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With the Permission of Microtubules: An Updated Overview on Microtubule Function During Axon Pathfinding

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During the establishment of neural circuitry axons often need to cover long distances to reach remote targets. The stereotyped navigation of these axons defines the connectivity between brain regions and cellular subtypes. This chemotrophic guidance process mostly relies on the spatio-temporal expression patterns of extracellular proteins and the selective expression of their receptors in projection neurons. Axon guidance is stimulated by guidance proteins and implemented by neuronal traction forces at the growth cones, which engage local cytoskeleton regulators and cell adhesion proteins. Different layers of guidance signaling regulation, such as the cleavage and processing of receptors, the expression of co-receptors and a wide variety of intracellular cascades downstream of receptors activation, have been progressively unveiled. Also, in the last decades, the regulation of microtubule (MT) assembly, stability and interactions with the submembranous actin network in the growth cone have emerged as crucial effector mechanisms in axon pathfinding. In this review, we will delve into the intracellular signaling cascades downstream of guidance receptors that converge on the MT cytoskeleton of the growing axon. In particular, we will focus on the microtubule-associated proteins (MAPs) network responsible of MT dynamics in the axon and growth cone. Complementarily, we will discuss new evidences that connect defects in MT scaffold proteins, MAPs or MT-based motors and axon misrouting during brain development.
| Guidance signaling downstream pathways involved in MT dynamics in the axon and GC I: MT-stabilizing, MT-destabilizing and MT-polymerization supporters. Netrin-1-DCC signaling produces MT stabilization via MAP1B phosphorylation through GSK3 and CDK5 activity (Del Río et al., 2004). Draxin binds DCC receptor and leads to MAP1B phosphorylation via GSK3β (Meli et al., 2015). Sema3A stimulates MAP1B mRNA local translation by promoting the proteasome-dependent degradation of the repressor FRMP (Takabatake et al., 2020). Sema3A, EphrinA5, RGMa or Sema4D inhibit MT polymerization by increasing CRMP2 phosphorylation via GSK3β and CDK5 (Arimura et al., 2005; Cole et al., 2006; Ito et al., 2006; Wang et al., 2013). Sema3A promotes MT destabilization by promoting DCX-fall off the MT lattice via CDK5-dependent phosphorylation of DCX (Bott et al., 2020). The combined action of EphB, laminin and L1 leads to MT overgrowth and buckling by reducing SCG10 protein levels (Suh, 2004). Sema3C increases tau protein levels (Moreno-Flores et al., 2004). EphrinB1-EphB2 signaling reduces tau hyperphosphorylation via PI3K-dependent inhibition of GSK3 (Jiang et al., 2015). Wnt5a promotes MT redistribution by stimulating CaMKII-dependent phosphorylation of tau at Ser262 (Li et al., 2014). Sema3A transiently increases tau phosphorylation at Ser202 and Thr205 via CDK5-dependent phosphorylation (Sasaki et al., 2002). MTs are shown as light purple tubes, F-actin as red lines. MAPs are represented in blue, kinases in yellow and MAP-interacting proteins in purple. Guidance cue receptors are in brown. Guidance-evoked responses are represented in green (attraction), red (repulsion) and orange (pause) arrows. MT advance and retraction are represented with green and red arrowheads, respectively.
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fnmol-14-759404 November 30, 2021 Time: 13:1 # 1
REVIEW
published: 02 December 2021
doi: 10.3389/fnmol.2021.759404
Edited by:
Joaquim Egea,
Biomedical Research Institute
of Lleida, Spain
Reviewed by:
Aurnab Ghose,
Indian Institute of Science Education
and Research, Pune, India
Carsten Theiss,
Ruhr University Bochum, Germany
*Correspondence:
Carlos Sánchez-Huertas
chuertas@umh.es
Specialty section:
This article was submitted to
Molecular Signalling and Pathways,
a section of the journal
Frontiers in Molecular Neuroscience
Received: 16 August 2021
Accepted: 01 November 2021
Published: 02 December 2021
Citation:
Sánchez-Huertas C and Herrera E
(2021) With the Permission
of Microtubules: An Updated
Overview on Microtubule Function
During Axon Pathfinding.
Front. Mol. Neurosci. 14:759404.
doi: 10.3389/fnmol.2021.759404
With the Permission of Microtubules:
An Updated Overview on Microtubule
Function During Axon Pathfinding
Carlos Sánchez-Huertas*and Eloísa Herrera
Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas-Universidad Miguel Hernández (CSIC-UMH),
Alicante, Spain
During the establishment of neural circuitry axons often need to cover long distances
to reach remote targets. The stereotyped navigation of these axons defines the
connectivity between brain regions and cellular subtypes. This chemotrophic guidance
process mostly relies on the spatio-temporal expression patterns of extracellular
proteins and the selective expression of their receptors in projection neurons. Axon
guidance is stimulated by guidance proteins and implemented by neuronal traction
forces at the growth cones, which engage local cytoskeleton regulators and cell
adhesion proteins. Different layers of guidance signaling regulation, such as the cleavage
and processing of receptors, the expression of co-receptors and a wide variety of
intracellular cascades downstream of receptors activation, have been progressively
unveiled. Also, in the last decades, the regulation of microtubule (MT) assembly, stability
and interactions with the submembranous actin network in the growth cone have
emerged as crucial effector mechanisms in axon pathfinding. In this review, we will
delve into the intracellular signaling cascades downstream of guidance receptors that
converge on the MT cytoskeleton of the growing axon. In particular, we will focus on
the microtubule-associated proteins (MAPs) network responsible of MT dynamics in the
axon and growth cone. Complementarily, we will discuss new evidences that connect
defects in MT scaffold proteins, MAPs or MT-based motors and axon misrouting during
brain development.
Keywords: microtubules, microtubule-associate proteins, growth cone, neuronal cytoskeleton, axon guidance
and pathfinding, +TIP
INTRODUCTION
The navigation of neural axons to find appropriate synaptic partners is one of the most
extraordinary events that take place during the development of the nervous system. Axon extension
is led by an amoeboid-like cytoplasmic enlargement at the tip, denominated the growth cone (GC).
This is a small but extremely dynamic and sensitive cellular structure that integrates extracellular
guidance information and transduces the mechanical forces necessary for the steering and
propulsion movements during axonal navigation. At the leading edge, the growth cone is composed
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
of motile sheet-like lamellipodia and narrow filopodia that are
sensitive to external guidance cues or ligands because they express
specific receptors at the surface. Ligand-receptor signaling
activates intracellular transduction pathways that primarily
converge over growth cone cytoskeleton remodeling, which in
coordination with substrate adhesions turnover and membrane
trafficking, orchestrates the steering movements of the axon
(Geraldo and Gordon-Weeks, 2009;Lowery and Vactor, 2009;
Vitriol and Zheng, 2012;Kerstein et al., 2015).
The highly conserved collection of axon guidance proteins
consists of attractive/repulsive membrane-anchored and secreted
molecules. Five large families of canonical guidance proteins
have been identified: netrins that signal through the deleted in
colorectal cancer (DCC), Neogenin and UNC-5 receptors; Slits,
that bind to their roundabout (Robo) receptors; Semaphorins,
that activate both Neuropilin and Plexin receptors; Ephrins
and Ephs; and Repulsive Guidance Molecule family (RGMs)
that bind to Neogenin. Besides these initially identified families
of guidance proteins, cell-adhesion molecules, growth factors
and morphogens, such as the Wnts, Sonic hedgehog (Shh),
TGF-β/BMP, neurotrophins or endocannabinoids have been
implicated in axonal navigation (Kolodkin and Tessier-Lavigne,
2011;Yam and Charron, 2013;Zhou et al., 2014;Short et al.,
2021). These guidance ligand-receptor modules have been
identified and reported as essential for the formation of the
commissural tracts in the spinal cord and forebrain, the
retinotopic maps, the thalamocortical connections or the sensory
motor innervation of the limbs, among other systems (Chedotal
and Richards, 2010;Leyva-Díaz and López-Bendito, 2013;
Chédotal, 2019;Herrera et al., 2019a).
During pathfinding GCs are simultaneously exposed to
various signaling proteins and the final guidance decision
relies on the spatial-temporal repertoire of guidance receptors
expressed at the surface and the computation of their
downstream signaling pathways. In addition, neuron-intrinsic
molecular mechanisms including response-modulating co-
receptors, receptor-receptor interactions, receptor clustering and
oligomerization, proteolytic processing of receptors, or the
trafficking of signaling receptors-carrying endosomes, contribute
to the diversification of axonal responses to a same guidance
cue (Dudanova and Klein, 2013;Pasterkamp and Burk, 2021;
Zang et al., 2021). These intracellular pathways ultimately
converge on proteins managing the cytoskeleton remodeling in
the axon and GC.
The major networks constituting the mature neuronal
cytoskeleton are formed by microtubules (MTs), actin fibers
(F-actin) and neurofilaments, but axon pathfinding is mainly
governed by F-actin and MTs acting coordinately in response
to extracellular guidance signaling (Geraldo and Gordon-Weeks,
2009;Lowery and Vactor, 2009;Dent et al., 2011;Coles and
Bradke, 2015). Seminal studies on the effects of F-actin disrupting
drugs over invertebrate neurons in culture, revealed the critical
role of the actin cytoskeleton in filopodia maintenance, GC
turning and axonal pathfinding (Bentley and Toroian-Raymond,
1986;Zheng et al., 1996). Thereafter, an intricate network of
actin-binding and regulatory proteins mediating the axonal
response to guidance molecules has been progressively disclosed
[see Table 1 in Dent et al. (2011) and Kolodkin and Pasterkamp
(2013) for detailed information on actin-binding proteins
steering the GC]. Most axon guidance pathways engage the Rho
family of small GTPases, mainly represented by RhoA, Rac1
and Cdc42, via their activating guanine nucleotide exchange
factors (RhoGEFs) and deactivating GTPase activating proteins
(RhoGAPs) (Hall and Lalli, 2010). RhoGTPases, in turn, drive
the activity of F-actin regulators, such as the nucleating Arp2/3
complex (Strasser et al., 2004;Shakir et al., 2008), the WASP
nucleation factors (Shekarabi et al., 2005;Shakir et al., 2008),
the polymerization regulators formins or the Ena/VASP family
(Goode and Eck, 2007), the molecular motor Myosin II (Amano
et al., 1998;Medeiros et al., 2006) or the severing protein
ADF/cofilin (Kuhn et al., 2000), to modulate the actin-based
filopodia and lamellipodia dynamics. The essential contribution
of actin dynamics to axon guidance signal transduction in the
GC has been reviewed recently by other authors (Omotade et al.,
2017;Niftullayev and Lamarche-Vane, 2019) and, therefore, will
not be the main focus of this review. For years, the long-standing
view in the field was that the GC turns as a result of the
stabilization/destabilization balance of the actin-rich filopodia
and lamellipodia in the presence of a guidance cue and the MT
cytoskeleton just provided structural support via MT-dependent
transport to consolidate the actin-dependent turning events.
However, the role of MTs in cellular functioning is continuously
expanding and accumulating evidence indicate that MTs are
not just passive regulators of GC dynamics. Instead, MTs can
actively control GC protrusion and steering and, along with MT-
associated proteins (MAPs), are direct targets of axon guidance
signaling pathways (Gordon-Weeks, 2004;Dent et al., 2011;
Kalil et al., 2011;Liu and Dwyer, 2014;Bearce et al., 2015;
Cammarata et al., 2016;Kahn and Baas, 2016).
In the first part of this review we provide an overview on the
configurations of the MT cytoskeleton in the axon and growth
cone and describe the cytoskeletal mechanisms that drive GC
directional responses, focusing on the contribution of MTs. In
the second half of this article, we highlight recent evidences
suggesting that guidance cues directly control the activity and
localization of MT-associated proteins (MAPs) and discuss how
alterations in genes encoding MT network regulators may cause
an abnormal development of neural networks in vivo.
THE NEURONAL MICROTUBULE
CYTOSKELETON
Microtubules are hollow cylindrical structures composed of 13
laterally-associated protofilaments of α-tubulin and β-tubulin
heterodimers assembled in a head-to-tail manner, conferring
an intrinsic polarity characterized by a stable/slow-growing
“minus-end” and a dynamic/fast-growing “plus-end” (Desai and
Mitchison, 1997). In eukaryotic cells, the MT nucleation – the
de novo MT formation from its minus end – is initiated by the
γ-tubulin ring complex (γTuRC) in cooperation with additional
proteins that regulate MT-nucleation kinetics. MT nucleation
events are spatially restricted to MT-organizing centers (MTOC),
which concentrate the γ-TuRCs, and the centrosome is a major
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MTOC in animal cells (Paz and Lüders, 2018). MT minus-ends
are stabilized by a γTuRC cap (Wiese and Zheng, 2000) or by
calmodulin-regulated spectrin-associated proteins (CAMSAPs)
(Jiang et al., 2014). Instead, the MT plus-ends are more dynamic,
alternating polymerization and shrinkage phases (catastrophes), a
property commonly referred to as “dynamic instability” (Walker
et al., 1989;Tran et al., 1997;Zhang et al., 2015). These MT-
intrinsic dynamic plus-end transitions between growth and
shrinkage can be externally regulated by the activity of other
MAPs, that control the supply of soluble tubulin-heterodimers,
the speed and duration of the polymerization/depolymerization
events and the plus-ends resilience to collapse (reviewed in
Akhmanova and Steinmetz, 2015).
At the onset of neuron differentiation, MTs nucleation
takes place mainly at the centrosome. To meet axon growth
needs, MTs are subsequently released from the centrosome,
sorted into the axon and anterogradely transported by means
of molecular motor forces (Kapitein and Hoogenraad, 2015).
During maturation the neuron centrosome progressively loses
its MT-nucleating and MT-organizing skills to such an extent
that axonal MT growth, axonal extension or overall neural
development do not require a centrosome (Basto et al., 2006;
Stiess et al., 2010;Nguyen et al., 2011). Indeed, during the last
years centrosome-independent MT nucleation activities has been
identified within the axon and dendrites of mammalian and
invertebrate neurons (Stiess et al., 2010;Ori-McKenney et al.,
2012;Nguyen et al., 2014;Sánchez-Huertas et al., 2016;Cunha-
Ferreira et al., 2018;Qu et al., 2019;Liang et al., 2020). MT
configurations display differently in axons and dendrites. In
mammalian neurons, MTs are uniformly oriented in the axons,
with their plus-ends toward the tip, while in dendrites MTs are
arranged with mixed polarity. The uniform MT plus-end-out
polarity of axons is mainly established and maintained by motor-
dependent MT sliding mechanisms (Miller and Suter, 2018) and
the spatial-temporal control of non-centrosomal MT nucleation
(Wilkes and Moore, 2020). For example, the cytoplasmic dynein
motor promotes bulk forward translocation of MTs into the axon
and clears the minus-end-out MTs from axons by soma-directed
sliding (Roossien et al., 2014;Rao et al., 2017). In addition, new
plus-end-out MTs are locally generated from the lateral surface
of pre-existing axonal MTs by the Augmin-γTuRC module
(Sánchez-Huertas et al., 2016;Cunha-Ferreira et al., 2018) and
TRIM46 bundles these plus-end-out MTs (van Beuningen et al.,
2015), to strengthen the axonal identity.
Neuronal MTs show specific physical and dynamic features
enabled by their differential tubulin isotype composition,
assorted post-translational tubulin modifications – including
tyrosination, acetylation or polyglutamylation – and a neuron-
specific MAP network unevenly distributed over the axonal and
somatodendritic compartments (Moutin et al., 2021). Within the
axon shaft, MTs are heavily stabilized and organized in dense,
parallel and overlapping bundles. This longitudinally aligned MT
network enable the directional transport of organelles, vesicles,
other MTs and cargoes along the axon, mediated by MT-based
motor proteins of the kinesin superfamily and cytoplasmic
dynein (Hirokawa et al., 2010;Leterrier et al., 2017). By virtue of
the differential MT layouts in neuron compartments, uniform in
axons and mixed in dendrites, specific motor-driven cargos are
selectively sorted, determining axonal specification, maturation
and navigation (Guillaud et al., 2020).
MICROTUBULE CYTOSKELETON IN THE
GROWTH CONE
At the axon tip, the growth cone (GC) can be subdivided
in several areas: a mobile peripheral (P) domain, containing
filopodia and lamellipodia, a central (C) domain and a transition
zone (TZ) in between. The cytoskeletal networks are organized
in a highly segregated fashion among these GC subdomains
(Figure 1A). The P-domain is mainly made of actin fibers
organized in dense bundles or loose F-actin meshworks,
originating the filopodia and lamellipodia, respectively. The
C-domain is populated by thickly bundled MTs, which are
continuously pushed by actin-based Myosin II-dependent
rearwards forces working at the TZ. Only isolated MTs can
pass this actomyosin-mediated barrier to MT assembly and
invade the actin-rich P-domain, where MTs display “dynamic
instability” (Figures 1A,B) (Letourneau, 1983;Forscher and
Smith, 1988;Dent and Kalil, 2001;Schaefer et al., 2002;Zhou
et al., 2002). F-actin bundles at the GC P-domain experience a
sustained retrograde flow (RF) product of: (i) the continuous
F-actin polymerization at the submembranous cortex, (ii) the
retrograde Myosin-dependent pulling forces and (iii) the F-actin
depolymerizing activity of ADF/Cofilin at the transition zone.
Actomyosin arcs contribute to MT bundling and advance into
the C-domain by exerting forces along the side of the neck
of the GC (Figure 1B) (Medeiros et al., 2006;Burnette et al.,
2008). In addition, once entering the P-domain, MTs tend to
unfasciculate, bend and loop due to a dynamic MT-F-actin
interplay enabled by MT-F-actin coupling proteins, including
MAPs or MT plus-tip interacting proteins (+TIPs) (Coles and
Bradke, 2015;Cammarata et al., 2016). This transient MT-F-actin
coupling mechanism mediated by MAPs enables MT capture and
guidance by F-actin bundles at the P-domain, but also exposes
MTs to the continuous F-actin retrograde flow, influencing the
orientation and speed of MT growth. This F-actin retrograde flow
drags MTs backward and clears the GC periphery of MTs, by
attenuating and buckling the MT trajectories. Opposing to these
retrograde forces over MT dynamics, the cytoplasmic dynein
motor exerts anterograde MT-sliding movements to introduce
MTs in the P-domain (Figure 1B) (Forscher and Smith, 1988;
Zhou et al., 2002;Suter et al., 2004;Myers et al., 2006;Grabham
et al., 2007;Schaefer et al., 2008;Marx et al., 2013). Therefore, MT
distribution in the GC is partially determined by F-actin network
at the same time that the F-actin-based filopodia and lamellipodia
dynamics is also known to be strongly influenced by MT capture
and stabilization at the GC periphery (Sabry et al., 1991;Tanaka
et al., 1995;Gallo, 1998). Such intense reciprocal regulation
between MT and F-actin networks in the GC highlights the
importance of an expanding family of MT-actin crosslinking
proteins in axon guidance decisions, but the precise nature of
MT-actin interlinking mechanisms remain to be elucidated.
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FIGURE 1 | The growth cone cytoskeleton. (A) High-resolution image of an axonal growth cone labeled with phalloidin (red) and α-tubulin (blue) from a hippocampal
neuron culture. Actin filaments (F-actin) and microtubules (MTs) are segregated amid the peripheral (P-domain) and central regions (C-domain), respectively.
Arrowhead marks isolated MTs invading the P-domain aligned with F-actin bundles. P-domain and C-domain are outlined using a dotted or dashed lines,
respectively. TZ, transition zone. Scale bar, 5 µm. (B) The clutch model for growth cone protrusion and steering. F-actin (red lines), microtubules (blue thick tubes),
MT plus-ends (+), point contacts of adhesion (green). Filopodia is formed by F-actin bundles and lamellipodia by F-actin meshworks. The F-actin retrograde flow at
the P-domain is balanced between F-actin polymerization and depolymerization rates, and myosin-based pulling forces at the TZ. Actomyosin forces at the TZ
restrain MT entry into the P-domain. The engagement of F-actin to adhesion point contacts slows the F-actin retrograde flow rate and facilitates MT invasion to the
growth cone periphery, determining outgrowth and steering.
Traction forces that propel axon outgrowth rely on clutching
forces exerted at substrate adhesion points, which generally
assemble within growth cone filopodia. At these point contact
adhesions, extracellular matrix (ECM) proteins activate integrin
receptors that recruit scaffolding and signaling proteins, which
physically link to the F-actin cytoskeleton (Suter et al., 1998;
Woo and Gomez, 2006;Bard et al., 2008;Shimada et al., 2008;
Myers and Gomez, 2011;Toriyama et al., 2013). This molecular
“clutch” restrains myosin-II mediated F-actin contractile forces
and increases the pushing forces of actin polymerization toward
the leading edge membrane, producing GC protrusion. Then,
taking advantage of the attenuated F-actin retrograde flow,
pioneer MTs invade the filopodia and eventually get captured and
stabilized. The stabilized MTs enable the entry of organelles and
vesicles to the GC periphery, powered by MT-based motor forces,
and the GC stepwise progresses toward the engorgement and
consolidation stages (Figure 1B) (Suter et al., 1998;Zhou et al.,
2002;Gomez and Letourneau, 2014;Kerstein et al., 2015). This
is the currently accepted mechanistic model for axon outgrowth,
although recent data has revealed some inconsistencies (Santos
et al., 2020;Turney et al., 2020). By analyzing GC protrusion
and axon growth over three-dimensional (3D) matrices, Santos
et al. (2020) showed that actomyosin forces do not restrain MTs
at the C-domain of GCs in this environment. Instead, MTs
widely populate the P-domain of the GCs, enabling a rapid
axon elongation. In addition, the authors showed that axons can
polarize and extend in adhesion-inert 3D matrices, suggesting
that cell adhesions may be dispensable for axon growth in a
3D environment (Santos et al., 2020). GC motility and axon
advance has also been analyzed over non-adhesive substrate gaps
in a 2D environment. This study revealed that axons transiently
stop at gaps, but GC protrusive activity continued, with MTs
entering the filopodium extending across the gap. These MTs
were powering the necessary molecular forces for GC to pass over
the non-adhesive substrate (Turney et al., 2020). Experimental
evidence has extensively validated the clutch hypothesis during
in vivo axon wiring (Berezin and Walmod, 2014), so this new
data suggests the existence of additional regulatory levels in the
mechanism of GC motility.
A classic study analyzing the in vivo GC morphology of
retinal axons performed in the 80’s described that retinal axons
display long and slender GCs when navigating the optic nerve,
while at the guidance decision point of the optic chiasm GCs
become shorter, wider and grow multiple filopodia (Bovolenta
and Mason, 1987). The study revealed that GC behaviors, and
the underlying mechanisms, can differ between bulk navigation
regions and guidance decision environments. This sets a
model out where GCs may either waive or activate substrate
adhesion mechanisms according to extracellular guidance factors
or neuron-intrinsic commands (Padmanabhan and Goodhill,
2018), integrating the a priori antagonistic evidences previously
exposed. However, further work is needed to fully understand the
mechanotransduction events during in vivo axon navigation.
Microtubules as the Mechanistic
Effectors of Growth Cone Turning
The reorganization of the GC cytoskeleton during axon steering
is ultimately enabled by a tight regulation of the MT-actin
interplay (Gordon-Weeks, 2004;Lowery and Vactor, 2009;Dent
et al., 2011). The targeting of the actin cytoskeleton, either by
downregulation of actin isoforms or by depolymerization of
F-actin using Cytochalasin B, reduces the size of the growth
cone, hinders filopodial dynamics and abolishes the axonal
turning response (Forscher and Smith, 1988;Zheng et al., 1996;
Moradi et al., 2017). Despite the requirement of F-actin for axon
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steering, it has been shown that neurons can still elongate their
axons after F-actin depolymerization or inhibition of myosin
II (Bentley and Toroian-Raymond, 1986;Zheng et al., 1996;
Bradke and Dotti, 1999;Hur et al., 2011b), indicating that F-actin
filaments remodeling is essential for motility and steering but
dispensable for axon extension. In contrast, MT dynamics is
necessary for axon growth (Yamada et al., 1970;Bamburg et al.,
1986;Mansfield and Gordon-Weeks, 1991;Tanaka et al., 1995;
Yu and Baas, 1995;Rochlin et al., 1996;Tischfield et al., 2010) but
the role of MTs in GC steering is yet a matter of discussion.
More than 30 years ago, pioneer studies already showed
that MTs asymmetrically invade the P-domain of the GC in
the direction of the turn and their reorganization is essential
during GC maneuvers (Forscher and Smith, 1988;Sabry et al.,
1991;Tanaka et al., 1995;Williamson et al., 1996). In addition,
MTs entering the filopodia were found to be captured and
stabilized by preventing shrinkage. These stabilized MTs allowed
the flow of cytoplasmic organelles and increased the filopodia
lifetimes, enabling directional axon outgrowth (Gordon-Weeks,
1991;Sabry et al., 1991;Geraldo et al., 2008). Ever since,
different works have demonstrated that axon guidance signaling
proteins influence MT dynamics at the GCs. For instance,
bath incubation of Sema3A and netrin-1 in neuron cultures
revealed that Sema3A treatments result in the collapse of MT
networks, while netrin-1 incubation stimulates MT splaying
and exploration in the GC peripheral region (Dent et al.,
2004;Shao et al., 2017). Nerve growth factor (NGF) signaling
in sensory neurons also redistributed the MTs in the distal
part of axons (Zhou et al., 2004;Turney et al., 2016), and
Wnt3a or Wnt5a treatments changed the organization and
directionality of MT polymerization in the GC (Purro et al., 2008;
Li et al., 2014). Micro-gradients of Brain-derived neurotrophic
factor (BDNF) or Sema3A over GCs of sensory axons also
biased the direction of MT growth toward the treated GC
side or the opposite side, respectively (Pavez et al., 2019).
In addition, genetic studies in Caenorhabditis elegans showed
that UNC-6/Netrin signaling hampers MT accumulation in the
GC and inhibits protrusion, through a molecular pathway that
involves the repulsive Netrin receptor UNC-5, RHO-1/RhoA
and UNC-33/CRPM (Gujar et al., 2019). Consistently with
these findings, the inhibition of MT dynamics blunts GC
turning in response to guidance cues. The exposure of GCs
and axons to drugs that disrupt MT dynamics, such as taxol or
nocodazole, reduced the GC activity and abolished the turning
of GCs exposed to netrin-1 and glutamate gradients or substrate
boundaries (Tanaka and Kirschner, 1995;Challacombe et al.,
1997;Buck and Zheng, 2002;Suter et al., 2004). Yet, because
the spatial distribution of MTs in the GC periphery is strongly
influenced by actomyosin forces and F-actin dynamics, MTs have
been classically relegated to simple supporters of actin-guided
movements during axon navigation.
Now, mounting evidences support the idea that MTs and
MAPs can also instruct axon pathfinding even when actin
dynamics are not directly perturbed. Seminal studies performed
20 years-ago showed that the focal application of the MT
stabilizing drug taxol to one side of the GC induces turning
toward the drug source, whereas the application of the MT
destabilizing drug nocodazole triggers GC turning away from the
application side (Buck and Zheng, 2002). Experimental evidence
also demonstrated that MT-initiated GC turning engages F-actin
remodeling during the movement, since the inhibition of actin
polymerization abolished the taxol-evoked attractive GC turning
(Buck and Zheng, 2002). Subsequent works employing micro-
scale chromophore-assisted laser inactivation (micro-CALI) have
also revealed that several MAPs can trigger GC steering. Micro-
CALI technique has been exploited to address the role of
specific proteins in GC steering (Chang et al., 1995;Diefenbach
et al., 2002), although it may have caveats, such as a rapid
recovery dependent on protein diffusion or trafficking. The
asymmetric inactivation of the MT-stabilizing protein MAP1B
or the Adenomatous polyposis coli (APC) protein – a +TIP
that stimulates MT polymerization – by micro-CALI in one
side of the GC, led to the collapse of the irradiated side
followed by GC turning toward the opposite direction (Mack
et al., 2000;Koester et al., 2007). In contrast, the asymmetric
inactivation of the MT extension-modulator CRMP2 or the
MT-sliding motor Kinesin-5 in half of the GC by micro-CALI,
resulted in GC turning toward the irradiated side (Higurashi
et al., 2012;Nadar et al., 2012). Overall, these experiments
support the idea that MT dynamics are not just required for
guidance-evoked GC steering movements, but they play an
instructive role in GC turning. Corroborating their instructive
role in axon guidance signal transduction, evidences from
Liu’s laboratory have proven that the neuron-specific tubulin
isotype TUBB3 – polymerized in MTs – is a direct target
of netrin-1 signaling (Qu et al., 2013;Huang et al., 2015;
Shao et al., 2017). Netrin-1 is a dual guidance cue that can
evoke attractive and repulsive responses by binding to its high-
affinity GC receptors DCC and UNC5, respectively (Alcántara
et al., 2000). It was reported that the exposure of cortical
neuron cultures to netrin-1 induces GC chemoattraction and
stimulates the MT dynamics in the GC. Both of these netrin-
1 effects required the direct interaction of its receptor DCC
with the neuron-specific tubulin isotype TUBB3, integrated in
the MT polymer. Indeed, the interaction of TUBB3 with DCC
was greatly increased by exogenous netrin-1 addition and this
interaction was dependent on MT dynamics (Qu et al., 2013).
Conversely, the GC repulsion induced by netrin-1 exposure
through UNC5C receptor signaling also relied on TUBB3-
UNC5C binding. UNC5C directly interacts with polymerized
TUBB3 in vitro and both partially colocalize in the GC periphery
of primary neurons. The focal application of Netrin-1 was found
to disengage UNC5C-TUBB3 interaction in GCs and stimulate
MT polymerization in the GC region distally to the netrin-1
source, promoting the repulsive response (Shao et al., 2017).
Missense mutations of TUBB3 in humans are associated to an
abnormal development of the corpus callosum, the anterior
commissure, the corticospinal tracts or optic nerves in human
patients (Table 1). Additionally, it was found that TUBB3
mutations impaired MT dynamics and abolished both netrin-
1-evoked attractive and repulsive responses of cortical axons
in vitro (Poirier et al., 2010;Tischfield et al., 2010;Whitman
et al., 2016;Huang et al., 2018;Shao et al., 2019). Although
the deficits caused by TUBB3 loss-of-function in neural circuits
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
TABLE 1 | Links of microtubule-associated proteins (MAPs) with axon guidance.
Roles in MT
networks
MAP Function on GC motility Axon tract development in
animal models
Guidance pathways
participated
Nerve tract associated
pathology in humans
Actin crosstalk
Structural TUBB3 Interaction with DCC or UNC5
increase/decrease upon netrin-1
signaling, interfering with MT dynamics
and promoting both the attractive and
repulsive responses of the GC.
Disease-associated Tubb3
mutant mice show abnormal
AC, CC and cranial nerves
(Tischfield et al., 2010;
Latremoliere et al., 2018)
Netrin-1-DCC (Qu et al.,
2013); Netrin-1-UNC5
(Shao et al., 2017)
CFEOM3. Defects in the
CC, AC or corticospinal
tracts. Asymmetric cortical
dysplasia and gyral
disorganization (Poirier
et al., 2010;Tischfield
et al., 2010)
Structural TUBA1A Loss-of-function hindered neurite
outgrowth in cortical neurons and
altered GC cytoskeleton
Tuba1a ko mice show
abnormal development of
forebrain commissures
(Buscaglia et al., 2020)
Abnormalities of the CC
and basal ganglia/internal
capsule. Lissencephaly and
other cortical and cerebellar
dysgenesis (Romaniello
et al., 2018)
Structural TUBB
(TUBB5)
Altered MT dynamics and MT-based
transport in patient’s fibroblasts
Hypoplasia or partial
agenesis of the CC, and
other cortical and cerebellar
dysgenesis (Romaniello
et al., 2018)
Nucleation
modulator
TPX2 Localizes to neurite tips together with
RanGTP to promote local MT
nucleation in hippocampal neurons
– –
Stability MAP1B Phospho-MAP1B stabilizes MTs at the
GC periphery. Phosphorylated by
GSK3βand CDK5 upon guidance
signaling
Map1b ko mice display
defective cortical and
thalamocortical wiring, and
CFEOM (Meixner et al., 2000;
Del Río et al., 2004;Cheng
et al., 2014)
Netrin-1 (Del Río et al.,
2004); Draxin-DCC (Meli
et al., 2015); Sema3A
(Takabatake et al., 2020)
White matter deficit,
hypoplasia of the CC
(Walters et al., 2018)
Binds F-actin in vitro.
Coordinates MTs and F-actin
remodeling in DRG GCs
(Villarroel-Campos and
Gonzalez-Billault, 2014)
Stability Tau Hyperphosphorylated tau detaches
from MTs and compromises MT stability
in the GC. Phosphorylated by GSK3β,
CDK5, or CaMKII upon guidance
signaling
No phenotype in tau ko mice
likely due to function
overlapping with MAP1B
Sema3A (Sasaki et al.,
2002); Wnt5a (Li et al.,
2014;Biswas and Kalil,
2018); EphrinB1-EphB2
(Jiang et al., 2015);
Sema3C (Moreno-Flores
et al., 2004)
Crosslinks MT-F-actin in vitro
(Elie et al., 2015).
Couples MT and F-actin in GCs
of cortical neurons (Biswas and
Kalil, 2018)
Polymerization/
stability
CRMP2 Non-phosphorylated CRMP2
transports tubulin heterodimers to distal
axons via kinesin-1, to support MT
growth. Phosphorylated sequentially by
CDK5 and GSK3βupon guidance
signaling
Crmp2 ko mice display
abnormal development of
peripheral nerves and CC (Ziak
et al., 2020)
Sema3A (Goshima et al.,
1995); Sema4D-plexinB1
(Ito et al., 2006): RGMa
(Wang et al., 2013);
EphrinA5 (Arimura et al.,
2005)
Binds the actin regulators:
cytoskeleton a2-chimaerin and
Sra-1/WAVE1 complex in
axons (Brown et al., 2004;
Kawano et al., 2005)
Stability DCX Stabilizes MT in the GC periphery.
Phosphorylated by CDK5 upon
Sema3A signaling, resulting in MT
destabilization
Dcx/Dclk1 ko mice show
widespread defects in brain
axon tracts (Deuel et al., 2006;
Koizumi et al., 2006)
Netrin-1 (Fu et al., 2013);
Sema3A (Bott et al.,
2020)
Lissencephaly and double
cortex syndrome (laminar
heterotopias) (Bahi-Buisson
et al., 2013)
Binds the actin-binding protein
Spinophilin to organize F-actin.
Coordinates MTs and F-actin in
GCs (Tsukada et al., 2005;Tint
et al., 2009)
(Continued)
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
TABLE 1 | (Continued)
Roles in MT
networks
MAP Function on GC motility Axon tract development in
animal models
Guidance pathways
participated
Nerve tract associated
pathology in humans
Actin crosstalk
Instability SCG10 Active (non-phosphorylated) SCG10
destabilizes MTs, stimulating MT
dynamics and promoting axon
outgrowth and regeneration
EphB (Suh, 2004);
Sema4D-PlexinB1?
(Oinuma et al., 2004;Li
Y.-H. et al., 2009)
– –
Severing Spastin Spastin isoform M1 represses BMP
guidance signaling during spinal motor
axon pathfinding in developing
zebrafish
BMP (Jardin et al., 2018) Hereditary spastic
paraplegia (Roll-Mecak and
Vale, 2008)
Severing Fignl1 Involved in spinal motor axons wiring
during zebrafish development
– –
Polymerization
inhibition
KIF21A Decreases MT polymerization and
suppresses catastrophes, modulating
the GC morphology, axon growth and
pathfinding
Mutant Kif21a mice show
defects in oculomotor nerves
development (Cheng et al.,
2014)
Sema3F (van der Vaart
et al., 2013)
CFEOM1 (Yamada et al.,
2003)
Binds and regulates the
localization of Kank1, an F-actin
polymerization inhibitor
(Kakinuma and Kiyama, 2009)
Pausing KIF21B Accumulates in MT plus-ends and acts
as autonomous pausing factor
Kif21b ko mice display thinner
CC (Kannan et al., 2017)
Agenesis of the CC and
microcephaly (Asselin et al.,
2020)
Associates with ELMO1, a
Rac1 regulator (Morikawa et al.,
2018)
Polymerization
inhibition
KIF2A Prevents MT overstabilization in the GC. Kif2a ko mice show aberrant
overextension of hippocampal
axons (Homma et al., 2003)
Malformations of cortical
development, including
microcephaly and gyration
phenotypes (Poirier et al.,
2013)
Transport Dynein
motor
complex
Retrograde transport of signaling
endosomes. Antiparallel MT sliding
NGF (Sainath and Gallo,
2015)
Polymicrogyria and
Charcot-Marie-Tooth
disease type2 (Poirier et al.,
2013)
Transport Kinesin-5 Antiparallel MT sliding. Blocks MT
invasion into the GC periphery and
determines GC turning. Required for
evoked-turning response
Microcephaly and
chorioretinopathy (Jones
et al., 2014)
Transport Kinesin-1
motor
complex
Axonal transport of CB1R in
hippocampal neurons
Klc1 ko mice show pathfinding
defects in corticofugal axons
(Saez et al., 2020)
Endocannabinoids (Saez
et al., 2020)
Kif5C: microcephaly,
gyration phenotypes and
white matter dysgenesis
(Poirier et al., 2013;Michels
et al., 2017)
Transport KIF13B Transports the F-actin-based motor
Myosin X and its cargo DCC
anterogradely along axons upon
guidance signaling
Netrin-1-DCC (Yu et al.,
2020)
– –
Transport KIF1BP – Kif1bp ko mice show defects in
the anterior commissure and
sympathetic innervation, but
not in CC (Hirst et al., 2017)
Microcephaly, peripheral
neuropathy
(Goldberg-Shprintzen
syndrome) (Drévillon et al.,
2013)
(Continued)
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
TABLE 1 | (Continued)
Roles in MT
networks
MAP Function on GC motility Axon tract development in
animal models
Guidance pathways
participated
Nerve tract associated
pathology in humans
Actin crosstalk
Transport KIF1BβAxonal transport of IGF1R to mediate
IGF-1-induced axon growth
Kif1b ko mice show abnormal
development of the CC (Zhao
et al., 2001)
IGF1-IGF1R (Xu et al., 2018) Charcot-Marie-Tooth disease
type 2A (Zhao et al., 2001)
Polymerization/
scaffold
EB1, EB3 Guidance signaling instructs the
asymmetric invasion of EB-labeled MT
plus-ends or the MT polymerization
dynamics
Sema4D-plexin (Laht et al.,
2012, 2014); SDF1-CXCR4 via
EB1/Drebrin module (Shan
et al., 2021); BDNF, Sema3A
via EB3/STIM1 module (Pavez
et al., 2019)
EB3/drebrin coordinates MT-actin and
regulates F-actin dynamics (Geraldo
et al., 2008;Mizui et al., 2009;Mikati
et al., 2013;Grintsevich and Reisler,
2014)
Stability CLASP Phosphorylation by Abl and GSK3βupon
guidance signaling determines MT
plus-end binding
Slit-Robo (Lee et al., 2004).
PDGF (Engel et al., 2014).
Binds F-actin in vitro and regulates
F-actin networks in sensory GCs (Marx
et al., 2013)
Stability/
RNA transport
APC Asymmetric accumulation of APC in the
GC anticipates the steering movement.
Guidance signaling modulates APC MT
plus-end binding via PI3K-GSK3βactivity
Apc ko mice show widespread
white matter defects (Yokota
et al., 2009)
NGF (Zhou et al., 2004;Villarin
et al., 2016); Wnt3a (Purro
et al., 2008)
Regulates mDia and IQGAP1 (Watanabe
et al., 2009;Okada et al., 2010).
Required for MT-dependent F-actin
assembly in hippocampal GCs (Efimova
et al., 2020)
Stability APC2 Defines the guidance of retinal ganglion
cell axons at the chiasm midline
EphrinA2 (Shintani et al.,
2009); Wnt5a (Morenilla-Palao
et al., 2020)
Regulates actin dynamics through the
formin DIA in Drosophila (Zhou et al.,
2011)
Crosslink/
arrangement
MACF1 Links MTs and F-actin. Coordinates MTs
and F-actin interaction to organize the
axonal cytoskeleton
Midline axon guidance in flies
(Lee et al., 2007). Macf1 ko mice
show widespread white matter
defects (Chen et al., 2006;Ka
and Kim, 2016)
Wnt-βcatenin (Chen et al.,
2006)
Thin CC and AC, with
lissencephaly (Dobyns et al.,
2018)
Binds, stabilizes and organizes F-actin
configurations (Kodama et al., 2003)
Stability/
crosslink
NAV1 Stabilizes paused MT plus-ends. Couples
MTs and F-actin in the GC of
hippocampal neurons
Netrin-1 (Martínez-López et al.,
2005;Sánchez-Huertas et al.,
2020)
Binds F-actin in vitro, crosslinks
MT-F-actin. Recruits the Trio to MT
plus-ends (van Haren et al., 2014;
Sánchez-Huertas et al., 2020)
Polymerization/
nucleation
modulator
XMAP215 Promotes MT entry in filopodia, regulates
GC morphology and axon outgrowth in
Xenopus neurons
EphrinA5 (Slater et al., 2019) Co-aligns MTs and F-actin in GCs (Slater
et al., 2019)
Polymerization TACC3 Forms a complex with XMAP215.
Phosphorylated by Abl.
Phospho-mutants interfere with axon
pathfinding
Slit2, EphrinA5 (Erdogan et al.,
2017, 2020)
– –
Crosslink Gas2L1 Regulates axon outgrowth and branching Stabilizes F-actin upon MT-F-actin
interaction (Willige et al., 2019)
Stability/
crosslink
DAAM Actin assembly factor involved in axon
growth and guidance. Regulates GC
filopodia dynamics also via interaction
with +TIPs
Wnt5 (Gombos et al., 2015) Crosslinks MT and F-actin in vitro and
coordinates the GC cytoskeleton in
Drosophila neurons (Szikora et al., 2017)
Stability/
crosslink
mDia1,
mDia3
Actin assembly factor involved in axon
growth and guidance. Binds and
stabilizes MTs
Double mDia ko mice show
midline crossing defects in the
spinal cord (Toyoda et al., 2013)
EphrinA5, EphrinB3, Sema3A
(Toyoda et al., 2013); SDF1-α
(Arakawa et al., 2003)
Play dual roles in actin and MT dynamics
(Thurston et al., 2012)
Stability/
crosslink
FMN2 Enables MT capture by F-actin bundles
and focal adhesion-based traction in
filopodia
Fmn2 depletion impairs midline
crossing in chick spinal cord
(Sahasrabudhe et al., 2016)
Wnt (Lian et al., 2016) Couples MTs and F-actin in GCs (Kundu
et al., 2021)
MT, microtubules; F-actin, actin fibers; GC, growth cone; +TIPS, MT plus-end interacting proteins; CFEOM, congenital fibrosis of the extraocular muscles; CC, corpus callosum; AC, anterior commissure; GC, growth
cone; NGF, ner ve growth factor; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; DRG, dorsal root ganglia.
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
development may be compensated by the remaining β-tubulin
isotypes (Latremoliere et al., 2018).
Other mutations in human α-and β-tubulin-encoding genes –
such as TUBA1A, TUBB2B, TUBA8, TUBB4A, TUBB2A, TUBB
– are linked to severe brain malformations and motor-
cognitive disabilities, collectively refereed as tubulinopathies.
These syndromes present gross brain malformations and an
abnormal development of various nerve tracts, suggesting a
putative role in axon guidance (Romaniello et al., 2018).
Recent analysis performed on TUBA1A loss-of-function mice
and cultured fibroblasts from TUBB-associated tubulinopathy
patients have revealed an impaired MT dynamics and aberrant
cytoskeleton configurations in the axonal GCs (Buscaglia et al.,
2020;Sferra et al., 2020). However, it remains to be uncovered
whether and which guidance molecules are involved in these
MT-associated axon tract malformations.
MICROTUBULE-ASSOCIATED
PROTEINS IN AXON GUIDANCE
The assembly, stability and remodeling of MT networks
during axon navigation mostly relies on the localization and
activity of a wide range of MAPs located in the axon and
GC compartments. MAPs manage many aspects of the MT
cytoskeleton, including the spatial-temporal control of MT
nucleation, polymerization, depolymerization, stability, pausing,
bundling, severing, trafficking or interaction with other cellular
structures (Goodson and Jonasson, 2018). Therefore, MAPs play
a pivotal role in the transduction of attractive and repulsive
guidance signaling over MT dynamics in axons and growth
cones. Consistently, mutations in human MAP-coding genes
have been associated to a wide spectrum of neurodevelopmental
disorders linked to axon misrouting (Poirier et al., 2013;Chilton
and Guthrie, 2017;Lasser et al., 2018;Romaniello et al., 2018).
In this section we will summarize the intracellular pathways
downstream axon guidance signaling that directly control the
activity, localization or expression of MAPs to achieve GC
protrusion and steering, as well as MAPs requirement for axon
tract development in vivo. For clarity, we have classified the
MAPs as: (1) MT-nucleation MAPs, (2) MT-stabilizing and
polymerization-supporting MAPs, (3) MT-severing, destabilizing
and polymerization-inhibitory MAPs, (4) MT-tracking motor
proteins and (5) MT plus-tip interacting proteins (+TIPs).
Microtubule-Nucleation Microtubule-Associated
Proteins
In the shaft of cortical axons, MTs are formed de novo
nucleated – locally in an acentrosomal manner, by a mechanism
involving the Augmin/HAUS complex and the γ-tubulin ring
complex (γTuRC) that ensures the uniform polarity of the MT
network (Sánchez-Huertas et al., 2016;Cunha-Ferreira et al.,
2018). Although local events of MT nucleation have not been
yet reported in the axonal GCs, acentrosomal γTuRC-dependent
MT nucleation has been recently observed over endosomes in
the dendritic GCs of invertebrate neurons (Liang et al., 2020;
Yoong et al., 2020). These evidences, in combination with the
following recent findings, allow to put forward the hypothesis of
axon guidance signaling influencing local MT nucleation events
in the distal axon.
The γTuRC-dependent MT nucleation in eukaryotic cells
undergoes spatial and temporal regulation by means of additional
MAPs, such as TPX2 and its activator RanGTP, and both proteins
have been found to be enriched at neuritic tips (Chen et al.,
2017;Huang et al., 2020;Liu et al., 2021). MT-bound TPX2
participates in MT nucleation at elongating neurite tips in
cultured hippocampal neurons (Chen et al., 2017). RanGTP is
transported anterogradely along the axons through actin waves,
it colocalizes with actin-based structures in the axonal GC and
enables local nucleation events at neurite tips (Chen et al.,
2017;Huang et al., 2020). Actin waves (also known as growth
cone-like waves) are dynamic cytoskeletal structures traveling
anterogradely along the axon shaft. These waves are associated
to transient MT generation activity along the axons, including an
increase in MT polymerization and MT-based transport (Winans
et al., 2016). Therefore, it is possible that RanGTP and TPX2
are transported to the GC, jointly with other MT nucleation
machinery such as γ-TuRCs, to trigger local short-lived MT
nucleation events.
New results also suggest that Wnt signaling could shape
axonal MT configurations via regulation of local MT nucleation
mechanisms. Weiner et al. (2020) showed that in Drosophila,
some Wnt signaling proteins, such as Fz, LRP5/6 or Axin,
recruit the MT core-nucleation protein γ-Tubulin to endosomes
in the dendritic branch points, enabling local MT nucleation
and indicating that extracellular Wnt signaling can regulate
local MT nucleation in dendrites. In addition, two other recent
studies have revealed that the Wnt pathway controls axon
specification in developing neurons by organizing the polarity
of MT networks both in the axon (Stanganello et al., 2019)
and in non-axonal neurites (Puri et al., 2021). Plus, it is known
that local MT nucleation contribute the MT arrangements
in these compartments (Sánchez-Huertas et al., 2016;Cunha-
Ferreira et al., 2018). Overall, these results convey a putative
mechanism whereby extracellular Wnt signaling might control
MT architecture in axons and dendrites via spatial-temporal
control of MT nucleation in developing neurons. Hence, we
believe that the contribution of local MT nucleation events in
distal axons to guidance cue-instructed navigation should be
further investigated.
Microtubule-Stabilizing and
Polymerization-Supporting Microtubule-Associated
Proteins
Microtubules are heavily stabilized in the axonal shaft, whereas
in the GC they are very dynamic. The stability status and
polymerization rate of MTs in the axons rely on the activity
of specific MAPs, such as MAP1B, tau or CRMP2, whose
activities are directly regulated by axon guidance signaling
pathways (Figure 2). MAP1B is a MT-stabilizing protein that
associates with the lattice of dynamic MTs in the most distal
region of the axon and in the GC. Studies of asymmetric laser
inactivation in GCs together with genetic analyses revealed
that the phosphorylated form of MAP1B is a direct effector
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
FIGURE 2 | Guidance signaling downstream pathways involved in MT dynamics in the axon and GC I: MT-stabilizing, MT-destabilizing and MT-polymerization
supporters. Netrin-1-DCC signaling produces MT stabilization via MAP1B phosphorylation through GSK3 and CDK5 activity (Del Río et al., 2004). Draxin binds DCC
receptor and leads to MAP1B phosphorylation via GSK3β(Meli et al., 2015). Sema3A stimulates MAP1B mRNA local translation by promoting the
proteasome-dependent degradation of the repressor FRMP (Takabatake et al., 2020). Sema3A, EphrinA5, RGMa or Sema4D inhibit MT polymerization by increasing
CRMP2 phosphorylation via GSK3βand CDK5 (Arimura et al., 2005;Cole et al., 2006;Ito et al., 2006;Wang et al., 2013). Sema3A promotes MT destabilization by
promoting DCX-fall off the MT lattice via CDK5-dependent phosphorylation of DCX (Bott et al., 2020). The combined action of EphB, laminin and L1 leads to MT
overgrowth and buckling by reducing SCG10 protein levels (Suh, 2004). Sema3C increases tau protein levels (Moreno-Flores et al., 2004). EphrinB1-EphB2 signaling
reduces tau hyperphosphorylation via PI3K-dependent inhibition of GSK3 (Jiang et al., 2015). Wnt5a promotes MT redistribution by stimulating CaMKII-dependent
phosphorylation of tau at Ser262 (Li et al., 2014). Sema3A transiently increases tau phosphorylation at Ser202 and Thr205 via CDK5-dependent phosphorylation
(Sasaki et al., 2002). MTs are shown as light purple tubes, F-actin as red lines. MAPs are represented in blue, kinases in yellow and MAP-interacting proteins in
purple. Guidance cue receptors are in brown. Guidance-evoked responses are represented in green (attraction), red (repulsion) and orange (pause) arrows. MT
advance and retraction are represented with green and red arrowheads, respectively.
of axon turning because selectively stabilizes MTs at the GC
periphery (Black et al., 1994;Mack et al., 2000;Bouquet
et al., 2004). MAP1B phosphorylation levels are increased in
cortical neurons after Netrin1 treatment via GSK3βand CDK5
kinase activity. Consistently, growing axons from MAP1B-
deficient CNS explants are irresponsive to netrin-1-induced
chemoattraction. MAP1B mutant mice are viable but exhibit
misguided cortical, thalamocortical and hippocampal axons
(Table 1) (Meixner et al., 2000;Del Río et al., 2004). These
dramatic axon wiring defects suggest that MAP1B is involved
in additional axon guidance pathways, other than netrin-1.
Indeed, the repulsive axonal guidance responses evoked by
Draxin and Sema3A treatments also involve MAP1B in their
downstream pathways. Draxin, which is an essential guidance cue
for the development of forebrain commissural tracts, interacts
with the netrin receptor DCC and activates the GSK3β-MAP1B
pathway in order to induce a repulsive response in cortical axons
(Meli et al., 2015). On the other hand, Sema3A treatment of
hippocampal neurons increases MAP1B levels in distal axons
in a local translation-dependent manner (Campbell and Holt,
2001;Li C. et al., 2009). Specifically, Sema3A induces the
local degradation of the translational suppressor FMRP via the
ubiquitin-proteasome pathway, which results in the increase
of MAP1B mRNA-coding translation in the GC (Takabatake
et al., 2020). Thus, it appears that MAP1B is a downstream
mediator of both attractive and repulsive guidance cues. This
high degree of MAP1B tunability could be entailed by its multiple
phosphorylation sites (Kawasaki et al., 2018), sensitive to CDK5
and GSK3βactivity, but further work is needed to understand the
molecular mechanisms whereby MAP1B promotes GC steering.
Tau is another phospho-MAP that binds the MT lattice and
stabilizes the MTs in the axon shaft, playing a critical role in axon
specification, growth and branching. Tau hyperphosphorylation
generally correlates with impaired MT binding and axonal MT
cytoskeleton disruption (Cleveland et al., 1977;Binder et al., 1985;
Drubin and Kirschner, 1986;Grundke-Iqbal et al., 1986). Similar
to MAP1B, Tau is a downstream target of GSK3βand CDK5
- among other kinases (Guo et al., 2017). In particular, it was
found that Sema3A treatment transiently increases phospho-tau
levels in the GC of chick neuron cultures via CDK5-dependent
phosphorylation previously to GC collapse (Sasaki et al., 2002).
Also, Wnt5a promotes the reorganization of MTs in the GC of
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
cortical neurons through CaMKII-dependent phosphorylation
of tau within its MT-binding site (Ser262) in order to evoke
a repulsive axonal response (Li et al., 2014). In opposition to
this, the activation of EphB2 receptor by ephrin B1 reduces tau
hyperphosphorylation through GSK3βinhibition in vivo in the
CA3 hippocampal region of tau transgenic mice (Jiang et al.,
2015). Moreover, Sema3C addition upregulates the total tau
protein levels in cultured cerebellar granule neurons, preserving
survival and stimulating neuritogenesis (Moreno-Flores et al.,
2004). Interestingly, it was uncovered that tau promotes the
co-alignment of MT and actin fibers in vitro, and stimulates
the coordinated polymerization of both cytoskeleton networks
(Elie et al., 2015). In line with this, it was recently reported
that tau does not only decorates the lattice of stabilized MTs
along the axon shaft but also associates to dynamic MTs aligned
with actin filaments in the GC periphery of cortical neurons.
Tau downregulation disrupted the MT bundling in the GC
central domain, prevented MT invasion into the periphery
and misoriented MT trajectories. Overall, tau loss-of-function
inhibited the turning of cortical axons exposed to Wnt5a
gradients (Biswas and Kalil, 2018).
The collapsin response mediator proteins (CRMPs) family
are cytosolic phospho-MAPs that play important roles in the
developing nervous system, including axon guidance (Nakamura
et al., 2020). CRMP family name was given because its first
member identified, CRMP2, was a molecular mediator of GC
collapse upon stimulation with Sema3A (originally known
as Collapsin) (Goshima et al., 1995). There are five human
CRMPs (CRMP1-5), displaying different subcellular localization
and cytoskeletal targets. Among them, CRMP2 localizes to
the axon and the C-domain of the GC and controls MT
polymerization/stability. Indeed, it has been observed that
CRMP2 participates in axon specification, elongation, branching
and guidance effect by several guidance cues (Inagaki et al., 2001;
Lin et al., 2011;Higurashi et al., 2012;Yamashita and Goshima,
2012). When CRMP2 monomers are non-phosphorylated, they
bind tubulin heterodimers and the complex is transported to
the distal part of growing axons, by kinesin-1-dependent motor
forces, to support MT polymerization and axon growth. Upon
Sema3A stimulation, CRMP2 is sequentially phosphorylated
at its C-terminal domain by CDK5 and GSK3βkinases,
hampering its tubulin-binding properties and leading to GC
collapse via MT destabilization (Figure 2). The Sema3A-induced
CRMP2 inactivation is achieved by phosphorylation at Ser522
by CDK5, followed by GSK3β-dependent phosphorylation at
Ser518, Thr514 and Thr509 (Fukata et al., 2002;Kimura et al.,
2005;Cole et al., 2006). In addition to Sema3A, other repulsive
guidance cues induced CRMP2 phosphorylation via GSK3 and/or
Rho kinase to achieve GC collapse, these include Sema4D, RGMa
and ephrinA5 (Arimura et al., 2005;Ito et al., 2006;Wang
et al., 2013). Consistently, CRMP2 has been demonstrated to be
essential for axon navigation in vivo because CRMP2KO mice
exhibit axon guidance defects in peripheral nerves and in the
corpus callosum (Ziak et al., 2020).
Mutations in the genes encoding the MAP doublecortin
(DCX) account for the majority of the human cases of
double cortex syndrome, which exhibits severe brain cortex
malformations primarily attributable to neuronal migration and
proliferation deficits (Gleeson et al., 1998;Bahi-Buisson et al.,
2013). DCX is a MT-stabilizing phospho-protein abundant in
the axonal GCs, which decorates the lattice of MTs invading the
F-actin rich peripheral region of the GC (Moores et al., 2004;Tint
et al., 2009). Interestingly, the double genetic deletion of DCX and
its closest homolog protein doublecortin-like kinase1 (DCLK1)
in mice led to widespread defects in axon tracts, affecting the
corpus callosum, anterior commissure, subcortical fiber tracts
and internal capsule. More specifically, the DCX mutant axons
exhibit impaired transport, growth and are irresponsive to netrin-
1-evoked chemoattraction, although the latter was suggested
to stem from DCX regulatory effects on actin configurations
(Deuel et al., 2006;Koizumi et al., 2006;Fu et al., 2013). This
data suggests that DCX is required for guidance signaling-
evoked axonal steering during nervous system development.
Indeed, a recent study uncovered that DCX mediates the
repulsive response of GCs upon Sema3A treatment (Bott et al.,
2020). Bott et al. (2020) showed that DCX forms a complex
with Nestin that enables DCX phosphorylation by CDK5/p35
downstream Sema3A signaling. They also demonstrated that
DCX phosphorylation by CDK5 decreased its MT affinity and
resulted in MT destabilization.
Microtubule-Destabilizing, Severing and
Polymerization-Inhibitory Microtubule-Associated
Proteins
In addition to MT polymerization and stability, MT
depolymerization and severing are also critical mechanisms
for the arrangement of MT networks. Several of these MAPs
have been involved in the transduction of axon guidance
signaling. SCG10 (superior cervical ganglion-10)/Stathmin-2 is a
neuron-specific member of the MT-destabilizing protein family
of the stathmins. Stathmins bind tubulin dimers, sequestering
them from growing plus-ends and thereby, promoting MT
depolymerization (Charbaut et al., 2001;Grenningloh et al.,
2004). SCG10 is considered an axon survival protein, highly
enriched in the GCs C-domain of developing neurons, and
its levels are dynamically regulated by local degradation and
KIF1B-dependent axonal transport toward the GC (Shin et al.,
2012;Drerup et al., 2016). Axon extension during neuron
differentiation requires SGC10 activity, since its downregulation
produces MT overstabilization and looping in the GC of
hippocampal neurons (Morii et al., 2006). The repulsive protein
EphB typically triggers GC collapse, but in the presence of
laminin and L1 leads to paused GCs, which retain their normal
filopodial dynamics and actin distribution. It was found that
this guidance cue combination specifically reduced SCG10 levels
in GC, which stimulated the invasion of long curved MTs into
the GC periphery and led to GC pause (Figure 2) (Suh, 2004).
Additionally, SCG10 interacts with the small RhoGTPase Rnd1,
and this interaction enhances SCG10 MT destabilizing activity in
neurons. Rnd1 is known to mediate the GC collapse induced by
Sema4D-Plexin-B1 signaling in hippocampal neurons (Oinuma
et al., 2004;Li Y.-H. et al., 2009), suggesting that SCG10 may also
function downstream of the Sema4D signaling pathway.
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On the other hand, the MT-severing enzymes cut MT
fibers into shorter fragments, creating new local MT seeds and
influencing axon branching (Sharp and Ross, 2012). Spastin
is a MT-severing protein required for axon morphogenesis,
associated to a degenerative disease of the corticospinal axon
tracts, named Hereditary spastic paraplegia (Roll-Mecak and
Vale, 2008). Recently, the alternative translation of spastin
mRNA transcripts has been found to influence both motor
neuron axon guidance and migration downstream of bone
morphogenic protein (BMP) and neuropilin-1 signaling during
zebrafish development (Jardin et al., 2018). Fidgetin-like-1
(Fignl1) is another MT-severing protein enriched in the growth
cone of zebrafish growing axons, whose downregulation led to
pathfinding defects in spinal motor axons and impaired larvae
locomotion (Fassier et al., 2018), although no specific guidance
proteins controlling Fignl1 activity have been identified.
Concerning MT polymerization inhibitors, the kinesin-
4 family members KIF21A and KIF21B and the immotile
kinesin-13 family member KIF2A, have been linked to
neurodevelopmental malformations associated with axon
growth and guidance defects in human patients (Table 1)
(Yamada et al., 2003;Poirier et al., 2013;Asselin et al., 2020).
More specifically, KIF21A is a gene risk factor for the CFEOM1
(congenital fibrosis of the extraocular muscles type-1), a
developmental oculomotor nerve disorder. CFEOM1-associated
Kif21a mutations in mice caused aberrant axon branching,
stalling and misorientation defects in oculomotor nerves
(Yamada et al., 2003;Cheng et al., 2014). It was reported that
KIF21A decreases MT polymerization rate and suppresses MT
plus-end catastrophes. KIF21A overexpression in hippocampal
neurons slendered the GC morphology, stimulated axon growth
and suppressed the repulsive axonal response to Sema3F (van
der Vaart et al., 2013). In turn, KIF2A has been proposed to
regulate axon pruning by preventing MT overstabilization in the
GC. It was found that Kif2a/mice exhibit an aberrant axonal
overextension in hippocampal neurons, due to reduced MT
depolymerization in the GCs (Homma et al., 2003;Maor-Nof
et al., 2013).
Microtubule-Tracking Motor Proteins
As aforementioned, dynein-driven motor forces facilitate the
entry of MTs into the GC periphery, influencing neurite
initiation, axon outgrowth and steering (Dehmelt et al., 2006;
Myers et al., 2006;Grabham et al., 2007). In support of dynein’s
role in guidance-evoked GC movements, dynein loss-of-function
experiments using RNAi or Cilibrevin D revealed an impairment
in NGF-evoked filopodia formation and in GC turning over
substrate boundaries. However, both dynein-driven MT-sliding
into the GC periphery or MT-based retrograde transport of
signaling endosomes could contribute to these instructed axon
movements (Myers et al., 2006;Sainath and Gallo, 2015).
Likewise, MT-based kinesin-dependent anterograde transport is
necessary for axonal extension and steering. For instance, the
MT-sliding activity of kinesin-5 – also called Eg5 or kif11 –
inhibits the MT invasion into the GC periphery and it is required
for GC turning in response to repulsive substrate boundaries. It
was found that an asymmetric accumulation in the GC of the
phosphorylated form of kinesin-5 precedes turning, and its acute
inactivation in one side of the GC elicits the MT invasion into the
hampered side and GC turning (Nadar et al., 2008, 2012).
A recent study has pinpointed the kinesin KIF13B as the
molecular motor responsible of Myo X localization to axons upon
netrin-1 stimulation. Myo X is an actin-based motor protein that
transports lipids and transmembrane receptors, such as DCC,
to the filopodia tip during axon pathfinding. It was found that
netrin-1 signaling increases Myo X-KIF13B interaction and its
anterograde MT-dependent transport along the axons, in order
to stimulate axon initiation and axon branching in the cortical
commissural projections (Yu et al., 2020). The kinesin family
member 1 binding protein (KIF1BP) is also necessary for a proper
development of the anterior commissures and the sympathetic
innervation of the gut (Hirst et al., 2017). Mutations in the
Kif1βgene, associated to the Charcot-Marie-Tooth peripheral
neuropathy, have been found to prevent KIF1Bβbinding to the
insulin-like growth factor 1 (IGF1) receptor IGF1R, involved
in sensory axon guidance. These mutations blocked the MT-
dependent axonal transport of IGF1R and inhibited IGF1-evoked
axon outgrowth (Scolnick et al., 2008;Xu et al., 2018).
The kinesin-1 motor complex has also been suggested
to participate in the netrin-1-evoked repulsive response in
invertebrate motor axons and is a phosphorylation target
of GSK3β, a major transduction hub of various guidance
signaling pathways (Teulière et al., 2011;Banerjee et al., 2018).
Furthermore, mutations in gene encoding the subunit KIF5C
of the kinesin-1 complex (encoded by the Kif5 genes) have
been linked to an abnormal development of the axon tracts
of the corpus callosum and the internal capsule (Table 1)
(Poirier et al., 2013;Michels et al., 2017). The recent analysis
of a mutant mice lacking the kinesin-1 light chain KLC1 has
revealed hypoplasia of the internal capsule tract, that includes
corticofugal and thalamocortical axons. The innervation defects
were found to be caused by an impaired kinesin-1-dependent
axonal transport of the cannabinoid type-1 receptors (CB1R),
and the subsequent axon unresponsiveness to endocannabinoids
signaling (Saez et al., 2020).
Microtubule Plus-Tip Interacting Proteins (+TIPs)
Plus-end tracking proteins (+TIPs) regulate MT plus-end
polymerization and stability, and mediate interactions between
the MT ends and actin fibers, organelles and plasma membrane
(van de Willige et al., 2016). Evidences obtained during the
last 15 years have demonstrated that axon guidance signaling
pathways directly target via regulation of +TIPs’ activity and
localization (Figure 3) (Bearce et al., 2015;Cammarata et al.,
2016;Voelzmann et al., 2016).
Microtubule end-binding (EB) proteins are the most abundant
+TIPs in cells. EBs (EB1, EB2, and EB3) directly associate
with MT plus-ends through their N-terminal calponin homology
(CH) domain and are autonomous regulators of plus-end
dynamics. MT tip-tracking of EBs mainly correlates with
MT polymerization episodes, since it favors a continuous
polymerization and reduces the number of catastrophes.
Importantly, EBs and are also scaffold-providers for other
+TIPs through their C-terminal domain, and for this reason
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Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
FIGURE 3 | Guidance signaling downstream pathways involved in MT dynamics in the axon and GC II: +TIPs. SDF1/CXCR4 signaling activates the EB1/Drebrin
module for MT remodeling (Shan et al., 2021). Sema4D/plexin signaling inhibits EB3-labeled MT polymerization (Laht et al., 2012, 2014). BDNF and Sema3A
promote asymmetric MT invasion via STIM1-EB3 interaction (Pavez et al., 2019). NGF stimulates APC-dependent MT plus-end stabilization via local inhibition of
GSK3βactivity (Zhou et al., 2004). Wnt3a alters MT polymerization direction by misslocating APC from the MT plus-ends (Purro et al., 2008). Slit/Robo signaling
promotes MT growth arrest by dissociating CLASP from the MT plus-end via Abl-dependent CLASP phosphorylation (Lee et al., 2004). High GSK3 kinase activity
(poorly phosphorylated) dissociates CLASP from plus-ends, low GSK3 activity (highly phosphorylated) misslocates CLASP from plus-ends to the MT lattice,
moderate GSK3 activity allows CLASP plus-end binding, MT stabilization and growth (Hur et al., 2011a). MTs are shown as light purple tubes, F-actin as red lines.
+TIPs are represented in green, kinases in yellow, actin-interacting/regulatory proteins in orange and other +TIP-interacting proteins in pink. Guidance cue receptors
are in brown. Guidance-evoked responses are represented in green (attraction), red (repulsion) or orange (pause) arrows. Empty arrows were used when downstream
transduction pathways are unclear or guidance cues unknown. MT advance and retraction are represented with green and red arrowheads, respectively.
EBs are considered master regulators of +TIP network. EB1
protein is ubiquitous, whereas EB3 is predominantly expressed
by neurons, and both EB1 and EB3 are necessary for axon
extension (Akhmanova and Steinmetz, 2015;van de Willige et al.,
2016). Semaphorin4D can influence the EB3-labeled MT plus-
ends polymerization dynamics in hippocampal neurons, and it
was found that both EB1 and EB3 interact with the intracellular
domains of the Plexin-A2, Plexin-B1, and Plexin-B3 Semaphorin
family receptors (Laht et al., 2012, 2014). This data suggest that
the semaphorin-plexin-EB pathway may regulate MT dynamics
during axon pathfinding. When MT plus-ends enter the actin-
rich P-domain of GCs, EB3 recruits the F-actin-binding protein
Drebrin to couple growing MT tips to actin filaments. Hence, the
EB3-Drebrin module facilitates the invasion of exploratory MTs
into the GC periphery, and enables growth cone formation and
neuritic elongation (Geraldo et al., 2008). It has been recently
proposed that the EB1-Drebrin module at plus-ends interacts
with the chemokine receptor type 4 (CXCR4) upon stromal
cell-derived factor-1 (SDF-1) signaling. The chemokine SDF-1
can regulate axonal elongation and branching, and the SDF-
1/CXCR4/Drebrin/EB1 pathway appears to be critical for SDF-
1-induced MT cytoskeleton remodeling during neuronal motility
(Pujol et al., 2005;Shan et al., 2021). Furthermore, EB3 colocalizes
at the MT plus-ends within the GC filopodia with the stromal
interacting molecule (STIM1), which is a calcium-sensing protein
that mediates GC steering in response to various axon guidance
cues (Mitchell et al., 2012;Pavez et al., 2019). New data revealed
that EB3-STIM1 interaction at MT plus-ends is calcium-sensitive
and STIM1 instructs the asymmetric invasion of EB3-labeled
MT plus-end into the motile GC side downstream of BDNF or
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Sema3A signaling in sensory neuron cultures. Moreover, in vivo
experiments in zebrafish showed that the EB3-STIM pathway
regulates the axon guidance of spinal motor neurons (Pavez
et al., 2019). In addition, EB1/3 proteins can bind MAP1B and
Tau, and this interaction sequesters EBs from MT plus-ends
and jeopardizes MT growth (Tortosa et al., 2013;Sayas et al.,
2015). Because MAP1B and tau are downstream effectors of
several axon guidance pathways, their interaction with EBs could
indirectly influence the MT dynamics and the assembly of the
+TIP complex on account of its dependence on EB scaffold.
One of the pioneer studies that assigned +TIPs a prominent
role in axon guidance refers to the cytoplasmic linker associated
protein (CLASP) and the Slit-evoked repellent response (Lee
et al., 2004). CLASP decorates the MT plus-ends that polymerize
over F-actin bundles in the GC periphery, and its overexpression
causes MT overstabilization, looping and prevents their extension
beyond the transition zone (Hur et al., 2011a). Orbit/MAST, the
CLASP ortholog in invertebrates, is an Abelson tyrosine kinase
(Abl) target downstream of Slit/Robo signaling that mediates
repulsion. These evidences served the authors to propose that
focal Slit stimulation in one side of the GC provokes asymmetric
activation of the Abl-CLASP pathway and MT growth arrest,
entailing a GC movement away of the source of Slit (Lee
et al., 2004). CLASP is recruited to MT plus-ends through
EB binding, although it contains tumor overexpressed gene
(TOG) domains which can serve as tubulin-binding modules
(Mimori-Kiyosue et al., 2005;Al-Bassam and Chang, 2011).
Indeed, it has been observed that CLASP localization in the
MTs can alternate between the plus-end and the MT lattice,
based on its phosphorylation by GSK3β. These MT-binding
activities determine the degree of MT protrusion and subsequent
axon growth in an opposing manner. A high GSK3 kinase
activity promotes CLASP dissociation from MT plus-ends,
leading to MT destabilization and impaired axon growth, while
a moderate GSK3 activity allows CLASP plus-end binding,
promoting MT stabilization and axon extension. A low GSK3
activity leads to CLASP localization to the MT lattice, producing
MT overstabilization and looping in the GCs, and axon growth
attenuation (Akhmanova et al., 2001;Hur et al., 2011a). Given
that GSK3 kinase activity is fine-tuned by many downstream
axon guidance pathways, CLASP may also act as transducing
factor of other extracellular guidance cues (Hur and Zhou, 2010).
APC (Adenomatous Polyposis Coli Protein) is a critical
tumor suppressor, initially reported as Wnt-signaling regulator.
In the Wnt pathway, APC forms a complex with GSK3 and
other proteins to target and degrade the oncoprotein β-catenin
(Stamos and Weis, 2013). In addition to this function, APC
is an EB-binding +TIP that stabilizes the MT plus-ends and,
similar to CLASP, this activity is abolished by GSK3β-mediated
phosphorylation (Nakamura et al., 2001;Zumbrunn et al., 2001).
In neurons, APC is transported toward the distal region of
the growing axon by kinesin-1 motor forces, and distributes
asymmetrically within the GC. Indeed, the local accumulation of
APC in one side of the GC anticipates the steering movement
of the axon in this axial direction (Koester et al., 2007;Ruane
et al., 2016). It was demonstrated that the focal stimulation of
GCs with Nerve Growth Factor (NGF) produces the localized
inactivation of GSK3βvia PI3K activity, which enables APC-
dependent stabilization of MT plus-ends in GCs and rapid
axon elongation (Zhou et al., 2004). Additionally, the treatment
with the GC-pausing guidance cue Wnt3a led to altered MT
growth directionality in the GC by misallocating APC from the
MT plus-ends at the P-domain (Purro et al., 2008). In vivo,
despite initial contradictory results obtained in Drosophila, APC
has been shown to play an important role in neural circuits
formation. APC mutant mice exhibit gross misrouting defects
in the internal capsule, posterior commissure or thalamocortical
axons, and APC-deficient neurons displayed an abnormal axonal
arborization and curling at the tips (Rusan et al., 2008;Yokota
et al., 2009;Jin et al., 2018). Besides its MT-stabilizing role at the
plus-end, APC participates in the MT-based transport of mRNAs,
such as those encoding β-actin, Tubb2b or the dynein complex
subunit Lis1, toward the axon. Importantly, APC association with
their mRNA targets to transport them along sensory axons is
triggered by exogenous stimulation with NGF (Preitner et al.,
2014;Villarin et al., 2016;Baumann et al., 2020).
APC2, APC’s brain specific homolog, is a MT-binding
protein and contains a C-terminal region with MT tip-tracking
properties. APC2 localizes to GCs of chick retinal axons and
participates in retinotectal axon guidance through regulation of
MT stability. Apc2-knockdown display an attenuated response
to ephrin-A2 in retinal ganglion cells (Shintani et al., 2009;
Kahn et al., 2018). Also in retinal neurons, APC2 has been
identified as a direct target of the transcription factor Zic2,
the main determinant of axon midline avoidance, which also
regulates the guidance receptors EphB1 and Unc5c (Herrera
et al., 2003, 2019b;Escalante et al., 2013;Kridsada et al., 2018;
Murcia-Belmonte et al., 2019). In ipsilaterally projecting neurons,
Apc2 expression is intrinsically downregulated by Zic2 likely to
facilitate Wnt5a and ephrinB2-mediated axon repulsion at the
optic chiasm (Morenilla-Palao et al., 2020).
Microtubule-actin crosslinking factor 1 (MACF1), also known
as actin-crosslinking factor 7 (ACF7), is a large multidomain
protein of the spectraplakin family, highly expressed in the
nervous system. MACF1 interacts with MT plus-ends and
enables MT capture by F-actin, facilitating MT polymerization
over F-actin bundles at the cellular periphery (Kodama et al.,
2003;Wu et al., 2008). MACF1 can directly interact with
MTs through its C-terminal Gas2-related (GAR) domain or
indirectly by EB binding, and simultaneously binds F-actin
through its N-terminal calponin-homology (CH) domains. In
addition, MACF1 has a C-terminal AAA-ATPase domain that
can exert molecular forces over the MT cytoskeleton (Moffat
et al., 2017). Genetic studies in Drosophila showed that MACF1
homolog protein Shot is required for axon extension and midline
guidance, and that its MT plus-tip tracking enabled by EB1-
binding is necessary to maintain an organized MT network in
axons (Lee et al., 2007;Alves-Silva et al., 2012). Consistently,
mammalian MACF1 also regulates neuronal MTs configurations
and filopodia formation, a role dependent on both MACF1
F-actin- and MT-binding domains (Sanchez-Soriano et al., 2009).
MACF1 mediates Wnt/GSK3βsignaling, and its loss-of-function
in mice phenocopied the early developmental defects observed
in Wnt3/embryos. Specifically, the conditional deletion
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of MACF1 in neural progenitors produced the agenesis of
the anterior commissure and an abnormal development of
the thalamocortical fibers and the hippocampal commissure
in neonatal mice (Chen et al., 2006;Goryunov et al., 2010).
Moreover, MACF1 downregulation in cortical early postmitotic
neurons interfered with the normal arrangement of MTs
and F-actin networks in neurites, inhibited neuron radial
migration and disrupted callosal axon innervation (Ka et al.,
2014;Ka and Kim, 2016). Interestingly, heterozygous missense
mutations in the MT-binding GAR domain of MACF1 have
been recently identified in human individuals exhibiting axonal
midline crossing phenotypes, among other defects (Table 1)
(Dobyns et al., 2018).
Neuron navigator-1 (NAV1) belongs to the +TIP family of
Navigators (NAVs), which is represented by NAV1, NAV2, and
NAV3 in mammals. NAVs are large proteins, carrying N-terminal
calponin-homology (CH) domains and an intriguing C-terminal
ATPase domain, which have been associated to axon outgrowth
(Martínez-López et al., 2005;van Haren et al., 2009;McNeill
et al., 2010;Abe et al., 2014). In particular, NAV1 expression was
found to be largely restricted to the developing nervous system
being enriched in the neuritic tips and GCs. Hindbrain neurons
lacking NAV1 do not respond to Netrin-1, which suggested
a function downstream of Netrin-1 signaling (Martínez-López
et al., 2005;van Haren et al., 2014). It was recently described
that, similar to CLASP or MACF1, NAV1 is an EB-dependent
+TIP that can directly bind actin fibers in vitro, and data suggest
that it crosslinks MT plus-ends to the F-actin network within
the GCs from mammalian cortical neurons (Sánchez-Huertas
et al., 2020). In the proposed model, EB proteins recruit NAV1
to the MT tip during polymerization inside F-actin-enriched
regions. Following EB-complex disassembly and MT growth
arrest, NAV1 switches to an EB-independent form of association
with the MT plus-end and stabilizes it, reducing the frequency of
MT shrinkage. Thereafter, paused plus-ends undergo retrograde
translocation coupled to F-actin retrograde flow via MT-NAV1-
F-actin crosslinking (Sánchez-Huertas et al., 2020). However,
NAV1 sequence does not possess a CH domain for actin binding,
neither GAR nor TOG domains for direct MT interaction. Hence,
the specific NAV1 domains responsible for direct F-actin binding
and whether NAV1-MT interaction requires an intermediary
autonomous MT-binding protein, still remain to be elucidated.
NAV1 was also found to mediate the chemoattractive response
of cortical axons toward a source of netrin-1 and the radial
migration of pyramidal neurons during in vivo corticogenesis
(Sánchez-Huertas et al., 2020). NAV1 mRNA and protein levels
are highly enriched in developing cortical layer V, mainly
populated by projection neurons innervating subcortical targets,
such as the brainstem or the spinal cord (Martínez-López et al.,
2005;Sorensen et al., 2015). This observation suggests that NAV1
might be required for axonal navigation by layer V projection
neurons in particular, and allows to hypothesize that ad hoc
neuron cytoskeletal machinery may transduce guidance signaling
differently in specific neuron subtypes.
Recent evidences suggest that the module formed by the
+TIPs XMAP215 (chTOG or CKAP5 in mammalian cells) and
transforming acidic coiled-coil 3 (TACC3) protein represent
an unconventional EB-independent regulatory mechanism of
MT plus-end dynamics downstream axon guidance signaling.
XMAP215 is a conserved processive MT polymerase that
catalyzes tubulin addition into the polymer while it tracks the
MT plus-ends (Gard and Kirschner, 1987;Brouhard et al., 2008).
Although XMAP215 and EB1 can act synergistically to promote
MT growth, XMAP215 does not require EB proteins to track
MT plus-ends because it binds MTs directly through its five
N-terminal TOG domains. Indeed, XMAP215 locates to the
extreme MT plus-end several tens of nanometers ahead of the
region bound by EB1 and remains attached to the MT plus-
end even during shrinkage events (Nakamura et al., 2012;Zanic
et al., 2013;Maurer et al., 2014). XMAP215 downregulation
greatly increases MT catastrophe frequency throughout the
neuron cell body and compromises hippocampal axon growth
(van der Vaart et al., 2012). While in most cellular contexts
XMAP215 downregulation decreases MT plus-end growth, in
GCs it accelerates MT plus-end velocities. This increase was
proposed to arise from higher MT anterograde translocation rates
in the GCs, likely due to the uncoupling between MT plus-ends
and the F-actin retrograde flow in the absence of XMAP215
(Lowery et al., 2013). More recently, it was reported that
XMAP215 directly binds actin fibers and it is necessary for MT-F-
actin alignment in the GCs. Indeed, it has been demonstrated that
XMAP215 regulates MT invasion into GC filopodia, influences
GC morphology and protrusion, and mediates the repulsive
response to ephrinA5 (Slater et al., 2019).
TACC3, first identified as a regulator of astral and spindle MT
length, has been classified as +TIP on account of its binding
to MT plus-ends through its TACC domain and assigned a
role in plus-end dynamics and axon outgrowth (Gergely et al.,
2000;Nwagbara et al., 2014). TACC3 interacts with XMAP215
in the distal region of MT plus-ends, and they are important
for each other’s localization to the plus-end. Indeed, TACC3 and
XMAP215 can rescue each other’s downregulation phenotypes
in axon elongation, and it has been suggested that TACC3
strengthens the XMAP215-TACC3 complex binding to MTs in
order to drive polymerization activity (Nwagbara et al., 2014;
Erdogan et al., 2017). TACC3 is a phosphorylation target of
the kinase Abl, whose activity is known to be regulated by
axon guidance signaling (Kannan and Giniger, 2017). A TACC3
phospho-null mutant failed to localize at MT plus-ends in GCs,
leading to an increase of MT invasion into the filopodia and
impaired axon pathfinding. Interestingly, the overexpression
of TACC3 interfered with the responsiveness of axons from
Xenopus neurons explants upon Slit2 and Ephrin-A5 signaling
(Erdogan et al., 2017, 2020).
MICROTUBULES INSTRUCT F-ACTIN
REMODELING IN THE GROWTH CONE
The interaction of MTs with actin filaments and the involvement
of MAPs in this crosstalk is a matter of study since more
than 40 years (Griffith and Pollard, 1978;Selden and Pollard,
1983). This body of work has established that axonal navigation
responses to guidance signals demand an intense and coordinated
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cytoskeleton remodeling, during which both MT and F-actin
influence each other’s dynamics. As aforementioned, F-actin
dynamics influence MT advance and retrograde translocation
in the GC periphery (Schaefer et al., 2002;Zhou et al., 2002).
Even along the axonal shaft, F-actin structures contribute to the
maintenance and dynamics of the MT networks (Winans et al.,
2016;Qu et al., 2017). Conversely, the entry of MT plus-ends into
the actin-rich cortical regions promotes changes in actin-based
structures of the growth cone. Seminal works reported that drugs
that inhibit MT dynamics, without appreciable depolymerization,
halt the bundling and splaying movements in the peripheral
GC domain. At higher concentrations, MT drugs resulted in
the loss of lamellipodia and an increase in filopodial length
but not filopodial number in the GCs (Tanaka et al., 1995;
Gallo, 1998). MT dynamics were also found to be necessary
for the maintenance of the F-actin foci that formed in GCs in
response to substrate adhesions. In particular, it was found that
dampening MT dynamics with drugs suppressed focal F-actin
assembly upon laminin signal detection, while the washout
of the drug restored these foci, indicating that extracellular
signaling can influence F-actin in the GC via MTs (Grabham
et al., 2003;Suter et al., 2004). More recently, live microscopy
experiments on hippocampal cultures exposed to MT-targeting
drugs, revealed that decreasing MT stability significantly reduced
F-actin treadmilling in the GC periphery of the nascent axons.
Conversely, increasing the MT stability or the MT density in
axons resulted in an increase in F-actin dynamics in GCs (Zhao
et al., 2017). Together, this data showed that MT dynamics
influence F-actin turnover in the GC periphery and revealed the
critical role of MTs in the maintenance of the actin-based lamellar
and filopodial structures of GCs.
The MT-stabilizing MAPs MAP1B and Tau can
simultaneously bind actin filaments and contribute to MT-
actin coalignment in the GC. Additionally, MAP1B and tau
can stimulate F-actin polymerization and bundling (Villarroel-
Campos and Gonzalez-Billault, 2014;Elie et al., 2015;Biswas
and Kalil, 2018). However, F-actin and MTs crosstalk mainly
takes place at the MT plus-ends and the most suitable candidates
to assemble both networks are the +TIPs (Bearce et al., 2015;
Cammarata et al., 2016). A minimal engineered version of
the +TIP MACF1, containing N-terminal CH domains and
C-terminal EB-binding motifs – denominated TipAct – showed
efficient MT plus-end tracking and binding to F-actin structures
at the cell periphery. TipAct showed low F-actin binding affinity
in vitro, but its local concentration at MT plus-ends allowed
MT tips to link actin fibers. Therefore, when TipAct was
added to mixed preparations of purified tubulin and actin, it
enabled MTs to transport, pull and bundle actin fibers, globally
arranging F-actin configurations (Preciado López et al., 2014).
The +TIP CLIP170 also exhibited capacity to stimulate in vitro
F-actin elongation in MT-actin re-constitution experiments
via CLIP170 interaction with the formin mDia1. It was shown
that CLIP170-mDia1 complexes are recruited to growing MT
ends by EB1 and stimulate F-actin polymerization from the MT
surface. The actin fibers remained attached to MTs until they
spontaneously detached or were released by a MT catastrophe
event (Henty-Ridilla et al., 2016). Furthermore, a recent study
performed in hippocampal neurons uncovered that MT plus-
ends assemble F-actin networks in the GC periphery in an
APC-dependent manner (Efimova et al., 2020). APC modulates
the activity of various actin regulators, such as the formin mDia
or IQGAP1, which is a downstream effector of Rac1 and Cdc42
GTPases (Watanabe et al., 2009;Okada et al., 2010). In support
of this data, electron microscopy analysis reported that APC
targets MT plus-ends at the MT-actin interphase in the GC
periphery of hippocampal neurons, and that APC is necessary
for the local assembly of branched actin filaments in these GCs
and also for filopodial protrusions. Importantly, encounters
of dynamics APC-positive MT tips with the membranous cell
cortex induced local actin-rich protrusions (Efimova et al.,
2020). These experiments demonstrate that MTs are important
regulators of actin configurations in the GC, either by controlling
F-actin treadmilling and polymerization, or by templating
F-actin organization.
Other +TIPs have also been shown to bind actin fibers
in vitro and/or influence F-actin configurations in the GC or
filopodial dynamics. CLASP directly binds F-actin in vitro and
its downregulation alters the F-actin networks in the GC of
invertebrate neurons. It was described that CLASP-depleted GCs
lack a dense F-actin meshwork and contain less actin bundles,
and that lamellipodial architecture relies on CLASP interaction
with MTs. Interestingly, CLASP binding to both MTs and F-actin
was found to be regulated by Abl-dependent phosphorylation
upon serum or platelet-derived growth factor (PDGF) signaling
(Marx et al., 2013;Engel et al., 2014). Growing MT plus-ends
that enter F-actin-rich areas of the GC are decorated with
EB1-NAV1 complexes, and NAV1 transiently crosslinks MTs
to F-actin. It has been shown that NAV1 restrains filopodial
dynamics and compacts the GC morphology, suggesting a role
in F-actin remodeling perhaps through recruiting the RhoGEF
Trio to MT plus-ends invading the GC periphery. In addition,
NAV1 protein mediates the netrin-1-evoked chemoattraction
over cortical axons (van Haren et al., 2014;Sánchez-Huertas et al.,
2020). Similarly, the EB3-Drebrin module also contributes to
MT-actin coordination and moreover, drebrin inhibits myosin II
activity, reduces cofilin-induced severing of F-actin and stabilizes
F-actin (Geraldo et al., 2008;Mizui et al., 2009;Mikati et al., 2013;
Grintsevich and Reisler, 2014;Zhao et al., 2017). Drebrin’s F-actin
bundle-binding activity is controlled via CDK5 phosphorylation,
and CDK5 is a molecular hub downstream various guidance
signaling pathways (Gordon-Weeks, 2017). Yet, the specific
guidance cues leading to Debrin’s phosphorylation via CDK5
remain to be identified. In addition, the protein Growth arrest-
specific 2-like 1 (Gas2L1) has a domain composition similar
to MACF1 and a recent study revealed that it performs as a
MT-F-actin cytolinker. The simultaneous interaction of Gas2L1
with MTs and actin fibers in vitro released its autoinhibition.
Thus, it was proposed that MT-F-actin crosslinking via Gas2L1
in actin-rich regions promotes local F-actin stabilization and
influences axon outgrowth and branching. In contrast, MT
dynamics were unaffected in neurons following Gas2L1 depletion
(Willige et al., 2019).
Other emerging players of MT-actin crosstalk in the GC of
navigating axons are the formins, a protein family composed
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fnmol-14-759404 November 30, 2021 Time: 13:1 # 17
Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance
by F-actin assembly factors. Formins may also display MT
stabilizing and organizing activities, in some cases independently
of their actin polymerization roles, to regulate axon pathfinding
(Kawabata Galbraith and Kengaku, 2019). For instance, mDia1
and mDia3 appear to mediate the axonal response to ephrinA5,
ephrinB3, Sema3A or SDF1-αin different neuron types, and
knockout mice models demonstrate that they are required for
spinal cord midline crossing (Arakawa et al., 2003;Thurston et al.,
2012;Toyoda et al., 2013). In Drosophila, Disheveled-associated
activator in morphogenesis (DAAM) is a downstream effector
of Wnt5 signaling that exhibits MT-F-actin crosslinking activity
during axonal development. It has been proposed that DAAM
reshapes filopodia and actin structures in GCs via interaction
with +TIPs at MT plus-ends (Gombos et al., 2015;Szikora
et al., 2017). Another member of the formin family, FMN2, also
participates in the stability of focal adhesions and the generation
of traction forces in filopodia and facilitates MT capture by
F-actin bundles in the GC of spinal neurons. Interestingly, chick
FMN2-depleted spinal commissural neurons exhibited midline
crossing defects (Sahasrabudhe et al., 2016;Kundu et al., 2021).
FUTURE DIRECTIONS
During the last years, our understanding of the molecular
mechanisms and proteins involved in the cytoskeletal
transduction of axon guidance signaling has greatly progressed.
While the list of upstream guidance cues and receptor families
has not significantly grown, novel combinatorial mechanisms
involved in signal transduction and cytoskeleton-regulatory
proteins recipient of guidance information are continuously
emerging (Stoeckli, 2018;Zang et al., 2021). Among the latter,
Microtubule-Associated Proteins (MAPs) represent a significant
group. Yet, the role of numerous MAPs in axon guidance is
still unexplored and the intricate mechanisms of MT-F-actin
coordination in the GC remain unclear.
Despite significant advances, experimental designs performed
in non-neuronal cells or limited to few cytoskeleton-regulatory
proteins and guidance cues, may not reflect the full scope
of cytoskeletal changes triggered by extracellular guidance
signaling during axon pathfinding. As a sign of the complex
regulation of physiological MT dynamics in cells, recent data
has demonstrated that MAP combinations exert collective effects
on MTs and MAPs must follow certain hierarchies in their MT
recruitment to achieve specific functions (Niu et al., 2019;Hahn
et al., 2021). Besides, in addition to stereotyped mechanisms
of guidance signal transduction - including regulated guidance
receptor expression, dimerization or trafficking - other molecular
mechanisms underlying axon guidance decisions are being
characterized (Harada et al., 2020;Klein and Pasterkamp,
2021). For instance, it was recently shown that retinal ganglion
cell (RGC) axons exhibit an intrinsic pathfinding program
in absence of any paracrine signaling from the surrounding
tissue (Harada et al., 2020). This sort of cell-autonomous
guidance mechanism could act in coordination with extrinsic
guidance cues to enable divergent axonal responses to the same
guidance information. Indeed, mathematical models predict that
extracellular signaling may instruct axon guidance by simply
controlling neuron-intrinsic stochastic transitions between GC
states (Padmanabhan and Goodhill, 2018).
In summary, we believe that further experiment
conceptualization approaching the molecular mechanisms of
axon guidance should keep in mind that: (i) downstream
guidance pathways may simultaneously target both actin and MT
regulatory proteins, enabling an intricate cytoskeletal crosstalk
in the GC, (ii) the expanding and diverse MAP network can
exert combined effects on MT dynamics, (iii) GC-intrinsic
states (stalled/dynamic) and ad hoc cytoskeletal machinery may
influence axon behavior in specific neuron subtypes, and (iv)
GCs navigate a three-dimensional environment and transduction
pathways described in the literature may not perfectly match
with those operating in living organisms. Furthermore, the use
of transcriptomics and proteomics techniques applied to the
GC fraction of specific neuron subpopulations (Poulopoulos
et al., 2019), high-resolution cytoskeleton imaging (Jung
et al., 2020;Katrukha et al., 2021) or 3D microfluidic assays
(Spijkers et al., 2021) will expand our understanding of the
steered GC locomotion mechanisms and reveal new molecular
specificities in the long-range growing axons accounting for
neural circuits development.
AUTHOR CONTRIBUTIONS
CS-H wrote the article and made the figures. EH edited the
article. Both authors contributed to the article and approved the
submitted version.
FUNDING
CS-H acknowledges the financial support of the “Severo Ochoa”
Program for Centers of Excellence in R&D (SEV-2013-0317). EH
laboratory was funded by the Spain’s National Grant Research
Program (PID2019-110535GB-100) and Prometeo Program
(2020/007) from Generalitat Valenciana.
ACKNOWLEDGMENTS
We are grateful to Jens Lüders and Augusto Escalante for
comments on the manuscript. We apologize to those of our
colleagues whose contributions could not be acknowledged due
to space limitations.
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Frontiers in Molecular Neuroscience | www.frontiersin.org 17 December 2021 | Volume 14 | Article 759404