Sigua Robilyn, Tripathy Suvranta, Anand Preetha, Gross Steven P
Department of Developmental and Cell Biology, School of Biosciences, University of California, Irvine, USA.
J Vis Exp. 2012 Apr 27(62):3501. doi: 10.3791/3501.
Motor proteins move cargoes 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 °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.
驱动蛋白沿着微管移动货物，并将它们运输到特定的亚细胞位置。由于运输改变被认为是多种神经退行性疾病的基础，因此了解基于微管的驱动蛋白运输及其调节可能最终会带来改进的治疗方法。驱动蛋白-1是一种真核驱动蛋白，它沿着微管（MTs）以顺行（正端）方向移动，由ATP水解提供动力。在这里，我们报告了一种详细的纯化方案，用于从果蝇胚胎中分离活性全长驱动蛋白，从而将果蝇遗传学与单分子生物物理研究相结合。从大约50个产卵杯开始，每个杯中有大约1000只雌性果蝇，我们进行了过夜收集。这提供了大约10毫升压实的胚胎。胚胎经过漂白去壳处理（得到大约9克胚胎），然后进行匀浆。匀浆后，先用低速离心然后高速离心使匀浆液澄清。澄清的上清液用GTP和紫杉醇处理以使微管聚合。在室温下加入ATP类似物5'-腺苷酰亚胺二磷酸，将驱动蛋白固定在聚合的微管上。驱动蛋白结合后，通过在蔗糖垫层上高速离心使微管沉淀。然后将微管沉淀重新悬浮，并重复此过程。最后，加入ATP以从微管中释放驱动蛋白。高速离心然后使微管沉淀，将驱动蛋白留在上清液中。将这种驱动蛋白使用截留分子量为100 KD的过滤器进行离心过滤以进一步纯化，分装，在液氮中速冻，并储存在-80°C。使用纯化的样品进行SDS凝胶电泳和蛋白质免疫印迹。使用体外单分子微管测定法评估最终离心过滤步骤前后纯化样品的运动活性。离心过滤前后的驱动蛋白组分显示出如先前文献报道的持续性。正在进行进一步的实验以评估驱动蛋白与其他运输相关蛋白之间的相互作用。
