We would like to thank all members of the Chipuk Laboratory for their colleagueship and support. In addition, we would like to give appreciation to Tomomi Kuwana and Donald Newmeyer for developing the experimental foundation for this work. This work was supported by: NIH CA157740 (to J.E.C.), and a pilot project from NIH P20AA017067 (to J.E.C.). This work was also supported in part by a Research Grant 5-FY11-74 from the March of Dimes Foundation (to J.E.C.).

1. Combine Lipids and Create a Lipid Film
<ol>
	<li>Working in a fume hood with minimal lighting, chloroform-solubilized lipids are combined into a 1.5 ml amber glass vial using a chloroform-rinsed Hamilton gas-tight glass syringe. This combination will yield approximately 4 mg of lipid (Figure 1A).
	<ul>
		<li>Cardiolipin 10 mg/ml (CL) - 63 &mu;l</li>
		<li>Phosphatidylcholine 10 mg/ml (PC) - 84 &mu;l</li>
		<li>Phosphatidylethanoloamine 10 mg/ml (PE) - 125 &mu;l</li>
		<li>Phosphatidylinositol...

<ol>
	<li>Chipuk, J. E., Moldoveanu, T., Llambi, F., Parsons, M. J., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BCL-2+family+reunion.">The BCL-2 family reunion.</a> Mol. Cell. 37, 299-310 (2010).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+pathways:+ten+minutes+to+dead.">Apoptotic pathways: ten minutes to dead.</a> Cell. 121, 671-674 (2005).</li><li>Gavathiotis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAX+activation+is+initiated+at+a+novel+interaction+site.">BAX activation is initiated at a novel interaction site.</a> Nature. 455, 1076-1081 (2008).</li><li>Dewson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+trigger+apoptosis,+Bak+exposes+its+BH3+domain+and+homodimerizes+via+BH3:groove+interactions.">To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.</a> Mol. Cell. 30, 369-380 (2008).</li><li>Dewson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bak+activation+for+apoptosis+involves+oligomerization+of+dimers+via+their+alpha6+helices.">Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices.</a> Mol. Cell. 36, 696-703 (2009).</li><li>Gavathiotis, E., Reyna, D. E., Davis, M. L., Bird, G. H., Walensky, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-triggered+structural+reorganization+drives+the+activation+of+proapoptotic+BAX.">BH3-triggered structural reorganization drives the activation of proapoptotic BAX.</a> Mol. Cell. 40, 481-492 (2010).</li><li>Kuwana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3+domains+of+BH3-only+proteins+differentially+regulate+Bax-mediated+mitochondrial+membrane+permeabilization+both+directly+and+indirectly.">BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.</a> Mol. Cell. 17, 525-535 (2005).</li><li>Chipuk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+apoptosis+induction+by+inhibition+of+the+anti-apoptotic+BCL-2+proteins.">Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20327-20332 (2008).</li><li>Letai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+BH3+domains+either+sensitize+or+activate+mitochondrial+apoptosis,+serving+as+prototype+cancer+therapeutics.">Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics.</a> Cancer Cell. 2, 183-192 (2002).</li><li>Kuwana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lipids+cooperate+to+form+supramolecular+openings+in+the+outer+mitochondrial+membrane.">lipids cooperate to form supramolecular openings in the outer mitochondrial membrane.</a> Cell. 111, 331-342 (2002).</li><li>Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+binding+by+tBid+initiates+an+ordered+series+of+events+culminating+in+membrane+permeabilization+by+Bax.">Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax.</a> Cell. 135, 1074-1084 (2008).</li><li>Hsu, Y. T., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonionic+detergents+induce+dimerization+among+members+of+the+Bcl-2+family.">Nonionic detergents induce dimerization among members of the Bcl-2 family.</a> J Biol Chem. 272, 13829-13834 (1997).</li><li>Suzuki, M., Youle, R. J., Tjandra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Bax:+coregulation+of+dimer+formation+and+intracellular+localization.">Structure of Bax: coregulation of dimer formation and intracellular localization.</a> Cell. 103, 645-654 (2000).</li><li>Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3+domains+other+than+Bim+and+Bid+can+directly+activate+Bax/Bak.">BH3 domains other than Bim and Bid can directly activate Bax/Bak.</a> J Biol Chem. 286, 491-501 (2011).</li></ol>

No conflicts of interest declared.

The described method for generating LUVs enables a rapid and efficient means to test the function of various BCL-2 family proteins, peptides, and related reagents in a biochemically-defined membrane environment similar to the OMM. If using end point values to determine LUV permeabilization, multiple plates can be set-up to analyze hundreds of conditions within a single day. We find that the limiting reagents in these assays tend to be the quality and quantity of recombinant proteins, so dedicating sufficient time and res...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine "PE"</td>
			<td>Avanti</td>
			<td>850725C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-&alpha;-Phosphatidylcholine (Egg, Chicken) "PC"</td>
			<td>Avanti</td>
			<td>840051C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-&alpha;-Phosphatidylinositol (Liver, Bovine) "PI"</td>
			<td>Avanti</td>
			<td>840042C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-&alpha;-Phosphatidylserine (Brain, Porcine) "PS"</td>
			<td>Avanti</td>
			<td>8400320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cardiolipin (Heart, Bovine - Sodium Salt) "CL"</td>
			<td>Avanti</td>
			<td>840012C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mini-extruder set</td>
			<td>Avanti</td>
			<td>610023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PC membrane 0.2 &mu;M</td>
			<td>Avanti</td>
			<td>610006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Costar black 96 well plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-590</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Caspase-8 cleaved human BID</td>
			<td>R&amp;D Systems</td>
			<td>882-B8-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human BCL-xL (minus C-terminus)</td>
			<td>R&amp;D Systems</td>
			<td>894-BX-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BID/PUMA BH3 domain peptides</td>
			<td>Anaspec</td>
			<td>61711/62404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Synergy H1 hybrid multi-mode microplate reader</td>
			<td>BioTek</td>
			<td>None</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

examining bcl-2 family function with large unilamellar vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

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Examining BCL-2 Family Function with Large Unilamellar Vesicles

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

examining-bcl-2-family-function-with-large-unilamellar-vesicles

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JoVE Journal

Biology

7.8K Görüntüleme.  Mount Sinai School of Medicine. Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.