Proteoglycans in Growth Factor Signaling and Development

说明(申请人提供)：硫酸乙酰肝素蛋白多糖(HSPGs)是存在于细胞表面和细胞外基质中的丰富分子。在过去的十年中，这些分子被证明在控制细胞对分泌的生长因子的反应以及信号分子在基质中的分布方面发挥了关键作用。我们的长期目标是了解这些分子在发育过程中如何影响细胞行为和组织的组装。最近，我的实验室使用模式生物果蝇进行的研究表明，HSPGs在塑造无翼(Wg)形态原的梯度、控制视觉系统中的轴突引导以及影响神经肌肉突触的形式和功能方面发挥了作用。我们提出的研究有三个广泛但相关的目标，1)确定HSPGs是否通过调节内吞作用和细胞内转运来控制Wg的分布，2)确定HSPGs是否通过控制内吞作用和生长因子信号来影响突触组装，以及3)确定HSPGs是否控制生长锥体对指导线索的反应，或者指导线索在脑细胞外基质中的分布。使用针对Wg的抗体和不同内体隔间的标记，我们将评估Wg在影响特定HSPG(Dally-like)和分泌酶Notom(从细胞表面移除Dip)的突变体中的运输情况。我们认为Wg梯度的区域控制是通过发展翼中的Dip和Notom活动相结合的方式实现的。我们对突触的研究采用了直接测量内吞作用的方法，包括使用掺入内吞小泡的荧光染料，可视化对突触发育至关重要的生长因子，以及基因研究以确定突触中受HSPG影响的信号系统。最后，我们将使用遗传策略来确定HSPG必须在哪些细胞类型中表达才能允许视觉系统中正常的轴突引导，以及哪些轴突引导系统需要这些分子。HSPGs控制由多种生长因子介导的信号传递，这些生长因子影响免疫系统功能中的肿瘤发展、血管生成、组织修复和趋化因子反应。硫酸乙酰肝素的生物合成是正常骨骼发育所必需的，而特定的HSPG会影响人类的生长调节。因此，在影响人类疾病的许多基本过程中，对这些不同分子的详细细胞和分子理解是至关重要的。

DESCRIPTION (provided by applicant): Heparan sulfate proteoglycans (HSPGs) are abundant molecules found on cell surfaces and in the extracellular matrix. In the last decade these molecules have been shown to play critical roles in controlling responses of cells to secreted growth factors, and the distributions of signaling molecules in the matrix. Our long-term objectives are to understand how these molecules affect cellular behavior and the assembly of tissues during development. Recent work in my lab using the model organism Drosophila has demonstrated roles for HSPGs in shaping the gradient of the Wingless (Wg) morphogen, controlling axon guidance in the visual system, and influencing the form and function of the neuromuscular synapse. The research we propose has three broad but related aims, 1) determine if HSPGs control the distribution of Wg by regulating endocytosis and intracellular trafficking, 2) establish if HSPGs affect synapse assembly by controlling endocytosis and growth factor signaling, and 3) determine if HSPGs govern responses of growth cones to guidance cues, or the distributions of guidance cues in the extracellular matrix of the brain. Using antibodies directed against Wg and markers for different endosomal compartments, we will assess the trafficking of Wg in mutants affecting a specific HSPG, Dally-like, and a secreted enzyme, Notum, that removes Dip from cell surfaces. We propose that regional control of Wg gradients are achieved by a combination of Dip and Notum activity in the developing wing. Our studies of the synapse employ direct measures of endocytosis using fluorescent dyes that are incorporated into endocytosed vesicles, visualization of growth factors critical for synapse development, and genetic studies to identify the signaling systems affected by HSPGs in the synapse. Finally, we will use genetic strategies to determine in what cell types HSPG must be expressed to permit normal axon guidance in the visual system, and what axon guidance systems require these molecules. HSPGs control signaling mediated by numerous growth factors that affect tumor development, angiogenesis, tissue repair, and chemokine responses in immune system function. Heparan sulfate biosynthesis is required for normal bone development and specific HSPGs affect growth regulation in humans. A detailed cellular and molecular understanding of these diverse molecules is therefore of fundamental importance in a number of fundamental processes affecting human disease.