Name: recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography
Uploaded: 2010-08-06T17:30:00
Description: Watch this Scientific Journal Video about Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography at JoVE.com

Esta pesquisa foi possível graças a um prêmio de carreira National Science Foundation (MCB-0746066, caracterização de ativadores de transcrição que regulam o comportamento emergente) para RDW Somos gratos a LJ Shimkus, Goldman BS, Suen G., Singer M., Welch LG, Murphy KA, e Taylor HG para discussões úteis e comentários sobre o manuscrito.

Suprimentos necessários: <ul><li> Klett metros </li><li> Pipeta e dicas </li><li> 2,5 ml tubos de microcentrífuga </li><li> Microcentrífuga </li><li> CTTYE mídia: <ul><li> Casitone 1,0% (Difco Laboratories), extrato de levedura 0,5% (Difco Laboratories), </li><li> 10,0 mM Tris-HCl (pH 8,0), 1,0 mM KH 2 PO 4, 8,0 mM MgSO 4 </li></ul></li><li> TPM mídia: <ul><li> 10,0 mM Tris-HCl (pH 8,0), 1,0 mM KH 2 PO 4, 8,0 mM MgSO 4 </li></ul></li><li> Agarose <...

<ol>
	<li>Taylor, R. G., Welch, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+as+an+emergent+property+of+a+swarm.">Chemotaxis as an emergent property of a swarm.</a> J Bacteriol. 190, 6811-6816 (2008).</li><li>Curtis, P. D., Taylor, R. G., Welch, R. D., Shimkets, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Myxococcus+xanthus+During+Fruiting+Body+Formation.">Spatial Organization of Myxococcus xanthus During Fruiting Body Formation.</a> J Bacteriol. 189, 9126-9130 (2007).</li><li>Mignot, T., Merlie, J. P., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+pole-to-pole+oscillations+of+a+bacterial+gliding+motility+protein.">Regulated pole-to-pole oscillations of a bacterial gliding motility protein.</a> Science. 310, 855-857 (2005).</li><li>Stoodley, P., Hall-Stoodley, L., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+surface+migration,+and+other+dynamic+behavior+in+bacterial+biofilms+revealed+by+digital+time-lapse+imaging.">Detachment surface migration, and other dynamic behavior in bacterial biofilms revealed by digital time-lapse imaging.</a> Methods Enzymol. 337, 306-319 (2001).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu Rev Microbiol. 54, 49-79 (2000).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+and+twitching+motility+are+necessary+for+Pseudomonas+aeruginosa+biofilm+development.">Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.</a> Mol Microbiol. 30, 295-304 (1998).</li><li>Dalton, H. M., Poulsen, L. K., Halasz, P., Angles, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substratum-induced+morphological+changes+in+a+marine+bacterium+and+their+relevance+to+biofilm+structure.">Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.</a> J Bacteriol. 176, 6900-6906 (1994).</li><li>Dworkin, M., Eide, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxococcus+xanthus+does+not+respond+chemotactically+to+moderate+concentration+gradients.">Myxococcus xanthus does not respond chemotactically to moderate concentration gradients.</a> J Bacteriol. 154, 437-442 (1983).</li><li>Dworkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactic+behavior+of+Myxococcus+xanthus.">Tactic behavior of Myxococcus xanthus.</a> J Bacteriol. 154, 452-459 (1983).</li><li>Wu, S. S., Kaiser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+expression+of+the+pilA+gene+in+Myxococcus+xanthus.">Regulation of expression of the pilA gene in Myxococcus xanthus.</a> J Bacteriol. 179, 7748-7758 (1997).</li><li>Bustamante, V. H., Mart&#237;nez-Flores, I., Vlamakis, H. C., Zusman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Frz+signal+transduction+system+of+Myxococcus+xanthus+shows+the+importance+of+the+conserved+C-terminal+region+of+the+cytoplasmic+chemoreceptor+FrzCD+in+sensing+signals.">Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals.</a> Mol Microbiol. 53, 1501-1513 (2004).</li></ol>

Lapso de tempo microcinematography (TM) tornou-se uma abordagem padrão para estudar a motilidade procarióticas 2-7. Tradicionalmente, a TM é realizado usando mechas de papel de filtro, pastilhas de ágar fina, ou placas de ágar como substrato 8-11. Estes métodos são adequados e de custo eficaz quando usado para gerar seqüências de imagens para ilustrações geral do movimento bacteriano. No entanto, se seqüências de imagens deve resultar na geração de dados reprodutíveis e quantitativa...

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<td>1.0% Casitone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
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<td>0.5% yeast extract</td>
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<td>Difco Laboratories</td>
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<td>Micro-sampling pipette</td>
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<td>Fisher</td>
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<td>100 &mu;l glass disposable tip</td>
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<td>Fisher</td>
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<td>2 x 2 cm, 0.5-mm-thick silicone rubber gasket</td>
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<td>Grace Bio-Lab Inc.</td>
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recording multicellular behavior in myxococcus xanthus biofilms using time-lapse microcinematography

Um enxame da δ-proteobacterium Myxococcus xanthus contém milhões de células que funcionam como um movimento coletivo, coordenando através de uma série de sinais para criar complexos, padrões dinâmicos como uma resposta a estímulos ambientais. Estes padrões estão a organizar-auto e emergentes, não pode ser prevista observando o comportamento das células individuais. Usando um lapso de tempo de ensaio de rastreamento microcinematography, identificamos um padrão distinto emergente em M. xanthus chamado quimiotaxia, definido como o movimento dirigido de um enxame de um gradiente de nutrientes em direção a sua fonte de 1. A fim de caracterizar de forma eficiente via quimiotaxia lapso de tempo microcinematography, desenvolvemos um altamente complexo placa modificáveis ​​(figura 1) e construído um cluster de 8 de microscópios (Figura 2), cada um capaz de capturar vídeos de lapso de tempo. O ensaio é rigoroso o suficiente para permitir a replicação consistente de dados quantificáveis, e os vídeos resultantes nos permitem observar e acompanhar as mudanças sutis no comportamento enxame. Uma vez capturados, os vídeos são transferidos para um computador de análise / armazenamento com memória suficiente para processar e armazenar milhares de vídeos. A flexibilidade desta configuração tem se mostrado útil para vários membros da M. xanthus comunidade.

Watch this Scientific Journal Video about Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography at JoVE.com

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Para estudar<em&gt; Myxococcus xanthus</em&gt; Swarm comportamento, criamos um protocolo microcinematography lapso de tempo que podem ser modificados para ensaios diferentes. Emprega condições de crescimento padrão adaptado para microscopia, e produz resultados reproduzíveis pelo uso de barato, juntas de silicone reutilizável. Temos utilizado este método para quantificar a quimiotaxia multicelulares.

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A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

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Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

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