GENES ESSENTIAL TO MOTOR AXON GUIDANCE IN DROSOPHILIA

描述：结构类比强烈表明DLAR作为一种 未知导航提示的接收器。这项提议旨在澄清 DLAR在制导中正确发挥作用的结构要求 决策，以描述DLAR和组件之间的交互作用 更好地表征信号通路，并表征一种新的突变 DLAR的表观复制突变。 在第一个具体目标中，DLAR中的一些工程更改将是 测试它们拯救DLAR突变体中DLAR功能的能力 背景资料。构件将由P-Element设计成多行 插入，然后与表达GAL4的DLAR突变体系杂交 有丝分裂后神经元。GAL4激活一个驱动程序，该驱动程序导致 这样就可以测试它的救援活动了。营救行动已经 已经在完整的DLAR下实现了。要测试的构造包括： 胞外区被删除，胞内区被删除，每一个都被删除 两个磷酸酶结构域的分别，以及失活点 每个磷酸酶结构域内的突变。只要工程设计的 结构得到适当的表达，这些实验很可能 确定DLAR是否作为受体，以及其磷酸酶是否 它的引导功能需要活动。在……的建议下 之前的审查者，DLAR将通过以下方式直接测试磷酸酶活性 一个合适的化验方法。这里唯一的困难是很难知道 DLAR的合适底物是什么？ 具体目标2有两个独立的部分。第一部分取决于 观察到DLAR表型与RAC中的弱突变协同作用 DLAR的人类亲属通过一种 鸟嘌呤核苷酸交换因子命名为Trio。因此，建议将 进一步研究显性消极和结构性积极(或 Rho、Rac和CD42在运动神经元推进、引导和 DLAR表型。GAL4-UAS系统将被用来驱动表达 适当地构造。第二组拟议的实验是 原因是发现DLAR表型被突变抑制 在ABL酪氨酸激酶中。DLAR的磷酸酶活性可能 在某种程度上与Able信号中的激酶活性竞争 路径。这一可能的相互作用将由 用DLAR和ABL其他已知成员构建双突变体 路径。激活型和非激活型ABL也将 比较它们抑制DLAR表型的能力。这些 实验提供了更好地描述信号的可能性 参与DLAR功能的通路。 具体目标#3涉及对新突变的进一步分析， 在圆周上，这是DLAR。几个重要的问题将是 解决：突变将被映射，导航的特殊性 它引起的错误将被更好地描述，新的等位基因将被 并将执行镶嵌分析以确定是否 环状突起细胞的自主作用。这最后一个实验是非常 很重要。如果运动神经元需要环行，那么它 可能是DLAR信号通路的成员。如果外面需要的话 运动神经元，它可能是DLAR仍然难以捉摸的配体。所有的 这些研究将使用该领域的标准技术，并将 对这个调查员来说是可行的。

DESCRIPTION: Structural analogies strongly suggest that DLAR acts as a receptor for an unknown navigational cue. This proposal aims to elucidate the structural requirements for DLAR to function correctly in guidance decisions, to characterize interactions between DLAR and components of better characterized signaling pathways, and to characterize a new mutation that phenocopies mutations in DLAR. In the first specific aim a number of engineered alterations in DLAR will be tested for their ability to rescue DLAR function in a DLAR mutant background. Constructs will be engineered into multiple lines by P-element insertion and then crossed into a DLAR mutant line that expresses GAL4 in postmitotic neurons. GAL4 activates a driver that causes the expression of the construct so that it can be tested for rescuing activity. Rescue has already been achieved with intact DLAR. Constructs to be tested include: extracellular domain deleted, cytoplasmic domain deleted, deletion of each of the two phosphatase domains individually, and inactivating point mutations within each of the phosphatase domains. So long as the engineered constructs are appropriately expressed, these experiments will likely determine whether DLAR acts as a receptor and whether its phosphatase activity is required for its guidance function. At the suggestion of a previous reviewer, DLAR will be tested directly for phosphatase activity by an appropriate assay. The only difficulty here is that it is hard to know what the appropriate substrate is for DLAR to act upon. There are two separate parts to specific aim #2. The first depends upon the observation that the DLAR phenotype is synergized by a weak mutation in Rac and that a human relative of DLAR affects Rho and Rac activity through a guanine nucleotide exchange factor named Trio. It is therefore proposed to further study the effects of dominant negative and constitutively active (or overexpressed) Rho, Rac, and CD42 on motor neuron advance, guidance, and the DLAR phenotype. A GAL4-UAS system would be used to drive expression of the constructs appropriately. The second set of proposed experiments are motivated by the finding that the DLAR phenotype is suppressed by a mutation in the abl tyrosine kinase. It is possible that DLAR's phosphatase activity is somehow in competition with kinase activity in the able signaling pathway. This possible interaction will be further examined by the construction of double mutants with DLAR and other known members of the abl pathway. Kinase-active and kinase-inactive forms of abl will also be compared for their ability to suppress the DLAR phenotype. These experiments offer the potential of better characterizing the signaling pathway involved in DLAR function. Specific aim #3 involves the further analysis of a new mutation, circumfirential, that phenocopies DLAR. Several important issues will be addressed: the mutation will be mapped, the specificity of the navigation errors it evokes will be better characterized, new alleles will be collected, and a mosaic analysis will be performed to determine if circumfirential acts cell autonomously. This last experiment is very important. If circumfirential is required in the motor neuron, then it could be a member of the DLAR signaling pathway. If it is required outside the motor neuron, it could be the still elusive ligand for DLAR. All of these studies will use techniques that are standard in the field and are feasible for this investigator.