GENES ESSENTIAL TO MOTOR AXON GUIDANCE IN DROSOPHILIA

描述：结构类比强烈表明，DLAR作为一个 接收未知的导航信号 这项建议旨在阐明 DLAR在指导下正确运行的结构要求 决策，以表征DLAR与 更好地表征信号通路，并表征新的突变， 模拟DLAR突变的基因 在第一个具体目标中，将在DLAR中进行一些工程改造。 测试了它们在DLAR突变体中拯救DLAR功能的能力 背景 构建体将通过P-元件被工程化为多个线 插入，然后与表达GAL 4的DLAR突变株系杂交， 有丝分裂后神经元 GAL 4激活了一个驱动程序，导致表达 这样就可以测试它的救援活动。 救援已 在DLAR完好的情况下已经实现。 待测结构包括： 细胞外结构域缺失，细胞质结构域缺失， 单独的两个磷酸酶结构域，以及失活点 每个磷酸酶结构域内的突变。 只要工程师 适当表达构建体，这些实验将可能 确定DLAR是否作为受体起作用，以及其磷酸酶是否 活动是其指导职能所必需的。 在一个 之前的评审员，DLAR将直接检测磷酸酶活性， 适当的测定。 这里唯一的困难是很难知道 DLAR的作用基础是什么。 具体目标#2有两个单独的部分。 第一个取决于 观察到DLAR表型与Rac中的弱突变协同作用， 并且DLAR的人类亲属通过一种新的途径影响Rho和Rac活性。 鸟嘌呤核苷酸交换因子，命名为Trio。 因此拟 进一步研究显性消极和组成性积极（或 过度表达）Rho、Rac和CD 42对运动神经元前进、引导和运动神经元分化的影响。 DLAR表型。 GAL 4-UAS系统将用于驱动 适当地构建。 第二组拟议的实验是 其动机是发现DLAR表型受到突变的抑制， 在ABL酪氨酸激酶中。 DLAR的磷酸酶活性可能与DLAR的磷酸酶活性有关。 在某种程度上与能够信号传导中的激酶活性竞争 通路 这种可能的相互作用将由 用DLAR和abl的其它已知成员构建双突变体 通路 激酶活性和激酶非活性形式的abl也将被 比较它们抑制DLAR表型的能力。 这些 实验提供了更好地表征信号的潜力， 参与DLAR功能的通路。 具体目标#3涉及新突变的进一步分析， 环周肌，模仿DLAR的表现。 几个重要问题将是 解决：突变将被映射，导航的特异性 它引起的错误将被更好地描述，新的等位基因将被 收集，并将进行马赛克分析，以确定是否 细胞自主活动。 最后一个实验非常 重要. 如果在运动神经元中需要环绕运动， 可能是DLAR信号通路的一员。 如果外面需要 运动神经元，它可能是DLAR的配体。 所有 这些研究将使用该领域的标准技术， 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.