by ‘homing’, i.e. a conversion to a VDE‐containing allele. We found that upon VDE expression, homing of a marker gene at an extragenic locus occurs only when a 45 bp element containing the VRS is inserted at its allelic site, while mutants of VDE with no endonuclease activity lack authentic extragenic homing activity. Thus, both the VRS and VDE are required for homing. Insertion of the VRS in a homozygous diploid significantly lowered the spore germination ability, indicating that a template for gene repair at its allelic locus is essential for efficient homing and survival of yeast cells. Copyright

Estimation of presampling modulation transfer function in synchrotron radiation microtomography

The spatial resolution achieved by recent synchrotron radiation microtomographs should be estimated from the modulation transfer function (MTF) on the micrometer scale. Step response functions of a synchrotron radiation microtomograph were determined by the slanted edge method by using high-precision surfaces of diamond crystal and ion-milled aluminum wire. Tilted reconstruction was introduced to enable any edge to be used as the slanted edge by defining the reconstruction pixel matrix in an arbitrary orientation. MTFs were estimated from the step response functions of the slanted edges. The obtained MTFs coincided with MTF values estimated from square-wave patterns milled on the aluminum surface. Although X-ray refraction influences should be taken into account to evaluate MTFs, any flat surfaces with nanometer roughness can be used to determine the spatial resolutions of microtomographs.

Submicrometer tomographic resolution examined using a micro-fabricated test object.

To estimate the spatial resolution of microtomographs, a test object on the submicrometer scale was prepared by focused ion beam milling and subjected to microtomographic analysis. Since human tissues are composed of cells and extracellular matrices with micrometer and submicrometer structures, it is important to investigate the three-dimensional spatial resolution of microtomographs used to visualize microstructures of human tissues. The resolutions along the direction within the tomographic slice plane (in-plane resolution) and perpendicular to it (through-plane resolution) were determined from the modulation transfer function of square-wave patterns. The in-plane resolution was estimated to be 1.2 microm from the modulation transfer function of the non-zoom image. In contrast, the zoom image gave the in-plane resolution of 0.8 microm. This in-plane resolution is comparable to the through-plane resolution, which was estimated to be 0.8 microm. Although the two-dimensional radiographs were taken with the pixel width of half the X-ray optics resolution, these three-dimensional resolution analyses indicated that the zoom reconstruction should be performed to achieve the in-plane resolution comparable to the X-ray optics resolution. The submicrometer three-dimensional analysis was applied in the structural study of human cerebral tissue stained with high-Z elements and the obtained tomograms revealed that the microtomographic analysis allows visualization of the subcellular structures of the cerebral tissue.

Element-specific microtomographic imaging of Drosophila brain stained with high-Z probes

An application of X-ray microtomography to the Drosophila adult brain stained with colloidal gold and a platinum compound is described. The transparency of biological tissue to hard X-rays enables tomographic visualization of the three-dimensional structure of tissue entrails. Each high-Z element was visualized as a three-dimensional structure from the difference absorption coefficient image at the corresponding L(III) absorption edge. The cortex of the optic lobe was selectively visualized by the specific adsorption of colloidal gold. The entire structure revealed by the platinum impregnation allowed the anatomical assignment of the gold-stained structures. Selective staining and specific visualization of biological tissues at micrometer resolution should elucidate the three-dimensional cellular organization essential for the understanding and application of biological microstructures.