Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Published: July 09, 2021

doi:

Lindsey R. Hayes, Lauren Duan, Svetlana Vidensky, Petr Kalab

¹Department of Neurology,Johns Hopkins School of Medicine, ²Whiting School of Engineering,Johns Hopkins University

Summary

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

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.

Introduction

Disruption of nucleocytoplasmic transport, the regulated trafficking of proteins and RNA between the nucleus and cytoplasm, is increasingly implicated in the pathogenesis of neurodegenerative diseases (recently reviewed¹). We and others have reported structural and functional disruption of the nucleocytoplasmic transport apparatus in postmortem tissue and animal models of C9orf72 amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), Alzheimer’s disease, and Huntington's disease²^,³^,⁴^,⁵. The mechanisms and functional consequences of nucleocytoplasmic transport disruption for neurodegeneration, and approaches for therapeutic rescue, are areas of ongoing investigation.

Nuclear pores are large transmembrane complexes of ~30 nucleoporin proteins that permit diffusion of small molecules across the nuclear membrane but increasingly restrict the passage of cargoes >40 kD via a permeability barrier of phenylalanine-glycine (FG)-rich nucleoporins in the central channel⁶. Larger cargoes containing nuclear localization signal (NLS) or nuclear export signal (NES) sequences undergo active, receptor-mediated transport across the pore via nuclear transport receptors (importins and exportins) and a steep gradient of the small GTPase Ran across the nuclear membrane (recently reviewed⁷). A wide array of methods has been developed to analyze nuclear transport dynamics in cultured cells, including the trafficking of endogenous cargoes and tagged reporter constructs that serve as substrates for the major subclasses of transport receptors. Such approaches have been readily adapted to neurons²^,⁵^,⁸ and provide a readout of nuclear transport perturbations in the context of an intact, living cell. However, in live cell assays, the ability to directly manipulate nuclear transport reactions or investigate them in isolation from other cellular processes is limited.

In permeabilized cell assays, the plasma membrane is selectively perforated, and the cytoplasm is released, leaving the nuclear envelope and nuclear pore complexes intact and able to perform either passive or energy-dependent bidirectional transport⁹^,¹⁰. Such transport reactions can be readily reconstituted by adding whole-cell lysates, cytoplasmic fractions, or purified recombinant nuclear transport proteins and their cargos. Thus, permeabilized cell assays permit a broad range of biochemical or biophysical investigations, including delivery of recombinant or synthetic proteins and RNAs relevant to the study of neurodegenerative diseases.

Given reports of cell-specific differences in nuclear pore complex structure and transport dynamics¹¹^,¹², we aimed to adapt the permeabilized cell assay for use in primary neuronal cultures. Although widely used to analyze nuclear transport in immortalized cell lines, despite exhaustive literature search, we found no published reports of neuronal plasma membrane permeabilization that verified preservation of nuclear membrane integrity. Most protocols rely on digitonin, a detergent that targets the unique cholesterol composition of the plasma membrane, to perforate the nuclear membrane while leaving the nuclear membrane intact¹³. Our initial attempts using digitonin in primary mouse cortical neurons showed immediate loss of nuclear membrane integrity, evidenced by diffusion of a 70 kD fluorescent dextran into the nucleus. We hypothesized that nuclear envelope rupture might be caused by mechanical perturbation from loss of cytoplasmic support, and tested multiple methods of optimization, including molecular crowding, cytoskeletal stabilization, and alternate methods of cell lysis. Here, we detail a method of rapid hypotonic permeabilization using a concentrated bovine serum albumin (BSA) cushion to protect neuronal nuclei and facilitate downstream analysis of neuronal nuclear import. We recently used this method to evaluate the mechanism of dipeptide repeat protein disruption in C9orf72-ALS/FTD¹⁴ and anticipate that it will be broadly applicable to future studies of passive and active nuclear transport in primary neurons.

Protocol

First, the protocol describes the generation of primary neuronal cultures (step 1) and preparation of materials for the transport assay (step 2), followed by the transport assay itself (steps 3-4) and image acquisition and analysis (step 5). All methods described here were approved by the Animal Care and Use Committee (ACUC) of Johns Hopkins University. 1. Primary mouse cortical neuron cultures Prepare the stock solutions. Dissection buffer: Combine 50 mL of 10x HBSS, 1…

Representative Results

Selective permeabilization of the plasma membrane (Figure 1A) is the most critical step in the protocol and must be verified prior to proceeding with the analysis of nuclear import. Due to variations between each culture preparation, an initial titration plate is routinely run to identify the optimal, batch-specific concentrations of hypotonic Tris-HCl buffer and BSA cushion, as described in step 3. Under- and over-permeabilized cells are readily identified by confocal microscopy (<strong cl…

Discussion

The protocol detailed above provides a reliable and reproducible method for selectively permeabilizing the plasma membrane of primary mouse cortical neurons for nuclear import analysis. Here, an application of the method for nuclear import analysis of a direct importin β cargo (Rango) is shown, but this same approach can be used to analyze the passive and active import of a wide range of fluorescent cargoes. Permeabilization enables precise manipulation of the transport reaction in ways that are not feasible in inta…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was supported by NINDS K08NS104273 (to L.R.H.).

Materials

1 M HEPES	Gibco	15630-080
10x HBSS	Gibco	14185-052
32% paraformaldehyde	Electron Microscopy Sciences	15714-S
96-well optical glass plates	CellVis	P96-1.5H-N
ATP lithium salt	Millipore Sigma	11140965001
B27	Gibco	17504-044
Bio-Rad Protein Assay Kit II	Bio-Rad	5000002
BL21(DE3) E. coli	NEB	C2527H
Bovine serum albumin fraction V, heat shock, fatty acid free	Sigma-Aldrich	3117057001
Chromatography columns	Bio-Rad	7311550
Creatine kinase	Millipore Sigma	10127566001
Creatine phosphate	Millipore Sigma	10621722001
Dextran, Texas Red, 70,000 MW	Thermo Fisher	D1864
DNase I	Sigma-Aldrich	DN25
E15-16 timed pregnant C57BL/6J female mice	Jackson Laboratory	000664
Excel	Microsoft	N/A
Fetal bovine serum	Hyclone	SH30070.03
Glutamax	Gibco	35050-061
Glycerol	Thermo Fisher	15514011
GTP lithium salt	Millipore Sigma	11140957001
HALT protease inhibitor (100x)	Thermo Fisher	78439
HEK293T cells	ATCC	CRL-3216
HIS-Select HF Nickel affinity gel	Sigma-Aldrich	HO537
Hoechst 33342	Thermo Fisher	H1399
ImageExpress Micro Confocal High-content Imaging System	Molecular Devices	N/A	Used for time-lapse imaging
Imidazole	Millipore	I3386
Importazole	Sigma-Aldrich	SML0341
IPTG	Corning	46-102-RF
Laminin	Sigma-Aldrich	L2020
LB broth	Grainger	31FZ62
LSM800 confocal microscope	Zeiss	N/A	Used for dextran imaging
MetaXpress High Content Image Analysis Software	Molecular Devices	N/A
Neurobasal medium	Gibco	21103
Papain	Worthington	LS003126
Penicillin-streptomycin	Gibco	15140-122
Poly-D-Lysine	Sigma-Aldrich	P6407
Protease inhibitor cocktail	Millipore Sigma	11873580001
Rango Plasmid (pRSET Rango2/a1 + linkers)	N/A	N/A	pK44, containing N-terminal 6-His tag
SOC (super optimal broth with catabolite repression) media	Quality Biological	340-031-671
Sodium pyruvate	Gibco	11360-070
Spin-X UF concentrators (30K MWCO)	Corning	CLS431484
Trypsin-EDTA (0.05%)	Gibco	25300054

Referencias

Hutten, S., Dormann, D. Nucleocytoplasmic transport defects in neurodegeneration – Cause or consequence. Seminars in Cell & Developmental Biology. 5, 151-162 (2019).
Zhang, K., et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 525 (7567), 56-61 (2015).
Grima, J. C., et al. Mutant Huntingtin disrupts the nuclear pore complex. Neuron. 94 (1), 93-96 (2017).
Eftekharzadeh, B., et al. Tau protein disrupts nucleocytoplasmic transport in Alzheimer’s disease. Neuron. 99 (5), 925-927 (2018).
Coyne, A. N., et al. G4C2 repeat RNA initiates a POM121-mediated reduction in specific nucleoporins in C9orf72 ALS/FTD. Neuron. 107 (6), 1124-1140 (2020).
Timney, B. L., et al. Simple rules for passive diffusion through the nuclear pore complex. The Journal of Cell Biology. 215 (1), 57-76 (2016).
Paci, G., Caria, J., Lemke, E. A. Cargo transport through the nuclear pore complex at a glance. Journal of Cell Science. 134 (2), 247874 (2021).
Ding, B., Akter, M., Zhang, C. -. L. Differential Influence of Sample Sex and Neuronal Maturation on mRNA and Protein Transport in Induced Human Neurons. Frontiers in Molecular Neuroscience. 13, 46 (2020).
Adam, S. A., Marr, R. S., Gerace, L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. The Journal of Cell Biology. 111 (3), 807-816 (1990).
Brownawell, A. M., Holaska, J. M., Macara, I. G., Paschal, B. M. The use of permeabilized cell systems to study nuclear transport. Methods in Molecular Biology. 189, 209-229 (2002).
Raices, M., D’Angelo, M. A. Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions. Nature Reviews. Molecular Cell Biology. 13 (11), 687-699 (2012).
Ori, A., et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Molecular Systems Biology. 9, 648 (2013).
Colbeau, A., Nachbaur, J., Vignais, P. M. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochimica et Biophysica Acta. 249 (2), 462-492 (1971).
Hayes, L. R., Duan, L., Bowen, K., Kalab, P., Rothstein, J. D. C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import. eLife. 9, 51685 (2020).
Grote, P., Ferrando-May, E. In vitro assay for the quantitation of apoptosis-induced alterations of nuclear envelope permeability. Nature Protocols. 1 (6), 3034-3040 (2006).
Lowe, A. R., et al. Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner. eLife. 4, 04052 (2015).
Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R., Weis, K. Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature. 440 (7084), 697-701 (2006).
Soderholm, J. F., et al. Importazole, a small molecule inhibitor of the transport receptor importin-β. ACS Chemical Biology. 6 (7), 700-708 (2011).
Newmeyer, D. D., Forbes, D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 52 (5), 641-653 (1988).
Woerner, A. C., et al. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA. Science. 351 (6269), 173-176 (2016).
Khosravi, B., et al. Cytoplasmic poly-GA aggregates impair nuclear import of TDP-43 in C9orf72 ALS/FTLD. Human Molecular Genetics. 26 (4), 790-800 (2016).
Chou, C. -. C., et al. TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nature Neuroscience. 21 (2), 228-239 (2018).