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

Destek İsviçre Ulusal Bilim Vakfı'ndan aldığı; İsviçre Sinergia programı; Lehçe-İsviçre araştırma programı (2010/PSPB-094); NCCR Kimya Biyoloji; ve LipidX İsviçre SystemsX.ch girişimi üzerinden değerlendirilir (J. G. için) İsviçre Ulusal Bilim Vakfı tarafından. O. M. bir EMBO uzun vadeli Bursu (ALTF-516-2012) tarafından desteklenmiştir.

1. çözümleri ve ürünleri Not: tüm arabellekleri ve çözümleri (dd) H çift distile 2 ' O. hazırlanmalıdır Sükroz hidrasyon durumu değiştiğinden, tüm Sükroz çözümler son konsantrasyonu bir Refraktometre kullanarak belirlenmelidir.
<ol><li>Hazırla fosfat tamponlu tuz olmadan divalent katyonlar (PBS-): 137 mM NaCI, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ve 6.5 mM Na 2 HPO 4. PH 7.4 için ayarlayın. Isıyla (15dk için 121 ° C...

<ol>
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Aktin, endosome membran dynamics4,14' te önemli bir rol oynar. Biz daha önce aktin çekirdekleşme ve polimerizasyon erken endosomes üzerinde oluşan küçük F-aktin yamalar veya ağları kurma bildirdi. Bu F-aktin ağlar membran taşıma düşmesine yol boyunca erken endosomes ötesinde için kesinlikle gereklidir. Bu çekirdekleşme ve polimerizasyon sürecinin herhangi bir aşamasında müdahale endosome olgunlaşması ve böylece geç endosomes andlysosom...

Daha yüksek ökaryotik hücrelerde, proteinler ve yağlar sıralama oluştuğu erken endosomes içselleştirilmiş. Bazı proteinler ve yağlar, reutilized kaderinde vardır, erken endosomes borulu bölgelere dahil ve plazma zarı veya trans-Golgi ağ (TGN)1,2nakletti. Buna karşılık, diğer proteinler ve yağlar multivesicular bir görünüm sergileyen erken endosomes bölgelere seçmeli olarak paketlenir. Bu bölgeleri genişler ve erken endosomal membran üzerinden dekolmanı sonunda kart ücretsiz endosomal taşıyıcı veziküller veya multivesicular organları (ECV/MVBs), hangi kargo taşımacılığı geç endosomes1, karşı sorumlu değil 2.
Aktin endosomal sıralama kapasite ve endosome Biyogenez ile ilişkili membran tadilat sürecinde önemli bir rol oynar. Plazma zarı veya TGN geri dönüşüm yolları sıralama protein retromer karmaşık ve ilişkili proteinler bağlıdır. Bu sıralama makine oluşumu geri dönüşüm tübüllerin yolu ile etkileşimleri karmaşık, retromer, eşek ARISI ve yara izi homologue (yıkama) karmaşık ve dallı aktin3,4,5 ile birleştiğinde olabilir gibi görünüyor . Buna ek olarak, moleküller bozulması için mukadder, özellikle sinyal reseptörleri aktive, intraluminal veziküller (ILVs) taşıma (ESCRT)2,6içingerekli kompleksleri sıralama endosomal göre sıralanır, 7. Aktin ESCRT bağlı sıralama sürecinde olası rolü bilinen değil iken, F-aktin ECV/MVBs Biyogenez ve taşıma erken endosomes ötesinde önemli bir rol oynar. Özellikle, A2 annexin kolesterol zenginleştirilmiş bölgeler erken endosome ve spire1 ile birlikte bağlar, F-aktin polimerizasyon nucleates bulduk. Endosomes üzerinde gözlenen dallı aktin ağ oluşumu aktin ile ilgili protein (ARP) 2/3 dallanma etkinliğin gerektirdiği kompleksi, hem de ERM protein moesin ve aktin bağlayıcı protein cortactin8,9.
Burada, biz çekirdekleşme ve F-aktin polimerizasyon test tüpleri erken endosomal membranlar üzerinde reconstitutes mikroskobu tabanlı vitro tahlil açıklayın. Bu tahlil A2 F-aktin çekirdekleşme içinde annexin ve moesin ve cortactin endosomal aktin ağları8,9oluşumunda rol araştırmak için daha önce kullanılmış. Bu vitro protokolü ile endosomes aktin polimerizasyon sırasında ortaya çıkan tepkiler karmaşık dizi aktin çekirdekleşme, doğrusal bir işlemi, sıralı adımları biyokimyasal ve moleküler analiz mükellef haline polimerizasyon, dallanma ve çapraz.

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			<td height="20" style="height:20px;width:477px;">NaCl</td>
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			<td style="width:145px;">71380</td>
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			<td height="20" style="height:20px;">KCl</td>
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			<td>196770010</td>
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			<td height="20" style="height:20px;">KH2PO4</td>
			<td>AppliChem</td>
			<td>A1042</td>
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			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Acros Organics</td>
			<td>424370025</td>
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			<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>
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			<td>63047</td>
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			<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>
			<td></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>
			<td></td>
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			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Roche</td>
			<td>10236624001</td>
			<td></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>
			<td></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>
			<td></td>
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		<tr height="20">
			<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>
			<td>Millex&#160;</td>
			<td>SL6V033RS</td>
			<td></td>
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		<tr height="20">
			<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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		<tr height="20">
			<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>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Micro glass slides&#160;</td>
			<td>Assistent</td>
			<td>2406</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">18X18-mm glass coverslip</td>
			<td>Assistent</td>
			<td>1000/1818</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">SW60 centrifuge tube</td>
			<td>Beckman coulter</td>
			<td>344062</td>
			<td></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>
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		<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>
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		<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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			<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Refractometer</td>
			<td>Carl Zeiss</td>
			<td>79729</td>
			<td></td>
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		<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>
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		<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

Birçok erken endosome işlev, özellikle kargo protein sıralama ve membran deformasyon, endosomal membran parçalanmayabilir kısa F-aktin filamentleri yamalari bağlıdır. Biz böylece reaksiyonlar karmaşık bu dizi genetik ve biyokimyasal mükellef işleme çekirdekleşme ve F-aktin polimerizasyon test tüpleri, erken endosomal membranlar üzerinde reconstitutes mikroskobu tabanlı vitro tahlil kurduk manipülasyonlar. Endosomal kesirler flotasyon erken endosomal protein GFP-RAB5 ifade hücrelerden Sükroz Degradelerde tarafından hazırlanır. Sitozolik kesirler hücreler ayrı gruplar halinde hazırlanır. Hem endosomal hem sitozolik kesirler sıvı azot içinde donmuş gerekirse depolanabilir. Tahlil, endosomal ve sitozolik kesirler karıştırılır ve karışım uygun koşullarda (çevre azaltılmasıÖrneğin, iyonik gücü,) 37 ° C'de inkübe. İstenen zamanda tepki karışımı sabit ve F-aktin phalloidin ile ortaya çıkar. Aktin çekirdekleşme ve polimerizasyon Floresans mikroskobu tarafından incelenir. Burada, bu tahlil ya aktin çekirdekleşme membran veya sonraki uzama, dallanma veya F-aktin filamentleri polietilenin söz konusu faktörler rol araştırmak için kullanılabilir raporu.

F-aktin yamalar üzerinde erken endosome membran oluşumu anlayışlar kazanmak için Şekil 2' de belirtilen iletişim kuralı takip ettik. Kısaca, hücreleri GFP-RAB5 ile transfected ve o zaman erken endosomes hücre altı ayırma tarafından hazırlanmıştır. Bu saf erken endosomes kendisi gibi diğer faktörler tepki muhtemelen katılan aktin sağlamak sitozol ile inkübe. Kuluçka dönemi sonunda, reaksiyon karışımı fiksasyonu tarafından durdurul...

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

Erken endosome fonksiyonları F-aktin polimerizasyon üzerinde bağlıdır. Burada, böylece reaksiyonlar karmaşık bu dizi biyokimyasal ve genetik mükellef işleme çekirdekleşme ve F-aktin polimerizasyon test tüpleri, erken endosomal membranlar üzerinde reconstitutes mikroskobu tabanlı vitro tahlil açıklamak manipülasyonlar.

Vitro F-aktin üzerinde erken Endosomes polimerizasyon

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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