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.
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.
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.
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
2. Preparation of lysis buffer and sucrose gradients
3. Lysis of iPSNs and postmortem human CNS tissue
4. Isolation of nuclei from iPSNs and postmortem human CNS tissue
5. Immunostaining of the isolated nuclei
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.
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.
The authors have nothing to disclose.
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.
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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