Diese Arbeit wurde unterstützt durch die Finanzierung von DZNE und ein Marie-Curie-International Wiedereingliederungsbeihilfe (322120, D.C.D.)

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Hier berichten wir über eine Methodik zu isolieren, hoch-unlösliche Aggregate aus Altern C. Elegans RNAi durch Massenspektrometrie und Western blotting für Analyse unterzogen. Wir zeigen, dass Verbesserung Proteostase durch Reduzierung der IIS stark altersabhängig Proteinaggregation verhindert. Wenn Sie spezifische Aggregation neigen Proteine, in C. Elegansoverexpress auswählen, ist es möglich, weitere Mechanismen, die Modulation der inhärenten Proteinaggregation sezieren.
<p class="jove_cont...

Protein misfolding und Aggregation sind ein Markenzeichen von mehreren neurodegenerativen Erkrankungen wie AD, PD, Amyotrophe Lateralsklerose (ALS), frontotemporale Demenz (FTD) und viele andere anerkannte. Α-Synuclein Baugruppen zu Amyloid-Fibrillen anzusammeln, die beispielsweise als Lewy-Körper vor allem in der Substantia Nigra von Parkinson-Patienten, während bei ALS-Patienten TDP-43 oder FUS Misfold Form zytoplasmatischen Aggregaten in degenerierenden Motoneuronen. In jedem dieser neurodegenerativen Erkrankungen nicht Mechanismen, die Aufrechterhaltung der Homöostase Protein oder Proteostase verhindern die Ansammlung von fehlgefaltete Proteine, folglich zu Krankheit führt.
Proteostase ist entscheidend für die Zellfunktionen zu gewährleisten und unter normalen Bedingungen diese Regulationsmechanismen genau steuern die Rate der Proteinsynthese, Faltung und Abbau. Mehrere Studien zeigen, dass mit der Alterung, allmählich die Fähigkeit vieler Zellen und Organe, Protein Homöostase zu bewahren gefährdet ist und die physiologische Verschlechterung der Proteostase Netze mit zunehmendem Alter ein wichtiger erschwerender Faktor für ist Neurodegenerative Erkrankungen (rezensiert in Referenzen1,2,3). Die Tatsache, dass mit zunehmendem Alter der Protein-Qualitätskontrolle und die zelluläre Reaktion auf unfolded Protein Stress beeinträchtigt werden legt nahe, dass Protein misfolding und Aggregation eine allgemeine Folge des Alterns sein könnte. In der Tat haben wir und andere gezeigt, dass Proteinaggregation beschränkt sich nicht auf Krankheit und stattdessen Teil des Proteoms hoch Waschmittel-unlöslich in Alter Tiere4,5,6,7 wird ,8,9,10. Informatik und in Vivo Analyse ergab, dass diese physiologischen altersbedingte Aggregate Krankheit Aggregate in mehreren Aspekten5ähneln. Die Entdeckung der endogenen, altersabhängigen Proteinaggregation gibt uns die Möglichkeit, die molekularen und zellulären Mechanismen zu zergliedern, die Proteinaggregation, ohne Verwendung von ectopically ausgedrückt menschliche Krankheit-assoziierte Proteine regulieren. Derzeit gibt es nur begrenzte Informationen über die Regelung der weit verbreitete Protein Unlöslichkeit und über die Auswirkungen dieser Störungen auf die Gesundheit des Organismus.
Der Fadenwurm C. Elegans gehört zu den am häufigsten untersuchten Modellorganismen in der Alternsforschung, wie diese Tiere eine relativ kurze Lebensdauer haben und viele charakteristische Alterung Funktionen in höheren Organismen beobachtet zeigen. Die Auswirkungen des Alterns auf Protein Unlöslichkeit sind untersucht worden, in C. Elegans durch sequentielle biochemische Fraktionierung basierend auf unterschiedliche Löslichkeit, die weit verbreitet ist, extrahieren Sie Krankheit Aggregate im Bereich der Neurodegeneration Research11 . Durch quantitative Massenspektrometrie wurden mehrere hundert Proteine Aggregation-anfällig in C. Elegans in der Abwesenheit von Krankheit5gezeigt. Hier beschreiben wir ausführlich das Protokoll um große Anzahl von Würmern in Flüssigkultur und die sequentielle Extraktion aggregierte Proteine für die Quantifizierung von Massenspektrometrie und Analyse von Western-Blot isolieren zu wachsen. Denn fehlgefaltete und Aggregation neigen Proteine im Alter ansammeln C. Elegans Gonaden und Masken Veränderungen in anderen somatischen Gewebe5,12,13, verwenden wir eine Mutante Gonade-weniger Protein die Analyse darauf Unlöslichkeit in nicht-reproduktiven Geweben. Die vorgestellte Methode ermöglicht die Analyse von hoch-unlösliche, große Aggregate, die unlöslich in 0,5 % SDS und gebeizte durch relativ niedrige zentrifugale Geschwindigkeit. Alternativ wurde eine weniger strenge Extraktion-Protokoll auch kleiner und besser löslichen Aggregate sammeln10an anderer Stelle veröffentlicht. Darüber hinaus beschreiben wir die Methode zur Aggregation in Vivo in C. Eleganszu beurteilen.
Alles in allem können diese Methoden in Kombination mit RNA-Interferenz (RNAi) die Rolle eines Gens von Interesse an der Modulation der altersabhängigen Proteinaggregation auswerten. Dafür beschreiben wir die Analyse von Auszügen aus jungen und alten Würmer mit und ohne Zuschlag eines spezifischen Proteins des Interesses mit RNAi. Diese Methoden sollte ein leistungsstarkes Tool zum bestimmen, welche Komponenten des Netzwerks Proteostase Protein Unlöslichkeit regulieren. Mehrere Eingriffe reduziert wie Insulin, Insulin-ähnlichen Wachstumsfaktoren (IGF) 1 Signalisierung (IIS) haben gezeigt, dass C. Elegans Altern14erheblich verzögern. Langlebigkeit-Wege induzieren oft Protein-Qualitätskontrolle-Mechanismen und damit diese Wege könnten aktiv beeinflussen die Rate der Proteinaggregation. Als Beispiel zeigen wir Ihnen reduzierte inhärenten Proteinaggregation in langlebigen Tiere auf Hemmung der IIS-Pfad-7.

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		<col style="width:92pt" width="122" />
		<col style="width:258pt" width="344" />
		<col style="width:125pt" width="166" />
	</colgroup>
	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="width:161pt;height:30.0pt" width="215">Fernbach culture flask&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Corning</td>
			<td class="xl19" style="width:258pt" width="344">4425-2XL</td>
			<td class="xl18" style="width:125pt" width="166">Pyrex, Capacity 2,800 ml, with 3 baffle indents</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Membrane Screw Cap&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Schott</td>
			<td class="xl19">1088655</td>
			<td class="xl17">GL45</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Nutating Mixer</td>
			<td class="xl18" style="width:92pt" width="122">VWR</td>
			<td class="xl19">444-0148</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Separatory funnel</td>
			<td class="xl18" style="width:92pt" width="122">Nalgene</td>
			<td class="xl19">4300-1000</td>
			<td class="xl17">Capacity 1,000 ml</td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">1 ml syringe&#160;</td>
			<td class="xl18" style="width:92pt" width="122">BD Plastipak</td>
			<td class="xl19">300013</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Gray needle, 27 G x &#189; &#34;, 0.4 mm x 13 mm</td>
			<td class="xl18" style="width:92pt" width="122">BD Microlance 3</td>
			<td class="xl19">300635</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Membrane filters 0.025 &#181;M</td>
			<td class="xl18" style="width:92pt" width="122">Millipore</td>
			<td class="xl19">VSWP04700</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">pH strip</td>
			<td class="xl18" style="width:92pt" width="122">Machery-Nagel</td>
			<td class="xl19">92110</td>
			<td class="xl17">pH-Fix 0-14</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Protease Inhibitor Cocktail</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">4693132001</td>
			<td class="xl16" style="width:125pt" width="166">Complete Mini EDTA-free tablets&#160;</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Octoxynol-9&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1388</td>
			<td class="xl18" style="width:125pt" width="166">Triton X-100</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">4-Morpholineethanesulfonic acid (MES)</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">M1317</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Nonylphenylpolyethylenglycol</td>
			<td class="xl18" style="width:92pt" width="122">Applichem</td>
			<td class="xl19">A1694</td>
			<td class="xl18" style="width:125pt" width="166">Nonidet P40 (NP40)</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">DNaseI</td>
			<td class="xl18" style="width:92pt" width="122">Roche</td>
			<td class="xl19">04716728001</td>
			<td class="xl18" style="width:125pt" width="166">recombinant, RNase free</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">RNaseA</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl19">A7973</td>
			<td class="xl18" style="width:125pt" width="166">solution</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein blot staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S11791</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein blot stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Total protein gel staining</td>
			<td class="xl18" style="width:92pt" width="122">Thermofisher</td>
			<td class="xl19">S12001</td>
			<td class="xl18" style="width:125pt" width="166">Sypro Ruby protein gel stain</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">TCEP (tris (2-carboxyethyl) phosphine hydrochloride)</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">36970</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Iodoacetamide</td>
			<td class="xl18" style="width:92pt" width="122">Serva</td>
			<td class="xl19">26710</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Ammoniumbicarbonate</td>
			<td class="xl18" style="width:92pt" width="122">Sigma-Aldrich</td>
			<td class="xl19">09830</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Sequencing Grade Modified Trypsin</td>
			<td class="xl18" style="width:92pt" width="122">Promega</td>
			<td class="xl25" style="width:258pt" width="344">V5111</td>
			<td class="xl18" style="width:125pt" width="166"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Isobaric tags for relative and absolute quantitation</td>
			<td class="xl18" style="width:92pt" width="122">Sciex</td>
			<td class="xl19">4352135</td>
			<td class="xl18" style="width:125pt" width="166">iTRAQ Reagents Multiplex Kit</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Avanti J-26XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393126</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Ultracentrifuge Optima Max-XP</td>
			<td class="xl18" style="width:92pt" width="122">Beckmann Coulter</td>
			<td class="xl19">393315</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5424R</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5404000413</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge 5702</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5702000329</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Centrifuge Megafuge 40R</td>
			<td class="xl18" style="width:92pt" width="122">Thermo Scientific</td>
			<td class="xl19">75004518</td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Concentrator Plus</td>
			<td class="xl18" style="width:92pt" width="122">Eppendorf</td>
			<td class="xl19">5305000304</td>
			<td class="xl17">Centrifugal evaporator</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Fluorescent stereo-microscope M165 FC&#160;</td>
			<td class="xl18" style="width:92pt" width="122">Leica</td>
			<td class="xl19"></td>
			<td class="xl18" style="width:125pt" width="166">With Planapo 2.0x objective</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Dissection microscope</td>
			<td class="xl18" style="width:92pt" width="122">Leica&#160;</td>
			<td class="xl19"></td>
			<td class="xl17">Leica S6E</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="80" rowspan="2" style="width:161pt;height:60.0pt" width="215">High magnification microscope Zeiss Axio Observer Z1</td>
			<td class="xl18" height="80" rowspan="2" style="width:92pt;height:60.0pt" width="122">Zeiss</td>
			<td class="xl19" height="80" rowspan="2" style="width:258pt;height:60.0pt" width="344"></td>
			<td class="xl16" height="80" rowspan="2" style="width:125pt;height:60.0pt" width="166">With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">Software</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl32"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">Image analysis software</td>
			<td class="xl18" style="width:92pt" width="122">ImageJ</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="215">Analysis of mass spectrometry data</td>
			<td class="xl18" style="width:92pt" width="122">Protein Prospector</td>
			<td class="xl136" style="width:258pt" width="344"></td>
			<td class="xl137">http://prospector.ucsf.edu/prospector/mshome.htm</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">E.coli strain</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">OP50</td>
			<td class="xl18" style="width:92pt" width="122">CGC</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl32" height="40" style="height:30.0pt">RNAi bacteria</td>
			<td class="xl33" style="width:92pt" width="122"></td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl17" height="40" style="height:30.0pt">L4440</td>
			<td class="xl18" style="width:92pt" width="122">Julie Ahringer RNAi library</td>
			<td class="xl19"></td>
			<td class="xl17"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl32" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans mutants</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl17" height="42" style="height:31.5pt">CF2253</td>
			<td class="xl18" style="width:92pt" width="122">CGC, strain name: EJ1158&#160;</td>
			<td class="xl40" style="width:258pt" width="344"></td>
			<td class="xl17">Genotype: gon-2(q388)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl33" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">C. elegans transgenics</td>
			<td class="xl33" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122"></td>
			<td class="xl32" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl19" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD214</td>
			<td class="xl25" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="42" rowspan="2" style="width:161pt;height:31.5pt" width="215">DCD215</td>
			<td class="xl87" height="42" rowspan="2" style="width:92pt;height:31.5pt" width="122">Della David&#39;s lab at DZNE T&#252;bingen</td>
			<td class="xl40" height="42" rowspan="2" style="width:258pt;height:31.5pt" width="344"></td>
			<td class="xl68" height="42" rowspan="2" style="width:125pt;height:31.5pt" width="166">Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]</td>
		</tr>
		<tr height="21" style="height:15.75pt">
		</tr>
	</tbody>
</table>

methods to study changes in inherent protein aggregation with age in caenorhabditis elegans

In den letzten Jahrzehnten ist die Prävalenz von neurodegenerativen Erkrankungen wie Alzheimer-Krankheit (AD) und der Parkinson-Krankheit (PD), gewachsen. Diese altersassoziierter Erkrankungen zeichnen sich durch das Auftreten von Protein-Aggregate mit fibrillary Struktur in den Gehirnen von Patienten. Genau warum normalerweise löslichen Proteine zu unterziehen, bleibt ein Aggregationsprozess schlecht verstanden. Die Entdeckung, dass Proteinaggregation beschränkt sich nicht auf Krankheitsprozesse und stattdessen Teil des normalen Alterungsprozesses die Untersuchung der molekularen und zellulären Mechanismen, die Proteinaggregation, zu regulieren ermöglicht, ohne Verwendung von ectopically zum Ausdruck menschlicher Krankheit-assoziierte Proteine. Hier beschreiben wir Methoden um inhärente Proteinaggregation in Caenorhabditis Elegans durch komplementäre Ansätze zu untersuchen. Zunächst untersuchen wir wie zahlreicher Alter synchronisiert C. Elegans wachsen im Alter von Tieren zu erhalten und wir präsentieren die biochemische Verfahren um hoch-unlöslich-große Aggregate zu isolieren. In Kombination mit einer gezielten genetischen Knockdown, ist es möglich, die Rolle eines Gens von Interesse an der Förderung oder altersabhängig Proteinaggregation zu verhindern, indem Sie entweder eine umfassende Analyse mit quantitativen Massenspektrometrie sezieren oder eine Kandidaten-basierte Analyse mit Antikörpern. Diese Erkenntnisse werden dann mit transgenen Tieren ausdrücken Leuchtstoff-Tags Aggregation neigen Proteine durch in-Vivo -Analyse bestätigt. Diese Methoden sollen erklären, warum bestimmte Proteine sind anfällig für Aggregat mit zunehmendem Alter und letztlich wie diese Proteine voll funktionsfähig zu halten.

Wir verwendet um wie langlebigen Tiere mit bewerten hier vorgestellten Methoden reduzierte IIS modulieren altersabhängigen Proteinaggregation. Durch Western blot (siehe Punkt 2.2, schnell unlöslichen Protein Extraktion für Western-Blot Analyse), analysiert die Summe und die unlösliche Eiweißgehalt von Young (Tag 3 des Erwachsenenalters) und im Alter von (18. Tag des Erwachsenenalters) Würmer auf Kontrolle RNAi und RNAi gezielt das Insulin / IGF-1-Like-Rezeptor Daf-2. Wir be...

Watch this Scientific Journal Video about Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans at JoVE.com

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

Das Ziel der hier vorgestellten Methode ist Proteinaggregation während der normalen Alterung bei Modellorganismus C. Eleganszu erkunden. Das Protokoll stellt ein leistungsfähiges Werkzeug, sehr unlöslichen großen Aggregaten, die mit zunehmendem Alter bilden zu studieren und zu klären, wie Änderungen in Proteostase Proteinaggregation auswirken.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

methods-to-study-changes-inherent-protein-aggregation-with-age

Research

JoVE Journal

Biology

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

8.4K Aufrufe.  German Center for Neurodegenerative Diseases (DZNE). Das Ziel der hier vorgestellten Methode ist Proteinaggregation während der normalen Alterung bei Modellorganismus C. Eleganszu erkunden. Das Protokoll stellt ein leistungsfähiges Werkzeug, sehr unlöslichen großen Aggregaten, die mit zunehmendem Alter bilden zu studieren und zu klären, wie Änderungen in Proteostase Proteinaggregation auswirken.

Serie: Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Solide Plate-basierte diätetische Einschränkung in Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Forschung

Biologie

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Zeitraffer-Mikroskopie der frühen Embryogenese in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D-Bildgebung von Protein Aggregation in lebenden Zellen

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandin Extraktion und Analyse in<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In-vitro-Assays mit Aggregation hyperphosphoryliertem Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

der Anbau von<em&gt; Caenorhabditis elegans</em&gt; In drei Dimensionen im Labor

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Kombinierte Nukleotid- und Protein Extraktionen in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantifizierung des gewebespezifischen proteostatischen Rückgangs bei Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

Zusammenfassung

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Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

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