Assembly and maintenance of nodes of ranvier rely on distinct sources of proteins and targeting mechanisms.
ABSTRACT: We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are freely diffusible within the axon membrane and accumulate at forming nodes from local sources, whereas ion channels and cytoskeletal components are largely immobile and require transport to the node. We further characterize targeting of NF186, an adhesion molecule that pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. Its initial accumulation and clearance from the internode require extracellular interactions, whereas targeting to mature nodes, i.e., those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus, nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells, whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport.
Project description:Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.
Project description:Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na+ channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies.
Project description:Rapid action potential propagation in myelinated axons requires Na? channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na? channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na? channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na? channel clustering.
Project description:Intricate molecular interactions between neurons and glial cells underlie the creation of unique domains that are essential for saltatory conduction of action potentials by myelinated axons. Previously, the cell surface adhesion molecule Neurofascin (Nfasc) has been shown to have a dual-role in the establishment of axonal domains from both the glial and neuronal interface. While the neuron-specific isoform of Neurofascin (NF186) is indispensable for clustering of voltage-gated sodium channels at nodes of Ranvier; the glial-specific isoform of Neurofascin (NF155) is required for myelinating glial cells to organize the paranodal domain. Although many studies have addressed the individual roles of NF155 and NF186 in assembling paranodes and nodes, respectively; critical questions about their roles in the maintenance and long-term health of the myelinated axons remain, which we aimed to address in these studies. Here using spatiotemporal ablation of Neurofascin in neurons alone or together with myelinating glia, we report that loss of NF186 individually from postnatal mice leads to progressive nodal destabilization and axonal degeneration. While individual ablation of paranodal NF155 does not disrupt nodes of Ranvier; loss of NF186 combined with NF155 causes more accelerated nodal destabilization than loss of NF186 alone, providing strong evidence regarding a supporting role for paranodes in nodal maintenance. In both cases of NF186 loss, myelinating axons show ultrastructural changes and degeneration. Our studies reveal that long-term maintenance of nodes and ultimately the health of axons is correlated with the stability of NF186 within the nodal complex and the presence of auxiliary paranodes.
Project description:Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.
Project description:Two major isoforms of the cell adhesion molecule neurofascin NF186 and NF155 are expressed in the central nervous system (CNS). We have investigated their roles in the assembly of the node of Ranvier and show that they are targeted to distinct domains at the node. At the onset of myelination, NF186 is restricted to neurons, whereas NF155 localizes to oligodendrocytes, the myelin-forming glia of the CNS. Coincident with axon ensheathment, NF155 clusters at the paranodal regions of the myelin sheath where it localizes in apposition to the axonal adhesion molecule paranodin/contactin-associated protein (Caspr1), which is a constituent of the septate junction-like axo-glial adhesion zone. Immunoelectron microscopy confirmed that neurofascin is a glial component of the paranodal axo-glial junction. Concentration of NF155 with Caspr1 at the paranodal junctions of peripheral nerves is also a feature of Schwann cells. In Shiverer mutant mice, which assemble neither compact CNS myelin nor normal paranodes, NF155 (though largely retained at the cell body) is also distributed at ectopic sites along axons, where it colocalizes with Caspr1. Hence, NF155 is the first glial cell adhesion molecule to be identified in the paranodal axo-glial junction, where it likely interacts with axonal proteins in close association with Caspr1.
Project description:Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3K?-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin ?4?1 on lymph node lymphatic endothelium. Activated integrin ?4?1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)(+) metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of ?4?1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin ?4?1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin ?4?1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin ?4?1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.
Project description:Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.
Project description:Nodes of Ranvier in the axons of myelinated neurons are exemplars of the specialized cell surface domains typical of polarized cells. They are rich in voltage-gated sodium channels (Nav) and thus underpin rapid nerve impulse conduction in the vertebrate nervous system . Although nodal proteins cluster in response to myelination, how myelin-forming glia influence nodal assembly is poorly understood. An axoglial adhesion complex comprising glial Neurofascin155 and axonal Caspr/Contactin flanks mature nodes . We have shown that assembly of this adhesion complex at the extremities of migrating oligodendroglial processes promotes process convergence along the axon during central nervous system (CNS) node assembly . Here we show that anchorage of this axoglial complex to the axon cytoskeleton is essential for efficient CNS node formation. When anchorage is disrupted, both the adaptor Protein 4.1B and the cytoskeleton protein ?II spectrin are mislocalized in the axon, and assembly of the node of Ranvier is significantly delayed. Nodal proteins and migrating oligodendroglial processes are no longer juxtaposed, and single detached nodal complexes replace the symmetrical heminodes found in both the CNS and peripheral nervous system (PNS) during development. We propose that axoglial adhesion complexes contribute to the formation of an interface between cytoskeletal elements enriched in Protein 4.1B and ?II spectrin and those enriched in nodal ankyrinG and ?IV spectrin. This clusters nascent nodal complexes at heminodes and promotes their timely coalescence to form the mature node of Ranvier. These data demonstrate a role for the axon cytoskeleton in the assembly of a critical neuronal domain, the node of Ranvier.
Project description:Fast neural conduction requires accumulation of Na(+) channels at nodes of Ranvier. Dedicated adhesion molecules on myelinating cells and axons govern node organization. Among those, specific laminins and dystroglycan complexes contribute to Na(+) channel clustering at peripheral nodes by unknown mechanisms. We show that in addition to facing the basal lamina, dystroglycan is found near the nodal matrix around axons, binds matrix components, and participates in initial events of nodogenesis. We identify the dystroglycan-ligand perlecan as a novel nodal component and show that dystroglycan is required for the selective accumulation of perlecan at nodes. Perlecan binds the clustering molecule gliomedin and enhances clustering of node of Ranvier components. These data show that proteoglycans have specific roles in peripheral nodes and indicate that peripheral and central axons use similar strategies but different molecules to form nodes of Ranvier. Further, our data indicate that dystroglycan binds free matrix that is not organized in a basal lamina.