DOCSLIB.ORG

The Assembly and Interactions of Mreb in the Maintenance of Cell Shape in Caulobacter Crescentus

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Assembly and Interactions of Mreb in the Maintenance of Cell Shape in Caulobacter Crescentus THE ASSEMBLY AND INTERACTIONS OF MREB IN THE MAINTENANCE OF CELL SHAPE IN CAULOBACTER CRESCENTUS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Natalie Anne Dye May, 2010 © 2010 by Natalie Anne Dye. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/bg008yn0701 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Julie Theriot, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Lucille Shapiro, Co-Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. James Spudich I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Aaron Straight Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT This work focuses on the mechanism by which MreB contributes to the maintenance of cell shape in the gram-negative alpha-proteobacterium Caulobacter crescentus. The gene mreB encodes a protein that resembles actin, a eukaryotic cytoskeletal protein. Previously, it was shown that mreB is required to maintain a rod-like shape and localizes to a helical pattern near the cytoplasmic membrane. Here, we show that MreB is associated with regions of active growth in Caulobacter, as mutant strains that mislocalize MreB to the cell poles direct new growth at or near the poles. We present evidence to suggest that MreB contributes to the determination of proper length, width, and curvature through partially distinct mechanisms. The determination of proper width involves the essential proteins MreC and Pbp2, which are encoded in the mreB operon. While MreB and MreC are both required to position the cell wall transpeptidase Pbp2 along the lateral sidewalls and away from midcell, the two do not colocalize and each can maintain its localization in the absence of the other. When MreB is mislocalized to the poles, MreC and Pbp2 do not follow. These data argue against the idea that MreB provides a scaffold-like structure to localize enzymes that directly modify the cell wall. The determination of proper curvature, involves the intermediate filament-like protein, Crescentin. We identify a putative binding site on MreB for Crescentin or other curvature-mediating factors. We also show that the extent to which the subcellular localization of MreB changes over the cell cycle is correlated with cell size, indicating that MreB is involved in the coordination between elongation and division. In addition, we show that in vitro purified MreB spontaneously forms very stable polymers in the presence or absence of nucleotide. These polymers are globular or amorphous and only filamentous when placed on a highly positively charged surface of Poly-L-lysine. These in vitro data suggest that MreB is likely to be regulated at the disassembly step in the cell and that the cellular environment may influence the structure of MreB polymers. Lastly, we present biochemical evidence to support the existence of a disassembly factor in cytoplasmic Caulobacter extract. Together our data suggest that the maintenance of the crescent-rod cell shape in Caulobacter is the result of a complicated balance between MreB’s dynamic subcellular localization, polymeric structure, and communication with cellular components. iv For my always loving and supportive parents John and Donna Dye v ACKNOWLEDGEMENTS This thesis is the culmination of almost seven years of work. During this time, I have had the pleasure of working with so many truly brilliant, creative, and knowledgeable scientists. I would not be the scientist I am today without the generous help of my mentors and colleagues. I would like to take this opportunity to thank the following persons. First and foremost, I must thank my joint thesis advisors, Julie Theriot and Lucy Shapiro. These two women are phenomenal scientists and individuals, and it has been a privilege and a pleasure to work with them. While they have very different scientific perspectives, styles, and interests, they share an inspiring enthusiasm for scientific research and a genuine care for their students. Thank you both for the guidance, mentorship, motivation and inspiration you have given me during this process. In addition to my advisors, I received considerable guidance from the other members of my committee Aaron Straight and Jim Spudich. I very much appreciate the advice and feedback that I received from each of them over the years. I am also thankful for KC Huang, who agreed to be my chair and has given me significant feedback on this work. Harley McAdams, though not an official committee member or advisor, has also provided an interesting and important point of view on this project. I’d also like to thank John Perrino at the EM facility in the Beckman center here at Stanford for guidance with the electron microscopy. I must acknowledge my collaborators in this project. Zachary Pincus was a graduate student with Julie when I first started this project, and even after he left Julie’s lab to become a postdoc thousands of miles away, he remained an active participant in this research. His contributions to the work presented in Chapters 2 and 3 were significant, and these stories would not have been as mature as they are now without his expertise in computation and statistics. Isabelle Fisher contributed to this project as a high school student. Though lacking the many years of formal training in biology, Isabelle was able to grasp complicated concepts and make me think about this subject in new ways. She was a joy to teach and work with, and I hope to see her continue in science. Enrique de la Cruz and his graduate student Kendra Frederick hosted me in their lab at Yale vi University for a short time to work with purified MreB. While this collaboration was brief and did not end up yielding publishable data, it was a valuable experience for me to learn specific techniques from them and get their perspective on this project. I have received countless ideas, feedback, reagents, protocols, and emotional support from the members of the Theriot and Shapiro labs, and as such I would like to thank everyone with whom I overlapped. In particular in the Shapiro/McAdams lab: Zemer Gitai, who was my rotation advisor, Antonio Iniesta, Grant Bowman, Erin Goley, Jerod Ptacin, Esteban Toro, Joseph Chen, Patrick McGrath, and Virginia Kalogeraki. In the Theriot lab: Kinneret Keren, Karine Gibbs, Aretha Fiebig, Guy Ziv, David Hallidan, Greg Allen, Mark Tsuchida, and Steph Weber. A very special thank you goes to Susanne Rafelski, who was my mentor in the Theriot lab when I first started and has remained a dear friend. I owe a huge huge huge debt of gratitude to Matthew Footer. He has taught me so much about biochemistry, protein purification, optics, cooking, and more. Thank you for always being so friendly and generous with your time. I think all of us agree that the lab would fall apart without you! I must also especially thank my friend and labmate Erin Barnhart, who has sat next to me almost my entire time in graduate school. She has been my sounding board, my colleague, my friend, and at times my entertainment . Erin also read almost this entire thesis and gave me thoughtful feedback, all while writing her own thesis. You are a talented, fun, intelligent, courageous, and goofy individual and it’s been a pleasure sharing this experience with you. Thank you. I would also like to thank all of the past and present members of the Biochemistry and Developmental Biology departments here at Stanford for making it a collegial and enjoyable place to work. I must specifically acknowledge the friendly, patient, and helpful support staff in these departments, in particular Joella Ackerman, Karen Butzman, Tara Trim, Todd Galitz, and Shedrick Watts. Special thanks to my classmates and friends in the department Ryan Nottingham, Ian Brennan, Kirstin Milks, Eric Espinosa, and especially Kristina Godek. Kristina has been a dear friend and colleague. Her endurance and dedication to science is an inspiration, and I wish her all the success her hard work deserves. I will miss having her vii right down the hall. She also provided significant feedback on this thesis. I’d also like to thank my friends Dina Finan, Duane Baxter, Michael Costa, and Corey Meyer, who provided me with much wine and amazing home-cooked meals. In my time at Stanford, I have been a part of a wonderful network of female scientists and engineers, and I would like to thank the following for their considerable advice and support in both professional and personal manners: Megan Young, Jennifer Zamanian, Hyejun Ra, Davie Yoon, Misty Davies, Esther Chen, Liv Walter, Sabina Stefania, and Lisa Moore. Through my involvement with the Alpine club, I became part of a fantastic climbing community, which has enriched my experience here at Stanford.
Load more

Recommended publications
  • The Cytoskeleton in Cell-Autonomous Immunity: Structural Determinants of Host Defence
    Mostowy & Shenoy, Nat Rev Immunol, doi:10.1038/nri3877 The cytoskeleton in cell-autonomous immunity: structural determinants of host defence Serge Mostowy and Avinash R. Shenoy Medical Research Council Centre of Molecular Bacteriology and Infection (CMBI), Imperial College London, Armstrong Road, London SW7 2AZ, UK. e‑mails: [email protected] ; [email protected] doi:10.1038/nri3877 Published online 21 August 2015 Abstract Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. 1 Mostowy & Shenoy, Nat Rev Immunol, doi:10.1038/nri3877 Cell-autonomous immunity, which is defined as the ability of a host cell to eliminate an invasive infectious agent, is a first line of defence against microbial pathogens 1 . It relies on antimicrobial proteins, specialized degradative compartments and programmed host cell death 1–3 . Cell- autonomous immunity is mediated by tiered innate immune signalling networks that sense microbial pathogens and stimulate downstream pathogen elimination programmes. Recent studies on host– microorganism interactions show that components of the host cell cytoskeleton are integral to the detection of bacterial pathogens as well as to the mobilization of antibacterial responses (FIG.
    [Show full text]
  • Cell Division in Corynebacterineae
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector REVIEW ARTICLE published: 10 April 2014 doi: 10.3389/fmicb.2014.00132 Cell division in Corynebacterineae Catriona Donovan* and Marc Bramkamp* Department of Biology I, Ludwig-Maximilians-University, Munich, Planegg-Martinsried, Germany Edited by: Bacterial cells must coordinate a number of events during the cell cycle. Spatio-temporal Wendy Schluchter, University of regulation of bacterial cytokinesis is indispensable for the production of viable, genetically New Orleans, USA identical offspring. In many rod-shaped bacteria, precise midcell assembly of the division Reviewed by: machinery relies on inhibitory systems such as Min and Noc. In rod-shaped Actinobacteria, Julia Frunzke, Jülich Research Centre, Germany for example Corynebacterium glutamicum and Mycobacterium tuberculosis, the divisome Andreas Burkovski, Friedric assembles in the proximity of the midcell region, however more spatial flexibility is University of Erlangen-Nuremberg observed compared to Escherichia coli and Bacillus subtilis. Actinobacteria represent a (FAU), Germany group of bacteria that spatially regulate cytokinesis in the absence of recognizable Min and *Correspondence: Noc homologs. The key cell division steps in E. coli and B. subtilis have been subject to Catriona Donovan and Marc Bramkamp, Department of Biology I, intensive study and are well-understood. In comparison, only a minimal set of positive and Ludwig-Maximilians-University, negative regulators of cytokinesis are known in Actinobacteria. Nonetheless, the timing Munich, Großhaderner Str. 2-4, of cytokinesis and the placement of the division septum is coordinated with growth as 82152 Planegg-Martinsried, well as initiation of chromosome replication and segregation.
    [Show full text]
  • Crystal Structure of Ftsa from Staphylococcus Aureus
    FEBS Letters 588 (2014) 1879–1885 journal homepage: www.FEBSLetters.org Crystal structure of FtsA from Staphylococcus aureus Junso Fujita a, Yoko Maeda a, Chioko Nagao c, Yuko Tsuchiya c, Yuma Miyazaki a, Mika Hirose b, ⇑ Eiichi Mizohata a, Yoshimi Matsumoto d, Tsuyoshi Inoue a, Kenji Mizuguchi c, Hiroyoshi Matsumura a, a Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan c National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan d Laboratory of Microbiology and Infectious Diseases, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan article info abstract Article history: The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA Received 5 February 2014 and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crys- Revised 2 April 2014 tal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer Accepted 2 April 2014 units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the Available online 18 April 2014 half of S12–S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate Edited by Kaspar Locher in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA. Structured summary of protein interactions: Keywords: Bacterial divisome SaFtsA and SaFtsZ bind by comigration in non denaturing gel electrophoresis (View interaction) Staphylococcus aureus SaFtsZ and SaFtsA bind by molecular sieving (View interaction) FtsA SaFtsA and SaFtsA bind by x-ray crystallography (View interaction) FtsZ Ó 2014 Federation of European Biochemical Societies.
    [Show full text]
  • Soft Matter PAPER
    View Article Online / Journal Homepage / Table of Contents for this issue Soft Matter Dynamic Article LinksC< Cite this: Soft Matter, 2012, 8, 7446 www.rsc.org/softmatter PAPER Growth of curved and helical bacterial cells Hongyuan Jiang and Sean X. Sun* Received 26th February 2012, Accepted 17th May 2012 DOI: 10.1039/c2sm25452b A combination of cell wall growth and cytoskeletal protein action gives rise to the observed bacterial cell shape. Aside from the common rod-like and spherical shapes, bacterial cells can also adopt curved or helical geometries. To understand how curvature in bacteria is developed or maintained, we examine how Caulobacter crescentus obtains its crescent-like shape. Caulobacter cells with or without the cytoskeletal bundle crescentin, an intermediate filament-like protein, exhibit two distinct growth modes, curvature maintenance that preserves the radius of curvature and curvature relaxation that straightens the cell (Fig. 1). Using a proposed mechanochemical model, we show that bending and twisting of the crescentin bundle can influence the stress distribution in the cell wall, and lead to the growth of curved cells. In contrast, after crescentin bundle is disrupted, originally curved cells will slowly relax towards a straight rod over time. The model is able to quantitatively capture experimentally observed curvature dynamics. Furthermore, we show that the shape anisotropy of the cross-section of a curved cell is never greater than 4%, even in the presence of crescentin. 1. Introduction forces applied by external constraints generate curved cells. Strikingly, the growth modes of the cell with or without cres- Bacterial cell walls are built through a complex biochemical centin are different17,18 as shown in Fig.
    [Show full text]
  • A Metabolic Assembly Line in Bacteria
    NEWS AND VIEWS A metabolic assembly line in bacteria Matthew T. Cabeen and Christine Jacobs-Wagner The bacterial cytoplasm is rich in filament-forming proteins, from homologues of eukaryotic cytoskeletal elements to other scaffolding and segregation proteins. We now learn that even the metabolic enzyme CTP synthase forms cytoplasmic filaments that affect bacterial cell shape. Bacteria keep surprising us. It was not so long in mediating cell curvature in Caulobacter cres- and analysing their function later. Using high- ago that they were thought to be mere bags of centus9; subsequent characterization revealed resolution electron cryotomography (ECT), an chemicals, possessing only the cell wall as a sort its intermediate filament-like properties9. But unbiased method which uses no labels, Jensen of exoskeleton to hold everything together. As what about proteins with functions that would and colleagues uncovered several filament-like it turns out, bacterial cells have a sophisticated never suggest any polymerizing property? structures in the cytoplasm of C. crescentus internal organization. They possess counter- Recent work has approached the discovery of that could not be identified by disrupting or parts of tubulin, actin and intermediate fila- subcellular structures from the opposite direc- eliminating known cytoskeletal structures10. ment proteins, suggesting that a cytoskeleton tion by searching for filamentous structures first Meanwhile, in another unbiased approach, first evolved in bacteria. Moreover, in recent years the known bacterial filament-forming proteins have expanded beyond the traditional cytoskeleton to include DNA segregators, structural scaffolds and proteins, the function of which are still unknown. On page 739 of this TubZ issue, Ingerson-Mahar et al.
    [Show full text]
  • The Bacterial Actin Mreb Rotates, and Rotation Depends on Cell-Wall Assembly
    The bacterial actin MreB rotates, and rotation depends on cell-wall assembly Sven van Teeffelena,1, Siyuan Wanga,b, Leon Furchtgottc,d, Kerwyn Casey Huangd, Ned S. Wingreena,b, Joshua W. Shaevitzb,e,1, and Zemer Gitaia,1 aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; bLewis–Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; cBiophysics Program, Harvard University, Cambridge, MA 02138; dDepartment of Bioengineering, Stanford University, Stanford, CA 94305; and eDepartment of Physics, Princeton University, Princeton, NJ 08854 Edited by Lucy Shapiro, Stanford University School of Medicine, Palo Alto, CA, and approved July 27, 2011 (received for review June 3, 2011) Bacterial cells possess multiple cytoskeletal proteins involved in a tently in a nearly circumferential direction. Interestingly, this wide range of cellular processes. These cytoskeletal proteins are MreB rotation is not driven by its own polymerization, but rather dynamic, but the driving forces and cellular functions of these requires cell-wall synthesis. These findings indicate that a motor dynamics remain poorly understood. Eukaryotic cytoskeletal dy- whose activity depends on cell-wall assembly rotates MreB. namics are often driven by motor proteins, but in bacteria no mo- Furthermore, the coupling of MreB rotation to cell-wall synthesis tors that drive cytoskeletal motion have been identified to date. suggests that MreB may not merely act upstream of cell-wall Here, we quantitatively study the dynamics of the Escherichia coli assembly. Indeed, computational simulations suggest that cou- actin homolog MreB, which is essential for the maintenance of pling MreB rotation to cell-wall synthesis can help cells maintain rod-like cell shape in bacteria.
    [Show full text]
  • Structural and Genetic Analyses Reveal the Protein Sepf As a New
    Structural and genetic analyses reveal the protein PNAS PLUS SepF as a new membrane anchor for the Z ring Ramona Dumana,1, Shu Ishikawab,1, Ilkay Celikc,1, Henrik Strahlc, Naotake Ogasawarab, Paulina Troca, Jan Löwea,2, and Leendert W. Hamoenc,d,2 aMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom; bGraduate School of Information Science Functional Genomics, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan; cCentre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle NE2 4AX, United Kingdom; and dSwammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH, Amsterdam, The Netherlands Edited by Richard Losick, Harvard University, Cambridge, MA, and approved October 11, 2013 (received for review July 26, 2013) A key step in bacterial cell division is the polymerization of the N-terminal transmembrane domain (13). The necessity for ZipA tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the can be bypassed by a gain-of-function mutation in FtsA (14). cell membrane by FtsA and are required for the assembly of all Gram-positive bacteria contain EzrA, which shows a similar to- other cell division proteins. In Gram-positive and cyanobacteria, pology to that of ZipA, with an N-terminal transmembrane helix FtsZ filaments are aligned by the protein SepF, which in vitro pol- and a large C-terminal domain that binds to the FtsZ C terminus ymerizes into large rings that bundle FtsZ filaments. Here we de- (15). It therefore seemed likely that EzrA functions as an al- scribe the crystal structure of the only globular domain of SepF, ternative membrane anchor for the Z ring in B.
    [Show full text]
  • Arxiv:1105.2423V1 [Physics.Bio-Ph] 12 May 2011 C
    Cytoskeleton and Cell Motility Thomas Risler Institut Curie, Centre de Recherche, UMR 168 (UPMC Univ Paris 06, CNRS), 26 rue d'Ulm, F-75005 Paris, France Article Outline C. Macroscopic phenomenological approaches: The active gels Glossary D. Comparisons of the different approaches to de- scribing active polymer solutions I. Definition of the Subject and Its Importance VIII. Extensions and Future Directions II. Introduction Acknowledgments III. The Diversity of Cell Motility Bibliography A. Swimming B. Crawling C. Extensions of cell motility IV. The Cell Cytoskeleton A. Biopolymers B. Molecular motors C. Motor families D. Other cytoskeleton-associated proteins E. Cell anchoring and regulatory pathways F. The prokaryotic cytoskeleton V. Filament-Driven Motility A. Microtubule growth and catastrophes B. Actin gels C. Modeling polymerization forces D. A model system for studying actin-based motil- ity: The bacterium Listeria monocytogenes E. Another example of filament-driven amoeboid motility: The nematode sperm cell VI. Motor-Driven Motility A. Generic considerations B. Phenomenological description close to thermo- dynamic equilibrium arXiv:1105.2423v1 [physics.bio-ph] 12 May 2011 C. Hopping and transport models D. The two-state model E. Coupled motors and spontaneous oscillations F. Axonemal beating VII. Putting It Together: Active Polymer Solu- tions A. Mesoscopic approaches B. Microscopic approaches 2 Glossary I. DEFINITION OF THE SUBJECT AND ITS IMPORTANCE Cell Structural and functional elementary unit of all life forms. The cell is the smallest unit that can be We, as human beings, are made of a collection of cells, characterized as living. which are most commonly considered as the elementary building blocks of all living forms on earth [1].
    [Show full text]
  • Identification and Characterization of Novel Filament-Forming Proteins In
    www.nature.com/scientificreports OPEN Identifcation and characterization of novel flament-forming proteins in cyanobacteria Benjamin L. Springstein 1,4*, Christian Woehle1,5, Julia Weissenbach1,6, Andreas O. Helbig2, Tal Dagan 1 & Karina Stucken3* Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel flament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative flament-forming proteins, we observed a higher proportion of CCRPs in flamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identifed nine protein families with putative intermediate flament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed flaments in vivo in several organisms. Additionally, we identifed a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into flaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form flaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into flaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic flament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity. Species in the phylum Cyanobacteria present a wide morphological diversity, ranging from unicellular to mul- ticellular organisms.
    [Show full text]
  • Bacterial Actin Mreb Forms Antiparallel Double Filaments Fusinita Van Den Ent1*†, Thierry Izoré1†, Tanmay AM Bharat1, Christopher M Johnson2, Jan Löwe1
    RESEARCH ARTICLE elifesciences.org Bacterial actin MreB forms antiparallel double filaments Fusinita van den Ent1*†, Thierry Izoré1†, Tanmay AM Bharat1, Christopher M Johnson2, Jan Löwe1 1Structural Studies Division, Medical Research Council - Laboratory of Molecular Biology, Cambridge, United Kingdom; 2Protein and Nucleic Acid Chemistry Division, Medical Research Council - Laboratory of Molecular Biology, Cambridge, United Kingdom Abstract Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments ofCaulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: 10.7554/eLife.02634.001 *For correspondence: fent@mrc- lmb.cam.ac.uk Introduction †These authors contributed Cell shape is a characteristic and hereditary feature that is crucial for existence and its regulation is a equally to this work common challenge for organisms across all biological kingdoms.
    [Show full text]
  • Investigating the Actin Regulatory Activities of Las17, the Wasp Homologue in S. Cerevisiae Liemya E. Abugharsa
    Investigating the actin regulatory activities of Las17, the WASp homologue in S. cerevisiae A thesis submitted for the degree of Doctor of Philosophy By Liemya E. Abugharsa Department of Molecular Biology and Biotechnology University of Sheffield March 2015 Abstract Investigating the actin regulatory activities of Las17, the WASp homologue in S. cerevisiae Clathrin mediated endocytosis (CME) in S. cerevisiae requires the dynamic interplay between many proteins at the plasma membrane. Actin polymerisation provides force to drive membrane invagination and vesicle scission. The WASp homologue in yeast, Las17 plays a major role in stimulating actin filament assembly during endocytosis. The actin nucleation ability of WASP family members is attributed to their WCA domain [WH2 (WASP homology2) domain, C central, and A (acidic) domains] which provides binding sites for both actin monomers and the Arp2/3 complex. In addition, the central poly-proline repeat region of Las17 is able to bind and nucleate actin filaments independently of the Arp2/3 complex. While Las17 is a key regulator of endocytic progression and has been found to be phosphorylated in global studies, the mechanism behind regulation of Las17 actin-based function is unclear. Therefore, the aims of this study were to investigate the post-translation modification of Las17 by phosphorylation, and to determine how this modification impacts on Las17 function both in vivo and in vitro. Mass Spec analysis was employed and allowed identification of further phosphorylation sites in Las17. Through the studies described here I was able to demonstrate that Las17 is phosphorylated, and that one specific phosphorylation event was of importance in endocytosis.
    [Show full text]
  • Cytoskeleton and Cell Motility Thomas Risler
    Cytoskeleton and Cell Motility Thomas Risler To cite this version: Thomas Risler. Cytoskeleton and Cell Motility. Robert A. Meyers. Encyclopedia of Complexity and System Science, Springer, pp.1738-1774, 2009, 978-0-387-75888-6. 10.1007/978-0-387-30440-3_112. hal-00961037 HAL Id: hal-00961037 https://hal.archives-ouvertes.fr/hal-00961037 Submitted on 22 Mar 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Cytoskeleton and Cell Motility Thomas Risler Institut Curie, Centre de Recherche, UMR 168 (UPMC Univ Paris 06, CNRS), 26 rue d'Ulm, F-75005 Paris, France Article Outline C. Macroscopic phenomenological approaches: The active gels Glossary D. Comparisons of the different approaches to de- scribing active polymer solutions I. Definition of the Subject and Its Importance VIII. Extensions and Future Directions II. Introduction Acknowledgments III. The Diversity of Cell Motility Bibliography A. Swimming B. Crawling C. Extensions of cell motility IV. The Cell Cytoskeleton A. Biopolymers B. Molecular motors C. Motor families D. Other cytoskeleton-associated proteins E. Cell anchoring and regulatory pathways F. The prokaryotic cytoskeleton V. Filament-Driven Motility A. Microtubule growth and catastrophes B.
    [Show full text]