Name: super-resolution imaging of the bacterial division machinery
Uploaded: 2013-01-21T13:00:00
Description: Watch this Scientific Journal Video about Super-resolution Imaging of the Bacterial Division Machinery at JoVE.com

Grant: 5RO1GM086447-02.

1. Prøvefremstilling <ol><li> Inokulere LB-medier med en enkelt koloni af stammen JB281 [BW25113 / pJB042 (P Lac: FtsZ-mEos2)]. Vokse natten over på et rysteapparat ved 37 ° C. </li><li> Fortynd kulturen 1:1000 i M9 + minimale medier [M9-salte, 0,4% glucose, 2 mM MgSO4, 0,1 mM CaCl2, MEM aminosyrer og vitaminer] og vokser til mid-log fase (OD600 = 0,2 til 0,3) i nærværelse af chloramphenicol (150 ug / ml) ved stuetemperatur (RT). </li><li> Fremkald kultur med...

<ol>
	<li>Buddelmeijer, N., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+cell+division+proteins+at+the+E.+coli+cell+center.">Assembly of cell division proteins at the E. coli cell center.</a> Curr. Opin. Microbiol. 5, 553-557 (2002).</li><li>de Boer, P., Crossley, R., Rothfield, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+bacterial+cell-division+protein+FtsZ+is+a+GTPase.">The essential bacterial cell-division protein FtsZ is a GTPase.</a> Nature. 359, 254-256 (1992).</li><li>Lutkenhaus, J. F., Wolf-Watz, H., Donachie, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+genes+in+the+ftsA-envA+region+of+the+Escherichia+coli+genetic+map+and+identification+of+a+new+fts+locus+(ftsZ).">Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus (ftsZ).</a> J. Bacteriol. 142, 615-620 (1980).</li><li>Bi, E. F., Lutkenhaus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FtsZ+ring+structure+associated+with+division+in+Escherichia+coli.">FtsZ ring structure associated with division in Escherichia coli.</a> Nature. 354, 161-164 (1991).</li><li>Erickson, H. P., Taylor, D. W., Taylor, K. A., Bramhill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cell+division+protein+FtsZ+assembles+into+protofilament+sheets+and+minirings,+structural+homologs+of+tubulin+polymers.">Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 519-523 (1996).</li><li>Osawa, M., Anderson, D. E., Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+contractile+FtsZ+rings+in+liposomes.">Reconstitution of contractile FtsZ rings in liposomes.</a> Science. 320, 792-794 (2008).</li><li>Li, Z., Trimble, M. J., Brun, Y. V., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+FtsZ+filaments+in+vivo+suggests+a+force-generating+role+in+cell+division.">The structure of FtsZ filaments in vivo suggests a force-generating role in cell division.</a> Embo J. 26, 4694-4708 (2007).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nat. Methods. 6, 131-133 (2009).</li><li>Fu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+FtsZ-ring+structure+revealed+by+Photoactivated+Localization+Microisocpy+(PALM).">In-vivo FtsZ-ring structure revealed by Photoactivated Localization Microisocpy (PALM).</a> Plos One. , (2010).</li><li>Huecas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+and+geometry+of+FtsZ+polymers:+nucleated+self-assembly+of+single+protofilaments.">Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments.</a> Biophys. J. , (2007).</li><li>Ma, X., Ehrhardt, D. W., Margolin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+cell+division+proteins+FtsZ+and+FtsA+to+cytoskeletal+structures+in+living+Escherichia+coli+cells+by+using+green+fluorescent+protein.">Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 12998-13003 (1996).</li><li>Dai, K., Lutkenhaus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proper+ratio+of+FtsZ+to+FtsA+is+required+for+cell+division+to+occur+in+Escherichia+coli.">The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.</a> J. Bacteriol. 174, 6145-6151 (1992).</li><li>Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U., Radenovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clustering+artifacts+in+photoactivated+localization+microscopy.">Identification of clustering artifacts in photoactivated localization microscopy.</a> Nat. Meth. 8, 527-528 (2011).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319, 810-813 (2008).</li></ol>

PALM billeder indeholder information om molekyle tæller og positioner inden for en celle, hvilket tillader detaljeret analyse af fordelingen og placeringen af ​​mål-proteinmolekyler, som er vanskeligt at opnå på anden måde. Nedenfor skitserer vi forholdsregler, der skal træffes for at udtrække præcis kvantitativ information og samtidig opretholde den biologiske relevans af PALM billeder. Vi har også udforske oplysninger, der kan bedst opnås ved anvendelse af levende vs faste celler. Endelig foreslår vi mu...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 50 x MEM aminosyrer </td><td> Sigma </td><td> M5550 </td><td></td></tr><tr><td> 100 x MEM Vitaminer </td><td> Sigma </td><td> M6895 </td><td></td></tr><tr><td> IPTG </td><td> Mediatech </td><td> 46-102-RF </td><td></td></tr><tr><td> 16% Paraformaldehyd </td><td> Electron Micrsocopy Sciences </td><td> 15710-S </td><td></td></tr><tr><td> SeaPlaque GTG Agarose </td><td> Lonzo </td><td> 50.111 </td><td></td></tr><tr><td> 50 nm guldperler </td><td> Mikrokugler-nanosfærer </td><td> 790113-010 &lt;/ Td&gt; <td></td></tr><tr><td> FCS2 Imaging Chamber </td><td> Bioptechs </td><td></td><td></td></tr><tr><td> Stage Adaptor </td><td> ASI </td><td> I-3017 </td><td></td></tr><tr><td> Omvendt mikroskop </td><td> Olympus </td><td> IX71 </td><td></td></tr><tr><td> 1,45 NA, 60x Formål </td><td> Olympus </td><td></td><td></td></tr><tr><td> IXON EMCCD Camera </td><td> Andor Technology </td><td> DU897E </td><td></td></tr><tr><td> 488-nm Sapphire Laser </td><td> Sammenhængende </td><td></td><td></td></tr><tr><td> 561-nm Sapphire Laser </td><td> Sammenhængende </td><td></td><td></td></tr><tr><td> 405-nm CUBE Laser </td><td> Sammenhængende </td><td></td><td></td>
 </tr>
 </tbody>
 </table>

super-resolution imaging of the bacterial division machinery

Bakteriel celledeling kræver den koordinerede samling af mere end ti essentielle proteiner ved midcell 1,2. Centralt for denne proces er dannelsen af en ringlignende suprastructure (Z-ring) af FtsZ proteinet ved opdelingsplaner 3,4. Z-ring består af multiple enkeltstrengede FtsZ protofilamenter, og forståelse af arrangementet af protofilamenter inde i Z-ringen, vil give indsigt i mekanismen for Z-ringsamlingen og dets funktion som force generator 5,6. Denne information er forblevet undvigende grund af de nuværende begrænsninger i konventionelle fluorescens mikroskopi og elektronmikroskopi. Konventionel fluorescens-mikroskopi er i stand til at tilvejebringe et billede med høj opløsning af Z-ring på grund af diffraktion grænsen af ​​lys (~ 200 nm). Electron cryotomographic billeddannelse har registreret spredt FtsZ protofilamenter i små C. crescentus celler 7, men er vanskelig at anvende til større celler, såsomE. coli eller B. subtilis. Her beskriver vi anvendelsen af en super-opløsning fluorescensmikroskopi metode, til fotoaktiveret Localization Microscopy (PALM), kvantitativt karakterisere den strukturelle organisering af E. coli Z-ring 8. PALM billedbehandling tilbyder både høj rumlig opløsning (~ 35 nm) og særlig mærkning for at muliggøre entydig identifikation af target-proteiner. Vi mærkede FtsZ med den fotoaktiverbare fluorescerende protein mEos2, som skifter fra grøn fluorescens (excitation = 488 nm) til rød fluorescens (excitation = 561 nm) efter aktivering ved 405 nm 9. Under en PALM eksperiment, er enlige FtsZ-mEos2 molekyler stokastisk aktiveret og de tilsvarende centroid positioner af de enkelte molekyler bestemmes med &lt;20 nm præcision. En super-opløsning billede af Z-ringen bliver så rekonstrueret ved at sammenføje det geometriske tyngdepunkt positioner for alle detekterede FtsZ-mEos2 molekyler. &lt;p class = &quot;jove_content&quot;&gt; anvendelse af denne fremgangsmåde, har vi fundet, at Z-ringen har en fast bredde på ~ 100 nm og består af en løs bundt FtsZ protofilamenter der overlapper hinanden i tre dimensioner. Disse data giver et afsæt til yderligere undersøgelser af cellecyklus afhængige ændringer i Z-ringen 10 og kan anvendes på andre proteiner af interesse.

Vist i fig 3Aiv er et todimensionalt, super opløsning præstationen af Z-ringen dannet fra PALM imaging beskrevet ovenfor. Nedenfor har vi opsummere kvalitative og kvantitative oplysninger, der kan opnås fra dem. Kvalitativt vi observeret, at Z-ringen er en uregelmæssig struktur, der anvender multiple konfigurationer (enkelt bånd eller spiralformede bue), der ikke kan skelnes i konventionelle fluorescensbilleder (sammenlign figur 3A-Dii og iv).</...

Watch this Scientific Journal Video about Super-resolution Imaging of the Bacterial Division Machinery at JoVE.com

Super-resolution Imaging of the Bacterial Division Machinery

Vi beskriver en super-opløsning billeddannelse metoden til undersøgelse af den strukturelle organisation af det bakterielle FtsZ-ring, en væsentlig apparat til celledeling. Denne metode er baseret på kvantitative analyser af fotoaktiverede lokalisering mikroskopi (PALM) billeder og kan anvendes på andre bakterielle cytoskeletproteiner.

Super-opløsning Imaging af Bakteriel Division Machinery

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Research

JoVE Journal

Biology

Test Samples for Optimizing STORM Super-Resolution Microscopy

Prøverne, til Optimering STORM Super-opløsning Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Prøve forberedelse til single virion Atomic Force mikroskopi og superopløsning fluorescens Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Super-opløsning Imaging af Cytokinetic Z Ring i levende bakterier Brug af Fast 3D-strukturerede Illumination Mikroskopi (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Forberedelse, Imaging, og kvantificering af bakteriel Surface Motilitet assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Tre-dimensionel Super Resolution Mikroskopi af F-actinfilamenter af Interferometrisk fotoaktiverede Lokalisering Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Open source Single-partikel Analyse for Super-resolution Microscopy med VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Grundtilstand udtynding super-resolution Imaging i pattedyrceller

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

Anvendelsen af højhastigheds super-resolution hastighed mikroskopi i Live primære Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Korrektion med høj nøjagtighed af 3D-kromatisk forskydninger i en alder af biologisk billedbehandling i superopløsning ved hjælp af Chromagnon

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to ...

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Time-lapse Imaging af bakterielle sværme og den kollektive stress respons

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...