Postsynaptic ß1 spectrin maintains Na+ channels at the neuromuscular junction.

Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (ß4 spectrin) were reported to cause both neuropathy and myopathy. Although the role of ß4 spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific ß4 spectrin conditional knockout mouse, we show that ß4 spectrin does not contribute to muscle function. In addition, we show ß4 spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, ß1 and ß2 spectrins are found in skeletal muscle, with α2 and ß1 spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and ß2 spectrins had no effect on muscle health, function or the enrichment of ß1 spectrin at the NMJ. Muscle specific deletion of ß1 spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na+ channels. Together, our results suggest that muscle ß1 spectrin functions independently of an associated α spectrin to maintain Na+ channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. KEY POINTS: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding ß4 spectrin) is neurogenic in origin. ß1 spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na+ channels. By contrast to the canonical view of spectrin organization and function, we show that ß1 spectrin can function independently of an associated α spectrin. Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons.

A PITX2-HTR1B pathway regulates the asymmetric development of female gonads in chickens.

All female vertebrates develop a pair of ovaries except for birds, in which only the left gonad develops into an ovary, whereas the right gonad regresses. Previous studies found that the transcription factor Paired-Like Homeodomain 2 (PITX2), a key mediator for left/right morphogenesis in vertebrates, was also implicated in asymmetric gonadal development in chickens. In this study, we systematically screened and validated the signaling pathways that could be targeted by Pitx2 to control unilateral gonad development. Integrated chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analyses indicated that Pitx2 directly binds to the promoters of genes encoding neurotransmitter receptors and leads to left-biased expression of both serotonin and dopamine receptors. Forcibly activating serotonin receptor 5-Hydroxytryptamine Receptor 1B (HTR1B) signaling could induce ovarian gene expression and cell proliferation to partially rescue the degeneration of the right gonad. In contrast, inhibiting serotonin signaling could block the development of the left gonad. These findings reveal a PITX2-HTR1B genetic pathway that guides the left-specific ovarian growth in chickens. We also provided new evidence showing neurotransmitters stimulate the growth of nonneuronal cells during the early development of reproductive organs well before innervation.

High Performance Epoxy Composites Containing Nanofiller Modified by Plasma Bubbles.

The thermal conductivity of polymer materials is a fundamental parameter in the field of high-voltage electrical insulation. When the operating frequency and power for electrical equipment or electronic devices increase significantly, the internal heat will increase dramatically, and the accumulation of heat will further lead to insulation failure and serious damage of the whole system. The addition of filler with high thermal conductivity into polymer is a common solution. However, the interfacial thermal resistance between filler and bulk materials is the major obstacle to improve thermal conductivity. Herein, in order to reduce the interfacial thermal resistance, nanofillers are modified by plasma technology. The surface modification of nano-Al2O3 is carried out using plasma bubbles with three atmospheres (Ar, Ar+O2, air) as well as coupling agent. The situation of surface grafting before and after the modification is characterized using FTIR, XPS, and SEM. The effect of the mechanism of modification on the thermal conductivity and reaction pathway is investigated. The results showed that the thermal conductivity after plasma modification is increased significantly. Especially, the thermal conductivity is increased by 35% for the sample modified by Ar+O2 atmosphere. This results because more hydroxyl is introduced on the filler surface by the plasma bubbles, which enhance the interface compatibility between filler and epoxy. In addition, surface insulation performance for the modified samples also is enhanced by 14%. This is associated with the change of surface resistance and trap distribution. These results provide potential support for the development of fabrication for high performance epoxy composites.

Spectrin-beta 2 facilitates the selective accumulation of GABAA receptors at somatodendritic synapses.

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, ß and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. ß2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while ß4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating ß2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.

Ankyrin-dependent Na+ channel clustering prevents neuromuscular synapse fatigue.

Skeletal muscle contraction depends on activation of clustered acetylcholine receptors (AchRs) and muscle-specific Na+ channels (Nav1.4). Some Nav1.4 channels are highly enriched at the neuromuscular junction (NMJ), and their clustering is thought to be essential for effective muscle excitation. However, this has not been experimentally tested, and how NMJ Na+ channels are clustered is unknown. Here, using muscle-specific ankyrinR, ankyrinB, and ankyrinG single, double, and triple-conditional knockout mice, we show that Nav1.4 channels fail to cluster only after deletion of all three ankyrins. Remarkably, ankyrin-deficient muscles have normal NMJ morphology, AchR clustering, sarcolemmal levels of Nav1.4, and muscle force, and they show no indication of degeneration. However, mice lacking clustered NMJ Na+ channels have significantly reduced levels of motor activity and their NMJs rapidly fatigue after repeated nerve-dependent stimulation. Thus, the triple redundancy of ankyrins facilitates NMJ Na+ channel clustering to prevent neuromuscular synapse fatigue.

Chromosome-level genome assembly of the Arctic fox (Vulpes lagopus) using PacBio sequencing and Hi-C technology.

The Arctic fox (Vulpes lagopus) is the only fox species occurring in the Arctic and has adapted to its extreme climatic conditions. Currently, the molecular basis of its adaptation to the extreme climate has not been characterized. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first V. lagopus genome assembly, which is assembled into chromosome fragments. The genome assembly has a total length of 2.345 Gb with a contig N50 of 31.848 Mb and a scaffold N50 of 131.537 Mb, consisting of 25 pseudochromosomal scaffolds. The V. lagopus genome had approximately 32.33% repeat sequences. In total, 21,278 protein-coding genes were predicted, of which 99.14% were functionally annotated. Compared with 12 other mammals, V. lagopus was most closely related to V. Vulpes with an estimated divergence time of ~7.1 Ma. The expanded gene families and positively selected genes potentially play roles in the adaptation of V. lagopus to Arctic extreme environment. This high-quality assembled genome will not only promote future studies of genetic diversity and evolution in foxes and other canids but also provide important resources for conservation of Arctic species.

Nano-sized composite improving the insulating performance of insulating paper using low-temperature plasmas.

Nanostructured dielectric composite has been considered as a promising manner in improving the flashover performance of oil-paper which has been widely used in power systems. In this paper, plasma-enhanced chemical vapor deposition (PECVD) is used to deposit SiO2 on the ceramic fiber-reinforced insulating paper. Scanning electron microscope images show a large number of SiO2 nanoparticles with diameters of 100 nm-250 nm uniformly attached to the fiber surface after the plasma deposition. The surface flashover voltage of the insulating paper was tested in the air and the transformer oil, respectively. Results show that the corresponding DC surface flashover voltages increased by 15.1% in the air and breakdown between liquid and solid interface increased by 24.6% after the PECVD. It is believed that nanoparticles constructed in ceramic fibers change the electron injection barrier which inhibits the injection of negative charges and hinders the accumulation of charges in the dielectric. Nanoparticles can capture electric charges formed in the transformer oil which affects the generation and development of streamers, resulting in an increased dielectric strength. This study provides a new method to comprehensively improve the surface insulating property which has the prospect of promoting other dielectric materials.

Low-temperature plasma polymerized fluorocarbon coating promotes surface charge dissipation in polystyrene.

Polystyrene (PS) is a common insulating material in pulsed power devices, which has excellent and reliable insulation properties. However, the charge accumulation on the insulator surface seriously threatens its surface insulation property. Surface modification has been verified as an effective way for inhibiting surface charge accumulation. In this paper, plasma polymerized fluorocarbon (PPFC) coating was prepared by low-temperature plasma polymerization in the mixture of methyl-methacrylate and dodecafluoroheptyl-methacrylate (DFHMA). Compared with the untreated PS, the surface charge dissipation rate of PPFC coating is increased by more than 6 times. The introduction of DFHMA makes the coating have no obvious ageing effect after the storage, and has good reusability after the surface flashover. This work provides a new method for modification of polymer dielectrics and a novel way for the preparation of high-charge-dissipation polymers in other related fields.

Optimization of Ethanol Detection by Automatic Headspace Method for Cellulose Insulation Aging of Oil-immersed Transformers.

The method using ethanol to evaluate the cellulose insulation aging condition of oil-immersed transformers has been proposed. At present, the dominating method for detecting ethanol in insulating oil is to use headspace-gas-chromatography-mass-spectrometry (HS-GC-MS). However, the problem of quantitative inaccuracy will be sometimes encountered in the actual detection process due to improper instrument parameter setting and improper manual operation. In this study, as an aging marker, ethanol in transformer insulating oil was separated by using VF-624 ms capillary column. The effects of gas-chromatography-mass-spectrometry (GC-MS) optimization conditions, headspace equilibrium temperature, headspace equilibrium time and standard solution preparation method on the determination of ethanol content in oil were discussed, and optimized measures were proposed. The experimental results showed that the measurement can be more accurate under the headspace temperature of 80 °C and the headspace time of 40 min, and relative standard deviation percentage (RSD%) could reach to 4.62% under this condition. It was also pointed out that, for the preparation of standard solution, the method which controlled the sampling volume of anhydrous ethanol by microliter syringe could make the peak area of ethanol chromatogram have a better linear relationship with the standard curve. Under the similar linear range, the goodness of fitting curve without diluting process could be as high as 0.9993, while the method of preparing the stock solution and diluting stepwise to obtain the fitting curve only had a goodness of 0.9910. The method was validated by standard addition recovery test, and the recovery values obtained were between 90.3% and 95.8%. The optimized method is of great significance for the measurement of ethanol dissolved in insulating oil.

Profiling of RNA N6-methyladenosine methylation during follicle selection in chicken ovary.

Ovarian follicle selection is the critical step which determines the oocyte development and ovulation. In avian species, the somatic cells in the follicles decide the process of follicle selection but the precise molecular regulation is not well defined. N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in the gene expression regulation and cell functions. In this study, we profiled transcriptome-wide m6A methylation in chicken follicles during follicular selection process in order to identify key factors involved in the follicle selection. The chicken follicle transcriptome was extensively methylated by m6A and a negative correlation was found between the m6A methylation enrichment and gene expression levels. Interestingly, both the m6A methylation peaks and the m6A modified transcripts increased during follicle selection, which lead to the dynamic expression of many folliculogenesis relevant genes. Functional enrichment analysis indicated that m6A modification of key factors in Wnt pathway could play a major role in regulating follicle selection. This study is the first to comprehensively characterize the m6A patterns in the chicken transcriptome, and provides deep insights into the m6A topology and relevant molecular mechanisms underlying follicle selection.

Correction to: Hair follicles transcriptome profiles in Bashang long-tailed chickens with different plumage colors.

The words 'hair follicles' was replaced with 'feather follicles' in the title and the main text.

Feather follicles transcriptome profiles in Bashang long-tailed chickens with different plumage colors.

Despite the rich variety in plumage color found in nature, genetic studies on how feather follicles affect pigmentation are often limited to animals that have black and white pigment. To test how gene expression influences plumage color, transcriptomes of chicken feather follicles with white, black, hemp, reed catkins, silvery grey, and landscape plumage colors were generated using Illumina sequencing. We generated six RNA-Seq libraries with over 25 million paired-end clean reads per library with percentage of paired-end clean reads ranging from 96.73 to 96.98%. 78% of the reads mapped to the chicken genome, and approximately 70% of the reads were mapped to exons and 6% mapped to introns. Transcriptomes of feather follicles producing hemp and land plumage were similar, but these two showed moderate differences compared with gray and reed colored plumage. The black and white follicle transcriptomes were most divergent from the other colors. We identified several candidate genes, including GPNMB, PMEL, TYRP1, GPR143, OCA2, SOX10, SLC45A2, KRT75, and TYR. All of these genes are known to induce pigment formation in mice. White feathers result from the lack of pigment formation, and our results suggest that the white chickens due to the recessive insertion mutation of TYR. The formation of black area size and color depth may be due to the expression levels of GPNMB, PMEL, TYRP1, GPR143, OCA2, SOX10, SLC45A2, KRT75, and TYR. The GO analysis of the differentially expressed genes (DEGs) revealed that DEGs in our transcriptome analysis were enriched in cytoskeleton and cell structure related pathways. The black plumage transcriptome showed significant differences in melanogenesis, tyrosine metabolism, and riboflavin metabolism compared with transcriptomes of other plumage colors. The transcriptome profiles of the different chicken plumage colors provide a valuable resource to understand how gene expression influences plumage color, and will be an important resource for identifying candidate genes in breeding programs.

Genome-wide differential expression of long noncoding RNAs and mRNAs in ovarian follicles of two different chicken breeds.

Bashang long-tail chickens are an indigenous breed with dual purpose in China (meat and eggs) but have low egg laying performance. To improve the low egg laying performance, a genome-wide analysis of mRNAs and long noncoding RNAs (lncRNAs) from Bashang long-tail chickens and Hy-Line brown layers was performed. A total of 16,354 mRNAs and 8691 lncRNAs were obtained from ovarian follicles. Between the breeds, 160 mRNAs and 550 lncRNAs were found to be significantly differentially expressed. Integrated network analysis suggested some differentially expressed genes were involved in ovarian follicular development through oocyte meiosis, progesterone-mediated oocyte maturation, and cell cycle. The impact of lncRNAs on cis and trans target genes, indicating some lncRNAs may play important roles in ovarian follicular development. The current results provided a catalog of chicken ovarian follicular lncRNAs and genes for further study to understand their roles in regulation of egg laying performance.

[Identification of the core promoter of the pmel gene of Bashang long-tail chickens].

To explore the activity of the pmel core promoter of Bashang long-tail chickens, we constructed dual-luciferase expression vectors and transiently transfected into DF1 cells with Lipofectamine 2000. We measured the luciferase activity with the dual-luciferase detection kit. The 1 268 bp fragment in 5-flanking region of the pmel gene in Bashang long-tail chickens was cloned. The region from -1 200 bp to +68 bp included 2 CpG islands and multiple transcription factor binding sites. We constructed 9 expression vectors with different promoter regions and a mutant vector of the core promoter region of the pmel gene of Bashang long-tail chickens. The core promoter region from -840 bp to +68 bp was identified in the pmel gene. The region from -590 to -525 bp negatively regulated the pmel gene during the transcription process. The -840--590 bp and -525--266 bp regions were positive regulatory regions. The polymorphic sites (-456, -435, -410, -374 and -341) had a significant effect on the promoter activity of the pmel gene.

Glial ßII Spectrin Contributes to Paranode Formation and Maintenance.

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na+ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. ßII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell ßII spectrin is highly enriched at paranodes. To elucidate the roles of glial ßII spectrin, we generated mutant mice lacking ßII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions.SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of ßII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). RESULTS: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. CONCLUSIONS: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.

αII Spectrin Forms a Periodic Cytoskeleton at the Axon Initial Segment and Is Required for Nervous System Function.

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse ß subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and ßIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all ß spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system.SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with ßIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology.

An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with ßIV and ßII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K+ channels. We show that the density of nodal ßIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier.SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in maintenance of axon integrity. We demonstrate the role of the periodic spectrin-dependent cytoskeleton in axons and show that loss of αII spectrin from PNS axons causes preferential degeneration of large-diameter myelinated axons. We show that nodal αII spectrin is found at greater densities in large-diameter myelinated axons, suggesting that nodes are particularly vulnerable domains requiring a specialized cytoskeleton to protect against axon degeneration.

The paranodal cytoskeleton clusters Na+ channels at nodes of Ranvier.

A high density of Na+ channels at nodes of Ranvier is necessary for rapid and efficient action potential propagation in myelinated axons. Na+ channel clustering is thought to depend on two axonal cell adhesion molecules that mediate interactions between the axon and myelinating glia at the nodal gap (i.e., NF186) and the paranodal junction (i.e., Caspr). Here we show that while Na+ channels cluster at nodes in the absence of NF186, they fail to do so in double conditional knockout mice lacking both NF186 and the paranodal cell adhesion molecule Caspr, demonstrating that a paranodal junction-dependent mechanism can cluster Na+ channels at nodes. Furthermore, we show that paranode-dependent clustering of nodal Na+ channels requires axonal ßII spectrin which is concentrated at paranodes. Our results reveal that the paranodal junction-dependent mechanism of Na+channel clustering is mediated by the spectrin-based paranodal axonal cytoskeleton.

Cytoskeletal control of axon domain assembly and function.

Neurons are organized and connected into functional circuits by axons that conduct action potentials. Many vertebrate axons are myelinated and further subdivided into excitable domains that include the axon initial segment (AIS) and nodes of Ranvier. Nodes of Ranvier regenerate and propagate action potentials, while AIS regulate action potential initiation and neuronal polarity. Two distinct cytoskeletons control axon structure and function: 1) a submembranous ankyrin/spectrin cytoskeleton that clusters ion channels and provides mechanical support, and 2) a microtubule-based cytoskeleton that controls selective trafficking of dendritic and axonal cargoes. Here, we review recent studies that provide significant additional insight into the cytoskeleton-dependent mechanisms controlling the functional organization of axons.