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
and that of 23EGFP (Figure 1D). We conclude that the diffu-
sion of EB1-EGFP in the cytoplasm of LLCPK1 cells is consis-
tent with a dimer, but not with a monomer, indicating that
EB1-EGFP exists as a dimer in vivo.
An Average of 270 EB1 Dimers Are Bound to the Growing
MT Plus End In Vivo
To set a lower bound on the GTP cap size, we then determined
the number of EB1 dimers in an EB1 comet at the tip of
a growing MT. First, the brightness of a single EGFP molecule
was calibrated using the known packing density of ab-tubulin
in the MT (Figure 2), w1,625 tubulin subunits per mm of MT
[23, 24]. In the stable EGFP-a-tubulin LLCPK1 pig epithelial
cell line (LLCPK1a), it has been previously determined via
both quantitative western blotting and fluorescence speckle
analysis that 17% of the a-tubulin molecules are labeled with
EGFP [25, 26]. The integrated fluorescence intensity (FI),
measured directly as digital camera electron counts, from
a background-subtracted image of a MT, was calculated (Fig-
ure2A)andthen convertedinto countsperEGFP perexposure
(Figure 2B). For our imaging conditions, one EGFP molecule
yielded 44 6 0.15 counts , EGFP21, exposure21(mean 6
SEM, n = 106 MTs), where one exposure was 300 ms. As
density of tubulin in the MT lattice provides a convenient stan-
dard for in vivo calibration of EGFP fluorophore brightness.
Figure 1. EB1-EGFP Exists as a Dimer in LLCPK1 Epithelial Cells
(A) EB1-EGFP labels growing microtubule (MT) plus ends in LLCPK1 epithelial cells. An example of the EB1-EGFP LLCPK1 stable cell line transiently ex-
pressing mCherry-a-tubulin is shown. Upper left: individual growing MTs observed near the periphery of the cell have EB1-EGFP comets at their tips. Upper
right: merged kymograph with tubulin and EB1 in the red and green channels, respectively. Bottom: kymographs in the mCherry-a-tubulin and EB1-EGFP
channels corresponding to the MT in the white box.
(B) Montage of a fluorescence recovery after photobleaching (FRAP) experiment in the cytoplasm of an EB1-EGFP-expressing LLCPK1 cell. Individual
examples of experimental FRAP recovery curves for EGFP, 23EGFP, EB1-EGFP, and EGFP-a-tubulin are shown.
(C and D) The diffusion coefficient of EB1-EGFP is consistent with a dimer.
(C) Diffusion coefficients were determined from simulations of FRAP experiments by quantitatively comparing experimental and simulated recovery half-
times. The theoretical dependence of the diffusion coefficient on the molecular weight (MW) is based on Stokes-Einstein-Sutherland theory for a spherical
particle. The diffusion coefficient of EB1-EGFP is similar to that of EGFP-a-tubulin (MW = 140 kDa) but different from that of 23EGFP (MW = 60 kDa).
(D) Further FRAP experiments show that cytoplasmic EB1-EGFP and 23EGFP diffuse at significantly different rates, consistent with EB1-EGFP diffusing as
a dimer in LLCPK1 cells.
Error bars represent SEM. See also Table S2.
Current Biology Vol 22 No 18
TheEGFP brightness wasthen usedtocalculate thenumber
of EB1 dimers in an EB1-EGFP comet at the MT tip (Figure 2C).
For the EB1 comet analysis, the region of interest size was
fixed to 24 pixels 3 5 pixels (1 mm 3 500 nm; Supplemental
Experimental Procedures). In an EB1 comet, we found that
there are 270 6 18 EB1 dimers (mean 6 SEM, n = 63 comets).
Because comet lengths are w1 mm, this tip-associated signal
rises significantly above the background lattice intensity of
36 6 5.2 EB1 dimers per mm of MT lattice (mean 6 SEM,
n = 16 MTs, 11 cells; Figure S1). This value of 270 EB1 dimers
represents a lower bound on the GTP cap size, which would
require that EB1 completely saturate the GTP-tubulin binding
sites at the growing MT tip. This lower-bound estimate of
270 GTP-tubulins, or equivalently at least 20 layers, indicates
that the GTP cap in vivo is much larger than a single layer of
tubulin (13 subunits) and even larger than the most extreme
upper limit estimate from the literature of 200 GTP-tubulin
subunits for MTs in vitro . Thus, our lower-bound estimate
of the GTP cap size in vivo exceeds the previous upper-bound
estimate for cap size in vitro and is 20-fold larger than previous
estimates of a single-layer cap.
The MT GTP Cap In Vivo Is Composed of 750 GTP-Tubulin
Subunits on Average
After establishing a lower bound for the GTP cap of 270 GTP-
tubulin subunits, we wanted to determine the average cap size
in vivo. To estimate the average GTP cap size, we used the
brightness of the EB1 comet and the known tubulin packing
in a MT to establish the percent occupancy of GTP-tubulin
sites by EB1 at the tip of the MT (Figure 3A). On average,
there were 22 6 1.6 EB1 dimers (mean 6 SEM, n = 36) at the
brightest pixel (42 nm/pixel = 68.25 subunits/pixel) on the
comet, which means the GTP cap is 32% saturated with EB1
dimers (assuming that the tip is composed of nearly 100%
GTP-tubulin; see Supplemental Results and Discussion for
justification). With 270 EB1 dimers occupying the GTP cap
at 32% of saturation, this corresponds to 270/0.32 z 800
tubulin subunits in the GTP cap (95% confidence interval of
650–1,000 GTP-tubulin subunits), which corresponds to
a >65-layer distributed GTP cap that decays exponentially
in vivo (Figure 3B).
To independently estimate the average GTP cap size, we
used the EB1 comet decay length (l) and the known tubulin
packing in a MT to calculate the number of tubulin subunits
in the GTP cap (Figure 3C). On average, an EB1 comet had
a half-length (L1/2) of 310 6 18 nm (mean 6 SEM, n = 36), which
is related to the exponential decay length (l = L1/2/ln2) of
440 nm. The exponential decay was then integrated from the
growing tip toward the minus end of the MT, under the
assumption that the growing tip is composed exclusively of
GTP-tubulin whose concentration drops exponentially at a
rate given by the EB1 comet decay length. Using this method,
we estimate that there are w700 GTP-tubulin subunits in
a GTP cap (95% confidence interval of 620–780 GTP-tubulin
subunits), which corresponds to a >50-layer distributed GTP
Figure 2. An Average of 270 EB1 Dimers Are at the Growing MT Plus End
(A) Individual MTs observed near the periphery of LLCPK1a cells. Left: the
local background (yellow box) near the MT is subtracted to yield a section
of MT lattice that is free of MT-unrelated signal. Right: a section of the back-
ground-subtracted MT (red box) is analyzed in (B).
(B) Calibrating the fluorescence intensity (FI) of a single EGFP molecule
in vivo. A line scan along the MT (blue box), encompassing the width of
the MT, is taken to measure the integrated FI of the MT region. The
corresponding line scan and integrated FI are shown below. From
the known MT packing and percentage of labeled tubulin in the cell (17%),
the average brightness of a single EGFP molecule was calculated to be
44 FI , EGFP21, exposure21. Relevant experimental parameters for cali-
brating the brightness of a single EGFP molecule are shown in the table.
(C) Determining the number of EB1 dimers in an EB1-EGFP comet. A region
used to calculate the integrated FI of the comet, which is then used to deter-
mine the number of EB1 dimers based on the EGFP brightness calibration.
The example EB1-EGFP comet shown has 216 EB1 dimers. A histogram of
the number of EB1 dimers in a comet is shown below, with the mean and
See also Figure S1 and Table S1.
Estimating the Microtubule GTP Cap Size In Vivo
cap that decays exponentially in vivo. This independent esti-
mate of the in vivo MT GTP cap size corroborates our large
GTP cap estimate, which utilized the EGFP fluorophore bright-
Asacheck onour GTPcap sizeestimate, wethen compared
the KDof EB1 binding to previous estimates of MAP binding to
MTs in vivo . We estimated the KDof EB1 binding to GTP-
tubulin in the MT lattice, assuming equilibrium, via
where [EB1free]eqis the free EB1 concentration at equilibrium,
[GTP 2 Tubulin]eqis total concentration of available MT GTP-
tubulin binding sites at equilibrium, and [EB1bound]eqis the
concentration of MT-bound EB1 at equilibrium. We also
measured the free EB1 dimer concentration to be 1.2 mM using
the EGFP brightness calibration (Figure 2B). From the EB1
dimer free concentration and the fractional occupancy, the
KD was calculated to be 3.8 mM. This corresponds to
a moderate binding affinity in vivo, which has been previously
reported for in vitro EB1-tubulin binding to paclitaxel-stabi-
lized MTs . From this, we can also estimate koff, assuming
a typical konvalue of 1–10 mM21s21. We estimate koffto be
3.8–0.38 s21, which is consistent with previous fluorescence
recovery after photobleaching (FRAP) experiments . The
binding affinity of EB1-EGFP to the MT tip is comparable to
the apparent affinity of ensconsin for MTs in vivo, estimated
to be KD= 11 mM . Therefore, our KDfor EB1 in vivo is
comparable to another MAP in vivo and consistent with
previous FRAP experiments for EB3 and CLIP-170 .
Comparing our in vivo estimate of the MT GTP cap size to
previous in vitro estimates (Table S1), we find that our in vivo
estimates of both the lower bound and average MT GTP cap
size are much larger than any in vitro estimates. However, in
EB1-EGFP-expressing LLCPK1 epithelial cells, we found that
Figure 3. The MT GTP Cap Is Composed of 750
GTP-Tubulin Subunits In Vivo
(A) Determining the number of GTP-tubulin
subunits in the GTP cap. The EB1 percent occu-
pancy was measured at the tip of the MT plus
end. First, the brightest pixel in an experimental
EB1-EGFP comet was found using a custom
MATLAB code. Then, a 1 3 7 pixel ROI (red
box) was centered on the brightest pixel, and
the integrated FI within the region was calcu-
lated. Next, using the brightness calibration of
a single EGFP, the corresponding number
of EB1 dimers within the ROI was calculated.
The EB1 percent occupancy was then calculated
by dividing the number of EB1 dimers by the
tubulin packing density. Finally, the number of
GTP-tubulin subunits was calculated by dividing
the average number of EB1 dimers in an EB1
comet (Figure 2C) by the fractional occupancy
to yield w800 GTP-tubulin subunits in the
(B) MT animation showing the distributed GTP
cap. Only the first 50 layers of the 800-subunit
cap are shown. Purple and white dimers repre-
sent GTP-tubulin subunits; green and white
dimers represent GDP-tubulin subunits. The cor-
responding model-convolved fluorescent image
is shown below. The red box indicates the same
size region as shown in (A). The blue box corre-
sponds to the same length indicated by the
blue box in (A) and in the model-convolved
(C) Example EB1 comet from an EB1-EGFP
LLCPK1 cell and the corresponding FI line scan
(blue line). Exponential decay fit (red line) to
EB1 comet signal is shown. The average EB1
comet decay length (l) and half-length (L1/2) are
shown with the calculation of the GTP cap size
using the EB1 comet half-length. This alternative
method for estimating the MT GTP cap size
yields an estimate that is consistent with the
GTP cap size estimate from the EGFP calibration
method in (A).
See also Figures S2 and S3.
Current Biology Vol 22 No 18
MTs grow at 156 6 13 nm/s (mean 6 SEM, n = 29) with 6.9 mM
tubulin (measured using the EGFP brightness calibration from
Figure 2B), whereas MTs grow at w20 nm/s in vitro at nearly
the same tubulin concentration (w5–10 mM GTP-tubulin; [29–
31]). This w7-fold disparity in MT growth rate from in vitro to
in vivo conditions predicts an w7-fold larger GTP cap size
in vivo. Upon further examination of the previous in vitro esti-
mates of the GTP cap size (Table S1), we find that, although
mated a one- to three-layer cap, the largest in vitro estimate
tent in vivo GTP cap size estimate. A 7-fold increase in the
upper-bound estimate from Walker et al. would predict
a GTP cap size range of 700–1,400 GTP subunits, which is
consistent with our average in vivo estimate of w750 GTP
subunits (95% confidence interval of 670–830 GTP-tubulin
subunits; see further discussion in Supplemental Results and
Discussion). Therefore, although our estimate is 65-fold larger
than the single-layer cap theory, it is still consistent with the
largest in vitro prediction.
There have been mechanisms proposed for EB1 tip tracking
alternative to GTP-tubulin recognition, including MT seam
binding and recognition of an unknown tubulin conformation
such as a sheet [32, 33]. The EB1homolog, Mal3, was reported
EB1 were to only bind along the MT seam, this would result in
a uniformly dim signal along the length of the MT rather than
a comet-like signal, because the seam is not confined to the
MT plus end. Also, a seam-binding model does not explain
the MT lattice. If EB1 binds to an unknown conformation
confined to the tip of the MT, we would predict the EB1-
EGFP comet decay length to be small, on the order of our
recent estimates of MT tip structures in LLCPK1 cells ,
which have an average length of w180 nm, much less than
the 1/e decay length of EB1 comets we measured here to be
w440 nm. Given these considerations and the recent demon-
tubulin in the MT lattice [12–14], the overall conclusion from
previous work is simply that EB1 recognizes the GTP-tubulin
cap in growing MTs. Based on this assumption, we conclude
that the GTP cap in LLCPK1 cells comprises w750 subunits
Frequent MT Rescue, More Than 150 Layers Away from the
MT Tip, Is Correlated with the Presence of GTP-Tubulin
One prediction of a large GTP cap is that GTP-tubulin will exist
far from the tip (i.e., more than one decay length, >440 nm). In
this case, a postcatastrophic MT tip must first shorten through
the tail of the GTP cap. A key prediction is that the shortening
rate in the GTP cap tail region will be slower than in more prox-
imal GDP-rich regions. To test this prediction, we divided MTs
into two sections, the first 500–2,000 nm away from the MT tip
and the second >2,000 nm from the MT tip, and compared the
MT shortening rates within these two sections. We did not use
the first 500 nm from the MT tip because we wanted to be very
conservative in our estimate of the limit of detectability of
a catastrophe event (see Supplemental Experimental Proce-
dures). As predicted, we found that MTs shorten significantly
more slowly near the MT tip as compared to regions
>2,000 nm away from the MT tip (p = 0.003). Near the MT tip,
MTs shorten on average 610 6 34 nm/s as compared to 790
6 54 nm/s (mean 6 SEM, n = 31 MTs) >2,000 nm away from
the MT tip (Figure 4A).
Another prediction of a large GTP cap is that rescues should
be common in the tail of the cap and less frequent in more
proximal regions. As shown in Figure 4B, we found that
rescues were largely limited to the first 2,000 nm of shortening
and that more proximal rescues were not detected. For 34
observed rescues, we found rescues occurred on average
1,200 6 95 nm (mean 6 SEM, n = 34) or w150 tubulin layers
from the MT catastrophe site, based on the mean tip position
(Figure 4B). The minimum distance from the MT tip that
a rescue event could be confidently resolved was 500 nm.
However, this 500 nm threshold is larger than two point spread
functions and is w12 pixels in length, which is greater than
a single layer and about double our lower-bound estimate for
the GTP cap size. Together, these data demonstrate that MT
rescues occurred in the tail regions of the cap, located on
average 150 tubulin layers away from the MT plus end, and
very rarely in more proximal regions of the MT.
Furthermore, the large GTP cap predicts that the rescues
should be due to residual low levels of local GTP-tubulin,
and those rescue sites should therefore have slightly higher
EB1-EGFP signal than more proximal regions. To test this
prediction, we compared the EB1-EGFP FI at rescue sites to
regions that were proximal and distal to the rescue site (Fig-
ure 4C). In order to reliably determine the rescue site location,
we established EB1-EGFP as an accurate basis for automated
tracking of growing MT tips in vivo. We found that our single-
time-point accuracy of MT tip tracking via digital image anal-
ysis of EB1-EGFP was 13 nm, or about 1.6 dimer layers (1
layer = 8 nm). This now establishes a method for near-molec-
ular-resolution tracking of MT growth in vivo and eclipses our
previous method for high-accuracy tracking (36 nm via EGFP-
tubulin in LLCPK1 cells using the same microscope ) by 3-
fold. Thus, we can track MT tip dynamics at 5 Hz and l/40 nm,
demonstrating powerful superresolution analysis in vivo using
conventional microscopy and conventional digital image anal-
ysis. As shown in Figure 4D, rescue sites within the MT lattice
have a statistically 2.0-fold brighter EB1-EGFP FI than the
EB1-EGFP signal on more proximal MT regions. However,
rescue sites were not statistically brighter than more distal
regions, which is consistent with a large distributed MT GTP
cap but not consistent with an island or remnant of GTP-
tubulin . These results indicate that a large GTP cap, de-
caying over 1 mm, influences postcatastrophe shortening
If we assume that the MT tip is completely saturated with
GTP-tubulin subunits (for further discussion, see Supple-
mental Results and Discussion) and use the measured EB1-
EGFP comet decay length, we can calculate the percentage
of tubulin subunits at the mean rescue site that are GTP. We
measured the EB1-EGFP comet decay length (1/e) in EB1-
EGFP-expressing LLCPK1 epithelial cells to be 440 6 83 nm
(mean 6 SEM, n = 26 MTs; Figure 3C), with an equivalent
EB1 comet L1/2 of 310 nm, 440 nm , ln(2). Based on the
measured EB1-EGFP comet decay length, we predict the
average rescue site location to be 6.5% saturated with GTP-
tubulin subunits, or about one GTP-tubulin per layer. It is
remarkable that such a small fraction of GTP subunits is suffi-
cient to influence MT dynamics. Again, these data are consis-
tent with a large GTP cap in vivo where multiple opportunities
for rescue are available in the tail of the GTP cap but are not
consistent with a small GTP cap that is only a few layers deep.
Collectively, these in vivo estimates of the MT GTP cap
size indicate the presence of a very large GTP cap that is on
the order of 750 tubulin subunits, with a lower bound of
Estimating the Microtubule GTP Cap Size In Vivo
270 subunits, and decays with increasing distance from
the tip. Furthermore, stabilizing features of the cap can be
detected at distances greater than 1 mm (150 layers from tip;
Figure 4), which agrees with observed EB1 comet lengths.
These results indicate that the GTP cap size in vivo is 4- to
65-fold larger than published in vitro estimates and functions
to promote rescue and suppress shortening.
Potential Functional Implications for Large MT GTP Caps
A large GTP-tubulin cap on growing MTs has important poten-
tial implications for the cell. First, the large GTP cap provides
multiple opportunities for depolymerizing MTs to rescue in
the tail of the GTP cap, >100 layers away from the MT plus
end. As previously reported , and as we have observed in
LLCPK1 cells (Movie S1), MTs that reach the cell periphery
undergo multiple cycles of growth and shortening before
finally depolymerizing back toward the cell center. Thus,
a MT is more persistent in the face of barriers that tend to
suppress net assembly , because the long tail of the large
As a result, a MT has multiple opportunities to deliver cargo or
find abinding partner, rather than asingle attempt as would be
expectedfrom asingle-layer (orsmall)GTP cap.Ifthe MT were
to participate in signaling pathways, by either delivering cargo
necessary for signaling , or binding to the actin cortex ,
then the MT will benefit from an extended GTP cap, with GTP-
tubulin subunits >2 mm away from the MT plus end, to provide
additional opportunities for rescue in the GTP cap tail. This
‘‘tail rescue’’ phenomenon will prolong the duration of interac-
tion at the cortex, which can then amplify a MT-mediated
A large GTP cap, extending over several hundred GTP-
tubulin subunits in vivo, can potentially serve as a highly
concentrated point source for biochemical activity. We esti-
mate that the large GTP cap in vivo allows EB1 dimers in
a comet to be highly concentrated (>100 mM) locally, far above
the cytoplasmic free concentration of 1.2 mM. In this way,
a growing MT plus end with a high concentration of proteins
bound to the large GTP cap through EB1 can act as a potent
localized signal and more effectively participate in signaling
cascades than would be possible with a small (e.g., single-
layer) GTP cap. An example of this type of signaling occurs
during Drosophila embryogenesis, where DRhoGEF2 is deliv-
ered to a localized region of the cell membrane to affect
myosin-mediated cell contractility . In general, the large
GTP cap makes MTs more robust to catastrophe in face of
Figure 4. Suppressed MT Shortening and MT Rescues Are Correlated with
(A) MTs shorten more slowly through the GTP cap. Kymographs from time-
lapse movies of EGFP-a-tubulin MTs were used to determine that
MTs shorten more slowly 500–2,000 nm away from the MT tip (610 6
34 nm/s, blue line) as compared to >2,000 nm away from the MT tip
(790 6 54 nm/s, red line; mean 6 SEM, n = 31 MTs; p = 0.003 by paired
Student’s t test). The MT region <500 nm away from the MT tip was consid-
ered to be within the GTP-tubulin-rich region (i.e., within approximately one
decay length of the average GTP cap size) and was not included in the anal-
ysis (green triangles). An example trace of a shortening MT is plotted
(orange circles) on the corresponding kymograph (see inset). The start of
the 500–2,000 nm section (white arrow) and the >2,000 nm (black arrow)
section are indicated.
(B) MT rescues occur on average 1,200 6 95 nm (mean 6 SEM, n = 34 MTs)
from the plus end of the MT. The mean rescue site is w150 layers away from
the tip of the MT, which is inconsistent with a single-layer GTP cap but
consistent with a large GTP cap with a long tail.
(C) Measuring EB1-EGFP FI signal during MT rescue events. An example
EB1-EGFP comet kymograph from a live time-lapse movie is shown. It
contains a catastrophe (arrow head) followed by a rescue event 714 nm
away and 5 s later (arrow indicates time point of rescue). To determine
whether MT rescue sites have more GTP-tubulin than nonrescue sites, the
imal region (2, dashed purple box) and distal region (5, green box). For
further analysis, the proximal site (2) was divided into left (3) and right (4)
(D) MT rescues occur at sites with relatively high levels of EB1-EGFP. The
rescue site was significantly brighter than the proximal regions but not
brighter than the distal region by multiple-comparison test, which is consis-
tent with a large GTP cap but inconsistent with a GTP remnant or a single-
layer cap. Error bars represent SEM.
See also Figure S4 and Movie S1.
Current Biology Vol 22 No 18
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