DOCSLIB.ORG

Barcoding the Actin Track: Differential Regulation of Myosin Motors by Tropomyosin Joseph Emerson Clayton University of Vermont

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Barcoding the Actin Track: Differential Regulation of Myosin Motors by Tropomyosin Joseph Emerson Clayton University of Vermont University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2016 Barcoding the actin track: Differential regulation of myosin motors by tropomyosin Joseph Emerson Clayton University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Molecular Biology Commons Recommended Citation Clayton, Joseph Emerson, "Barcoding the actin track: Differential regulation of myosin motors by tropomyosin" (2016). Graduate College Dissertations and Theses. 638. https://scholarworks.uvm.edu/graddis/638 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. BARCODING THE ACTIN TRACK: DIFFERENTIAL REGULATION OF MYOSIN MOTORS BY TROPOMYOSIN A Dissertation Presented by Joseph E. Clayton to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Specializing in Cellular, Molecular, and Biomedical Sciences October, 2016 Defense Date: June 10, 2016 Dissertation Examination Committee: Teresa Ruiz, Ph.D., Advisor Karen Lounsbury, Ph.D., Chairperson David Warshaw, Ph.D. Jason Stumpff, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Myosins and tropomyosins represent two types of actin filament-associated proteins that often work together in contractile and motile processes in the cell. While the role of thin filament troponin-tropomyosin complexes in regulating striated muscle myosin II is well characterized, the role of tropomyosins in non-muscle myosin regulation is not well understood. Fission yeast has recently proved to be a useful model with which to study regulation of myosin motors by tropomyosin owing to its tractable genetics, well-defined actin cytoskeleton, and established actin biochemistry. A hallmark of type V myosins is their processivity – the ability to take multiple steps along actin filaments without dissociating. However, the fission yeast type V myosin (Myo52) is a nonprocessive motor whose activity is enhanced by the sole fission yeast tropomyosin (Cdc8). The molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52 transport was investigated using a combination of in vitro and in vivo approaches. Single molecules of Myo52, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8 was present on actin filaments. Small ensembles of Myo52 bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8 for continuous motion. Although a truncated form of Myo52 that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin- dependent movement to polarized growth sites. This result suggests that truncated Myo52 lacking cargo, or single molecules of wild-type Myo52 with small cargoes, can undergo processive movement along actin-Cdc8 cables in vivo. These findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. To understand the broader implications of actomyosin regulation by tropomyosin we examined the role of two mammalian tropomyosins (Tpm3.1 and Tpm4.2) recently implicated in cancer cell proliferation and metastasis. As previously observed with Cdc8, Tpm3.1 and Tpm4.2 isoforms significantly enhance non-muscle myosin II (Myo2). Additionally, the mammalian tropomyosins enable Myo52 processive movement along actin tracks. In contrast to the positive regulation of Myo2 and Myo52, Cdc8 and the mammalian tropomyosins potently inhibit skeletal muscle myosin II, while having negligible effects on the highly processive mammalian myosin- Va. Thus, different motor outputs favoring functional specification within the same myosin class are possible in the presence of certain tropomyosins. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1 rescued normal contractile ring dynamics, cytokinesis, and fission yeast cell growth in the absence of functional Cdc8. This work has broad implications with regard to regulation of non-muscle and muscle actomyosin function in complex cellular environments such as developing muscle tissue and metastatic cancer cells. CITATIONS Material from this dissertation has been published in the following form: Clayton, J. E., Pollard, L. W., Murray, G. G., & Lord, M.. (2015). Myosin motor isoforms direct specification of actomyosin function by tropomyosins. Cytoskeleton. 72(3), 131-145. Clayton, J. E., Pollard, L. W., Sckolnick, M., Bookwalter, C. S., Hodges, A. R., Trybus, K. M., & Lord, M.. (2014). Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables. Mol Biol Cell, 25(1), 66-75. ii DEDICATION This is dedicated to my parents, who have never wavered in their love and support. And to Diana, Caroline, and Wilder, who make everything brighter. With all my love – thank you. iii ACKNOWLEDGEMENTS I am grateful to my advisors, Dr. Matt Lord, for the experience and training I gained while working in his lab, and Dr. Teresa Ruiz, for the support and guidance that got me across the finish line. I also want to thank my committee members, Dr. Karen Lounsbury, Dr. David Warshaw, and Dr. Jason Stumpff for all of the thoughtful discussions and advice. Finally, I would like to thank the members of the Molecular Physiology and Biophysics Department, and everyone who has contributed to the work presented in this dissertation. iv TABLE OF CONTENTS Page CITATIONS ........................................................................................................... ii DEDICATION ....................................................................................................... iii ACKNOWLEDGEMENTS ................................................................................... iv LIST OF TABLES ................................................................................................ ix LIST OF FIGURES ................................................................................................ x CHAPTER 1: INTRODUCTION ...........................................................................1 Actin ...................................................................................................................2 Actin Polymerization ...................................................................................4 Actin filament structure ...............................................................................6 Myosin Motors ...................................................................................................7 Myosin Structure ........................................................................................10 of Type V Myosins ....................................................................................11 Tropomyosin ....................................................................................................13 Tropomyosin Expression and Localization ...............................................14 Functional Roles of Tropomyosin .............................................................18 Role of Tropomyosin During Development ..............................................20 Involvement of Tropomyosin in Cancer ....................................................21 The Benefits of Using Fission Yeast to Study the Actin Cytoskeleton ...........23 Scope and Purpose ...........................................................................................24 CHAPTER 2: FISSION YEAST TROPOMYOSIN SPECIFIES DIRECTED TRANSPORT OF MYOSIN-V ALONG ACTIN CABLES ................................28 Abstract ............................................................................................................29 Introduction ......................................................................................................30 Results ..............................................................................................................32 Fission Yeast Tropomyosin Converts Myo52p into a Processive Motor....................................................................................................32 Tropomyosin Increases Myo52p Run Frequency and Run Length at Low ATP Concentration ......................................................................34 Myo52p Motors Work in Small Clusters that Rely on Tropomyosin for Effective Transport ..............................................................................35 Myo52p Molecules Lacking a Cargo-Binding Domain Undergo Continuous Motility Along Actin Cables In Vivo ...............................37 v Discussion ........................................................................................................39 Tropomyosin Promotes Myo52p Processivity by Altering The Kinetics of the Motor .........................................................................................39 Alternative Modes of Cdc8p Regulation and Their Relationship to Established Models ..............................................................................41 Materials and Methods .....................................................................................43 Fission Yeast Strains and Genetic Approaches .........................................43
Load more

Recommended publications
  • Engineering of the Myosin-I Nucleotide-Binding
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 44, Issue of October 29, pp. 31373–31381, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Engineering of the Myosin-Ib Nucleotide-binding Pocket to Create Selective Sensitivity to N6-modified ADP Analogs* (Received for publication, April 30, 1999, and in revised form, July 21, 1999) Peter G. Gillespie‡§¶, Susan K. H. Gillespie‡i, John A. Mercer**, Kavita Shah‡‡, and Kevan M. Shokat‡‡ §§ From the Departments of ‡Physiology and §Neuroscience, The Johns Hopkins University, Baltimore, Maryland 21205, **McLaughlin Research Institute, Great Falls, Montana 59405, and the ‡‡Departments of Chemistry and Molecular Biology, Princeton University, Princeton, New Jersey 08544 Distinguishing the cellular functions carried out by and amplifies small movements within the head, and diverse enzymes of highly similar structure would be simplified tail domains that couple myosin molecules to other cellular by the availability of isozyme-selective inhibitors. To structures (2). determine roles played by individual members of the Although roles have been identified for conventional myosin large myosin superfamily, we designed a mutation in isozymes (the myosin-II class), elucidating cellular functions myosin’s nucleotide-binding pocket that permits bind- for unconventional myosin isozymes has been notably difficult. 6 ing of adenine nucleotides modified with bulky N sub- Cells may express a dozen or more myosin isozymes simulta- stituents. Introduction of this mutation, Y61G in rat my- neously (5, 6), and many of these isozymes have similar biochem- osin-Ib, did not alter the enzyme’s affinity for ATP or ical properties.
    [Show full text]
  • Rho Kinase Proteins—Pleiotropic Modulators of Cell Survival and Apoptosis
    ANTICANCER RESEARCH 31: 3645-3658 (2011) Review Rho Kinase Proteins—Pleiotropic Modulators of Cell Survival and Apoptosis CATHARINE A. STREET1 and BRAD A. BRYAN2 1Ghosh Science and Technology Center, Department of Biology, Worcester State University, Worcester, MA, U.S.A.; 2Center of Excellence in Cancer Research, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, U.S.A. Abstract. Rho kinase (ROCK) proteins are Rho-GTPase regulators of actin cytoskeletal dynamics, and therefore activated serine/threonine kinases that function as modulators control cell migration and motility (1). Specifically, ROCK of actin-myosin cytoskeletal dynamics via regulation of Lin11, proteins promote the formation of stress fibers and focal Isl-1 & Mec-3 domain (LIM) kinase, myosin light chain (MLC), adhesions (Figure 1), but have also been implicated in and MLC phosphatase. A strong correlation between diverse processes such as cell junction integrity and cell cytoskeletal rearrangements and tumor cell invasion, metastasis, cycle control (2). ROCK activity is responsible for and deregulated microenvironment interaction has been stabilization of actin microfilaments as well as promoting reported in the literature, and the utilization of pharmacological cellular contraction and cell substratum contact. ROCK inhibitors of ROCK signaling for the treatment of cancer is stimulates actin polymerization via an inhibitory actively being pursued by a number of pharmaceutical phosphorylation of the actin severing LIM kinase (Figure 2). companies. Indeed, in many preclinical models ROCK inhibitors ROCK promotes cellular contraction and attachment via an have shown remarkable efficacy in reducing tumor growth and activating phosphorylation of myosin light chain (MLC) to metastasis.
    [Show full text]
  • Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer
    cancers Review Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer Nayden G. Naydenov 1, Susana Lechuga 1, Emina H. Huang 2 and Andrei I. Ivanov 1,* 1 Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA; [email protected] (N.G.N.); [email protected] (S.L.) 2 Departments of Cancer Biology and Colorectal Surgery, Cleveland Clinic Foundation, Cleveland, OH 44195, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-216-445-5620 Simple Summary: Colorectal cancer (CRC) is a deadly disease that may go undiagnosed until it presents at an advanced metastatic stage for which few interventions are available. The develop- ment and metastatic spread of CRC is driven by remodeling of the actin cytoskeleton in cancer cells. Myosins represent a large family of actin motor proteins that play key roles in regulating actin cytoskeleton architecture and dynamics. Different myosins can move and cross-link actin filaments, attach them to the membrane organelles and translocate vesicles along the actin filaments. These diverse activities determine the key roles of myosins in regulating cell proliferation, differ- entiation and motility. Either mutations or the altered expression of different myosins have been well-documented in CRC; however, the roles of these actin motors in colon cancer development remain poorly understood. The present review aims at summarizing the evidence that implicate myosin motors in regulating CRC growth and metastasis and discusses the mechanisms underlying the oncogenic and tumor-suppressing activities of myosins. Abstract: Colorectal cancer (CRC) remains the third most common cause of cancer and the second most common cause of cancer deaths worldwide.
    [Show full text]
  • Non-Muscle Myosin 2A (NM2A): Structure, Regulation and Function
    cells Review Non-Muscle Myosin 2A (NM2A): Structure, Regulation and Function Cláudia Brito 1,2 and Sandra Sousa 1,* 1 Group of Cell Biology of Bacterial Infections, i3S-Instituto de Investigação e Inovação em Saúde, IBMC, Universidade do Porto, 4200-135 Porto, Portugal; [email protected] 2 Programa Doutoral em Biologia Molecular e Celular (MCBiology), Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-002 Porto, Portugal * Correspondence: [email protected] Received: 19 May 2020; Accepted: 29 June 2020; Published: 1 July 2020 Abstract: Non-muscle myosin 2A (NM2A) is a motor cytoskeletal enzyme with crucial importance from the early stages of development until adulthood. Due to its capacity to convert chemical energy into force, NM2A powers the contraction of the actomyosin cytoskeleton, required for proper cell division, adhesion and migration, among other cellular functions. Although NM2A has been extensively studied, new findings revealed that a lot remains to be discovered concerning its spatiotemporal regulation in the intracellular environment. In recent years, new functions were attributed to NM2A and its activity was associated to a plethora of illnesses, including neurological disorders and infectious diseases. Here, we provide a concise overview on the current knowledge regarding the structure, the function and the regulation of NM2A. In addition, we recapitulate NM2A-associated diseases and discuss its potential as a therapeutic target. Keywords: non-muscle myosin 2A (NM2A); NM2A activity regulation; NM2A filament assembly; actomyosin cytoskeleton; cell migration; cell adhesion; plasma membrane blebbing 1. Superfamily of Myosins The cell cytoskeleton is an interconnected and dynamic network of filaments essential for intracellular organization and cell shape maintenance.
    [Show full text]
  • Relationship Among Fibre Type, Myosin Atpase Activity and Contractile Properties
    Histochemical Journal 14, 981-997 (1982) Relationship among fibre type, myosin ATPase activity and contractile properties LEO C. MAXWELL 1, JOHN A. FAULKNER 2and RICHARD A. MURPHY 3 1Departmen t of Physiology, The University of Texas Health Science Center, San An tonio, Texas 78284, U.S.A. 2Department of Physiology, The University of Michigan, Ann Arbor, Michigan 48109, U.S.A. 3Department of Physiology, The University of Virginia, Charlottesville, Vird~inia 22901, U.S.A. Received 25 February 1982 and in revised form 18 May 1982 Sunlmal'y At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types, Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.
    [Show full text]
  • Distinct Structures of ATP and GTP Complexes in the Myosin Atpase a Variety of Ntps Other Than ATP (6-16). How Ever, GTP And
    J. Biochem. 96, 155-162 (1984) Distinct Structures of ATP and GTP Complexes in the Myosin ATPase Toshiaki HIRATSUKA Department of Chemistry, Asahikawa Medical College Asahikawa, Hokkaido 078-11 Received for publication, February 10, 1984 The active site of the myosin subfragment-1 ATPase was affinity-labeled with ribose modified fluorescent analogs of ADP, dADP, CDP, UDP, IDP, and GDP in com bination with vanadate, forming a stable myosin-nucleoside diphosphate-vanadate complex that is analogous to the normal myosin-ADP-Pi intermediate [Hiratsuka, T. (1984) J. Biochem. 96, 147-154]. Labeled enzyme was isolated free of unbound analog and vanadate, and fluorescent properties of the fluorophore at the active site were examined. Fluorescence emission and acrylamide quenching studies re vealed that the hydrophobicity of environment around the fluorophore and the degree of its burial in the protein vary with the base structure of NDP. It was found that the fluorophore of ADP analog is most buried into the protein, while that of the GDP analog is least buried. The results suggest that the deep burial of ATP into the myosin active site is essential for muscle contraction. Energy transducing involving nucleoside triphos a variety of NTPs other than ATP (6-16). How phate (NTP) hydrolysis is usually linked to ATP ever, GTP and other NTPs cannot substitute for hydrolysis. However, this is not always the case. ATP completely in its ability to support super Energy from GTP is also used to control the con precipitation of actomyosin (12-14), to contract formation of proteins in a large number of bio and relax myofibrils (6, 14), and to protect the chemical processes, including protein synthesis (1), active site of myosin against thermal denaturation assembly of tubulin (2, 3), activation and inhibi (7).
    [Show full text]
  • Triphosphate and Its Cr(H20)4 and Co(NH 3)4 Complex Derivatives Are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K +-Atpase
    Physiol. Res. 46: 345-349, 1997 2’(3’)-0-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate and its Cr(H20)4 and Co(NH 3)4 Complex Derivatives are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K +-ATPase H. LINNERTZ, I. LASTRES BECKER, R. KRUMSCHEID1, E. AMLER2, D. THOENGES, W. SCHONER Institute of Biochemistry and Endocrinology, ‘Biochemical Institute, Justus-Liebig-University Giessen, Giessen, Germany and 2 Institute of Physiology, Czech Academy o f Sciences, Prague, Czech Republic Receh’ed March 5, 1997 Accepted May 14, 1997 Summary 2’(3’)-0-[N - [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K +- ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na + /K + -ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, K<j = 2001MM) and the high (Ei, Kd = 22,«M) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Kj = (KdEl*KdE2)1/2, the experimentally measured inhibition constant of 6 6,wM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium (III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na + /K + -ATPase in a specific way. Key words Na + /K +-ATPase - Complex derivatives
    [Show full text]
  • Sodium Dysregulation Coupled with Calcium Entry Leads to Muscular
    Sodium dysregulation coupled with calcium entry leads to muscular dystrophy in mice A dissertation submitted to the Division of Research and Advanced Studies Of the University of Cincinnati In partial fulfillment of the Requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the department of Molecular and Developmental Biology of the College of Medicine 2014 Adam R. Burr B.S. University of Minnesota, 2007 i Abstract Duchenne Muscular Dystrophy (DMD) and many of the limb girdle muscular dystrophies form a family of diseases called sarcoglycanopathies. In these diseases, mutation of any of a host of membrane and membrane associated proteins leads to increased stretch induced damage, aberrant signaling, and increased activity of non-specific cation channels, inducing muscle necrosis. Due to ongoing necrosis, DMD follows a progressive clinical course that leads to death in the mid-twenties. This course is slowed only modestly by high dose corticosteroids, which cause a plethora of harsh side effects. Targeted therapies are needed to ameliorate this disease until a more permanent therapy such as replacement of the mutated gene can be routinely performed. Here, we identified sodium calcium exchanger 1 (NCX1) as a potential therapeutic target. We started from the observation that sodium calcium exchanger 1 (NCX1) was upregulated during the necrotic phase of the disease in Sgcd-/- mice, which have similar pathology and mechanism of disease to boys with DMD. To test the causal effect of NCX1 overexpression on disease, we generated mice that overexpress NCX1 specifically in skeletal muscle. By Western blotting and immunofluorescence, we showed that NCX1 transgenic mice express more NCX1 protein in a similar localization pattern as endogenous NCX1.
    [Show full text]
  • Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders
    1521-0081/68/2/476–532$25.00 http://dx.doi.org/10.1124/pr.115.010652 PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:476–532, April 2016 Copyright © 2016 The Author(s) This is an open access article distributed under the CC-BY Attribution 4.0 International license. ASSOCIATE EDITOR: STEPHANIE W. WATTS Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders F.V. Brozovich, C.J. Nicholson, C.V. Degen, Yuan Z. Gao, M. Aggarwal, and K.G. Morgan Department of Health Sciences, Boston University, Boston, Massachusetts (C.J.N., Y.Z.G., M.A., K.G.M.); Department of Medicine, Mayo Clinic, Rochester, Minnesota (F.V.B.); and Paracelsus Medical University Salzburg, Salzburg, Austria (C.V.D.) Abstract ...................................................................................478 I. Introduction . ..............................................................................478 A. Scope and Limitations..................................................................478 B. Overview of Regulation of Blood Pressure/Vascular Tone. ...............................478 1. Guyton View of Regulation Blood Pressure, Kidney Role, Volume Regulation. .......478 2. Recent Direct Confirmation of Changes in Vascular Tone/Resistance Related to Changes in Systemic Vascular Resistance and Blood Pressure and Downloaded from the Importance of Vascular Smooth Muscle Contraction in both Normal Physiology and Pathophysiology—Hypertension......................................479 3. Racial Differences/Personalized
    [Show full text]
  • Catalytic Strategy Used by the Myosin Motor to Hydrolyze
    Catalytic strategy used by the myosin motor to PNAS PLUS hydrolyze ATP Farooq Ahmad Kiani and Stefan Fischer1 Computational Biochemistry, Interdisciplinary Center for Scientific Computing, University of Heidelberg, D-69120 Heidelberg, Germany Edited by Donald G. Truhlar, University of Minnesota, Minneapolis, MN, and approved June 6, 2014 (received for review January 30, 2014) − + Myosin is a molecular motor responsible for biological motions triphosphate4 /Mg2 moiety of ATP. A structure of myosin such as muscle contraction and intracellular cargo transport, for bound to the dissociated product (ADP/Pi) has not been which it hydrolyzes adenosine 5’-triphosphate (ATP). Early steps of available so far. This product state is important, because the the mechanism by which myosin catalyzes ATP hydrolysis have inorganic phosphate (Pi) and ADP products are only released been investigated, but still missing are the structure of the final later by myosin, upon rebinding to the actin filament during the ADP·inorganic phosphate (Pi) product and the complete pathway power stroke. This rebinding can only take place when the nu- leading to it. Here, a comprehensive description of the catalytic cleotide is in the ADP/Pi product state. Some residues thought strategy of myosin is formulated, based on combined quantum– to be catalytically relevant are Glu459 (22), Ser181 (23), Ser236 classical molecular mechanics calculations. A full exploration of (24–25), and Gly457 (26) (using the residue numbering of Dic- catalytic pathways was performed and a final product structure tyostelium discoideum), which are all strictly conserved. Whereas was found that is consistent with all experiments. Molecular movies Glu459 has been proposed to act as a general base in the catalysis, of the relevant pathways show the different reorganizations of the exact role played by the other residues remained unclear.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Rho Gtpase Signalling Pathways in the Morphological Changes Associated with Apoptosis
    Cell Death and Differentiation (2002) 9, 493 ± 504 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 www.nature.com/cdd Review Rho GTPase signalling pathways in the morphological changes associated with apoptosis ML Coleman1 and MF Olson*,1 membrane blebs and apoptotic bodies (reviewed in refer- ence3). Ultimately, the dead cell is packaged into membrane- 1 Cancer Research Campaign Centre for Cell and Molecular Biology, Institute of clad apoptotic bodies that facilitate uptake by neighbouring Cancer Research, 237 Fulham Road, London SW3 6JB, UK cells or by specialised phagocytic cells. Dynamic re- * Corresponding author: MF Olson, Abramson Family Cancer Research arrangements of the actin cytoskeleton in the phagocyte are Institute, Room 411, BRB II/III, 421 Curie Boulevard, University of required for the internalisation of apoptotic cell fragments. Pennsylvania, Philadelphia, PA 19104-6160, USA. Tel: +1 215 746 6798; Fax: +1 215 746 5525; E-mail: [email protected] Recent research has revealed the importance of signal transduction pathways controlled by Rho family GTPases in Received 23.8.01; revised 26.10.01; accepted 5.11.01 regulating the marked changes in cell morphology observed Edited by M Piacentini in the processes of apoptosis and phagocytosis. The Rho GTPases are a family of proteins (RhoA, RhoB, RhoC, RhoD, RhoE/Rnd3, RhoG, RhoH/TTF, Rnd1, Rnd2, Abstract Rac1, Rac2, Rac3, Cdc42/G25K, Wrch-1, TC10, TCL, Chp, Rif) that act as molecular switches in intracellular signal The killing and removal of superfluous cells is an important 4 step during embryonic development, tissue homeostasis, transduction pathways (reviewed in reference ).
    [Show full text]