Shootin1 Acts in Concert with KIF20B to Promote Polarization of Migrating Neurons
Shootin1 Acts in Concert with KIF20B to Promote Polarization of Migrating Neurons
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DOI：
10.1523/jneurosci.5425-12.2013
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发表时间：
2013-07
期刊：
影响因子：
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通讯作者：
T. Sapir;Talia Levy;Akira Sakakibara;A. Rabinkov;T. Miyata;O. Reiner
中科院分区：
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作者：
T. Sapir;Talia Levy;Akira Sakakibara;A. Rabinkov;T. Miyata;O. Reiner
Shootin 1被认为在调节原代海马神经元的极化中起作用。为了更好地了解Shootin 1在发育中的大脑中可能发挥的作用，我们确定了驱动蛋白超家族的成员KIF 20 B，作为一种新的Shootin 1相互作用蛋白和Shootin 1与微管相互作用的潜在介体。将KIF 20 B/Shootin 1结合定位于57 aa KIF 20 B序列，其用作显性阴性片段。通过基于表面等离子体共振的技术证实了该肽（MBD）与Shootin 1之间的直接相互作用，并在10−7 m范围内测定了亲和力。这些蛋白质在发育中的大脑中表达，并在体内形成复合物，基于免疫共沉淀实验和原代神经元中的免疫共染色。在原代海马神经元中，Kif 20 b敲低减少了Shootin 1向发育中轴突的动员，如通过光漂白分析后的免疫染色和荧光恢复所证明的，这表明Shootin 1是一种新的KIF 20 B货物。靶向Shootin 1的shRNA减少了生长锥中PIP 3的积累，Kif 20 b shRNA也是如此。在发育中的小鼠大脑中，Kif 20 b敲低或Kif 20 B最小结合结构域的表达抑制神经元迁移，体内迁移试验表明Shootin 1/Kif 20 b在相同的遗传途径中起作用。心室下区多极细胞的延时成像显示，下调Shootin 1或Kif 20 b的水平阻碍了多极细胞向双极细胞的转变。总的来说，我们的数据表明Shootin 1/KIF 20 B相互作用的锥体神经元极化和迁移的动态过程的重要性。
Shootin1 has been ascribed a role in regulating polarization of primary hippocampal neurons. To better understand the possible role of Shootin1 in the developing brain, we identified a member of the kinesin superfamily, KIF20B, as a novel Shootin1 interacting protein and a potential mediator of Shootin1 interaction with microtubules. KIF20B/Shootin1 binding was mapped to a 57 aa KIF20B sequence, which was used as a dominant-negative fragment. Direct interaction between that peptide (MBD) and Shootin1 was confirmed by surface plasmon resonance-based technology and the affinity was determined in the 10−7 m range. The proteins are expressed in the developing brain and formed a complex in vivo based on coimmunoprecipitation experiments and coimmunostaining in primary neurons. In primary hippocampal neurons Kif20b knockdown reduced Shootin1 mobilization to the developing axon, as evidenced by immunostaining and fluorescence recovery after photobleaching analysis, suggesting that Shootin1 is a novel KIF20B cargo. shRNA targeting of Shootin1 reduced PIP3 accumulation in the growth cone, as did Kif20b shRNA. In the developing mouse brain, Kif20b knockdown or expression of the KIF20B minimal binding domain inhibited neuronal migration, and in vivo migration assays suggested that Shootin1/Kif20b acts in the same genetic pathway. Time-lapse imaging of multipolar cells in the subventricular zone revealed that downregulating levels of either Shootin1 or Kif20b hindered the transition from multipolar to bipolar cells. Collectively, our data demonstrate the importance of the Shootin1/KIF20B interaction to the dynamic process of pyramidal neuronal polarization and migration.