Biology Reference
In-Depth Information
One of the most striking properties of DCX is its ability to specifically nucleate
and bind MTs with a 13-protofilament (13-pf) architecture (
Bechstedt & Brouhard,
2012; Moores et al., 2004
). This is particularly important because although the
number of pfs within MTs polymerized
in vitro
varies between 8 and 19, the almost
exclusive architecture found
in vivo
is the 13-pf MT (
McIntosh, Morphew, Grissom,
Gilbert, & Hoenger, 2009; Tilney et al., 1973; Wade & Chr´tien, 1993
). Cryo-
electron microscopy (cryo-EM) continues to be an invaluable method for elucidating
mechanism and function in the MT cytoskeleton and its binding partners (
Amos &
Hirose, 2007; Hoenger & Gross, 2008
), and the stabilization mechanism and archi-
tecture specificity of DCX were in large part explained by cryo-EM reconstructions.
Our early, low-resolution (
30
˚
) reconstruction was calculated using helical
analysis of a few, rare 14-pf paclitaxel-stabilized MTs that could be decorated with
a truncated construct of DCX (t-DCX, DCX 1-275, lacking the S/P-rich domain;
Moores et al., 2004
). Because of its architectural preference, FL DCX could not
be analyzed using this method. This first reconstruction showed a globular density
wedged between pfs and in contact with four tubulin monomers. The DCX density
corresponds to only one DC domain while additional weak density that may be at-
tributable to the rest of the protein was observed at higher radius. It was immediately
obvious that DCX's binding between pfs represents an excellent way to “staple” pfs
together and thus increases MT stability. By binding at the junction between four
tubulin monomers, DCX has the potential to strengthen both lateral contacts between
pfs and also longitudinal contacts along the MT. In addition, this unique binding
mode suggests the key to DCX's specificity for 13-pf MTs: the width of the inter-
pf valley varies with pf number and DC domain may have evolved to fit this binding
site in the 13-pf MT wall (
Fig. 3.2
A).
This binding site does not overlap with those of the MT-based motors kinesin and
dynein and, thus, would be predicted not to impede movement of these motors
(
Fig. 3.2
B). Indeed, in an ensemble gliding assay, DCX-MTs were found to support
kinesin motility and with only slightly decreased speed (
Moores et al., 2006
). This
suggests that,
in vivo
, MTs stabilized by DCX (and its relatives) may act as tracks for
intracellular transport; the ramifications of this for cargo delivery control are an ac-
tive area of ongoing research (
Deuel et al., 2006; Liu et al., 2012
).
Yet, after this initial work, several key questions remained and have been the fo-
cus of our more recent research efforts. For example, the biochemical stoichiometry
of binding to tubulin for both t-DCX and FL DCX is estimated to be 1:1, suggesting
that each density in our reconstruction corresponds to a single DC domain, with the
rest of the molecule disordered and therefore invisible in the reconstruction. How-
ever, it is possible that adjacent binding sites are occupied by N-DC and C-DC from
the same molecule—the inter-DC linker is long enough that either a longitudinal or
lateral configuration is possible. Our hypothesis was that in working with t-DCX—in
the absence of the S/P-rich domain that is critical for conferring MT architecture
specificity—on non-13-pf MTs at low resolution, we were compromising our struc-
tural experiment. In addition, the low resolution of the reconstruction prevented us
from determining whether DCX binds between two dimers or at the corner of four
