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-/- or ripk3-/- mlkl-/-&#160;mouse embryonic fibroblasts (MEFs) obtained from the respective mice available in the Green laboratory by transient transfection with SV40-large antigen expressing plasmid. Select immortalized cells in ~1&#8211;2 weeks, and maintain in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and streptomycin, 1 mM sodium pyruvate, and nonessential amino acids at 37 &#176;C and 5% CO2 in standard tissue culture incubators.</li>
	<li>Transduce SV40-immortalized mlkl-/- MEFs with the reverse tetracycline controlled transactivator (rtTA)-containing retrovirus expressed from a plasmid containing the blasticidin resistance gene (our modified plasmid) and treat for 1 week with 5 &#956;g/mL blasticidin to select for cells stably expressing rtTA28.</li>
	<li>Transduce the rtTA-expressing MEFs with the Dox-inducible retroviral vector containing the chimeric MLKL (pRetroX-NBB140-2xFV-Venus-puro) and select using 4 &#956;g/mL puromycin for one week, 2 days after transduction.</li>
</ol>
2. Live-Cell Microscopy Imaging of MLKL-mediated Necroptosis
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 50,000 cells per well (1 mL/well) in 24 well plates, and incubate overnight (Figure 2A). Use live-cell staining for automated cell counting (see Table of Materials).</li>
	<li>Treat cells for 12 h with Dox (0.5 &#956;g/mL) to induce the expression of NBB140-2xFV-Venus.</li>
	<li>Induce necroptosis with 25 nM FKBP dimerizer (Dim) in addition to&#160;25 nM membrane-impermeable green fluorescent dye (see Table of Materials) in standard DMEM (see 1.2). Cells undergoing necroptosis accumulate the dye as their plasma membrane integrity is compromised by NBB140-mediated rupture. Perform assays in triplicate or quadruplicate.</li>
	<li>To monitor necroptosis using single- or dual-color imaging systems (see Table of Materials), place the vessels in the respective imaging unit housed in a mammalian tissue culture incubator.</li>
	<li>Open the software (see Table of Materials) and select the tab Schedule Upcoming Scans under the Tasks List. 
	NOTE: This protocol is for imaging using single-color systems, but a similar software is available for&#160;dual-color systems.</li>
	<li>Select the &#34;Tray, Vessel, Scan Types&#34;, and the &#34;Scan Pattern&#34; in the &#34;Physical Layout&#34; tab and &#34;Fast Fluorescence&#34; in the Properties tab.</li>
	<li>Add the &#34;Scan Time&#34; by clicking on the &#34;Timeline&#34; window and the total number of time points and frequency of acquisition (typically 1 acquisition every 0.5 h to 1 h for 6 h to 12 h, or every 5 min for fast-kinetics necroptosis) and start image acquisition by selecting &#34;Apply&#34; (red button).</li>
	<li>Quantify necroptosis using the image analysis software (see Table of Materials) by selecting from the &#34;Tasks Pane&#34;, &#34;Object Counting New Analysis with Fixed Segmentation Threshold above the background level&#34;, which can be determined by hovering the mouse cursor over background regions in several images used in the analysis.</li>
	<li>Examine fluorescent objects by selecting Previewing using current image and refine the Threshold levels as needed.</li>
	<li>Integrate green fluorescence counts by opening&#160;the &#34;Launch New Analysis&#34; tab, choose the &#34;Time Range&#34;, select the wells to be analyzed, enter the &#34;Analysis Job Name&#34;, and press &#34;OK&#34;.</li>
	<li>Access analyzed data from the &#34;Analysis Jobs&#34; tab by double-clicking on the respective job name and exporting from the Graph/Export window for additional analysis in external graphing software (see Table of Materials).</li>
	<li>Quantitate data as green fluorescence positive (+ve) counts/mm2 or normalize the counts by confluence and express the data as green fluorescence +ve counts/confluence.</li>
	<li>Optionally, at the experimental endpoint, stain cells with 100 nM of the membrane-permeant green fluorescent dye (see Table of Materials). Normalize data to % necroptotic cells as the ratio of green fluorescence/green fluorescence at endpoint.</li>
</ol>
3. Live-cell Confocal Microscopy Imaging of Plasma Membrane Recruitment and Permeabilization by MLKL
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 20,000 cells per well (0.5 mL/well) in a glass bottom 4-well microscopy chamber pretreated with 50 &#181;g/mL fibronectin&#160;in PBS at 37 &#176;C for 30 min and incubate for ~12 h (Figure 2B).</li>
	<li>Treat the cells with Dox (0.5 &#956;g/mL) for ~12 h to induce the expression of NBB140-2xFV-Venus.</li>
	<li>Induce necroptosis by incubating with fresh medium (1 mL/well) containing 25 nM Dim to induce NBB140-2xFV-Venus activation and translocation to the plasma membrane.</li>
	<li>Place the chamber on a spinning disc laser scanning confocal microscope built on an inverted stand equipped with environmental control and a 63 1.4 NA objective and begin imaging acquisition using software of choice (see Table of Materials).</li>
	<li>Initialize the software, select &#34;Focus&#34; icon and YFP filter configuration (excitation wavelength 515 nm).</li>
	<li>Locate the field of view and focal plane, and engage the focus maintenance system using the &#34;Definite Focus&#34; tab.</li>
	<li>To begin acquisition, select the &#34;Capture&#34; tab followed by assignment of filter configuration, appropriate EMCCD camera exposure time and intensification gain, and time-lapse acquisition parameters including interval and length of acquisition.</li>
	<li>Monitor cellular redistribution of the fluorescent NBB140-2xFV-Venus protein during necroptosis by acquiring images every 10 s by a EMCCD camera using a 515 nm laser (Movie 1). 
	NOTE: Photobleaching is a concern with fluorescent protein imaging. Venus is one of the more resistant fluorescent proteins to photobleaching29. If fluorescent proteins that are more susceptible to photobleaching are used, image every 30 s or longer to allow recovery of signal between imaging time points.</li>
	<li>To monitor plasma membrane association of NBB140-2xFV-Venus during necroptosis, image every 10 s the sample prepared in 3.3 by total internal reflection fluorescence (TIRF) microscopy using a microscope equipped with an automated TIRF slider and a 100X 1.4 NA objective and an EMCCD camera (Movie 2). Follow steps 3.5&#8211;3.7, but adjust the TIRF angle within the TIRF focus tab according to sample and bottom glass thickness of the microscopy chamber. 
	NOTE: Plasma membrane markers such as LCK-C-RFP may be used as controls for plasma membrane localization in TIRF analysis.</li>
	<li>Perform image processing to enhance quality by convolution with the negative normalized second derivative of a Gaussian function (Marr algorithm).</li>
</ol>
4. Electron Microscopy
<ol>
	<li>Plate mlkl-/- MEFs expressing NBB140-2xFV-Venus at a density of 5 million cells in a plasma-coated 150 mm cell culture dish and incubate for 12 h (Figure 3).</li>
	<li>Treat the cells with Doxycycline (0.5 &#956;g/mL) to induce the expression of NB1-140-2xFV-Venus for 12 h.</li>
	<li>Induce NBB140-2xFV-Venus activation and translocation to the plasma membrane with 25 nM of homodimerizer for 5 min. This time is established from the kinetics of necroptosis observed in live-cell imaging.</li>
	<li>Remove the media&#160;and fix cells using 10 mL of 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (cacodylate buffer) pre-warmed at 37 &#176;C.</li>
	<li>Collect the sample by scraping and spin the samples for 10 min at 500 x g. Discard the supernatant.</li>
	<li>Postfix the sample for 1.5 h in reduced 2% osmium tetroxide with 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer. Osmium tetroxide is a staining agent widely used in TEM to provide contrast. We used TEM as preliminary analysis prior to SEM of cell undergoing necroptosis.</li>
	<li>Rinse the sample 5 times in ultrapure water for 5 min each at 500&#160;x g, followed by rinses in buffer (see 4.6), water, and ethanol on an automatic processor. Discard the supernatant.</li>
	<li>For the SEM, stain the previously fixed samples with 1% uranyl acetate and lead aspartate heavy-metal stain31.</li>
	<li>Dehydrate the samples through a graded series of alcohol and propylene oxide solutions.</li>
	<li>Embed the sample in hard resin32 to obtain a resin block.</li>
	<li>Cut thick sections of 0.5 &#956;m to determine the correct area of analysis.</li>
	<li>Coat the samples with an ultra-thin film of electrically-conducting metal (Iridium).</li>
	<li>Image and analyze the samples (Figure 3). For quantification, a low-magnification image of the resin block surface with exposed cells is scanned at 5 keV using an electron microscope.</li>
	<li>Visualize cells across individual images captured by SEM or imaging software may be utilized to join multiple fields-of-view into one contiguous image30. Roughly 100 cells may be used to evaluate the fraction of cells undergoing necroptosis in this manner by generating a high-resolution montage in software of choice (see Table of Materials). 
	NOTE: Scoring necrotic morphology by SEM compares normal cell features with the progression of pre-necrotic or necrotic phenotypes. These features include plasma membrane alterations including microvilli disappearance, flattening, ruptures in cells ranging from 10 nm to 1 &#181;m in size, or loss of organelle structure across at least 30&#8211;40% of the cytosolic cell area.</li>
</ol>
5. Lipid Binding of MLKL by Nuclear Magnetic Resonance (NMR) Spectroscopy
<ol>
	<li>Prepare 15N-labeled NBB1-156 by standard protein expression and purification as previously described24.</li>
	<li>Dissolve 500 &#181;g of 1,2-distearoyl-sn-glycero-3-phosphoinositol ammonium salt (18:0 PI) and 1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol) ammonium salt (18:1 PI) in 500 &#181;L of ice-cold&#160;CHCl3 each for final concentrations of 1 mg/mL.</li>
	<li>Sonicate 1 mg/mL 18:0 and 18:1 PI in a sonication water bath for 1&#160;min at room temperature or up to 5 min in an ice-water mixture.</li>
	<li>Aliquot 1 mg/mL 18:0 and 18:1 PI into clean glass vials for individual NMR titration series (Figure 4). Transfer lipids in organic solvents using glass air-tight syringes.
	<ol>
		<li>Aliquot 132 &#181;L of 1 mg/mL 18:1 PI (molar mass = 880.137 g/mol) in CHCl3 for a final resuspension volume of 1,200 &#181;L at 125 &#181;M concentration. 
		NOTE: For 5 mm NMR tubes, prepare serial dilution volumes of &#62;1,000 &#181;L and final NMR sample volumes of &#62;500 &#181;L. For 3 mm NMR tubes, prepare serial dilution volumes of &#62;300 &#181;L and final NMR sample volumes of &#62;150 &#181;L.</li>
	</ol>
	</li>
	<li>Aliquot 1 mg/mL porcine brain L-&#945;-phosphatidylinositol-4,5-bisphosphate ammonium salt in 20:9:1 CHCl3/MeOH/H2O (brain PI(4,5)P2) into clean glass vials.</li>
	<li>Place vials under a gaseous stream of argon (Ar) or nitrogen (N2) with moderate flow to evaporate organic solvent without splashing against the glass vial walls. Five to thirty&#160;min is typically sufficient for volumes of 10&#8211;200 &#181;L organic solvent.</li>
	<li>Arrange glass vial(s) at bottom of a B&#252;chner or vacuum flask using large tweezers, seal with a rubber stopper, and attach plastic tubing connected to a vacuum line. Evacuate flask under mild vacuum overnight to remove residual organics.</li>
	<li>Overlay lipid film aliquots with inert gas, seal with an airtight lid, and store at -20 &#176;C for use within one month or -80 &#176;C for longer-term storage.</li>
	<li>Prepare NMR sample buffers without detergent (-Det) containing 20 mM NaxHyPO4 pH 6.8, 10% D2O and with detergent (+Det) containing 20 mM NaxHyPO4 pH 6.8, 10% D2O, 0.34 mM n-dodecyl-&#946;-D-maltopyranoside (DDM).</li>
	<li>Add +Det buffer to glass vial with lipid film to achieve desired concentration and sonicate in water bath for up to 30 min, alternating sonication for 10 min followed by visual inspection. 
	NOTE: Sonication may warm sample. Allow to cool to room temperature for 15 min before final protein sample reconstitution.</li>
	<li>Perform serial dilution of lipid-detergent micelles in +Det buffer using sonication of 1:1 mixtures for the desired concentration series. Starting at 125 &#181;M lipid concentration, three repetitions will yield 62.5 &#181;M, 31.3 &#181;M, and 15.6 &#181;M lipid in 0.34 mM DDM.</li>
	<li>Prepare control and reference NMR samples of protein and additives in -Det and +Det buffers, respectively. Add protein and additives to each dilution of lipid in +Det buffer. 
	NOTE: For 15N NBB156, protein is added to a final concentration of 40 &#181;M and supplemented with 2 mM deuterated dithiothreitol to keep Cys residues reduced.</li>
	<li>Transfer the appropriate volume of samples to 5 mm or 3 mm NMR tubes (see note in step 5.4).</li>
	<li>Manually load samples in NMR instrument or arrange in an automation platform such as the SampleCase loader.</li>
	<li>Acquire composite NMR spectra using standard pulse programs for 1H proton spectra as quality control followed by 2D 1H-15N correlation spectra either as transverse relaxation optimized spectroscopy (TROSY) or heteronuclear multiple quantum coherence (SOFAST-HMQC)33. 
	NOTE: Usage of 3 mm NMR tubes requires 64 scans for 1H-15N TROSY (~3 h) or 1H-15N SOFAST-HMQC (~90 min). Scan numbers are halved for 5 mm NMR tubes.</li>
	<li>Processed 2D NMR spectra may be added to a CARA repository34 for analysis or other software of choice to the user.</li>
	<li>Analyze software by annotating 2D NMR resonances for three well-dispersed and resolved peaks for each protein state. Record the peak amplitudes for each of the six peaks for each buffer condition containing NBB156 (Figure 5A).
	<ol>
		<li>Use CARA to prepare a batch-integration list (Click Main Menu heading &#34;Spectrum | Setup Batch List&#34;&#8230;) of all spectra collected and analyze&#160;using a single peak assignment file (Click Main Menu heading &#34;Peaks | Import Peaklist&#34;&#160;&#8230;and choose a user-defined .peaks file with 6 total peaks, 3 for each protein state, defined within).</li>
		<li>Tune the settings (&#34;Integrator | Tune Peak Model&#34; &#8230;) of X-width 0.06 ppm and Y-width 0.30 ppm (Click Main Menu heading &#34;Integrator | Tune Peak Model&#34; &#8230;) before recording the final output in two menu selections (1. Click Main Menu heading &#34;Integrator | Integrate Batch List&#34;) (2. Click Main Menu heading &#34;Peaks | Export Integration Table&#34;) (Figure 5B). 
		NOTE: The backbone amide resonances for residues M21, V53, and L105 have been assigned for the NBB156&#160;closed-brace state. The open-brace state was assigned to backbone amide resonances of residues G130 and A141 and the epsilon proton side-chain resonance of W133 using 3D NMR experiments24.</li>
	</ol>
	</li>
	<li>Normalize samples for direct comparison from different magnets or sample conditions by averaging the three open-brace amplitudes of the reference samples and calculating a normalization factor relating the two references. Calculate the scaled amplitudes for NBB156 resonances using the normalization factor and setting negative amplitudes to zero (Table 2). 
	Examples: 1) Comparing samples from different magnets: Magnet A, NBB156+DDM and Magnet B, NBB156+DDM. 2) Detergent comparison: 0.34 mM DDM and 1.7 mM DDM.</li>
	<li>Calculate for each resonance the scaled fraction of closed- or open-brace by dividing with the range of minimum to maximum amplitude of all spectra collected containing appropriate references of free NBB156 and fully open-brace NBB156 in detergent.</li>
	<li>Plot the normalized NMR amplitudes as a function of lipid concentration and compare the fraction of open-brace NBB156 in different conditions (Figure. 5C). 
	NOTE: Alternatively, analysis of the fraction of closed-brace conformation against lipid concentration can be performed to compare lipid binding to NBB156. We prefer the former analysis because it is quicker and better reports on the fraction fully-engaged by lipids.</li>
</ol>

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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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=15N+and+13C-+SOFAST-HMQC+editing+enhances+3D-NOESY+sensitivity+in+highly+deuterated,+selectively+[1H,13C]-labeled+proteins.">15N and 13C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [1H,13C]-labeled proteins.</a> Journal of Biomolecular NMR. 66 (4), 259-271 (2016).</li><li>Keller, 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+computer+aided+resonance+assignment+tutorial.">The computer aided resonance assignment tutorial.</a> Cantina Verlag. , (2004).</li><li>Chen, W., 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=Diverse+sequence+determinants+control+human+and+mouse+receptor+interacting+protein+3+(RIP3)+and+mixed+lineage+kinase+domain-like+(MLKL)+interaction+in+necroptotic+signaling.">Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling.</a> Journal of Biological Chemistry. 288 (23), 16247-16261 (2013).</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=Evolutionary+divergence+of+the+necroptosis+effector+MLKL.">Evolutionary divergence of the necroptosis effector MLKL.</a> Cell Death &amp; Differentiation. 23 (7), 1185-1197 (2016).</li></ol>

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 ...

characterization-mlkl-mediated-plasma-membrane-rupture

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10.7K Views.  St. Jude Children's Research Hospital. 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.

Video: Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

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 ...

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 ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...