Biology Reference
In-Depth Information
3.3 Discussion......................................................................................................... 43
Acknowledgments ..................................................................................................... 44
References ............................................................................................................... 45
Abstract
Doublecortin (DCX) is a microtubule (MT)-stabilizing protein essential for neuronal
migration during human brain development. Missense mutations in DCX cause
severe brain defects. This implies that the many other MT-stabilizing proteins in
neurons cannot compensate for DCX function. To understand the unusual properties
of DCX, we expressed the recombinant human DCX in Sf9 cells and undertook struc-
tural characterization of its interaction with MTs using cryo-electron microscopy.
DCX specifically nucleates 13-protofilament (13-pf) MTs, the architecture of human
MTs
in vivo
. Cryo-electron tomography (cryo-ET) of DCX-nucleated MTs showed
that they are primarily built from B-lattice contacts interrupted by a single discon-
tinuity, the seam. Because of this asymmetry, we used single-particle reconstruction
and determined the 8
˚
structure of DCX-stabilized 13-pf MTs in the absence of a
stabilizing drug. The DCX-binding site, at the corner of four tubulin dimers, is ide-
ally suited to stabilize both lateral and longitudinal tubulin lattice contacts. Its precise
geometry suggests that DCX is sensitive to the angle between pfs, and thereby pro-
vides insight into the specificity of DCX for 13-pf MT architecture. DCX's precise
interaction at the corner of four tubulin dimers also means that DCX does not bind the
MT seam. Our work has provided mechanistic insight into the evolutionarily con-
served DCX family of MT-stabilizing proteins and also into more general regulatory
mechanisms of the MT cytoskeleton.
INTRODUCTION
The neuronal microtubule (MT)-associated protein (MAP) doublecortin (DCX) was
first characterized in 1998, when it was discovered that mutations in the X-linked dou-
blecortin (
DCX
) gene cause a syndrome called double cortex (or subcortical
band heterotopia, SBH) in females and lissencephaly in males (
des Portes et al.,
1998; Gleeson et al., 1998
). Lissencephaly and SBH patients exhibit a range of
symptoms—including epilepsy, intellectual impairment, and infant death—that
result from abnormal development of the cerebral cortex. Mutation of the DCX protein
causes defective neuronal migration, such that the precisely structured layers of the cor-
tex are poorlyorganized.Because the
DCX
gene is on theXchromosome, femaleswith a
DCX
genotype exhibit random inactivation of one of the two X chromosomes; this
ensures that half of the cells have a functional copy of the gene and migrate correctly
into a layered cortex. In contrast, the other half lack a functional copy and subsequently
