Structure And Function Of Cytoplasmic Motors

鱿鱼巨轴突一直是研究神经元功能的重要模型系统。最近，我们已经启动了EST（表达序列标签）项目，旨在获得鱿鱼神经系统的基因组信息。到目前为止，我们已经对6，912个随机选择的cDNA克隆进行了测序，并确定了1，935个在星状神经节中表达的独特基因，其中包含巨大轴突的细胞体。到目前为止，四个额外的马达蛋白，包括第二个驱动蛋白和三个肌球蛋白以前未知的鱿鱼已被确定。此外，我们还发现了淀粉样前体蛋白（APP）的EST，APP是一种在阿尔茨海默病中起作用的跨膜蛋白。疾病（AD）。使用该序列合成肽，它是可能的，以显示一个特定的鱿鱼（和人类）APP的胞质结构域是足以诱导顺行运输荧光珠注射到鱿鱼巨大的轴突。这一发现表明APP在轴突运输中起着至关重要的作用。NINDS的三个独立小组以及伍兹霍尔海洋生物实验室的研究人员正在利用其他EST数据进行基因鉴定、肽序列分析、抗体生产和蛋白质组学。正在进行的工作预计将创建一个几乎完整的鱿鱼神经系统中表达的蛋白质库。与克隆库直接相关的这一信息将随时向科学界公开提供，预计在科学界将有广泛的用处。 在我们的实验室开发的抗体的帮助下，对各种肌球蛋白在鱿鱼巨大的轴突，我们已经能够分离出特定的肌球蛋白，肌球蛋白II，负责在轴突切断后的荷包收缩。荷包收缩被认为是最小化轴突损伤后的变性和功能丧失。使用肌球蛋白II抗体，我们目前正在可视化的位置和时间过程中的肌球蛋白轴突减少其直径， 在与NIDCR的Catherine Galbraith博士的合作中，我们也在研究轴突的寻路和文化指导。通过开发一种新的技术，用于标记的肌动蛋白细胞骨架的亚群，使用photoactivatable-EGFP-actin，我们能够可视化以前未观察到的横向运动的新聚合的肌动蛋白丝。我们已经观察到，这种新描述的肌动蛋白运动是伴随着同步的横向运动和β 1整合素的聚集。整联蛋白聚集沿着成纤维细胞板层和生长锥丝状伪足的前缘产生引发的但未连接的β 1整联蛋白沿着。这些结果表明，肌动蛋白定位激活整合素，为细胞寻找新的粘附位点和首选的迁移方向提供了一种机制。

The squid giant axon has been and remains an important model system for studying neuronal function. Recently, we have initiated an EST (Expressed Sequence Tag) project aimed at obtaining genomic information on the squid nervous system. To date, we have sequenced 6,912 randomly selected cDNA clones and identified 1,935 unique genes expressed in the stellate ganglion, which contains the cell bodies for the giant axons. So far four additional motor proteins, including a second kinesin and three myosins previously unknown in squid have been identified. Additionally, we have found an EST for Amyloid Precursor Protein (APP), a transmembrane protein with a role in Alzheimer?s Disease (AD). Using this sequence to synthesize peptides it was possible to show that a particular cytoplasmic domain of squid (and human) APP is sufficient to induce anterograde transport of fluorescent beads injected into the squid giant axon. This finding suggests a crucial role for APP in axonal transport. Other EST data is being utilized by three separate groups at NINDS as well as by investigators at the Marine Biological Laboratory, Woods Hole, for gene identification, peptide sequence analysis, antibody production and proteomics. Work in progress is expected to create a nearly complete library of proteins expressed in the squid nervous system. This information directly linked to a library of clones will be made readily and openly available to the scientific community, where it is expected to be broadly useful. With the help of antibodies developed in our laboratory against a variety of myosins in the squid giant axon we have been able to isolate the specific myosin, myosin II, responsible for the purse string contraction in axons following transection. The purse string contraction is thought to minimize degeneration and loss of function after axonal injury. Using the myosin II antibodies we are currently visualizing the location and time course of the myosins as the axon reduces its diameter, In collaboration with Dr. Catherine Galbraith of NIDCR we are also investigating axonal pathfinding and guidance in cultures. By developing a new technique for labeling a subpopulation of the actin cytoskeleton using photoactivatable-EGFP-actin, we were able to visualize previously unobserved lateral movement of newly polymerized actin filaments. We have observed that this newly described motion of actin is accompanied by synchronous lateral movement and clustering of beta 1 integrins. The integrin clustering produces primed but unligated beta1 integrins along the leading edge of fibroblast lamella and growth cone filipodia. These results suggest that actin positioning activates integrins to provide a mechanism for cells to seek new adhesion sites and preferred directions of migration.