1. Cloning and Cell Line Generation
<ol>
	<li>PCR amplify the NBB region, corresponding to amino acid residues 1-140 (NBB140), from human MLKL cDNA for in frame standard restriction enzyme-based cloning with the oligomerization domain 2x FK506 binding protein (2xFKBP or 2xFV) and Venus fluorescent protein into the Doxycycline (Dox)-inducible retroviral vector pRetroX-TRE3G to obtain NBB140-2xFV-Venus (Table 1, Figures 1A-B).</li>
	<li>Immortalize primary mlkl-/-</sup...

<ol>
	<li>Weinlich, R., Oberst, A., Beere, H. M., 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=Necroptosis+in+development,+inflammation+and+disease.">Necroptosis in development, inflammation and disease.</a> Nature Reviews Molecular Cell Biology. 18 (2), 127-136 (2017).</li><li>Kaiser, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP3+mediates+the+embryonic+lethality+of+caspase-8-deficient+mice.">RIP3 mediates the embryonic lethality of caspase-8-deficient mice.</a> Nature. 471 (7338), 368-372 (2011).</li><li>Murphy, J. 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+pseudokinase+MLKL+mediates+necroptosis+via+a+molecular+switch+mechanism.">The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.</a> Immunity. 39 (3), 443-453 (2013).</li><li>Oberst, A., 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=Catalytic+activity+of+the+caspase-8-FLIP(L)+complex+inhibits+RIPK3-dependent+necrosis.">Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.</a> Nature. 471 (7338), 363-367 (2011).</li><li>Zhang, H., 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=Functional+complementation+between+FADD+and+RIP1+in+embryos+and+lymphocytes.">Functional complementation between FADD and RIP1 in embryos and lymphocytes.</a> Nature. 471 (7338), 373-376 (2011).</li><li>He, 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137 (6), 1100-1111 (2009).</li><li>Dondelinger, Y., Hulpiau, P., Saeys, Y., Bertrand, M. J. M., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionary+perspective+on+the+necroptotic+pathway.">An evolutionary perspective on the necroptotic pathway.</a> Trends in Cell Biology. 26 (10), 721-732 (2016).</li><li>Newton, K., Manning, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+and+Inflammation.">Necroptosis and Inflammation.</a> Annual Review of Biochemistry. 85, 743-763 (2016).</li><li>Kaiser, W. J., Upton, J. W., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+modulation+of+programmed+necrosis.">Viral modulation of programmed necrosis.</a> Current Opinion in Virology. 3 (3), 296-306 (2013).</li><li>Newton, K., 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=RIPK3+deficiency+or+catalytically+inactive+RIPK1+provides+greater+benefit+than+MLKL+deficiency+in+mouse+models+of+inflammation+and+tissue+injury.">RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.</a> Cell Death &amp; Differentiation. 23 (9), 1565-1576 (2016).</li><li>Kearney, C. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inflammatory+Perspective+on+Necroptosis.">An Inflammatory Perspective on Necroptosis.</a> Molecular Cell. 65 (6), 965-973 (2017).</li><li>Pasparakis, M., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+and+its+role+in+inflammation.">Necroptosis and its role in inflammation.</a> Nature. 517 (7534), 311-320 (2015).</li><li>Sun, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+kind+of+cell+suicide:+mechanisms+and+functions+of+programmed+necrosis.">A new kind of cell suicide: mechanisms and functions of programmed necrosis.</a> Trends in Biochemical Sciences. 39 (12), 587-593 (2014).</li><li>Grootjans, S., Vanden Berghe, T., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+and+execution+mechanisms+of+necroptosis:+an+overview.">Initiation and execution mechanisms of necroptosis: an overview.</a> Cell Death &amp; Differentiation. 24 (7), 1184-1195 (2017).</li><li>Sun, L., 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=Mixed+lineage+kinase+domain-like+protein+mediates+necrosis+signaling+downstream+of+RIP3+kinase.">Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.</a> Cell. 148 (1-2), 213-227 (2012).</li><li>Wang, H., 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=Mixed+lineage+kinase+domain-like+protein+MLKL+causes+necrotic+membrane+disruption+upon+phosphorylation+by+RIP3.">Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.</a> Molecular Cell. 54 (1), 133-146 (2014).</li><li>Cai, Z., 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=Plasma+membrane+translocation+of+trimerized+MLKL+protein+is+required+for+TNF-induced+necroptosis.">Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.</a> Nature Cell Biology. 16 (1), 55-65 (2014).</li><li>Chen, X., 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=Translocation+of+mixed+lineage+kinase+domain-like+protein+to+plasma+membrane+leads+to+necrotic+cell+death.">Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death.</a> Cell Research. 24 (1), 105-121 (2014).</li><li>Dillon, C. P., 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=RIPK1+blocks+early+postnatal+lethality+mediated+by+caspase-8+and+RIPK3.">RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.</a> Cell. 157 (5), 1189-1202 (2014).</li><li>Rickard, J. A., 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=RIPK1+regulates+RIPK3-MLKL-driven+systemic+inflammation+and+emergency+hematopoiesis.">RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.</a> Cell. 157 (5), 1175-1188 (2014).</li><li>Kaiser, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+3-mediated+necrosis+via+TRIF,+RIP3,+and+MLKL.">Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.</a> Journal of Biological Chemistry. 288 (43), 31268-31279 (2013).</li><li>Upton, J. W., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAI+Another+Way:+Necroptotic+Control+of+Viral+Infection.">DAI Another Way: Necroptotic Control of Viral Infection.</a> Cell Host &amp; Microbe. 21 (3), 290-293 (2017).</li><li>Tanzer, M. 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=Necroptosis+signalling+is+tuned+by+phosphorylation+of+MLKL+residues+outside+the+pseudokinase+domain+activation+loop.">Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop.</a> Biochemical Journal. 471 (2), 255-265 (2015).</li><li>Quarato, G., 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=Sequential+Engagement+of+Distinct+MLKL+Phosphatidylinositol-Binding+Sites+Executes+Necroptosis.">Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.</a> Molecular Cell. 61 (4), 589-601 (2016).</li><li>Davies, K. A., 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+brace+helices+of+MLKL+mediate+interdomain+communication+and+oligomerisation+to+regulate+cell+death+by+necroptosis.">The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.</a> Cell Death &amp; Differentiation. , (2018).</li><li>Su, L., 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=A+plug+release+mechanism+for+membrane+permeation+by+MLKL.">A plug release mechanism for membrane permeation by MLKL.</a> Structure. 22 (10), 1489-1500 (2014).</li><li>Dondelinger, Y., 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=MLKL+compromises+plasma+membrane+integrity+by+binding+to+phosphatidylinositol+phosphates.">MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.</a> Cell Reports. 7 (4), 971-981 (2014).</li><li>Llambi, F., 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=BOK+Is+a+Non-canonical+BCL-2+Family+Effector+of+Apoptosis+Regulated+by+ER-Associated+Degradation.">BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.</a> Cell. 165 (2), 421-433 (2016).</li><li>Malkani, N., Schmid, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+secrets+of+fluorescent+proteins:+distinct+bleaching+in+various+mounting+fluids+and+photoactivation+of+cyan+fluorescent+proteins+at+YFP-excitation.">Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.</a> PLoS One. 6 (4), 18586 (2011).</li><li>Perez, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+workflow+for+the+automatic+segmentation+of+organelles+in+electron+microscopy+image+stacks.">A workflow for the automatic segmentation of organelles in electron microscopy image stacks.</a> Frontiers in Neuroanatomy. 8, 126 (2014).</li><li>Walton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lead+aspartate,+an+en+bloc+contrast+stain+particularly+useful+for+ultrastructural+enzymology.">Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.</a> Journal of Histochemistry &amp; Cytochemistry. 27 (10), 1337-1342 (1979).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biology. 2 (11), 329 (2004).</li><li>Rossi, P., Xia, Y., Khanra, N., Veglia, G., Kalodimos, C. 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We provide protocols for techniques that we combined to implicated MLKL as the putative executioner of plasma membrane rupture24. In addition to deciphering the regulatory network that regulates MLKL-mediated necroptosis, these techniques can be used independently to characterize other suitable biological systems. Practically speaking, these techniques are medium- to low-throughput discovery tools.
We have routinely used live-cell imaging of NBB140-2xFV-Venus...

Identifying genetic components of necroptosis has facilitated the use of animal models to test the implication of necroptosis in physiology and disease1,2,3,4,5. Knockout of RIPK3 or MLKL in mice had minimal implication in development and adult homeostasis suggesting that necroptosis is not essential for life3,6. Moreover, certain species do not contain either RIPK3 or MLKL genes, supporting the non-essential role of necroptosis in animals7,8. On the other hand, challenging knockout animal models with various pathologies induced in the laboratory has revealed an important role of necroptosis in inflammation, innate immunity, and viral infection9,10,11,12.
Necroptosis can be activated in several ways by signaling through different innate immunity sensors, all of which result in the activation of RIPK31,13,14. Active RIPK3 in turn phosphorylates and activates MLKL3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18. The most studied, and perhaps the most complex way, leading to activation of RIPK3 involves death receptor ligation, which bifurcates based on the downstream composition of the signaling complexes to either induce apoptosis or necroptosis1. Necroptosis ensues when signaling through RIPK1 is favored and results in engagement of RIPK319,20. This outcome is easily favored upon pharmacological inhibition or genetic deletion of caspase 8, a putative endogenous inhibitor of necroptosis that keeps necroptosis at bay. RIPK1 binds to and activates RIPK3. Another way to activate necroptosis is through Toll-like receptors TLR3/TLR4 signaling, which engages and activates RIPK3 through TIR-domain-containing adapter-inducing interferon-&#946; (TRIF)21. Yet another way to die by necroptosis is by activation of the DNA sensor DAI, which directly engages and activates RIPK322.
MLKL is a cytosolic protein comprised of an N-terminal helix bundle (NB) domain and a C-terminal pseudokinase domain (psKD) linked by a regulatory brace region3. In normal cells, MLKL is found in the cytosol where it is thought to be in an inactive complex with RIPK314. Activation of necroptosis triggers RIPK3 phosphorylation of MLKL in the activation loop of the psKD, and potentially additional sites in the NB and brace3,15,23. Phosphorylation induces a conformational change in MLKL that results in dissociation from RIPK314. Poorly understood conformational changes release the brace from the psKD24. The brace, which contains 2 helices, mediates oligomerization of MLKL into a putative trimer through the C-terminal helix25. The N-terminal helix of the brace inhibits the NB domain, which is essential for membrane permeabilization24,26. In isolation, NB domain is sufficient to induce plasma membrane permeabilization and necroptosis16,24,27. The pro-necroptotic activity of NB was reconstituted in mouse embryonic fibroblasts deficient in MLKL (mlkl-/- MEFs). NB is a lipid binding domain that preferentially engages the phospholipid phosphatidylinositol 4,5 diphosphate (PIP2). We proposed a stepwise mechanism of activation of MLKL, wherein brace oligomerization facilitates recruitment of MLKL to the plasma membrane via weak interactions of NB with the PIP2 polar head group24. At the membrane, the NB undergoes regulated exposure of an additional high-affinity binding site for PIP2, which is masked by the brace in inactive MLKL. Overall, the multiple interactions of NB with PIP2 destabilize the plasma membrane leading to its rupture, although the molecular mechanism of these events have not been elucidated.
Here we illustrate specific methods used to characterize the function of MLKL as executioner of necroptosis24. In particular, we focus on the most minimal domain of MLKL, the NB and brace (NBB), which is regulated by brace inhibition and can be activated through enforced dimerization to induce plasma membrane rupture and necroptosis. We describe our inducible expression system combined with enforced drug-induced FKBP-mediated dimerization for live-cell imaging, and electron microscopy of cells undergoing necroptosis. Additionally, we illustrate our in vitro NMR analysis of the interactions of NBB with phosphatidylinositols (PIPs).

<table border="0" cellpadding="0" cellspacing="0" style="width:1175px;" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Cloning and cell line generation</td>
			<td style="width:229px;"></td>
			<td style="width:323px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pRetroX-TRE3G</td>
			<td>Clontech</td>
			<td>631188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tet-On transactivator plasmid</td>
			<td></td>
			<td></td>
			<td>Llambi et al., 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Embryonic Fibroblasts (MEFs) mlkl-/-</td>
			<td></td>
			<td></td>
			<td>Dillon et al., 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP2647100 CAS#3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell death quantification and live-cell microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline</td>
			<td>Clontech</td>
			<td>631311 CAS# 24390-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B/B Homodimerizer AP20187</td>
			<td>Takara</td>
			<td>635059 CAS# 195514-80-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syto16</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15N Ammonium Chloride</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-467-10 CAS# 12125-02-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterated DTT</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-2622-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium Oxide</td>
			<td>Sigma Aldrich</td>
			<td>617385-1 CAS# 7789-20-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310 CAS# 69227-93-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X CAS# 383907-42-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)</td>
			<td>Avanti Polar Lipids</td>
			<td>850143 CAS# 849412-67-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol) (ammonium salt) (18:1)</td>
			<td>Avanti Polar Lipids</td>
			<td>850149 CAS# 799268-53-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specialized Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte FLR or ZOOM</td>
			<td>Essen BioScience, Inc.</td>
			<td></td>
			<td>Live-cell microscopy imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios NanoLab 660 DualBeam&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Electron microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte 2011A Rev2 v20111.3.4288 (FLR)</td>
			<td>Essen BioScience, Inc.</td>
			<td>http://www.essenbioscience.com</td>
			<td style="width:193px;">Imaging analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FEI MAPS</td>
			<td>Thermo Fisher Scientific</td>
			<td>https://www.fei.com/software/maps/</td>
			<td style="width:193px;">EM analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopSpin v3.2</td>
			<td>Bruker BioSpin</td>
			<td>http://www.bruker.com</td>
			<td style="width:193px;">NMR data collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CARA v1.9.1.7</td>
			<td></td>
			<td>http://cara.nmr.ch/&#160;</td>
			<td style="width:193px;">NMR data analysis</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:429px;">Slidebook</td>
			<td style="width:229px;">3i (Intelligent Imaging Innovations)</td>
			<td style="width:323px;">https://www.intelligent-imaging.com/slidebook</td>
			<td style="width:193px;">Confocal microscopy</td>
		</tr>
	</tbody>
</table>

characterization of mlkl-mediated plasma membrane rupture in necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

Visualizing regulated necroptosis execution in live cells has been possible through inducible expression of a minimal truncated MLKL construct, NBB140-2xFV-Venus. This construct maintains the ability to induce plasma membrane permeabilization and is activated through Dim-induced oligomerization of the FKBP cassette (2xFV). We observe and quantify necroptosis by live-cell microscopy imaging, monitoring kinetically (every 5 min) the uptake of a cell impermeable green fluorescence...

Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

These methods can help answer key questions about the role of the pseudokinase mixed-lineage kinase-domain like or MLKL in necroptosis such as membrane translocation and permeabilization in lipid binding. The main advantage of these techniques is that they directly implicate MLKL as the executioner of plasma membrane rupture in necroptosis. Live-cell imaging has revolutionized the field of cell-death research, especially for MLKL-mediated necroptosis.Nuclear magnetic resonance is a powerful technique for monitoring lipid binding to proteins as demonstrated here for MLKL. Begin by plating five times 10 to the fourth MLKL knockout mouse embryonic fibroblasts or MEFs expressing the executioner domain of MLKL fused to an oligomerization cassette and Venus in one milliliter of supplemented DMEM in 24-well plates for their overnight incubation in a cell-culture incubator. The next morning, treat the cells with doxycycline for 12 hours to induce expression of the transgene, followed by the addition of standard DMEM supplemented with FK506-binding protein dimerizer and membrane-impermeable green fluorescent dye to induce necroptosis.To monitor the cell death, place the plate in an appropriate imaging unit housed in a mammalian-tissue culture incubator and open the imaging system software. Select the Schedule Upcoming Scans tab and select the tray, vessel, scan types, and scan pattern in the Physical Layout tab. Select fast fluorescence in the Properties tab and click on the Timeline window and the total number of time points and frequency of acquisition to add the scan time.Then, click Apply to start the image acquisition. To quantify the necroptosis, select Tasks pane and hover the mouse cursor over the background of several images to set the object counting new analysis with fixed segmentation threshold above the background level. Then, select Previewing to examine the fluorescent objects in the current image and refine the threshold levels as needed.Careful selection of the threshold could be performed observing multi wells on the plate and using the Preview feature on the software. Just be aware to use the same threshold setting for all the wells in the plate. To integrate the green fluorescence counts, under the Launch New Analysis tab select time range and the wells to be analyzed and enter the analysis job name.Then, click Okay and double-click on the job name to export the data for additional analysis in an appropriate graphing software program. For lipid binding of the MLKL by nuclear magnetic resonance spectroscopy or NMR, use airtight syringes to aliquot one milligram per milliliter of the phospholipids of interest into clean glass vials for individual NMR titration series and place the vials under a gaseous stream of argon or nitrogen for five to 30 minutes. Dispensing lipids from volatile solvents such as chloroform or methanol must be performed quickly from stockpiles kept on dry ice in order to minimize inconsistencies in lipid concentrations between samples due to solvent evaporation.At the end of the evaporation period, use large tweezers to arrange the vials at the bottom of a Buchner flask. Seal the flask with a rubber stopper and attach plastic tubing connected to a vacuum line to evacuate the vial contents under mild vacuum overnight to remove any residual organics. The next morning, add NMR buffer supplemented with detergent at the appropriate experimental concentration to each glass vial with a lipid film and sonicate the vial in a water bath for up to 30 minutes, stopping the sonication every 10 minutes to check the solubilization of the lipid film.At the end of the sonication, allow the vials to cool for 15 minutes and perform a serial dilution of the lipid-detergent micelles in detergent-positive NMR buffer, sonicating the 1:1 mixtures for the desired concentration series. Prepare control and reference NMR samples of protein and additives in detergent-negative and detergent-positive NMR buffers respectively, adding protein and additives to each lipid dilution in detergent-positive buffer. Then, transfer the appropriate volume of samples to suitably-sized NMR tubes and load the samples onto the NMR instrument.Complete necroptosis of MLKL-knockout MEFs can be observed within one hour of dimer-mediated oligomerization of doxycycline pre-incubated cells expressing the fused transgene. This fast kinetics necroptosis supports the role of MLKL as the putative executioner of plasma membrane rupture, resulting in the production of plasma-membrane-associated Venus puncta within one to two minutes of treatment with the dimer and Venus coding of the cell periphery and gradual cell rounding within two to three minutes. Under fast necroptosis, the cells change morphology from their normal elongated shape to rounded and swelled within five minutes of oligomerization, partially ruptured at 10 minutes, and extensively dismantled with vanished cytosol at 20 minutes.In vitro lipid binding experiments monitored by NMR spectroscopy reveal major structural changes in the MLKL executioner domain protein upon lipid binding to displace the inhibitory brace region from the closed N-helical to the open and intrinsically disordered conformation as well as per-residue information on mixtures of both conformers. The percentages of closed and open conformers in a given sample can also be monitored, providing supporting evidence for the interaction of the MLKL executioner domain with phospholipids and suggesting a direct link between MLKL and the plasma membrane. So while attempting to quantify cell death using this procedure, it is important to know and remember that the DNA-binding dye have higher threshold fluorescence than normally genetically-encoding fluorescence protein.For this reason, they can be used in parallel. Also note that micellar lipids are temperature sensitive and therefore the NMR sample temperature may need to be optimized. So following this procedure, other technique could be used like total internal reflection microscopy and electromicroscopy to address how fast the cell ruption happens on cells undergoing necroptosis and where the rupture are localized on the plasma membrane.In addition, microscale thermophoresis can be performed to orthogonally validate MLKL binding to micellar lipids. The techniques described in this video paved the way for researchers in the field of necroptosis to explore the role of MLKL as the executioner of plasma membrane rupture. Don't forget that working with chloroform can be extremely hazardous and that precautions such as wearing gloves and working in a well-ventilated area should always be taken when performing this procedure.

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10.7K vues.  St. Jude Children's Research Hospital. These methods can help answer key questions about the role of the pseudokinase mixed-lineage kinase-domain like or MLKL in necroptosis such as membrane translocation and permeabilization in lipid binding. The main advantage of these techniques is that they directly implicate MLKL as the executioner of plasma membrane rupture in necroptosis. Live-cell imaging has revolutionized the field of cell-death research, especially for MLKL-mediated necroptosis.Nuclear magnetic resonance is a pow...