Name: monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp
Uploaded: 2015-09-06T14:00:00
Description: Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

1. In Vitro analyse: Oppdager SERCaMP Løslatelse fra Stable SH-SY5Y cellelinje <ol><li> Plate SH-SY5Y-Gluc-ASARTDL (SERCaMP) i vevskultur behandlede plater på 150.000 celler pr cm2 overflateareal. I 96 brønners plater, eksempelvis frø 50.000 celler per brønn (figur 1A). Grow SH-SY5Y celler i DMEM (høy glukose, Glutamax, pyruvat) + 10% storfe vekst serum + 1x penicillin / streptomycin. <ol><li> Passasje celler inntil 15 ganger (figur 1B). Høyere passasje anta...

<ol>
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Denne protokollen fremhever in vitro og in vivo nytten av Gluc-SERCaMP å overvåke utarming av ER kalsium. Selv om protein modifikasjon til å generere SERCaMP ser ut til å generalisere til andre reporter proteiner 12, valgte vi Gaussia luciferase for sin robuste (200-1000 ganger større) Bioluminescens sammenlignet med andre luciferases 18. Vi viser påvisbar thapsigargin-indusert Gluc-SERCaMP frigivelse over en 100-ganger doseområdet Gluc-SERCaMP virus levert til pri...

Endoplasmatisk retikulum (ER) fungerer på mange cellulære kapasiteter, inkludert proteinfolding, protein sekresjon, lipid homeostase, og intracellulær signale en. Sentralt i normal ER funksjon er å opprettholde luminale kalsiumkonsentrasjoner på ~ 5000 ganger de funnet i cytoplasma 2-4. Denne energikrevende prosess er regulert av Sarco / endoplasmatiske retikulum kalsium ATPase (SERCA), en pumpe som flytter kalsiumioner inn på akuttmottaket. Utstrømming av kalsium fra ER medieres primært av ryanodine (RyR) og inositol trifosfat (IP3R) reseptorer. Fordi mange ER prosesser er avhengige av kalsium, kan forstyrre butikken føre til ER stress og eventuell celledød. ER kalsium dysregulation har blitt observert i sykdommer inkludert kardiomyopati, diabetes, Alzheimers og Parkinsons 5. På grunn av den progressive karakter av disse sykdommene er det blitt vanskelig å avgrense årsak-virkning relationship mellom patogenesen og endringer i ER kalsium butikken. En rekke teknologier har tillatt for betydelige fremskritt i vår forståelse av ER kalsium dynamikk, inkludert fargestoffer og genetisk kodede kalsium indikatorer (GECIs). Lavaffinitets kalsiumfargestoffer, som øker i fluorescens når de er bundet til Ca 2+, kan lastes inn i celler for å undersøke subcellulære lommer med høy konsentrasjon av kalsium 6. GECIs, slik som D1ER og Catcher tillate overvåkning av kalsium svingninger med mer presis styring av subcellulære lokalisering 7-9. Nylig, en annen klasse av GECIs kalles kalsium-måling organelle-omsluttet protein indikatorer (CEPIA) har blitt beskrevet 10. En tredje metode som kombinerer genetikk og lite molekyl kjemi er rettet-esterase fargestoff laste (TED), som benytter en genetisk kodet karboksylesterase (målrettet til ER) med en ester-basert kalsium fargestoff 11. Mens aforementioned tilnærminger har iboende styrker og svakheter, de kan gi verdifull innsikt i ER kalsium dynamikk gjennom akutte målinger av fluorescens. De er imidlertid ikke optimal for de langsgående studier ofte nødvendig å undersøke sykdomsprogresjon. Med mål om å utarbeide en metode for å overvåke kalsium dynamikk over lengre tid, har vi identifisert og utviklet et protein modifikasjon for å lage de skilles ER kalsium overvåkings proteiner (SERCaMPs) 12. SERCaMP omgår flere begrensninger forbundet med andre metoder, ved å gi en minimal invasiv tilnærming til gjentatte ganger avhøre ER kalsium butikken. Vi har tidligere demonstrert at den karboksy-terminale peptid ASARTDL (alanin-serin-alanin-arginin-treonin-aspartinsyre-leucin) er tilstrekkelig til å fremme ER retensjon; imidlertid, under betingelser som fører til reduksjon i ER kalsium, er peptidsekvensen ikke lenger i stand til å beholde ER localization og proteinet blir utskilt 13. Grunnlaget for SERCaMP teknologi er vedheng av ASARTDL til den karboksy-terminale ende av et utskilt protein (f.eks Gaussia luciferase, eller Gluc), slik at sekresjon utløses av ER kalsiummangel, og dermed skape en robust reporter av ER kalsium dysregulering 12. Ekspresjon av Gluc-SERCaMP via transgene metoder gjør det mulig for biologiske væsker, inkludert cellekulturmedium og plasma som skal analyseres for endringer i Gluc aktivitet som en indikator på ER kalsium homeostase. Fremgangsmåten har anvendelser for den langsgående studium av progressive endringer i ER kalsium butikken både in vitro og in vivo. Følgende protokoll er skrevet som en generell oversikt for bruk Gluc baserte SERCaMP å studere ER kalsiumhomeostase, men protokollen kan fungere som en guide for alternative reporter SERCaMPs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.5mL tubes</td>
			<td>Fisher&#160;</td>
			<td>02-682-550</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40 solution&#160;</td>
			<td>Pierce</td>
			<td>28324</td>
			<td>for intracellular GLuc assays</td>
		</tr>
		<tr>
			<td>1mL luer-lok syringes</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL filter tips</td>
			<td>Rainin</td>
			<td>RT-L200F</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 surgical sutures</td>
			<td>Fisher</td>
			<td>NC9598192</td>
			<td></td>
		</tr>
		<tr>
			<td>30g needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microplate sealing sheets</td>
			<td>Thermo</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>Fisher</td>
			<td>22-246-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Auto Flow System</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-AF9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal recovery chamber</td>
			<td>Lyon Vet</td>
			<td>ICU-912-004</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher</td>
			<td>NC9386574</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine growth serum</td>
			<td>Thermo</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine, Native</td>
			<td>Regis Technologies</td>
			<td>1-361204-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting needles 3/8 circle sutures</td>
			<td>WPI</td>
			<td>501803</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital ultrasconic cleaner</td>
			<td>Fisher Scientific</td>
			<td>FS60D</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose, GlutaMAX, pyruvate</td>
			<td>Life Technologies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA mass ladder</td>
			<td>Life Technologies</td>
			<td>10496-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase (recombinant protein)</td>
			<td>Nanolight</td>
			<td>321-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td>
			<td>New England Biolabs</td>
			<td>E8023S</td>
			<td>1:2000 (WB)</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>CellPoint Scientific</td>
			<td>DS-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluc assay plates (96 well, opaque)</td>
			<td>Fisher</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Allmedtech</td>
			<td>63323-276-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butler Schein</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>995-2949</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik Stop Styptic powder</td>
			<td>Butler Schein</td>
			<td>5867</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>a452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 22R Centrifuge</td>
			<td>Bekman Colter</td>
			<td>368831</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Fisher</td>
			<td>19-898-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Gel and PCR Cleanup</td>
			<td>Machery-Nagel</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet</td>
			<td>Rainin</td>
			<td>L-200XLS+</td>
			<td></td>
		</tr>
		<tr>
			<td>p24 Lenti-X rapid titer kit</td>
			<td>Clontech</td>
			<td>632200</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR film seal</td>
			<td>Fisher</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>ReFresh Charcoal Filter canister</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades, #10</td>
			<td>Fine Science tools Inc</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats 150-200g</td>
			<td>Charles River</td>
			<td>Rats</td>
			<td>rats ordered at 150-200g.&#160; Surgery 5 days after arrival</td>
		</tr>
		<tr>
			<td>Small animal ear tags</td>
			<td>National Band and Tag co</td>
			<td>1005-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drapes</td>
			<td>Braintree Scientific</td>
			<td>SP-MPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 2 plate reader</td>
			<td>BioTek</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma</td>
			<td>T9033</td>
			<td>harmful to human health</td>
		</tr>
		<tr>
			<td>Virapower lentiviral packaging mix</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xfect Transfection reagent</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Valley Vet</td>
			<td>468RX</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp

Det endoplasmatiske retikulum (ER) inneholder det høyeste nivået av intracellulært kalsium, ved konsentrasjoner omtrent 5000 ganger større enn cytoplasmatiske nivåer. Stram kontroll over ER kalsium er viktig for proteinfolding, modifikasjon og trafficking. Perturbasjoner til ER kalsium kan resultere i aktivering av den ufoldede protein respons, en tre-spiss ER stress svar mekanisme, og bidrar til patogenese i en rekke sykdommer. Evnen til å overvåke ER kalsium forandringer i løpet av sykdomsutbruddet og progresjon er viktig i prinsippet, men likevel vanskelig i praksis. For tiden tilgjengelige metoder for overvåking ER kalsium, for eksempel kalsium-avhengige fluorescerende fargestoffer og proteiner, har gitt innsikt i ER kalsium dynamikken i cellene, men disse verktøyene er ikke godt egnet for in vivo studier. Vårt laboratorium har vist at en modifikasjon av den karboksy-terminale ende av Gaussia luciferase confers sekresjon av reporter i respons tilER kalsium uttømming. Metodene for anvendelse av en luciferase basert, utskilte ER kalsium overvåkning protein (SERCaMP) for in vitro og in vivo anvendelser er beskrevet heri. Denne videoen fremhever lever injeksjoner, farmakologisk manipulering av Gluc-SERCaMP, blod innsamling og prosessering, og analyseparametere for langsgående overvåking av ER kalsium.

Den Gluc-SERCaMP metoden gjør for vurdering av ER kalsiumhomøostase ved prøvetaking ekstracellulære væsker. Flere kontroller kan inkluderes i eksperimentell design for å forbedre tolkning av resultater. For det første kan bruk av et konstitutivt utskilt reporter (f.eks Gluc uten den C-terminale ASARTDL eller &quot;Gluc-No-ID&quot;) bli anvendt for å vurdere effektene av eksperimentelle behandlinger på den sekretoriske vei (global cellulær sekresjon) og transgen ekspresjon. For eksempel vil en økning ...

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Overvåking endoplasmic nettet kalsiumhomeostase Ved hjelp av en<em&gt; Gaussia</em&gt; Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Research

JoVE Journal

Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transkripsjon og tildekking av Gaussia luciferase mRNA Etterfulgt av Jakten Cell Transfeksjon

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Overvåking Cell-autonome biologiske klokke Rhythms of Gene Expression Bruke luciferase Bioluminescens Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Visualisering av endoplasmatisk retikulum Lokalisert mRNAs i pattedyrceller

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Høy gjennomstrømming Funksjonell Screening ved hjelp av en hjemmelaget Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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

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 ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Visualisering av endoplasmatiske retikulum underdomener i dyrkede celler

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

RAW264.7 transfektere celler med en luciferasereportergenet

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Måling av kalsium svingninger i løpet av sarkoplasmatisk retikulum av dyrkede glatte muskelceller Bruke FRET basert Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...