Name: using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones
Uploaded: 2014-09-07T15:00:00
Description: Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

We thank Dr. Gaudenz Danuser and members of his lab for creating the plusTipTracker software and for helpful discussion regarding using the software, in particular Maria Bagonis and Sebastien Besson. We especially thank the Boston College Media Center for their assistance and support in the creation and editing of the video. We also thank members of the Lowery Lab for useful discussions and constructive criticism, and Abigail Antoine for proof-reading the manuscript. This work was funded by an NIH R00 MH095768 award to LAL.

Denne protokol og video er beregnet til at tjene som en følgesvend til den oprindelige papir, der beskriver softwarepakken nærmere 4, samt den tekniske rapport, der kommer med software download på Danuser Labs websted. Læserne opfordres til at gennemgå disse dokumenter omhyggeligt, hvis der er yderligere spørgsmål vedrørende brugen af ​​softwaren. 1. Forud for billedanalyse <ol><li> Konverter hver time-lapse film til en sekvens af TIFF (Tagged Image File Format) bill...

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	<li>Stepanova, T., 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=Visualization+of+microtubule+growth+in+cultured+neurons+via+the+use+of+EB3-GFP+(end-binding+protein+3-green+fluorescent+protein).">Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 2655-2664 (2003).</li><li>Lee, H., 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=The+microtubule+plus+end+tracking+protein+Orbit/MAST/CLASP+acts+downstream+of+the+tyrosine+kinase+Abl+in+mediating+axon+guidance.">The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.</a> Neuron. , 913-926 (2004).</li><li>Purro, S. A., 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=Wnt+regulates+axon+behavior+through+changes+in+microtubule+growth+directionality:+a+new+role+for+adenomatous+polyposis+coli.">Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28, 8644-8654 (2008).</li><li>Applegate, K. T., 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=plusTipTracker:+Quantitative+image+analysis+software+for+the+measurement+of+microtubule+dynamics.">plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.</a> Journal of structural biology. 176, 168-184 (2011).</li><li>Lowery, L. A., Faris, A. E., Stout, A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Explant+Cultures+from+Xenopus+laevis.">Neural Explant Cultures from Xenopus laevis.</a> Journal of visualized experiments : JoVE. (68), e4232 (2012).</li><li>Long, J. B., 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=Multiparametric+analysis+of+CLASP-interacting+protein+functions+during+interphase+microtubule+dynamics.">Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.</a> Molecular and cellular biology. 33, 1528-1545 (2013).</li><li>Myers, K. A., Applegate, K. T., Danuser, G., Fischer, R. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ECM+mechanosensing+pathways+regulate+microtubule+dynamics+to+control+endothelial+cell+branching+morphogenesis.">Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis.</a> The Journal of cell biology. 192, 321-334 (2011).</li><li>Nishimura, Y., Applegate, K., Davidson, M. W., Danuser, G., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+microtubule+growth+dynamics+identifies+MARK2+as+a+regulator+of+leading+edge+microtubules+downstream+of+Rac1+in+migrating+cells.">Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.</a> PLoS One. 7, e41413 (2012).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+ends:+a+dynamic+protein+network+controls+the+fate+of+microtubule+tips.">Tracking the ends: a dynamic protein network controls the fate of microtubule tips.</a> Nature reviews. Molecular cell biology. 9, 309-322 (2008).</li><li>Schuyler, S. C., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+'plus-end-tracking+proteins':+The+end+is+just+the+beginning.">Microtubule 'plus-end-tracking proteins': The end is just the beginning.</a> Cell. 105, 421-424 (2001).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312, 237-242 (1984).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Current biology : CB. 10, 865-868 (2000).</li><li>Dixit, R., 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=Microtubule+plus-end+tracking+by+CLIP-170+requires+EB1.">Microtubule plus-end tracking by CLIP-170 requires EB1.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 492-497 (2009).</li><li>Li, W., 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=EB1+promotes+microtubule+dynamics+by+recruiting+Sentin+in+Drosophila+cells.">EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells.</a> The Journal of cell biology. 193, 973-983 (2011).</li><li>Maurer, S. P., 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=EB1+accelerates+two+conformational+transitions+important+for+microtubule+maturation+and+dynamics.">EB1 accelerates two conformational transitions important for microtubule maturation and dynamics.</a> Current biology : CB. 24, 372-384 (2014).</li><li>Zanic, M., Widlund, P. O., Hyman, A. A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nature cell biology. 15, 688-693 (2013).</li><li>Lowery, L. A., 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=Growth+cone-specific+functions+of+XMAP215+in+restricting+microtubule+dynamics+and+promoting+axonal+outgrowth.">Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth.</a> Neural development. 8, 22 (2013).</li><li>Marx, A., 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=Xenopus+cytoplasmic+linker-associated+protein+1+(XCLASP1)+promotes+axon+elongation+and+advance+of+pioneer+microtubules.">Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.</a> Molecular biology of the cell. 24, 1544-1558 (2013).</li></ol>

PlusTipTracker giver en enkel, grafisk brugergrænseflade til hurtigt og automatisk at registrere næsten alle synlige EB1-GFP-kometer i en celle eller vækst kegle, knytte de kometer i spor, og beregne MT parametre. Andre publikationer har rapporteret design af lignende typer af software (for eksempel Marx et al. Også brugt kvantitativ analyse af taggede EB1 dynamik i vækstkegler 18). Men synes denne software til at være enestående i sin lette adgang, som det frit kan downloades fra hjemmesiden ...

Målet med denne metode er at få kvantitative oplysninger om mikrotubuli (MT) plus-end-sporing protein (+ tip) dynamik i levende vækstkegler. MT + Drikkepenge er en gruppe af proteiner, der lokaliserer til plus-ender MTS 9,10. De udfører en række funktioner til at regulere parametre for MT dynamisk ustabilitet 11, herunder satserne for polymerisation, katastrofe, og redning. En godt brugt metode til at analysere MT dynamik er at spore opførsel + TIP EB1, som binder specifikt til voksende MT plus-ender 1,12. EB1 er kendt for at rekruttere flere andre proteiner til voksende MT plus-ender 13,14, og er for nylig blevet etableret som en MT modning faktor 15, fremme både MT vækst og katastrofe frekvens 15,16. Mange studier af MT dynamik inden vækstkegler har udnyttet hånd-tracking metoder til at måle ændringer i EB1-GFP-dynamik over tid 1-3, som EB1 localizatipå MT plus-ender kan anvendes som en markør for MT polymerisation. En vigtig fordel for behandlingen EB1-GFP-kometer som en proxy for MT vækst er, at der kan måles MT dynamik selv i områder af væsentlig MT overlapning. Mens metoden af hånd-tracking EB1-GFP-kometer har givet en nyttig indsigt i MT adfærdsmønstre 1-3, det er tidskrævende og kan være tendentiøst. Derudover, som afvigende vækst kegle adfærd er sandsynligvis et resultat af meget små forskydninger i cytoskeletale dynamik, analysere kun en lille delmængde af MTS (normalt nødvendigt, når hånd-sporing) kan gå glip af vigtige oplysninger. Således måler vi globale MT dynamics parametre ved hjælp af softwarepakken, plusTipTracker 4, efter opkøbet af høj opløsning, levende billeder af tagget EB1 i dyrkede embryonale vækstkegler 5. Denne software er udviklet i Danuser Lab, har været anvendt i flere studier, der kendetegner MT dynamik i forskellige celletyper 6-8. Det er en open-source, osER børn, MATLAB-baserede pakke, der inkluderer automatiseret detektion, sporing, visualisering og analyse for film af fluorescens-mærket + tips. En lang liste af specifikke parametre for MT dynamik beregnes ved denne software (se reference 4 for detaljer), men til analyse af MT dynamik i vækstkegler, de mest nyttige parametre er MT vækstsporet hastighed (i mikron / minut), vækstsporet levetid (i sekunder), og væksten banelængde (i mikrometer). Softwaren kan downloades direkte fra Danuser Lab hjemmeside (under &quot;Software&quot;). Mens Danuser Lab understøtter i øjeblikket en nyere interface til + TIP sporing analyse, som er indarbejdet i en software-pakke kaldet U-bane 2.0, den oprindelige, stand-alone-software vil forblive tilgængelige. De underliggende algoritmer mellem de to programmer er de samme (i det mindste fra 2014), med kun en forskel på grænsefladen og analyse udgange. For nybegynderen med lidt Matlab og / eller beregningsmæssige analyse erfaning, plusTipTracker har flere brugervenlige funktioner, herunder automatiserede statistiske parameter udgange. Her beskriver vi de skridt til at analysere billeder af EB1-GFP-dynamik i dyrkede Xenopus laevis vækstkegler. Denne protokol blev udnyttet i et nyligt papir undersøger MT dynamik 17. Se også Lowery et al. 2012 5 for detaljerede anvisninger vedrørende dyrkning vækstkegler udtrykker EB1-GFP. Mens dette dokument primært fokuseret på at undersøge EB1-GFP-dynamik i vækstkegler, kan den samme protokol bruges til andre celletyper 17. For alle celletyper, skal tidsintervallet mellem frames være mellem 0,5-2 sek til optimal + TIP sporing. Et tidsinterval på op til 4 sekunder mellem rammer er muligt, men denne øgede interval tid resulterer i yderligere sporingsfejl.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">plusTipTracker software</td>
			<td style="width:116px;">Danuser Lab</td>
			<td style="width:345px;"><a href="http://lccb.hms.harvard.edu/software.html">http://lccb.hms.harvard.edu/software.html</a></td>
			<td style="width:172px;">This software may be hosted by another website in the future.&#160; If the listed site does not exist, search &#34;Danuser Lab Software&#34; on a web search engine to find the site.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Matlab software</td>
			<td style="width:116px;">Mathworks</td>
			<td><a href="http://www.mathworks.com/products/matlab/">http://www.mathworks.com/products/matlab/</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones

Mikrotubuli (MT) plus-end-tracking proteiner (+ tips) lokalisere til de voksende plus-ender MTS og regulere MT dynamik 1,2. En af de mest kendte og udbredte udnyttede + tips til at analysere MT dynamik er slutningen protein, EB1, som binder alle voksende MT plus-ender, og dermed er en markør for MT polymerisation 1. Mange studier af EB1 adfærd inden vækstkegler har brugt tidskrævende og forudindtagede computer-assisteret, hånd-tracking metoder til at analysere de enkelte MTS 1-3. Vores tilgang er at kvantificere de globale parametre for MT dynamik ved hjælp af software-pakke, plusTipTracker 4, efter købet af høj opløsning, levende billeder af tagget EB1 i dyrkede embryonale vækstkegler 5. Denne software er en Matlab-baseret, open source, brugervenlig pakke, der kombinerer automatiseret detektion, sporing, visualisering og analyse af film af fluorescens-mærket + tips. Her præsenterer vi en protokol for at bruge plusTipTracker til analyse af fluorescens-mærket + TIP kometer i dyrkede Xenopus laevis vækstkegler. Imidlertid kan denne software også bruges til at karakterisere MT dynamikken i forskellige celletyper 6-8.

Ved hjælp af denne software, som beskrevet her, vil give flere filer af information, der kvantificerer + tip dynamik i levende celler. De funktionelle plusTipGetTracks identificerer spor (ved hjælp af eksempler på forskellige indstillinger er vist i figur 1), og derefter giver parametre vedrørende + tip spor. Hvis du vil se de oplysninger, som softwaren har opnået, gå ind i roi_X bibliotek, der blev oprettet i trin 2.2. Mappen &quot;feat&quot; indeholder &quot;overlayI...

Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Matlab-baseret, open source software-pakke, plusTipTracker, kan bruges til at analysere billede serie af fluorescens-mærket + tips til at kvantificere mikrotubuli dynamikken.

Brug plusTipTracker Software til måling mikrotubulære Dynamics i<em&gt; Xenopus laevis</em&gt; vækstkegler

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

Research

JoVE Journal

Biology

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

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