The brain contains hundred types of neurons, but they are all variations on the same basic structure. Each neuron consists of a cell body that is covered in short protrusions called dendrites and a long thin structure called the axon. The dendrites receive incoming signals from neighboring neurons and they transmit these signals via the cell body to the axon, which in turn relays them to the dendrites of the next neuron (or neurons).

Like all cells, neurons maintain their structure with the help of an internal cytoskeleton made up of many different proteins. However, it was discovered recently that axons have an additional lattice-like structure underneath their outer membrane. This structure, which consists of rings of actin filaments separated by molecules of a protein called spectrin, is preferentially formed in axons and is found much less frequently in dendrites.

Now Zhong, He et al., who are members of the research group that discovered the axonal skeleton, have used ‘super-resolution imaging’ to figure out how this skeleton forms and why it predominantly forms in axons. In brief, a basic version of the sub-membrane periodic skeleton is laid down early in development, starting next to the cell body before gradually spreading down the axon. The skeleton then continues to mature throughout development with the incorporation of several additional types of proteins.

The periodic skeleton only forms in regions which contain enough βII spectrin. Under normal conditions, dendrites contain too little βII spectrin to support the growth of such a periodic skeleton. However, artificially increasing the amount of βII spectrin present by overexpressing the corresponding gene, or by knocking out ankyrin B (a molecule that is important for establishing the preferential distribution of βII spectrin in axons), is sufficient to trigger periodic skeleton formation in dendrites. Given that axons and dendrites have distinct roles in neuronal signaling, this uneven distribution of spectrin is likely to be one way in which these regions maintain the specific structures that support their individual functions.

Neurons are highly polarized cells with their somatodendritic regions receiving synaptic inputs and axons propagating electrical signals and sending synaptic outputs to target cells. Cytoskeletal proteins are important for maintaining the polarity of neurons. For example, actin and microtubules are essential for the growth and stabilization of axons, the trafficking of cargos to specific neurites, and the stabilization and plasticity of synapses (Luo, 2002; Dent and Gertler, 2003; Cingolani and Goda, 2008; Barnes and Polleux, 2009; Kapitein and Hoogenraad, 2011; Stiess and Bradke, 2011). Transient destabilization of actin at the tip of a neurite is sufficient to induce a dendrite to become an axon (Bradke and Dotti, 1999). Increasing evidence also suggests an important role for spectrin in the maintenance of neuronal polarization, as well as the development and stabilization of axons (Hammarlund et al., 2007; Galiano et al., 2012). αII and βII spectrin are enriched in axons (Riederer et al., 1986; Galiano et al., 2012). Spectrin is known to be important for providing the mechanical stability for axons (Hammarlund et al., 2007) and protecting them from mechanical stress (Krieg et al., 2014), for axon path finding (Hulsmeier et al., 2007), for the stabilization of pre-synaptic terminals (Pielage et al., 2005), and for maintaining specific membrane domains in axons (Susuki and Rasband, 2008). Mice lacking either αII or βII spectrin die in the embryo, highlighting the crucial function of these proteins (Tang et al., 2003; Stankewich et al., 2011; Galiano et al., 2012). Spectrin has also been shown to play a role in human neurological diseases (Ikeda et al., 2006; Writzl et al., 2012).

Recently, we discovered a periodic sub-membrane lattice structure made of actin, spectrin, and other associated molecules in the axons of mammalian neurons (Xu et al., 2013). In this membrane skeleton, actin filaments form a ring-like structure that wraps around the circumference of axons. These actin rings are evenly spaced along the axon shaft with a period of ∼190 nm and show a remarkably long-range order. Actin filaments in the rings are capped by adducin. Adjacent actin rings are connected by spectrin, likely in the form of αII-βII-spectrin heterotetramers, given the observations that the periodic βII spectrin rings alternate with the actin–adducin rings along axons and that the observed 190 nm period matches the length of the spectrin tetramer. The ultrastructural organization of this quasi-one-dimensional, periodic lattice structure is different from the previously observed, two-dimensional polygonal membrane skeletal structure found in red blood cells (Byers and Branton, 1985; Liu et al., 1987; Bennett and Lorenzo, 2013), whereas an erythrocyte-like polygonal membrane skeletal was observed in the axon terminals at the Drosophila neuromuscular junction (Pielage et al., 2008). Interestingly, this periodic structure preferentially forms in axons, with the actin in dendrites primarily adopting the form of long filaments running along the dendrite shaft (Xu et al., 2013). In Caenorhabditis elegans that lack β spectrin or carry a β spectrin mutant, axons break more easily during animal movement (Hammarlund et al., 2007) and exhibit impaired touch sensation (Krieg et al., 2014), suggesting that this structure may be important for the mechanical stability of axons and for sensing mechanical stimuli. This periodic lattice also organizes the axonal membrane by placing important membrane proteins, such as the voltage-gated sodium channels, into a periodic distribution (Xu et al., 2013).

However, it is unknown how this highly regular membrane skeleton structure develops in axons and how its formation is regulated. For example, it is unclear whether the periodic lattice develops during early or late stages of axon differentiation. Although protein factors previously identified to be important for axon differentiation tend to be enriched and function at the growing tips of axons (Arimura and Kaibuchi, 2007; Barnes and Polleux, 2009; Stiess and Bradke, 2011; Cheng and Poo, 2012), it is unknown whether the actin–spectrin lattice also initiates at the distal ends of axons or instead forms first in the proximal region near the cell body. Finally, the molecular mechanism that regulates the specific formation of this periodic structure in axons, instead of dendrites, remains a mystery. In this study, we addressed these important questions concerning the development of this newly discovered neuronal structure. We found that the periodic membrane skeleton initiated early during axon differentiation. The lattice structure originated in the axonal region adjacent to the cell body and propagated to the distal ends of axons. The lattice structure further matured by recruiting other components, and the matured membrane skeleton was highly stable. Multiple molecular factors played roles in regulating the formation of this structure. The lattice structure depended on intact microtubules. The high local concentration of βII spectrin in axons was the key determining factor for the specific formation of the lattice structure in axons, and artificially increasing the concentration of βII spectrin in dendrites was sufficient to induce the formation of the periodic lattice structure in dendrites. Remarkably ankyrin B was important for the polarized distribution of βII spectrin in neurites; in ankyrin B knockout mice, βII spectrin was evenly distributed in axons and dendrites, giving rise to a highly regular, periodic membrane skeleton in both dendrites and axons.

Actin, spectrin, and associated molecules form a periodic lattice structure with long-range order underneath the axonal membrane. Many molecular components, including actin, βII spectrin, adducin, ankyrin B, βIV spectrin, ankyrin G, and sodium channels, are present in this structure. We have observed this periodic membrane skeleton with STORM imaging of fixed cultured neurons (Figure 1 and Figure 1—figure supplements 1–3) and fixed brain tissue slices (Xu et al., 2013) using actin-binding phalloidin and immunolabeling of endogenously expressed proteins, as well as in live cultured neurons using fluorescent fusion proteins (Figure 5). Recently, this periodic structure has also been observed in live neurons using a cell-permeable actin-binding dye and STED imaging (Lukinavicius et al., 2014). Since a single actin filament can interact with multiple spectrin tetramers, and a single spectrin tetramer can bind to two actin filaments, one at each end of the symmetric tetramer (Bennett and Lorenzo, 2013), we reason that these crosslinking interactions are responsible for the formation of the lattice structure. Indeed, depolymerizing actin filaments disrupted the periodic distribution of βII spectrin, and knocking-down of βII spectrin disrupted the periodic distribution of actin (Figure 1—figure supplements 2–3). Because this lattice structure is associated with the axonal membrane, proper preservation of the membrane structure is essential for observing this structure. For example, a recent electron microscopy study of the AIS in neurons that has been subjected to detergent extraction of membrane before fixation did not show such periodic membrane skeleton (Jones et al., 2014). Indeed when we applied the same ‘fixation after membrane extraction’ protocol to neurons, the structure was destroyed and not observed in STORM images (Figure 1—figure supplement 6). In this study, we investigated the developmental mechanism of this newly discovered axonal membrane skeleton.

We found that this periodic membrane skeleton started to form early during axon development. In stage 3 neurons at DIV 2, when one neurite just broke the symmetry and became an axon (typically several times longer than other neurites), the periodic pattern of βII spectrin already emerged (Figure 1). It originated in the proximal axon regions near the cell bodies and gradually propagated to the distal ends of axons (Figure 1). This spatial distribution is in contrast to most previously identified signaling molecules involved in axon differentiation and development, which are enriched and function at the growing tip of axons (Arimura and Kaibuchi, 2007; Barnes and Polleux, 2009; Stiess and Bradke, 2011; Cheng and Poo, 2012). After its initial appearance, the lattice structure continued to mature with the actin filaments becoming more stable, potentially because of capping by adducin (Figures 2, 3). Once matured, the structure appeared highly stable with little movement and extremely slow turnover of its molecular components was observed in live neurons (Figure 5). This highly stable membrane skeleton may function to provide a stable mechanical support for axons. Indeed, deletion of β spectrin from C elegans causes axons to break when the animals move (Hammarlund et al., 2007).

The emergence of this periodic lattice during early axon development and its origination in the proximal axon region near the cell body suggest that the periodic membrane skeleton may function as an independent mechanism for establishing or maintaining neuronal polarization in addition to the previously identified pathways that function at the distal ends of axons. However, the periodic membrane skeleton is not required for the initiation of axon differentiation because the periodic structure only started to form in stage 3, but not stage 2, a stage at which most signaling molecules for axon initiation exhibit high activity (Arimura and Kaibuchi, 2007; Cheng and Poo, 2012). Moreover, neurons depleted of βII spectrin are also capable of forming long axons (Galiano et al., 2012), though these axons may not be fully functional. Indeed, removal of αII or βII spectrin is embryonically lethal in mice (Tang et al., 2003; Stankewich et al., 2011) and AIS, the structure important for action potential generation, fails to assemble properly in neurons that are depleted of βII spectrin (Galiano et al., 2012).

Interestingly, molecules important for the specification of AIS, ankyrin G, βIV spectrin, and sodium channels were all incorporated into this periodic membrane skeleton. During the early stages of axon development, the expression levels of ankyrin G and βIV spectrin were low in the proximal axon region, which was instead occupied by the periodic lattice comprising βII spectrin (Figure 1). Ankyrin G and βIV spectrin began to enrich in the proximal axon region later during axon development, at ∼DIV 8 (Figure 4—figure supplement 1), consistent with previous observations (Galiano et al., 2012). The incorporation of ankyrin G and βIV spectrin into the periodic lattice was observed even later, at around DIV 12, replacing βII spectrin from the structure in the AIS (Figure 4). The enrichment and periodic assembly of the AIS components appeared to depend on βII spectrin (Figure 4—figure supplement 2). Our data suggest the following developmental course for the AIS formation. Before the AIS is formed, actin and βII spectrin form a cohesive periodic lattice structure that covers the entire axonal shaft including the proximal axonal region. The AIS then forms by the enrichment of ankyrin G and βIV spectrin in the proximal axonal region and the replacement of βII spectrin by βIV spectrin in the periodic membrane skeleton. Potentially, ankyrin G, the master regulator of AIS that recruits other AIS components (Zhou et al., 1998; Jenkins and Bennett, 2001; Yang et al., 2007), is first enriched in the proximal axon region. Ankyrin G then recruits βIV spectrin to the same region and causes it to be incorporated in the periodic lattice. βIV spectrin in turn anchors ankyrin G into a periodic pattern as well. As an adaptor, ankyrin G then places sodium channels into a periodic distribution pattern in the AIS.

Finally, we addressed the question why the cohesive, periodic lattice structure preferentially formed in axons, with dendrites showed at most isolated patches of periodic structure with much less regularity (Figure 1—figure supplement 5). We found multiple molecular factors participate in this regulation. The periodic lattice structure depended on intact microtubules. Treatment with a microtubule-depolymerizing drug disrupts the structure in axons, whereas treatment with microtubule-stabilizing drugs induces the formation of the lattice structure in multiple neurites (Figure 6). Importantly, we found that the local concentration of βII spectrin is a determining factor for the formation of the lattice structure. The local concentration of βII spectrin is ∼2 times higher in axons than in dendrites (Figure 7). Remarkably, increasing the dendritic concentration of βII spectrin by overexpression induced the formation of the periodic lattice structure in all dendrites (Figure 8). Interestingly, ankyrin B was critical for maintaining the polarized distribution of βII spectrin. Knocking out ankyrin B led to an even distribution of βII spectrin in dendrites and axons and the formation of a highly regular and cohesive periodic lattice structure in all dendrites (Figure 7). Consistent with the notion that the increased concentration of βII spectrin in dendrites is responsible for inducing the formation of the periodic lattice structure in dendrites, knocking-down βII spectrin from ankyrin B knockout neurons disrupted the lattice structure (Figure 7—figure supplement 2). These results indicate that ankyrin B is critical for establishing a polarized distribution of βII spectrin in neurites with a higher concentration of βII spectrin in axons than in dendrites, which in turn promotes the formation of the periodic membrane skeleton in axons.

It is interesting to speculate how ankyrin B may establish such a polarized distribution of βII spectrin. The predominant form of ankyrin B during early neuronal development is a 440-kDa splice variant that is preferentially targeted to axons (Kunimoto et al., 1991; Chan et al., 1993; Kunimoto, 1995). Given that ankyrin B specifically binds to βII spectrin, we speculate that the distribution of ankyrin B in axons may help establishing the enrichment of βII spectrin in axons during early neuronal development. Moreover, we recently found that ankyrin B is a major cargo adaptor for dynactin and promotes axonal transport of proteins and organelles and that disruption of ankyrin B–dynactin interaction significantly impairs the axonal transport of many proteins (Lorenzo et al., 2014). It is thus possible that ankyrin B may also preferentially transport βII spectrin into axons instead of dendrites. By maintaining a polarized distribution of βII spectrin in neurons, ankyrin B functions as a negative regulator for preventing the formation of the periodic membrane skeleton in dendrites. The exact mechanism by which ankyrin B maintains a polarized distribution of βII spectrin remains an interesting question for future investigation.

All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard University.

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Author details

1. Guisheng Zhong

   Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States

   Contribution

   GZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Jiang He

   Competing interests

   No competing interests declared.

2. Jiang He

   Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States

   Contribution

   JH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Contributed equally with

   Guisheng Zhong

   Competing interests

   No competing interests declared.

3. Ruobo Zhou

   Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States

   Contribution

   RZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Damaris Lorenzo

   1. Department of Biochemistry, Duke University, Durham, United States
   2. Department of Neurobiology, Duke University, Durham, United States

   Contribution

   DL, Provided the dissociated neurons from the ankyrin B knockout and control mice

   Competing interests

   No competing interests declared.

5. Hazen P Babcock

   Center for Brain Sciences, Harvard University, Cambridge, United States

   Contribution

   HPB, Acquisition of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

6. Vann Bennett

   1. Department of Neurobiology, Duke University, Durham, United States
   2. Department of Biochemistry, Howard Hughes Medical Institute, Duke University, Durham, United States

   Contribution

   VB, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

7. Xiaowei Zhuang

   1. Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States
   2. Department of Physics, Harvard University, Cambridge, United States

   Contribution

   XZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   zhuang@chemistry.harvard.edu

   Competing interests

   XZ: Reviewing editor, eLife.

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