우리는 그의 그룹 이후 회사에 의해 조정되었다 글라이딩 운동성 분석을위한 기본 프로토콜을 개발 조나단 하워드에 크게 빚을 수 있습니다. NSF 그랜트 DMR0645023의 재정 지원은 기꺼이 인정받고 있습니다.

1.) 버퍼 및 시약 이러한 솔루션은 사전에 준비하고 편리하게 크기 aliquots에 보관해야합니다. 나누어지는는 전형적인 실험과 신선한 나누어지는 각 운동성 분석을 위해 사용되어야에 대한 충분한 해결책을 포함해야합니다. 저장 조건 및 전형 나누어지는 크기는 다음과 같은 프로토콜에 언급하고 있습니다. 1. BRB80 버퍼 (80 MM 파이프, 1 MM MgCl 2, deionized 소주 1 MM EGTA (DD) 물, 코에서 6.9로 조정 PH) <ul><li> DD 물에 0.5 M EGTA의 100 ML 주식 솔루션을 확인하십시오. 2 M NaOH 용액을 사용하여 7.0으로 산도를 조정합니다. </li><li> DD 물 1 M MgCl 2 100 ML 주식 솔루션을 확인하십시오. 솔루션을 압력솥. </li><li> 파이프와 DD의 물을 약 800 ML에 3.1 g 코 알약의 24.2 g을 추가하고 디졸브로 저어. 1 M 코 솔루션을 사용하여 6.9으로 산도를 조정합니다. 0.5 M EGTA 주식 솔루션과 1 M MgCl 2 주식 솔루션 1 ML 2 ML을 추가합니다. DD 물로 볼륨 1000 ML로 가져 오십시오. </li><li> 나중에 사용하기 위해 -20 ° C 50 ML 팔콘 튜브 및 동결로 나누어지는. BRB80 튜브 현재 사용중인 4 ° C 또는 상온에서 저장할 수 있습니다. </li></ul> 2. 마그네슘 염화물 MgCl 2 (DD 물을 100 ㎜) <ul><li> 100 mm의 최종 농도를 달성하기위한 DD의 물에 희석. </li><li> 나중에 사용하기 위해 -20 ° C에서 0.5 ML microcentrifuge 튜브 및 저장소로 나누어지는 (10 μL 볼륨) </li></ul> 3. 구아 노신 5&#39; - 삼인산 -, 나트륨 소금, GTP (NaOH에 의해 7 조정 DD 물, pH를 25 ㎜) <ul><li> 밖으로 무게와 DD 물에 용해 2 M NaOH 용액에 의해 7 산도를 조정합니다. </li><li> 260 nm의 UV 흡광도에서 측정하여 농도를 확인합니다. (11.7 X 103 M -1 cm -1의 흡광 계수를 사용하여). </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (10 μL 볼륨). </li></ul> 4. 디메틸 sulfoxide, DMSO <ul><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (10 μL 볼륨). </li></ul> 5. Taxol (DMSO 1 ㎜) <ul><li> 밖으로 무게 1 mm의 최종 농도를 달성하기 퓸 후드 아래에 DMSO에 용해. </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 6. D - (+) - 포도당 (DD 물 2 M) <ul><li> 밖으로 무게 2 M.의 최종 농도를 달성하기위한 DD의 물에 용해 </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 7. 포도당 산화 효소 (BRB80 2 MG / ML) <ul><li> 2 MG / ML의 최종 농도를 달성하기 위해 BRB80에 디졸브. </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 8. Dithiothreitol, DTT (DD 물 1 M) <ul><li> 1 M.의 최종 농도를 달성하기 퓸 후드 아래에 DD 물에 용해 </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 9. 카탈 라 아제 (BRB80의 0.8 MG / ML) <ul><li> 0.8 MG / ML의 최종 농도를 달성하기위한 최소한 2 단계에서 BRB80에 용해. 276 NM 및 406 nm의 (276 NM에서 3.1 X 10 5 M -1 cm -1 및 2.2 X 10 5 M -1 cm 406 nm의에서 -1의 흡광 계수를 사용하여 UV 흡광도에서 측정하여 각 단계에서 농도를 확인하고 방정식 A = CL). </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 10. 아데노신 5&#39; - 삼인산 -, ATP (100mM MgCl 2 100 ㎜) <ul><li> DD 물 100 MM MgCl 2 주식 솔루션을 준비합니다. 건조 분말을 무게와 100mM의 최종 농도를 달성하기위한이 주식 솔루션 디졸브. </li><li> 260 nm의 UV 흡광도에서 측정하여 농도를 확인합니다. (15.4 × 10 3 M -1 cm -1의 흡광 계수를 사용하여). </li><li> 0.5 ML microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장소로 나누어지는 (20 μL 볼륨). </li></ul> 11. 카제인 용액 (BRB80 20 MG / ML 카제인) <ul><li> 50 ML 팔콘 튜브 30 ML의 DD 물을 약 3g 카제인을 추가합니다. 약 1 시간 동안 소용돌이 솔루션까지 두꺼운 일관성을 개발하고 있습니다. </li><li> 30 분 정도 1만5천그램에서 원심 분리기. 0.5 μm의 및 0.2 μm의 주사기 필터를 통해 뜨는을 필터링합니다. </li><li> 280 NM (0.67 ML MG -1 cm -1의 흡광 계수를 사용)에서 UV 흡광도를 측정하여 표면에 뜨는의 농도를 결정합니다. 20 BRB80의 MG / ML로 희석. </li><li> 나누어지는 (20 μL 볼륨)에0.5 ML의 microcentrifuge 튜브 및 향후 사용을 위해 -20 ° C에서 저장하는. </li></ul> 2.) 표준 솔루션 이들은 실험 당일 준비하고 실험이 끝난 후 폐기하여야한다. 각 1 ML을 준비합니다. 1. BRB80CS0.5 <ul><li> BRB80 얼음 이상 0.5 MG / ML 및 저장소의 최종 농도 카제인 솔루션을 희석. 이 솔루션은 이전 kinesin에 흐름 세포에 도입하여 표면에 흡착 후 kinesin 활동을 유지하는 데 도움이됩니다. </li></ul> 2. BRB80CA <ul><li> BRB80 얼음 이상의 저장소에있는 0.2 MG / ML 카제인 1 MM ATP를 준비합니다. Kinesin은 더욱 흐름 세포에 도입하기 전에이 솔루션을 사용하여 희석합니다. </li></ul> 3. BRB80T <ul><li> BRB80에 상온에서 10 μm의 및 저장소의 최종 농도 taxol 솔루션을 희석. 이 솔루션은 microtubules을 안정화하는 데 사용됩니다. </li></ul> 4. BRB80CT <ul><li> 10 μm의의 taxol과 BRB80 0.2 MG / ML 카제인과 실온에서 저장을 준비합니다. 이것은 더욱 antifade와 미세 소관 솔루션을 준비하는 데 사용됩니다. </li></ul> 5. BRB80AF <ul><li> 20 MM D - 글루코오스, 20 μg / ML 포도당 산화 효소, 카탈 라 아제 8 μg / ML, 10 MM의 DTT, 그리고 BRB80CT 20 μm의 ATP와 실온에서 저장을 준비합니다. 이 솔루션은 streptavidin과 nanospheres을 희석하기 위해 사용되며 흐름 세포에있는 여분의 streptavidin &quot;를 씻어&quot;입니다. kinesin 속도는이 솔루션의 ATP 농도를 조절하여 제어할 수 있습니다. </li></ul> 3.) Kinesin 준비 <ul><li> 익스프레스 kinesin는 야생 형, 전체 길이 Drosophila melanogaster kinesin 중쇄 및 C - 터미널 대장균에서 그의 태그 구성된를 구축 6에서 설명한 니켈 NTA 칼럼을 사용하여 정화. </li><li> -80에서 0.5 ML의 microcentrifuge 튜브 및 저장소에 aliquots (10 μL 각)를 확인하면 ° C 나중에 사용하기 위해. 이러한 aliquots에 적극적으로 kinesin의 농도는 약 200 nm의 수 있습니다. 7 </li><li> 전형적인 실험은 kinesin 솔루션에게 BRB80CA 20 배 희석. 얼음을 통해 솔루션 KIN20하고 저장을 라벨. </li></ul> 4.) 미세 소관 준비 <ul><li> 0.5 ML의 microcentrifuge 관에서 성장 솔루션을 25 μL를 준비 : 4 MM에게 MgCl 2, 1 MM GTP와 5% DMSO (V / V)를 BRB80 버퍼 인치 </li><li> 동결 건조된 biotinylated tubulin의 20 μg의 나누어지는이 솔루션의 6.25 μL를 추가합니다. </li><li> 37 열 욕탕에 소용돌이 다음 장소 ° 중합 30 분 C. 부드럽게 BRB80T과 와동 100 배 희석. 상온에서 솔루션 MT100하고 저장을 라벨. </li><li> BRB80AF에 MT100의 10 배 희석하십시오. 솔루션 MT1000을 라벨. </li></ul> 5.) Streptavidin 및 Nanosphere 솔루션 BRB80AF 100 NM의 농도에 AlexaFluor568 - 라벨 streptavidin을 준비합니다. 얼음 위에는 STV100 및 저장 라벨. 마찬가지로, BRB80AF 솔루션 nanospheres 5,000 배 희석. 얼음 통해 NS5000하고 저장을 라벨. 6.) 플로우 셀 건설 양면 테이프로 분리된 두개의 유리 coverslips를 사용하여 flowcell을 구성. 이 흐름 세포는 1cm 폭 및 100 μm의 높이 약 2cm 길이이며, 약 20 μL의 볼륨을했다. 솔루션은 종이 필터를 사용하여 다른에서 피펫과 악한 아웃를 사용하여 한쪽의 흐름 세포에 도입하고 있습니다. 7.) 인버티드 분석 어셈블리 유리 표면 처음 kinesin가 흡착시 그 기능을 유지할 수 카제인과 코팅입니다. kinesin가 adsorbed되면 microtubules은 kinesin에 의해 개최되는, 소개하고 있습니다. Microtubules 그때 형광 streptavidin과 함께 코팅하고 있습니다. 초과 streptavidin을 세척 후 biotinylated 폴리스티렌 플루오레신 nanospheres (40 나노미터 직경)을 소개합니다. 표면 adsorbed 고정 nanospheres은 이동 microtubules과 충돌하고 (그림 1)에로드하십시오. 다음 솔루션의 도입 전에 허용되는 솔루션과 시간의 흐름의 순서는 다음과 같습니다. <ul><li> BRB80CS0.5 5 분 </li><li> KIN20 5 분 </li><li> MT1000 5 분 </li><li> STV100 5 분 </li><li> BRB80AF, 배 </li><li> NS5000 </li></ul> 8.) 현미경 즉시 nanosphere 소개 후 현미경 단계에있는 흐름 전지를 탑재합니다. 본 실험에서는 이클립스 TE2000 - U 형광 현미경 (니콘, 멜빌, NY) 100X 오일 목표 (NA 1.45), X - 인용 120 램프 (EXFO, 온타리오, 캐나다)과 iXon EMCCD 카메라 (ANDOR을 갖춘 사우스 윈저, CT)가 사용되었다. FITC 필터 큐브 (# 48001)과 TRITC 필터 큐브는 (# 48002, 채도 테크놀로지, 로킹엄, VT) 이미지 nanospheres와 microtubules respecti하는 데 사용되었다vely 흐름 세포의 바닥 표면에. 노출 사이의 시간 2 S. 동안 노출 시간, 0.2s되었습니다 <img src="/files/ftp_upload/2006/2006fig1.jpg" alt="그림 1" /> 그림 1. 분자 셔틀의 개략도 그림.

<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>

관심 없음 충돌 선언하지 않습니다.

사소한 수정이 프로토콜은 성공적으로 kinesin - 미세 소관 운동성 기반의 assays를 조립하는 그룹의 다양한 의해 사용되고 있습니다. 최종 운동성 솔루션 10 MM의 DTT는 0.5 % β - 메르 캅 토 에탄올로 대체 수 있습니다. 2 시간 이상 된 표준 솔루션 (BRB80AF, KIN20 및 MT1000) 사용해서는 안됩니다. 특히 taxol과 microtubules를 포함한 모든 솔루션은 얼음에 배치해서는 안됩니다. 기능 구성 요소 photodamage의 UV 여기 ?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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>
		</div>

cargo loading onto kinesin powered molecular shuttles

세포는화물의 적극적인 세포내 수송을 지원하기 위해, 이러한 kinesin 모터 단백질 및 미세 소관의 필라멘트로 복잡한 분자 기계를 발전시켜 왔습니다. kinesins 꼬리 도메인화물의 다양한 바인딩 있지만, kinesins 머리 도메인은 미세 소관을 따라 격자 단계로 ATP 분자에 저장된 화학 에너지를 활용. 길고 뻣뻣한 microtubules는 장거리 세포내 전송을위한 트랙으로 제공하고 있습니다. 이러한 모터 및 필라멘트는 분자 셔틀 하나의 구성 요소로서 microfabricated 합성 환경에서 근무하실 수 있습니다. 자주 사용되는 디자인, kinesin 모터 자신의 꼬리를 통해 트랙 표면에 고정하고, 기능화 microtubules이 모터에 의해 추진 아르화물 운송 요소로 제공하고 있습니다. 이 셔틀은 비오틴과 streptavidin 사이의 강력하고 선택 바인딩을 이용하여화물로 로드할 수 있습니다. 주요 구성 요소 (biotinylated tubulin, streptavidin과 biotinylated화물) 상업적으로 사용할 수 있습니다. 고전 거꾸로 운동성 분석 2 빌딩 분자 셔틀 건설 여기서 자세한 내용입니다. Kinesin 모터 단백질은 카제인과 precoated 표면에 adsorbed되며 microtubules는 biotinylated tubulin에서 polymerized kinesin을 준수하고 이후 rhodamine - 라벨 streptavidin과 함께 코팅하고 있습니다. ATP 농도는화물 3 로딩을위한 최적의 미세 소관 글라이딩 속도를 달성하는 subsaturating 농도에서 관리하고 있습니다. 마지막으로, 플루오레신 - 표시 nanospheres은화물로 추가됩니다 biotinylated. Nanospheres은 표면에 준수 글라이딩 microtubules와 nanospheres 사이의 충돌의 결과로 microtubules을 첨부합니다. 프로토콜은 쉽게 이러한 biotinylated DNA 4, 양자 점 5 biotinylated 항체 4-6 통해 항원의 다양한으로화물의 다양한 로드할 수 수정할 수 있습니다.

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

Cargo Loading onto Kinesin Powered Molecular Shuttles

표면 준수 kinesin 모터 단백질이 nanoscale 교통 시스템으로 제공할 수에 글라이딩 기능화 microtubules로 구성된 분자 셔틀. 여기 전형적인 셔틀 시스템의 조립이 설명되어 있습니다.

Kinesin 전원 분자 셔틀로화물 로딩

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

Research

JoVE Journal

Biology

10.4K 조회수.  University of Florida. 표면 준수 kinesin 모터 단백질이 nanoscale 교통 시스템으로 제공할 수에 글라이딩 기능화 microtubules로 구성된 분자 셔틀. 여기 전형적인 셔틀 시스템의 조립이 설명되어 있습니다.

비디오: 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.

텐든의 전 생체내 기계 중

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

연구

생물학

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.

칼슘 지표로 로딩 Drosophila 신경 터미널

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.

트레 Recombinase의 분자 진화

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.

Matrigel에 MEFs에서 분리 hESCs : MEFs에서 Matrigel 2

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.

MEFs에서 Matrigel 3 : Matrigel에 Matrigel에서 Passaging hESCs

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.

조립, 로딩 및 분석 초원 심 분리기 샘플 셀 정렬

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.

Kinesin의 분리 및 정제에서 Drosophila 태아

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.

대상 - 에스테르 가수 분해 효소와 ER 칼슘의 직접 이미징 유발 염료 로딩 (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.

&quot;인간 답게&quot;를 생성<em&gt; 초파리</em키네신-5의 약리 억제에&gt; S2 세포주 민감성

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.

형광 현미경을 사용 키네신 -1-화물 식별

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