The role of RNA methylation in cytoskeleton regulation during axon development

RNA修饰N6-甲基腺苷（m6 A）已被发现在轴突和突触定位的mRNA，其局部翻译是轴突生长，突触发生和突触可塑性所需的。然而，负责m6 a介导的有丝分裂后神经元发育的调节的分子机制还不完全清楚。在JSPS奖学金期间，我们的目标是通过APC翻译调节来揭示m6 A epitranscriptomic修饰在轴突生长锥中微管和肌动蛋白动力学调节中的作用。到目前为止，我们已经证实了APC蛋白在发育神经元中在m6 A阅读器YTHDF 1蛋白敲低后下调。我们对轴突生长锥中的EB 3-GFP彗星进行了实时成像，以评估YTHDF 1敲低对微管动力学的影响。与对照组相比，我们观察到MT生长速度的增加以及YTHDF 1-KD神经元生长锥中EB 3彗星的累积距离的增加。与此同时，我们进行免疫荧光染色的酪氨酸化微管和F-肌动蛋白在培养的神经元转染的shControl或shYTHDF 1，以确定如何YTHDF 1的失调影响典型的生长锥细胞骨架结构。我们观察到YTHDF 1的下调导致动态酪氨酸化MT对生长锥的侵袭增加，并且生长锥周围的F-肌动蛋白染色面积减少。

RNA modification N6-methyl-adenosine (m6A) has been found in axonal and synaptically localized mRNAs whose local translation is required for axon growth, synaptogenesis, and synaptic plasticity. However, the molecular mechanisms responsible for m6a-mediated regulation of postmitotic neuronal development are not completely understood. During the JSPS fellowship, we aim to uncover the role of m6A epitranscriptomic modification in the regulation of microtubule and actin dynamics in the axonal growth cone through APC translation regulation. So far, we have confirmed the down-regulation of APC protein in developing neurons upon m6A reader YTHDF1 protein knock-down. We conducted live-imaging of EB3-GFP comets in the axonal growth cone to evaluate the consequences of YTHDF1 knock-down on microtubule dynamics. We observed an increase in the MT growth speed as well as an increase in the cumulative distance of EB3 comets in the growth cone of YTHDF1-KD neurons in comparison to control. In parallel, we performed immunofluorescence stainings of tyrosinated microtubules and F-actin in cultured neurons transfected with either shControl or shYTHDF1 to determine how the deregulation of YTHDF1 affects the typical growth cone cytoskeleton architecture. We observed that the downregulation of YTHDF1 leads to an increase of the invasion of the growth cone by the dynamic tyrosinated MTs and a decrease of the F-actin staining area in the periphery of the growth cone.