Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

Alyssa N. Coyne; Jeffrey D. Rothstein

doi:10.3791/62789

JoVE Journal > Neuroscience

Neuroscienze

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

Published: September 10, 2021

doi:

10.3791/62789

Alyssa N. Coyne^1,2, Jeffrey D. Rothstein^1,2

¹Brain Science Institute,Johns Hopkins University School of Medicine, ²Department of Neurology,Johns Hopkins University School of Medicine

Summary

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

Abstract

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD)/Frontotemporal Dementia (FTD), and Huntington's Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

Introduction

The prevalence of age-related neurodegenerative diseases is increasing as the population ages¹. While the genetic underpinnings and pathologic hallmarks are well characterized, the precise molecular events leading to neuronal injury remain poorly understood²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹². Recently, a G₄C₂ hexanucleotide repeat expansion in the first intron of the C9orf72 gene was identified as the most common genetic cause of the related neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)¹³^,¹⁴. Several studies now support a central role for disruptions in the nuclear transport machinery, including nuclear pore complexes (NPCs) and nuclear transport receptors (NTRs, karyopherins), as being causative of C9orf72 ALS¹⁵^,¹⁶. In non-dividing cells within the rat brain, scaffold nucleoporins (Nups) are extremely long-lived. As a result, alterations in NPCs and NCT have been reported during aging¹⁷^,¹⁸^,¹⁹^,²⁰. Moreover, some nucleoporins or transportins, when mutated, are linked to specific neurological diseases²¹^,²². For example, mutations in Nup62 have been linked to Infantile Bilateral Striatal Necrosis (IBSN), a neurological disorder affecting the caudate nucleus and putamen²³; mutations in Gle1 have been implicated in the fetal motor neuron disease Human Lethal Congenital Contracture Syndrome-1 (LCCS1)²⁴; and mutations in Aladin are causative of Triple-A Syndrome²⁵. Alterations in functional NCT are exacerbated in age-related neurodegenerative diseases such as ALS, Huntington's Disease (HD), and Alzheimer's Disease (AD)¹⁶^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹. In addition, specific Nups and NTRs have been reported as modifiers of C9orf72 mediated toxicity in the Drosophila eye²⁸ or biochemically modify the aggregation state of disease-linked proteins such as FUS and tau²⁷^,³²^,³³^,³⁴. Collectively, these early studies suggest that altered NCT may be a primary and early pathological feature of ALS and FTD. Studies in overexpression-based model system have suggested that mislocalization of specific Nups and karyopherins may impact NCT¹⁶^,³⁵^,³⁶^,³⁷^,³⁸. However, these pathology studies do not actually link cytoplasmic accumulations of NPC proteins to defects in the structure or function of the NPC. For example, this pathology may simply reflect the dysregulation of cytoplasmic pools of Nup proteins with little impact on NPC composition and function. In contrast, a recent study employing super resolution structured illumination microscopy (SIM) demonstrates the emergence of a significant injury to the NPC itself characterized by reduction in specific Nup levels within the nucleoplasm and NPCs of human C9orf72 ALS/FTD neurons ultimately leading to altered NPC function as an early initiating event in pathogenic disease cascades¹⁵.

The passage of macromolecules between the nucleus and cytoplasm is governed by the nuclear pore complex (NPC). The NPC is a large macromolecular complex embedded in the nuclear envelope comprised of multiple copies of 30 nucleoporin proteins (Nups)³⁹^,⁴⁰^,⁴¹. Although Nup stoichiometry varies among cell types⁴²^,⁴³^,⁴⁴, maintenance of overall NPC composition is critical for NCT, genome organization, and overall cellular viability³⁹^,⁴¹^,⁴⁵^,⁴⁶. As a result, altered NPC composition and subsequent defects in functional transport are likely to impact a myriad of downstream cellular functions. The Nup constituents of the NPC are highly organized into multiple subcomplexes, including the cytoplasmic ring and filaments, central channel, outer ring, inner ring, transmembrane ring, and nuclear basket. Collectively, scaffold Nups of the inner, outer, and transmembrane rings anchor NPCs within the nuclear envelope and provide anchor points for Nups of the cytoplasmic ring, central channel, and nuclear basket. While small molecules (<40-60 kD) can passively diffuse through the NPC, the active transport of larger cargoes is facilitated by interactions between nuclear transport receptors (NTRs, karyopherins) and the FG Nups of the cytoplasmic filaments, central channel, and nuclear basket³⁹^,⁴⁰^,⁴¹^,⁴⁵. Also, a handful of Nups can additionally function outside of the NPC, within the nucleoplasm, to regulate gene expression⁴⁶^,⁴⁷.

Given that the lateral dimension of a single human NPC is approximately 100-120 nm⁴⁰, standard widefield or confocal microscopy is insufficient to resolve individual NPCs⁴⁸. Electron microscopy (EM) techniques such as TEM or SEM are often used to evaluate the overall structure of NPCs³⁹^,⁴⁰. Despite the advantages of these techniques for resolving NPC ultrastructure, they are less commonly used to evaluate the presence of individual Nup proteins within the NPC. The technical limitations of combining antibody or tag-based labeling with these state-of-the-art technologies, TEM and SEM, do not always allow for an accurate and reliable assessment of individual Nups themselves within NPCs or the nucleoplasm. Further, these techniques can be technically challenging and are not yet widely accessible to all researchers. However, recent advances in light and fluorescence microscopy have increased the accessibility of super-resolution imaging technologies. Specifically, SIM affords the unparalleled opportunity to image individual Nups with a resolution that approaches the lateral dimensions of one human NPC⁴⁰^,⁴⁸^,⁴⁹^,⁵⁰^,⁵¹. In contrast to other super-resolution approaches such as stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED), SIM is compatible with conventional antibody-based immunostaining⁴⁹. Thus, SIM allows for a comprehensive analysis of all Nups for which a specific Nup antibody is available. The ability to sample and image multiple different Nups in the same preparation provides significant advantages to other imaging methods when surveying the many proteins that comprise the NPC. The following procedure details an optimized protocol for evaluating individual Nup components of the NPC using nuclei isolated from induced pluripotent stem cell (iPSC) derived neurons (iPSNs) and postmortem human central nervous system (CNS) tissues.

Protocol

All blood samples for iPSC generation and autopsied tissue collections are approved by Johns Hopkins IRB with Johns Hopkins ethics oversight. All patient information is HIPPA compliant. The following protocol adheres to all Johns Hopkins biosafety procedures.

1. Preparation of slides for immunostaining and imaging

Position a positively charged glass microscope slide in an empty cytofunnel and draw a circle with a hydrophobic barrier pen to outline an area to deposit the nuclei.
Add 50 μL of 1 mg/mL collagen solution (diluted in 1x PBS) to the center of the circle drawn in step 1.1 and incubate at room temperature for 5 min.
Aspirate the collagen solution and let the slides air dry at room temperature for about 1 h.

2. Preparation of lysis buffer and sucrose gradients

Prepare the lysis buffer and sucrose gradient solutions according to the nuclei isolation kit protocol.
1. For each sample, prepare a 50 mL conical tube of lysis buffer by combining 11 mL of the supplied lysis buffer, 110 μL of the supplied 10% Triton X-100 solution, and 11 μL of freshly prepared or thawed 1 M DTT (Dithiothreitol).
2. For each sample, use a 50 mL conical tube to prepare a 1.85 M sucrose cushion solution by combining 27.75 mL of the supplied 2 M sucrose cushion solution, 2.25 mL of the supplied sucrose cushion buffer, and 30 μL of freshly prepared or thawed 1 M DTT.
  NOTE: A 1.85 M sucrose cushion solution is optimal for iPSNs and postmortem human CNS tissue, but adjust it for other cell types or tissue samples. Additional details for HEK293 cells, iPSC-derived astrocytes, and enrichment of oligodendrocyte nuclei from postmortem CNS tissues have recently been published¹⁵.
Gently mix lysis buffer and sucrose cushion solutions by inverting conical tubes and store it on ice.

3. Lysis of iPSNs and postmortem human CNS tissue

Before proceeding with the lysis, place the ultracentrifuge rotor (without sample holders) in the ultracentrifuge and allow it to pre-cool to 4 °C.
Lysis of iPSNs.
NOTE: Lysis protocols differ for iPSNs and postmortem human CNS tissue. Follow the protocol for the sample type specified below (step 3.2: iPSNs, step 3.3: postmortem human CNS tissue).
1. Remove the iPSNs from the incubator and aspirate the media. Rinse briefly with 1x PBS and add the lysis buffer directly to the well or plate.
  NOTE: Take care to adequately rinse iPSNs with a sufficient quantity of 1x PBS to remove debris and dead cells. The volume of lysis buffer to be added to iPSN plates will vary. Please refer to Table 1. Ensure that the starting material is >2.5 million iPSNs.
2. Scrape the iPSNs with a cell scraper and transfer them to the remaining lysis buffer in the 50 mL conical tube.
3. Cap each conical tube and vortex the samples for 20 s to facilitate iPSN lysis.
4. Let the samples sit on ice for 1-2 min before proceeding.
Lysis of postmortem human CNS tissues.
1. Weigh out ~100 mg of frozen postmortem human CNS tissue by cutting with a razor blade in a Petri dish placed on a bed of dry ice. Be sure to work on a surface surrounded by disposable pads to avoid local tissue contamination. Pay attention to specific gray matter versus white matter dissections (e.g., cortical mantel versus underlying white matter) visible at the time of tissue dissection.
  NOTE: Wear the appropriate PPE, including eye protective wear and gloves when handling frozen postmortem human tissue samples. This step can also be completed ahead of time and tissue aliquots stored at -80 °C. 50 mg is a sufficient quantity for nuclei isolation. However, more than 100 mg tissue often yields incomplete isolation of nuclei, as evidenced by the presence of intact cytoplasm surrounding some nuclei.
2. Prepare the Dounce homogenizer by cleaning and thoroughly rinsing with distilled water. Chill the homogenizer by placing it on ice.
3. Add 100 mg of tissue to freshly cleaned, rinsed, and chilled (on ice) Dounce homogenizer containing 2 mL of the prepared lysis buffer.
4. Homogenize in Dounce homogenizer using the standard procedure on ice.
  NOTE: Typically, 10-20 strokes per pestle is sufficient to move through the sample without resistance.
5. Transfer 2 mL of postmortem human brain homogenate to the remaining 9 mL of lysis buffer in conical tubes.
6. Vortex vigorously for 30 s and let it sit on ice for 5 min to facilitate lysis.

4. Isolation of nuclei from iPSNs and postmortem human CNS tissue

Layer 10 mL of 1.85 M sucrose cushion solution (made in step 2.1.2) at the bottom of an ultracentrifuge tube and add 18 mL of 1.85 M sucrose cushion solution to each lysate.
Gently mix the lysate/sucrose cushion solution (combined in step 4.1) by inverting the 50 mL conical tube.
Slowly add 28 mL of lysate/sucrose cushion solution mix to the top of the 10 mL of 1.85 M sucrose cushion solution in the ultracentrifuge tube (from step 4.1).
NOTE: There is a total of 29 mL lysate/sucrose cushion solution mix in the 50 mL conical tube (steps 4.1-4.2). Leave 1 mL of this lysate/sucrose cushion solution mix in the 50 mL conical tube. This ensures accurate and consistent pipetting among all samples due to the viscosity of the sucrose cushion solution.
Place the ultracentrifuge tubes in the holders and add an additional 1.85 M sucrose cushion solution (made in step 2.1.2) as needed to balance the samples.
Place the sample holders in the rotor in the chilled ultracentrifuge and spin at 30,000 x g, 4 °C for 45 min.
Remove the ultracentrifuge tubes from the sample holders and discard the supernatants. Nuclei will be visible as pellets on the bottom sides of the ultracentrifuge tube.
Resuspend the nuclei pellets in 1 mL of the supplied nuclei storage buffer by vortexing and transfer to a microcentrifuge tube.
Centrifuge the microcentrifuge tubes at 2,500 x g, 4 °C for 5 min.
Remove the supernatant and resuspend the nuclei by vortexing in fresh 1 mL of the supplied nuclei storage buffer.
Proceed with immunostaining or store the nuclei at -80 °C.
NOTE: Nuclei can be stored at -80 °C for up to 6 months and be subjected to two freeze/thaw cycles before structural integrity is compromised.

5. Immunostaining of the isolated nuclei

Take a 10 μL aliquot of the nuclei suspension and count using a hemacytometer or an automated cell counter.
Assemble the prepared slides (from step 1.3) into the cytofunnels.
To each cytofunnel, layer 200 μL of fresh nuclei storage buffer and ~100,000 nuclei.
Gently spin the nuclei onto the slides by placing the cytofunnels into a cytospin and centrifuging for 3 min at 100 x g.
Unclip the cytofunnels and immediately add ~100 μL of 4% PFA (paraformaldehyde) to the slides and incubate for 15 min at room temperature.
NOTE: Nuclei can also be fixed with methanol. Use fixation method appropriate for each antibody. If using methanol, skip the permeabilization step (5.7). Cytofunnels are single-use; discard them at this point.
Wash the nuclei 3x for 5 min with 1x PBS.
Permeabilize the nuclei with 0.1% Triton X-100 in 1x PBS for 10 min at room temperature.
Block the nuclei in block solution (10% goat or donkey serum in 1x PBS) for 30 min at room temperature.
Incubate the nuclei with primary antibodies diluted in block solution overnight at 4 °C.
Wash the nuclei 3x for 5 min with 1x PBS.
Incubate the nuclei with secondary antibodies diluted in block solution for 1 h at room temperature.
NOTE: Alexa Fluor 488, 568, and 647 secondary antibodies are preferred.
Wash the nuclei 3x for 5 min with 1x PBS.
OPTIONAL: Incubate the nuclei for 5 min with DAPI or Hoechst followed by two additional 5 min washes with 1x PBS.
Hold a lint-free wipe at the edge of the circle containing nuclei to remove the last PBS wash completely.
Add 1 drop (~10 μL) of a hard mount antifade mounting media (without DAPI) to each slide and gently place high tolerance 18 mm x 18 mm square coverslips on each slide.
NOTE: When using a hard-mount media, slide sealing is not necessary if slides are imaged within an appropriate time frame. Nup immunoreactivity has typically remained stable on unsealed slides stored at 4 °C for ~6 months. However, the edges of the coverslip can be sealed with a thin layer of nail polish if a wet-mount media is used or for prolonged storage.
Keep the slides protected from light and let them cure overnight at room temperature.
Image the nuclei can by super-resolution structured illumination microscopy (SIM).
NOTE: Zeiss, Nikon, and GE Healthcare all manufacture SIM microscopes. Follow the system-specific protocols for image acquisition and processing. Use imaging parameters (laser power, filter sets, exposure time) appropriate for each Nup and fluorophore. When imaging, avoid edges of the circle (from step 1.1) as these nuclei are typically flattened and spread out as a result of the centrifugation step (5.4). Fully deconvolved and processed images can be subjected to a number of analyses, including spot detection and volume measurements using standard 3D analysis modules in FIJI or Imaris image analytics software. Additional details on analysis methods have been recently published¹⁵.

Representative Results

To examine the NPC and nucleoplasmic distribution and expression of POM121 in human neuronal nuclei, control, and C9orf72 iPSNs were differentiated as previously described¹⁵. Postmortem human motor cortex and day 32 iPSNs were lysed and subjected to nuclei isolation and immunostaining as described above. NeuN positive isolated nuclei were imaged by super-resolution structured illumination microscopy (SIM) using a super-resolution structured illumination microscope (Zeiss) and processed using default structured illumination deconvolution parameters as previously described¹⁵ (see also Figure 1 for workflow). Images were acquired using a 63x oil immersion objective, 1x magnification, an HR Diode 448 laser at 1% power, exposure time of 100 ms, BP495-550/LP750 emission filter, 110 nm thick z sections, and 5 grid rotations. As shown in 3D maximum intensity projections in Figure 2, individual POM121 spots were resolved in both iPSN and postmortem neuronal nuclei. These images were subsequently subjected to spot detection using the 3D suite plugin in FIJI or Imaris as previously described¹⁵. Consistent with prior analyses¹⁵, this methodology was sufficient to detect a substantial decrease in the number of POM121 spots in C9orf72 iPSN and postmortem motor cortex nuclei compared to controls (Figure 2).

Figure 1: Schematic representation of nuclei isolation, immunofluorescent staining, super-resolution imaging, and analysis workflow. Please click here to view a larger version of this figure.

Figure 2: Representative structured illumination microscopy (SIM) images and quantification. (A–B) Maximum intensity projection SIM images and quantification of POM121 spots in NeuN+ nuclei isolated from control and C9orf72 iPSNs. n = 3 control and 3 C9orf72 iPSC lines, 50 nuclei per line. Student's t-test was used to calculate statistical significance. **** p < 0.0001. (C–D) Maximum intensity projection SIM images and quantification of POM121 spots in NeuN+ nuclei isolated from control and C9orf72 postmortem motor cortex tissue. n = 3 control and 3 C9orf72 patient cases, 50 nuclei per case. Student's t-test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm. Statistical analyses were performed whereby the average number of spots from 50 nuclei per iPSC line were used to calculate statistical significance as previously described ¹⁵. Please click here to view a larger version of this figure.

Culture Vessel	Volume of Lysis Buffer
12 well plate	0.5 mL per well
6 well plate	1 mL per well
10 cm dish	3 mL
T25 flask	2 mL

Table 1: Lysis buffer volumes. The table contains the information of lysis buffer volume to be used for lysis of iPSNs or cultured cells grown in different culture vessels.

Discussion

Given the recent identification of NCT deficits as an early and prominent phenomenon in multiple neurodegenerative diseases¹⁶^,²⁷^,²⁸^,³⁰^,³¹, there exists a critical need to thoroughly examine the mechanism by which this pathology occurs. As the NPC and its individual Nup proteins critically control functional NCT³⁹^,⁴¹, an investigation of NPC composition in neurodegenerative disease models is essential. However, standard widefield and confocal microscopy techniques are insufficient to resolve individual NPCs⁴⁰^,⁴⁸^,⁴⁹^,⁵¹. Thus, to date, analysis of Nups at the resolution of a single NPC has not been adopted as a routine experimental approach.

In recent years, super-resolution imaging technologies such as SIM have become more accessible. These methodologies couple conventional immunofluorescence staining with advanced image processing and deconvolution to provide increased resolution sufficient to resolve an individual NPC⁴⁰^,⁴⁸^,⁴⁹^,⁵¹. Although compatible with standard immunostaining protocol, thereby affording researchers the ability to detect any Nup for which a specific anti-Nup antibody is available at high resolution⁴⁸^,⁴⁹^,⁵⁰, SIM does not come without its technical challenges or limitations as outlined below.

The success of any super-resolution imaging experiment is highly dependent upon sample quality. Specifically, for SIM, incomplete nuclei isolation or over lysis of samples can lead to retention of cytoplasm and cell membranes or fragmented or collapsed and shriveled nuclei, respectively. The lysis time should be adjusted, accordingly, to overcome this issue. While the protocol detailed above has been optimized for iPSNs and postmortem human CNS tissue, it is easily amended to produce high-quality nuclei from a variety of cell types and tissues. This is accomplished by adjusting the concentration of the sucrose gradient and/or sample lysis time and method. Additional details regarding sucrose gradient adjustments for HEK293 cells, iPSC-derived astrocytes, and enrichment of oligodendrocyte nuclei from postmortem human CNS tissue have been recently published¹⁵.

The process of nuclei isolation itself may disrupt the association of less stably attached Nups or NTRs that transiently associate with the NPC. As a result, to verify the results obtained from SIM imaging of isolated nuclei, researchers should consider employing additional techniques such as immunostaining and confocal imaging of intact cells or tissues as has been previously described¹⁵. However, it is noted that routine confocal imaging of Nups does not provide the resolution to resolve individual NPC spots⁴⁸ and thus, can only provide semi-quantitative information regarding overall Nup intensity. For those Nups which are also normally present within cytoplasmic pools⁵²^,⁵³^,⁵⁴^,⁵⁵, western blot analyses from whole cell lysates are not recommended as a validation method for NPC composition due to contamination from Nups that may be localized to the cytoplasm. However, western blot analyses from isolated nuclei samples have been employed to validate data obtained from imaging technologies¹⁵.

The resolution of SIM images can vary among fluorophores depending on the individual point spread functions of the specific imaging system⁵¹. This should be experimentally tested by the user for each sample type before proceeding. SIM imaging is not ideal for samples with a thickness greater than 10-15 μm⁴⁸^,⁵¹. As a result, nuclei isolation enables imaging of the full thickness of human nuclei and therefore provides for the most accurate 3D reconstruction and quantification of Nup spots and volume. However, in instances where nucleus thickness remains >15 μm, Airy Scan imaging can be used as an alternative. Although the resolution is decreased, Airy Scan imaging is not limited by sample thickness⁵⁶.

It should also be taken into consideration that the images presented in Figure 2 represent maximum intensity projections following 3D reconstruction of individual nuclei imaged by SIM. Therefore, in such an image, it is difficult to accurately distinguish between spots truly associated with the NPC as opposed to those within the nucleoplasm. As a result, when analyzing 3D images, it is critical to analyze and segment each z section or small series of z sections independently to distinguish between spots associated with the nuclear envelope as opposed to the nucleoplasm. The quantifications presented in Figure 2 represent only those spots detected along the outside faces of the analyzed nuclei. In other words, the representative quantitation indicates the number of POM121 spots associated with the nuclear envelope.

Lastly, given nuclear heterogeneity and variability¹⁵^,⁴², it is important to image and evaluate a large number of nuclei per sample regardless of the super-resolution imaging method employed.

In contrast to bulk proteomics and western blot assays, imaging methodologies provide the unparalleled opportunity to examine the distribution and expression of Nups within subsets of human CNS cells identified by specific cell types or nuclear markers (e.g., NeuN, Olig2). SIM is one of many super-resolution imaging techniques that can be employed to study the NPC and its individual Nup components⁵⁰^,⁵⁷^,⁵⁸. Each methodology (SIM, STORM, EM) will yield its own insights into NPC structure and composition. While SIM is capable of evaluating each Nup for which an anti-Nup antibody is available at the resolution of a single NPC, STORM is capable of resolving the octet structure of individual NPCs, and EM techniques provide a detailed view of the overall NPC structure at high resolution⁵⁰. STORM and Immuno EM approaches to evaluate individual Nups within NPCs are technically challenging. Specifically, STORM often requires the use of endogenous fluorescent tags to overcome steric hinderance provided by conventional antibody-based staining. To date, only a handful of Nups in non-neuronal cell lines have been imaged by this technology. Furthermore, conventionally STORM images NPCs on a single surface of nuclei, thus eliminating the opportunity for full 3D reconstruction of an entire nucleus to evaluate the spatial distribution and NPC heterogeneity⁵⁰. As a result, SIM is the preferred light microscopy methodology for robustly evaluating Nups within NPCs and the nucleoplasm at high resolution. A recent study used both SEM and SIM technologies to conclude that the overall NPC structure was intact, but specific Nups were reduced from the NPC and nucleoplasm of C9orf72 ALS/FTD neuronal nuclei¹⁵. This work highlights the critical importance of combining multiple high-resolution imaging approaches to yield novel insights into both NPC composition and structure as well as the strength of SIM for examining 23 individual human Nups.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Postmortem human CNS tissues were provided by the Johns Hopkins ALS Autopsy Bank and the Target ALS Postmortem Tissue Core. This work was supported by the ALSA Milton Safenowitz Postdoctoral Fellowship (ANC), as well as funding from NIH-NINDS, Department of Defense, ALS Association, Muscular Dystrophy Association, F Prime, The Robert Packard Center for ALS Research Answer ALS Program, and the Chan Zuckerberg Initiative.

Materials

50 mL conical tubes	Fisher Scientific	14-959-49A
Beckman Ultracentrifuge	Beckman Coulter
Cell Scrapers	Sarstedt	83.183
Collagen	Advanced Biomatrix	5005
Coverslips	MatTek	PCS-170-1818
Cytofunnel	Thermo Fisher Scientific	A78710020
Cytospin 4	Fisher Scientific	A78300003
Dounce Homogenizers	DWK Life Sciences	357542
DTT	Sigma Aldrich	D0632
Eppendorf tubes	Fisher Scientific	05-408-129
Goat Anti-Chicken Alexa 647	Thermo Fisher Scientific	A-21449
Goat Anti-Mouse Alexa 488	Thermo Fisher Scientific	A-11029
Goat Anti-Mouse Alexa 568	Thermo Fisher Scientific	A-11031
Goat Anti-Mouse Alexa 647	Thermo Fisher Scientific	A-21236
Goat Anti-Rabbit Alexa 488	Thermo Fisher Scientific	A-11034
Goat Anti-Rabbit Alexa 568	Thermo Fisher Scientific	A-11036
Goat Anti-Rabbit Alexa 647	Thermo Fisher Scientific	A-21245
Goat Anti-Rat Alexa 488	Thermo Fisher Scientific	A-11006
Goat Anti-Rat Alexa 568	Thermo Fisher Scientific	A-11077
Goat Anti-Rat Alexa 647	Thermo Fisher Scientific	A-21247
Hemacytometer	Fisher Scientific	267110
Microscope Slides	Fisher Scientific	12-550-15
Normal Goat Serum	Vector Labs	S-1000
Nuclei PURE Prep Nuclei Isolation Kit	Sigma Aldrich	NUC201	Contains Lysis Buffer, 10% Triton X-100, 2 M Sucrose Gradient, Sucrose Cushion Solution, and Nuclei Storage Buffer; Referenced in protocol as "nuclei isolation kit"
PBS	Thermo Fisher Scientific	10010023
PFA	Electron Microscopy Sciences	15714-S
Prolong Gold Antifade	Invitrogen	P36930	Referenced in protocol as "hard mount antifade mounting media"
SW 32 Ti Ultracentrifuge Rotor	Beckman Coulter	369694	Referenced in protocol as "ultracentrifuge rotor"
Triton X-100	Sigma Aldrich	T9284
Trypan Blue	Thermo Fisher Scientific	15-250-061
Ultracentrifuge Tubes	Beckman Coulter	344058
Nucleoporin Primary Antibodies			Primary antibodies suitable for immunofluorescent detection of invidual nucleoporins are available from multiple companies

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