Roche's Publication Grant covered the Free Access publication and production of this article. The author also thanks E. Premkumar Reddy, Richard V. Mettus, Stephen C. Cosenza, Sau Ying Yip and Sol D. Gloria for their support and critical reading of the manuscript.

1. tdTomato标记野生型和无电机KIF5B蛋白质的克隆<ol><li>扩增的cDNA为使用引物在表1中的人野生型和无电机KIF5B蛋白质，Taq DNA聚合酶（5单位为100微升），dNTP混合物（2mM的每种脱氧核苷酸）和30个循环的10×缓冲液。每个循环由一个变性步骤（95℃，30秒），退火步骤（45℃，30秒）和延伸步骤（72℃，3分钟）的。 </li><li>提取扩增DNA产物与等体积的苯酚/氯仿（1:1）。 注:苯酚是易燃，可致灼伤。三氯甲烷是危险的。避免与他们直接接触，化学通风柜下使用它们。可替代地，PCR产物可通过各种试剂盒进行纯化。 <ol><li>自旋18000 XG在微量，在室温下放置1分钟。水溶液转移到新的试管中，用等体积的氯仿萃取。 </li><li>纺在室温下微量离心0.5分钟18000 xg离心。转移水溶液至新管。 </li><li>混合使用的3米乙酸钠十分之一体积和无水乙醇两体积的水溶液中。 </li><li>自旋18000 XG在微量在室温下5分钟。丢弃水溶液。 </li><li>两个体积的75％乙醇洗涤DNA沉淀。丢弃水溶液和空气干燥，在室温下将DNA沉淀5分钟。重悬34微升水DNA沉淀。 </li></ol></li><li>消化的扩增产物中的40微升用限制性终体积内切酶SalI（10单位）和BamHI（10单位），在37℃下4微升的10X缓冲液两小时。解决在琼脂糖凝胶含溴化乙锭（0.2微克/毫升）的消化产物（在100V 1.0％）。 注意:溴化乙锭是一种强效的诱变剂，并且可以通过皮肤被吸收。因此，为了避免直接或间接W接触是很重要的第i溴化乙锭。 </li><li>按列削减了净化正确的波段。称重含有该DNA片段的琼脂糖凝胶。然后将其溶解在37℃的溶解缓冲液（300微升为0.1克）约20分钟。 </li><li>在室温下添加所得到的溶液，以一列和自旋在微量5秒。丢弃的流量通过。 </li><li>通过重复步骤1.5洗用0.5ml溶解缓冲液的柱。重复步骤1.5 0.75 1ml洗涤缓冲液洗一遍列。旋转2分钟摆脱剩余的洗涤缓冲液的列。 </li><li>通过重复步骤1.5洗脱用50μl水的DNA片段。通过在含有溴化乙锭（0.2微克/毫升）的琼脂糖凝胶（在100V 1.0％）运行它针对DNA大小梯等分试样估计的DNA片段的浓度。 </li><li>先前与日消化ptdTomato-C1载体17（约100纳克）结扎纯化的产品（约100纳克）È相同限制性内切酶在使用T4DNA连接酶（2000单位）和1微升10X连接缓冲液在室温下搅拌16小时10微升。 注意:野生型和无电机突变体分别含有氨基酸2至963以及584至963。先前，较大的无电机KIF5B突变体被用来确定其功能9。用较小的无动力KIF5B突变本研究，以提高其表达水平。 </li></ol> 2.免疫荧光显微镜<ol><li>对活细胞或具有等于或低于40X放大率间接荧光成像，种子0.2-0.3百万HeLa细胞在1ml完全培养基[Dulbecco氏改良Eagle培养基（DMEM）中，用10％胎牛血清（FBS）〕中的各孔6孔板。在37℃下生长，用5％ 的 CO 2。 </li><li>对于上述40X放大倍率间接免疫荧光研究，洗盖玻片，在无水乙醇短暂（18毫米×18毫米1.5厚）风干他们一个6孔板的孔中，一个生物安全柜内以避免污染。 <ol><li>种子0.2-0.3百万个细胞的1ml在六孔板的每个孔中完成的DMEM培养基。 </li></ol></li><li>转染复合物的制备<ol><li>第二天，一个生物安全柜内，稀0.6微克为在存在或不存在的表达质粒的用于货物的候选蛋白质tdTomato标记的野生型或无电机KIF5B表达质粒的（总）（pTagCFP-c-Myc的5 ）在1.6 ml离心管0.1毫升转染培养基。 注:矢量pTagCFP-c-Myc的表达TagCFP标记的c-MYC。 </li><li>添加1.8微升转染试剂按0.1ml转染培养基中另外16 ml离心管。通常情况下，准备稀释转染试剂足够12个样品的主结构。 </li><li>孵育5分钟，在室温下。 </li><li>稀释0.1毫升转染试剂添加到稀释的0.1毫升的DNA。弥反相管的X含量。旋管用于在微量离心5秒。 </li><li>孵育45分钟，在室温。 </li></ol></li><li>细胞洗涤<ol><li>洗接种的细胞用磷酸缓冲盐水（PBS）三次潜伏期间的转染复合物的形成。 </li><li>在各替换接种的细胞的培养基以及用0.8mL预热转染培养基。返回所述板的孵化器在37℃。 </li></ol></li><li>细胞转染<ol><li> 45分钟温育后，增加±毫升的DNA /转染试剂复合物（在步骤2.3中制备）滴加至板的各孔中。 </li><li>轻轻摇动6孔板5秒钟，它们在37℃下放回培养箱之前。 </li><li> 6-8小时后，更换完全DMEM培养基的媒介。 </li></ol></li><li>活细胞荧光成像18 <ol><li>孵化一个额外16小时后吨37℃时，DNA嵌入污点的Hoechst 33342加入到培养基中，以1μM的终浓度。 注意:赫斯特33342是一种细胞渗透性，DNA嵌入染料染色细胞核。 </li><li>通过使用40倍物镜的荧光显微镜检查它们的荧光蛋白质的表达之前孵育在孵化10分钟的转染细胞在37℃。过滤套的DAPI，CFP，FITC和Cy3的是（Ex350纳米/ Em460纳米），（Ex436纳米/ Em480纳米），（Ex470纳米/ Em525毫微米）和（Ex545纳米/ Em605纳米），分别。 注意:如果背景是高，由于完整的DMEM培养基的自体荧光，无酚红培养基中。 </li></ol></li></ol> 3.间接免疫荧光研究<ol><li>多聚甲醛溶液的制备<ol><li>称重多聚甲醛粉末（每100毫升PBS中的4克）和将其添加到PBS中。 注意:多聚甲醛是一种易燃固体和潜在的癌症哈萨ð。它会刺激眼睛，呼吸系统和皮肤。因此，为了避免多聚甲醛的接触或吸入它是重要的。 </li><li>添加NaOH，使溶液（150微升10的1N NaOH / 100毫升）中。 </li><li>保持在37溶液至42℃2-3小时，偶尔晃动。 </li><li>通过加入冰醋酸至多聚甲醛粉末后的溶液（约75微升/ 100毫升）溶解pH调整到7.0。 </li></ol></li><li>靶蛋白染色<ol><li>在37℃下进一步温育16小时后，由漩六孔板5秒洗在用PBS室温一次转染的细胞。 </li><li>修复细胞用1毫升每孔新鲜配制，4％多聚甲醛溶液中。在室温下孵育30分钟。 </li><li>一旦通过涡旋5秒的6孔板用PBS洗细胞。弃去溶液。 </li><li>孵育所述细胞在室温下将1ml 0.1％的Triton-X 100（在PBS中）处理30分钟。该DETergent的Triton-X 100将透化细胞膜以允许抗体访问它们的细胞内的目标。 </li><li>由旋流六孔板的每个时间5秒清洗细胞用PBS四次。每个丢弃后洗净解决方案。 </li><li>通过摇动4小时，在室温下的PBS溶液中的10％胎牛血清;孵育用1ml的初级抗体的细胞（0.1微克/毫升的c-MYC兔抗体或p53的小鼠抗体）。 </li><li>由旋流六孔板的每个时间5秒，用PBS洗涤四次。每个丢弃后洗净解决方案。 </li><li>通过摇动2小时，在PBS中的10％FBS的在黑暗中于室温;（0.5微克/毫升的Alexa Fluor 488缀合的抗兔或抗小鼠IgG抗体）用1ml荧光染料缀合的次级抗体孵育。 </li><li>由旋流六孔板的每个时间5秒，用PBS洗涤四次。每个丢弃后洗净解决方案。 </li></ol></li><li>核染色和安装<ol><lI&gt;孵育1毫升DNA嵌入染料4&#39;，6-二脒基-2-苯基的细胞（DAPI; 0.5微克/毫升）的PBS在室温下，在黑暗中进行10分钟。继续执行步骤3.4被利用无盖眼镜时。 注意:DAPI是DNA嵌入染料，即污迹细胞核。 </li><li>应用10微升安装解决方案到每个显微镜载玻片。挂载每个玻璃盖在载玻片上的安装解决方案。 </li><li>在黑暗中过夜孵育在室温下。 </li><li>密封的玻璃盖用指甲油的边缘。在通风橱内的暗放置过夜以除去指甲油烟雾。 </li></ol></li><li>荧光显微镜19 <ol><li>第二天，研究使用40倍物镜荧光显微镜细胞。过滤套的DAPI，CFP，FITC和Cy3的是（Ex350纳米/ Em460纳米），（Ex436纳米/ Em480纳米），（Ex470纳米/ Em525毫微米）和（Ex545纳米/ Em605纳米），分别。 </li></ol></li></ol>

<ol>
	<li>Hirokawa, N., Niwa, S., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+in+neurons:+transport+mechanisms+and+roles+in+brain+function,+development,+and+disease.">Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.</a> Neuron. 68, 610-638 (2010).</li><li>Yu, Y., Feng, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+kinesin+family+proteins+in+tumorigenesis+and+progression:+potential+biomarkers+and+molecular+targets+for+cancer+therapy.">The role of kinesin family proteins in tumorigenesis and progression: potential biomarkers and molecular targets for cancer therapy.</a> Cancer. 116, 5150-5160 (2010).</li><li>Verhey, K. J., Hammond, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+control:+regulation+of+kinesin+motors.">Traffic control: regulation of kinesin motors.</a> Nat Rev Mol Cell Biol. 10, 765-777 (2009).</li><li>Hirokawa, N., Noda, Y., Tanaka, Y., Niwa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+superfamily+motor+proteins+and+intracellular+transport.">Kinesin superfamily motor proteins and intracellular transport.</a> Nat Rev Mol Cell Biol. 10, 682-696 (2009).</li><li>Lee, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+c-MYC+by+Kinesin-1+for+proteasomal+degradation+in+the+cytoplasm.">Transport of c-MYC by Kinesin-1 for proteasomal degradation in the cytoplasm.</a> Biochim Biophys Acta. 1843, 2027-2036 (2014).</li><li>Bittins, C. M., Eichler, T. W., Hammer, J. A., Gerdes, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant-negative+myosin+Va+impairs+retrograde+but+not+anterograde+axonal+transport+of+large+dense+core+vesicles.">Dominant-negative myosin Va impairs retrograde but not anterograde axonal transport of large dense core vesicles.</a> Cell Mol Neurobiol. 30, 369-379 (2010).</li><li>Kimura, T., Watanabe, H., Iwamatsu, A., Kaibuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin+and+CRMP-2+complex+is+transported+via+Kinesin-1.">Tubulin and CRMP-2 complex is transported via Kinesin-1.</a> J Neurochem. 93, 1371-1382 (2005).</li><li>Lindsay, A. J., McCaffrey, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+is+required+for+the+transport+of+fragile+X+mental+retardation+protein+(FMRP)+granules.">Myosin Va is required for the transport of fragile X mental retardation protein (FMRP) granules.</a> Biol Cell. 106, 57-71 (2014).</li><li>Rivera, J., Chu, P. J., Lewis, T. L., Arnold, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Kif5B+in+axonal+localization+of+Kv1+K(+)+channels.">The role of Kif5B in axonal localization of Kv1 K(+) channels.</a> Eur J Neurosci. 25, 136-146 (2007).</li><li>Roland, J. T., Kenworthy, A. K., Peranen, J., Caplan, S., Goldenring, 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=Myosin+Vb+interacts+with+Rab8a+on+a+tubular+network+containing+EHD1+and+EHD3.">Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3.</a> Mol Biol Cell. 18, 2828-2837 (2007).</li><li>Uchida, A., Alami, N. H., Brown, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+functional+coupling+of+kinesin-1A+and+dynein+motors+in+the+bidirectional+transport+of+neurofilaments.">Tight functional coupling of kinesin-1A and dynein motors in the bidirectional transport of neurofilaments.</a> Mol Biol Cell. 20, 4997-5006 (2009).</li><li>Zadeh, A. D., 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=Kif5b+is+an+essential+forward+trafficking+motor+for+the+Kv1.5+cardiac+potassium+channel.">Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel.</a> J Physiol. 587, 4565-4574 (2009).</li><li>Su, Y. Y., 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=KIF5B+promotes+the+forward+transport+and+axonal+function+of+the+voltage-gated+sodium+channel+Nav1.8.">KIF5B promotes the forward transport and axonal function of the voltage-gated sodium channel Nav1.8.</a> J Neurosci. 33, 17884-17896 (2013).</li><li>Lalioti, V. S., Vergarajauregui, S., Tsuchiya, Y., Hernandez-Tiedra, S., Sandoval, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daxx+functions+as+a+scaffold+of+a+protein+assembly+constituted+by+GLUT4,+JNK1+and+KIF5B.">Daxx functions as a scaffold of a protein assembly constituted by GLUT4, JNK1 and KIF5B.</a> . J Cell Physiol. 218, 416-426 (2009).</li><li>Cho, K. I., 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=Association+of+the+kinesin-binding+domain+of+RanBP2+to+KIF5B+and+KIF5C+determines+mitochondria+localization+and+function.">Association of the kinesin-binding domain of RanBP2 to KIF5B and KIF5C determines mitochondria localization and function.</a> Traffic. 8, 1722-1735 (2007).</li><li>Diefenbach, R. J., Diefenbach, E., Douglas, M. W., Cunningham, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+receptor,+p180,+interacts+with+kinesin+heavy+chain,+KIF5B.">The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B.</a> Biochem Biophys Res Commun. 319, 987-992 (2004).</li><li>Shaner, N. 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat Biotechnol. 22, 1567-1572 (2004).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+fluorescence+imaging.">Live-cell fluorescence imaging.</a> Methods Cell Biol. 114, 125-150 (2013).</li><li>Sanderson, M. J., Smith, I., Parker, I., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Cold Spring Harb Protoc. 2014, (2014).</li><li>Hopkins, S. C., Vale, R. D., Kuntz, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+kinesin+activity+from+structure-based+computer+screening.">Inhibitors of kinesin activity from structure-based computer screening.</a> Biochemistry. 39, 2805-2814 (2000).</li><li>Zhang, Y., Xiong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+p53+ubiquitination+and+nuclear+export+by+MDM2+and+ARF.">Control of p53 ubiquitination and nuclear export by MDM2 and ARF.</a> Cell Growth Differ. 12, 175-186 (2001).</li><li>Michael, D., Oren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p53-Mdm2+module+and+the+ubiquitin+system.">The p53-Mdm2 module and the ubiquitin system.</a> Semin Cancer Biol. 13, 49-58 (2003).</li><li>Kneussel, M., Wagner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+motors+at+neuronal+synapses:+drivers+of+membrane+transport+and+actin+dynamics.">Myosin motors at neuronal synapses: drivers of membrane transport and actin dynamics.</a> Nat Rev Neurosci. 14, 233-247 (2013).</li><li>Maravillas-Montero, J. L., Santos-Argumedo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myosin+family:+unconventional+roles+of+actin-dependent+molecular+motors+in+immune+cells.">The myosin family: unconventional roles of actin-dependent molecular motors in immune cells.</a> J Leukoc Biol. 91, 35-46 (2012).</li></ol>

这篇文章的免费接入出版和生产由罗氏公司支付。

所描述的方法利用无电机KIF5B突变体，它缺乏沿微管移动的能力的性质，但保留以形成具有野生型KIF5B二聚体和，由此，允许所述四聚体蛋白具有相同货物蛋白质相互作用的能力作为野生型KIF5B。无电机KIF5B，因此，作为一个显性负突变体和形式的错误定位，其货物聚集。这种方法被证明识别驱动蛋白1货物的c-MYC（图1）5。在本文中，相同的无电机KIF5B突变体来鉴定的p53作为驱动蛋白1的另一个潜在的货物（ 图2）。这表明，该方法是确定驱动蛋白1的其它货物可行的。此外，该突变体的特异性是由缺乏对阴性对照蛋白c-Fos蛋白（图3）的突变体的效果提供。 在这个协议中，tdTomato标记的野生TYPE或无电机KIF5B蛋白共表达与另一种荧光蛋白标记的候选蛋白质的货物。在这种情况下，活细胞的荧光显微镜和成像中进行。聚集体的形成可通过时间推移成像进行跟踪。可替代地，tdTomato标记蛋白可以单独表达，并在其生理水平的候选货物蛋白是通过使用特异性抗体的间接免疫荧光显微镜观察。荧光蛋白tdTomato被选择用于其亮度和光17。如果背景是由于完全的DMEM培养基自动荧光高，则使用不含酚红介质。 无电机KIF5B突变体形成具有野生型KIF5B化学计量二聚体。因此，它是表达无马达KIF5B突变体的足够量的，以形成与野生型对应二聚体以抑制其功能，并形成聚集体的关键。为了解决这个问题，expr的优化无电机突变体的分裂国家是必需的。它是通过使用含有二聚结构域的小无马达的突变来实现。此外，适当的转染试剂和协议的优化也是必要的。 HeLa细胞的培养用DNA /转染试剂的持续时间在HeLa细胞中被优化为外源蛋白质和细胞活力的表达。孵育持续时间可以具有用于其它细胞系进行优化。 对于4X之间的放大倍数40X的，不需要盖眼镜。细胞可以直接检查在孔中。因此，在这种情况下，该协议是便宜和方便。对于以上40X放大倍数，将细胞生长在盖玻片在孔中和，染色后，被安装在显微镜载片上为下油浸物镜检查。 无电机KIF5B蛋白质的聚集体的观察由细胞质的大小的限制。这是很容易检测的聚集体在许多哺乳动物细胞。然而，这是相对难以观察中的神经元细胞的聚集体时，细胞质的体积大约是细胞核和在突起小。 该协议是用于显示除了在体内酵母双杂交和生化下拉实验13-16 KIF5B及其货物之间的关联。所有这些试验确定不同的身体状况下，联想及其结果可以互相补充。此外，该荧光测定在其它测定的优点在于，它可以显示由KIF5B的货物的亚细胞定位的调节（ 图 1和2）。 的协议不局限于KIF5B，可用于识别其它马达蛋白的货物如其他一些驱动蛋白马达 3和一些在货物3的胞内运输中使用的肌球蛋白马达23,24的。这些马达蛋白还包含用于沿微管驱动蛋白电机和肌球蛋白马达微丝，以及用于低聚卷曲螺旋区段移动电机域。他们中的大多数形成二聚体3,23。因此，类似的策略可以通过创建自己的无动力显性负突变体，以确定他们的货物适用于他们。

驱动蛋白1是介导其货物1,2-顺行输送马达蛋白。它是驱动蛋白轻链1（KLC1）和驱动蛋白重链的两个亚基（KHCs）的两个亚单位的异四聚体。 KIF5B 1，KHC，包含在其N-末端，其水解ATP和化学能转换为机械能用于沿微管运动的电机领域。其C-末端区包含与KLC1，其与货物相关联的交互二聚化结构域。驱动蛋白-1运输货物，如囊泡，细胞器和mRNA的3,4。最近，KIF5B显示出运送核转录因子c-Myc的对蛋白酶体降解在细胞质5。三种方法（化学抑制剂的siRNA / shRNA的和显性负突变体）被用来抑制驱动蛋白1功能。它们都诱导的c-MYC的聚集在细胞质中。在过去的方法，c-Myc的仅受主导negat香港专业教育学院KIF5B的突变体，但不是由另一相关KIF5A马达的蛋白质，这表明该突变不会对细胞内的部件或一般的效果（如微管中断）对蛋白聚集。 KIF5B的显性负突变体也没有影响另一个转录因子，这表明它不会对转录因子的一般效果。相反，它表明，显性负突变体上施加其货物特异性作用。 使用显性负突变体的是在电动机的蛋白质的领域常见的。驱动蛋白和肌球蛋白的类似无电机突变体以前使用。它们主要是用来展示在他们的货物的亚细胞定位或对细胞功能的6-12突变体的效果。不太重视被提上了突变体和受其影响的货物之间的空间关系。然而，在一些发生率，观察到突变体与其CA到共定位RGOS 6,10。 KIF5B及其相关蛋白质之间的相互作用以前由体内酵母双杂交测定和生化下拉测定法诸如免疫共沉淀和体外下拉实验13-16证实。在这篇文章中，一个额外的视觉方法使用荧光显微镜描述识别KIF5B货物蛋白质。该方法利用充当显性负突变体一种无马达KIF5B突变体。它聚合在细胞质和诱导其货物的聚集。 野生型和无电机KIF5B突变体与荧光蛋白tdTomato 17的标记通过荧光显微镜实现其可视化。加标签KIF5B蛋白可以与融合到与来自KIF5B标签适当地分离的光谱特性的不同荧光蛋白的候选蛋白共表达。该标记的蛋白在活细胞中直接观察在荧光显微镜下。由无电机KIF5B突变候选蛋白质的聚集的诱导将确认该候选蛋白是KIF5B 的体内的货物。此外，tdTomato标记KIF5B蛋白可以单独表达在细胞中，研究了内源性货物蛋白它们的影响。以后，免疫荧光显微镜下进行，其中转染的细胞固定并用抗内源性候选蛋白的特异性抗体染色，接着用荧光染料缀合的适当的二级抗体。在这种情况下，在其生理水平的内源候选蛋白质进​​行了研究。其他马达蛋白的类似无动力突变体可以准备识别他们的货物。

<table><tbody><tr><td>absolute ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>Opti-MEM-I (transfection medium)</td><td>Life Technologies</td><td>51985</td><td></td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Life Technologies</td><td>P36961</td><td></td></tr><tr><td>formaldehyde, para</td><td>Fisher Scientific</td><td>O4042-500</td><td></td></tr><tr><td>Triton-X100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>X-tremeGENE 9 DNA Transfection Reagent</td><td>Roche</td><td>6365787001</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>Life Technologies</td><td>10342020</td><td></td></tr><tr><td>PCR Grade Nucleotide Mix (dNTP Mix)</td><td>Roche</td><td>12111424</td><td></td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12-541A</td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA ladder</td><td>Life Technologies</td><td>SM1333</td><td></td></tr><tr><td>PureLink Quick Gel Extraction kit</td><td>Life Technologies</td><td>K210012</td><td></td></tr><tr><td>BamHI</td><td>New England Biolab</td><td>R0136</td><td></td></tr><tr><td>SalI</td><td>New England Biolab</td><td>R0138</td><td></td></tr><tr><td>T4 DNA ligase&amp;nbsp;</td><td>New England Biolab</td><td>M0202T</td><td></td></tr><tr><td>Ethidium bromide</td><td>Thermo Scientific</td><td>17898</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>c-MYC rabbit antibody</td><td>Cell Signaling</td><td>5606</td><td></td></tr><tr><td>p53 mouse antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated anti-rabbit IgG antibody</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated anti-mouse IgG antibody</td><td>Life Technologies</td><td>A11059</td><td></td></tr><tr><td>fluorescent microscope&amp;nbsp;</td><td>Olympus IX71_Fluoview</td><td></td><td></td></tr><tr><td>computer software for imaging</td><td>&amp;nbsp;cellSens&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

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.

外源性表达的野生型KIF5B在细胞质中均匀出现，而c-myc的主要出现在细胞核中（图1A）。但是，无电机KIF5B突变体形成在细胞质中（图1A）的聚集体。无电机KIF5B聚集诱导的c-MYC的聚集。细胞比例合计将表达TagCFP标记的c-MYC表达野生型KIF5B细胞是很低的。然而，这是当无电机KIF5B被共表达（图1A）显著更高。用C-MYC突变KIF5B观察到的共定位（图1）表明，KIF5B调节的c-Myc的亚细胞定位和c-MYC是驱动蛋白1的货物。在该构建体单独表达阴性对照被包括在图1A的下面板。结果表明:有荧光emissio的显著渗出通过无ñ。无电机KIF5B也诱导内源性c-Myc的（图1B）和转录因子p53的（图2）的聚集，这表明 c-myc和P53都是驱动蛋白-1的货物和KIF5B调节内源性蛋白的亚细胞定位。一起的结果是，驱动蛋白1抑制剂玫瑰红内酯（RBL）20诱导形成高分子量物种的两个c-myc和p53的5，p53是有可能驱动蛋白-1的货物。有趣的是，要注意的是，核转录因子的p53，如c-MYC，也从核输出到细胞质为蛋白酶体降解21,22。转录因子由KIF5B运动似乎是特定因为无电机KIF5B突变体的表达没有影响的亚细胞定位的c-Fos蛋白或使其聚集（ 图 3）。上述数据表明，方法具本出版物中使用ð允许驱动蛋白1货物的高特异性识别。类似的策略可以应用到其它动力蛋白为好。 <img alt="图1" src="/files/ftp_upload/53632/53632fig1.jpg" /> 图1:无电机KIF5B突变体的表达诱导在细胞质中的c-Myc聚合 （A）中的HeLa细胞用tdTomato标记的野生型（WT）KIF5B（红色）和TagCFP标记的c-MYC（蓝色）转染。 tdTomato标记WT KIF5B主要出现在细胞质中，而TagCFP标记的c-MYC主要出现在细胞核中。然而，tdTomato标记无电机KIF5B突变体（红色）形成在细胞质丝状或点状的聚集体。引起无电机KIF5B表达的c-MYC的聚集。突变KIF5B和c-MYC共定位在一起（粉红色）。说明CFP-C-MYC（％）的聚集细胞（％）百分比显示。结果示如平均值±SD（N = 3）。与tdTomato标记KIF5B蛋白质或空载体（V）的沿TagCFP标记的c-MYC表达的阴性对照示于下面板。 （B）中类似的结果与内源性c-Myc的（绿色）时表达tdTomato标记WT或无电机KIF5B蛋白（红色）获得。所述tdTomato标记无电机KIF5B突变体形成在细胞质聚集体和诱导的内源c-Myc的聚集。两种聚集体在细胞质中（橙色）共定位在一起。细胞核用染料赫斯特33342或DAPI染色。比例尺; 20微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53632/53632fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53632/53632fig2.jpg" /> 图2:无动力KIF5B突变表达诱导在T内源性p53的聚集他细胞质中。在HeLa细胞中，外源tdTomato标记的野生型（WT）KIF5B（红色）主要出现在细胞质中，而内源性p53蛋白（绿色）主要出现在细胞核中。与此相反，tdTomato标记无电机KIF5B突变体（红色）形成在细胞质聚集体。诱导无电机KIF5B的表达p53的聚集，从而导致KIF5B的共定位和p53在细胞质中（黄色）。该实验进行三次。细胞核用染料DAPI染色。比例尺; 20微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53632/53632fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53632/53632fig3.jpg" /> 图3:无电机KIF5B突变体的表达不诱导的c-Fos蛋白的聚集在细胞质 tdTomato标记的野生型（WT）KIF5B（红色）出现主要在HeLa细胞的细胞质中，而EGFP-c-fos（绿色）主要出现在细胞核中。与此相反，在tdTomato标记无电机KIF5B突变体（红色）形成在细胞质聚集体。无电机KIF5B的表达并没有诱导c-​​Fos蛋白的聚集。该实验进行四次。细胞的细胞核与染料DAPI染色。比例尺; 20微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53632/53632fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td>常见的反向引物</td><td> 5&#39;-AGAGGATCCTTACACTTGTTTGCCTCCTC-3&#39; </td></tr><tr><td>野生型KIF5B正向引物</td><td> 5&#39;AGAGTCGACGCGGACCTGGCCGAGTGCAACATCAAAGT-3&#39; </td></tr><tr><td>无动力KIF5B正向引物</td><td> 5&#39;AGAGTCGACGATGAAGAGTTCACTGTTGC-3&#39; </td></tr></tbody></table>表1:用于克隆野生型和无电机KIF5B蛋白的引物序列。

Watch this Scientific Journal Video about Identification of Kinesin-1 Cargos Using Fluorescence Microscopy at JoVE.com

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

在这里，提出一个协议，以确定驱动蛋白-1的货物。该驱动蛋白1重链（KIF5B）的无电机突变体聚集在细胞质和诱导其货物的聚集。两个聚集体在荧光显微镜下检测。类似的策略可以用于识别其他马达蛋白的货物。

使用荧光显微镜驱动蛋白-1的货物的识别

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

identification-of-kinesin-1-cargos-using-fluorescence-microscopy

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.8K 次观看.  Icahn School of Medicine at Mount Sinai. 在这里，提出一个协议，以确定驱动蛋白-1的货物。该驱动蛋白1重链（KIF5B）的无电机突变体聚集在细胞质和诱导其货物的聚集。两个聚集体在荧光显微镜下检测。类似的策略可以用于识别其他马达蛋白的货物。 

视频: Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

科研

化学

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

发育生物学

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

由可逆连接的运动素提供动力的分子穿梭机的组装

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

工程学

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

生物化学

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

表征分子马达的组成对运动轴突货物用"货物映射"分析

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

神经科学

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

货物装载到驱动蛋白供电分子梭

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

生物学

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

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-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. ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

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的分离和纯化果蝇胚胎

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

使用荧光显微镜驱动蛋白-1的货物的识别

摘要

探索更多视频

此视频中的章节

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

固定化的线虫线虫分析神经元内细胞的转运

由可逆连接的运动素提供动力的分子穿梭机的组装

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

表征分子马达的组成对运动轴突货物用"货物映射"分析

货物装载到驱动蛋白供电分子梭

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

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

单分子成像核运输

kinesin的分离和纯化果蝇胚胎

使用荧光显微镜驱动蛋白-1的货物的识别

摘要

探索更多视频

此视频中的章节

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

固定化的线虫线虫分析神经元内细胞的转运

由可逆连接的运动素提供动力的分子穿梭机的组装

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

表征分子马达的组成对运动轴突货物用"货物映射"分析

货物装载到驱动蛋白供电分子梭

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

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

单分子成像核运输

kinesin的分离和纯化果蝇胚胎

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性