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

得到瑞士国家科学基金会的支持;瑞士 Sinergia 计划;波兰-瑞士研究方案 (PSPB-094/2010);化学生物学中的 NCCR;LipidX 来自瑞士 SystemsX.ch 倡议, 由瑞士国家科学基金会 (j.g) 评估。o.m. 得到了 EMBO 长期研究金 (ALTF-516-2012) 的支持。

1. 解决方案和准备工作 注意: 所有的缓冲区和解决方案都应在双 (dd) H 2 O 中准备。由于蔗糖的水合状态不同, 所有蔗糖溶液的最终浓度必须用折射仪来确定. 
<ol> <li> 准备不含价阳离子的磷酸盐缓冲盐水 (PBS): 137 毫米盐、2.7 毫米氯化钾、1.5 mm 2 PO 4 和 6.5 mm Na 2 HPO 4 。调整 pH 值为7.4。灭菌 (1 循环在121和 #176; C 15 分钟) 和室温下?...

<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>

肌动蛋白在 endosome 膜动力学中起关键作用4,14。我们以前报道, 肌动蛋白成核和聚合发生在早期体, 形成小的 f-肌动蛋白补丁或网络。这些 f-肌动蛋白网络是绝对需要的膜运输超越早期体沿降解途径。干扰此成核和聚合过程的任何步骤, 防止 endosome 成熟, 从而对后期的体 andlysosomes9,11,15...

在更高的真核细胞中, 蛋白质和脂质被内化为早期的体, 在那里进行分类。一些蛋白质和脂质, 这是注定要利用, 被纳入到管状区域的早期体, 然后运到等离子膜或转移到高尔基体网络 (TGN)1,2。相比之下, 其他的蛋白质和脂质被选择性地包装到早期体的区域, multivesicular 出现。这些地区扩展, 并在脱离早期内涵膜, 最终成熟成自由内涵载体囊泡或 multivesicular 体 (ECV/MVBs), 负责货物运输对晚体1, 2。
肌动蛋白在与内涵分选能力和 endosome 生物相关的膜重塑过程中起着至关重要的作用。蛋白质的分类沿着回收途径的等离子膜或 TGN 取决于 retromer 复合物和相关的蛋白质。这种分拣机械似乎是耦合到回收小管通过相互作用的 retromer 复合体, 与黄蜂和疤痕同系物 (洗涤) 复杂和分支肌动蛋白3,4,5.相反, 注定要降解的分子, 特别是活化的信号受体, 被分类成腔内囊泡 (ILVs) 的内涵分拣复合物所需的运输 (ESCRT)2,6, 7。尽管肌动蛋白在 ESCRT 依赖性分选过程中可能扮演的角色尚不清楚, 但 F-肌动蛋白在 ECV/MVBs 的生物和早期体的运输中起着重要作用。特别是, 我们发现蛋白 A2 结合胆固醇丰富地区早期 endosome, 并与 spire1, 核 F-肌动蛋白聚合。在体上观察到的分支肌动蛋白网络的形成, 需要蛋白 (ARP) 2/3 复合体的分支活性, 以及蛋白质 moesin 和肌动蛋白结合蛋白 cortactin8,9。
在这里, 我们描述了一个 microscopy-based 的体外测定, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管。该方法已被用于研究蛋白 A2 在 f-肌动蛋白成核和 moesin 和 cortactin 形成内涵肌动蛋白网络中的作用8,9。通过这种体外协议, 在肌动蛋白聚合过程中体发生的一系列复杂反应, 可以对过程的顺序步骤进行生物化学和分子分析, 包括肌动蛋白成核、线性聚合、分枝和交联。

<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

许多早期的 endosome 功能, 特别是货物蛋白分选和膜变形, 依赖于内涵膜上有核的短的 f-肌动蛋白纤维的补丁。我们已经建立了一个 microscopy-based 的体外检测, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管, 从而呈现这一系列复杂的反应, 可遗传和生物化学操作.从表达早期内涵蛋白 GFP-RAB5 的细胞中浮选蔗糖梯度制备内涵分数。胞浆组分是由分离的细胞分批制备的。如果需要的话, 内涵和胞浆组分都可以在液氮中冷冻保存。在试验中, 内涵和胞浆组分混合, 在适当的条件下 (例如,离子强度, 还原环境) 在37° c 下孵育混合物。在所需的时间, 反应混合物是固定的, 和 f-肌动蛋白是揭示与笔。然后用荧光显微镜对肌动蛋白成核和聚合进行分析。在这里, 我们报告, 这种方法可以用来研究的作用, 涉及的因素, 在膜上的肌动蛋白成核, 或在随后的伸长, 分支, 或交联的 f-肌动蛋白丝。

为了深入了解早期 endosome 膜上的 F 肌动蛋白补丁的形成, 我们遵循了图 2中概述的协议。简单地, 细胞被转染与 GFP-RAB5, 然后早期体是由亚单位分离制备的。这些纯化早期体的培养与胞, 以提供肌动蛋白本身以及其他因素可能涉及的反应。在潜伏期结束时, 通过固定的混合物停止反应。然后采集样品并在显微镜下沉积。最后, 笔 (图 3A</str...

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

早期 endosome 功能依赖于 F-肌动蛋白聚合。在这里, 我们描述了一个 microscopy-based 的体外测定, 每天的核化和聚合的 f-肌动蛋白在早期内涵膜的试管, 从而呈现这一系列复杂的反应, 可生化和遗传操作.

体外F-肌动蛋白在早期体的聚合

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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In Vitro Polymerization of F-actin on Early Endosomes

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The Cytoskeleton I: Actin and Microfilaments

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