Name: in vitro polymerization of f-actin on early endosomes
Uploaded: 2017-08-28T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Polymerization of F-actin on Early Endosomes at JoVE.com

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>
	<li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> EMBO J. 30 (17), 3481-3500 (2011).</li><li>Scott, C. C., Vacca, F., 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=Endosome+Maturation,+Transport+and+Functions.">Endosome Maturation, Transport and Functions.</a> Semin. Cell Dev Biol. 31, 2-10 (2014).</li><li>Puthenveedu, 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=Sequence-dependent+sorting+of+recycling+proteins+by+actin-stabilized+endosomal+microdomains.">Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains.</a> Cell. 143 (5), 761-773 (2010).</li><li>Seaman, M. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+intermediates+in+the+pathway+of+intercompartmental+transport+in+a+cell-free+system.">Sequential intermediates in the pathway of intercompartmental transport in a cell-free system.</a> Cell. 39 (3 Pt 2), 525-536 (1984).</li></ol>

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.

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			<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>
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			<td height="20" style="height:20px;">KCl</td>
			<td>Acros Organics</td>
			<td>196770010</td>
			<td></td>
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			<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>AppliChem</td>
			<td>A3724</td>
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			<td height="20" style="height:20px;">Magnesiun acetate tetrahydrate</td>
			<td>Fluka</td>
			<td>63047</td>
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			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A2948</td>
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			<td height="20" style="height:20px;">Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>10125</td>
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			<td height="20" style="height:20px;">NaOH</td>
			<td>Fluka</td>
			<td>71690</td>
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			<td height="20" style="height:20px;">Sucrose</td>
			<td>Merck Millipore</td>
			<td>107687</td>
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			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Roche</td>
			<td>11017101001</td>
			<td></td>
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			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Roche</td>
			<td>10253286001</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
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			<td height="20" style="height:20px;">Paraformaldehide</td>
			<td>Polysciences. Inc</td>
			<td>380</td>
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			<td height="20" style="height:20px;">Alexa Fluor 555 phalloidin</td>
			<td>Molecular Probes</td>
			<td>A34055</td>
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			<td height="20" style="height:20px;">Actin rhodamine</td>
			<td>Cytoskeleton. Inc</td>
			<td>APHR-A</td>
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			<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>
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			<td height="20" style="height:20px;">DABCO</td>
			<td>Sigma-Aldrich</td>
			<td>D-2522</td>
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			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1086</td>
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			<td height="20" style="height:20px;">&#946;-casein</td>
			<td>Sigma-Aldrich</td>
			<td>C6905</td>
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			<td height="20" style="height:20px;">Filter 0.22um</td>
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			<td>SL6V033RS</td>
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			<td height="20" style="height:20px;">Round 10cm dishes for cell culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>150350</td>
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			<td height="20" style="height:20px;">Plastic Pasteur pipette</td>
			<td>Assistent</td>
			<td>569/3 40569003</td>
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			<td height="20" style="height:20px;">15-ml polypropylen tube&#160;</td>
			<td>TPP</td>
			<td>91015</td>
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			<td height="20" style="height:20px;">Hypodermic Needle 22G Black 30mm&#160;</td>
			<td>BD Microlance</td>
			<td>300900</td>
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			<td height="20" style="height:20px;">Sterile Luer-slip 1ml Syringes without needle</td>
			<td>BD Plastipak</td>
			<td>300013</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
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			<td>2406</td>
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		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
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			<td>1000/1818</td>
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			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
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		<tr height="20">
			<td height="20" style="height:20px;">TLS-55 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>343778</td>
			<td></td>
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		<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>
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		<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>
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			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
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		<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>
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		<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>
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		</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 ...

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Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

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