The function of the scaffolding proteins Fez1 and Fez2 in polarized transport and neuronal migration

神经元高度极化结构的建立和维持对神经元网络的形成和功能至关重要，并依赖于特定物质的选择性运输到轴突和树突。在轴突形成之前，运输过程的极化和囊泡和线粒体进入未来轴突的运输增加。连接分子马达及其货物的支架蛋白是连接信号通路和运输过程的重要因素。然而，调节货物和分子马达的联系并将它们引导到特定细胞位置的分子机制仍然知之甚少。驱动蛋白-1是调节囊泡和线粒体运输的分子马达之一。在没有货物的情况下，Kinesin-1呈自抑制构象。Kinesin-1被支架蛋白JNK相互作用蛋白1(Jip1)和伸缩伸长Zeta 1(Fez1)结合激活。海马神经元Fez1基因的敲除影响轴突的形成和线粒体的运输。令人惊讶的是，Fez1基因敲除小鼠是存活的，并且在神经系统发育方面没有表现出缺陷。没有重大发育缺陷的原因可能是Fez2基因的冗余，但到目前为止，Fez2的生理功能还没有被研究过。运输过程不仅在轴突形成过程中很重要，而且在神经元迁移过程中也很重要。我们对条件性Rap1a；Rap1b双基因敲除小鼠的分析表明，Rap1 GTP酶对于放射状胶质细胞和神经元的极化是必不可少的。Rap1 GTP酶的一个功能是引导钙粘附素在迁移神经元中的运输。因此，我们已经开始分析Fez1和Fez2的功能，以了解极化运输的分子机制。我们的初步实验表明，Fez2定位于线粒体，就像已经报道的Fez1一样。在培养的海马神经元中，Fez2基因的敲除导致轴突形成的缺陷。此外，我们观察到在体外电穿孔Fez2基因敲除结构后，皮层切片培养中的神经元迁移缺陷。在这个项目中，我们将研究FEZ蛋白在线粒体和特定囊泡运输中的功能及其在神经元迁移中的作用。我们将使用分离的海马神经元的原代培养，切片准备中神经元迁移的活细胞成像，以及Fez2基因敲除小鼠进行这些实验。

The establishment and maintenance of the highly polarized architecture of neurons is essential for the formation and functionality of neuronal networks and depends on the selective transport of specific cargos into axons and dendrites. Axon formation is preceded by the polarization of transport processes and an increased transport of vesicles and mitochondria into the future axon. Scaffolding proteins that link molecular motors and their cargo are important factors that connect signaling pathways and transport processes. However, the molecular mechanisms that regulate the association of cargo and molecular motors and direct them to a specific cellular location are still poorly understood. One of the molecular motors that mediate the transport of vesicles and mitochondria is kinesin-1. Kinesin-1 assumes an autoinhibited conformation in the absence of cargo. Kinesin-1 is activated by the binding of the scaffolding proteins Jnk interacting protein 1 (Jip1) and Fasciculation elongation zeta 1 (Fez1). Knockdown of Fez1 in cultured hippocampal neurons affects axon formation and the transport of mitochondria. Surprisingly, Fez1 knockout mice are viable and do not show defects in the development of the nervous system. The absence of major developmental defects is attributed to a possible redundancy with Fez2 but the physiological function of Fez2 has not been explored so far. Transport processes are important not only during axon formation but also during neuronal migration. Our analysis of conditional Rap1a;Rap1b double knockout mice showed that Rap1 GTPases are essential for the polarization of radial glia and neurons. One function of Rap1 GTPases is to direct cadherin trafficking in migrating neurons. We have therefore begun to analyze the function of Fez1 and Fez2 to understand the molecular mechanisms underlying polarized transport. Our preliminary experiments show that Fez2 localizes to mitochondria as already reported for Fez1. Knockdown of Fez2 in cultured hippocampal neurons results in defects in axon formation. In addition, we observed neuronal migration defects in cortical slice cultures after ex vivo electroporation of a Fez2 knockdown construct. In this project, we will investigate the function of Fez proteins in the transport of mitochondria and specific vesicles and its role in neuronal migration. We will use primary cultures of dissociated hippocampal neurons, live cell imaging of neuronal migration in slice preparations, and Fez2 knockout mice for these experiments.