With the permission of microtubules: an updated overview on microtubule function during axon pathfinding

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 [email protected] 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 Frontiers in Molecular Neuroscience | www.frontiersin.org 1December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 2 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 coreceptors, 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 MTassociated 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 Frontiers in Molecular Neuroscience | www.frontiersin.org 2December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 3 Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance 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 MTintrinsic 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;CunhaFerreira 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 motordependent 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 neuronspecific 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. Frontiers in Molecular Neuroscience | www.frontiersin.org 3December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 4 Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance 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 Frontiers in Molecular Neuroscience | www.frontiersin.org 4December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 5 Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance 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 microscale chromophore-assisted laser inactivation (micro-CALI) have also revealed that several MAPs can trigger GC steering. MicroCALI 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 highaffinity 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 netrin1 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 TUBB3UNC5C 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 netrin1-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 Frontiers in Molecular Neuroscience | www.frontiersin.org 5December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 6 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) Frontiers in Molecular Neuroscience | www.frontiersin.org 6December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 7 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) Frontiers in Molecular Neuroscience | www.frontiersin.org 7December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 8 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, nerve growth factor; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; DRG, dorsal root ganglia. Frontiers in Molecular Neuroscience | www.frontiersin.org 8December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 9 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 motorcognitive 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;CunhaFerreira 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 Frontiers in Molecular Neuroscience | www.frontiersin.org 9December 2021 | Volume 14 | Article 759404 fnmol-14-759404 November 30, 2021 Time: 13:1 # 16 Sánchez-Huertas and Herrera Role of Microtubules in Axon Guidance 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 MTactin coalignment in the GC. Additionally, MAP1B and tau can stimulate F-actin polymerization and bundling (VillarroelCampos 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 plusends 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 arrestspecific 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 Frontiers in Molecular Neuroscience | www.frontiersin.org 16 December 2021 | Volume 14 | Article 759404 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. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Molecular Neuroscience | www.frontiersin.org 25 December 2021 | Volume 14 | Article 759404