The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).
The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.
The axon initial segment (AIS) is a short, uniquely specialized region of the proximal axon of vertebrate neurons1. The AIS comprises a transport filter and diffusion barrier essential in maintaining neuronal polarity by sorting somato-dendritic cargo2,3,4,5,6,7. In addition, the unique structure of the AIS allows it to accommodate clusters of voltage-gated ion channels that facilitate its function as the site of action potential initiation8. A highly stable structural complex underlies the unique functions of the AIS. Research within the last decade has revealed the presence of a membrane periodic skeleton (MPS) containing actin rings connected by spectrin and providing a scaffold for anchoring various AIS proteins9,10.
The distance between actin rings in the MPS (~190 nm)9,10 is under the resolution limit of conventional light microscopy. Early attempts to use electron microscopy to visualize the MPS were not successful, as the harsh preparation procedures involved failed to preserve the structure of the MPS. Thus, super-resolution microscopy techniques have proven invaluable in elucidating some of the structural details of the MPS11. However, the understanding of the AIS structural complex, the identity and functions of its components, and its spatiotemporal regulation are still incomplete. Recent proteomic studies succeeded in creating a sizeable list of proteins that localize to the AIS close to structural components of the AIS12,13. Still, details of their function and precise place in the AIS complex are lacking. Thus, super-resolution microscopy techniques serve as an essential tool to examine the accurate positions of these proteins relative to other MPS components and investigate their functions. Several light microscopy techniques can achieve resolutions higher than the diffraction limit of light, some even capable of localizing single molecules. However, many of these techniques typically require specialized fluorophores or imaging buffers, and image acquisition is often time-intensive14.
3D structured illumination microscopy (3D-SIM), owing to its ease of use and simple sample preparation requirements, requires no special reagents for imaging or sample preparation, works well with a wide array of fluorophores and samples, can be readily implemented in multiple colors, and is capable of live-cell imaging15. While the best possible resolution SIM offers (~120 nm) is low compared to many other super-resolution techniques, it is sufficient for many applications (for example, for resolving the components of the MPS in neurons). Thus, it is crucial to consider the requirement for specific applications to determine if SIM is a suitable choice or if an even higher resolution is necessary. Here, a protocol is described for using cultured hippocampal neurons and 3D-structured illumination microscopy (3D-SIM) to examine the position and organization of putative AIS proteins relative to actin rings in the MPS, as implemented in Abouelezz et al.16
Primary hippocampal neurons used in these experiments were harvested16 from embryonic day 17 Wistar rat embryos of either sex under the ethical guidelines of the University of Helsinki and Finnish law.
1. Sample preparation
2. Imaging
3. Image analysis
Using cultured rat hippocampal neurons and 3D-SIM, a protocol is described to visualize and measure actin rings and other components of the MPS in the AIS. Reconstructions of image stacks showed clear periodicity of actin rings (Figure 2A). In our hands, the mean inter-peak distance of actin rings in the MPS, visualized using Alexa 488-tagged phalloidin, was 190.36 ± 1.7 nm (mean ± SEM). This is in line with the previously reported average distance of ~190 nm between actin rings in the MPS. Similarly, an anti-ankyrin G antibody was used to visualize ankyrin G (Figure 2B). The colocalization of ankyrin G and F-actin was tested in the AIS using a colocalization analysis procedure to calculate the PCC of co-localization19,20,21. The PCC of colocalization of ankyrin G and F-actin fluorescence was 0.36 ± 0.03 (mean ± SEM, Figure 2B). As ankyrin G and actin rings bind βIV-spectrin at different domains, they do not show significant colocalization. These data were adapted from Abouelezz et al.16
Figure 1: Schematic representation of the protocol for sample preparation for 3D-SIM imaging. Hippocampal neurons are harvested from rat embryos, dissociated, and allowed to grow on glass coverslips in culture for 14 days. Cells are then fixed and stained using tagged phalloidin and appropriate antibodies, then mounted on glass slides for 3D-SIM imaging. Please click here to view a larger version of this figure.
Figure 2: 3D-SIM reconstruction of the membrane periodic skeleton (MPS) in the axon initial segment (AIS). (A) F-actin (green) and ankyrin G (magenta) show a regular distribution in the AIS, visualized by 3D-SIM. Scale bar = 1 µm. (B) Pearson's coefficients of correlation (PCC) of colocalization of ankyrin G with F-actin in the MPS in the AIS. The mean PCC of ankyrin G was 0.36 (black circle). Gray diamonds represent individual cells (n = 16), mid-line represents the median, error bars represent 25th and 75th percentiles. The data is adapted from Abouelezz et al.16 Please click here to view a larger version of this figure.
The protocol described here provides a method for visualizing and measuring MPS proteins using the super-resolution technique. As actin rings and other MPS components display a periodicity of ~190 nm9,10, conventional diffraction-limited imaging approaches cannot reveal the details of the MPS. Several microscopy setups may resolve diffraction-limited structures in super-resolution, and SIM represents a robust and uncomplicated option. Importantly, SIM is compatible with the most widely-used fluorophores, providing significant flexibility. Furthermore, SIM is effective in visualizing potentially dim structures in the MPS, such as actin rings in latrunculin-treated neurons22, as well as live neurons15.
An essential aspect for the success of this protocol is preserving the integrity of the MPS in the first place. The most critical steps to successful SIM imaging occur during sample preparation. For example, moderate fixation (4% PFA for 12 min at room temperature) and strong permeabilization (1% Triton-X for 10 min) provide the best results. It is crucial to keep in mind that harsh treatments that may be required for specific preparations may have a negative effect on the structural integrity of the MPS. Therefore, it is perhaps best to consider modifying such treatments to maximize the preservation of the MPS.
The other crucial factor to consider when preparing samples for imaging is to maximize the labeling density. Ideally, every molecule would be tagged and detected. For example, the anti-ankyrin G antibody used in this experiment is commonly used to label the AIS. It provides outstanding performance in conventional fluorescence microscopy, even when used at a dilution of 1:1000 and incubated for just 1 h at room temperature. However, to obtain good labeling density for super-resolution microscopy, it is highly effective to use 5-fold that concentration (1:200) and incubate the antibody overnight at 4 °C. While the specific requirements for each antibody or labeling technique will vary and need to be determined experimentally, it is perhaps helpful to keep this in mind.
In addition, it is essential to note that achieving a high signal-to-noise ratio lends itself well to accurate and successful SIM reconstructions. A good rule of thumb is that the grid pattern should be visible in the individual images once the SIM modality is engaged. However, this is not always possible.
Finally, it is essential to note that SIM is among the weakest super-resolution techniques in resolving power14. Thus, while it is generally sufficient for revealing periodicity and overall organization of MPS proteins, it is significantly less capable of providing details about their interactions than stochastic optical reconstruction microscopy (STORM)10. Furthermore, the technique’s utility described here is limited to the study of proteins for which a specific, well-performing antibody is available. However, this can partly be circumvented through exogenous expression of tagged proteins15.
The authors have nothing to disclose.
Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain & Mind (A.A.)
24-well plates | Corning | 3524 | |
4% Paraformaldehyde | |||
Alexa-488 Phalloidin | ThermoFisher | A12379 | |
Alexa-647 anti-mouse | ThermoFisher | A31571 | |
Anti-Ankyrin G antibody | UC Davis/NIH NeuroMab Facility, Clone 106/36 | 75-146 | |
Anti-MAP2 antibody | Merck Millipore | AB5543 | |
B-27 | Invitrogen | 17504044 | |
Bovine Serum Albumin (BSA) | BioWest | P6154 | |
Deltavision OMX SR | GE Healthcare Life Sciences | N/A | |
Fiji software package | ImageJ | ||
GNU Octave | GNU | ||
High performance coverslips | Marienfeld | 117530 | |
Immersion Oil Calculator | Cytiva Life Sciences | https://tinyurl.com/ImmersionOilCalculator | |
L-Glutamine | VWR | ICNA1680149 | |
MATLAB R2020a | Mathworks | ||
Neurobasal media | Invitrogen | 21103049 | |
OMX SR | Delta Vision OMX | ||
Primocin | InvivoGen | ant-pm-1 | |
ProLong Gold mounting media | Invitrogen | P10144 | |
softWoRx Deconvolution | Cytiva Life Sciences | ||
Superfrost Slides | Epredia | ISO 8037/1 | |
TetraSpeck microspheres 0.1 µm | ThermoFisher | T7279 | |
Triton-X | Sigma | X100 |