Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
J Cell Sci. 2021 Mar 11;134(6):jcs256180. doi: 10.1242/jcs.256180.
Ranbp2 (also known as Nup358) is a member of the nucleoporin family, which constitutes the nuclear pore complex. Ranbp2 localizes at the nuclear membrane and was recently reported at the axon initial segment (AIS). However, we show that the anti-Ranbp2 antibody used in previous studies is not specific for Ranbp2. We mapped the antibody binding site to the amino acid sequence KPLQG, which is present in both Ranbp2 and neurofascin (Nfasc), a well-known AIS protein. After silencing neurofascin expression in neurons, the AIS was not stained by the antibody. Surprisingly, an exogenously expressed N-terminal fragment of Ranbp2 localizes at the AIS. We show that this fragment interacts with stable microtubules. Finally, using CRISPR/Cas9 in primary cultured neurons, we inserted an HA-epitope tag at N-terminal, C-terminal or internal sites of the endogenously expressed Ranbp2. No matter the location of the HA-epitope, endogenous Ranbp2 was found at the nuclear membrane but not the AIS. These results show that endogenously expressed Ranbp2 is not found at AISs.This article has an associated First Person interview with the first author of the paper.
Ranbp2（也称为 Nup358）是核孔复合体的核孔蛋白家族的成员。Ranbp2 定位于核膜，最近在轴突起始段（AIS）中也有报道。然而，我们发现以前研究中使用的抗 Ranbp2 抗体并非特异性针对 Ranbp2。我们将抗体结合位点定位到氨基酸序列 KPLQG，该序列存在于 Ranbp2 和神经束蛋白（Nfasc）中，后者是一种众所周知的 AIS 蛋白。在神经元中沉默神经束蛋白的表达后，该抗体不再对 AIS 进行染色。令人惊讶的是，Ranbp2 的 N 端片段在体外表达后可以定位到 AIS。我们表明该片段与稳定的微管相互作用。最后，我们在原代培养神经元中使用 CRISPR/Cas9，在内源性表达的 Ranbp2 的 N 端、C 端或内部插入了一个 HA 表位标签。无论 HA 表位的位置如何，内源性 Ranbp2 都位于核膜上，而不是 AIS。这些结果表明内源性表达的 Ranbp2 不在 AIS 上。本文附有该论文第一作者的第一人称采访。
