T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 171, No. 2, October 24, 2005 197–200
The Rockefeller University Press$8.00
TIP maker and TIP marker; EB1 as a master
controller of microtubule plus ends
Kevin T. Vaughan
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556
The EB1 protein is a member of the exciting and enig-
matic family of microtubule (MT) tip-tracking proteins.
EB1 acts as an exquisite marker of dynamic MT plus ends
in some cases, whereas in others EB1 is thought to di-
rectly dictate the behavior of the plus ends. How EB1
differentiates between these two roles remains unclear;
however, a growing list of interactions between EB1 and
other MT binding proteins suggests there may be a single
mechanism. Adding another layer of complexity to these
interactions, two studies published in this issue implicate
EB1 in cross-talk between mitotic MTs and between MTs
and actin filaments (Goshima et al., p. 229; Wu et al.,
p. 201). These results raise the possibility that EB1 is a
central player in MT-based transport, and that the activity
of MT-binding proteins depends on their ability or inabil-
ity to interact with EB1.
EB1 is a MT tip-tracking protein
EB1 was first described as an adenomatous polyposis coli
(APC)–interacting protein whose binding domain was affected
by APC mutations implicated in colon cancer (Su et al., 1995).
Subsequent localization revealed that EB1 binds to and labels a
subset of the total microtubule (MT) population and that it dis-
plays some preference for the very plus end of these MTs (Mor-
rison et al., 1998). Live-cell imaging of transfected GFP fusion
proteins further revealed specificity for the tips of MTs under-
going elongation (Mimori-Kiyosue et al., 2000). This tendency
to bind elongating MT plus ends and to appear to “track” with
these ends as long as they extend is known as “tip tracking,”
and an impressive number of MT-binding proteins display this
tip-tracking activity in live-cell imaging assays (for review see
Vaughan, 2004). In most cases, high resolution imaging sug-
gests that these proteins “treadmill” at the elongating plus ends
rather than translocate or “surf” with the growing tip. This con-
trasts, to some extent, the behavior of homologues in yeast and
filamentous fungi that appear to translocate toward the plus end
via a kinesin motor or other mechanisms (Xiang et al., 2000;
Liakopoulos et al., 2003). In mammalian cells, tip tracking is
best characterized in transfection assays where fluorescent fu-
sion proteins are overexpressed at some level. Often the degree
of tip specificity is influenced by the amount of expressed pro-
tein. Although tip specificity is evident at low levels of expres-
sion, a transition to decoration along the length of MTs becomes
prominent as the level of expression increases. For some tip
trackers, this tendency is thought to reflect a regulatory cycle at
the MT plus end (Vaughan et al., 2002). However, MTs can
tolerate high levels of EB1 better than the other tip trackers,
suggesting something unique and intriguing about EB1.
Tip tracking and EB1 function
Despite an explosion of recent work on EB1, the precise func-
tion and location of endogenous EB1 at native levels remains
unclear. The MT-associated population of EB1 represents a
small subset of total EB1, but this subset has received the most
attention. The remainder is thought to be largely soluble, simi-
lar to the form that dominates the GFP-EB1 expression studies.
Immunofluorescence microscopy images suggest that native
EB1 is punctate, resembling vesicles or large protein com-
plexes (Morrison et al., 1998; Faulkner et al., 2000; Mimori-
Kiyosue et al., 2000). This would be consistent with the exten-
sive colocalization with other membrane-associated proteins
such as CLIP-170 and dynactin. However, nonmembranous
protein complexes including binding partners such at APC are
also described, and the function of these is under investigation
(Mimori-Kiyosue et al., 2000; Wen et al., 2004). In these
settings, direct interactions between EB1 and other proteins
, CLIP-170, and CLASPs) have been interpreted as
recruitment mechanisms (Fig. 1). The fact that these binding
partners can also bind tubulin directly suggests some transition
or sequential loading process at plus ends that will require
more work to resolve.
Related to the recruitment function of EB1, another model
is focused on the ability of EB1 to regulate MT growth. A series
of elegant biochemical, immunodepletion, and small interfering
RNA (siRNA)/rescue assays indicate that EB1 can stimulate
growth at MT plus ends (Fig. 1) and that this is critical in situa-
tions where search-capture requires long and dynamic MTs
(Tirnauer and Bierer, 2000; Rogers et al., 2002; Tirnauer et al.,
Correspondence to Kevin T. Vaughan: Vaughan.firstname.lastname@example.org
Abbreviations used in this paper: APC, adenomatous polyposis coli; C-MT,
centrosomal MT; K-fibers, kinetochore fibers; MT, microtubule; siRNA, small
JCB • VOLUME 171 • NUMBER 2 • 2005198
2002b; Ligon et al., 2003). Current limitations make it difficult
to define how EB1 arrives at the MT plus end, but the substantial
soluble pool and the ability to add soluble EB1 experimentally
suggests it either coassembles with the elongating MT tip or is
recruited soon after tubulin polymer is exposed.
New tip trackers suggest additional
roles for EB1
In this issue, Wu et al. (p. 201) expand the list of tip trackers to
include melanophillin, which is recognized as a linker between
the myosin V motor and Rab 27a required for myosin-driven
transport of melanosomes in pigmented cells (Wu et al., 2002).
MT-based transport is also an essential component of melano-
some delivery in pigmented cells (Rogers et al., 1997), and the
mechanisms that allow transfer of melanosomes from the MT
network to microfilaments have been unclear. Using overexpres-
sion of fluorochrome-tagged melanophillin, Wu et al. (2005) re-
port robust tip tracking of the expressed protein and demonstrate
that this behavior requires EB1. This association of melanophil-
lin with EB1 does not require either myosin V or rab27a, be-
cause tip tracking is evident in melanocytes from
mice, and interaction-disrupting melanophillin mutants
tip-track as well. Although melanophillin depends on EB1 for
MT binding, EB1 does not appear to require melanophillin for
normal function. Only a subset of EB1 comets contains detect-
able melanophillin in melanocytes, and EB1 is expressed in
many cell types that do not express melanophillin.
The conceptual challenge of this work is to determine the
function of EB1 and MT binding for melanophillin. Is this a
nuance of overexpression or an important clue into the role
melanophillin plays in melanosome transport? Interestingly,
the authors point out that melanosomes do not tip-track nor-
mally, and that the movements of melanosomes are very differ-
ent from tip-tracking proteins. Furthermore, expression of the
tagged melanophillin constructs reveals both MT- and actin-
associated structures in the cell periphery. Perhaps this dichot-
omy is the crucial finding for melanophillin. The authors
propose the enticing possibility that melanophillin uses a com-
bination of MT binding (via EB1) and actin binding (via myo-
sin V) to build a transient transfer station in the cell periphery
where melanosomes can be efficiently handed from MTs to mi-
crofilaments (Fig. 2). This is consistent with the known be-
haviors of melanosomes (Rogers and Gelfand, 1998) and un-
covers a functional aspect of melanophillin that would be
difficult to examine due to the transient nature of these inter-
mediates. In common with other EB1 studies, it remains un-
clear if EB1 serves a role as a marker for MT plus ends that
have reached the cell periphery, or if EB1 actively preserves
particular MT plus ends long enough to allow the hand-off.
However, this work provides compelling evidence that EB1
plays a larger role than previously anticipated.
Adding further weight to the possibility that EB1 coordi-
nates transfer between cytoskeletal systems, this issue also in-
cludes a study from Goshima et al. (p. 229) that reports a
new role for EB1 in mitotic spindle function. Using
S2 cells as a model together with siRNA-driven depletion, this
group dissects the contribution of
in the formation and motility of kinetochore-linked (K-fibers)
and centrosome-linked (C-MTs) MT bundles during spindle
pole focusing. Depletion of cytoplasmic dynein primarily im-
pacted the ability of K-fibers to move toward the spindle poles,
whereas depletion of
affected the focusing of the k-fibers
into a tight spindle. Although these motors share some functional
redundancy, time-lapse sequences highlight the phenotypic
distinction between k-fiber focusing and transport.
To better understand the specific function of each motor,
-GFP was accomplished through depletion of
by siRNA coupled with expression of
GFP. Focusing on cells with almost normal levels of functional
, Goshima et al. (2005) report accumulation of
spindle poles and along K-fibers. This finding is consistent
with the bivalent nature of
playing a role in MT cross-linking in the spindle. However,
FRAP analysis revealed that
these MTs and displays enrichment at the plus ends of MTs
and cytoplasmic dynein
-MT interactions, potentially
-GFP is highly dynamic on
MT plus ends at multiple cellular sites where MT search-capture occurs.
(A) EB1 has been implicated in search-capture of ER-Golgi transport vesi-
cles and in MT capture at the cell cortex during cytoskeletal reorientation.
In these cases, EB1 highlights locations where search-capture is in
progress or has identified capture sites. (B) EB1 has also been linked to
search-capture sites during mitosis including the kinetochores of chromo-
somes during prometaphase and locations at the cell cortex where astral
MTs can attach. Depletion of EB1 has been shown to induce short MTs
that fail to reach the cell cortex. The consequences include mis-orientation
of the spindle.
Functions for EB1 during interphase and mitosis. GFP-EB1 labels
EB1 AND PLUS ENDS • VAUGHAN199
emanating from the spindle poles similar to tip tracking.
-GFP during interphase also revealed tip
to the list of plus end–binding proteins.
Although it was unclear how
would target to plus ends, previous work on EB1 (Rogers et al.,
2002, 2004) suggested a mechanism. siRNA-mediated deple-
tion of EB1 induced a transition from tip-specific binding to
more uniform labeling of MTs. This was coupled with a reduc-
(a minus end–directed motor)
tion in MT dynamics that correlated with a loss of K-fiber fo-
cusing similar to
As a tool to predict how MT plus end binding of
contribute to K-fiber focusing, Goshima et al. (2005) use molec-
ular modeling of a minimal spindle and compare the outcomes of
two scenarios. The first scenario assumes that an
plex targets the motor domain of
-terminus) to the plus ends of C-MTs via the EB1 inter-
action. The second scenario has the opposite orientation with the
motor binding C-MTs and the tail binding K-fibers via EB1.
Simulations support the second model with the
ciating with the plus ends of C-MTs and moving toward the
poles (Fig. 2). In this arrangement, the
to draw the K-fibers toward the poles.
Given the proposed role of EB1 in imparting plus end specificity
, this outcome is somewhat counterintuitive. However,
EB1 could contribute by ensuring that the K-fiber/
teraction occurs only at the plus ends of C-MTs. Similar to the
melanophillin story above, EB1 could represent a marker for
transient interactions between distinct filament systems.
to K-fibers and the tail of
tail and EB1 attach to
Insights into EB1 function
Although the studies of melanophillin and
issue appear to focus on very different questions, the overlap-
ping contributions of EB1 to both stories suggests a fundamen-
tally new model for EB1 function and the potential role of tip
tracking. If one adds the other locations where EB1 is thought
to function, a theme emerges that supports a more central role
for EB1 in interactions between large multi-subunit complexes.
Although not completely understood, in interphase cells MT
plus ends contain EB1 and other tip-trackers where they appear
to mark locations of search-capture between MTs and mem-
branes (Pierre et al., 1992; Valetti et al., 1999; Vaughan et al.,
2002; Wen et al., 2004). An overlapping class of EB1-associated
proteins are found at kinetochores during prometaphase where
search-capture of chromosomes occurs (Dujardin et al., 1998;
Faulkner et al., 2000; Tirnauer et al., 2002a). If we add transfer
of melanosomes from MTs to actin filaments (Wu et al., 2005)
and linkage of K-fibers and C-MTs (Goshima et al., 2005) to
the list, one could propose that EB1 is the master integrator of
protein complex assembly on MTs. It remains unclear if this
function is related to the impression conveyed by tip-tracking
assays, or if tip tracking simply reflects the fact that EB1–MT
interactions are tightly regulated (Vaughan, 2004). Future work
and the identification of new EB1-binding partners will shed
light on this intriguing question.
reported in this
The growing list of EB1-interacting proteins and functions
suggests that our understanding of EB1 and MT tip tracking
is incomplete. The addition of two new candidates to the plus
end–binding protein family implies that EB1 plays a funda-
mental role in coordinating movement along MTs by defining
locations where specific conditions have been met for trans-
port. This model represents a conceptual advance for analysis
of tip-tracking proteins and provides the framework for further
dissection of their function.
Models proposed by studies in this issue (Goshima et al., 2005; Wu et
al., 2005) suggest a more global role for EB1 in cytoskeletal transport.
(A) MT tip tracking of melanophillin in an EB1-dependent manner coupled
with binding of melanophillin to myosin Va in melanocytes identifies a
new function for EB1. In contrast to other models that implicate EB1 in re-
cruitment of proteins involved in MT-based transport, this work potentially
identifies a role in transfer of cargo from one cytoskeletal system to an-
other (MTs to actin). (B) Parallel work on MT motors involved in mitotic
spindle formation identify a connection between EB1 and ncd at sites
where kinetochore fibers (k-fibers) interact with centrosomal MTs (C-MTs)
emanating from the spindle poles. EB1 is proposed to play a role in deter-
mining where ncd initiates contact between these two MT populations—
a role that molecular modeling predicts is essential for the formation of a
functional and focused spindle.
EB1 as a multi-functional protein involved in cargo transfer.
JCB • VOLUME 171 • NUMBER 2 • 2005 200
The author thanks Edward Hinchcliffe (University of Notre Dame) for helpful
comments. Space limitations prevent the citation of additional EB1 studies that
contribute to our understanding of EB1 function.
This work was supported by funding from National Institutes of Health
(grant GM60560) and the American Cancer Society.
Submitted: 26 September 2005
Accepted: 4 October 2005
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