Stability of the tumor suppressor merlin depends on its ability to bind paxillin LD3 and associate with ß1 integrin and actin at the plasma membrane.

The NF2 gene encodes a tumor suppressor protein known as merlin or schwannomin whose loss of function causes Neurofibromatosis Type 2 (NF2). NF2 is characterized by the development of benign tumors, predominantly schwannomas, in the peripheral nervous system. Merlin links plasma membrane receptors with the actin cytoskeleton and its targeting to the plasma membrane depends on direct binding to the paxillin scaffold protein. Exon 2 of NF2, an exon mutated in NF2 patients and deleted in a mouse model of NF2, encodes the merlin paxillin binding domain (PBD1). Here, we sought to determine the role of PBD1 in regulation of merlin stability and association with plasma membrane receptors and the actin cytoskeleton in Schwann cells. Using a fluorescence-based pulse-chase technique, we measured the half-life of Halo-tagged merlin variants carrying PBD1, exon 2, and exons 2 and 3 deletions in transiently transfected Schwann cells. We found that PBD1 alone was necessary and sufficient to increase merlin's half-life from approximately three to eleven hours. Merlin lacking PBD1 did not form a complex with surface ß1 integrins or associate with the actin cytoskeleton. In addition, direct binding studies using purified merlin and paxillin domains revealed that merlin directly binds paxillin LD3 (leucine-aspartate 3) domain as well as the LD4 and LD5 domains. Together these results demonstrate that a direct interaction between merlin PBD1 and the paxillin LD3-5 domains targets merlin to the plasma membrane where it is stabilized by its association with surface ß1 integrins and cortical actin.

The actin-severing protein cofilin is downstream of neuregulin signaling and is essential for Schwann cell myelination.

Myelination is a complex process requiring coordination of directional motility and an increase in glial cell size to generate a multilamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin-1 (NRG1) expressed on the axon surface. Here we identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG1 signaling in rat Schwann cells (SCs). In isolated SCs, NRG1 promotes dephosphorylation of cofilin1 and its upstream regulators, LIM kinase (LIMK) and Slingshot-1 phosphatase (SSH1), leading to cofilin1 activation and recruitment to the leading edge of the plasma membrane. These changes are associated with rapid membrane expansion yielding a 35-50% increase in SC size within 30 min. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG1, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs cocultured with sensory neurons do not myelinate. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 h of coculturing with neurons, cofilin1-deficient SCs do not align or elongate on axons and often form adhesions with the underlying substrate. This study identifies cofilin1 and its upstream regulators, LIMK and SSH1, as end targets of a NRG1 signaling pathway and demonstrates that cofilin1 is necessary for dynamic changes in the cytoskeleton needed for axon engagement and myelination by SCs.