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Dynamic microtubules at the synapse
Erik W Dent
Microtubules (MTs) are a fundamental cytoskeletal component
that give neurons structure and are the primary polymer system
for long distance transport of cargo throughout the cytoplasm.
Although neurons are highly polarized and their structure is
often maintained throughout the life of an organism, MTs can
remain dynamic in axons and dendrites, undergoing bouts of
polymerization and depolymerization, referred to as dynamic
instability. Furthermore, MTs can be nucleated outside of the
centrosome or MT organizing center (MTOC) that is located in
the cell body, allowing dynamic formation and branching of MT
polymers throughout the neuron. Together, these recent
findings point to a much more dynamic landscape of
microtubules in developing and mature neurons than was
previously appreciated. Here we will focus on recent studies
that show MT dynamics are playing a role at the synapse, both
post-synaptically in dendrites and pre-synaptically in axons.
Address
Neuroscience Department, University of Wisconsin-Madison, Madison,
WI 53705, USA
Corresponding author: Dent, Erik W (ewdent@wisc.edu)
Current Opinion in Neurobiology 2020, 63:9–14
This review comes from a themed issue on Cellular neuroscience
Edited by Thomas Schwarz and Holly Cline
https://doi.org/10.1016/j.conb.2020.01.003
0959-4388/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
Microtubules (MTs) are one of four polymer systems
present in neurons. The other three include actin fila-
ments, intermediate/neurofilaments and septin polymers.
All of these polymers are capable of polymerizing and
depolymerizing. Moreover, MTs are polarized polymers
composed of alpha/beta tubulin dimers that assemble in a
head to tail fashion, resulting in a polymer with a plus and
minus end. The minus end is fairly stable [1], but recent
work indicates that free MT minus ends are capable of
growth in neurons [2]. However, at the plus ends MTs can
be very dynamic, polymerizing and depolymerizing over
tens of micrometers through a stochastic process termed
dynamic instability [3,4]. This stochastic polymerization
and depolymerization allows MTs to explore the entire
intracellular landscape of cells [5].
www.sciencedirect.com
In addition to exhibiting plus end dynamics, MTs in neu-
rons are generally not attached to the centrosome as in many
other cell types. Instead, they are cut by severing enzymes
[6] and are transported by motor proteins as polymers
throughout axonal and dendritic arbors [7,8]. Additionally,
the more recently described local nucleation of MTs in both
axons and dendrites is likely to play crucial roles in the
development and maintenance of the neuronal MT cyto-
skeleton [9–12]. It was long thought that MTs, unlike actin
filaments, were incapable of branching. However, recent
studies have shown that MTs are indeed capable of branch-
ing in neurons [13,14]. One of the primary functions of MTs
is to transport material both anterogradely and retrogradely
throughout both axons and dendrites through rapid move-
ment of both kinesin and dynein motor proteins. Kinesins
generally transport cargo toward the plus end of MTs, while
cytoplasmic dynein transports toward the minus end.
Recent studies indicate that motor proteins are sensitive
to posttranslational modifications of tubulin and bound MT
associated proteins (MAPs) as they transport material
throughout the neuron [15,16]. Such modifications to
MTs direct motors to bind and unbind in distinctive regions
of the neuron, allowing for targeted transport of cargoes.
Most cell types have their MTs positioned with plus ends
oriented toward the cell membrane. However, neurons
develop long, branched axons and dendrites. In axons,
MTs are positioned with their plus ends oriented distally,
either toward the extending growth cone in developing
neurons or toward the distal process in mature neurons. In
contrast, MTs develop an anti-parallel orientation in
dendrites where individual MTs are either positioned
plus end distal or minus end distal. The percentage of
minus end distal MTs can vary from approximately 50–
95% depending on the location in the dendrite or the
organism studied [17,18]. It should be noted that MTs of
both orientations appear to be capable of undergoing
dynamic instability although a recent study suggests that
minus-end distal MTs are more stable than plus-end
distal MTs [19]. Although it is still unclear why dendrites
develop such a MT orientation it obviously affects trans-
port properties in the dendrite. Together, MT dynamic
instability, branching, orientation, posttranslational mod-
ifications and MAP associations all affect how MTs func-
tion at synapses. For this review we will focus on new
studies that illuminate the increasingly important func-
tion MTs play both postsynaptically at the dendritic spine
and presynaptically in the axonal bouton.
Postsynaptic microtubules
For many years it was not appreciated how dynamic MTs
were in mature neurons. Labeling tubulin with
Current Opinion in Neurobiology 2020, 63:9–14
10 Cellular neuroscience

fluorescent compounds or through genetically encoded
fluorescent proteins labeled all of the MTs in neurons,
resulting in a highly fluorescent outline of the entire
neuron. It was not until growing MT ends were labeled
with end binding (EB) proteins that MT dynamics in
mature neurons were first appreciated [20]. When only
growing ends of MTs were visualized with EB3 it was
clear that MTs maintained dynamic instability through-
out the life of the neuron [21]. Several groups discovered
that MTs were capable of polymerizing into and out of all
regions of neurons, including dendritic spines [21–23]
(Figure 1). These studies also showed that MT entry
into dendritic spines was activity-dependent, occurring
after NMDA receptor activation and calcium influx
[24,25]. Chemically induced, long-term potentiation
(cLTP) increased MT invasions of spines, while chemi-
cally induced long-term depression (cLTD) decreased
MT invasions [24–26]. These results suggest that MTs
are sensitive to the intrinsic activity of neurons and target
dendritic spines undergoing synaptic plasticity.
Since dendritic spines are oriented perpendicular to the
dendrite it was curious that MTs polymerizing antero-
gradely or retrogradely throughout the dendritic arbor
would suddenly change their direction of polymerization
from parallel to the dendritic shaft to perpendicular and
enter dendritic spines. This behavior suggested that MTs
were being directed to enter specific spines, but not
others, along the dendritic arbor. As it turned out, MT
entry into dendritic spines is entirely dependent on actin
filaments [25]. More recent studies confirmed that MT
entry of dendritic spines requires actin filaments in the
dendritic shaft [27]. One group has suggested that the
developmentally regulated brain protein (drebrin) is
important for MT entry of spines by showing that knock-
down of drebrin decreased MT invasions, while over-
expression of drebrin increased invasions [25]. However,
recent work from another group suggests that knockdown
of drebrin and several other actin-associated proteins do
not affect MT invasion of spines [27]. This group showed
that only knockdown of the actin-associated protein cor-
tactin or inhibition of the Arp2/3 complex inhibited MT
entry of spines [27]. Although these two studies came to
different conclusions in regard to the importance of
drebrin in MT entry of dendritic spines it is clear that
actin filaments in the dendrite shaft play an instrumental
role in guiding MTs into dendritic spines. Mechanical
deflection of MTs from the dendritic shaft into the spine
is the most parsimonious explanation for how MTs are
guided to enter spines. However, these studies cannot
rule out direct interactions between MT-associated and
actin-associated proteins.
When MTs enter spines generally they are only present
in spine for seconds to minutes before depolymerizing out
of spines [21,23]. However, MTs can target individual

Figure 1

Axon

+ Microtubule
F-actin
Synaptic Vesicle
Dense Core
Vesicle/Syt-4

KIF1A motor

Augmin complex/
Gamma-tubulin
Dendrite

Current Opinion in Neurobiology

Dynamic microrubules play important roles both presynaptically and postsynaptically.

Presynaptically in the axon, microtubules preferentially nucleate via the augmin/gamma-tubulin complex as well as concentrate their plus ends in
the en passant dialation termed the synaptic bouton. Both synaptic vesicles and dense core vesicles are transported along microtubules by the
motor protein KIF1A and concentrate at boutons. Postsynaptically in the dendrite, microtubules polymerize directly into the dendritic spine and
transport vesicles via KIF1A as well. Microtubule polymerization into the dendritic spine is dependent on filamentous actin (F-actin) concentrating
at the base of spines.

Current Opinion in Neurobiology 2020, 63:9–14 www.sciencedirect.com


spines a number of times over extended time periods.
Because one of the primary functions of MTs is to
transport material throughout cells these transient excur-
sions into dendritic spines suggest they may be transport-
ing cargo into spines. Since MT-based transport is gen-
erally 5–10 times faster than MT polymerization, even
transient MT invasion of spines would allow transport of
cargo into spines. To determine if MTs could transport
material into dendritic spines McVicker and colleagues
imaged the motor protein KIF1A and one of its cargoes,
synaptotagmin IV (SytIV), in addition to MTs, and dis-
covered that MTs were capable of transporting this
motor/cargo pair into dendritic spines [28]. By using
pHlourin-labeled SytIV they were able to show that
SytIV-containing vesicles exocytosed in dendritic spines
after delivery by MTs. Paradoxically, this study also
showed that knockdown of KIF1A resulted in more
exocytosis of SytIV in dendritic spines and in the dendrite
shaft, and increased mobility of SytIV puncta in the plane
of the plasma membrane. These data suggest that KIF1A,
in addition to serving to transport cargo, is also important
for sequestering cargo along MTs so that unregulated
exocytosis does not occur throughout the dendritic arbor.
Further studies have shown that the actin-associated
proteins TANC2 and liprin-alpha recruit KIF1A-associ-
ated vesicles into dendritic spines [29].
Taken together, the studies outlined above indicate that
there are dynamic MTs in mature dendrites and they
appear to target dendritic spines undergoing plasticity in
an NMDA, calcium and actin-dependent manner.
Although the exact function of MT entry into dendritic
spines is still unclear, they target synaptically active
spines and are capable of transporting cargo into those
spines. Thus, in addition to diffusion of material in the
plane of the membrane [30] and actomyosin-based trans-
port of material into spines [31], MTs provide an addi-
tional mechanism by which dendritic cargo can be tar-
geted to specific spines.
Presynaptic microtubules
Axonal MTs, like dendritic MTs, are important for motor-
based transport of material and delivery of cargo. Early
electron microscopy studies from George Gray implicated
MT involvement in presynaptic architecture. Specifi-
cally, he showed that MTs were often associated with
synaptic vesicles at presynaptic sites [32,33]. These stud-
ies suggested that presynaptic MTs may be functioning in
synaptic vesicle release, but there has been a relative
dearth of studies on how MTs may be functioning pre-
synaptically in mammalian neurons.
Much of the research on MT involvement in presynaptic
function has focused on the Drosophila neuromuscular
junction (NMJ) [34] (Figure 2). In the Drosophila NMJ,
MTs form a looped structure in the terminal boutons, that
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Microtubules at the synapse Dent 11
Figure 2
Microtubule Futsch Ankyrin DAAM Brp
(MAP1B) (Active Zone)
Current Opinion in Neurobiology
Microtubules associate with multiple components at the Drosophila
neuromuscular junction.
A number of recent studies have shown that there is a complex of
proteins, including Futsch/MAP1B, Ankyrin proteins (Ankyrin2-L,
Ankyrin2-XL), and formin proteins (DAAM shown, Dia not shown) that
associate with microtubules and with active zones (shown as red
circles of bruchpilot (Brp)). These proteins appear to be important for
organizing synaptic architecture, regulating synaptic microtubules and
potentially guiding the delivery of synaptic vesicles to active zones.
Synaptic vesicles and actin are not shown for simplicity. Microtubules
are shown as continuous structures but are likely bundles of shorter
dynamic microtubules.
dynamically reorganize to form new boutons [35–37]. The
MT-associated protein Futsch/MAP1B is especially
important in presynaptic MT organization [38] and acts
as a link between MTs and the active zone [39]. This
work also showed that Futsch/MAP1B may link MTs to
active zones to regulate neurotransmitter release and
active zone density [39]. Further studies implicated giant
Drosophila Ankyrin2 (Ank2) isoforms working synergis-
tically with Futsch/MAP1B to regulate synaptic geometry
and release properties [40]. A recent study has implicated
another member of this MTs/Futsch/Ank2 complex.
Migh and colleagues discovered that a MT- and actin-
associated formin family protein, Disheveled associated
activator of morphogenesis (DAAM), functions together
with MTs/Futsch/Ank2 in the active zone scaffold at the
Drosophila NMJ [41]. Considering the merits of
electrophysiological data the authors suggest that DAAM
plays a role in synaptic vesicle release. Interestingly, it is
mostly through its MT-related, rather than actin-related,
interactions that DAAM acts in developing presynaptic
boutons. Together these studies provide ample data that
MTs, functioning through a number of MT-associated
proteins, play important roles in presynaptic physiology at
the Drosophila NMJ. Do MTs play similar roles in
mammalian presynaptic function in central synapses?
Interestingly, a recent study indicates that local axonal
MT dynamics and kinesin-based transport is important
for delivery of synaptic vesicle precursors (SVPs) to en
passant synapses in cultured mammalian hippocampal
neurons [42] (Figure 1). This work showed that MT
polymerization often initiates and terminates at synapses,
Current Opinion in Neurobiology 2020, 63:9–14
12 Cellular neuroscience
suggesting there are localized regions of MT dynamics in
the axon and pharmacologically blocking MT dynamics
was sufficient to inhibit anterograde delivery of SVPs to
synapses. Moreover, they discovered that the motor pro-
tein KIF1A was important for delivery of SVPs through a
mechanism whereby KIF1A unloaded cargo at synapses
because of its decreased affinity for the GTP-rich tubulin
lattice that comprises the plus end of growing MTs. As
the authors point out, this mechanism of KIF1A-depen-
dent transport and release of SVPs is likely working in
coordination with other mechanisms that regulate KIF1A
activity. Previous work in C. elegans has shown that the
ARF-like small GTPase Arl8 promotes SVP movement
by relieving KIF1A from an autoinhibited state [43].
Thus, coordination of kinesin motor activity and the
ability of KIF1A to detach from growing MT ends that
are positioned close to synapses helps replenish synaptic
vesicles to presynaptic sites.
A similar system appears to guide dense core vesicles
(DCVs) to synapses. DCVs were shown to bind KIF1A
through SytIV and phosphorylation of SytIV by JNK desta-
bilizes this interaction, allowing capture of DCVs at synap-
ses by actin [44] (Figure 1). Interestingly, these authors
showed synaptic activity increases DCV capture at presyn-
aptic sites. Although Bharat and colleagues did not study
MT dynamics, it is likely release of DCVs occurs at MT
plus ends presynaptically, as has been shown postsynapti-
cally [28]. How DCVs and SVPs possibly coordinate their
delivery and capture at boutons awaits further research.
The idea that MT plus ends, localized to synapses, allow for
the unloading of cargo in the form of synaptic vesicle
precursors is a compelling model. However, very recent
work suggests there is an additional MT-dependent mech-
anism upon which synaptic vesicles rely as they make their
way to synapses. This work demonstrated that MT nucle-
ation occurs preferentially at en passant synapses in mature
hippocampal neurons in both culture and slices [45]
(Figure 1). Non-centrosomal MT nucleation is a relatively
newly discovered phenomenon in neurons that allow MTs
to locally nucleate de novo or nucleate a new MT branch
from an existing MT [11,13]. Interestingly, Qu and collea-
gues [45] revealed that nucleation of MTs at synapses is
activity-dependent and requires gamma-tubulin to nucle-
ate MTs and the augmin complex to direct MT growth to
the distal axon. These nucleated MTs at synapses serve as
tracks for bidirectional movement of synaptic vesicles
between boutons. Importantly, loss of de novo nucleated
MTs at boutons impairs high-frequency evoked neuro-
transmitter release, suggesting these nucleated MTs serve
an important role in synapse function.
Together, these recent studies suggest that MTs are
playing a heretofore unappreciated role in SVP and
DCV transport and synaptic function at en passant synap-
ses in hippocampal neurons [42,44,45]. Results from
Current Opinion in Neurobiology 2020, 63:9–14
these studies are complementary and suggest that MT
nucleation, polymerization and motor protein-dependent
transport and release at MT plus ends serve important
roles in SVP and DCV targeting to presynaptic regions of
the axon.
Concluding remarks
Within the last ten years great strides have been made in
determining the function of MTs in both the pre and
post-synaptic compartments, but questions remain. Does
MT nucleation and branching function post-synaptically,
as it has been shown to function pre-synaptically [45]?
Are these MT dynamics disrupted in disease states, such
as Alzheimer’s or epilepsy? How exactly are MT-based
and actin-based polymerization and transport linked at
synapses? It is difficult to study MT dynamics at synapses
because temporal and spatial control of polymerization
and depolymerization is not trivial and MTs and actin
filaments contain an entire host of associated proteins that
are likely to play important roles at the synapse as well.
Continued developments in high/super resolution live-
cell microscopy will likely play an important role in
answering these and other important questions regarding
cytoskeletal function at the synapse.
Conflict of interest statement
Nothing declared.
Acknowledgements
Apologies for all of the excellent work that could not be included in this
article due to space constraints. This work was supported by N.I.H. grant
NS098372 to E.W.D.
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14 Cellular neuroscience
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16:2129-2141
This study shows that the size and density of synapses in C. elegans DA9
neurons is regulated by the activity of the motor protein UNC104/KIF1A.
Overactivation of UNC104/KIF1A results in smaller synapses, decreased
density of synapses and ectopic synapse formation. Additionally, they
Current Opinion in Neurobiology 2020, 63:9–14
show that the small GTPase ARL-8, which binds to synaptic vesicles in its
active state is responsible for releasing the autoinhibition of UNC104/
KIF1A.
44. Bharat V, Siebrecht M, Burk K, Ahmed S, Reissner C, Kohansal-
 Nodehi M, Steubler V, Zweckstetter M, Ting JT, Dean C: Capture
of dense core vesicles at synapses by JNK-dependent
phosphorylation of synaptotagmin-4. Cell Rep 2017, 21:2118-
2133
Using a variety of techniques, this study shows that synaptotagmin-4
(Syt-4) binds to dense core vesicles (DCVs), which are trafficked in axons
by the motor protein KIF1A. Activity-dependent phosphorylation of Syt-4
at presynaptic sites by JNK disrupts Syt-4/KIF1A association, leading to
release of DCVs from microtubules and capture by presynaptic actin.
These data, along with results from Ref. [28], show that Syt-4 and KIF1A
are functioning in similar ways both presynaptically and postsynaptically.
45. Qu X, Kumar A, Blockus H, Waites C, Bartolini F: Activity-
 dependent nucleation of dynamic microtubules at presynaptic
boutons controls neurotransmission. Curr Biol 2019, 29:4231-4240
This study provides an additional, complementary mechanism to that
outlined in Ref. [42] by which microtubules function to deliver synaptic
vesicles to excitatory en passant synapses. Here they show that micro-
tubules are preferentially nucleated at synapses via gamma-tubulin and
augmin. This nucleation and polymerization of microtubules are crucial for
release of vesicles at presynaptic sites and is regulated by synaptic
activity.
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