DOCSLIB.ORG

ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN Αiibβ3 TRAFFICKING, PLATELET SPREADING, and CLOT RETRACTION

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN Αiibβ3 TRAFFICKING, PLATELET SPREADING, and CLOT RETRACTION University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2015 ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION Yunjie Huang University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Huang, Yunjie, "ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION" (2015). Theses and Dissertations--Molecular and Cellular Biochemistry. 20. https://uknowledge.uky.edu/biochem_etds/20 This Doctoral Dissertation is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Molecular and Cellular Biochemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Yunjie Huang, Student Dr. Sidney W. Whiteheart, Major Professor Dr. Michael D. Mendenhall, Director of Graduate Studies ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Yunjie Huang Lexington, KY Director: Dr. Sidney W. Whiteheart, Professor of Molecular and Cellular Biochemistry Lexington, Kentucky 2015 Copyright © Yunjie Huang 2015 ABSTRACT OF DISSERTATION ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, -/- in response to thrombin, were unaffected. Arf6 mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase- activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent- insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets. KEYWORDS: Platelets, Arf6, GIT1, Integrin αIIbβ3, Trafficking Yunjie Huang March 16th, 2015 ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION By Yunjie Huang Dr. Sidney W. Whiteheart Director of Dissertation Dr. Michael D. Mendenhall Director of Graduate Studies March 16th, 2015 To Fanmuyi, Francis, Irene Acknowledgements I have to say it has been hard but invaluable experience during my graduate study. From the days I arrived in Lexington to the point where I am now, I have been showering in love from many people, including my mentor, my colleges, my friends, my family and beyond. Without these love, it is impossible for me to get to this point. The first person I would like to thank is my mentor and thesis advisor, Dr. Sidney “Wally” Whiteheart. He is absolutely a great person and scientist, and it is my greatest honor to have him as my mentor. His generous willingness gave me the opportunity to touch bench work and to science, an experience that still influences me. As many Ph.D. student may know, there are a lot of hurdles that have to be passing through during this trip, such as reading papers, writing a proposal, taking the qualify exam, giving a scientific presentation, designing experiments, analyzing data, and so on. Dr. Whiteheart is such a nice mentor who is always there for my questions with patient, knowledge, positive and optimistic view, and encouragements. More importantly, he knows what is good for me at a specific level, and feeds me what I need as I grew up in science. His impact will last for the rest of my career. It is still my honor to have Dr. Douglas A. Andres, Dr. Rebecca E. Dutch, and Dr. Susan Smyth in my thesis committee. I appreciate very much their encouragements and comments that are surely indispensable to make my projects progress. I would also thank my outside examiner, Dr. Yinan Wei for her time and comments in the final exam. Additionally, I would like to thank faculty members from the Biochemistry department for their support. Particularly, I would like to thank Dr. Louis B. Hersh (in addition to Dr. Douglas A. Andres, Dr. Susan Smyth), for his recommendation for my successful pre- doctoral fellowship application. I also thank Dr. Michael D. Mendenhall for his support during my preparation for my final exam; thank Dr. Sabire Ozcan, Dr. Craig Vander Kooi, and Dr. Daniel Noonan, as my student seminars advisors, for their comments; thank Dr. Qingjun Wang for her support during collaboration; and thank Dr. Zhenyu Li for his sharing with reagents and tools. iii I am always feeling blessed to have been working in the Whiteheart lab because it is surrounded by such a group of people who make the research life even more fun. Specially, I want to thank Dr. Wangsun Choi and Dr. Zubair Karim, the pioneers of Arf6 project, for their training and support. I also want to thank Dr. Shaojing Ye, Dr. Chunxia Zhao, Dr. Qiansheng Ren, Dr. Garland Crawford, Dr. Rania Al. Hawas, Dr. Deepa Jonnalagadda, Dr. Elena A. Matveeva, Dr. Mike C. Chicka, Jinchao Zhang, Meenakshi Banerjee, and Smita Joshi for their support and for being like a family to me. I also appreciate the Biochemistry students, postdocs and staff for their support and friendship. My Arf6 project would not be able to make progress without the help from Dr. Yasunoki Kanaho at University of Tsukuba in Japan, who provided me the Arf6-floxed mice strain, and from Dr. Richard T. Premont at Dr. Duke University, who provided me the GIT1-floxed mice strain. Their generosity is greatly appreciated. I would like to thank my friends in Lexington, particularly Brothers and Sisters in Lexington Chinese Christian Church, for their support during my living in Lexington. I would like to thank my family for their love and support. First of all, I want to express my most sincere acknowledgement to my wife Fanmuyi Yang. She is a great life partner who I could rely on to overcome each difficulty encountered in life and who bring to family the joy and a positive attitude. I really appreciate her hard work and the sacrifices that she has made for the sake of my work and career. The most importantly, I thank her for her love that leads me to become a better person. Secondly, I would like to thank my parents- in-law, Jiemin Yang and Yu Fang, for their love and great support, particularly during the times when my son and my daughter were born.
Load more

Recommended publications
  • Abl Family Tyrosine Kinases Govern Igg Extravasation in the Skin in a Murine Pemphigus Model
    ARTICLE https://doi.org/10.1038/s41467-019-12232-3 OPEN Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model Sachiko Ono1, Gyohei Egawa1, Takashi Nomura1, Akihiko Kitoh1, Teruki Dainichi 1, Atsushi Otsuka1, Saeko Nakajima1, Masayuki Amagai2, Fumi Matsumoto3, Mami Yamamoto 3, Yoshiaki Kubota4, Toshiyuki Takai5, Tetsuya Honda1 & Kenji Kabashima 1,6 1234567890():,; The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody- mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood- circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle for- mation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders. 1 Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan. 2 Department of Dermatology, Keio University Graduate School of Medicine, Tokyo, Japan. 3 Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Japan. 4 Department of Anatomy, Keio University School of Medicine, Tokyo, Japan.
    [Show full text]
  • Clathrin-Independent Pathways of Endocytosis
    Downloaded from http://cshperspectives.cshlp.org/ on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Clathrin-Independent Pathways of Endocytosis Satyajit Mayor1, Robert G. Parton2, and Julie G. Donaldson3 1National Centre for Biological Sciences, Tata Institute of Fundamental Research, and Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India 2The University of Queensland, Institute for Molecular Bioscience and Centre for Microscopy and Microanalysis, Queensland 4072, Brisbane, Australia 3Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Correspondence: [email protected] There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. he identification of many of the components Consequently, CME has remained a pre- Tinvolved in clathrin-mediated endocytosis dominant paradigm for following the uptake (CME) and their subsequent characterization of material into the cell.
    [Show full text]
  • Antibodies to Snare Complex Proteins
    ANTIBODIES TO SNARE COMPLEX PROTEINS Antibodies to SNARE Complex Proteins Synaptophysin VAMP/Synaptobrevin SNAP-25 Syntaxin CDCrel-1 Synaptotagmin-1 Munc18-1 Synapsin-1 INTERNATIONAL VERSION www.cedarlanelabs.com/SNARE CEDARLANE® is an ISO 9001 and ISO 13485 registered company Antibodies to SNARE Complex Proteins SNARE proteins are a large protein superfamily consisting of more than 60 members in yeast and mammals. The primary role of these proteins is to mediate fusion of vesicles with their target membrane-bound compartments (such as lysosomes). The most well studied SNARE proteins are those involved in mediating synaptic vesicle docking at the pre-synaptic membrane of neurons. During this process, syntaxin-1, SNAP-25 and munc18-1 associate and form a complex at the pre-synaptic membrane. This complex interacts with synaptobrevin-2 and synaptotagmin-1 located in synaptic vesicles and initiates docking, priming and fusion at the membrane. This fusion event leads to release of the vesicle's cargo into the synaptic cleft, where it can ultimately interact with the post-synaptic neuron. Antibodies to: Synaptophysin Synaptophysin is a 38 KDa synaptic vesicle (SV) glycoprotein containing four transmembrane domains. It is present in SVs of the neuroendocrine system, brain, spinal cord, retina, adrenal medulla and at neuromuscular junctions. Synaptophysin acts as a marker for neuroendocrine tumours and has been used to study the distribution of synapses within the brain due to its ubiquity at these regions. Although the exact function of synaptophysin is still unknown, several lines of evidence suggest it may have many important roles in SV exo and endocytosis. These include regulation of SNARE assembly, fusion pore formation initiating neurotransmitter release, and activation of SV endocytosis.
    [Show full text]
  • 43Rd Annual Scientific and Standardization Committee Meeting
    43rd Annual Scientific and Standardization Committee Meeting June 6­7, 1997 Florence, Italy SCIENTIFIC SUBCOMMITTEE MINUTES 6‐7 June 1997, Florence, Italy Table of Contents Registry of Animal Models of Thrombotic and Hemorrhagic Disorders ....................................................... 2 Biorheology ................................................................................................................................................... 4 Contact Activation ......................................................................................................................................... 7 Control of Anticoagulation ............................................................................................................................ 9 Exogenous Hemostatic Factors Subcommittee: Registry .......................................................................... 15 Factor VIII and Factor IX .............................................................................................................................. 18 Factor XIII .................................................................................................................................................... 22 Fibrinogen and DIC ...................................................................................................................................... 25 Fibrinolysis .................................................................................................................................................. 28 Hemostasis and Malignancy
    [Show full text]
  • SNARE Proteins and the Timing of Neurotransmitter Release
    Molecular Psychiatry (1998) 3, 293–297 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 NEWS & VIEWS SNARE proteins and the timing of neurotransmitter release The SNARE complex proteins have been implicated in exocytotic neurotransmitter release and other forms of membrane fusion. Recent work shows that NSF, the ATPase of the SNARE complex, regulates the kinetics of neurotransmitter release and can thereby control the inte- grative properties of synapses. Time is one of the most critical parameters in the func- hydrolyzes ATP. Because SNAREs are found on both tioning of the brain. Information transfer on the time- the synaptic vesicle membrane and the plasma mem- scale of milliseconds (10−3 seconds) is typical through- brane, it has been postulated that the various SNARE out the brain and in certain brain regions, such as the complexes mediate the interaction between the two auditory brainstem, time differences on the order of membranes before fusion and thus may be necessary microseconds (10−6 seconds) are used to define the fre- for neurotransmitter release.2 quency and location of perceived sounds. Thus infor- Evidence for a role for SNARE proteins in neuro- mation processing not only depends on a fast underly- transmitter release has come from a variety of sources. ing process but also on the precise timing of synaptic The most compelling indication of the central impor- activity. Such high temporal fidelity must rely upon tance of the three membrane SNARE proteins is that very finely-regulated molecular mechanisms. However, these proteins are remarkably specific targets of tetanus until recently the identity of these mechanisms has and botulinum toxins, a group of potent neurotoxins been remarkably elusive.
    [Show full text]
  • Defining the Kv2.1–Syntaxin Molecular Interaction Identifies a First-In-Class Small Molecule Neuroprotectant
    Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant Chung-Yang Yeha,b,1, Zhaofeng Yec,d,1, Aubin Moutale, Shivani Gaura,b, Amanda M. Hentonf,g, Stylianos Kouvarosf,g, Jami L. Salomana, Karen A. Hartnett-Scotta,b, Thanos Tzounopoulosa,f,g, Rajesh Khannae, Elias Aizenmana,b,g,2, and Carlos J. Camachoc,2 aDepartment of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; bPittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; cDepartment of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; dSchool of Medicine, Tsinghua University, Beijing 100871, China; eDepartment of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724; fDepartment of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and gPittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Lily Yeh Jan, University of California, San Francisco, CA, and approved June 19, 2019 (received for review February 27, 2019) + The neuronal cell death-promoting loss of cytoplasmic K follow- (13). The Kv2.1-dependent cell death pathway is normally initiated ing injury is mediated by an increase in Kv2.1 potassium channels in by the oxidative liberation of zinc from intracellular metal-binding the plasma membrane. This phenomenon relies on Kv2.1 binding to proteins (14), leading to the sequential phosphorylation of syntaxin 1A via 9 amino acids within the channel intrinsically disor- Kv2.1 residues Y124 and S800 by Src and p38 kinases, respectively dered C terminus. Preventing this interaction with a cell and blood- (15–17).
    [Show full text]
  • Identification of Synaptic Proteins and Their Isoform Mrnas In
    Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12487-12491, December 1994 Cell Biology Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells (exocytosis/secretion/insulin/diabetes) GUNILLA JACOBSSON*, ANDREW J. BEANt, RICHARD H. SCHELLERt, LISA JUNTTI-BERGGRENt, JUDE T. DEENEYt, PER-OLOF BERGGRENt AND BJORN MEISTER*§ *Department of Neuroscience and tRolf Luft's Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 77 Stockholm, Sweden; and tDepartment of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA 94305 Communicated by Tomas Hokfelt, August 30, 1994 ABSTRACT Several proteins that are of importance for clostridial neurotoxins, including tetanus toxin and botuli- membrane trafficking in the nerve terminal have recently been num neurotoxin B, whereas botulinum neurotoxins D and F characterized. We have used Western blot and immunohis- are capable of cleaving both forms of VAMP (10-12). tochemistry to show that synaptotagmin, synaptobrevin/VAMP VAMP-1 and VAMP-2 are encoded by two distinct genes (13) (vesicle-associated membrane protein), SNAP-25 (synaptosom- and are differentially expressed in the nervous system (14). al-associated protein of 25 kDa), and syntaxin proteins are Cellubrevin is a homologue of VAMP, which is present in a present in cells of the islets of Langerhans in the endocrine wide variety of tissues and may be a membrane trafficking pancreas. Synaptotagmin-like immunoreactivity (-LI) was lo- protein of a constitutively recycling pathway (15). calized to granules within the cytoplasm of a few endocrine cells In contrast to synaptotagmin and VAMP, the synaptoso- located in the periphery of the islets, identified as somatostatin- mal-associated protein of 25 kDa (SNAP-25) is located at the containing cells, and in many nerve fibers within the islets.
    [Show full text]
  • Hras Intracellular Trafficking and Signal Transduction Jodi Ho-Jung Mckay Iowa State University
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 HRas intracellular trafficking and signal transduction Jodi Ho-Jung McKay Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Cancer Biology Commons, Cell Biology Commons, Genetics and Genomics Commons, and the Medical Cell Biology Commons Recommended Citation McKay, Jodi Ho-Jung, "HRas intracellular trafficking and signal transduction" (2007). Retrospective Theses and Dissertations. 13946. https://lib.dr.iastate.edu/rtd/13946 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. HRas intracellular trafficking and signal transduction by Jodi Ho-Jung McKay A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Janice E. Buss, Co-major Professor Linda Ambrosio, Co-major Professor Diane Bassham Drena Dobbs Ted Huiatt Iowa State University Ames, Iowa 2007 Copyright © Jodi Ho-Jung McKay, 2007. All rights reserved. UMI Number: 3274881 Copyright 2007 by McKay, Jodi Ho-Jung All rights reserved. UMI Microform 3274881 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O.
    [Show full text]
  • Variable Priming of a Docked Synaptic Vesicle PNAS PLUS
    Variable priming of a docked synaptic vesicle PNAS PLUS Jae Hoon Junga,b,c, Joseph A. Szulea,c, Robert M. Marshalla,c, and Uel J. McMahana,c,1 aDepartment of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Physics, Stanford University School of Humanities and Sciences, Stanford, CA 94305; and cDepartment of Biology, Texas A&M University, College Station, TX 77845 Edited by Thomas S. Reese, National Institutes of Health, Bethesda, MD, and approved January 12, 2016 (received for review November 30, 2015) The priming of a docked synaptic vesicle determines the proba- transition. Biochemical and electrophysiological approaches have bility of its membrane (VM) fusing with the presynaptic membrane provided evidence that priming is mediated by interactions be- (PM) when a nerve impulse arrives. To gain insight into the nature tween the SNARE proteins and their regulators (7, 12–14, 24) and of priming, we searched by electron tomography for structural can involve differences in positioning of docked SVs relative to + relationships correlated with fusion probability at active zones of Ca2 channels (25). Biochemistry has also led to the suggestion axon terminals at frog neuromuscular junctions. For terminals that primed SVs may become deprimed (26). fixed at rest, the contact area between the VM of docked vesicles We have previously shown by electron tomography on frog and PM varied >10-fold with a normal distribution. There was no neuromuscular junctions (NMJs) fixed at rest that there are, for merging of the membranes. For terminals fixed during repetitive docked SVs, variations in the extent of the VM–PM contact area evoked synaptic transmission, the normal distribution of contact and in the length of the several AZM macromolecules linking areas was shifted to the left, due in part to a decreased number of the VM to the PM, the so-called ribs, pegs, and pins (2, 27).
    [Show full text]
  • The Mir-199–Dynamin Regulatory Axis Controls Receptor-Mediated Endocytosis Juan F
    © 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3197-3209 doi:10.1242/jcs.165233 RESEARCH ARTICLE The miR-199–dynamin regulatory axis controls receptor-mediated endocytosis Juan F. Aranda1,2, Alberto Canfrán-Duque1,2, Leigh Goedeke1,2, Yajaira Suárez1,2 and Carlos Fernández-Hernando1,2,* ABSTRACT mechanism for the selective uptake of essential nutrients such as Small non-coding RNAs (microRNAs) are important regulators of low-density lipoprotein (LDL), through the LDL receptor (LDLR) gene expression that modulate many physiological processes; (Brown and Goldstein, 1986), or iron, through transferrin receptor however, their role in regulating intracellular transport remains (TfR) (Harding et al., 1983). Thus, factors that affect RME have a largely unknown. Intriguingly, we found that the dynamin (DNM) direct effect on these receptors, and, in the case of LDLR, to regulate genes, a GTPase family of proteins responsible for endocytosis in intracellular cholesterol levels. In both the LDLR and TfR eukaryotic cells, encode the conserved miR-199a and miR-199b internalization processes, clathrin plays a key role during the family of miRNAs within their intronic sequences. Here, we formation of coated vesicles (Moore et al., 1987). Once vesicles are demonstrate that miR-199a and miR-199b regulate endocytic internalized, their passage through a broad endosomal compartment transport by controlling the expression of important mediators of system is required; first they are rapidly transported into early endocytosis such as clathrin heavy chain (CLTC), Rab5A, low- endosomes, where Rab5A is a key regulator (Nielsen et al., 1999), density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1).
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • ADP-Ribosylation Factor, a Small GTP-Binding Protein, Is Required for Binding of the Coatomer Protein Fl-COP to Golgi Membranes JULIE G
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6408-6412, July 1992 Biochemistry ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein fl-COP to Golgi membranes JULIE G. DONALDSON*, DAN CASSEL*t, RICHARD A. KAHN*, AND RICHARD D. KLAUSNER* *Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, and tLaboratory of Biological Chemistry, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Marc Kirschner, April 20, 1992 (receivedfor review February 11, 1992) ABSTRACT The coatomer is a cytosolic protein complex localized to the Golgi complex, although their functions have that reversibly associates with Golgi membranes and is Impli- not been defined. Distinct among these proteins is the ADP- cated in modulating Golgi membrane transport. The associa- ribosylation factor (ARF), originally identified as a cofactor tion of 13-COP, a component of coatomer, with Golgi mem- required for in vitro cholera toxin-catalyzed ADP- branes is enhanced by guanosine 5'-[v-thioltriphosphate ribosylation of the a subunit of the trimeric GTP-binding (GTP[yS]), a nonhydrolyzable analogue of GTP, and by a protein G, (G,.) (19). ARF is an abundant cytosolic protein mixture of aluminum and fluoride ions (Al/F). Here we show that reversibly associates with Golgi membranes (20, 21). that the ADP-ribosylation factor (ARF) is required for the ARF has been shown to be present on Golgi coated vesicles binding of (-COP. Thus, 13-COP contained in a coatomer generated in the presence of GTP[yS], but it is not a com- fraction that has been resolved from ARF does not bind to Golgi ponent of the cytosolic coatomer (22).
    [Show full text]