Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
Joni Vanneste,
Thomas Vercruysse,
Philip Van Damme,
Ludo Van Den Bosch,
Dirk Daelemans
Affiliations
Joni Vanneste
Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI) KU Leuven – University of Leuven, Leuven, BelgiumCenter for Brain & Disease Research – Laboratory of Neurobiology, VIB, Leuven, Belgium
Thomas Vercruysse
Department of Microbiology, Immunology and Transplantation – Laboratory of Virology and Chemotherapy, KU Leuven – Rega Institute for Medical Research, Leuven, Belgium
Philip Van Damme
Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI) KU Leuven – University of Leuven, Leuven, BelgiumCenter for Brain & Disease Research – Laboratory of Neurobiology, VIB, Leuven, Belgium, Department of Neurology, University Hospitals Leuven, Leuven, Belgium
Ludo Van Den Bosch
Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI) KU Leuven – University of Leuven, Leuven, BelgiumCenter for Brain & Disease Research – Laboratory of Neurobiology, VIB, Leuven, Belgium
Dirk Daelemans
Department of Microbiology, Immunology and Transplantation – Laboratory of Virology and Chemotherapy, KU Leuven – Rega Institute for Medical Research, Leuven, Belgium
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.
