# Mechanisms of Node of Ranvier Assembly

> **NIH NIH R01** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2022 · $469,840

## Abstract

The axon initial segment (AIS) and nodes of Ranvier are sites of action potential generation and regeneration,
respectively and are thus critical for the proper function of the nervous system. Their key roles in electrogenesis
results from the striking enrichment of a macromolecular complex of voltage-gated sodium (NaV) and potassium
(KCNQ) channels, cell adhesion molecules (NF186, NrCAM), and a cytoskeletal scaffold of ankyrin G (AnkG)
and beta-IV (βIV) spectrin. Prior to myelination, nodal components are diffusely distributed along axons
consistent with their continuous conduction of action potentials. With myelination, the axon reorganizes into
discrete domains, culminating in node assembly, thus enabling saltatory conduction. A key question is what
drives this reorganization? We have found two complementary mechanisms broadly contribute: i) recruitment
signals that target and stabilize this complex at nodes and ii) active clearance that removes nodal proteins from
everywhere else along the axon. Here, we examine the contribution of both mechanisms to node formation.
Recruitment signals at PNS and CNS nodes culminate in assembly of an AnkG/βIV spectrin cytoskeleton scaffold
to which all other nodal components bind. This scaffold is further tethered to and likely stabilized by regularly
spaced, sub-membranous actin rings at both the AIS and nodes. We recently reported actin rings at the AIS and
nodes are specifically modified by contractile myosin II. In particular, phosphorylated myosin light chain (pMLC)
- the regulatory subunit that activates the contractile function of myosin II - is enriched at and an early marker of
the AIS and nodes. Strategies that increase or decrease pMLC levels/myosin II activity, drive AIS assembly and
disassembly, respectively. These results implicate contractile NMII as a novel regulator of the AIS and suggest
a conserved role at nodes, a notion strongly supported by MLC knockdown studies. We have also found that
just prior to myelination, glial cells drive clearance of nodal components from the internode by clathrin-mediated
endocytosis (CME). Our results suggest cleared proteins are not linked to the cytoskeleton and can therefore be
clustered for endocytosis. In agreement expression of a mutant NF186 construct engineered to bind to the
internodal cytoskeleton, is not endocytosed but rather is persistently expressed along the axon. Strikingly, this
construct delays myelination. This latter finding suggests clearance has dual roles in sculpting the node and
preparing the axon for myelination thereby coordinating assembly of the node of Ranvier with myelination of
axons. Here, we test key aspects of this model, including the role of MLC/myosin II in node assembly/stability
and actin ring integrity by knockdown and knockout strategies. We will also examine the mechanisms and
consequences of this glia-driven clearance of axonal proteins, including modeling defective CME in mice to
broadly perturb the axon surface pr...

## Key facts

- **NIH application ID:** 10447728
- **Project number:** 5R01NS043474-17
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** JAMES SALZER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $469,840
- **Award type:** 5
- **Project period:** 2002-04-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10447728

## Citation

> US National Institutes of Health, RePORTER application 10447728, Mechanisms of Node of Ranvier Assembly (5R01NS043474-17). Retrieved via AI Analytics 2026-06-10 from https://api.ai-analytics.org/grant/nih/10447728. Licensed CC0.

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