Name: detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay
Uploaded: 2015-01-20T13:00:00
Description: Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Vi takker Bloomington Drosophila Stock Center for å gi flue aksjer. Vi takker også utviklingsstudier Hybridoma Bank og Dr. Lynn Cooley (Yale University) for å gi antistoffer. En spesiell takk går til AhHyun Yoo for hennes hjelp på manuskriptet. Dette arbeidet ble støttet med tilskudd fra de naturvitenskapelige og Engineering Research Council of Canada (Krieger), William og Ada Isabelle Steel Fund (Krieger), og den kanadiske Institutes of Health Research (Harden).

1. Body Wall Forberedelse MERK: Utarbeidelse av tredje stadium larve kroppens vegger (for studiet av NMJs som innerverer kroppen veggen muskler) ble utført som tidligere beskrevet i Brent et al 10, eller Ramachandran og Budnik 11,12, men med noen endringer.. <ol><li> Disseksjon <ol><li> Heve flue aksjer og kors ved 25 ° C i fem til seks dager ved hjelp av standard prosedyrer 13. </li><li> Plukk kryp tredje stadium larver fra ampuller ell...

<ol>
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(24), (2009).</li><li>Ramachandran, P., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+staining+of+Drosophila+larval+body-wall+muscles.">Immunocytochemical staining of Drosophila larval body-wall muscles.</a> Cold Spring Harb Protoc. 2010, (2010).</li><li>Ramachandran, P., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Drosophila+larval+body-wall+muscles.">Dissection of Drosophila larval body-wall muscles.</a> Cold Spring Harb Protoc. , (2010).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Manual. , (1989).</li><li>Brent, J., Werner, K., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+immunohistochemistry.">Drosophila larval NMJ immunohistochemistry.</a> J Vis Exp. (25), (2009).</li><li>Parnas, D., Haghighi, A. P., Fetter, R. D., Kim, S. W., Goodman, C. S. <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+postsynaptic+structure+and+protein+localization+by+the+Rho-type+guanine+nucleotide+exchange+factor+dPix.">Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix.</a> Neuron. 32, 415-424 (2001).</li><li>Petrella, L. N., Smith-Leiker, T., Cooley, 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+Ovhts+polyprotein+is+cleaved+to+produce+fusome+and+ring+canal+proteins+required+for+Drosophila+oogenesis.">The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis.</a> Development. 134, 703-712 (2007).</li><li>Pielage, J., Bulat, V., Zuchero, J. B., Fetter, R. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hts/Adducin+controls+synaptic+elaboration+and+elimination.">Hts/Adducin controls synaptic elaboration and elimination.</a> Neuron. 69, 1114-1131 (2011).</li><li>Spradling, A. 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=The+Berkeley+Drosophila+Genome+Project+gene+disruption+project:+Single+P-element+insertions+mutating+25%+of+vital+Drosophila+genes.">The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.</a> Genetics. 153, 135-177 (1999).</li><li>Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+p21-Activated+Kinases.">Biology of the p21-Activated Kinases.</a> Annu Rev Biochem. 72, 743-781 (2003).</li><li>Bahri, 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=The+leading+edge+during+dorsal+closure+as+a+model+for+epithelial+plasticity:+Pak+is+required+for+recruitment+of+the+Scribble+complex+and+septate+junction+formation.">The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.</a> Development. 137, 2023-2032 (2010).</li><li>Albin, S. D., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+structural+and+functional+synapse+development:+postsynaptic+p21-activated+kinase+independently+specifies+glutamate+receptor+abundance+and+postsynaptic+morphology.">Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.</a> J Neurosci. 24, 6871-6879 (2004).</li><li>Koles, K., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+neuromuscular+junction+development.">Wnt signaling in neuromuscular junction development.</a> Cold Spring Harb Perspect Biol. 4, (2012).</li><li>Packard, M., 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+Drosophila+Wnt,+wingless,+provides+an+essential+signal+for+pre-+and+postsynaptic+differentiation.">The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation.</a> Cell. 111, 319-330 (2002).</li><li>Weibrecht, I., 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=Proximity+ligation+assays:+a+recent+addition+to+the+proteomics+toolbox.">Proximity ligation assays: a recent addition to the proteomics toolbox.</a> Expert Rev Proteomics. 7, 401-409 (2010).</li><li>Thymiakou, E., Episkopou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+signaling+effector-complexes+downstream+of+bmp4+using+PLA,+a+proximity+ligation+assay.">Detection of signaling effector-complexes downstream of bmp4 using PLA, a proximity ligation assay.</a> J Vis Exp. 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Rapporten viser hvordan PLA kan anvendes til Drosophila larve NMJ. Analysen utføres på larvekroppsveggmuskelpreparater i den hensikt å detektere endogene protein-protein interaksjoner tilstede på NMJ. Med denne teknikk blir DLG og Hts vist seg å være i umiddelbar nærhet til hverandre, og således foreligge i en kompleks, spesielt på den postsynaptiske region 27. Til støtte for dette resultatet, har en tidligere studie gitt bevis for sin forening med følgende data: 1) de immunoreaktive distr...

Drosophila plater store (DLG) er en konservert medlem av membran-assosiert guanylat-kinase familien av stillaser proteiner som bidrar til å organisere montering av store proteinkomplekser på bestemte steder av plasmamembranen. Opprinnelig identifisert som en tumor suppressor protein, DLG tjener som en viktig determinant for epitelisk apicobasal polaritet 1,2,3. DLG fungerer også som et stort stillas modul ved nevromuskulære krysset (NMJ) av glutamaterge motoriske nerveceller i løpet av larveutvikling fire. DLG spiller ulike roller på larve NMJ, og dens pleiotropism avhengig av sin evne til å assosiere med flere proteiner 5,6. Et slikt protein er Hu-li tai Shao (HTS), en homolog til de pattedyr adducins som i hovedsak har vært beskrevet i forhold til sine roller i regulering av aktin-spektrin cytoskjelettet 7. Det har tidligere blitt vist at DLG og Hts kan danne et kompleks med hverandre basert på in vitro </em&gt; co-immunoutfellingsstudier forsøk med hel voksne fluelysatene 8. En brist av disse resultatene, er imidlertid at de ikke indikere hvor dette komplekse former. Med bruk av immunhistokjemi, blir fordelingen av DLG og Hts observert å overlappe på den postsynaptiske membran av larve NMJs, men de er i et kompleks i denne regionen 8? Som nylig vist og beskrevet nærmere her, er Proximity Ligation analysen (PLA) som brukes til å lete etter en in situ sammenheng mellom DLG og Hts spesielt på larve NMJ 27. PLA er en relativt ny teknikk som brukes for det meste i celler og vev kultur som kan oppdage protein-protein interaksjoner in situ 9. I denne analyse, er primære antistoffer mot de to proteiner av interesse påvises med et par av artsspesifikk sekundære antistoffer, betegnet PLA prober, som er konjugert til oligonukleotider (figur 1 A, B). Hvis de to proteins er i umiddelbar nærhet til hverandre (dvs. i løpet av få titalls nanometer), idet avstanden mellom de vedlagte PLA prober kan slås bro over ved hybridisering av to ytterligere forbindelses oligonukleotider (figur 1C). I denne konformasjon, de frie ender av kontakt oligonukleotider er nær nok til å gjøre kontakt med hverandre, og et lukket, sirkulært DNA-molekyl kan dannes ved in situ-ligering (figur 1D). Den sirkulære DNA-molekyl som virker som et templat for in situ rullende sirkel forsterkning, som er fylt av en av de oligonukleotider som er konjugert til PLA-prober (figur 1E). Sekvenser i de resulterende amplifiserte, concatemeric DNA-produkt kan deretter bli visualisert med fluoresceinmerkede, komplementære oligonukleotid-prober (figur 1F). Siden det amplifiserte DNA forblir festet til en av PLA-sonder, den subcellulære lokalisering av protein-protein interaksjon withina vevet lett kan fastslås. Flere metoder blir ofte brukt for å påvise protein-protein interaksjoner inkludert in vitro teknikker som co-immunoprecipitation, pull-down-analyser og gjær to-hybrid screening, og in vivo teknikker som Förster Resonance Energy Transfer (FRET) og bimolecular Fluorescens Komplemente ( BiFC). En fallgruve av in vitro teknikker er at de ikke identifisere hvor samspillet er endogent forekommende, mens den tidligere nevnte in vivo teknikker involvere kunstig uttrykk for fusjonsproteiner som viser ikke nødvendigvis den opprinnelige atferden til sine endogene kolleger. En stor fordel med PLA er at den er i stand til å bestemme i en vev av subcellulære lokalisering av endogene protein interactors som er i umiddelbar nærhet til hverandre, og sannsynligvis danner et kompleks, med graden av nærhet nødvendig for å generere et signal som kan sammenlignes med FRET og BiFC. PLA kan detektere interaksjoner med høy spesifisitet og sensitivitet som følge av koplingen av antistoffgjenkjenning og DNA-amplifisering. Således kan analysen generere diskrete, lyse signaler i form av puncta som viser den nøyaktige plasseringen av interaksjonen. I tillegg kan knapt synlige antigener påvises. Til slutt, er PLA en relativt enkel teknikk for å utføre, og det tar ikke noe lenger tid enn en standard immunhistokjemisk fremgangsmåte for å fullføre. Derfor gir PLA en teknisk fordel over andre protein-protein interaksjons analyser som ofte plaget med lange forberedelser ganger og omfattende feilsøking. Denne protokollen viser hvordan PLA kan anvendes til Drosophila larve NMJ for det formål å detektere endogene protein-proteininteraksjoner in situ. Her er PLA utført på larvekroppen veggmuskel preparater der DLG og Hts er vist å faktisk eksistere i et kompleks på den postsynaptiske regionen NMJs. PLA har ikke tidligere blitt brukt til å studere larve NMJ, og det er i dag bare en håndfull av publiserte artikler som har brukt denne analysen i Drosophila vev. Det er å håpe at ytterligere eksponering av PLA til Drosophila samfunnet vil resultere i sin økt bruk som et ekstra verktøy for å utfylle andre, mer vanlig brukte protein-protein interaksjons analyser.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
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			<td height="20" style="height:20px;width:211px;">Name of Material/Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Forceps (fine #5)</td>
			<td style="width:120px;">Almedic</td>
			<td>A10-704</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:211px;">Sylgard Disc</td>
			<td style="width:120px;">World Precision Instruments</td>
			<td>SYLG184</td>
			<td style="width:191px;">Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Minutien Pins (0.0125 mm tip diameter)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Microdissection Scissors (ultra fine)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td style="width:111px;">15200-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">Platform Slides</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">w1118</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">3605</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">hts01103</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">10989</td>
			<td style="width:191px;">Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Bouin&#39;s Solution</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">HT10132</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:211px;">4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Dlg (Discs large)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td style="width:191px;">Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">rabbit anti-HtsM (Hu-li tai shao)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">rabbit anti-Pak (p21-activated kinase)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Wg (Wingless)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:111px;">4D4</td>
			<td style="width:191px;">Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">goat anti-Hrp (Horseradish peroxidase)</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">123-065-021</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">FITC-conjugated donkey anti-goat</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">705-095-003</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92004</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Detection Reagents Red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Mounting Medium with DAPI</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82040</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

I villtype tredje stadium larve NMJs, er DLG hovedsakelig funnet på den postsynaptiske membran av type I glutamaterge boutons, med DLG immunoreaktivitets nivåer være mer uttalt hos type IB boutons enn type Er boutons (figur 3A) 4. Hts er til stede i hele muskelen, men konsentrerer seg på den postsynaptiske region med Hts immunoreaktivitets nivåer vises lik i både type I boutons, og er også funnet presynaptisk (figur 3A &#39;) 8,17. Legg merke til at fordelin...

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Påvisning av<em&gt; In Situ</em&gt; Protein-protein komplekser på<em&gt; Drosophila</em&gt; Larve Nevromuskulær Junction hjelp Proximity Ligation analysen

Denne protokollen demonstrerer hvordan Proximity Ligation analysen kan brukes til å oppdage in situ protein-protein interaksjoner på Drosophila larvenevromuskulære krysset. Med denne teknikken, er plater store og Hu-li tai Shao vist seg å danne et kompleks på den postsynaptiske region, en forening tidligere identifisert gjennom ko-immunoutfelling.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Research

JoVE Journal

Neuroscience

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sekvensiell Foto bleking å avgrense Enkelt Schwann celler på nevromuskulær Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Nevromuskulære krysset: Måling Synapse Størrelse, fragmentering og endringer i Synaptic Protein Tetthet Bruke Confocal Fluorescensmikroskopi

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Hvorfor Kvantifisering Matters: Karakterisering av fenotyper på<em&gt; Drosophila</em&gt; Larve Nevromuskulær Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Måle nevromuskulær Junction funksjonalitet

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

To Algoritmer for høy gjennomstrømming og Multi-parametrisk Kvantifisering av<em&gt; Drosophila</em&gt; Neuromuskulære Morfologi

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Fokal Macropatch opptak av Synaptic strøm fra Drosophila larver nevromuskulær krysset

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Disseksjon og immunhiistokjemi av Drosophila voksenben for å oppdage endringer ved neuromuskulært kryss for en identifisert motor neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...