Doublecortin (DCX) Mediates Endocytosis of Neurofascin Independently of Microtubule Binding

Abstract

Doublecortin (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX-neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons, and thereby the relative extent of accumulation between the axon initial segment, and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules, but is deficient in promoting NF endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.

Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons

“…However, knockdown of dynein heavy chain or Lis1 does not affect the cytoplasmic dilation formation, although inhibition of either protein perturbs nuclear forward movement , suggesting that another cellular event is involved in the Cdk5-mediated dilation formation. Although Dcx and Lis1, causative gene products of lissencephaly, share some common functions in the dynein-mediated regulation of centrosome positioning (Tanaka et al, 2004a), Dcx plays other roles in microtubule polymerization and endocytic trafficking (Francis et al, 1999;Gleeson et al, 1999;Friocourt et al, 2005;Moores et al, 2006;Yap et al, 2012), which are shown here to regulate dilation formation. These dynein-independent functions of Dcx may have important roles in dilation formation.…”

“…One of them, Doublecortin (Dcx), is involved in microtubule polymerization, membrane trafficking and actin organization (Francis et al, 1999;Gleeson et al, 1999;Friocourt et al, 2005;Tsukada et al, 2005;Moores et al, 2006;Yap et al, 2012;Fu et al, 2013). Another, p27 kip1 (Cdkn1b -Mouse Genome Informatics) controls microtubule and actin cytoskeletal organization (Kawauchi et al, 2006;Godin et al, 2012).…”

Different Doublecortin (DCX) Patient Alleles Show Distinct Phenotypes in Cultured Neurons

“…Dcx-303X behaves as a hypomorph in our assay, suggesting that AP binding might be additionally required for full Dcx function. We previously showed that Dcx promotes the endocytosis of a cell adhesion molecule (24), but further work is needed to explore which endocytic cargo might require Dcx for trafficking to support dendrite elaboration.…”

“…Detergent Extractions-Detergent extractions of live cells were carried out in BRB80 buffer (80 mM Pipes, 1 mM MgCl 2 , 1 mM EGTA, pH 6.8) with 0.3% Triton X-100 at 37°C for 3 min (24,25,26). Cells were washed twice in BRB80 buffer and fixed.…”

A critical and previously unsuspected role for doublecortin at the neuromuscular junction in mouse and human

“…These include the µ subunits of clathrin adaptor complexes [45] and two kinases, Cdk5 and Jnk [37,46]. Dcx has also been shown to interact with and regulate endocytosis and surface distribution of the phosphorylated neurofascin adhesion molecule [47,48]. Interestingly, this function of Dcx may not involve its microtubule-binding activity.…”