Biz yoğun olan grup, daha sonra bize adapte oldu bir kayma motilite tayini için temel protokolü geliştirildi Jonathon Howard, borçlu. NSF hibe DMR0645023 mali destek minnetle kabul edilmektedir.

1) Tamponlar ve Reaktifler Bu çözümler, önceden hazırlanmış ve uygun büyüklükteki alikotları saklanmalıdır. Bir kısım tipik bir deney ve taze bir kısım her motilite tayini için kullanılması gereken yeterli bir çözüm bulunmalıdır. Saklama koşulları ve tipik kısım boyutları da aşağıdaki protokollerden söz edilmektedir. 1. BRB80 tampon, (80 mM BORU, 1 mM MgCl 2, 1 mM EGTA deiyonize distile (dd) su, KOH ile 6.9 pH değeri ayarlanmış) <ul><li> Gg su EGTA 0.5 M 100 mL stok çözelti olun. 2 M NaOH çözeltisi ile 7.0 pH ayarlayın. </li><li> 1 M MgCl 2 gg su 100 mL stok çözelti olun. Çözüm Otoklav. </li><li> 24,2 g BORU ve gg su yaklaşık 800 ml, 3,1 g KOH pelet ekleyin ve erimesi için karıştırın. 1 M KOH çözeltisi kullanılarak 6.9 pH ayarlayın. 0.5 M EGTA stok solüsyonu ve 1 mL 1 M MgCl 2 stok solüsyonu 2 ml ekleyin. Gg su ile hacmi 1000 ml &#39;ye kadar getirin. </li><li> Ileride kullanmak üzere -20 ° C&#39;de 50 ml şahin tüpleri ve dondurma haline kısım. BRB80 tüp şu anda kullanılmakta olan 4 ° C ya da oda sıcaklığında saklanabilir. </li></ul> 2. Magnezyum Klorür, MgCl 2 (100 mM gg su) <ul><li> 100 mM son bir konsantrasyon elde etmek için gg su sulandırınız. </li><li> Ileride kullanmak üzere -20 ° C&#39;de 0.5 mL mikrosantrifüj tüpleri ve mağaza içine kısım (10 mcL hacmi) </li></ul> 3. Guanozin-5&#39;-trifosfat, disodyum tuzu, GTP (25 mM NaOH tarafından 7 ayarlanabilir gg su, pH) <ul><li> Tartılır ve gg suda çözülür ve 2 M NaOH çözeltisi ile pH ayarlamak 7. </li><li> 260 nm dalga boyunda UV absorbans ölçerek konsantrasyon olun. (11.7 x 103 M -1 cm -1 bir sönüm katsayısı kullanın). </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (10 mcL hacim). </li></ul> 4. Dimetil sülfoksit, DMSO <ul><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (10 mcL hacim). </li></ul> 5. Taxol (DMSO 1 mM) <ul><li> Tartılır ve 1 mM son bir konsantrasyon elde etmek için davlumbaz altında DMSO içinde çözülür. </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 6. D-(+)-Glikoz (gg su 2 M) <ul><li> Tartılır ve 2 M. nihai bir konsantrasyon elde etmek için gg su içinde çözülür </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 7. Glikoz Oksidaz, (BRB80 içinde 2 mg / ml) <ul><li> 2 mg / ml son bir konsantrasyon elde etmek için BRB80 içinde eritin. </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 8. Dithiothreitol, DTT (1 M gg su) <ul><li> 1 M. nihai bir konsantrasyon elde etmek için, davlumbaz altında gg suda çözülür </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 9 - Katalaz, (BRB80 içinde 0.8 mg / ml) <ul><li> 0.8 mg / ml son bir konsantrasyon elde etmek için en az 2 kademeli BRB80 içinde eritin. UV absorbansı 276 nm ve 406 nm (276 nm de 3.1 x 10 5 M -1 cm -1 ve 2.2 x 5 10 M 406 nm&#39;de -1 cm -1 bir sönüm katsayısı kullanın ölçerek her aşamada konsantrasyonunu belirlemek ve denklemi A = cL). </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 10 Adenozin-5&#39;-trifosfat, ATP (100 mM MgCl 2 100 mM) <ul><li> Gg su içinde 100 mM MgCl 2 stok solüsyonu hazırlayın. Kuru toz tartılır ve 100 mM son bir konsantrasyon elde etmek için bu stok solüsyonu çözülür. </li><li> 260 nm dalga boyunda UV absorbans ölçerek konsantrasyon olun. (15,4 x 10 3 M -1 cm -1 bir sönüm katsayısı kullanın). </li><li> 0.5 mL mikrosantrifüj tüpleri ve gelecekte kullanılmak üzere -20 ° C&#39;de muhafaza içine kısım (20 mcL hacim). </li></ul> 11. Kazein çözeltisi (20 mg / ml kazein BRB80 içinde) <ul><li> Yaklaşık 3 g kazein, 30 ml, 50 ml şahin tüp dd su ekleyin. Vorteks çözüm yaklaşık 1 saat kadar kalın tutarlılık geliştirir. </li><li> 30 dakika boyunca yaklaşık 15000 gr santrifüjleyin. Filtre 0.5 mikron ve 0.2 mikron şırınga filtreler aracılığıyla süpernatantı. </li><li> UV absorbansı 280 nm (0.67 mL mg -1 cm -1 bir sönüm katsayısı kullanın) ölçerek süpernatantı konsantrasyonu belirleyin. 20 mg / ml BRB80 seyreltilir. </li><li> Kısım (20 mcL hacmi)0.5 mL mikrosantrifüj tüpler ve ileride kullanmak üzere -20 ° C&#39;de saklayın. </li></ul> 2) Standart Çözümleri Bu deney gün hazırlanır ve deney bittikten sonra atılmalıdır. Her 1 mL hazırlayın. 1. BRB80CS0.5 <ul><li> BRB80, buz üzerinde 0.5 mg / ml ve mağaza nihai bir konsantrasyonu kazein çözüm seyreltilir. Bu çözüm, önce kinesin akış hücresi içine giriliyor ve yüzey adsorpsiyon sonra kinesin aktivite tutmak yardımcı olur. </li></ul> 2. BRB80CA <ul><li> 0.2 mg / ml kazein ve 1 mM ATP BRB80 ve buz üzerinde mağaza hazırlayın. Kinesin daha önce bu çözüm akış hücresi içine giriş kullanarak seyreltilir. </li></ul> 3. BRB80T <ul><li> BRB80 oda sıcaklığında 10 mcM ve mağaza nihai konsantrasyonu taksol çözüm sulandırınız. Bu çözüm, mikrotübül stabilize etmek için kullanılır. </li></ul> 4. BRB80CT <ul><li> 10 mcM taksol ve BRB80 0.2 mg / ml kazein ve oda sıcaklığında saklayın hazırlayın. Bu da antifade ve mikrotübül çözümler hazırlamak için kullanılır. </li></ul> 5. BRB80AF <ul><li> 20 mM D-glukoz, 20 mikrogram / ml glukoz oksidaz, katalaz 8 mcg / mL, 10 mM DTT ve BRB80CT 20 mcM ATP ve oda sıcaklığında saklayın hazırlayın. Bu çözüm streptavidin ve nanokürecikler sulandırmak için kullanılan ve akış hücresi aşırı streptavidin &quot;yıkama&quot;. Kinesin hızı bu çözüm ATP konsantrasyonu ayarlayarak kontrol edilebilir. </li></ul> 3.) kinesin Hazırlık <ul><li> Express kinesin yabani tip, tam uzunlukta Drosophila melanogaster kinesin ağır zincir ve C-terminal Onun Escherichia coli-tag oluşan inşa ve 6 açıklandığı gibi bir Ni-NTA sütun kullanarak arındırmak. </li><li> -80 0,5 mL mikrosantrifüj tüpleri ve mağaza alikotları (10 mcL her) ° C ileride kullanmak üzere. Bu alikotları aktif kinesin konsantrasyonu yaklaşık 200 nM. 7 </li><li> Tipik bir deneme için, kinesin çözüm BRB80CA 20 kat sulandırmak. Buz üzerinde çözüm KIN20 ve mağaza etiketleyin. </li></ul> 4.) Mikrotubul Hazırlık <ul><li> 0,5 mL&#39;lik bir mikrosantrifüj tüp, 25 mcL büyüme çözüm hazırlamak: 4 mM MgCl 2, 1 mM GTP ve% 5 DMSO (v / v) BRB80 tampon. </li><li> 20 mg liyofilize biotinlenmiş tubulin bir kısım için bu çözüm 6.25 mcL ekleyin. </li><li> 37 ısı banyo Vortex, sonra yerde ° C ila 30 dakika polimerize. Yavaşça BRB80T ve vorteks 100 kat sulandırınız. Oda sıcaklığında çözüm MT100 ve mağaza etiketleyin. </li><li> BRB80AF MT100, 10 kat seyreltme olun. Çözüm MT1000 Etiket. </li></ul> 5) Streptavidin ve Nanokürecikli Çözüm BRB80AF 100 nM bir konsantrasyon AlexaFluor568-etiketli streptavidin hazırlayın. Buz üzerinde STV100 ve mağaza etiket. Aynı şekilde, BRB80AF çözüm nanokürecikler 5000 kat sulandırmak. Buz üzerinde NS5000 ve mağaza Etiket. 6.) Akış Hücre İnşaat Çift taraflı bant ile ayrılmış iki cam lamelleri kullanarak flowcell oluşturun. Bu akış hücresi, yaklaşık 2 cm uzunluğunda 1 cm genişliğinde ve 100 mm yüksekliğinde ve yaklaşık 20 mcL bir hacmi vardır. Çözümleri filtre kağıdı kullanarak diğer bir pipet ve kötülerin dışarı kullanarak, bir taraftan akış hücresi içine tanıtıldı. 7) Ters Testi Meclisi Cam yüzey ilk kinesin adsorpsiyon üzerine, onun işlevselliğini korumak sağlar kazein ile kaplanmıştır. Kinesin adsorbe sonra, mikrotübül kinesin tarafından tutuldukları tanıtıldı. Mikrotübüller sonra floresan streptavidin ile kaplıdır. Aşırı streptavidin yıkadıktan sonra, biotinlenmiş polistiren floresein nanokürecikler (40 nm çapında) tanıtıldı. Yüzey adsorbe sabit nanokürecikler hareketli mikrotübül ile çarpışır ve onları (Şekil 1) yüklenir. Sonraki çözüm getirilmesi önce izin çözümler ve zaman akış sırasına göre aşağıda listelenmiştir. <ul><li> BRB80CS0.5, 5 dakika </li><li> KIN20, 5 dakika </li><li> MT1000, 5 dakika </li><li> STV100, 5 dakika </li><li> BRB80AF, 3x </li><li> NS5000 </li></ul> 8) Mikroskopi Nanokürecikli giriş hemen sonra mikroskop sahnede akış hücresi monte edin. Bu deneyde, bir 100X petrol hedefi (NA 1.45), bir X-cite 120 lambası (EXFO, Ontario, Kanada) ve bir IXON EMCCD kamera (ANDOR, Eclipse TE2000-U floresan mikroskop (Nikon, Melville, NY) ile donatılmış South Windsor, CT) kullanılmıştır. FITC filtresi küp (# 48001) ve TRITC filtre küp (# 48002, Chroma Teknolojileri, Rockingham, VT), görüntü nanokürecikler ve mikrotübül respecti için kullanılır.vely alt yüzey akış hücreleri. Maruz kalma arasındaki süre 2 sn iken maruz kalma süresi, 0.2s <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="Şekil 1" /> Şekil 1. Moleküler mekik şematik çizim.

<ol>
	<li>Agarwal, A., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science.">Biomolecular motors at the intersection of nanotechnology and polymer science.</a> Progress in Polymer Science. 35 (1-2), 252-252 (2010).</li><li>Howard, J., Hunt, A. J., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+microtubule+movement+driven+by+single+kinesin+molecules.">Assay of microtubule movement driven by single kinesin molecules.</a> Methods Cell Biol. 39, 137-137 (1993).</li><li>Agarwal, A., Katira, P., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles.">Millisecond curing time of a molecular adhesive causes velocity-dependent cargo-loading of molecular shuttles.</a> Nano Lett. 9 (3), 1170-1170 (2009).</li><li>Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins.">Stretching and Transporting DNA Molecules Using Motor Proteins.</a> Nano Lett. 3 (9), 1251-1251 (2003).</li><li>Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J., Liu, J., Bunker, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins.">Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.</a> Nano Lett. 4 (5), 817-817 (2004).</li><li>Coy, D. L., Wagenbach, M., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes.">Kinesin takes one 8-nm step for each ATP that it hydrolyzes.</a> J. Biol. Chem. 274 (6), 3667-3667 (1999).</li><li>Katira, P., Agarwal, A., Fischer, T., Chen, H. -. Y., Jiang, X., Lahann, J., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes.">Quantifying the performance of protein-resisting surfaces at ultra-low protein coverages using kinesin motor proteins as probes.</a> Advanced Materials. 19, 3171-3171 (2007).</li><li>Vigers, G. P. A., Coue, M., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Microtubules+Break+Up+Under+Illumination.">Fluorescent Microtubules Break Up Under Illumination.</a> J. Cell Biol. 107, 1011-1011 (1988).</li><li>Brunner, C., Hess, H., Ernst, K. -. H., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+of+biomolecules+in+hybrid+nanodevices.">Lifetime of biomolecules in hybrid nanodevices.</a> Nanotechnology. 15 (10), S540-S540 (2004).</li></ol>

Küçük değişiklikler ile bu protokolü kinesin-mikrotübül tabanlı motilite testleri başarıyla monte etmek için çeşitli gruplar tarafından kullanılan olmuştur. Son motilite çözüm 10 mM DTT,% 0.5 &#39;β-mercaptoethanol ile değiştirilebilir. 2 saatten fazla standart çözümler (BRB80AF KIN20 ve MT1000) olmamalıdır. Taksol ve özellikle mikrotübül içeren herhangi bir çözüm, buz üzerinde atÛlmamalÛdÛr. Işlevsel bileşenleri fotohasar UV uyarma ışık akışını hücrenin aşırı maruz ka...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
<tr>
<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
<tr>
<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
<tr>
<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol (DTT)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0610</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E-4378</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-8766</td>
<td>&nbsp;</td>
<tr>
<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7016</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-triphosphate (GTP)</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>106399</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
<tr>
<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
<tr>
<td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#x2032;-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Potassium hydroxide (KOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6310</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>480878</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin Alexa Fluor 568 conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11226</td>
<td>&nbsp;</td>		</tbody>
		</table>
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cargo loading onto kinesin powered molecular shuttles

Hücreler kinesin motor proteinlerin ve mikrotübül filamentler gibi aktif hücre içi kargo taşımacılığı desteklemek için, gelişmiş moleküler makineler gelişmiştir. Kinesins kuyruk etki alanı yüklerin çeşitli bağlar iken, kinesins baş etki alanı mikrotübül kafes boyunca adım ATP molekülleri depolanan kimyasal enerjiyi kullanır. Uzun, sert mikrotübül, uzun mesafe hücre içi ulaşım için parça olarak hizmet vermektedir. Bu motorlar ve filamentler, aynı zamanda moleküler mekik 1 bileşenleri olarak microfabricated sentetik ortamlarda istihdam edilebilir. Sık kullanılan bir tasarım, kinesin motorlar kendi kuyrukları ile parça yüzey demirlemiş ve Fonksiyonlu mikrotübüllerin bu motorlar ile hareketli yük taşıyan unsurları, olarak hizmet vermektedir. Bu mekik, biotin ve streptavidin arasında güçlü ve seçici bir bağlayıcılığı kullanan kargo ile yüklenebilir. Anahtar bileşenleri (biotinlenmiş tubulin, streptavidin ve biotinlenmiş kargo) ticari olarak kullanılabilir. Klasik ters motilite tayini 2 Bina, inşaat moleküler mekiklerinin burada ayrıntılı. Kinesin Motor proteinleri kazein ile kaplanmış bir yüzeye adsorbe; mikrotübül, biotinlenmiş tubulin polimerize kinesin yapıştırılır ve daha sonra rodamin etiketli streptavidin ile kaplı. ATP konsantrasyonu kargo 3 yükleme için optimum bir mikrotübül kayma hızı elde etmek için subsaturating konsantrasyon tutulur. Son olarak, biotinlenmiş floresein etiketli nanokürecikler kargo olarak eklenir. Nanokürecikler kayma yüzeyine yapışan mikrotübül ve nanokürecikler arasındaki çarpışmalar sonucu olarak mikrotübül eklemek. Protokol biotinlenmiş DNA 4, kuantum noktaları 5 veya biotinlenmiş antikorlar 4-6 aracılığıyla antijenleri geniş bir yelpazede çeşitli yüklerin kolayca yüklemek için modifiye edilebilir.

Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

Cargo Loading onto Kinesin Powered Molecular Shuttles

Fonksiyonlu mikrotübül üzerinde kayma yüzeyi yapıştırılır kinesin motorlu proteinlerinin nano ölçekli bir taşıma sistemi olarak hizmet verebilir oluşan Moleküler mekik. Burada, tipik bir servis sistemi montaj açıklanmıştır.

Kinesin Powered Moleküler minibüsleri üzerine Kargo Yükleniyor

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

cargo-loading-onto-kinesin-powered-molecular-shuttles

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Biology

10.4K Görüntüleme.  University of Florida. Fonksiyonlu mikrotübül üzerinde kayma yüzeyi yapıştırılır kinesin motorlu proteinlerinin nano ölçekli bir taşıma sistemi olarak hizmet verebilir oluşan Moleküler mekik. Burada, tipik bir servis sistemi montaj açıklanmıştır.

Video: Cargo Loading onto Kinesin Powered Molecular Shuttles

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Tendon ex vivo Mekanik Yükleniyor

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Biyoloji

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Kalsiyum Göstergeleri ile yükleme Drosophila Sinir Terminalleri

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Tre Recombinase Moleküler Evrim

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

MEFS Matrigel 2 Matrigel üzerine MEFS Yarma hESC:

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Matrigel MEFS Matrigel 3: Matrigel üzerine gelen Pasajlanması hESC

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Kurul, Yükleme ve Analitik ultrasantrifüjdeki Örnek Hücre Hizalama

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin İzolasyonu ve Saflaştırılması gelen Drosophila Embriyolar

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Hedefli-esteraz ile ER Kalsiyum Doğrudan Görüntüleme Kaynaklı Boya Yükleme (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Bir &quot;humanize&quot; oluşturuluyor<em&gt; Drosophila</emKinesin-5 Farmakolojik inhibisyonu için&gt; S2 Hücre Hattı Duyarlı

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Floresan mikroskobu kullanarak kinesin-1 Cargos Tanımlanması

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...