Sorbonne Universités UPMC Université Paris 06, INSERM U974, CNRS FRE3617, Center for Research in Myology, GH Pitié Salpêtrière, 47 Boulevard de l'Hôpital, 75013 Paris, France.
Institute of Medical Biology, Agency for Science, Technology and Research (A(∗)STAR), 8A Biomedical Grove, No. 06-06 Immunos, Singapore 138648, Singapore.
Curr Biol. 2017 Oct 9;27(19):2999-3009.e9. doi: 10.1016/j.cub.2017.08.031. Epub 2017 Sep 28.
The nucleus is the main microtubule-organizing center (MTOC) in muscle cells due to the accumulation of centrosomal proteins and microtubule (MT) nucleation activity at the nuclear envelope (NE) [1-4]. The relocalization of centrosomal proteins, including Pericentrin, Pcm1, and γ-tubulin, depends on Nesprin-1, an outer nuclear membrane (ONM) protein that connects the nucleus to the cytoskeleton via its N-terminal region [5-7]. Nesprins are also involved in the recruitment of kinesin to the NE and play a role in nuclear positioning in skeletal muscle cells [8-12]. However, a function for MT nucleation from the NE in nuclear positioning has not been established. Using the proximity-dependent biotin identification (BioID) method [13, 14], we found several centrosomal proteins, including Akap450, Pcm1, and Pericentrin, whose association with Nesprin-1α is increased in differentiated myotubes. We show that Nesprin-1α recruits Akap450 to the NE independently of kinesin and that Akap450, but not other centrosomal proteins, is required for MT nucleation from the NE. Furthermore, we demonstrate that this mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a nonsense mutation within the SYNE1 gene (23560 G>T) encoding Nesprin-1 [15, 16]. Finally, using computer simulation and cell culture systems, we provide evidence for a role of MT nucleation from the NE on nuclear spreading in myotubes. Our data thus reveal a novel function for Nesprin-1α/Nesprin-1 in nuclear positioning through recruitment of Akap450-mediated MT nucleation activity to the NE.
核是肌肉细胞中的主要微管组织中心（MTOC），这是由于中心体蛋白在核膜（NE）处的积累和微管（MT）成核活性[1-4]。中心体蛋白的重定位，包括 Pericentrin、Pcm1 和 γ-微管蛋白，取决于核膜外周蛋白（ONM）Nesprin-1，它通过其 N 端区域将核与细胞骨架连接[5-7]。Nesprins 还参与了驱动蛋白向 NE 的募集，并在骨骼肌细胞中发挥核定位的作用[8-12]。然而，MT 从 NE 成核在核定位中的作用尚未确定。使用邻近依赖性生物素鉴定（BioID）方法[13,14]，我们发现了几种中心体蛋白，包括 Akap450、Pcm1 和 Pericentrin，它们与 Nesprin-1α 的结合在分化的肌管中增加。我们表明，Nesprin-1α 独立于驱动蛋白将 Akap450 招募到 NE，并且只有 Akap450，而不是其他中心体蛋白，是从 NE 进行 MT 成核所必需的。此外，我们证明，在携带编码 Nesprin-1 的 SYNE1 基因（23560 G>T）无意义突变的先天性肌营养不良症患者肌管中，这种机制被破坏[15,16]。最后，我们使用计算机模拟和细胞培养系统，为 MT 从 NE 成核在肌管中的核扩展提供了证据。我们的数据因此揭示了 Nesprin-1α/Nesprin-1 通过招募 Akap450 介导的 MT 成核活性到 NE 在核定位中的新功能。
