Name: measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex
Uploaded: 2021-08-02T15:00:00
Description: Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

We acknowledge the Wilhelm Sander-Foundation (2017.008.02), the Center of Dynamic Systems (CDS), funded by the EU-program ERDF (European Regional Development Fund) and the DFG (LA 2386) for supporting our work. We thank Karina Guttek for supporting our experiments. We acknowledge Prof. Dirk Reinhold (OvGU, Magdeburg) for providing us primary T cells.

&#8203;T cell experiments were performed according to the ethical agreement 42502-2-1273 Uni MD.
1. Preparing cells for the experiment
NOTE: The average number of cells for this immunoprecipitation is 1 &#215; 107. Adherent cells have to be seeded one day before the experiment so that there are 1 &#215; 107 cells&#160;on the day of the experiment.
<ol>
	<li>Preparing adherent cells for the experiment
	<ol>
		<li>Seed 5-8 &#215; 10<...

<ol>
	<li>Lavrik, I. N., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD95/Fas+signaling+at+the+DISC.">Regulation of CD95/Fas signaling at the DISC.</a> Cell Death and Differentiation. 19 (1), 36-41 (2012).</li><li>Krammer, P. H., Arnold, R., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+and+death+in+peripheral+T+cells.">Life and death in peripheral T cells.</a> Nature Reviews Immunology. 7 (7), 532-542 (2007).</li><li>Dickens, L. 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=A+death+effector+domain+chain+DISC+model+reveals+a+crucial+role+for+caspase-8+chain+assembly+in+mediating+apoptotic+cell+death.">A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.</a> Molecular Cell. 47 (2), 291-305 (2012).</li><li>Fu, T. -. 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=Cryo-EM+structure+of+caspase-8+tandem+DED+filament+reveals+assembly+and+regulation+mechanisms+of+the+death-inducing+signaling+complex.">Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.</a> Molecular Cell. 64 (2), 236-250 (2016).</li><li>Scaffidi, C., Medema, J. P., Krammer, P. H., Peter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLICE+is+predominantly+expressed+as+two+functionally+active+isoforms,+caspase-8/a+and+caspase-8/b.">FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.</a> Journal of Biological Chemistry. 272 (43), 26953-26958 (1997).</li><li>Fox, J. 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=Cryo-EM+structural+analysis+of+FADD:Caspase-8+complexes+defines+the+catalytic+dimer+architecture+for+co-ordinated+control+of+cell+fate.">Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.</a> Nature Communications. 12 (1), 1-17 (2021).</li><li>Schleich, 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=Stoichiometry+of+the+CD95+death-inducing+signaling+complex:+experimental+and+modeling+evidence+for+a+death+effector+domain+chain+model.">Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.</a> Molecular Cell. 47 (2), 306-319 (2012).</li><li>Hughes, M. 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=Reconstitution+of+the+death-inducing+signaling+complex+reveals+a+substrate+switch+that+determines+CD95-mediated+death+or+survival.">Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.</a> Molecular Cell. 35 (3), 265-279 (2009).</li><li>Lavrik, I., 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+active+caspase-8+heterotetramer+is+formed+at+the+CD95+DISC+[2].">The active caspase-8 heterotetramer is formed at the CD95 DISC [2].</a> Cell Death and Differentiation. 10 (1), 144-145 (2003).</li><li>Hoffmann, J. C., Pappa, A., Krammer, P. H., Lavrik, I. N. <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+C-terminal+cleavage+product+of+procaspase-8,+p30,+defines+an+alternative+pathway+of+procaspase-8+activation.">A new C-terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation.</a> Molecular and Cellular Biology. 29 (16), 4431-4440 (2009).</li><li>Golks, 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+role+of+CAP3+in+CD95+signaling:+New+insights+into+the+mechanism+of+procaspase-8+activation.">The role of CAP3 in CD95 signaling: New insights into the mechanism of procaspase-8 activation.</a> Cell Death and Differentiation. 13 (3), 489-498 (2006).</li><li>Ozt&#252;rk, S., Schleich, K., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+FLICE-like+inhibitory+proteins+(c-FLIPs):+Fine-tuners+of+life+and+death+decisions.">Cellular FLICE-like inhibitory proteins (c-FLIPs): Fine-tuners of life and death decisions.</a> Experimental Cell Research. 318 (11), 1324-1331 (2012).</li><li>Golks, A., Brenner, D., Fritsch, C., Krammer, P. H., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-FLIPR,+a+new+regulator+of+death+receptor-induced+apoptosis.">c-FLIPR, a new regulator of death receptor-induced apoptosis.</a> Journal of Biological Chemistry. 280 (15), 14507-14513 (2005).</li><li>Hughes, M. 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=Co-operative+and+hierarchical+binding+of+c-FLIP+and+caspase-8:+A+unified+model+defines+how+c-FLIP+isoforms+differentially+control+cell+fate.">Co-operative and hierarchical binding of c-FLIP and caspase-8: A unified model defines how c-FLIP isoforms differentially control cell fate.</a> Molecular Cell. 61 (6), 834-849 (2016).</li><li>Hillert, L. 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=Long+and+short+isoforms+of+c-FLIP+act+as+control+checkpoints+of+DED+filament+assembly.">Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.</a> Oncogene. 39 (8), 1756-1772 (2020).</li><li>Yu, J. W., Jeffrey, P. D., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+procaspase-8+activation+by+c-FLIPL.">Mechanism of procaspase-8 activation by c-FLIPL.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (20), 8169-8174 (2009).</li><li>Micheau, O., 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+long+form+of+FLIP+is+an+activator+of+caspase-8+at+the+Fas+death-inducing+signaling+complex.">The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.</a> Journal of Biological Chemistry. 277 (47), 45162-45171 (2002).</li><li>Chang, D. 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=C-FLIPL+is+a+dual+function+regulator+for+caspase-8+activation+and+CD95-mediated+apoptosis.">C-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.</a> EMBO Journal. 21 (14), 3704-3714 (2002).</li><li>Hillert, L. 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=Dissecting+DISC+regulation+via+pharmacological+targeting+of+caspase-8/c-FLIPL+heterodimer.">Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.</a> Cell Death and Differentiation. 27 (7), 2117-2130 (2020).</li><li>Fricker, N., 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=Model-based+dissection+of+CD95+signaling+dynamics+reveals+both+a+pro-+and+antiapoptotic+role+of+c-FLIPL.">Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL.</a> Journal of Cell Biology. 190 (3), 377-389 (2010).</li><li>Arndt, B., 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=Analysis+of+TCR+activation+kinetics+in+primary+human+T+cells+upon+focal+or+soluble+stimulation.">Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.</a> Journal of Immunological Methods. 387 (1-2), 276-283 (2013).</li><li>Pietkiewicz, S., Eils, R., Krammer, P. H., Giese, N., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+treatment+of+CD95L+and+gemcitabine+in+pancreatic+cancer+cells+induces+apoptotic+and+RIP1-mediated+necroptotic+cell+death+network.">Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network.</a> Experimental Cell Research. 339 (1), 1-9 (2015).</li><li>Schleich, 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=Molecular+architecture+of+the+DED+chains+at+the+DISC:+regulation+of+procaspase-8+activation+by+short+DED+proteins+c-FLIP+and+procaspase-8+prodomain.">Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.</a> Cell Death and Differentiation. 23 (4), 681-694 (2016).</li><li>Lavrik, I. N., 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=Analysis+of+CD95+threshold+signaling:+Triggering+of+CD95+(FAS/APO-1)+at+low+concentrations+primarily+results+in+survival+signaling.">Analysis of CD95 threshold signaling: Triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling.</a> Journal of Biological Chemistry. 282 (18), 13664-13671 (2007).</li><li>Sprick, M. R., 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=Caspase-10+is+recruited+to+and+activated+at+the+native+TRAIL+and+CD95+death-inducing+signalling+complexes+in+a+FADD-dependent+manner+but+can+not+functionally+substitute+caspase-8.">Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.</a> EMBO Journal. 21 (17), 4520-4530 (2002).</li><li>Neumann, 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=Dynamics+within+the+CD95+death-inducing+signaling+complex+decide+life+and+death+of+cells.">Dynamics within the CD95 death-inducing signaling complex decide life and death of cells.</a> Molecular Systems Biology. 6 (1), 352 (2010).</li><li>Kischkel, F. 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=Cytotoxicity-dependent+APO-1+(Fas/CD95)-associated+proteins+form+a+death-inducing+signaling+complex+(DISC)+with+the+receptor.">Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.</a> EMBO Journal. 14 (22), 5579-5588 (1995).</li><li>Seyrek, K., Richter, M., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+sweet+regulation+of+apoptosis:+the+role+of+glycosylation+and+galectins+in+apoptotic+signaling+pathways.">Decoding the sweet regulation of apoptosis: the role of glycosylation and galectins in apoptotic signaling pathways.</a> Cell Death and Differentiation. 26 (6), 981-993 (2019).</li><li>Kallenberger, S. 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=Intra-+and+interdimeric+caspase-8+self-cleavage+controls+strength+and+timing+of+CD95-induced+apoptosis.">Intra- and interdimeric caspase-8 self-cleavage controls strength and timing of CD95-induced apoptosis.</a> Science Signaling. 7 (316), 23 (2014).</li></ol>

This approach was first described by Kischkel et al.27 and has successfully been developed since then by several groups. Several important issues have to be considered for efficient DISC-immunoprecipitation and monitoring caspase-8 processing in this complex.
First, it is essential to follow all washing steps during immunoprecipitation. Especially important are the final washing steps of the sepharose beads and the drying of the sepharose beads. This must be done correc...

One of the best-studied death receptors (DRs) is CD95 (Fas, APO-1). The extrinsic apoptotic pathway starts with the interaction of the DR with its cognate ligand, i.e., CD95L interacts with CD95 or TRAIL binds to TRAIL-Rs. This results in the formation of the DISC at the corresponding DR. DISC consists of CD95, FADD, procaspase-8/-10, and c-FLIP proteins1,2. Furthermore, the DISC is assembled by interactions between death domain (DD)-containing proteins, such as CD95 and FADD, and DED-containing proteins such as FADD, procaspase-8/-10, and c-FLIP (Figure 1). Procaspase-8 undergoes oligomerization via association of its DEDs, resulting in the formation of DED filaments, followed by procaspase-8 activation and processing. This triggers a caspase cascade, which leads to cell death (Figure 1)3,4. Thus, procaspase-8 is a central initiator caspase of the extrinsic apoptosis pathway mediated by CD95 or the TRAIL-Rs, activated at the corresponding macromolecular platform, DISC.
Two isoforms of procaspase-8, namely procaspase-8a (p55) and -8b (p53), are known to be recruited to the DISC5. Both isoforms comprise two DEDs. DED1 and DED2 are located at the N-terminal part of procaspase-8a/b followed by the catalytic p18 and p10 domains. Detailed cryo-electron microscopy (cryo-EM) analysis of procaspase-8 DEDs revealed the assembly of procaspase-8 proteins into filamentous structures called DED filaments4,6. Remarkably, the linear procaspase-8 chains were initially suggested to be engaged in the dimerization followed by procaspase-8 activation at the DISC. Now, it is known that those chains are only a substructure of the procaspase-8 DED filament, the latter comprising three chains assembled into a triple helix3,4,6,7.
Upon dimerization at the DED filament, conformational changes in procaspase-8a/b lead to the formation of the active center of procaspase-8 and its activation3,8. This is followed by procaspase-8 processing, which is mediated via two pathways: the first one goes via the generation of a p43/p41 cleavage product and the second one via the initial generation of a p30 cleavage product. The p43/p41 pathway is initiated by the cleavage of procaspase-8a/b at Asp374, resulting in p43/p41 and p12 cleavage products (Figure 2). Further, these fragments are auto-catalytically cleaved at Asp384 and Asp210/216, giving rise to the formation of the active caspase-8 heterotetramer, p102/p1829,10,11. In addition, it was shown that in parallel to the p43/p41 pathway of processing, procaspase-8a/b is also cleaved at Asp216, which leads to the formation of the C-terminal cleavage product p30, followed by its proteolysis to p10 and p1810 (Figure 2).
Procaspase-8a/b activation at the DED filament is strictly regulated by proteins named c-FLIPs12. The c-FLIP proteins occur in three isoforms: c-FLIPLong (c-FLIPL), c-FLIPShort (c-FLIPS), and c-FLIPRaji (c-FLIPR). All three isoforms contain two DEDs in their N-terminal region. c-FLIPL also has a C-terminal catalytically inactive caspase-like domain12,13. Both short isoforms of c-FLIP-c-FLIPS and c-FLIPR-act in an anti-apoptotic manner by disrupting DED filament formation at the DISC6,14,15. In addition, c-FLIPL can regulate caspase-8 activation in a concentration-dependent manner. This can result in both pro- and anti-apoptotic effects16,17,18. By forming the catalytically active procaspase-8/c-FLIPL heterodimer, c-FLIPL leads to the stabilization of the active center of procaspase-8 and its activation. The pro- or anti-apoptotic function of c-FLIPL is directly dependent on its amount at the DED filaments and the subsequent amount of assembled procaspase-8/c-FLIPL heterodimers19. Low or intermediate concentrations of c-FLIPL at the DISC result in sufficient amounts of procaspase-8/c-FLIPL heterodimers at the DED filament, which supports the activation of caspase-8. In contrast, increased amounts of c-FLIPL directly lead to its anti-apoptotic effects at the DISC20.
Taken together, the activation and processing of procaspase-8a/b at the DISC is a highly regulated process involving several steps. This paper discusses the measurement of procaspase-8 processing directly at the DISC as well as the analysis of the composition of this complex. This will be presented using CD95 DISC as the exemplary DR complex.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12.5% SDS gel</td>
			<td>self made</td>
			<td></td>
			<td>for two separating gels: 
			3.28 mL distilled H2O 
			2.5 mL Tris; pH 8.8; 1.5 M 
			4.06 mL acrylamide 
			100 &#181;L 10% SDS 
			100 &#181;L 10% APS 
			7.5 &#181;L TEMED 
			 
			for two collecting gels: 
			3.1 mL distilled H2O 
			1.25 mL Tris; pH 6.8; 1.5 M 
			0.5 mL acrylamide 
			50 &#181;L 10% SDS 
			25 &#181;L 10% APS 
			7.5 &#181;L TEMED</td>
		</tr>
		<tr>
			<td>14.5 cm cell dishes</td>
			<td>Greiner</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>acrylamide</td>
			<td>Carl Roth</td>
			<td>A124.1</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-actin Ab</td>
			<td>Sigma Aldrich</td>
			<td>A2103</td>
			<td>dilution: 1:4000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-APO-1 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by Enzo</td>
			<td>ALX-805-038-C100</td>
			<td>used only for immunoprecipitation</td>
		</tr>
		<tr>
			<td>anti-caspase-10 Ab</td>
			<td>Biozol</td>
			<td>MBL-M059-3</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-3 Ab</td>
			<td>cell signaling</td>
			<td>9662 S</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-caspase-8 Ab C15</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-242-C100</td>
			<td>dilution: 1:20 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-CD95 Ab</td>
			<td>Santa Cruz</td>
			<td>sc-715</td>
			<td>dilution: 1:2000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-c-FLIP NF6 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-804-961-0100</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-FADD 1C4 Ab</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ADI-AAM-212-E</td>
			<td>dilution: 1:10 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>anti-PARP Ab</td>
			<td>cell signaling</td>
			<td>9542</td>
			<td>dilution: 1:1000 in PBST + 1:100 NaN3</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Carl Roth</td>
			<td>9592.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Carl Roth</td>
			<td>4227.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford solution 
			Protein Assay Dye Reagent Concentrate 450ml</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>CD95L</td>
			<td>provided in these experiments by Prof. P. Krammer or can be purchased by ENZO</td>
			<td>ALX-522-020-C005</td>
			<td></td>
		</tr>
		<tr>
			<td>chemoluminescence detector 
			Chem Doc XRS+</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Protese Inhibitor Cocktail (PIC)</td>
			<td>Sigma Aldrich</td>
			<td>11 836 145 001</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>DPBS (10x) w/o Ca, Mg</td>
			<td>PAN Biotech</td>
			<td>P04-53500</td>
			<td>dilution 1:10 with H2O, storage in the fridge</td>
		</tr>
		<tr>
			<td>eletrophoresis buffer</td>
			<td>self made</td>
			<td></td>
			<td>10x electrophoresis buffer: 
			60.6 g Tris 
			288 g glycine 
			20 g SDS 
			ad 2 L H2O 
			1:10 dilution before usage</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG1 HRP</td>
			<td>SouthernBiotech</td>
			<td>1070-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG2b</td>
			<td>SouthernBiotech</td>
			<td>1090-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG-HRP</td>
			<td>SouthernBiotech</td>
			<td>4030-05</td>
			<td>dilution 1:10.000 in PBST + 5% milk</td>
		</tr>
		<tr>
			<td>Interleukin-2 Human(hIL-2)</td>
			<td>Merckgroup/ Roche</td>
			<td>11011456001</td>
			<td>for activation of T cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>loading buffer 
			4x Laemmli Sample Buffer,10 mL</td>
			<td>Bio Rad</td>
			<td>161-0747</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Luminata Forte Western HRP substrate</td>
			<td>Millipore</td>
			<td>WBLUFO500</td>
			<td></td>
		</tr>
		<tr>
			<td>lysis buffer</td>
			<td>self made</td>
			<td></td>
			<td>13.3 mL Tris-HCl; pH 7.4; 1.5 M 
			27.5 mL NaCl; 5 M 
			10 mL EDTA; 2 mM 
			100 mL Triton X-100 
			add 960 mL H2O</td>
		</tr>
		<tr>
			<td>medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine,&#160; 2,438 g/L</td>
			<td>PAN Biotech</td>
			<td>P04-41154</td>
			<td>adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium</td>
		</tr>
		<tr>
			<td>medium for primary T cells</td>
			<td>gibco by Life Technologie</td>
			<td>21875034</td>
			<td>adding 10% FCS and 1% Penicillin-Streptomycin to the medium</td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td>Carl Roth</td>
			<td>T145.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Carl Roth</td>
			<td>P030.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td>self made</td>
			<td></td>
			<td>20x PBST: 
			230 g NaCl 
			8 g KCl 
			56.8 g Na2HPO4 
			8 g KH2PO4 
			20 mL Tween-20 
			ad 2 L H2O 
			dilution 1:20 before usage</td>
		</tr>
		<tr>
			<td>PBST + 5% milk</td>
			<td>self made</td>
			<td></td>
			<td>50 g milk powder + 1 L PBST</td>
		</tr>
		<tr>
			<td>PHA</td>
			<td>Thermo Fisher Scientific</td>
			<td>R30852801</td>
			<td>for actavation of T ells</td>
		</tr>
		<tr>
			<td>Power Pac HC</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Standard All Blue</td>
			<td>Bio Rad</td>
			<td>161-0373</td>
			<td>use between 3-5 &#181;L</td>
		</tr>
		<tr>
			<td>Protein A Sepharose CL-4B beads</td>
			<td>Novodirect/ Th.Geyer</td>
			<td>GE 17-0780-01</td>
			<td>affinity resin beads prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>scraper</td>
			<td>VWR</td>
			<td>734-2602</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth</td>
			<td>4360.2</td>
			<td></td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Carl Roth</td>
			<td>K305.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot Turbo mini-size transfer stacks</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo 5x Transfer Buffer</td>
			<td>Bio Rad</td>
			<td>10026938</td>
			<td>prepared according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>TransBlot Turbo Mini-size nictrocellulose membrane</td>
			<td>Bio Rad</td>
			<td>170-4270</td>
			<td>used according to manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Trans-Blot-Turbo</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Chem Solute</td>
			<td>8,08,51,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Pan Reac Appli Chem</td>
			<td>A4974,1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring composition of cd95 death-inducing signaling complex and processing of procaspase-8 in this complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

To analyze caspase-8 recruitment to the DISC and its processing at the CD95 DISC, this paper describes a classical workflow, which combines IP of the CD95 DISC with western blot analysis. This allows the detection of several key features of caspase-8 activation at the DISC: the assembly of the caspase-8-activating macromolecular platform, recruitment of procaspase-8 to the DISC, and the processing of this initiator caspase (Figure 1 and Figure 2). This workflow ...

Watch this Scientific Journal Video about Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex at JoVE.com

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Apoptosis initiation is mediated by activation of caspases at marker molecular platforms. We present the detection of the key initiation cell death complex DISC, which serves as a platform for caspase-8 activation. The advantage of this technique is that it allows the detection of the assembly and the composition of the DISC complex along with providing information on caspase-8 processing in this complex.Begin the experiment with cells that are 80%to 90%confluent and adherent to the dish. Discard the medium and add fresh medium to the adherent cells. Then stimulate the cells with the selected concentration of CD95L by holding the plate at an angle and pipetting the ligand into the medium without touching the adherent cells.To harvest the cells, place the cell dish on ice. Add 10 milliliters of cold PBS to the cell suspension and scrape the attached cells off the plate. Collect the cell suspension in a 50 milliliter tube.Wash the cell dish with 10 milliliters of cold PBS twice and add the wash solution into the same 50 milliliter tube. Then centrifuge the cell suspension at 500 times G for five minutes at four degrees Celsius. After discarding the supernatant, resuspend the cell pellet in one milliliter of cold PBS.Then transfer the cell suspension into a 1.5 milliliter tube. Centrifuge the cell suspension and resuspend the cell pellet in one milliliter of cold PBS again. Repeat the centrifugation once again, then resuspend the cell pellet in one milliliter of lysis buffer and incubate it for 30 minutes on ice.After incubation, centrifuge the lysate at maximum speed for 15 minutes at four degrees Celsius, then transfer the supernatant to a clean tube. Discard the pellet and collect a 50 microliter sample of the lysate in another tube for determining the protein concentration. For immunoprecipitation, add two microliters of anti-APO1 antibody and 10 microliters of prepared Protein A Sepharose beads to the lysate.Add 10 microliters of the beads alone to a separate tube containing the lysate to serve as a bead control. Incubate the mixture containing the lysate, the antibodies, and Protein A Sepharose beads overnight at four degrees Celsius with gentle mixing. The following day, centrifuge the mixture at 500 times G for four minutes at four degrees Celsius.After discarding the supernatant, wash the beads by adding one milliliter of cold PBS and discarding the supernatant following centrifugation. Repeat this step at least three times. After discarding the last PBS wash, aspirate the beads preferably with a 50 microliter Hamilton syringe.To perform the Western blot, add 20 microliters of 4X loading buffer to the beads and heat at 95 degrees Celsius for 10 minutes. Simultaneously, heat the lysate controls at 95 degrees Celsius for five minutes. Load the lysates, immunoprecipitants, and a protein standard onto a 12.5%SDS gel and run with a constant voltage of 80 volts.After the gel run is complete, transfer the proteins from the SDS gel to a nitrocellulose membrane. After the transfer is complete, place the blotted membrane in a box and incubate it for an hour in blocking solution under gentle agitation, then wash the membrane three times for five minutes with PBST. To detect the proteins, add the first primary antibody at the indicated dilution to the membrane and incubate it overnight at four degrees Celsius with gentle agitation.The next day, wash the membrane with three five-minute PBST washes, then incubate the membrane with 20 milliliters of secondary antibody with gentle shaking for one hour at room temperature. Wash the membrane three times with PBST again. After discarding the last PBST wash, add approximately one milliliter of horseradish peroxidase substrate to the membrane and detect the chemoluminescence signal.Stimulation of cervical cancer HeLa-CD95 cells with CD95L resulted in a high level of CD95 DISC formation monitored via CD95 immunoprecipitation. CD95, FADD, procaspase-8, procaspase-10, and c-FLIPS were observed in these CD95 immunoprecipitations indicating efficient DISC formation. The cleavage products of p43-FLIP and p22-FLIP were detected in the immunoprecipitations, indicating caspase-8 activation.HeLa-CD95 cells that over-express c-FLIP long were used for analysis of CD95 DISC formation. Similar to parental HeLa-CD95 cells, no recruitment of FADD, procaspase-9, procaspase-10, c-FLIP proteins, and PARP1 was detected in the immunoprecipitations from these cells without CD95L treatment. Activated primary T-cells were characterized by high levels of CD95, FADD, procaspase-8, procaspase-10, and c-FLIPS in anti-CD95 immunoprecipitations.This technique allows the analysis of molecular DISC formation, including processing of caspase-8 at the DISC. All washing steps are very important. In addition, stimulation points and incubation times should be taken seriously.This is the only way to measure the assembly on the DISC complex. However, to measure caspase-8 activation, one can also implement caspase-8 activity assays. This approach allowed us to get new insights into apoptosis initiation.

measuring-composition-cd95-death-inducing-signaling-complex

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

CRAF-14-3-3 Interaction Analysis in Live Cells Using a BRET-Based Approach

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...