Koji Ikegami

University of Hamamatsu, Hamamatu, Shizuoka, Japan

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Publications (31)131.08 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.
    Biochimie 10/2014; · 3.14 Impact Factor
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    ABSTRACT: Imaging mass spectrometry (IMS) is a powerful tool for detecting and visualizing biomolecules in tissue sections. Most IMS instruments are equipped with a high-vacuum chamber for matrix-assisted laser desorption/ionization (MALDI). However, the use of high-vacuum conditions restricts the usage of the matrix to less-volatile substances. We recently developed an atmospheric pressure MALDI instrument, named the ‘mass microscope’, to avoid this problem. The atmospheric pressure condition enables us to use volatile matrices. In this study, we compared the reliability of a volatile matrix, 2,5-dihydroxyacetophenone (DHAP) with that of a conventionally used matrix, 2,5-dihydroxybenzoic acid (DHB). Compared with the results obtained with DHB, the mass spectra obtained with DHAP showed a variety of signal species in a mass region corresponding to phospholipid species in both the positive and negative ion modes without any matrix-derived signals. DHAP provided highly strong signals of ganglioside (GM1) species. We also compared two matrix deposition techniques for DHAP matrix: sublimation and spraying. We found that the sublimation method for applying DHAP matrix on a tissue surface can maintain ion images of lipid species, including ganglioside species only in a tissue. In contrast, the spraying method led the diffusion of GM1 species toward the outside of the tissue sections. Consequently, the sublimation method for applications of DHAP is adequate to analyze some types of lipid species including GM1 in atmospheric pressure MALDI IMS. Copyright © 2014 John Wiley & Sons, Ltd.
    Surface and Interface Analysis 06/2014; · 1.39 Impact Factor
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    ABSTRACT: Breast cancer is the most common cancer among women worldwide. The molecular characterization of breast tumor cells by using single-cell lipidomics remains relatively unexplored. Here, we introduce a time-of-flight secondary-ion mass spectrometry (TOF-SIMS) approach to visualize the lipids in individual breast cancer cells. The SKBR-3 breast cancer cell line was cultured and dispersed into individual cells. After attachment to a substrate, the cells were rinsed with ammonium acetate and were analyzed using TOF-SIMS. The instrument was operated with Bi32+ as the primary ion. The distributions of ions, including positively charged phosphocholine, and negatively charged phosphates and fatty acids, were simultaneously visualized. These ions were distributed predominantly at the cell attachment sites. The signal intensities of fatty acid ions were determined from the mass spectra at the regions-of-interest. The results of fatty acid analyses on breast cancer cells were consistent with those of our previous study in which prominent expression of stearoyl-CoA desaturase 1 in breast cancer cells was demonstrated. Static TOF-SIMS was shown to be an effective method for determining the lipid molecular signature of the plasma membrane of individual breast cancer cells. Copyright © 2014 John Wiley & Sons, Ltd.
    Surface and Interface Analysis 04/2014; · 1.39 Impact Factor
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    ABSTRACT: Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study we examined the activity of purified CCP5 towards synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multi glutamate residues from side chains and C-termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' gamma-linked residues. The rate of cleavage of alpha linkages by CCP5 is considerably slower than that of removal of a single gamma-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both alpha- and gamma-linked glutamate from tubulin.
    Journal of Biological Chemistry 09/2013; · 4.60 Impact Factor
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    ABSTRACT: BACKGROUND & AIMS: Several lipid synthesis pathways play important roles in development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and the alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS: Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and western blotting were carried out to examine the protein and mRNA levels of LPCATs which catalyze lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kind of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown LPCAT1 on cell proliferation, migration, and invasion in HCC cell lines. RESULTS: IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2 -position and the reduction of LPC with palmitic acid at the sn-1 -position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC-to-PC conversion, were more abundant in HCCs than in surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did vice versa. CONCLUSIONS: Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions for HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.
    Journal of Hepatology 04/2013; · 9.86 Impact Factor
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    ABSTRACT: Abdominal aortic aneurysm (AAA) is a common disease among elderly individuals. However, the precise pathophysiology of AAA remains unknown. In AAA, an intraluminal thrombus prevents luminal perfusion of oxygen, allowing only the adventitial vaso vasorum (VV) to deliver oxygen and nutrients to the aortic wall. In this study, we examined changes in the adventitial VV wall in AAA to clarify the histopathological mechanisms underlying AAA. We found marked intimal hyperplasia of the adventitial VV in the AAA sac; further, immunohistological studies revealed proliferation of smooth muscle cells, which caused luminal stenosis of the VV. We also found decreased HemeB signals in the aortic wall of the sac as compared with those in the aortic wall of the neck region in AAA. The stenosis of adventitial VV in the AAA sac and the malperfusion of the aortic wall observed in the present study are new aspects of AAA pathology that are expected to enhance our understanding of this disease.
    PLoS ONE 02/2013; 8(2):e57398. · 3.53 Impact Factor
  • American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California; 05/2012
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    ABSTRACT: Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.
    Nature Genetics 02/2012; 44(2):193-9. · 29.65 Impact Factor
  • Alu Konno, Mitsutoshi Setou, Koji Ikegami
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    ABSTRACT: 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.
    International review of cell and molecular biology 01/2012; 294:133-70. · 4.52 Impact Factor
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    ABSTRACT: Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.
    Journal of Biological Chemistry 12/2011; 287(8):5290-300. · 4.60 Impact Factor
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    ABSTRACT: The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.
    Journal of Biological Chemistry 12/2011; 287(9):6503-17. · 4.60 Impact Factor
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    ABSTRACT: In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins.
    Biophysical Journal 09/2011; 101(5):1270-6. · 3.83 Impact Factor
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    ABSTRACT: Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region.
    Medical Molecular Morphology 09/2010; 43(3):158-64. · 1.07 Impact Factor
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    ABSTRACT: Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.
    Journal of Biological Chemistry 07/2010; 285(30):22936-41. · 4.60 Impact Factor
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    ABSTRACT: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.
    PLoS ONE 07/2010; 5(7):e11755. · 3.53 Impact Factor
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    ABSTRACT: Airway epithelial cilia protect the mammalian respiratory system from harmful inhaled materials by providing the force necessary for effective mucociliary clearance. Ciliary beating is asymmetric, composed of clearly distinguished effective and recovery strokes. Neither the importance of nor the essential components responsible for the beating asymmetry has been directly elucidated. We report here that the beating asymmetry is crucial for ciliary function and requires tubulin glutamylation, a unique posttranslational modification that is highly abundant in cilia. WT murine tracheal cilia have an axoneme-intrinsic structural curvature that points in the direction of effective strokes. The axonemal curvature was lost in tracheal cilia from mice with knockout of a tubulin glutamylation-performing enzyme, tubulin tyrosine ligase-like protein 1. Along with the loss of axonemal curvature, the axonemes and tracheal epithelial cilia from these knockout (KO) mice lost beating asymmetry. The loss of beating asymmetry resulted in a reduction of cilia-generated fluid flow in trachea from the KO mice. The KO mice displayed a significant accumulation of mucus in the nasal cavity, and also emitted frequent coughing- or sneezing-like noises. Thus, the beating asymmetry is important for airway ciliary function. Our findings provide evidence that tubulin glutamylation is essential for ciliary function through the regulation of beating asymmetry, and provides insight into the molecular basis underlying the beating asymmetry.
    Proceedings of the National Academy of Sciences 06/2010; 107(23):10490-5. · 9.81 Impact Factor
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    ABSTRACT: Imaging mass spectrometry (IMS) provides a novel opportunity for visualization of molecular ion distribution. Currently, there are two major ionization techniques, matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS) are widely used for imaging of biomolecules in tissue samples. MALDI and SIMS-based IMS have the following features; measurable mass ranges are wide and small, and the spatial resolutions are low and high, respectively. To the best of our knowledge, this is a first report to identify the lipids in cultured mammalian neurons by MALDI-IMS. Further, those neurons were analyzed with SIMS-IMS in order to compare the distribution pattern of lipids and other derived fragments. The parameters which influence the identification of lipids in cultured neurons were optimized in order to get an optimum detection of lipid molecules. The combined spatial data of MALDI and SIMS supported the idea that the signals of small molecules such as phosphatidylcholine head groups and fatty acids (detected in SIMS) are derived from the intact lipids (detected in MALDI-IMS). Copyright © 2010 John Wiley & Sons, Ltd.
    Surface and Interface Analysis 05/2010; 42(10‐11):1606 - 1611. · 1.39 Impact Factor
  • K. Ikegami, S. Sato, K. Nakamura, M. Setou
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    ABSTRACT: Background / Purpose: Airway epithelial cilia protect mammalian respiratory organs from harmful small particles, e.g. dusts or viruses, by propelling mucus. The mucus flow is generated by vigorous ciliary beatings. Importance and molecular mechanisms of the ciliary beatings are well-studied. Ciliary beatings are asymmetric as they are clearly distinguished between effective and recovery strokes. Neither importance nor essential components of the ciliary beating asymmetry has been elucidated.We present that the beating asymmetry is crucial for ciliary function and requires tubulin polyglutamylation, a unique post-translational modification, which is highly abundant in cilia. Wild-type murine tracheal cilia had axoneme-intrinsic structural curvature independently of rigor dynein bridges. The curvature direction pointed the direction of effective strokes. The axonemal curvature was lost in tracheal cilia by a knockout of a tubulin polyglutamylation-performing enzyme. Along with the loss of axonemal curvature, the knockout axonemes and tracheal epithelial cilia lost beating asymmetry. This resulted in a slowdown of cilia-generated fluid flow in the knockout trachea. The knockout mice displayed severe respiratory defects; they frequently showed cough- and sneeze-like breath noises. Thus, the beating asymmetry is important for airway ciliary function. Main conclusion: Our findings provide evidence that beating asymmetry requires tubulin polyglutamylation and a new insight into the molecular basis underlying the beating asymmetry.
    American Society of Cell Biology Annual Meeting 2009; 03/2010
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    ABSTRACT: We have developed a mass microscopy technique, i.e., a microscope combined with high-resolution matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a powerful tool for investigating the spatial distribution of biomolecules without any time-consuming extraction, purification, and separation procedures for biological tissue sections. Mass microscopy provides clear images about the distribution of hundreds of biomolecules in a single measurement and also helps in understanding the cellular profile of the biological system. The sample preparation and the spatial resolution and speed of the technique are all important steps that affect the identification of biomolecules in mass microscopy. In this Award Lecture Review, we focus on some of the recent developments in clinical applications to show how mass microscopy can be employed to assess medical molecular morphology.
    Medical Molecular Morphology 03/2010; 43(1):1-5. · 1.07 Impact Factor
  • Koji Ikegami, Mitsutoshi Setou
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    ABSTRACT: Microtubules (MTs) play specialized roles in a wide variety of cellular events, e.g. molecular transport, cell motility, and cell division. Specialized MT architectures, such as bundles, axonemes, and centrioles, underlie the function. The specialized function and highly organized structure depend on interactions with MT-binding proteins. MT-associated proteins (e.g. MAP1, MAP2, and tau), molecular motors (kinesin and dynein), plus-end tracking proteins (e.g. CLIP-170), and MT-severing proteins (e.g. katanin) interact with MTs. How can the MT-binding proteins know temporospatial information to associate with MTs and to properly play their roles? Post-translational modifications (PTMs) including detyrosination, polyglutamylation, and polyglycylation can provide molecular landmarks for the proteins. Recent efforts to identify modification-regulating enzymes (TTL, carboxypeptidase, polyglutamylase, polyglycylase) and to generate genetically manipulated animals enable us to understand the roles of the modifications. In this review, we present recent advances in understanding regulation of MT function, structure, and stability by PTMs.
    Cell Structure and Function 02/2010; 35(1):15-22. · 2.35 Impact Factor

Publication Stats

488 Citations
131.08 Total Impact Points


  • 2014
    • University of Hamamatsu
      Hamamatu, Shizuoka, Japan
  • 2009–2014
    • Hamamatsu University School of Medicine
      • Division of Anatomy:Cell Biology and Anatomy
      Hamamatu, Shizuoka, Japan
  • 2010
    • Miyazaki University
      • Frontier Science Research Center
      Миядзаки, Miyazaki, Japan
    • Tokyo Institute of Technology
      • School of Bioscience and Biotechnology
      Tokyo, Tokyo-to, Japan