Current Biology 22, 1681–1687, September 25, 2012 ª2012 Elsevier Ltd All rights reservedhttp://dx.doi.org/10.1016/j.cub.2012.06.068
Estimating the Microtubule
GTP Cap Size In Vivo
Dominique Seetapun,1Brian T. Castle,1Alistair J. McIntyre,1
Phong T. Tran,2,3and David J. Odde1,*
1Department of Biomedical Engineering, University
of Minnesota, Minneapolis, MN 55455, USA
2Department of Cell & Developmental Biology, University
of Pennsylvania, Philadelphia, PA 19104, USA
3UMR 144 CNRS, Institut Curie, 75005 Paris, France
Microtubules (MTs) polymerize via net addition of GTP-
tubulin subunits to the MT plus end, which subsequently
hydrolyze to GDP-tubulin in the MT lattice. Relatively stable
GTP-tubulin subunits create a ‘‘GTP cap’’ at the growing MT
plus end that suppresses catastrophe. To understand MT
assembly regulation, we need to understand GTP hydrolysis
reaction kinetics and the GTP cap size. In vitro, the GTP
cap has been estimated to be as small as one layer [1–3]
(13 subunits) or as large as 100–200 subunits . GTP cap
size estimates in vivo have not yet been reported. Using
EB1-EGFP as a marker for GTP-tubulin in epithelial cells,
we find on average (1) 270 EB1 dimers bound to growing
MT plus ends, and (2) a GTP cap size of w750 tubulin
subunits. Thus, in vivo, the GTP cap is far larger than
layer cap. We also find that the tail of a large GTP cap
promotes MT rescue and suppresses shortening. We specu-
late that a large GTP cap provides a locally concentrated
scaffold for tip-tracking proteins and confers persistence
to assembly in the face of physical barriers such as the cell
Results and Discussion
but do not assemble from GDP-tubulin [5–8]. However, when
probed, only GDP-tubulin is detectable within the MT lattice
[9, 10]. Therefore, GTP hydrolysis must be occurring within
the lattice. After addition but before hydrolysis, the GTP-
tubulin subunits at the growing tip protect the MT from catas-
trophe byconstituting theso-calledGTP cap.The majority
of the in vitro estimates of the MT GTP cap size range from
about one to three layers (13–40 tubulin subunits; see Table
S1 available online), but to our knowledge, in vivo estimates
of the GTP cap size have not yet been reported. The questions
that we address here are (1) how large is the MT GTP cap
in vivo, and (2) what functional role, if any, does it play in sup-
pressing MT disassembly after catastrophe?
Multiple recent works have demonstrated that EB1, a MT
plus-end-tracking protein (+TIP), recognizes MT lattices
formed from analogs of GTP-tubulin, such as GMPCPP,
GTPgS, and GDP/BeF32, in preference to lattices composed
of GDP-tubulin [12–14]. These results, combined with the
fact that EB1 binding strongly correlates with the growth state
of the MT, indicate that EB1 recognizes the tubulin nucleotide
state . Therefore, the size of the GTP cap in vivo can poten-
tially be estimated by measuring the number of EB1-EGFP
molecules at the growing MT tip and the fractional occupancy
of EB1 binding to GTP-tubulin.
EB1-EGFP Exists as a Dimer In Vivo
To use EB1-EGFP as a quantitative readout of GTP-tubulin at
growing MT plus ends, we first determined whether EB1 exists
as a monomer or dimer in vivo. In LLCPK1 epithelial cells, as in
a wide range of cell types, EB1 binds to and rapidly turns over
on the MT lattice and is especially concentrated at growing
MT plus ends, forming a comet-like distribution behind the
polymerizing MT tip (Figure 1A; [15–18]). EB1 is an w30 kDa
monomer that is thought to exist as a homodimer [19, 20];
however, its dimerization has not been confirmed in a living
To test whether EB1 diffuses as a monomer or dimer in vivo,
we used a combination of fluorescence recovery after photo-
bleaching (FRAP) experiments and 3D Brownian dynamics
simulations [21, 22] to determine the diffusion coefficients of
23EGFP, EGFP, EB1-EGFP, and EGFP-a-tubulin in the cyto-
plasm of LLCPK1 cells (Figure 1B). The FRAP experiments
were then simulated with varying diffusion coefficients. For
each fluorescent species, simulated and experimental half-
times of recovery were quantitatively compared to determine
the underlying diffusion coefficient (Supplemental Experi-
mental Procedures; ). Once the diffusion coefficients of
all four species were determined, then the diffusion coefficient
of EB1-EGFP was compared to that of the other three species
based on molecular weight (MW).
From the Stokes-Einstein-Sutherland relationship, the diffu-
sion coefficient of a spherical particle is predicted to decrease
with the inverse cubed root of the MW,
where D is the diffusion coefficient, kB is Boltzmann’s
constant, T is temperature, h is the viscosity of the solution,
rH is the hydrodynamic radius, NA is Avogadro’s number,
MWisthemolecular weight,andristhe densityof the particle.
As shown in Figure 1C, a 2-fold increase in MW, from EGFP
(w30 kDa) to 23EGFP (w60 kDa), yields the theoretically pre-
dicted decrease in diffusion coefficient from 2.3 6 0.30 mm2/s
to 1.8 6 0.15 mm2/s (mean 6 SEM, n = 20; Table S2). The diffu-
sion coefficient of EGFP-a-tubulin (140 kDa; assumed to be in
a heterodimeric complex with b-tubulin) is also consistent with
theoretical predictions based on its MW (1.4 6 0.18 mm2/s;
n = 21). When the diffusion coefficient of EB1-EGFP (105 kDa
brightness-corrected effective MW for dimer; see Supple-
mental Experimental Procedures) was measured (1.4 6
0.25 mm2/s; n = 18), it was found to be similar to that of
EGFP-a-tubulin and significantly different from that of EGFP
barriers and potentially allows for more potent +TIP-mediated
Supplemental Information includes two tables, four figures, Supplemental
Results and Discussion, Supplemental Experimental Procedures, and one
movie and can be found with this article online at http://dx.doi.org/10.
We thank M.K. Gardner for technical assistance with the MT growth simula-
tion, P. Wadsworth for the LLCPK1a cell line, L. Cassimeris for the EB1-
EGFP LLCPK1 cell line, J. Mueller for the p23EGFP construct, and R.Y.
Tsien for the pmCherry-a-tubulin construct. Funding support was provided
by National Institutes of Health grants GM-071522 and GM-076177 and
National Science Foundation grant MCB-0615568.
Received: May 8, 2012
Revised: June 14, 2012
Accepted: June 27, 2012
Published online: August 16, 2012
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Estimating the Microtubule GTP Cap Size In Vivo