Schwannomin-interacting Protein 1 Isoform IQCJ-SCHIP1 Is a Multipartner Ankyrin- and Spectrin-binding Protein Involved in the Organization of Nodes of Ranvier*♦

The nodes of Ranvier are essential regions for action potential conduction in myelinated fibers. They are enriched in multimolecular complexes composed of voltage-gated Nav and Kv7 channels associated with cell adhesion molecules. Cytoskeletal proteins ankyrin-G (AnkG) and βIV-spectrin control the organization of these complexes and provide mechanical support to the plasma membrane. IQCJ-SCHIP1 is a cytoplasmic protein present in axon initial segments and nodes of Ranvier. It interacts with AnkG and is absent from nodes and axon initial segments of βIV-spectrin and AnkG mutant mice. Here, we show that IQCJ-SCHIP1 also interacts with βIV-spectrin and Kv7.2/3 channels and self-associates, suggesting a scaffolding role in organizing nodal proteins. IQCJ-SCHIP1 binding requires a βIV-spectrin-specific domain and Kv7 channel 1-5-10 calmodulin-binding motifs. We then investigate the role of IQCJ-SCHIP1 in vivo by studying peripheral myelinated fibers in Schip1 knock-out mutant mice. The major nodal proteins are normally enriched at nodes in these mice, indicating that IQCJ-SCHIP1 is not required for their nodal accumulation. However, morphometric and ultrastructural analyses show an altered shape of nodes similar to that observed in βIV-spectrin mutant mice, revealing that IQCJ-SCHIP1 contributes to nodal membrane-associated cytoskeleton organization, likely through its interactions with the AnkG/βIV-spectrin network. Our work reveals that IQCJ-SCHIP1 interacts with several major nodal proteins, and we suggest that it contributes to a higher organizational level of the AnkG/βIV-spectrin network critical for node integrity.

that it contributes to a higher organizational level of the AnkG/␤IV-spectrin network critical for node integrity.
Saltatory conduction in myelinated neurons depends on the compartmentalization of ion channels into polarized domains along the axons. Voltage-gated Na ϩ (Nav) 5 and K ϩ (Kv7.2/3) channels are clustered at axon initial segments (AIS) and nodes of Ranvier (NR), where they are responsible for the generation and propagation of action potentials and modulate axonal excitability. At NR and AIS, Nav and Kv7.2/3 are part of complexes, including cell adhesion molecules, NrCAM and neurofascin-186 (Nfasc186), and the scaffolding protein ankyrin-G (AnkG), which links membrane proteins to the underlying actin cytoskeleton through ␤IV-spectrin (1). Despite these molecular similarities, the mechanisms by which these complexes are concentrated are distinct at AIS and NR. Complex assembly at NR depends on both intrinsic axonal and extrinsic glial-controlled mechanisms, whereas AIS assembly depends only on intrinsic mechanisms (1). Several studies point to the ankyrin/ spectrin network as a master organizer of both AIS and NR, controlling the molecular and nanoscale organization of the complexes and providing an elastic and stable mechanical support to the plasma membrane (1,2). Mouse models highlight the essential role of ␤IV-spectrin in membrane stability (3)(4)(5)(6)(7).
SCHIP1 is a cytoskeleton-associated protein initially identified as a partner of schwannomin/merlin, the product of the tumor suppressor gene mutated in neurofibromatosis type 2 (NF2) (8,9). No strong association between SCHIP1 gene mutation and human nervous system disease has been reported with the exception of weak evidence for a possible link with autism spectrum disorders (10 -13). In contrast, mutation of the Drosophila orthologue, Schip1, whose sequence is only poorly conserved, is embryonic lethal (14). In mammals, six different SCHIP1 isoforms, encoded by the same gene, are expressed in the nervous system (8,15,16). They differ by their N-terminal part and share a common C-terminal domain, including a leu-cine zipper region predicted to adopt a coiled-coil conformation. We previously showed that IQ-containing J protein-SCHIP1 (IQCJ-SCHIP1), an isoform with a specific N-terminal domain, is a component of AIS and NR (15). During development, IQCJ-SCHIP1 accumulates at NR and AIS after Nav channels and AnkG, indicating that it could play a role in the organization and stabilization of mature NR and AIS (15). IQCJ-SCHIP1 interacts directly with AnkG and is lost at AIS in the absence of AnkG (15). The interaction with AnkG requires the SCHIP1 C-terminal leucine zipper and phosphorylation by protein kinase CK2 (15,17), which is enriched at NR and AIS, and regulates AnkG interactions with ␤IV-spectrin, Nav and Kv7 channels (17)(18)(19)(20). IQCJ-SCHIP1 is absent from NR and AIS in quivering-3J mice (qv 3J ) (15), which carry a mutation in the Spnb4 gene encoding ␤IV-spectrin and lack Kv7.2 clustering at peripheral NR (21,22). This suggests that IQCJ-SCHIP1 may associate with one or both of these proteins.
In this study we show that IQCJ-SCHIP1 is able to interact with both ␤IV-spectrin and Kv7.2/3 channels. Moreover, we provide evidence for IQCJ-SCHIP1 protein self-association, likely forming oligomers, suggesting a specific mechanism by which IQCJ-SCHIP1 participates in protein complex organization. We investigate the role of IQCJ-SCHIP1 in vivo by studying NR in peripheral myelinated fibers in Schip1 mutant mice (Schip1⌬10, named hereafter ⌬10), in which none of the six isoforms of SCHIP1 is expressed (16). We observe that the major nodal proteins, including AnkG, ␤IV-spectrin, and Kv7.2/3 channels, are normally present at NR in young and 14-month-old mutant mice, indicating that IQCJ-SCHIP1 is not required for their persistent nodal accumulation. However, ⌬10 mice present an altered nodal shape similar to that observed in ␤IV-spectrin mutant mice, indicating that IQCJ-SCHIP1 contributes to nodal membrane-associated protein organization. ⌬10 mice also display an increased accumulation of vesicles at NR as well as microtubule disorganization around mitochondria all along the axon, suggesting that SCHIP1 could play additional roles, possibly in relation with axon trafficking.
To identify more precisely the protein domains implicated in this interaction, we performed co-immunoprecipitations with ␤IV⌺6 proteins bearing various C-terminal deletions (see Fig.  1A). Deletion of either the PH and SD domains without (Myc-⌺6⌬1) or with deletion of part of the spectrin repeat 17 (Myc-⌺6⌬2) dramatically altered co-immunoprecipitation of FLAG-IQCJ (Fig. 1C). By contrast, FLAG-IQCJ was efficiently pulled down with a mutant ␤IV⌺6 protein lacking only the PH domain (Myc-⌺6⌬3) (Fig. 1D). These observations indicate that the interaction of ␤IV-spectrin with IQCJ-SCHIP1 requires its SD domain but not its PH domain. Interestingly and consistently, FLAG-IQCJ did not co-immunoprecipitate with GFP-tagged ␤II-spectrin (GFP-␤II), which displays high sequence similarity with ␤IV⌺1 but does not contain an SD domain (Fig. 1, A and E) (23). We then performed reciprocal experiments, testing the ability of IQCJ-SCHIP1 to interact with various C-terminal parts of ␤IV-spectrin (see Fig. 1F). A molecule encompassing only part of the spectrin repeat 17 and the SD and PH domains (GFP-tagged, GFP-␤IVCter) co-immunoprecipitated with FLAG-IQCJ (Fig. 1G), indicating that the C-terminal part of ␤IV-spectrin is sufficient for its association with IQCJ-SCHIP1. Co-immunoprecipitation was maintained or even increased when the PH domain was deleted (GFP-␤IVSR-SD) or with the SD domain alone (GFP-␤IVSD) (Fig. 1G). These results further support that the SD domain of ␤IV-spectrin is sufficient for the interaction and is the primary site of binding to IQCJ-SCHIP1, although a secondary binding site may exist within the spectrin repeat domain considering the low but consistent level of IQCJ-SCHIP1 binding to C-terminal truncated ␤IV⌺6 proteins (Fig.  1C, Myc-⌺6⌬1 and Myc-⌺6⌬2).
The specific 97-amino acid N-terminal domain of IQCJ-SCHIP1 contains an IQ CaM-binding motif , which is able to interact with CaM in the absence of a calcium (15). We further asked whether this domain was required for the association with HA-␤IVCter and Kv7.2, using another SCHIP1 isoform, SCHIP1a (FLAG-1a), which presents a distinct 22-amino acid N-terminal domain (Fig. 3A). Both HA-␤IVCter (Fig. 3F) and Kv7.2 ( Fig. 3G) co-immunoprecipitated with FLAG-1a, indicating that the interactions of IQCJ-SCHIP1 with ␤IV-spectrin and Kv7 channels are not strictly dependent on its IQ CaMbinding motif but rather require sequences located between residues 97 and 325.
IQCJ-SCHIP1 Self-associates and Forms Oligomers-Requirement of the C-terminal coiled-coil region for both Kv7 and ␤IV-spectrin interactions was intriguing because we previously showed that this region was also required for SCHIP1 association with AnkG (15) and schwannomin (8). Of interest, bioinformatics analyses of the IQCJ-SCHIP1 amino acid sequence revealed a high content of potentially unfolded regions in the N-terminal and central domains and a higher probability of structuring at the C terminus, including the predicted coiled-coil region (Fig. 4A, upper panel). This coiled-coil region presented a high probability to form trimeric coiledcoils (Fig. 4A, lower panel), suggesting that IQCJ-SCHIP1 could oligomerize. In support of this hypothesis, we had previously shown that SCHIP1 was able to self-associate in vitro (8). We thus wondered whether the C-terminal coiled-coil region could mediate IQCJ-SCHIP1 oligomerization in vivo. We first performed co-immunoprecipitation from lysates of COS-7 cells co-expressing HA-tagged IQCJ-SCHIP1 (HA-IQCJ) and FLAG-IQCJ or FLAG-IQCJ⌬C. HA antibodies pulled down FLAG-IQCJ but not FLAG-IQCJ⌬C (Fig. 4B), demonstrating the ability of IQCJ-SCHIP1 to self-associate through its coiledcoil region in a cell environment. We next analyzed cell lysates from transfected cells on native-polyacrylamide gels. Three protein bands (ϳ150, ϳ440, and Ͼ1000 kDa) were detected for FLAG-IQCJ, whereas only one protein band (ϳ140 kDa) was visible for FLAG-IQCJ⌬C (Fig. 4C). This observation supported the oligomerization of FLAG-IQCJ in cells through its coiled-coil region. Abnormal migration of FLAG-IQCJ and FLAG-IQCJ⌬C monomers (150/140 kDa versus 70/80 kDa on SDS-polyacrylamide gels) may be due to the high content of charged amino acids in IQCJ-SCHIP1 (ϳ16.3% Glu/Asp). The ϳ440-kDa band may correspond to trimers, whereas the highest molecular band (Ͼ1000 kDa) indicates that IQCJ-SCHIP1 can form more complex oligomers. The interactions of IQCJ-SCHIP1 with its partners therefore may require its oligomerization rather than a direct interaction with its C-terminal domain. In addition, IQCJ-SCHIP1 might play a role in clustering together several proteins with which it interacts through its self-association. Lysates IP αFLAG  ⌬10 Mice Display Locomotor and Sensory Dysfunctions-Altogether, our data showed that SCHIP1 associates with at least four nodal components as follows: AnkG, ␤IV-spectrin, and Kv7.2 and Kv7.3 channels. Our previous observations suggested that IQCJ-SCHIP1 could play a role in mature NR, potentially in NR organization and/or stabilization (15). We further explored SCHIP1 function in peripheral nerves by studying ⌬10 mutant mice previously described, in which we showed that none of the six isoforms of SCHIP1 is expressed (16).
We first characterized the behavioral phenotype of ⌬10 mice (4 -6 months old), using a battery of tests that may indicate peripheral deficits. Although mice moved around normally in their home cages, footprint pattern analysis revealed subtle walking problems, including abnormal hind limb spreading (Fig. 5A). Tests of motor ability function such as grid test (Fig.  5B) and hanging wire test (Fig. 5C) also revealed impairments. However, the muscular strength of ⌬10 mice evaluated by the grip test did not show any deficit (Fig. 5D). An increased reaction time was observed when mice were placed on a hot plate (Fig. 5E), indicating a slightly reduced pain sensitivity. Thus, mutant mice displayed some degree of ataxia and reduced pain sensitivity, which could reflect sensory defects.
Alterations of PNS Myelinated Fibers in ⌬10 Mice-Behavioral deficits in mutant mice suggested peripheral nerve defects, although a central nervous system origin cannot be excluded because ⌬10 mice also present some brain defects (16). We thus investigated peripheral nerve functions and morphology of ⌬10 mice. Electrophysiological measurements showed that sciatic nerve conduction was not significantly different between mutant and wild-type (WT) littermates ( Fig. 6A; Table 1;  . B, grid test. When walking on a grid ⌬10 mice slipped more often than WT mice (six mice/genotype). C, hanging wire test. The latency to fall down from the grid was significantly shorter for ⌬10 mice than for WT mice (6 -9 mice/genotype). D, grip strength analysis. The grip strength of ⌬10 mice was similar to that of WT mice (7-9 mice/genotype). E, hot plate test. On a heated plate at 52°C, paw withdrawal latency was increased for ⌬10 mice compared with WT mice (7-9 mice/genotype). Data are means Ϯ S.E. Statistical analyses: unpaired t test (A-C, **, p Ͻ 0.01; D, p ϭ 0.4643, non significant; E, *, p Ͻ 0.05).

TABLE 1
Electrophysiological characteristics of sciatic nerves of WT and ⌬10 mice CV V, max and CV V, 1 ⁄ 2 indicate conduction velocity at maximal amplitude and at halfmaximal amplitude, respectively. The duration was measured at half the maximal amplitude. n represents the number of nerves tested. Data are means Ϯ S.D. Statistical analyses: two-tailed t tests for two samples of equal variance. *, p Ͻ 0.01. in WT mice (Fig. 6F, inset). Furthermore, whereas in WT mice groups of unmyelinated fibers were regularly surrounded by a single Schwann cell process (Remak bundles) (Fig. 6F, arrowheads), in ⌬10 mice Schwann cell processes often made several turns around small caliber axons (Fig. 6F, arrowheads). Altogether, these observations suggest that ⌬10 mice present axonal loss with mild signs of neuropathy.
Ultrastructural Alterations of PNS NR in ⌬10 Mice-We further characterized the ultrastructure of NR by electron microscopy analysis on ultra-thin sections of sciatic and phrenic nerves. On longitudinal sections of sciatic nerves, the cytoplasmic glial loops contacting the axons at paranodes appeared globally normal in mutant mice (Fig. 8A). The transverse bands, the ultrastructural hallmark of paranodal junctions, were visible in mutant as well as in WT mice (Fig. 8A). Schwann cell microvilli filled the nodal gap and contacted the axon, and their organization was indistinguishable in mutant and WT mice (Fig. 8B). However, the presence of swollen and shorter NR was observed in ⌬10 mice (Fig. 8B). In addition, the electron-dense coat beneath the nodal plasma membrane appeared scalloped (Fig. 8B). These observations indicate that IQCJ-SCHIP1 is required to stabilize the structural organization of NR and are consistent with morphological abnormalities detected by immunolabeling. Further analysis suggested an increased number of intra-axonal vesicles randomly distributed in the nodal regions of ⌬10 mice as compared with WT mice (Fig. 8B,  arrows). Quantification on ultra-thin transversal sections of phrenic nerves showed a significantly increased number of ves- icles (Fig. 8C, arrows and arrowheads), some with an electrondense core (Fig. 8C, arrows) Moreover and interestingly, a detailed examination of the cytoskeleton in transverse sections revealed the presence of microtubules gatherings closely associated with mitochondria in nodal (Fig. 8D) and internodal regions (Fig. 8E) of mutant fibers. We quantified the number of microtubules localized 30 nm or closer to each mitochondria in the internodes. This number was significantly higher in ⌬10 mice than in WT mice (WT, 5.04 Ϯ 0.35; ⌬10, 7.89 Ϯ 0.43; Mann-Whitney test, n ϭ 48 mitochondria/genotype, three mice/genotype, 16 mitochondria/animal, ***, p Ͻ 0.0001), indicating peri-mitochondrial spatial disorganization in mutant mice. This phenotype was independent from any obvious abnormalities in neurofilament density or organization (Fig. 8, D, and E) or significant change in the expression levels of the neurofilament subunits NF-H (WT, 100 Ϯ 18%; ⌬10, 113 Ϯ 12%; mean Ϯ S.E., n ϭ 3 mice/genotype; t test, t ϭ 0.587, p ϭ 0.59), NF-M (WT, 100 Ϯ 17%, ⌬10, 106 Ϯ 10%; t ϭ 0.315, p ϭ 0.77), and NF-L (WT, 100 Ϯ 17%; ⌬10, 113 Ϯ 11%; t ϭ 0.638, p ϭ 0.56) (Fig. 8F). No change in neuronal tubulin (␤3-tubulin) expression was detected in mutant mice (WT, 100 Ϯ 7; ⌬10, 107 Ϯ 16; n ϭ 3 mice/genotype; t test, t ϭ 0.417, p ϭ 0.7) (Fig. 8F). These observations suggest additional roles for SCHIP1 in axons, possibly in axonal transport.

Discussion
We previously characterized IQCJ-SCHIP1 as a partner of two cytoskeleton-associated proteins, schwannomin/merlin and the nodal protein AnkG. We identify here three additional nodal partners for IQCJ-SCHIP1, ␤IV-spectrin, Kv7.2, and Kv7.3 channels, and we show that IQCJ-SCHIP1 likely forms oligomers. These protein-protein interactions suggest that IQCJ-SCHIP1 could play multiple roles in the organization and/or function of NR by linking important proteins. Ultrastructural analyses revealed an altered shape of NR in mutant mice, indicating that IQCJ-SCHIP1 contributes to the nodal architecture. Nonetheless, IQCJ-SCHIP1 appears dispensable for nodal protein accumulation because the major nodal proteins are similarly enriched in young and old ⌬10 mice.
IQCJ-SCHIP1 Associates with Multiple Nodal Proteins-Our previous work (15,17) and this study demonstrate the ability of IQCJ-SCHIP1 to associate with multiple proteins of NR and therefore strongly support that IQCJ-SCHIP1 is a component of nodal complexes, which include channels, cell adhesion molecules, and the cytoskeletal proteins ␤IV-spectrin and AnkG. In peripheral myelinated fibers, ankyrin-spectrin complexes are not restricted to NR, and ankyrin-B (AnkB) and ␤II-spectrin are enriched at paranodes (28,29). Importantly, we found that the interaction of IQCJ-SCHIP1 with ␤IV-spectrin requires the SD domain, which is absent in ␤II-spectrin or other ␤-spectrins (23). This provides a possible mechanism for the selective enrichment of IQCJ-SCHIP1 at AIS and NR where ␤IV-spectrin is localized. In addition, we previously showed that IQCJ-SCHIP1 interacts in vitro with the N-terminal ankyrin repeat domains of AnkG and AnkB with similar affinities, but these interactions require IQCJ-SCHIP1 phosphorylation by CK2, which is specifically concentrated in NR and also regulates AnkG interactions with ␤IV-spectrin, Nav, and Kv7 channels (17)(18)(19)(20). Thus, multiple IQCJ-SCHIP1 protein-protein interactions as well as phosphorylation by CK2 appear to contribute to the highly specialized molecular organization of NR.
IQCJ-SCHIP1 Stabilizes Nodal Axon Membrane-We found that ⌬10 mice present shorter and wider NR as compared with WT mice. These shape alterations were detectable in young as well as old mice, suggesting that IQCJ-SCHIP1 could contribute to nodal shape architecture. In mammalian cells, the mechanical support of the plasma membrane, as well as the microscale organization of membrane-spanning proteins, is provided by the association of spectrins and ankyrins, which promote the formation of an actin-based network coupled to the inner surface of the plasma membrane (30). Mutant mouse studies showed that ␤IV⌺6 plays a specific role in nodal Nav channel clustering, whereas ␤IV⌺1 stabilizes nodal membrane integrity (3,4,7). Mutant mice lacking ␤IV⌺1 present nodal shape alterations, which are very similar to those observed in ⌬10 mice (3,4). Some degree of nodal protrusion was also observed in qv 3J mice (5). This strongly suggests that IQCJ-SCHIP1 could contribute to the organization of the actin/␤IVspectrin/AnkG network to provide elastic and stable mechanical support of the plasma membrane.
Such a scaffolding role for IQCJ-SCHIP1 in cytoskeleton organization is supported by its ability to associate with both AnkG and ␤IV-spectrin. Moreover, we showed that the interactions with ␤IV-spectrin, AnkG, and Kv7 channels require N-terminal sequences of IQCJ-SCHIP1 but also its C-terminal region, which is likely to be implicated in its oligomerization (Fig. 9A). This raised the intriguing possibility that IQCJ-SCHIP1 could form complex oligomers through its C-terminal domain (see Fig. 4C) and expose its more N-terminal sequences to interact with its multiple partners, and thus contribute to organize protein networks. Protein flexibility and high surface charge have been found to be important properties for hub proteins, which interact with multiple proteins (31). Thus, the unfolded nature of IQCJ-SCHIP1 would fit well with a hub position in protein interaction networks.
The nanoscale architecture of AIS was recently resolved, showing that the actin/␤IV-spectrin/AnkG submembranous cytoskeleton is organized as a periodic lattice of proteins that alternate between actin rings and ␤IV-spectrin⅐AnkG com-  plexes (32)(33)(34)(35). NR appear to present a similar architecture (33,34). The periodic cytoskeleton lattice was also observed in distal axons, where actin rings alternate with ␤II-spectrin (32)(33)(34). In that case, actin rings were proposed to be connected by typical head-to-tail ␤II-spectrin/␣-spectrin tetramers. At AIS, the periodicity of the lattice was shown to result from longitudinal head-to-tail ␤IV-spectrin subunits connecting actin rings at their N-terminal extremities to AnkG⅐Nav channel complexes near their C-terminal SD domains (Fig. 9B) (35). However, no ␣-spectrin subunit has been reported in AIS or in NR. Because IQCJ-SCHIP1 interacts with the C-terminal ␤IV-spectrin SD domain, we propose that, through its oligomerization, IQCJ-SCHIP1 could serve to indirectly link ␤IV-spectrin molecules in a head-to-tail manner (Fig. 9B). This hypothesis implies that ␤IV-spectrin and IQCJ-SCHIP1 mutations would somehow lead to similar cytoskeleton disturbance. Thus, it is consistent with the fact ⌬10 and ␤IV-spectrin mutant mice present similar nodal shape alterations (Refs. 3-5 and this study).
The persistence of NrCAM, Nfasc186, and Nav and Kv7 channels in NR of ⌬10 mutant mice indicates that the interactions of IQCJ-SCHIP1 with ␤IV-spectrin and/or AnkG are not strictly required for nodal membrane protein clustering. However, because most of these membrane proteins are able to interact with AnkG, we suggest that IQCJ-SCHIP1 could contribute to their microscale organization within membrane complexes by controlling the AnkG/␤IV-spectrin network organization (Fig. 9B). The ability of IQCJ-SCHIP1 to associate with Kv7 channels could also serve this function for Kv7 channels by connecting them indirectly with ␤IV-spectrin.
Remarkably, we showed that the N-terminal IQCJ domain of IQCJ-SCHIP1 is not required for its interactions with ␤IVspectrin, AnkG, and Kv7 channels (Fig. 3, F and G) 9. A, schematic structure and interacting partners of IQCJ-SCHIP1. IQCJ-SCHIP1 is able to oligomerize through its C-terminal coiled-coil region and to interact with AnkG, ␤IV-spectrin, and Kv7 through more N-terminal sequences. Its interaction with AnkG requires its phosphorylation by CK2. Numbers correspond to amino acid residues. Dashed lines delineate the C-terminal domain, which is conserved in the six isoforms of SCHIP1 and found in almost all animals (annotated in Pfam as "SCHIP-1 domain," PF10148). B, schematic model of IQCJ-SCHIP1-containing molecular complexes at NR. IQCJ-SCHIP1 interacts with the intracellular C-terminal domain of Kv7 channels, the SD domain of ␤IV-spectrin, and the membrane-binding domain of AnkG (270/480 kDa), which by itself associates with Nav/Kv7 channels, cell adhesion molecules (CAM), and ␤IV-spectrin. The submembrane cytoskeleton is organized as a periodic lattice that alternates between F-actin rings and ␤IV-spectrin⅐AnkG complexes. Longitudinal head-to-tail ␤IV-spectrin subunits connect F-actin rings at their N-terminal extremities to AnkG⅐channels⅐CAM complexes near their SD domains. Through oligomerization, IQCJ-SCHIP1 could indirectly link ␤IV-spectrin subunits in a head-to-tail manner (1) and contribute to Nav/Kv7 channels and CAM microscale organization within membrane complexes by controlling the AnkG/␤IVspectrin network organization (2).

IQCJ-SCHIP1 at Nodes of Ranvier
FEBRUARY 10, 2017 • VOLUME 292 • NUMBER 6 questions whether this domain could mediate the association of IQCJ-SCHIP1 with additional non-identified nodal partners and/or whether it could contribute to regulate in vivo complex formations through its ability to interact with CaM in the absence of calcium. Of interest, Leterrier et al. (35) showed that an acute intracellular calcium increase has a strong effect on the periodicity of the AIS submembrane lattice. Additional Axonal and/or Glial Roles for SCHIP1?-NR are sites of vesicle membrane compartment accumulation, which results from local transport retardation and is thought to serve nodal membrane processing and/or turnover (36). A finding of our study is that ⌬10 mice present an increase number of nodal vesicles, which interestingly, was also observed in ␤IV⌺1 and qv 3J mutant mice (3,5) and could therefore result from abnormal nodal membrane processing and/or stability. Electron microscopy showed in addition an abnormal spatial organization of microtubules around mitochondria at NR and all along the axon. The functional relevance of this phenotype is not known because to our knowledge it was not described before. Interestingly, the coiled-coil region of SCHIP1 presents sequence homologies with a coiled-coil region within the C-terminal domain of the protein FEZ1 (8), which plays a role in kinesin-mediated anterograde transport of vesicles and mitochondria in axons (37)(38)(39). In addition, although we showed that the SD domain of ␤IV-spectrin is the primary binding site for SCHIP1, our results do not strictly exclude the possibility that SCHIP1 could interact with other ␤-spectrins such as ␤III-spectrin, which has been shown to interact with dynactin (40) and is implicated in dynein-mediated vesicular transport (41,42). This raises the intriguing hypothesis that SCHIP1 could play a role in anterograde and/or retrograde microtubule-based axonal transport. Axonal trafficking defects could thus contribute to vesicle accumulation at nodes in ⌬10 mice and possibly to the wrinkled aspect of the nodal membrane as a result of defects in the balance of membrane delivery/recovery. A role for SCHIP1 in axonal transport would also be consistent with the axonal loss observed in mutant mice.
Structural analyses also revealed an axon diameter decrease and a myelin thickness increase in ⌬10 mice. The mechanisms underlying this phenotype may be complex because the development of unmyelinated and myelinated fibers depends on communication between axons and Schwann cells, and both myelin and axon defects may therefore be of glial and/or axonal origin. However, schwannomin knockdown in either neurons or Schwann cells also results in hypermyelination in mice (43,44), thus raising the possibility that SCHIP1 could contribute to glial and/or neuronal functions of schwannomin in myelination.
In conclusion, we provide here evidence for the importance of SCHIP1 in the organization of peripheral myelinated fibers. We show that through its ability to interact with several nodal partners, IQCJ-SCHIP1 appears to be important for the organization of molecular complexes of peripheral NR. Through its oligomerization and multiple interactions, IQCJ-SCHIP1 may contribute to the high scale organization of the actin/AnkG/ ␤IV-spectrin network, thus providing elastic and stable mechanical support of the nodal plasma membrane.

Behavioral Tests
The behavioral phenotype of WT and ⌬10 littermate mice (4 -6 months old) was examined during footprinting, grid, grip, and hot plate tests. All tests were performed in sound-attenuated rooms, between 9 a.m. and 5 p.m. Mice were group-housed with ad libitum access to food and water and a 12-12 h lightdark cycle (light phase onset at 7 a.m.). For most experiments and initial quantitative analyses, the experimenter was blinded to the genotype of the mice tested and analyzed.
Footprint Pattern Analysis-The gait analysis method was modified from Refs. 51, 52. Mice (three of each genotype) were tested in a confined walkway 4.5 cm wide and 100 cm long with 10-cm high walls and a dark shelter at the end. Mice were trained several times to walk into the dark compartment. The footprints were obtained by dipping the hind paws into ink, before they walked down the corridor on white paper. The footprint patterns generated were scored for angles with regard to walking direction (ImageJ software, NCBI).
Grid Test-Coordination between forelimbs and hind limbs and accurate limb placement were examined by assessing the ability to walk on metal grid bars with 1.5-cm gaps on the bottom of a 30 ϫ 20 ϫ 20-cm box. The performance of each animal (six of each genotype) was analyzed by counting the number of errors in foot placement/total number of steps, during the 2-min sessions, once a day, for 3 consecutive days. On the day prior to data collection, each mouse was allowed to walk on the grid for 2 min.
Wire-hanging Test-The wire-hanging test was performed to measure neuromuscular strength. Mice (6 -9/genotype) were gently placed on a wire-cage lid, which was then slowly waved and turned upside down above the soft bedding. The hanging time for each mouse to fall onto the bedding below was measured with a cutoff time of 60 s.
Grip Strength Analysis-Forelimb grip strength was measured using a Grip Strength Meter (Bioseb). Mice (7-9/genotype) were held by the tail and allowed to grasp a trapeze bar with their forepaws. Once the mouse grasped the bar with both paws, the mouse was pulled away from the bar until the mouse released the bar. The digital meter displays the level of tension (in grams) exerted on the bar by the mouse.
Hot Plate-A standard hot plate (Bioseb), adjusted to 52°C, was used to assess motor reactions in response to noxious stimuli. Mice (7-9/genotype) were confined on the plate by a Plexiglas cylinder (diameter 19 cm, height 26 cm). The latency to a hind paw response (licking or shaking) or jumping, whatever happened first, was taken as the nociceptive threshold.

Immunohistochemistry and Image Analysis
Sciatic nerves of mice (2.5 months old, three of each genotype; 14 months old, four of each genotype) were dissected and fixed in 2% paraformaldehyde for 30 min at room temperature, teased apart to yield single fiber preparations, air-dried, and kept at Ϫ20°C. Immunofluorescent staining was performed as described previously (53). Briefly, slides were treated with 0.1 M glycine for 30 min, preincubated for 1 h at room temperature in 2 g/liter porcine skin gelatin and 2.5 ml/liter Triton X-100 in PBS (PGT buffer), before incubation with primary antibodies (diluted in PGT) overnight at 4°C. After washing with PBS, coverslips were incubated for 2 h at room temperature with secondary antibodies (diluted in PGT), washed again with PBS, and mounted in Vectashield. Images were acquired using a Leica SP5 confocal laser-scanning microscope (Leica Microsystems). Measurements of NR width (perpendicular to the axon axis) and length (parallel to the axon axis) were performed on images of NrCAM labeling acquired with a DM6000-2 Leica microscope equipped with a CCD camera, using ImageJ software (100 NR/animal).

Electron Microscopy and Morphometry
Mice (10 months old, three of each genotype) were anesthetized with pentobarbital and perfused with 9 g/liter NaCl, followed by 40 g/liter paraformaldehyde and 30 g/liter glutaraldehyde in 0.1 M phosphate buffer (PB). The sciatic and phrenic nerves were removed and placed in fresh fixative overnight at 4°C, rinsed in PB, post-fixed in 20 g/liter OsO 4 in PB, dehydrated in an ascending series of ethanol, and embedded in epoxy resin. Morphometric analyses were performed on 0.5m-thick semi-thin transversal sections of phrenic nerves stained with toluidine blue and visualized with a DM6000 Leica microscope. Fibers and axon diameters, and g ratios, defined as ratios of axonal to fiber's diameter, were measured with IQCJ-SCHIP1 at Nodes of Ranvier FEBRUARY 10, 2017 • VOLUME 292 • NUMBER 6

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DigitalMicrograph software (Gatan), for ϳ100 fibers/animal. Ultrastructural studies were performed on phrenic nerve transversal sections and sciatic nerve longitudinal sections. Ultrathin sections (40 nm) were stained with Reynold's lead citrate and uranyl acetate and examined with a Philipps CM-100 transmission electron microscope. Images were acquired using an Orius (Gatan) digital camera. Ten NR were examined in longitudinal sections of sciatic nerves from mice of each genotype. Quantification of the number of vesicles per NR section (14 sections/animal) and the distance between microtubules and mitochondria in the internodes (17 mitochondria/animal) was performed on phrenic nerve transversal sections using the DigitalMicrograph software (Gatan).

Sciatic Nerve Lysate Preparation and Immunoblotting
Sciatic nerves of mice (8 months old, three of each genotype) were dissected out and homogenized in a Dounce vessel containing 200 l of a lysis buffer containing 10 mM NaP i buffer, pH 7.8, 59 mM NaCl, 1 ml/liter Triton X-100 g/liter deoxycholate, 1 g/liter SDS, 100 ml/liter glycerol, 25 mM ␤-glycerophosphate, 50 mM NaF, 2 mM Na 3 VO 4 , and Complete protease inhibitors. Homogenates were centrifuged for 30 min at 4°C at 20,000 ϫ g, and protein concentration in the supernatants was determined by the bicinchoninic acid method (Sigma). Equal amounts of protein (40 g) were loaded in NuPAGE 8 -12% BisTris gels (Thermo Fisher Scientific), and immunoblots were performed as described above. The expression levels of the proteins were quantified using Odyssey Imaging System (LI-COR Biosciences) and normalized on clathrin expression, which was not expected to be affected in ⌬10 mice.

Electrophysiological Analysis
Mice (8 months old, 4 -5/genotype) were euthanized, and the sciatic nerves were quickly dissected out and transferred into artificial cerebrospinal fluid containing 126 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 2 mM MgSO 4 , 1.25 NaH 2 PO 4 , 26 mM NaHCO 3 , and 10 mM dextrose, pH 7.4 -7.5. The sciatic nerves (2-cm segments) were placed in a three-compartment recording chamber and perfused at 1-2 ml/min in medium equilibrated with 95% O 2 and 5% CO 2 . The distal end was stimulated supramaximally (40-s duration) through two electrodes isolated with petroleum jelly, and recordings were performed at the proximal ends. Signals were amplified and digitized at 500 kHz. The duration of the CAPs was calculated at the half-maximal amplitude (V1 ⁄ 2 ). The delay of the CAPs was measured at V1 ⁄ 2 and at the maximal amplitude (V max ). The conduction velocity was derived from the delay. For recruitment analysis, the CAP amplitude was measured and plotted as a function of the stimulation intensity. For the refractory period analysis, two successive stimuli were applied at different intervals, and the amplitude of the second CAP was measured and plotted as a function of the delay between the two stimuli.

Bioinformatics and Statistical Analysis
Secondary structure and oligomerization were predicted using the FoldIndex and MULTICOILS software, respectively. Statistical analyses were performed with GraphPad Prism 5 software. For variables that did not follow a normal distribu-tion, statistical analyses were carried out using the Mann-Whitney rank sum test to compare quantitative variables between WT and ⌬10 mice. Significant main effects were further analyzed by post hoc comparisons of means using t tests. The significance was established at a p value Ͻ0.05.