Name: simultaneous visualization of the dynamics of crosslinked and single microtubules in vitro by tirf microscopy
Uploaded: 2022-02-18T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy at JoVE.com

这项工作得到了NIH（编号1DP2GM126894-01）的资助，以及皮尤慈善信托基金和史密斯家庭基金会对R.S.的资助。作者感谢蒋硕博士对方案开发和优化的贡献。

1. 制备试剂
<ol><li>制备缓冲液和试剂，如 表1 和 表2所述。在实验过程中，除非另有说明，否则请将所有溶液保持在冰上。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>溶液</td>
			<td colspan="2">组件</td>
			<td>建议存储持续时间</td>
			<td colspan="2">笔记</td>
		</tr>
<tr>
<t...

<ol>
	<li>Subramanian, R., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+complexity:+insights+into+self-organized+assembly+of+microtubule-based+architectures.">Building complexity: insights into self-organized assembly of microtubule-based architectures.</a> Developmental Cell. 23 (5), 874-885 (2012).</li><li>Baas, P. W., Rao, A. N., Matamoros, A. J., Leo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+properties+of+neuronal+microtubules.">Stability properties of neuronal microtubules.</a> Cytoskeleton (Hoboken). 73 (9), 442-460 (2016).</li><li>Bitan, A., Rosenbaum, I., Abdu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+dynamic+microtubules+coordinately+determine+and+maintain+Drosophila+bristle+shape.">Stable and dynamic microtubules coordinately determine and maintain Drosophila bristle shape.</a> Development. 139 (11), 1987-1996 (2012).</li><li>Foe, V. E., von Dassow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+dynamic+microtubules+coordinately+shape+the+myosin+activation+zone+during+cytokinetic+furrow+formation.">Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation.</a> The Journal of Cell Biology. 183 (3), 457-470 (2008).</li><li>Pous, C., 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=Functional+specialization+of+stable+and+dynamic+microtubules+in+protein+traffic+in+WIF-B+cells.">Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells.</a> The Journal of Cell Biology. 142 (1), 153-165 (1998).</li><li>Uehara, R., Goshima, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+central+spindle+assembly+requires+de+novo+microtubule+generation+in+the+interchromosomal+region+during+anaphase.">Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase.</a> The Journal of Cell Biology. 191 (2), 259-267 (2010).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312 (5991), 237-242 (1984).</li><li>Bieling, P., 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=Reconstitution+of+a+microtubule+plus-end+tracking+system+in+vitro.">Reconstitution of a microtubule plus-end tracking system in vitro.</a> Nature. 450 (7172), 1100-1105 (2007).</li><li>Bieling, P., Telley, I. A., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimal+midzone+protein+module+controls+formation+and+length+of+antiparallel+microtubule+overlaps.">A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.</a> Cell. 142 (3), 420-432 (2010).</li><li>Shimamoto, Y., Forth, S., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pushing+and+braking+forces+generated+by+ensembles+of+Kinesin-5+crosslinking+two+microtubules.">Measuring pushing and braking forces generated by ensembles of Kinesin-5 crosslinking two microtubules.</a> Developmental Cell. 34 (6), 669-681 (2015).</li><li>Mani, N., Jiang, S., Neary, A. E., Wijeratne, S. S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+single+microtubules+and+bundles+by+a+three-protein+module.">Differential regulation of single microtubules and bundles by a three-protein module.</a> Nature Chemical Biology. 17 (9), 964-974 (2021).</li><li>Hyman, A. A., Salser, S., Drechsel, D., Unwin, N., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTP+hydrolysis+in+microtubule+dynamics:+information+from+a+slowly+hydrolyzable+analogue,+GMPCPP.">Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.</a> Molecular Biology of the Cell. 3 (10), 1155-1167 (1992).</li><li>Subramanian, R., 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=Insights+into+antiparallel+microtubule+crosslinking+by+PRC1,+a+conserved+nonmotor+microtubule+binding+protein.">Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein.</a> Cell. 142 (3), 433-443 (2010).</li><li>Rasnik, I., McKinney, S. A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonblinking+and+long-lasting+single-molecule+fluorescence+imaging.">Nonblinking and long-lasting single-molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li><li>Wijeratne, S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+of+antiparallel+microtubule+bundles+regulates+relative+sliding+and+stalling+by+PRC1+and+Kif4A.">Geometry of antiparallel microtubule bundles regulates relative sliding and stalling by PRC1 and Kif4A.</a> eLife. 7, 32595 (2018).</li><li>Mani, N., Wijeratne, S. S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micron-scale+geometrical+features+of+microtubules+as+regulators+of+microtubule+organization.">Micron-scale geometrical features of microtubules as regulators of microtubule organization.</a> eLife. 10, 63880 (2021).</li><li>Freal, 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=Feedback-driven+assembly+of+the+axon+initial+segment.">Feedback-driven assembly of the axon initial segment.</a> Neuron. 104 (2), 305-321 (2019).</li><li>Ledbetter, M., Porter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;microtubule&amp;#34;+in+plant+cell+fine+structure.">A &amp;#34;microtubule&amp;#34; in plant cell fine structure.</a> The Journal of Cell Biology. 19 (1), 239-250 (1963).</li><li>Wijeratne, S. S., Marchan, M. F., Tresback, J. S., Subramanian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+reveals+distinct+protofilament-scale+structural+dynamics+in+depolymerizing+microtubule+arrays.">Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.</a> Proceedings of the National Academy of Sciences of the United States of America. , 119 (2022).</li></ol>

这里描述的实验显着扩展了常规微管重构测定的范围和复杂性，传统上在单个微管或一种类型的阵列上进行。目前的测定提供了一种同时定量和比较两个群体（即单个微管和交联束）上的调节MAP活性的方法。此外，该测定允许检查两种类型的束：那些在动力学开始之前从稳定的种子预先形成的束，以及那些在两个生长端彼此相遇并交联时新形成的束（图3）。此外，除了传统...

微管是生物聚合物，形成对多种细胞过程至关重要的结构支架，从细胞内转运和细胞器定位到细胞分裂和伸长。为了执行这些不同的功能，单个微管被组织成微米大小的阵列，例如有丝分裂纺锤体，睫状轴突，神经元束，相间阵列和植物皮质阵列。在这些结构中发现的无处不在的建筑图案是一束沿其长度交联的微管1。几种基于微管的结构的一个有趣的特征是捆绑的微管和非交联的单个微管在紧密的空间接近中共存。这些微管亚群可以显示出彼此截然不同的聚合动力学，这取决于其正常功能的需要2，3，4，5。例如，在有丝分裂纺锤体内，稳定的交联束和动态单个微管存在于细胞中心的微米级区域内6。因此，研究如何指定共存微管群的动态特性是理解基于微管的结构的组装和功能的核心。
微管是动态聚合物，在聚合和解聚阶段之间循环，在称为灾难和救援的事件中在两个阶段之间切换7。细胞微管的动力学由无数的微管相关蛋白（MAPs）调节，这些蛋白调节微管聚合和解聚的速率以及灾难和救援事件的频率。由于光学显微镜中空间分辨率的限制，研究MAPs在细胞空间近端阵列上的活性具有挑战性，特别是在高微管密度的区域。此外，同一细胞区域中存在多个MAP阻碍了细胞生物学研究的解释。与全内反射荧光（TIRF）显微镜一起进行的 体外 重建测定规避了检查MAPs特定子集调节近端细胞微管阵列动力学的机制的挑战。在这里，在受控条件下，在一个或多个重组MAPs存在下检查 体外 组装的微管的动力学8，9，10。 然而，传统的重构测定通常在单个微管或一种类型的阵列上进行，从而排除了共存群体的可视化。
在这里，我们提出了体外 重建测定，可以在相同的溶液条件下同时可视化两个微管群体11。我们描述了一种同时观察多个MAP在单个微管和由有丝分裂纺锤体相关蛋白PRC1交联的微管束上的集体活性的方法。蛋白 PRC1 优先在反平行微管之间的重叠处结合，使它们交联9。简而言之，该协议包括以下步骤：（i）制备储备溶液和试剂，（ii）用于创建显微镜实验成像室的盖玻片的清洁和表面处理，（iii）制备稳定的微管“种子”，在实验过程中从中开始聚合，（iv）TIRF显微镜设置的规范以可视化微管动力学，（v）微管种子的固定和交联微管束的产生 在成像室中，并且（vi）在添加可溶性微管蛋白，MAP和核苷酸后，通过TIRF显微镜可视化成像室中的微管动力学。这些测定能够对MAP定位及其对两个微管群体动态的影响进行定性评估和定量检查。此外，它们有助于在广泛的实验条件下评估多个MAP对这些微管群体的协同作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)</td>
			<td >Sigma Aldrich</td>
			<td >238813</td>
			<td ></td>
		</tr>
		<tr >
			<td >1,4-piperazinediethanesulfonic acid (PIPES)</td>
			<td>Sigma Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr >
			<td >18x18 mm #1.5 coverslips&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>63787</td>
			<td></td>
		</tr>
		<tr >
			<td >2-Mercaptoethanol (BME)</td>
			<td>Sigma Aldrich</td>
			<td>M-6250</td>
			<td></td>
		</tr>
		<tr >
			<td >24x60 mm #1.5 coverslips</td>
			<td>Electron Microscopy Sciences</td>
			<td>63793</td>
			<td></td>
		</tr>
		<tr >
			<td >405/488/560/647 nm Laser Quad Band&#160;</td>
			<td>Chroma</td>
			<td>TRF89901-NK</td>
			<td></td>
		</tr>
		<tr >
			<td >Acetone</td>
			<td>Sigma Aldrich</td>
			<td>320110</td>
			<td></td>
		</tr>
		<tr >
			<td >Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma Aldrich</td>
			<td>A7699-5G</td>
			<td></td>
		</tr>
		<tr >
			<td >Avidin, NeutrAvidin&#174; Biotin-binding Protein (Molecular Probes&#174;)</td>
			<td>Thermo Fischer Scientific</td>
			<td>A2666</td>
			<td></td>
		</tr>
		<tr >
			<td >Bath sonicator: Branson 2800 Cleaner</td>
			<td>Branson</td>
			<td>CPX2800H</td>
			<td></td>
		</tr>
		<tr >
			<td >Beckman Coulter Polycarbonate Thickwall Tubes, 11 x 34 mm</td>
			<td>Beckman-Coulter&#160;</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr >
			<td >Beckman Coulter Polycarbonate Thickwall Tubes, 8 x 34 mm</td>
			<td>Beckman-Coulter&#160;</td>
			<td>343776</td>
			<td></td>
		</tr>
		<tr >
			<td >Biotin-PEG-SVA, MW 5,000</td>
			<td>Laysan Bio</td>
			<td>#Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>2905</td>
			<td></td>
		</tr>
		<tr >
			<td >Catalase</td>
			<td>Sigma Aldrich</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr >
			<td >Corning LSE Mini Microcentrifuge, AC100-240V</td>
			<td>Corning</td>
			<td>6670</td>
			<td></td>
		</tr>
		<tr >
			<td >Delicate Task Wipes</td>
			<td>Kimtech</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr >
			<td >Dithiothreitol (DTT)</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td></td>
		</tr>
		<tr >
			<td >Emission filter</td>
			<td>Chroma</td>
			<td>ET610/75m</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol (200-proof)</td>
			<td>Decon Labs</td>
			<td>2705</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethylene glycol tetraacetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr >
			<td >Glucose Oxidase</td>
			<td>Sigma Aldrich</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr >
			<td >GMPCPP</td>
			<td>Jena Bioscience&#160;</td>
			<td>NU-405</td>
			<td></td>
		</tr>
		<tr >
			<td >Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma Aldrich</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr >
			<td >Hellmanex III detergent&#160;</td>
			<td>Sigma Aldrich</td>
			<td>Z805939</td>
			<td></td>
		</tr>
		<tr >
			<td >Immersion oil, Type A</td>
			<td>Fisher Scientific</td>
			<td>77010</td>
			<td></td>
		</tr>
		<tr >
			<td >Kappa-casein</td>
			<td>Sigma Aldrich</td>
			<td>C0406</td>
			<td></td>
		</tr>
		<tr >
			<td >Lanolin</td>
			<td>Fisher Scientific</td>
			<td>S25376</td>
			<td></td>
		</tr>
		<tr >
			<td >Lens Cleaning Tissue</td>
			<td>ThorLabs</td>
			<td>MC-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium Chloride (MgCl2)</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr >
			<td >Methylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr >
			<td >Microfuge 16 Benchtop Centrifuge</td>
			<td>Beckman-Coulter&#160;</td>
			<td>A46474</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides, Diamond White Glass, 25 x 75mm, 90&#176; Ground Edges, WHITE Frosted</td>
			<td>Globe Scientific</td>
			<td>1380-50W</td>
			<td></td>
		</tr>
		<tr >
			<td >mPEG-Succinimidyl Valerate, MW 5,000&#160;</td>
			<td>Laysan Bio</td>
			<td>#NH2-PEG-VA-5K</td>
			<td></td>
		</tr>
		<tr >
			<td >Optima&#8482; Max-XP Tabletop Ultracentrifuge</td>
			<td>Beckman-Coulter&#160;</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraffin</td>
			<td>Fisher Scientific</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr >
			<td >PELCO Reverse (self-closing), Fine Tweezers</td>
			<td>Ted Pella</td>
			<td>5377-NM</td>
			<td></td>
		</tr>
		<tr >
			<td >Petrolatum, White</td>
			<td>Fisher Scientific</td>
			<td>18-605-050</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasma Cleaner, 115V</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Potassium Hydroxide (KOH)</td>
			<td>Sigma Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermal-Lok 1-Position Dry Heat Bath</td>
			<td>USA Scientific</td>
			<td>2510-1101</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermal-Lok Block for 1.5 and 2.0 mL Tubes</td>
			<td>USA Scientific</td>
			<td>2520-0000</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Scientific&#8482; Pierce&#8482; Bond-Breaker&#8482; TCEP Solution, Neutral pH; 500mM</td>
			<td>Thermo Fischer Scientific</td>
			<td>PI-77720</td>
			<td></td>
		</tr>
		<tr >
			<td >TIRF 100X NA 1.49 Oil Objective</td>
			<td>Nikon</td>
			<td colspan="2">CFI Apochromat TIRF 100XC Oil</td>
		</tr>
		<tr >
			<td >TIRF microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr >
			<td >TLA 120.1 rotor</td>
			<td>Beckman-Coulter&#160;</td>
			<td>362224</td>
			<td></td>
		</tr>
		<tr >
			<td >TLA 120.2 rotor</td>
			<td>Beckman-Coulter&#160;</td>
			<td>357656</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (&#62;99% pure): porcine brain</td>
			<td>Cytoskeleton</td>
			<td>T240</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin Protein (Biotin): Porcine Brain</td>
			<td>Cytoskeleton</td>
			<td>T333P</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (fluorescent HiLyte 647): porcine brain</td>
			<td>Cytoskeleton</td>
			<td>TL670M</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubulin protein (X-rhodamine): bovine brain</td>
			<td>Cytoskeleton</td>
			<td>TL620M</td>
			<td></td>
		</tr>
		<tr >
			<td >VECTABOND&#174; Reagent, Tissue Section Adhesion</td>
			<td>Vector Biolabs</td>
			<td>SP-1800-7</td>
			<td></td>
		</tr>
		<tr >
			<td >VWR&#174; Personal-Sized Incubator, 120V, 50/60Hz, 0.6A</td>
			<td>VWR</td>
			<td>97025-630</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous visualization of the dynamics of crosslinked and single microtubules in vitro by tirf microscopy

微管是αβ-微管蛋白异源二聚体的聚合物，在细胞中组织成不同的结构。基于微管的架构和网络通常包含动态属性不同的微管阵列子集。例如，在分裂细胞中，稳定的交联微管束与动态非交联微管紧密共存。基于TIRF显微镜 的体外 重建研究能够同时可视化这些不同微管阵列的动力学。在该测定中，成像室与表面固定的微管组装在一起，这些微管要么以单丝形式存在，要么组织成交联束。微管蛋白、核苷酸和蛋白质调节剂的引入允许直接可视化相关蛋白质以及单个和交联微管的动态特性。此外，可以实时监测动态单个微管组织成束时发生的变化。这里描述的方法允许系统地评估单个蛋白质的活性和定位，以及在相同的实验条件下蛋白质调节剂对两个不同微管子集的协同作用，从而提供其他方法无法获得的机制见解。

上述实验使用647nm荧光团标记的生物素化微管，560nm氟团标记的非生物素化微管和560nm氟团标记的可溶性微管混合物进行。微管通过交联蛋白PRC1（GFP标记）交联。在产生表面固定束和单个微管（步骤5.11）后，将成像室安装在TIRF 100X 1.49 NA油物镜上，并在560nm和647nm荧光通道中观察。通过它们在647 nm通道中的荧光信号鉴定单个微管。在两个通道中具有荧光信号的微管被鉴定为预先形成的束（<strong class=...

Watch this Scientific Journal Video about Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy at JoVE.com

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

在这里，提出了基于TIRF显微镜 的体外 重建测定，以同时量化和比较两个微管群体的动力学。描述了一种同时观察多个微管相关蛋白在交联微管束和单个微管上的集体活性的方法。

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

细胞微管阵列包含具有功能所需的不同动态特性的微管亚群。该协议解决了调节器的集体活动如何赋予近端微管亚群具有不同特性。这种自下而上的恢复测定使我们能够同时可视化近端微管群体（如单个微管和束）的组织和动态，并破译其自组织背后的机制。多组分重构需要优化pH、离子强度和蛋白质浓度等实验参数，同时保持每个参数的活性。在多蛋白测定之前对单个组分进行系统分析非常有帮助。首先，将4.5微升BRB ADDTT的混合物在一微升微管溶液中移液到显微镜载玻片上。用 18 x 18 毫米的盖玻片盖住，并用透明指甲油或 VALAP 密封剂密封边缘。将 TIRF 物镜放在盖玻片下方。在适合明亮混合物中荧光标记的微管蛋白的波长处可视化新聚合的微管，以确定在即将到来的实验中使用微管的稀释度。根据经验确定实验的激光强度，使得所有荧光蛋白都被照亮，但在实验的时间过程中不会经历显着的光漂白。将显微镜温度设置为28摄氏度以查看动态微管。使用镜头纸用70%乙醇清洁100倍物镜。使用滤光片立方体和发射滤光片的最佳组合，具体取决于要成像的荧光通道。设置成像序列。对于使用647纳米荧光团标记的生物素化微管，可溶性微管中的560纳米荧光团标记的非生物素化微管和感兴趣的GFP标记的蛋白质的实验，每10秒对488纳米，560纳米和647纳米通道进行成像，持续20分钟。为了在加入可溶性微管蛋白和微管相关蛋白之前捕获束的参考图像，在560纳米和647纳米波长通道中建立一个具有一个图像的序列。检查激光束在显微镜上方天花板上的位置，并使用软件对光束进行更改。确保激光照明器对齐。在成像之前，向物镜中加入一滴显微镜浸没油。准备可溶性微管蛋白混合物，同时将其保持在冰上并向下旋转混合物。为了通过生物素 - NeutrAvidin -生物素连接固定微管，首先在大约7.5微升的NeutrAvidin溶液中流动，直到腔室充满并孵育五分钟。用10微升MB冷水洗涤。在7.5微升的封闭蛋白KC中流动并孵育2分钟。用10微升MB温水洗涤，以准备引入微管的腔室。稀释BRB ADDTT中生物素化微管的储备，并将该稀释液的一微升加入九微升MB温热。将混合物倒入腔室并孵育10分钟。用10微升MB温洗掉未固定的微管。将7.5微升温的KC流入腔室并孵育两分钟。在孵育过程中，在KC中制备交联或蛋白质PRC1的两纳摩尔溶液并向下旋转溶液。将10微升该溶液流入成像室并孵育5分钟。要制成束，将10微升非生物素化微管流入腔室并孵育10分钟。用10微升MB温温洗涤腔室两次。在10分钟的孵育时间内，制备含有目标蛋白质，可溶性微管蛋白，核苷酸，除氧剂和抗氧化剂的10微升测定混合物并将其旋转下来。将混合物放在冰上。将准备好的成像室装入胶带到100X TIRF物镜上的载玻片支架上。使用 560 纳米和 647 纳米通道查找包含单个微管和束的最佳数量和密度的视场。确定视野后，拍摄参考图像。小心地在测定混合物中流动，而不会干扰成像室。用 VALAP 密封剂密封腔室的开口端。观察微管并开始成像序列。固定微管种子并产生束后，获得荧光图像。单个微管通过647纳米通道中的荧光信号进行鉴定，而当微管在两个通道中显示荧光信号时，微管被鉴定为预制束。在KIF4A和CLASP1等蛋白存在下研究了单个微管和PRC1交联束的动力学，结果表明单个微管在测定过程中伸长，而交联微管的生长停滞。小心将聚合微管保持在室温或室温以上。将可溶性微管蛋白保持在冰上，并保护成像室和荧光标记的微管和蛋白质免受光照。这些测定提供了在有丝分裂纺锤体中发现的蛋白质模块如何不同地调节在纺锤体中共存的两个不同微管群体的动力学的机制见解。

Research

JoVE Journal

Biochemistry

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

科研

生物化学

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

通过全内反射荧光（TIRF）显微镜 在体外 可视化肌动蛋白和微管耦合动力学

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

通过TIRF显微镜在体外同时可视化交联和单微管的动力学