Alu Konno

Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.

Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge.

Distribution and structural diversity of cilia in tadpole larvae of the ascidian Ciona intestinalis.

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan.

Microscopic visualization of testosterone in mouse testis by use of imaging mass spectrometry.

AbstractTestosterone is one of the androgens synthesized from cholesterol as a precursor in the Leydig cells of testes. Since the ionization efficiency of testosterone in matrix-assisted laser desorption ionization (MALDI) is quite low, visualization of testosterone by using MALDI-imaging mass spectrometry (MALDI-IMS) has been considered difficult. To overcome this problem, we used two types of on-tissue derivatization techniques, which were achieved by pyridine sulfur trioxide and Girard’s T (GT) reagent, to introduce a polar group into testosterone molecule with the aim to increase the sensitivity. Derivatization by use of GT reagent provided excellent results, superior to those obtained with pyridine sulfur trioxide, in terms of ionization efficiency, molecular specificity, and tissue damage. In GT derivatized testis tissues of mice treated with human chorionic gonadotropin (hCG), testosterone was broadly observed both inside and outside the seminiferous tubules by using an iMScope. To evaluate our imaging results, we performed quantification experiments of underivatized testosterone extracted from hCG-treated testes and control testes using LC-MS/MS. We confirmed the 256-fold concentration change between hCG-treated tissues and control tissues. We also confirmed the 228-fold change in detected peak intensities between hCG-treated tissue sections and control tissue sections in imaging results. We consider our tissue preparation methods for IMS provide high sensitivity with high precision. In addition, high-spatial definition IMS was also available, and we confirmed testosterone had mainly accumulated on the surface of the Leydig cells. Graphical abstractGirard’s T-testosterone (GT-Ts) provides the fragment ion at m/z 343.24. Clear GT-Ts signal was detected in hCG treated mouse testis not only as spectra but also as a mass image

Branchial Cilia and Sperm Flagella Recruit Distinct Axonemal Components

Eukaryotic cilia and flagella have highly conserved 9 + 2 structures. They are functionally diverged to play cell-type-specific roles even in a multicellular organism. Although their structural components are therefore believed to be common, few studies have investigated the molecular diversity of the protein components of the cilia and flagella in a single organism. Here we carried out a proteomic analysis and compared protein components between branchial cilia and sperm flagella in a marine invertebrate chordate, Ciona intestinalis. Distinct feature of protein recruitment in branchial cilia and sperm flagella has been clarified; (1) Isoforms of α- and β-tubulins as well as those of actins are distinctly used in branchial cilia or sperm flagella. (2) Structural components, such as dynein docking complex, tektins and an outer dense fiber protein, are used differently by the cilia and flagella. (3) Sperm flagella are specialized for the cAMP- and Ca2+-dependent regulation of outer arm dynein and for energy metabolism by glycolytic enzymes. Our present study clearly demonstrates that flagellar or ciliary proteins are properly recruited according to their function and stability, despite their apparent structural resemblance and conservation.

Network structure of projections extending from peripheral neurons in the tunic of ascidian larva

In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole‐larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 9+2 axonemes were accompanied by a vesicle‐containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle‐containing fibrous mass and no 9+2 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic. Developmental Dynamics 239:2278–2287, 2010.© 2010 Wiley‐Liss, Inc.

Multidimensional Analysis of Uncharacterized Sperm Proteins in Ciona intestinalis: EST-Based Analysis and Functional Immunoscreening of Testis-Expressed Genes

An extensive analysis of testis-expressed genes in the ascidian Ciona intestinalis explored a large number of genes of unknown function. Here we characterized these genes or gene products in a multidimensional manner. We analyzed genes both highly and uniquely expressed in the testis, as expected from the EST analysis. Immunolocalization of these proteins revealed that they are all expressed in sperm. Sperm membrane/matrix proteins play essential roles in cell responses and intracellular signaling at fertilization. By immunoscreening with antisera against the detergentsoluble and membrane fractions of sperm, we isolated 49 potential cDNA clones for membrane/matrix proteins. These included several unidentified genes, including a protein with sequence similarity to mammalian testicular cancer antigen Sp17. These data should facilitate exploration of the functions of uncharacterized sperm proteins and ultimately elucidate new molecular mechanisms in sperm physiology.

Ttll9-/- mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating.

ABSTRACT Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9−/−) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9−/− sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9−/− spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after anti-hook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns. Highlighted Article: A tubulin polyglutamylase, TTLL9, is involved in the establishment of normal interdoublet heterogeneity of sperm axoneme, and is required for structural integrity and fluid motility of murine sperm flagella.

Improved vessel painting with carbocyanine dye-liposome solution for visualisation of vasculature

Vessel painting is one of the most accessible and cost-effective techniques for visualising vasculature by fluorescence microscopy. In this method, the hydrophobic carbocyanine dye DiIC18 labels the plasma membrane via insertion of its alkyl chains into the lipid bilayer. A major disadvantage of this procedure is that it does not stain veins and some microvessels in mouse brain. Furthermore, DiIC18 molecules can aggregate during perfusion, thereby occluding arteries and reducing the success rate and reproducibility of the experiment. To overcome these problems, we developed an improved vessel painting procedure that employs neutral liposomes (NLs) and DiIC12. NLs prevented DiI aggregation under physiological conditions whereas DiIC12 showed enhanced dye incorporation into liposomes and consequently increased staining intensity. Using this method, we successfully labelled all major blood vessel types in the mouse brain, including both veins and microvessels. Thus, liposome-mediated vessel painting is a simple and efficient method for visualising vasculature.

Correction of spherical aberration in multi-focal multiphoton microscopy with spatial light modulator

We demonstrate that high-quality images of the deep regions of a thick sample can be obtained from its surface by multi-focal multiphoton microscopy (MMM). The MMM system incorporates a spatial light modulator to separate the excitation beam into a multi-focal excitation beam and modulate the pre-distortion wavefront to correct spherical aberration (SA) caused by a refractive index mismatch between the immersion medium and the biological sample. When fluorescent beads in transparent epoxy resin were observed using four SA-corrected focal beams, the fluorescence signal of the obtained images was ~52 times higher than that obtained without SA correction until a depth of ~1100 μm, similar to the result for single-focal multiphoton microscopy (SMM). The MMM scanning time was four times less than that for SMM, and MMM showed an improved fluorescence intensity and depth resolution for an image of blood vessels in the brain of a mouse stained with a fluorescent dye.

Erratum to: Microscopic visualization of testosterone in mouse testis by use of imaging mass spectrometry

1 Division of Advanced Science and Biotechnology, Graduate School of Engineering of Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Department of Medical Chemistry, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan 3 Department of Urology and Andrology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan 4 Department of Optical Imaging, Institute for Medical Photonics Research, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan