Name: assessment of dictyostelium discoideum response to acute mechanical stimulation
Uploaded: 2017-11-09T12:30:00
Description: Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

Dette arbejde blev støttet af nationale kontorer i sundhed grant R35 GM118177 på PND.

1. forberedelse af løsninger <ol><li>forberede HL-5 medier ved hjælp af følgende: 10 g/L dextrose, 10 g/L proteose pepton, 5 g/L gær extract, 0.965 g/L Na 2 HPO 4 ·7H 2 O, 0.485 g/L KH 2 PO 4, og medmindre andet er angivet, 0,03 g/L streptomycin i ionbyttet vand. Autoklave medium og opbevares ved stuetemperatur. 
	Bemærk: På grund af forskelle mellem proteose pepton og gær ekstrakt erhvervet fra forskellige leverandører samt mellem individuelle batch...

<ol>
	<li>Swaney, K. F., Huang, C. H., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+chemotaxis:+a+network+of+signaling+pathways+controls+motility,+directional+sensing,+and+polarity.">Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.</a> Annu Rev Biophys. 39, 265-289 (2010).</li><li>Sasaki, A. T., Chun, C., Takeda, K., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J Cell Biol. 167 (3), 505-518 (2004).</li><li>Jeon, T. J., Lee, D. -. J., Merlot, S., Weeks, G., Firtel, R. 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D., Marganski, W. A., Wu, L. F., Altschuler, S. J., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Actin-Based+Wave+Generator+Organizes+Cell+Motility.">An Actin-Based Wave Generator Organizes Cell Motility.</a> PLOS Biol. 5 (9), e221 (2007).</li><li>Devreotes, P., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+Networks+that+Regulate+Cell+Migration.">Signaling Networks that Regulate Cell Migration.</a> Cold Spring Harbor Perspectives in Biology. 7 (8), (2015).</li><li>Artemenko, Y., Lampert, T. J., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+towards+a+paradigm:+common+mechanisms+of+chemotactic+signaling+in+Dictyostelium+and+mammalian+leukocytes.">Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes.</a> Cell Mol Life Sci. 71 (19), 3711-3747 (2014).</li><li>Dalous, J., 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=Reversal+of+cell+polarity+and+actin-myosin+cytoskeleton+reorganization+under+mechanical+and+chemical+stimulation.">Reversal of cell polarity and actin-myosin cytoskeleton reorganization under mechanical and chemical stimulation.</a> Biophys J. 94 (3), 1063-1074 (2008).</li><li>Sato, M. J., 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=Switching+direction+in+electric-signal-induced+cell+migration+by+cyclic+guanosine+monophosphate+and+phosphatidylinositol+signaling.">Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.</a> Proc Natl Acad Sci U S A. 106 (16), 6667-6672 (2009).</li><li>Zhao, M., Pu, J., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids,+EGF+receptors,+and+intracellular+signals+colocalize+and+are+polarized+in+epithelial+cells+moving+directionally+in+a+physiological+electric+field.">Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.</a> Faseb j. 16 (8), 857-859 (2002).</li><li>Decave, 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=Shear+flow-induced+motility+of+Dictyostelium+discoideum+cells+on+solid+substrate.">Shear flow-induced motility of Dictyostelium discoideum cells on solid substrate.</a> J Cell Sci. 116 (Pt 21), 4331-4343 (2003).</li><li>Artemenko, Y., Axiotakis, L., Borleis, J., Iglesias, P. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+mechanical+stimuli+act+on+common+signal+transduction+and+cytoskeletal+networks.">Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks.</a> Proc Natl Acad Sci U S A. 113 (47), E7500-E7509 (2016).</li><li>Artemenko, Y., Swaney, K. F., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+development+and+chemotaxis+in+Dictyostelium+discoideum+mutants.">Assessment of development and chemotaxis in Dictyostelium discoideum mutants.</a> Methods Mol Biol. 769, 287-309 (2011).</li><li>Gaudet, P., Pilcher, K. E., Fey, P., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Dictyostelium+discoideum+with+plasmid+DNA.">Transformation of Dictyostelium discoideum with plasmid DNA.</a> Nat. Protocols. 2 (6), 1317-1324 (2007).</li><li>Veltman, D. M., Lemieux, M. G., Knecht, D. A., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIP3-dependent+macropinocytosis+is+incompatible+with+chemotaxis.">PIP3-dependent macropinocytosis is incompatible with chemotaxis.</a> J Cell Biol. 204 (4), 497-505 (2014).</li><li>Cai, H., Huang, C. H., Devreotes, P. N., Iijima, M. <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+chemotaxis+in+Dictyostelium.">Analysis of chemotaxis in Dictyostelium.</a> Methods Mol Biol. 757, 451-468 (2012).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Meth. 9 (7), 676-682 (2012).</li><li>Brenner, M., Thoms, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+blocks+activation+of+cyclic+AMP+synthesis+in+Dictyostelium+discoideum.">Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum.</a> Dev Biol. 101 (1), 136-146 (1984).</li><li>Bretschneider, 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=Dynamic+Actin+Patterns+and+Arp2/3+Assembly+at+the+Substrate-Attached+Surface+of+Motile+Cells.">Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells.</a> Curr Biol. 14 (1), 1-10 (2004).</li><li>Swaney, K. F., Borleis, J., Iglesias, P. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+protein+Callipygian+defines+the+back+of+migrating+cells.">Novel protein Callipygian defines the back of migrating cells.</a> Proc Natl Acad Sci U S A. 112 (29), E3845-E3854 (2015).</li><li>Meili, R., Ellsworth, C., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Akt/PKB-related+kinase+is+essential+for+morphogenesis+in+Dictyostelium.">A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.</a> Curr Biol. 10 (12), 708-717 (2000).</li><li>Westendorf, C., 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=Actin+cytoskeleton+of+chemotactic+amoebae+operates+close+to+the+onset+of+oscillations.">Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations.</a> Proc Natl Acad Sci U S A. 110 (10), 3853-3858 (2013).</li></ol>

De metoder, der beskrives her tilbyder en nem måde at vurdere både befolkningen og individuelle celle adfærd som svar for at vride flow. Vigtigere, mens tidligere undersøgelser analyseret lokalisering af førende og halter kant markører under overflytningen, tillader den nuværende tilgang undersøgelse af umiddelbare virkninger ved at anvende shear flow akut. Benytter indeværende metode viste vi, at i virkeligheden, den første reaktion af celler til shear flow ikke kræver celle migration. I stedet, den hurtige o...

Migration af eukaryote celler er forudindtaget af forskellige kemiske og fysiske signaler i miljøet, herunder forløb af opløselig eller substrat-bundne chemoattractants, variabel stivhed af substrater, elektriske felter eller shear flow. Selv om der er sket mange fremskridt i vores forståelse for de molekylære mekanismer kørsel chemotaxis, mindre vides om andre typer af styret migration og hvordan disse forskellige signaler er integreret på cellulært niveau at udarbejde en samlet vandrende svar.
Instrueret migration mod en stigende koncentration af en chemoattractant indebærer tre adfærdsmæssige komponenter: motilitet, retningsbestemt sensing og polaritet1. Motilitet refererer til tilfældige bevægelse af cellerne opnået ved pseudopod fremspring. Retningsbestemt sensing er en celle evne til at opdage kilden til en chemoattractant, hvilket kan forekomme selv i immobiliserede celler. Polaritet refererer til den mere stabile asymmetrisk distribution af intracellulære komponenter mellem førende og halter kanten af en celle, hvilket fører til øget persistens i bevægelse.
Cellulære reaktion på en chemoattractant afhænger af aktiviteten af fire begrebsmæssigt definerede regulerende net: receptor / G protein, signaltransduktion, actin cytoskelettet og polaritet1. Chemoattractant binding til G protein-koblet receptor sender signal via heterotrimeric G proteiner α og βγ til downstream signaltransduktion netværk, som forstærker directional signal. Flere veje inden for signaltransduktion net fungere parallelt og indgå i actin cytoskeleton netværk til bias actin polymerisering, og deraf følgende pseudopod fremspring, i retning af gradient. Blandt vigtige regulatorer af chemotaxis er Ras GTPase, TorC2, phosphoinositide 3-kinase (PI3K), fosfatase og tensin homolog (PT) og guanylyl cyclase. Feedback-mekanismer inden for signaltransduktion netværk og mellem signaltransduktion og aktin cytoskeleton netværk yderligere forstærke svaret. Endelig, dårligt defineret polaritet netværket modtager input fra actin cytoskeleton, og yderligere fordomme signaltransduktion netværk for at fremme vedvarende migration i retning af gradient.
Meget af vores mekanistiske forståelse af chemotaxis blev muliggjort på grund af udviklingen af fluorescently markeret biosensorer til forskellige komponenter af de regulerende net. Mange chemotaxis lovgivere har en asymmetrisk fordeling enten af lovgivningsmæssige molekylet selv eller dets aktivitet. For eksempel, biosensorer, som genkender aktiveret versioner af små GTPases Ras og Rap1-Ras-bindende domæner af Raf1 (refereret til som RBD her) og RalGDS, henholdsvis-lokalisere til forkanten af en chemotaxing celle2,3. På samme måde, PI3K og dets produkt phosphatidylinositol (3,4,5)-trisphosphate (PIP3), anerkendt af en pleckstrin Homologi (PH) domæne, også vise lokalisering på forsiden af en celle4,5. Derimod et 3-fosfatase PT, der omdanner PIP3 tilbage til phosphatidylinositol (4,5)-bisfosfat, lokaliserer på halter kanten af den celle6. Vigtigere, ændre disse biosensorer deres lokalisering som svar på globale stimulation med en chemoattractant. Førende mærker, som er cytosole eller på spidsen af fremspring i en resting celle, relocalize cortex, mens halter kant markører, som har kortikale lokalisering og er fraværende fra spidsen af fremspring i en resting celle blive cytosole efter stimulation. Analyse af biosensor distribution som svar på globale stimulation med en chemoattractant minimerer motilitet og polaritet, som ofte forvirre observationer bidrag. Globale eller ensartet stimulering af en cellesuspension med et chemoattractant bruges også som et redskab til at vurdere befolkning-wide ændringer i protein aktivering, ofte opdaget af protein fosforylering7,8,9 . Denne biokemiske assay er primært bruges til at få tidsmæssige oplysninger, hvorimod mikroskopi bruges til at indsamle både tidsmæssige og rumlige oplysninger om opførsel af forskellige komponenter af de regulerende net.
Signaltransduktion netværk inkorporerer elementer af en overgearet system10,11. Reaktioner på supra-tærskel kemotaktisk stimuli er "alt" og vise ildfaste perioder. Svar også udløses stochastically og kan vise oscillerende adfærd. Signaltransduktion begivenheder er lokaliseret til områder af cortex, som overføres som bølger12,13,14,15. Forside, tilbage, markører er rekrutteret til eller tage afstand fra de aktive zoner af formeringsmateriale bølger. På grund af ildfaste regionen trailing zonen aktive, tilintetgøre oppositely rettet bølger som de mødes. Formeringsmateriale signaltransduktion bølger ligge til grund for de cellulære fremspring, der mægle celle migration10.
Mange af de ovennævnte oplysninger på chemotaxis kom fra undersøgelser på den sociale amøbe Dictyostelium discoideum, selv om lignende reguleringsmekanismer finder også anvendelse på neutrofile og andre pattedyr celle typer16 . Dictyostelium er en veletableret model organisme, der har en robust kemotaktisk svar under sult, når tusindvis af enkelt celler vandrer mod en sammenlægning centrum, til sidst danner et flercellet frugtsætning krop der indeholder sporer. Chemotaxis er også afgørende i én celle vækst fase af denne organisme for at lokalisere bakteriel fødekilder. Vigtigere, er migration af enkelt Dictyostelium celler bemærkelsesværdigt ens til migration af pattedyr neutrofiler eller metastatisk kræftceller, som alle gennemgår meget hurtige amoeboid-type migration. Faktisk er begge samlede topologien for de regulerende net, samt mange af de enkelte signaltransduktionsveje involveret i chemotaxis bevaret mellem Dictyostelium og pattedyr leukocytter17. Derudover bruge andre celler, såsom fibroblaster, receptor tyrosin kinaser (RTK) i stedet for GPCRs; RTKs kan dog indgå i lignende netværk.
I modsætning til chemotaxis mangler grundig forståelse af de signaling mekanismer, der drev forskellige andre tilstande af styret migration. Tilsvarende celler migrerer i et chemoattractant forløb flere undersøgelser har rapporteret aktivering og/eller lokalisering af typiske førende mærker, herunder actin polymerisering, PIP3 og/eller ekstracellulær signal-reguleret kinase (ERK) 1/2, på den forsiden af celler gennemgår instrueret migration som svar på shear strømmen eller ændringer i elektriske felter18,19,20,21. Men i disse studier kontinuerlig eksponering til stimulus også resulterede i celle migration, forlader spørgsmålet om eksempelvis forkant markører lokalisere specielt som svar på en stimulus, eller om de blot lokalisere på forkant på grund af øget antal pseudopods på forsiden af en overflytning celle.
Vi udviklede assays, der tillader os at observere svar af celler til akut mekanisk undertrykkelse af netbårne leveret som shear flow både på befolkningsniveau samt enkelte celler22. Svarende til globale stimulation med en chemoattractant, akut stimulation med shear flow giver mulighed for undersøgelse af en cellulære reaktion på en mekanisk stimulus uden forstyrrende bidrag fra motilitet eller polaritet. Kombinere disse biokemiske og mikroskopiske assays med genetiske eller farmakologiske perturbationer tillader os at lære om hvordan mekaniske stimuli opfattes og overføres. Desuden, denne tilgang giver også en roman metode til at tappe system nedstrøms for chemoattractant receptoren i mangel af en chemoattractant, derved isolere signal transduktion og aktin cytoskeleton netværk fra receptor/G protein netværk.
Ved hjælp af de teknikker, der er beskrevet nedenfor vi for nylig viste, at akut shear stress fører til aktivering af flere komponenter af kemotaktisk signal transduktion og aktin cytoskeleton netværk22. Ved at anvende de akutte mekaniske stimuli med varierende mellemrum, viste vi, at på samme måde til chemoattractants, svar på mekaniske stimuli også udstiller funktioner af en overgearet system, herunder svar under alt adfærd mætte betingelser og tilstedeværelsen af en refraktær periode. Endelig, ved at kombinere mekaniske og kemiske stimulation viste vi, at de to stimuli del signal transduktion og aktin cytoskeleton netværk, som sandsynligvis giver mulighed for integration af flere stimuli til bias celle migration.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D16-1</td>
			<td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>Fisher</td>
			<td>DF0120-17-6</td>
			<td>Use to prepare HL-5 media (step 1.1)</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher</td>
			<td>50-550-445</td>
			<td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Fisher</td>
			<td>S373-500</td>
			<td>Use to prepare HL-5 media, and 10X Phosphate Buffer (steps 1.1, 1.3)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P386-500</td>
			<td>Use to prepare HL-5 media, SM plates, and 10X Phosphate Buffer (steps 1.1-1.3)</td>
		</tr>
		<tr>
			<td>Streptomycin (dihydrostreptomycin sulfate)</td>
			<td>Fisher</td>
			<td>ICN10040525</td>
			<td>Use to prepare HL-5 media (step 1.1)</td>
		</tr>
		<tr>
			<td>Peptone (Bacto)</td>
			<td>Fisher</td>
			<td>DF0118-17-0</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>P288-100</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td>Use to prepare DB Buffer (step 1.4)</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Use to prepare DB Buffer (step 1.4)</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Fisher</td>
			<td>O1728-500</td>
			<td>Use to prepare caffeine (step 1.5)</td>
		</tr>
		<tr>
			<td>cAMP (EMD Millipore Calbiochem Adenosine 3 ft.,5 ft.-cyclic Monophosphate, Sodium Salt)</td>
			<td>Fisher</td>
			<td>11-680-1100MG</td>
			<td>Use to prepare cAMP (step 1.6)</td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma</td>
			<td>F7876</td>
			<td>Use to prepare folic acid (step 1.7)</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher</td>
			<td>BP166-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Bio-Rad</td>
			<td>1610610</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>1610404</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Fisher</td>
			<td>S299-100</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Fisher</td>
			<td>50-994-911</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate decahydrate</td>
			<td>Fisher</td>
			<td>S390-500</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail (Roche)</td>
			<td>Sigma</td>
			<td>11873580001</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Latrunculin A</td>
			<td>Enzo Life Sciences</td>
			<td>BML-T119-0100</td>
			<td>Use to prepare Latrunculin A (step 1.9)</td>
		</tr>
		<tr>
			<td>4-15% Tris-HCl polyacrylamide gel</td>
			<td>Bio-Rad</td>
			<td>3450029</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Polyvinylidene fluoride membrane</td>
			<td>Bio-Rad</td>
			<td>1620177</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit antibody</td>
			<td>Cell Signaling</td>
			<td>2060</td>
			<td>Primary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibody</td>
			<td>Cell Signaling</td>
			<td>4370</td>
			<td>Primary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Rabbit IgG HRP Linked Whole Ab (from Donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-100UL</td>
			<td>Secondary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence substrate (Clarity Western ECL Substrate)</td>
			<td>Bio-Rad</td>
			<td>1705060</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Stripping buffer (Restore Plus Western blot stripping buffer)</td>
			<td>Pierce</td>
			<td>PI46430</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue</td>
			<td>Fisher</td>
			<td>PI20278</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>K. aerogenes strain (non-pathogenic)</td>
			<td>Dicty Stock Center (dictybase.org)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker (model G-33, or a comparable alternative)</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td>Use to grow K. aerogenes (step 2.1.1), and to basalate and stimulate D. discoideum cells (step 3). The radius of gyration for this shaker is 9.5 mm.</td>
		</tr>
		<tr>
			<td>10 cm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-51B</td>
			<td>Use to count D. discoideum cells (step 2.1.2)</td>
		</tr>
		<tr>
			<td>35 mm dish (Corning Falcon Easy-Grip Tissue Culture Dish)</td>
			<td>Fisher</td>
			<td>08-772A</td>
			<td>Use to plate D. discoideumcells for mechanical stimulation followed by cell lysis (step 3.1.1)</td>
		</tr>
		<tr>
			<td>Polystyrene cup (5 mL)</td>
			<td>VWR</td>
			<td>13915-985</td>
			<td>Use to stimulate D. discoideum cell suspension with a chemoattractant (step 3.2.2)</td>
		</tr>
		<tr>
			<td>Fluidic unit with pump (ibidi Pump System)</td>
			<td>Ibidi</td>
			<td>10902</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>Slide with a channel (&#956;-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized)</td>
			<td>Ibidi</td>
			<td>80316</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by a fluidic device (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>50 mL reservoirs (Ibidi Reservoir Holder for Fluidic Unit)</td>
			<td>Ibidi</td>
			<td>10978</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>Lines for the fluidic unit (Perfusion Set YELLOW-and-GREEN)</td>
			<td>Ibidi</td>
			<td>10964</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5). Note: the two lines can also be cut from the 1.6 mm tubing (catalog number 10842).</td>
		</tr>
		<tr>
			<td>Line for the drain (1.6 mm ID tubing)</td>
			<td>Ibidi</td>
			<td>10842</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5).</td>
		</tr>
		<tr>
			<td>Zeiss Observer.Z1 inverted microscope equipped with a 40X/1.3 oil objective and a 20X/0.3 air objective (or a comparable alternative)</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used to image D. discoideum cells with phase-contrast or fluorescence illumination (steps 4 and 5)</td>
		</tr>
		<tr>
			<td>UltraView spinning disk confocal microscope equipped with a 40&#215;/1.25&#8211;0.75 oil objective (or a comparable alternative)</td>
			<td>Perkin-Elmer</td>
			<td>DM 16000</td>
			<td>An alternative used to image D. discoideum cells (step 4.1.4)</td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector (Eppendorf; model 5247, or a comparable alternative) with Eppendorf InjectMan</td>
			<td>Eppendorf</td>
			<td>5252000021D and 5192000027</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3). Note: catalog number are for the newest version of the equipment (4i).</td>
		</tr>
		<tr>
			<td>Micropipette (Femtotip; 1 &#956;m outside diameter, 0.5 &#956;m inside diameter )</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3)</td>
		</tr>
		<tr>
			<td>Microloader tips (Eppendorf Femtotips Microloader Tips for Femtojet Microinjector)</td>
			<td>Eppendorf</td>
			<td>5242956.003</td>
			<td>Use to fill the micropipette (femtotip) with buffer (step 4.3.1)</td>
		</tr>
		<tr>
			<td>1-well chamber (Thermo Scientific Nunc Lab-Tek Chambered Coverglass)</td>
			<td>Fisher</td>
			<td>12-565-472</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by a micropipette (step 4.3.2)</td>
		</tr>
		<tr>
			<td>8-well chamber (Thermo Scientific Nunc Lab-Tek II Chambered Coverglass)</td>
			<td>Fisher</td>
			<td>12-565-338</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software (Fiji)</td>
			<td>NIH</td>
			<td>https://fiji.sc/</td>
			<td>Use to quantify response to mechanical stimulation (step 4.5)</td>
		</tr>
		<tr>
			<td>Migration analysis software (Tracking Tool PRO)</td>
			<td>Gradientech</td>
			<td>http://gradientech.se/tracking-tool-pro/</td>
			<td>Use to quantify cell speed and persistence (step 5.4)</td>
		</tr>
	</tbody>
</table>

assessment of dictyostelium discoideum response to acute mechanical stimulation

Chemotaxis, eller migration op et forløb af en chemoattractant, er den bedste forstået tilstand af styret migration. Undersøgelser ved hjælp af sociale amøbe Dictyostelium discoideum viste, at en kompleks signaltransduktion netværk af parallelle veje forstærker svar på chemoattractants, og fører til tendentiøse actin polymerisation og fremspring af en pseudopod i den retningen af et farveforløb. Molekylære mekanismer kører andre typer af styret migration, for eksempel på grund af eksponering for shear flow eller elektriske felter, er derimod ikke kendt. Mange regulatorer af chemotaxis udstille lokalisering på førende eller halter kanten af en overflytning celle, samt vise forbigående ændringer i lokalisering eller aktivering efter globale stimulation med en chemoattractant. For at forstå de molekylære mekanismer af andre typer af styret migration udviklede vi en metode, der giver mulighed for undersøgelse af cellulære reaktion på akut mekanisk stimulation baseret på kort (2-5 s) eksponering for shear flow. Denne stimulation kan leveres i en kanal mens imaging celler, der udtrykker fluorescently mærket biosensorer til at undersøge enkelte celle adfærd. Derudover kan celle befolkning stimuleres i en plade, mængden, og immunoblotted ved hjælp af antistoffer, der genkender aktive versioner af proteiner af interesse. Ved at kombinere begge assays, kan man undersøge en bred vifte af molekyler aktiveres ved ændringer i subcellulært lokalisering og/eller fosforylering. Benytter indeværende metode vi fast besluttet på at akutte mekaniske stimulation udløser aktivering af kemotaktisk signal transduktion og aktin cytoskeleton netværk. Mulighed for at undersøge cellulære svar til akut mekanisk stimulation er vigtigt for at forstå de igangsættende begivenheder nødvendige for shear flow-induceret motilitet. Denne tilgang giver også et redskab til at studere kemotaktisk signaltransduktion netværk uden forstyrrende indflydelse af chemoattractant receptoren.

Tidsmæssige svar af chemotaxis regulatorer til akut mekanisk stimulation
For at vurdere svar af D. discoideum celler til mekanisk stimulation, blev vedhængende sammenlægning-kompetente celler udsat for en kort puls af shear flow. Sammenlægning-kompetente D. discoideum celler udskiller cAMP, der kan sanses af tilstødende celler. For at overvinde bidrag af celle-celle signalering, blev celler behandlet med koff...

Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Vurdering af Dictyostelium discoideum svar på akutte mekaniske Stimulation

Her beskriver vi metoder til vurdering af cellulære reaktion på akut mekanisk stimulation. I analysen mikroskopi-baserede undersøger vi lokalisering af fluorescently mærket biosensorer efter korte stimulation med shear flow. Vi tester også aktivering af forskellige proteiner af interesse som reaktion på akut mekanisk stimulation biokemisk.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

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Biology

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

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