Name: characterization of mlkl-mediated plasma membrane rupture in necroptosis
Uploaded: 2018-08-07T15:00:00
Description: Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

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.

characterization-mlkl-mediated-plasma-membrane-rupture

Research

JoVE Journal

Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...