Vanneste Joni, Vercruysse Thomas, Van Damme Philip, Van Den Bosch Ludo, Daelemans Dirk
Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI) KU Leuven - University of Leuven, Leuven, Belgium.
Center for Brain & Disease Research - Laboratory of Neurobiology, VIB, Leuven, Belgium.
Bio Protoc. 2020 Jun 20;10(12):e3659. doi: 10.21769/BioProtoc.3659.
Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these experiments can be performed without the need for expensive equipment and have the potential to be upscaled for high-throughput screening purposes.
核质运输缺陷被认为在神经退行性疾病中起作用，包括肌萎缩侧索硬化症（ALS）。鉴于这一过程的重要性和复杂性，了解这些异常何时发生以及涉及哪些途径至关重要。在这里，我们利用CRISPR-Cas9技术设计稳定表达荧光蛋白的细胞系，这些荧光蛋白通过选择的核转运蛋白在细胞核和细胞质之间穿梭。为了验证该方案，我们在存在疾病相关肽多聚-PR的情况下测量了一条与ALS相关的核质运输途径。该技术允许在神经退行性疾病相关背景下测量完整细胞中特定的活性核质运输途径。此外，这些实验无需昂贵设备即可进行，并且有扩大规模用于高通量筛选目的的潜力。
