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Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons
- Source :
- Journal of visualized experiments : JoVE. (173)
- Publication Year :
- 2021
-
Abstract
- Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.
- Subjects :
- Nuclear Envelope
General Chemical Engineering
Active Transport, Cell Nucleus
Digitonin
General Biochemistry, Genetics and Molecular Biology
chemistry.chemical_compound
Mice
medicine
Animals
Humans
Nuclear pore
Nuclear membrane
Cell Nucleus
Neurons
General Immunology and Microbiology
Chemistry
General Neuroscience
Neurodegeneration
medicine.disease
Cell biology
Membrane
medicine.anatomical_structure
Cytoplasm
Nucleocytoplasmic Transport
Nuclear Pore
Nuclear transport
HeLa Cells
Subjects
Details
- ISSN :
- 1940087X
- Issue :
- 173
- Database :
- OpenAIRE
- Journal :
- Journal of visualized experiments : JoVE
- Accession number :
- edsair.doi.dedup.....e8905d00fd57cf7f1b2fa7b8669cb5d6