Stephen Lambert

AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.

We have characterized a new ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended “tail” domain. We term this gene ankyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific ankyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

Physiological roles of axonal ankyrins in survival of premyelinated axons and localization of voltage-gated sodium channels

Abstract440 kD ankyrin-B and 480/270 kD ankyrin-G are membrane skeletal proteins with closely related biochemical properties yet distinctive physiological roles in axons. These proteins associate with spectrin-actin networks and also bind to integral membrane proteins including the L1 CAM family of cell adhesion molecules and voltage-gated sodium channels. 440 kD ankyrin-B is expressed with L1 in premyelinated axon tracts, and is essential for survival of these axons, at least in the case of the optic nerve. 440 ankyrin-B may collaborate with L1 in transcellular structures that mediate axon fasciculation and mechanically stabilize axon bundles, although these proteins may also be involved in axon pathfinding. Ankyrin-B (−/−) mice exhibit loss of L1 from premyelinated axon tracts and a similar, although much more severe, phenotype to L1 (−/−) mice and humans with L1 mutations. Ankyrin-B and L1 thus are candidates to collaborate in the same structural pathway and defects in this pathway can lead to nervous system malformations and mental retardation. 480/270 kD ankyrin-G are highly concentrated along with the L1CAM family members neurofascin and NrCAM at nodes of Ranvier and axon initial segments. Voltage-gated sodium channels bind directly to ankyrins, and are likely to associate in a ternary complex containing neurofascin/NrCAM, and ankyrin-G. Mice with ankyrin-G expression abolished in the cerebellum exhibit loss of ability of Purkinje neurons to fire action potentials, as well as loss of restriction of neurofascin/NrCAM to axon initial segments. Ankyrin-G thus is a key component in assembly of functional components of the axon initial segment and possibly the node of Ranvier.

Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier

Nodes of Ranvier are specialized, highly polarized axonal domains crucial to the propagation of saltatory action potentials. In the peripheral nervous system, axo–glial cell contacts have been implicated in Schwann cell (SC) differentiation and formation of the nodes of Ranvier. SC microvilli establish axonal contact at mature nodes, and their components have been observed to localize early to sites of developing nodes. However, a role for these contacts in node formation remains controversial. Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth cone–like behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar component–enriched “caps” and influences the efficiency of node formation.

Schwannomin/merlin promotes Schwann cell elongation and influences myelin segment length.

The Neurofibromatosis type 2 tumor suppressor, schwannomin (Sch) is a plasma membrane-cytoskeleton linking protein that regulates receptor signaling and actin dynamics. We examined Schs role in specifying morphological changes needed for Schwann cell (SC) function in vitro. Isolated Sch-GFP-expressing SCs extended bipolar processes 82% longer than those formed by GFP-expressing cells. In contrast, SCs expressing dominant negative Sch-BBA-GFP extended bipolar processes 16% shorter than controls and 64% shorter than Sch-GFP-expressing SCs. nf2 gene inactivation caused isolated mouse SCs to transition from bipolar to multipolar cells. Live imaging revealed that SCs co-expressing Sch-GFP and dominant negative RacN17 behaved similarly in dorsal root ganglion explant cultures; they quickly aligned on axons and slowly elongated bipolar processes. In contrast, SCs expressing constitutively active RacV12 underwent continuous transitions in morphology that interfered with axon alignment. When co-cultured with neurons under myelin-promoting conditions, Sch-GFP-expressing SCs elaborated longer myelin segments than GFP-expressing SCs. In contrast, Sch-BBA-GFP-expressing SCs failed to align on or myelinate axons. Together, these results demonstrate that Sch plays an essential role in inducing and/or maintaining the SCs spindle shape and suggest that the mechanism involves Sch-dependent inhibition of Rac activity. By stabilizing the bipolar morphology, Sch promotes the alignment of SCs with axons and ultimately influences myelin segment length.

Asymmetric ERM activation at the Schwann cell process tip is required in Axon‐Associated motility

Axon‐associated Schwann cell (SC) motility and process dynamics are crucial in the development and regeneration of the peripheral nervous system (PNS). The bipolar morphology of SCs represents an unexplored conundrum in terms of directed motility. Using fluorescence time‐lapse microscopy of transfected SCs within myelinating dorsal root ganglion (DRG) explants, we demonstrate cycling of SCs between bipolar and highly motile, unipolar morphologies as a result of asymmetric process retraction and extension. Unipolar SC motility appears nucleotaxic in nature, similar to the movement of neurons on radial glia during cortical development. We also show that asymmetric process retraction is associated with the activation of ERM (ezrin/radixin/moesin) proteins and subsequent recruitment of ezrin‐binding phospho‐protein 50 kDa (EBP50) at the retracting process tip. This activation occurs in response to localized synthesis of phosphatidylinositol (4,5)‐bisphosphate (PIP2) at this site. Finally, we demonstrate that the activation of ERM proteins at the SC process tip is essential for motility and the maintenance of SC polarity, as ERM disruption yields a dysfunctional, multi‐polar cell. These results demonstrate that specializations at the tips of SC processes regulate their dynamics, which in turn is associated with directed motility in these cells. J. Cell. Physiol. 210: 122–132, 2007.

Chapter 7 Axonal Ankyrins and Ankyrin-Binding Proteins: Potential Participants in Lateral Membrane Domains and Transcellular Connections at the Node of Ranvier

Publisher Summary This chapter examines the roles of axonal ankyrins and ankyrin-binding proteins in the formation of lateral-membrane domains and transcellular connections at the node of Ranvier. Molecular interactions at the interface between extracellular and cytoplasmic compartments are believed to be of basic importance for establishment of lateral-membrane domains and formation of specialized sites of cell–cell contact. This interface is especially important for nerve axons, which require mechanisms for appropriate organization of ion channels involved in initiation and conductance of action potentials, and recognition systems for synaptogenesis during early development as well as to form specific contacts with glial cells along the length of axons. Nodes of Ranvier are flanked by intricate glial-cell processes and contain high concentrations of voltage-dependent sodium channels. Nodes of Ranvier of myelinated nerves are of considerable clinical interest because of their involvement in pathological conditions, including diabetic peripheral neuropathy and trauma; they are also the sites of regeneration of damaged peripheral nerve axons. Myelinated axons and nodes of Ranvier also exemplify several basic issues for cell biologists: formation of polarized cell domains, assembly of integral proteins into lateral-membrane domains, and formation of morphological structures that require cooperation between distinct types of cells.