Support was received from the Swiss National Science Foundation; the Swiss Sinergia program; the Polish-Swiss Research Programme (PSPB-094/2010); the NCCR in Chemical Biology; and LipidX from the Swiss SystemsX.ch initiative, evaluated by the Swiss National Science Foundation (to J. G.). O. M. was supported by an EMBO long-term fellowship (ALTF-516-2012).

1. Solutions and Preparations
NOTE: All buffers and solutions should be prepared in double-distilled (dd) H2O. Because the hydration state of sucrose varies, the final concentration of all sucrose solutions must be determined using a refractometer.
<ol>
	<li>Prepare phosphate-buffered saline without divalent cations (PBS-): 137 mM NaCI, 2.7 mM KCl, 1.5 mM KH2PO4, and 6.5 mM Na2HPO4. Adjust the pH to 7.4. Sterilize by autoclaving (1 cycle...

<ol>
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N., Gautreau, A., Billadeau, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retromer-mediated+endosomal+protein+sorting:+all+WASHed+up!.">Retromer-mediated endosomal protein sorting: all WASHed up!.</a> Trends Cell Biol. 23 (11), 522-528 (2013).</li><li>Burd, C., Cullen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retromer:+a+master+conductor+of+endosome+sorting.">Retromer: a master conductor of endosome sorting.</a> Cold Spring Harb Perspect Biol. 6 (2), a016774 (2014).</li><li>Woodman, P. G., Futter, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivesicular+bodies:+co-ordinated+progression+to+maturity.">Multivesicular bodies: co-ordinated progression to maturity.</a> Curr Opin Cell Biol. 20 (4), 408-414 (2008).</li><li>Henne, W. M., Buchkovich, N. J., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ESCRT+pathway.">The ESCRT pathway.</a> Dev Cell. 21 (1), 77-91 (2011).</li><li>Morel, E., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A2+binding+to+endosomes+and+functions+in+endosomal+transport+are+regulated+by+tyrosine+23+phosphorylation.">Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation.</a> J Biol Chem. 284 (3), 1604-1611 (2009).</li><li>Muriel, O., Tomas, A., Scott, C. C., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moesin+and+cortactin+control+actin-dependent+multivesicular+endosome+biogenesis.">Moesin and cortactin control actin-dependent multivesicular endosome biogenesis.</a> Mol Biol Cell. 27 (21), 3305-3316 (2016).</li><li>Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J., Zerial, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+membrane+domains+on+endosomes+in+the+recycling+pathway+visualized+by+multicolor+imaging+of+Rab4,+Rab5,+and+Rab11.">Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.</a> J Cell Biol. 149 (4), 901-914 (2000).</li><li>Morel, E., Parton, R. G., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A2-dependent+polymerization+of+actin+mediates+endosome+biogenesis.">Annexin A2-dependent polymerization of actin mediates endosome biogenesis.</a> Dev Cell. 16 (3), 445-457 (2009).</li><li>Kamentsky, 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=Improved+structure,+function+and+compatibility+for+CellProfiler:+modular+high-throughput+image+analysis+software.">Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software.</a> Bioinformatics. 27 (8), 1179-1180 (2011).</li><li>Meijering, E., 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=Design+and+validation+of+a+tool+for+neurite+tracing+and+analysis+in+fluorescence+microscopy+images.">Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.</a> Cytometry A. 58 (2), 167-176 (2004).</li><li>Granger, E., McNee, G., Allan, V., Woodman, P. <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+the+cytoskeleton+and+molecular+motors+in+endosomal+dynamics.">The role of the cytoskeleton and molecular motors in endosomal dynamics.</a> Semin Cell Dev Biol. 31, 20-29 (2014).</li><li>Mayran, N., Parton, R. G., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+II+regulates+multivesicular+endosome+biogenesis+in+the+degradation+pathway+of+animal+cells.">Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells.</a> EMBO J. 22 (13), 3242-3253 (2003).</li><li>Gorvel, J. P., Chavrier, P., Zerial, M., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rab5+controls+early+endosome+fusion+in+vitro.">rab5 controls early endosome fusion in vitro.</a> Cell. 64 (5), 915-925 (1991).</li><li>Wandinger-Ness, A., Zerial, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab+proteins+and+the+compartmentalization+of+the+endosomal+system.">Rab proteins and the compartmentalization of the endosomal system.</a> Cold Spring Harb Perspect Biol. 6 (11), a022616 (2014).</li><li>Balch, W. E., Glick, B. S., Rothman, J. 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Actin plays a crucial role in endosome membrane dynamics4,14. We previously reported that actin nucleation and polymerization occur on early endosomes, forming small F-actin patches or networks. These F-actin networks are absolutely required for membrane transport beyond early endosomes along the degradation pathway. Interfering at any step of this nucleation and polymerization process prevents endosome maturation and thus downstream transport towards late endoso...

In higher eukaryotic cells, proteins and lipids are internalized into early endosomes where sorting occurs. Some proteins and lipids, which are destined to be reutilized, are incorporated into tubular regions of the early endosomes and then transported to the plasma membrane or to the trans-Golgi network (TGN)1,2. By contrast, other proteins and lipids are selectively packaged into regions of the early endosomes that exhibit a multivesicular appearance. These regions expand and, upon detachment from early endosomal membranes, eventually mature into free endosomal carrier vesicles or multivesicular bodies (ECV/MVBs), which are responsible for cargo transport towards late endosomes1,2.
Actin plays a crucial role in the membrane remodeling process associated with endosomal sorting capacity and endosome biogenesis. Protein sorting along the recycling pathways to the plasma membrane or to the TGN depends on the retromer complex and the associated proteins. This sorting machinery seems to be coupled to the formation of the recycling tubules via interactions of the retromer complex, with the WASP and Scar homologue (WASH) complex and branched actin3,4,5. In contrast, molecules destined for degradation, particularly activated signaling receptors, are sorted into intraluminal vesicles (ILVs) by the endosomal sorting complexes required for transport (ESCRT)2,6,7. While the possible role of actin in the ESCRT-dependent sorting process is not known, F-actin plays an important role in the biogenesis of ECV/MVBs and in transport beyond early endosomes. In particular, we found that annexin A2 binds cholesterol-enriched regions of early endosome, and together with spire1, nucleates F-actin polymerization. The formation of the branched actin network observed on endosomes requires the branching activity of the actin-related protein (ARP) 2/3 complex, as well as the ERM protein moesin and the actin-binding protein cortactin8,9.
Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes. This assay has been used previously to investigate the role of annexin A2 in F-actin nucleation and of moesin and cortactin in the formation of endosomal actin networks8,9. With this in vitro protocol, the complex series of reactions that occur on endosomes during actin polymerization become amenable to the biochemical and molecular analysis of the sequential steps of the process, including actin nucleation, linear polymerization, branching, and crosslinking.

<table border="0" cellpadding="0" cellspacing="0" width="1287">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:477px;">NaCl</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:145px;">71380</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Acros Organics</td>
			<td>196770010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>AppliChem</td>
			<td>A1042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Acros Organics</td>
			<td>424370025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>AppliChem</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesiun acetate tetrahydrate</td>
			<td>Fluka</td>
			<td>63047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A2948</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>10125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fluka</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Merck Millipore</td>
			<td>107687</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Roche</td>
			<td>11017101001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Roche</td>
			<td>10253286001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehide</td>
			<td>Polysciences. Inc</td>
			<td>380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 555 phalloidin</td>
			<td>Molecular Probes</td>
			<td>A34055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Actin rhodamine</td>
			<td>Cytoskeleton. Inc</td>
			<td>APHR-A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>poly(vinyl alcohol), Mw ~31 000&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DABCO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1086</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-casein</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter 0.22um</td>
			<td>Millex&#160;</td>
			<td>SL6V033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round 10cm dishes for cell culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Pasteur pipette</td>
			<td>Assistent</td>
			<td>569/3 40569003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15-ml polypropylen tube&#160;</td>
			<td>TPP</td>
			<td>91015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypodermic Needle 22G Black 30mm&#160;</td>
			<td>BD Microlance</td>
			<td>300900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Luer-slip 1ml Syringes without needle</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
			<td>Assistent</td>
			<td>2406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
			<td>Assistent</td>
			<td>1000/1818</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200-&#956;l yellow tip</td>
			<td>Starlab</td>
			<td>S1111-0706</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1000-&#956;l Blue Graduated Tip</td>
			<td>Starlab</td>
			<td>S1111-6801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5-ml test tube</td>
			<td>Axygen</td>
			<td>MCT-175-C 311-04-051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18-mm diameter round coverslip&#160;</td>
			<td>Assistent</td>
			<td>1001/18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35-mm diameter round dish with a 20-mm glass bottom (0.16-0.19&#160;mm)&#160;</td>
			<td>In vitro Scientific</td>
			<td>D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorvall WX80 Ultracentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>TL-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SW60 rotor</td>
			<td>Beckman coulter</td>
			<td>335649</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 rotor</td>
			<td>Beckman coulter</td>
			<td>346936</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscopy</td>
			<td>Carl Zeiss</td>
			<td>LSM-780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fugene HD transfection reagent</td>
			<td>Promega</td>
			<td style="width:145px;">E2311</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent A</td>
			<td>Bio-Rad</td>
			<td>500-0113</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent B</td>
			<td>Bio-Rad</td>
			<td>500-0114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein assay reagent S</td>
			<td>Bio-Rad</td>
			<td>500-0115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell scraper</td>
			<td>Homemade</td>
			<td></td>
			<td>Silicone rubber piece of about 2 cm, cut at a very sharp angle and attached to a metal bar or held with forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Minimum Essential Medium Eagle (MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids (NEAA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Meter 691</td>
			<td>Metrohm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td></td>
			<td></td>
			<td>NIH, Bethesda MD</td>
		</tr>
	</tbody>
</table>

in vitro polymerization of f-actin on early endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

To gain insights into the formation of F-actin patches on early endosome membranes, we followed the protocol outlined in Figure 2. Briefly, cells were transfected with GFP-RAB5 and then early endosomes were prepared by subcellular fractionation. These purified early endosomes were incubated with cytosol in order to provide actin itself as well as other factors possibly involved in the reaction. At the end of the incubation period, the reaction was stopped by ...

Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

The overall goal of this microscopy based in vitro assay is to study actin polymerization from endosomes in vitro. This method can help answer key questions in the membrane trafficking and actin fields, such as the world of actin in the acidic pathway. And the main advantage of this technique is to reconstitute the nucleation polymerization of F-actin on early endosomal membranes in the test tube.That's rendering this complex series of reactions amenable to my chemical manipulations. Demonstrating the procedure will be Jorge Larios and Olivia Muriel from the lab. Begin by placing dishes of hela cells onto a wet metal plate in a flat ice bucket.And washing the cells twice with five milliliters of ice cold PBS. After removing the PBS from the last wash, add three milliliters of ice cold PBS per dish. Then use a flexible rubber policeman to scrape the cells off the dishes in a quick circular motion around the outside of the dish.Followed by a downward motion in the middle of the dish. Scrape gently to obtain sheets of attached cells. Next use a plastic pasteur pipette with a wide opening to gently transfer the cells to 15 milliliter conical polypropylene tubes on ice.Centrifuge for five minutes at 175 times G and four degrees celsius. Gently remove the supernatant and add one to three milliliters of homogenization buffer to the pellets. Using a plastic pasteur pipette with a wide opening, gently pipette up and down once to re suspend the cells.Then centrifuge for seven to 10 minutes at 1, 355 times G and four degrees celsius. After removing the supernatant, add a known volume of homogenization buffer with protease inhibitors to each cell pallet. And gently pipette up and down without introducing air bubbles until the cells are re suspended.Rinse a one milliliter tuberculin syringe with homogenization buffer and ensure that it is free of air or bubbles. Then slowly fill the syringe with the cell suspension to avoid creating air bubbles. Place the bevel tip of the needle against the wall of the tube and expel the cell suspension rapidly to shear off the plasma membranes of the attached cells.Then take a small aliquot of each homogenate, dilute it into 50 microliters of homogenization buffer on a glass slide and mix. Then cover it with a glass cover slip, inspect the homogenates under a phase contrasting microscope equipped with a 20 times or 40 times objective. Under optimal conditions, most cells are broken, but nuclei which appear dark gray are round and are not.Repeat the shearing step until most cells are broken, usually three to six up and down strokes through the needle are necessary. Keep a small aliquot of each homogenate for protein determination. After centrifuging as before, carefully collect the post nuclear supernatant.Reserve small aliquots of each post nuclear supernatant and nuclear pellet for protein determination and then discard the nuclear pellets. Adjust the post nuclear supernatants to 40.6 percent sucrose by adding post nuclear supernatant to 62 percent sucrose solution at a ratio of one to one point one. Mix gently but thoroughly without creating air bubbles.Transfer the post nuclear supernatants in 40.6 percent sucrose to ultra centrifugation tubes. Overlay carefully with two point five milliliters of 35 percent sucrose solution, and fill the centrifuge tube with homogenization buffer. Ultra centrifuge the gradients for one hour at approximately 165, 000 times G and four degrees celsius.Following ultra centrifugation, carefully remove the white layer of lipids on top of the gradients. Next, use a 200 microliter micro pipette with a cut tip to collect the interface containing endosomes which is between the homogenization buffer and 35 percent sucrose. To collect the cytosol, pipette the post nuclear supernatant into an ultra centrifuge tube.And ultra centrifuge for 45 minutes at approximately 250, 000 times G and four degrees celsius. After, carefully remove the white layer on top, and collect the supernatant which is the cytosol fraction without disturbing the microsome pellet. Begin the assay by gently mixing ice cold purified GFP wrapped five endosomes with cytosol at a ratio of one to 10 protein concentration in an ice cold one point five milliliter conical test tube.Next, use potassium chloride stock solution to adjust the reaction mixture to final concentrations of 125 millimolar potassium chloride. 12.5 millimolar hepes and 1.5 millimolar magnesium acetate. Then adjust the mixture with protease inhibitors to final concentrations of 10 millimolar of leupeptin one millimolar of pepstatin a and 10 nanograms per milliliter aprotinin.Place the test tube containing the reaction mixture at 37 degrees celsius without shaking and incubate for the desired time. After the incubation period, stop the reaction by placing the test tube on ice and adding pre cooled three percent PFA solution at a volumetric ratio of one to 10. Add 200 micrograms per milliliter phalloidin conjugated to red orange dye at a volumetric ratio of zero point three to 10 to stain the polymerized actin.Pipette 12 microliters of the mixture onto 12 microliters of PVA mounting medium on a micro glass slide. Put an 18 by 18 milliliter glass cover slip on top. Then analyze the sample using fluorescence confocal microscopy.Begin by incubating the clean surface of microscopic imaging chambers with one percent beta casein for 20 minutes on ice to minimize protein binding to the glass. Following the incubation, wash twice with three millimolar imidazole. Add the rhodamine actin into a pre cooled one point five milliliter test tube containing the endosomes and cytosol mixture for the assay as previously demonstrated.Adjust the final refractive index of the mixture to one point three seven five by adding 39 percent sucrose solution to a final concentration of 26.5 percent. Dispense the mixture onto the pre treated 35 millimeter dish and cover it with the pre treated 18 millimeter cover slip. Place the chamber at 37 degrees celsius for three or thirty minutes as before.Then analyze by fluorescence confocal microscopy and quantify the actin network using the recommendations in the written protocol. The following images show early endosomes purified from cells expressing GFP-RAB5 that were incubated with cytosol as a source of actin. The assay mixture was fixed, sampled and placed on a microscopic slide and f actin was visualized with phalloidin conjugated to a red flora for.The actin nucleation and polymerization process was rapid since f actin could already be detected on some endosomes after one minute. These short actin structures rapidly grew in length over a period of 30 minutes and became interconnected and branched, eventually forming a filamentous network. Presumably reflecting unbalanced actin dynamics in vitro.These images were obtained when the assay was carried out with purified rhodamine labeled actin in addition to cell cytosol in glass bottomed dishes. So that structures could be imaged directly In the absence of any perturbation. As seen here, similar nucleation and polymerization rates were observed.In the assay described here, early endosomes nucleate de novo actin polymerization selectively. Since no actin polymerization is detected when either the endosome or the cytosol is omitted. These observations demonstrate that early endosomes had the intrinsic and specific capacity to trigger actin nucleation and polymerization.When attempting this procedure, it's important to remember that conditions of relatively low level of RAP5 expression in vivo are preferred. And that cellular membranes are relatively fragile and sensitive to biochemical or chemical shocks. Similarly, the actin network from in vitro is also very fragile and should be handled delicately to avoid collapse or breakage.After watching this video, you should have a good understanding of how to reconstitute the nucleation polymerization of f actin on early endosomal membranes in the test tube. With this technique you will be able to study the world of factors involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of f actin filaments.

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The Cytoskeleton I: Actin and Microfilaments

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8.9K vistas.  University of Geneva. The overall goal of this microscopy based in vitro assay is to study actin polymerization from endosomes in vitro. This method can help answer key questions in the membrane trafficking and actin fields, such as the world of actin in the acidic pathway. And the main advantage of this technique is to reconstitute the nucleation polymerization of F-actin on early endosomal membranes in the test tube.That's rendering this complex series of reactions amenable to my chemical manipulations. D...

Video: In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic...

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

labeling f-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed...

actin co-sedimentation assay; for the analysis of protein binding to f-actin

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules.  In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind...

measuring protein binding to f-actin by co-sedimentation

Measuring Protein Binding to F-actin by Co-sedimentation

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the...

the gliding actin filament assay: an in vitro motility assay to study translocation of actin filaments on immobilized myosins

In this video, we perform an in vitro motility assay to analyze the actin-myosin interactions. The gliding movement of actin filaments on immobilized myosin molecules is visualized using fluorescence...

The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Load 8 square coverslips onto a rack, and thoroughly wash them with approximately 2 to 5 milliliters of 200-proof ethanol, followed by 2 to 5 milliliters of distilled water. Then, dry the coverslips completely using a filtered air-line or...

high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin...

inverted motility assay: an in vitro technique to visualize myosin movement on immobilized actin filaments

Inverted Motility Assay: An In Vitro Technique to Visualize Myosin Movement on Immobilized Actin Filaments

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Wash the chamber with 10 microliters of motility buffer with DTT. Flow in 10 microliters of 1 milligram per milliliter BSA in motility buffer with DTT. Repeat this twice, waiting a minute after the third wash. Use tissue or filter paper to wick the solution through the channel by gently placing the corner of the paper at the flow chamber...

in vitro reconstitution of the actin cytoskeleton inside giant unilamellar vesicles

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

a time-efficient fluorescence spectroscopy-based assay for evaluating actin polymerization status in rodent and human brain tissues

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human...

reconstitution of membrane-tethered minimal actin cortices on supported lipid bilayers

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence...

visualizing actin and microtubule coupling dynamics in vitro by total internal reflection fluorescence (tirf) microscopy

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

in vitro culture strategy for oocytes from early antral follicle in cattle

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.

studying the motility of myosin-7a-tailored actin filament using an in vitro gliding assay

Studying the Motility of Myosin-7a-Tailored Actin Filament Using an In Vitro Gliding Assay

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory...

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...