Department of Pathology and Cell Biology, Columbia University Medical Center, New York City, NY 10032, USA; Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus of Gualtar, Braga 4710-057, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães 4710-057, Portugal.
Department of Pathology and Cell Biology, Columbia University Medical Center, New York City, NY 10032, USA.
Curr Biol. 2020 Aug 17;30(16):3116-3129.e4. doi: 10.1016/j.cub.2020.05.091. Epub 2020 Jul 2.
Vertebrate brain development depends on a complex program of cell proliferation and migration. Post-mitotic neuronal migration in the developing cerebral cortex involves Nesprin-2, which recruits cytoplasmic dynein, kinesin, and actin to the nuclear envelope (NE) in other cell types. However, the relative importance of these interactions in neurons has remained poorly understood. To address these issues, we performed in utero electroporation into the developing rat brain to interfere with Nesprin-2 function. We find that an ∼100-kDa "mini" form of the ∼800-kDa Nesprin-2 protein, which binds dynein and kinesin, is sufficient, remarkably, to support neuronal migration. In contrast to dynein's role in forward nuclear migration in these cells, we find that kinesin-1 inhibition accelerates neuronal migration, suggesting a novel role for the opposite-directed motor proteins in regulating migration velocity. In contrast to studies in fibroblasts, the actin-binding domain of Nesprin-2 was dispensable for neuronal migration. We find further that, surprisingly, the motor proteins interact with Nesprin-2 through the dynein/kinesin "adaptor" BicD2, both in neurons and in non-mitotic fibroblasts. Furthermore, mutation of the Nesprin-2 LEWD sequence, implicated in nuclear envelope kinesin recruitment in other systems, interferes with BicD2 binding. Although disruption of the Nesprin-2/BicD2 interaction severely inhibited nuclear movement, centrosome advance proceeded unimpeded, supporting an independent mechanism for centrosome advance. Our data together implicate Nesprin-2 as a novel and fundamentally important form of BicD2 cargo and help explain BicD2's role in neuronal migration and human disease.
脊椎动物大脑的发育依赖于一个复杂的细胞增殖和迁移的程序。在发育中的大脑皮层中，有丝分裂后神经元的迁移涉及到核膜（NE）上的细丝蛋白-2（Nesprin-2），它将细胞质动力蛋白、驱动蛋白和肌动蛋白募集到其他细胞类型的核包膜上。然而，这些相互作用在神经元中的相对重要性仍知之甚少。为了解决这些问题，我们在发育中的大鼠大脑中进行了体内电穿孔，以干扰 Nesprin-2 的功能。我们发现，一种大约 100kDa 的“迷你”形式的大约 800kDa 的 Nesprin-2 蛋白，它结合动力蛋白和驱动蛋白，足以显著支持神经元的迁移。与这些细胞中核向前迁移中动力蛋白的作用相反，我们发现驱动蛋白-1 的抑制加速了神经元的迁移，这表明相反方向的运动蛋白在调节迁移速度方面有一个新的作用。与成纤维细胞的研究不同，Nesprin-2 的肌动蛋白结合结构域对于神经元的迁移是可有可无的。我们进一步发现，令人惊讶的是，在神经元和非有丝分裂的成纤维细胞中，运动蛋白通过动力蛋白/驱动蛋白“适配器”BicD2 与 Nesprin-2 相互作用。此外，Nesprin-2 的 LEWD 序列的突变，在其他系统中被牵连到核包膜的驱动蛋白募集，干扰了 BicD2 的结合。尽管 Nesprin-2/BicD2 相互作用的破坏严重抑制了核的运动，但中心体的前进仍然不受阻碍，这支持了中心体前进的一个独立机制。我们的数据共同表明，Nesprin-2 是一种新型的、非常重要的 BicD2 货物形式，并有助于解释 BicD2 在神经元迁移和人类疾病中的作用。
