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DCX-mediated MT nucleation. A parallel possibility is that the missing
75% of the
DCX molecules are not sufficiently ordered for us to visualize using our current
averaging procedures. By computationally investigating the conformational variabil-
ity of DCX within our samples, we aim to gain further insight into its location
and structure, albeit with a likely compromise of resolution in the resulting
reconstructions.
Based on the availability of the structure C-DC of DCDC2 (PDB ID 2DNF), it
seems likely that the conserved DC fold is important for C-DC function and may in-
volve tubulin interaction and control of DCX cooperativity (
Bechstedt & Brouhard,
2012; Kim et al., 2003
). However, structural insight into the C-terminal S/P-rich do-
main of DCX—previously shown to be essential for DCX's highly specific binding of
13-pf MTs (
Moores et al., 2004
)—remains lacking. As it is predicted to be disordered
and highly charged due to phosphorylation (
Reiner et al., 2004
), one hypothesis is
that the C-terminal domain does not make specific contact with tubulin but rather
modulates the DCX-MT interaction indirectly. This would predict that single point
mutations in the S/P-rich domain would only indirectly affect the interaction and
in fact hardly any disease-causing mutations in this domain have been reported
(
Bahi-Buisson et al., 2013
). To further test this hypothesis, it would be interesting
to generate a DCX construct with a scrambled C-terminal sequence, keeping the same
phosphorylation sites and overall charge.
Our structural insight into the DCX-MT interface provides a framework to local-
ize and predict the severity of the gradually accumulating examples of disease-
causing point mutations in DCX. Our results suggest that a spectrum of cellular
effects would be seen, with some mutations totally disrupting the DCX-MT interac-
tion, while others might have only minor effects on the MT interface and exert their
mutagenic effects elsewhere within the network of DCX binding partners, including
via intracellular trafficking control (
Liu et al., 2012
). It will be informative to con-
tinue to test the effects of these mutations
in vitro
to investigate their consequences
on the DCX structure and function (
Bechstedt & Brouhard, 2012
). Clinically, the
rarity of these mutations presents a challenge for establishing a statistically robust
diagnostic link between genotype and phenotype;
in vitro
studies could provide func-
tional parameters to predict the exact nature and severity of the disease based on the
location and nature of the mutation (
Bahi-Buisson et al., 2013
). Multiple molecular
pathways to disease may emerge from such studies with DCX forming a MT-based
regulatory hub.
Acknowledgments
We thank Charles Sindelar (Yale University) for sharing his MT reconstruction scripts and
members of the Birkbeck EM group for helpful discussions and advice. We are supported
by The Wellcome Trust, New Life and F´d´ration pour la Recherche sur le Cerveau.
