Submembranous cytoskeletons stabilize nodes of Ranvier

    Submembranous molecular complex stabilizes nodes of Ranvier Keiichiro Susuki, Yoshinori Otani, Matthew N. Rasband PII: DOI: Reference:

S0014-4886(15)30125-4 doi: 10.1016/j.expneurol.2015.11.012 YEXNR 12159

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

3 September 2015 10 November 2015 23 November 2015

Please cite this article as: Susuki, Keiichiro, Otani, Yoshinori, Rasband, Matthew N., Submembranous molecular complex stabilizes nodes of Ranvier, Experimental Neurology (2016), doi: 10.1016/j.expneurol.2015.11.012

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Invited review, special issue on Myelin Repair, Experimental Neurology

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Submembranous Molecular Complex Stabilizes Nodes of Ranvier Running title: Nodal cytoskeleton

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Keiichiro Susukia, Yoshinori Otania, and Matthew N. Rasbandb

Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine, Wright State University, Dayton, OH

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Department of Neuroscience, Baylor College of Medicine, Houston, TX

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Address correspondence to:

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Keiichiro Susuki Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine, Wright State University 3640 Colonel Glenn Highway, Dayton, Ohio 45435 Tel: (937) 775-2292 Fax: (937) 775-3391 E-mail: [email protected]

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Matthew N. Rasband Department of Neuroscience, Baylor College of Medicine One Baylor Plaza, Houston, Texas 77030 Tel: 713-798-4494 Fax: 713-798-3946 E-mail: [email protected]

Word count: abstract 156; text, 3399. Number of figures, 2. Key words: node of Ranvier, paranode, spectrin, ankyrin, protein 4.1

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ABSTRACT Rapid action potential propagation along myelinated axons requires voltage-gated Na+ (Nav)

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channel clustering at nodes of Ranvier. At paranodes flanking nodes, myelinating glial cells

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interact with axons to form junctions. The regions next to the paranodes called juxtaparanodes

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are characterized by high concentrations of voltage-gated K+ channels. Paranodal axoglial junctions function as barriers to restrict the position of these ion channels. These specialized

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domains along the myelinated nerve fiber are formed by multiple molecular mechanisms

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including interactions between extracellular matrix, cell adhesion molecules, and cytoskeletal scaffolds. This review highlights recent findings into the roles of submembranous cytoskeletal

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proteins in the stabilization of molecular complexes at and near nodes. Axonal ankyrin-spectrin complexes stabilize Nav channels at nodes. Axonal protein 4.1B-spectrin complexes contribute to paranode and juxtaparanode organization. Glial ankyrins enriched at paranodes facilitate node

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formation. Finally, disruption of spectrins or ankyrins by genetic mutations or proteolysis is

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involved in the pathophysiology of various neurological or psychiatric disorders.

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Introduction Neurons are highly polarized cells that are divided into several domains. Somatodendritic

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domains of neurons receive input, whereas axonal domains support action potential initiation and

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propagation to the target. Glial cells, oligodendrocytes in the central nervous system (CNS) and

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Schwann cells in the peripheral nervous system (PNS), form myelin sheaths surrounding the axons. Axonal membranes along the myelinated nerve fibers are organized into highly

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specialized domains formed by interaction between neurons and myelinating glial cells including the nodes of Ranvier, short gaps between two adjacent myelin segments (reviewed in Normand

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and Rasband, 2015). At the nodal axolemma, highly accumulated voltage-gated Na+ (Nav)

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channels generate action potentials. Since the internodal segments of axons are covered and insulated by myelin, action potentials propagate rapidly in a saltatory manner. Thus, the molecular organization at and near nodes of Ranvier is required for proper nervous system

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function. The importance of the nodes is further underscored by the fact that the disruption of the nodal molecular organization is involved in the pathophysiology of various nervous system

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diseases and injuries (reviewed in Arancibia-Carcamo and Attwell, 2014; Faivre-Sarrailh and Devaux, 2013; Susuki, 2013). Recent studies revealed the molecules responsible for node of Ranvier formation. These include extracellular matrix molecules secreted by myelinating glial cells, cell adhesion molecules that promote neuron-glia interaction, and cytoskeletal and scaffolding proteins that stabilize the membrane protein complex (reviewed in Eshed-Eisenbach and Peles, 2013; Normand and Rasband, 2015). Here, we focus on the roles of submembranous cytoskeletal proteins including spectrins, ankyrins, and 4.1 proteins for the molecular organization of nodes, paranodes, and juxtaparanodes. We also review the disruption of the spectrins and ankyrins by

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genetic mutations or proteolysis that contributes to the pathophysiology of neurological or

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psychiatric disorders.

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Submembranous cytoskeleton formed by spectrins, ankyrins, and 4.1 proteins

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The evolution of animals required the development of diverse cellular systems including cell differentiation, membrane polarization, cell adhesion, signaling pathways, etc. The emergence of

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spectrins, ankyrins, and 4.1 proteins during evolution has been considered to be one of the

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adaptations to organize cells into multicellular tissues and organs. For detail, see other review articles (Baines, 2010; Baines et al., 2014; Bennett and Healy, 2009; Machnicka et al., 2014). In

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brief, these spectrin-ankyrin-4.1 complexes interact with membrane proteins including channels, cell adhesion molecules, receptors, and transporters, and stabilize these membrane proteins by linking them to the actin cytoskeleton. They also function as a platform for signal transduction.

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Mammals have seven spectrins: I and II spectrins; and I to V spectrins.  and  spectrins associate side-by-side and form antiparallel dimers, which in turn are associated head-to-head to

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form tetramers. The N-terminus of  spectrins contains an actin-binding domain that can also bind protein 4.1, whereas the ankyrin-binding site is located in spectrin repeats 14-15. Ankyrins are scaffolding proteins that interact with both membrane proteins and spectrins. Vertebrates have three ankyrins: ankyrinG, ankyrinB, and ankyrinR. 4.1 proteins include 4.1B, 4.1G, 4.1R, 4.1O, and 4.1N, and serve as adapters by binding to a wide variety of transmembrane proteins and spectrins. Spectrins, ankyrins, and 4.1 proteins are expressed in multiple cell types, tissues, and organs including erythrocytes, epithelial cells, heart muscle, skeletal muscle, and the nervous system. Increasing evidence suggests that these submembranous proteins are essential for multiple nervous system structures including myelinated axons.

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Transmembrane proteins at and near nodes of Ranvier

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Myelinated axons are highly polarized and divided into functionally and molecularly distinct

and Rasband 2015). The molecules responsible for action potential propagation are

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(Normand

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polarized domains including nodes of Ranvier, paranodes, juxtaparanodes, and internodes

concentrated in the short segments at and near nodes (Figure 1); the voltage-gated ion channels

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are highly accumulated at these domains (Figure 2A). Nodes are the short gaps (approximately 1

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m) between two adjacent myelin segments. The nodal voltage-gated ion channels include Nav1.6 (Caldwell et al., 2000), potassium channels KCNQ2 and KCNQ3 (Devaux et al., 2004;

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Pan et al., 2006), and Kv3.1b (Devaux et al., 2003). During node development, Nav1.2 channels are detected at newly forming nodes, but are then replaced by Nav1.6 (Boiko et al., 2001). The regions next to paranodes are called juxtaparanodes and contain highly clustered voltage-gated

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K+ (Kv1) channels (Wang et al., 1993).

How are voltage-gated ion channels restricted to the nodal or juxtaparanodal axolemma?

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Recent studies show that the robust Nav channel clustering at nodes is secured by multiple redundant molecular mechanisms promoted by neurons and myelinating glial cells (reviewed in Eshed-Eisenbach and Peles, 2013; Normand and Rasband 2015). During the early development of PNS nodes, extracellular proteins such as gliomedin secreted by Schwann cells interact with the cell adhesion molecule neurofascin (NF) 186 on the axolemma and promote its clustering at the edge of developing myelin sheaths, resulting in the accumulation of ankyrinG and Nav channels. In contrast, in the CNS, node assembly starts by the formation of axoglial junctions at paranodes that function as a diffusion barrier. At paranodes, the axonal proteins contactin and contactin-associated protein (Caspr) and the glial molecule NF155 form cell adhesion complex

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(Figures 1 and 2B) (Bhat et al., 2001; Boyle et al., 2001; Gollan et al., 2003; Pillai et al., 2009; Sherman et al., 2005; Zonta et al., 2008). Paranodal junctions also function as barriers that

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segregate the Kv1 channels to the juxtaparanodes. Loss of paranodal junctions cause

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mislocalization of juxtaparanodal Kv1 channels to paranodal regions and impaired conduction

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(Bhat et al., 2001; Boyle et al., 2001; Pillai et al., 2009). In addition to their function as a diffusion barrier, paranodal junctions contribute to nerve conduction by sealing the myelin sheath

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to the axon to prevent shunting of nodal action currents beneath the myelin sheath while still

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leaving a narrow channel interconnecting the internodal periaxonal space with the perinodal space (Rosenbluth, 2009). At juxtaparanodes next to the paranodal region, axonal Caspr2 and

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TAG -1 and glial TAG -1 form a cell adhesion complex that is thought to initiate the assembly of the Kv1 channel clusters, although the interactions between Caspr2/TAG-1 and Kv1 channels remain unclear (Poliak et al., 2003; Traka et al., 2003). These specialized domains of ion

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channels and cell adhesion molecules at nodes, paranodes, and juxtaparanodes are further

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stabilized by a submembranous cytoskeletal complex as described below.

The axonal spectrin-ankyrin complex stabilizes the nodal Nav channel complex Spectrins and ankyrins play essential roles for node formation. At the nodal axolemma, the scaffolding protein ankyrinG binds with multiple molecules including the cytoskeletal protein IV spectrin, NF186, and Nav and KCNQ channels (Berghs et al., 2000; Kordeli et al., 1995; Pan et al., 2006; Xu and Cooper, 2015) (Figures 1 and 2C). The ankyrin-binding motif in Nav channels is both necessary and sufficient for Nav channel clustering at nodes (Gasser et al., 2012). IV spectrin’s clustering at nodes also depends on its ankyrin-binding domain (Yang et al., 2007). Silencing ankyrinG expression by RNA interference blocks clustering of

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Nav channels, NF186, and IV spectrin at nodes in myelinating dorsal root ganglion-Schwann cell co-culture (Dzhashiashvili et al., 2007). However, mutant mice lacking ankyrinG in sensory

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neurons or in retinal ganglion neurons showed normal nodes, demonstrating that ankyrinG is

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dispensable for nodal Nav channel clustering in vivo (Ho et al., 2014). Similarly, IV spectrin

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mutant mice still cluster Nav channels at nodes, although the nodal clusters are mildly altered (Komada and Soriano, 2002; Yang et al., 2005). These contradictions to the widely accepted

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model of ankyrin-spectrin complex as a key for node formation may be explained by multiple

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redundant cytoskeletal proteins. In the ankyrinG-deficient axons in dorsal roots or optic nerves, ankyrinR (mainly expressed in erythrocytes) is highly enriched at the nodes, suggesting that

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ankyrinR can substitute for ankyrinG (Ho et al., 2014). Interestingly, in these ankyrinG-deficient axons, nodal IV spectrin was replaced with I spectrin which is a binding partner of ankyrinR in erythrocytes. Similarly, IV spectrin-deficient quivering 3J mice show robust I spectrin

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immunostaining and increased ankyrin R immunoreactivity at the nodes in optic nerves. Thus, ankyrinR-I spectrin complexes replace the ankyrinG-IV spectrin complex and function as a

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reserve mechanism to rescue nodal Nav channel clustering. The critical roles of ankyrins in node formation were confirmed by the observations of loss of Nav channel clustering in the sensory axons lacking both ankyrinG and ankyrinR. Furthermore, loss of giant ankyrinG (480 and 270 kDa) causes a dramatic reduction in the number of nodes of Ranvier and malformation of remaining nodes in the CNS (Jenkins et al., 2015). In this mutant, 190-kDa ankyrinG is increased and may rescue Nav channel clustering at nodes. These results demonstrate that ankyrins and spectrins at nodal axons are required for proper Nav channel clustering at the nodes of Ranvier. In addition to the reserve mechanism (the ankyrinR-I spectrin complex), extracellular matrix molecules and paranodal junctions may rescue Nav channel clustering along the axons

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with disrupted IV spectrin. This was shown by generating double mutant mice with disruption or loss of two mechanisms: extracellular matrix molecules and cytoskeletal scaffolds (IV

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spectrin), or the paranodal junctions and cytoskeletal scaffolds (Susuki et al., 2013). These double mutant mice have juvenile lethality, profound motor dysfunction, and significantly

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reduced Nav channel clustering in the CNS, while mice with a single disrupted mechanism have mostly normal nodes. Furthermore, double mutants of extracellular matrix molecules and

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paranodal junctions show a severe phenotype with reduced nodes in the PNS (Feinberg et al.,

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2010) and the CNS (Susuki et al., 2013). These results suggest that three mechanisms mediated by the extracellular matrix, paranodal junctions, and cytoskeletal scaffolds work together and can

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compensate for each other to form nodes of Ranvier. Thus, robust Nav channel clustering at nodes of Ranvier is secured by multiple redundant molecular mechanisms that include spectrins

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and ankyrins.

Axonal spectrin-protein 4.1 complexes stabilize paranodes and juxtaparanodes

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In addition to their role in Nav channel clustering, paranodal axoglial junctions have been proposed to serve as a membrane barrier that excludes juxtaparanodal components including Kv1 channels (Bhat et al., 2001; Boyle et al., 2001; Pillai et al., 2009; Rosenbluth, 2009). What is the molecular basis of the paranodal membrane barrier? Previous studies demonstrate that the axonal molecular components are important for the formation of paranodal junctions and membrane barrier function. Retention of the paranodal Caspr-contactin complex depends on the cytoplasmic domain of Caspr which interacts with protein 4.1B, whereas the extracellular domain of Caspr is sufficient for directing it to the paranodes (Gollan et al., 2002).Protein 4.1B is primaliry expressed by neurons and highly enriched at paranodes and juxtaparanodes (Buttermore et al.,

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2011; Cifuentes-Diaz et al., 2011; Einheber et al., 2013; Horresh et al., 2010; Ohara et al., 2000) (Figure 1). In transgenic mice expressing a 4.1-binding motif-deficient Caspr protein on a Caspr-

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null background, paranodal axoglial junctions were rescued, but the average area of Caspr-

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positive paranodes was smaller than control mice, suggesting that stable expression of Caspr

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requires its binding to protein 4.1B (Horresh et al., 2010). Kv1 channels were often detected at paranodes in this mutant, suggesting that the interaction of Caspr with protein 4.1B is necessary

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for efficient paranodal membrane barrier. Consistent with these observations, in “Shambling”

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mutant mice expressing Caspr lacking its transmembrane and cytoplasmic domains, the paranodal Caspr-contactin-NF155 complex was disorganized, and Kv1 channels were located at

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paranodes (Sun et al., 2009). Furthermore, in 4.1B mutant mice, clusters of the paranodal molecules Caspr and NF155 are disorganized (Buttermore et al., 2011; Cifuentes-Diaz et al., 2011), although paranodes are mostly preserved in other 4.1B mutant mice (Buttermore et al.,

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2011; Einheber et al., 2013; Horresh et al., 2010). The most striking feature of 4.1B mutant mice is the lack of a juxtaparanodal protein complex including Kv1 channels (Buttermore et al., 2011;

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Cifuentes-Diaz et al., 2011; Einheber et al., 2013; Horresh et al., 2010). Protein 4.1B interacts directly with Caspr2 at juxtaparanodes (Denisenko-Nehrbass et al., 2003), and Caspr 2 is required for the clustering of Kv1 channels (Poliak et al., 2003). These findings demonstrate that the protein 4.1B plays key roles in the molecular organization of paranodes and juxtaparanodes.  The protein 4.1B links Caspr with the underlying axonal cytoskeleton through the spectrin cytoskeleton. II and II spectrin are highly enriched at paranodes and juxtaparanodes (Figures 1 and 2D) and form a complex with protein 4.1B (Garcia-Fresco et al., 2006; Ogawa et al., 2006; Voas et al., 2007). Loss of II spectrin selectively in sensory axons does not disrupt Caspr immunostaining at paranodes or ultrastructures of paranodal axoglial junctions, suggesting that

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the spectrins are not required for the formation and maintenance of these junctions (Zhang et al., 2013). However, II spectrin-deficient axons show redistribution of juxtaparanodal molecules

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including Kv channels into paranodal regions, despite the preserved axoglial cell adhesion

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complex. The abnormal Kv channel localization increased with age, suggesting a role for axonal

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II spectrin in the maintenance of juxtaparanodes. These findings suggest that the molecular basis of the paranodal barrier is the submembranous axonal cytoskeleton formed by II spectrin.

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The observations in II spectrin or protein 4.1B mutant mice also provide some clues for

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the molecular basis of a diffusion barrier fencing the nodal Nav channel complex. For example, PNS axons lacking 4.1B (Einheber et al., 2013) and II spectrin-deficient sensory axons (Zhang

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et al., 2013) have broadening of nodes of Ranvier. In II spectrin mutant zebrafish, the node was abnormally long, and the Nav channel clusters were reduced in number and disrupted at early stages (Voas et al., 2007). Importantly, a submembranous axonal cytoskeleton consisting II

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spectrin, II spectrin, and ankyrinB defines a boundary in unmyelinated axons that precedes

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development of axon initial segments which contain molecules almost identical to nodes (Galiano et al., 2012). Furthermore, in 4.1B-null mice, ankyrinG in axon initial segment invades into the first myelin segment and overlaps with Caspr, suggesting that the protein 4.1B also contributes to the molecular barrier for axon initial segment assembly (Duflocq et al., 2011). Thus, the axonal spectrin-protein 4.1 complex may be involved in the diffusion barrier for the Nav channel complex at the nodes as well as the juxtaparanodal Kv1 channel complex.

Roles of ankyrins and protein 4.1 in myelinating glial cells Paranodal junctions are formed by the interaction between neurons and myelinating glial cells. Recently, we showed ankyrins are expressed in myelinating glia and facilitate assembly of

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paranodal axoglial junctions (Chang et al., 2014). Although ankyrinB was previously reported to be localized at the axonal side of paranodes (Ogawa et al., 2006), subsequent experiments

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revealed that ankyrinB is expressed by myelinating Schwann cells and enriched at the glial side

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of paranodes in the PNS (Figures 1 and 2D). In the CNS, ankyrinG is expressed by

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oligodendrocytes and is also enriched on the glial side of paranodal junctions. These glial ankyrins interact with NF155, a glial cell adhesion molecule that forms the paranodal axoglial

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adhesion complex with axonal contactin and Caspr (Chang et al., 2014). Conditional knockout of

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oligodendrocyte ankyrinG in mouse oligodendrocytes disrupted paranodal junction formation and caused nerve conduction slowing during early CNS development. However, in mature animals,

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the paranodal junctions eventually formed and nerve conduction velocity recovered, suggesting that ankyrinG facilitates rapid assembly of CNS paranodal junctions during development but is not essential for the maintenance of paranodal junctions. AnkyrinB is redistributed in CNS

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paranodes lacking ankyrinG and can compensate for the loss of ankyrinG. However, in double conditional knockout mice lacking both ankyrinG and ankyrinB, paranodes are severely affected

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but gradually form with increasing age, suggesting that compensation by ankyrinB remains only partial. In contrast to CNS paranodes, loss of Schwann cell ankyrinB does not disrupt overall paranodal architecture in sciatic nerves, indicating that ankyrinB is not essential for the PNS paranodes. Given the complexity and compensatory mechanisms of the submembranous cytoskeleton (Ho et al., 2014), it is possible that still unidentified submembranous molecules or mechanisms exist at paranodes and can modulate axoglial junction formation or compensate for the loss of ankyrinG and ankyrinB. Nevertheless, these findings suggest that glial ankyrins interact with NF155 and contribute to formation of paranodal junctions. Protein 4.1G is expressed by myelinating Schwann cells and is localized at the paranodal

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area, Schmidt-Lantermann incisures, and periaxonal, mesaxonal, and abaxonal membranes (Einheber et al., 2013; Ivanovic et al., 2012; Ohno et al., 2006). Deletion of 4.1G in mice results

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in the aberrant distribution of both Schwann cell and axonal proteins along internodes and altered

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Schmidt-Lantermann incisures, but nodes and paranodal junctions are formed properly (Ivanovic

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et al., 2012; Terada et al., 2012). Paranodal and juxtaparanodal proteins are abnormally aggregated at the juxtaparanodal region and along the internodes of 4.1G null mice, suggesting

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that Schwann cell protein 4.1G helps organize the underlying axolemma (Ivanovic et al., 2012). Protein 4.1B is expressed at low levels by Schwann cells (Einheber et al., 2013), and II spectrin

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and II spectrin are expressed in the cytoplasmic bands on the outer surface of mature

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myelinating Schwann cells (Susuki et al., 2011). The composition, localization, and roles of spectrin-ankyrin-4.1 proteins in myelinating glial cells remain unknown.

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Disrupted submembranous cytoskeleton in nervous system diseases and injuries Given the roles of spectrins and ankyrins on stabilization of membrane domains including nodes

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of Ranvier, it is not surprising that the disruption of these proteins contributes to the pathophysiology of nervous system diseases and injuries. Indeed, several mutations in genes encoding spectrins and ankyrins have been reported. Mutations in SPTAN1 which encodes II spectrin cause a distinct clinical syndrome of epileptic encephalopathy characterized by brainstem and cerebellar atrophy and cerebral hypomyelination (reviewed in Tohyama et al., 2015). Mutations in III spectrin cause spinocerebellar ataxia type 5 (Ikeda et al., 2006). Expression of mutant III spectrin found in patients with spinocerebellar ataxia type 5 in Pukinje cells caused mislocalization and dysfunction of metabotropic glutamate receptor 1 at dendritic spines (Armbrust et al., 2014). Mutations in ANK3 that encodes ankyrinG are associated with

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psychiatric disorders including bipolar disorder, schizophrenia, and autism spectrum disorders (Iqbal et al., 2013; Leussis et al., 2012; Shi et al., 2013; Yuan et al., 2012). The giant ankyrinG

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(480kDa) is essential to assemble axon initial segments and the nodes of Ranvier (Jenkins et al.,

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2015) and stabilizes somatodendritic GABAergic synapses (Tseng et al., 2015), whereas a shorter

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form of ankyrinG (190 kDa) is integral to AMPA receptor-mediated synaptic transmission and maintenance of spine morphology (Smith et al., 2014).

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Degradation of spectrins and ankyrins has been implicated in the pathophysiology of

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various nervous system injuries (reviewed in Czogalla and Sikorski, 2005; Saatman et al., 2010; Yan et al., 2012). Spectrins are major substrates for the calcium-dependent cysteine protease

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calpain. Degradation of II spectrin by calpain is especially well documented. Calpain can produce multiple breakdown products of II spectrin with distinct molecular sizes at 150 kDa and 145 kDa. These spectrin breakdown products in the cerebrospinal fluid can predict injury

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severity and mortality after severe traumatic brain injury in human patients (Mondello et al., 2010; Saatman et al., 2010). Calpain-mediated spectrin proteolysis may induce neuronal death in

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traumatic brain injuries, ischemic injury, and neurodegenerative diseases such as Alzheimer’s disease (Czogalla and Sikorski, 2005; Saatman et al., 2010; Yan et al., 2012). Furthermore, calpain-specific degradation products of II spectrin are increased in brains from patients with multiple sclerosis, an autoimmune disease primarily involving CNS myelin (Shields et al., 1999). In the ischemic brain injury model, ankyrinG and IV spectrin at the axon initial segments are degraded by calpain, leading to loss of Nav channel clusters (Schafer et al., 2009). In an animal model of traumatic diffuse brain axonal injury, calpain-mediated proteolysis of ankyrinG and αII spectrin was associated with nodal damage, suggesting a possible contribution of spectrin and ankyrin degradation to nodal disruption (Reeves et al., 2010). Thus, loss of spectrins or ankyrins

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by genetic mutations or proteolysis may disrupt membrane domains in myelinated axons and/or

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somatodendritic domains of neurons, leading to nervous system diseases.

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Conclusion

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As reviewed here, submembranous cytoskeletal complexes formed by spectrins, ankyrins, and 4.1 proteins play key roles in the formation of specialized membrane domains at and near nodes

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of Ranvier along myelinated axons. In addition, these proteins are involved in multiple nervous

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system structures such as axon initial segments (Galiano et al., 2012), somatodendritic domains (Armbrust et al., 2014; Smith et al., 2014), or myelin sheaths (Susuki et al., 2011; Terada et al.,

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2012). Disruption of these proteins contribute to the pathophysiology of various neurological and psychiatric disorders. Increased knowledge of the spectrin-ankyrin-4.1 complexes during nervous system development and injury will impact future studies to establish specific

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treatments not only to limit disease progression but also to facilitate nervous system repair.

Acknowledgement

The authors thank Leonid M. Yermakov (Wright State University, Dayton, OH) for technical assistance.

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Figure legends Figure 1.

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Molecular organization of the node, paranode, and juxtaparanode.

An ankyrinG-IV spectrin complex links Nav channels to the actin cytoskeleton. Loss or

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disruption of these molecules may be substituted by ankyrinR-I spectrin as a reserve mechanism. Paranodal junctions formed by cell adhesion molecules act as a diffusion barrier to

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restrict the mobility of nodal and juxtaparanodal molecules. Axonal II/II spectrin-4.1B

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complexes enriched at paranodes and juxtaparanodes link cell adhesion complexes between axons and glial cells to the actin cytoskeleton. AnkyrinG (CNS) or ankyrinB (PNS) is enriched at

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the glial paranode and interacts with NF155. Nodal proteins are also secured by the interaction between the axonal cell adhesion molecule NF186 and extracellular matrix molecules such as

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brevican (CNS).

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Figure 2. Immunofluorescence of proteins at and near nodes of Ranvier.

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Longitudinal sections of mouse sciatic nerves are stained as indicated.

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(A) Nav channels at nodes (red) and Kv1.2 channels at juxtaparanodes (green). Note the gap

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between Nav and Kv1.2 channel clusters where paranodal junctions are located. (B) Antibodies to the cell adhesion molecule neurofascin (NF) stains axonal NF186 at nodes

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strongly and glial NF155 at paranodes weakly (red). The axonal cell adhesion molecule Caspr is stained in green. Juxtaparanodes are shown in blue (Kv1.2).

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(C) Axonal cytoskeletal and scaffolding proteins at nodes. AnkyrinG (AnkG) in red, and IV

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spectrin (IV) in green. Nodes and paranodes are shown in blue (NF). (D) Axonal II spectrin (II, red) is highly enriched at paranodes and juxtaparanodes. Schwann cell ankyrinB (AnkB) is stained in green. Nodes and paranodes are shown in blue (NF).

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Scale bars = 5 m.