The structural organization and dynamic remodeling of the cytoskeleton is essential for neuronal development and survival. Axons and dendrites are filled with bundles of non-centrosomal microtubules, which are important for maintaining cellular structure as well as intracellular transport. Notably, the microtubule cytoskeleton plays a central role in neuronal development and polarization: axons have microtubules with their plus-end projecting away from the soma, while dendrites have either a mixed (vertebrates), or uniform minus-end out (invertebrates), microtubule organization (Baas et al., 1989; Kapitein and Hoogenraad, 2015). This difference in microtubule organization allows for selective cargo transport to either neurite by specific microtubule motors and is considered an evolutionary conserved characteristic of axon-dendrite polarity (Rolls and Jegla, 2015). Disorganized axonal and dendritic microtubules lead to deficient transport and subsequently to neuronal polarity defects (Harterink et al., 2018; Yogev et al., 2016). In addition, defects of the microtubule cytoskeleton are associated with a range of neuronal diseases (Falnikar and Baas, 2009; Franker and Hoogenraad, 2013) and the capacity of neurons to regenerate is intimately linked to microtubule stability and plus-tip dynamics (Blanquie and Bradke, 2018; Tang and Chisholm, 2016).

During early neuronal development, microtubules are relatively mobile and can be transported by microtubule motors. Studies in Drosophila and Caenorhabditis elegans indicate that the motor kinesin-1 has a role in setting up the characteristic axon-dendrite microtubule organization, by sliding microtubules against each other (Lu et al., 2013; Winding et al., 2016; Yan et al., 2013). At later stages of development, the entire microtubule cytoskeleton is largely immobilized (Kahn et al., 2018; Lu et al., 2013), which is important to maintain the specific microtubule organization. Various microtubule associated proteins (MAPs) bind to the microtubule lattice, which can stabilize the microtubule itself and can cross-link them together thus preventing microtubule-microtubule sliding (Bodakuntla et al., 2019). In addition, the microtubule bundles were found connected to other cortical cytoskeletal elements (Fréal et al., 2016; Liang et al., 2013; Qu et al., 2017). Although the importance of cortical anchoring of microtubule bundles to maintain neuronal microtubule organization and thereby maintain neuronal polarity and function is apparent, the evidence for this is largely lacking.

In this study, we use C. elegans to identify the molecular mechanisms underlying neuronal microtubule organization. Surprisingly, we found that the highly conserved protein UNC-119 forms a ternary complex with UNC-44 (Ankyrin) and UNC-33 (CRMP) in vitro and in vivo. We show that this complex forms a similar periodic arrangement as the spectrin cytoskeleton and that it is critical to anchor the microtubule cytoskeleton to the neuronal cortex. In the absence of cortical anchoring, UNC-116 (kinesin-1) induces massive microtubule cytoskeleton sliding in axons and dendrites, leading to loss of axon-dendrite microtubule polarity. Thus, we propose that the balance between kinesin-1 dependent microtubule sliding and cortical microtubule anchoring is essential for axon-dendrite microtubule polarity and thus for neuron development and functioning.

unc-119 is important for neuronal polarity and development

Genetic screens in C. elegans identified many unc-genes, which lead to impaired (‘uncoordinated’) locomotion (Brenner, 1974). Neuronal polarity defects and disorganized neuronal microtubules have been observed in several unc-mutants (Maniar et al., 2012; Yan et al., 2013). In this study, we analyzed neuronal development and axon-dendrite polarity in the highly polarized PVD neuron (Figure 1A), which is widely used as a model neuron (Sundararajan et al., 2019). We found that the unc-119 mutant ed3, which carries an early stop mutation, has a fully penetrant microtubule orientation defect in the PVD anterior dendrite (Figure 1B and C). Axons were occasionally affected as well (Figure 1—figure supplement 1A). The microtubule orientation defect is not specific to the PVD neuron, as we observed a similar phenotype in the PHC neuron (Figure 1—figure supplement 1B). In contrast, the ciliated URX and PQR neurons did not have obvious microtubule defects (Figure 1—figure supplement 1C and D), as is expected because these neurons have additional mechanisms to organize neuronal microtubules (Harterink et al., 2018). Furthermore, synaptic vesicles were consistently mislocalized to the cell body and dendrites in the unc-119 mutant (Figure 1D and Figure 1—figure supplement 1E–I), and we observed morphological defects for the PVD neuron; the axon and anterior dendrite are shorter and dendrite branching is reduced (Figure 1E and Figure 1—figure supplement 1J–L). The disorganized microtubules and vesicle mislocalization resemble defects previously observed in unc-33 (CRMP) and unc-44 (Ankyrin) mutants (Maniar et al., 2012). unc-119 is mainly expressed in neurons (Maduro and Pilgrim, 1995) and codes for a highly conserved protein (Maduro et al., 2000), homologous to human UNC119A and UNC119B. Indeed, by introducing UNC-119::GFP specifically in the PVD neuron we were able to suppress the dendritic microtubule defect (Figure 1C), which indicates that UNC-119 functions in a cell intrinsic manner. However, the molecular function of UNC-119 in controlling neuronal development is completely unknown.

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To study UNC-119 protein localization, we generated a C-terminal GFP knock-in line using the SEC-approach (Dickinson et al., 2015) and observed GFP expression in all neurons (Figure 1F), as previously reported (Lim et al., 1999; Maduro and Pilgrim, 1995). The GFP signal can be detected in axons (ventral nerve cord and nerve ring) as well as dendrites (amphid nerve). Strikingly, the protein is not enriched in the cell bodies, which suggests that the protein is not freely diffusing in the cytoplasm. To determine UNC-119 protein dynamics, we performed fluorescence recovery after photobleaching (FRAP) microscopy (Diegelman, 1998). We found that both in axons and dendrites the fluorescence hardly recovers and that the immobile UNC-119 pool is 70–80% (Figure 1G–I). Taken together, this suggests that UNC-119 is tightly associated with undefined structures in axons and dendrites, where it is important for neuronal development and microtubule organization.

UNC-119 forms a complex with UNC-44 (Ankyrin) and UNC-33 (CRMP)

To understand how UNC-119 functions in organizing neuronal polarity, we performed immunoprecipitations experiments from C. elegans using the unc-119::gfp knock-in animals followed by mass spectrometry to identify potential interaction partners of UNC-119 (Supplementary file 1). Interestingly, one of the top-scoring partners of UNC-119, UNC-33 (CRMP), was reported to be important for neuronal development and microtubule organization (Maniar et al., 2012) and mutations in unc-33 lead to similar microtubule defects (Harterink et al., 2018) as the unc-119 mutant (Figure 1 and Figure 1—figure supplement 1). UNC-33 is a putative microtubule binding protein, which has three isoforms in C. elegans: small (UNC-33S), medium (UNC-33M) and large (UNC-33L), but only the large isoform is able to rescue the neuronal polarity defects of the unc-33 mutant (Maniar et al., 2012; Tsuboi et al., 2005). To validate and map the interaction between UNC-33 and UNC-119, we performed pull-down experiments in cultured Hek293T cells and found that the UNC-33L, but not UNC33S, efficiently binds to UNC-119 (Figure 2A and B). Moreover, UNC-119 was able to pull down the UNC-33L specific N-terminus (Figure 2B), further proving that the UNC-119 interacts with the UNC-33L isoform.

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Previous work has shown that UNC-33 (CRMP) acts downstream of unc-44 (Ankyrin) and that the unc-44 mutant has very similar phenotypes as the unc-33 mutant (Harterink et al., 2018; Maniar et al., 2012; Meng et al., 2016). However, we failed to detect a direct interaction between UNC-44 and UNC-33 by immunoprecipitation from Hek293T cells (Figure 2D). In a yeast two-hybrid screen for C. elegans interacting proteins, UNC-119 was identified as a possible binding partner for UNC-44 (Li et al., 2004). This, together with our finding that UNC-119 interacts with UNC-33, suggests that UNC-119 bridges UNC-44 to UNC-33. To test the possible interaction between UNC-44 and UNC-119, we cloned UNC-44 into six fragments (Figure 2A). Using these fragments in Hek293T cells we found that the UNC-44 C-terminus specifically was able to bind to UNC-119 (Figure 2C), which is in agreement with the interaction domain identified by the yeast two-hybrid screen (Li et al., 2004). To test if UNC-44/UNC-119/UNC-33 form a ternary complex we performed pull-down experiments with the UNC-44 C-terminus mixed with either UNC-119::GFP, UNC-33L::GFP or both proteins. We again found that UNC-119 efficiently binds to the UNC-44 C-terminus, however UNC-33L only binds to UNC-44 in the presence of UNC-119 (Figure 2D), showing that UNC-33L, UNC-44, and UNC-119 together can form a ternary complex (Figure 2E). Since mutants for each of these three unc-genes have very similar phenotypes and that the corresponding proteins can form a complex strongly suggests that UNC-33L, UNC-44, and UNC-119 have a common function in neuronal development.

UNC-44 (Ankyrin) immobilizes UNC-33 (CRMP) and UNC-119 in neurons

To determine whether the ternary complex also exists in living organisms, we visualized the localization and dynamics of the individual proteins. Using CRISPR/Cas9, we inserted a GFP at the C-terminus of the giant AO13 Ankyrin splice isoform, which is essential for neuronal development (Boontrakulpoontawee and Otsuka, 2002; Otsuka et al., 2002). UNC-44::GFP mainly localizes to axons and dendrites and its expression pattern is nearly indistinguishable from the UNC-119::GFP knock-in (Figure 3B). We observed that UNC-44::GFP clearly localizes to the cortex (Figure 3C), and that it is highly immobile by FRAP in axons and dendrites (Figure 3D–F), as expected from a protein that is part of cortical cytoskeleton (Bennett and Lorenzo, 2016), Moreover, this immobility does not depend on either UNC-119 or UNC-33 (CRMP) (Figure 3G and H, Figure 3—figure supplement 1C and D), which is expected if UNC-44 (Ankyrin) acts upstream of these proteins. However, when we analyzed the dynamics of the UNC-119::GFP in the unc-44 mutant, we found that the UNC-119 recovers rapidly and seems to diffuse freely in the absence of UNC-44 (Figure 3I and J, Figure 3—figure supplement 1E and F). Notably, the UNC-119 immobility was not affected in the unc-33 mutant. Taken together, this data suggests that UNC-44 binds and stabilizes UNC-119.

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Since UNC-33 exists as three isoforms and that an N- or C-terminal GFP fusion of UNC-33L disrupts protein function (results not shown), it is not possible to endogenously tag specific isoforms. However, inserting an internal GFP resulted in a functional UNC-33L protein (Figure 3K and Figure 3—figure supplement 1G; Maniar et al., 2012). Therefore, we generated single copy (MosSCI) transgenes for each of the UNC-33 isoforms, which we expressed in the PVD neuron, and found that specifically UNC-33L is able to rescue the neuronal microtubule orientation defect of the unc-33 mutant (Figure 3M). We did not detect a specific subcellular localization for UNC-33L, nor did we observe obvious differences in localization between the three UNC-33 isoforms, except for UNC-33S and UNC-33M being a bit enriched in the cell body (Figure 3L; Maniar et al., 2012). However, FRAP analysis did reveal differences in dynamic properties between the UNC-33 isoforms. UNC-33L signal did hardly recover (immobile fraction is ~85%; Figure 3N–P), whereas UNC-33M and UNC-33S partially recovered (immobile fraction is ~60%). This suggests that the two smaller UNC-33 isoforms have a rapidly diffusing pool as well as an anchored pool. Furthermore, we found that the UNC-33L immobility is completely lost in the unc-44 or unc-119 mutants (Figure 3Q and R; Figure 3—video 1), which indicates that UNC-33 acts downstream of UNC-44 and UNC-119. Similarly, GFP::UNC-33S mobility increases in the unc-119 and unc-44 mutant backgrounds (Figure 3S-T). In conclusion, these results together with the biochemical interaction data suggest that UNC-44 (Ankyrin) immobilizes UNC-33L (CRMP) via UNC-119.

UNC-44 (Ankyrin), UNC-119 and UNC-33 (CRMP) form a periodic pattern along neurites

Ankyrin proteins are well-characterized component of the cortical actin-spectrin cytoskeleton (Bennett and Baines, 2001), which forms a characteristic periodic cytoskeleton in invertebrate (Krieg et al., 2017) and vertebrate neurons (Xu et al., 2013). We thus sought to investigate if UNC-44, similar to Ankyrin-G in mammalian and fly neurons (Leterrier et al., 2015; Pielage et al., 2008) forms periodic structures as an indicator for its integration into the cortical cytoskeleton. In order to super resolve the cytoskeleton, we resorted to TIRF-SIM on isolated primary neuron cultures (Materials and methods, Figure 4—figure supplement 1). We observed that UNC-44::GFP (Ankyrin) consistently forms a periodic assembly similar to the 180–200 nm pattern observed with YFP::UNC-70 (β-spectrin) (Figure 4A; p=0.17, pairwise Wilcoxon rank sum test). Importantly, we also observed a ~ 200 nm periodic pattern with the UNC-119::GFP, that corresponds well with the observed UNC-44 distribution (Figure 4B; p=0.82, pairwise Wilcoxon rank sum test). Next, we isolated primary neurons from transgenic animals for the full-length, long version of UNC-33L, which we found to interact with UNC-119, and as well the truncated UNC-33M version, which does not bind to UNC-119. Consistent with this, we observed the periodicity of UNC-33L, in striking similarity to UNC-119 and UNC-44 (Figure 4C–E; p=0.82; p=0.97; pairwise Wilcoxon rank sum test, respectively). In contrast, we did not observe a periodic pattern for the UNC-33M isoform (Figure 4E; p=4e-3, pairwise Wilcoxon rank sum test with UNC-119). The partial periodicity could be explained by the immobile fraction we also observed in the FRAP assay, that associates with microtubules. Taken together, the ternary UNC-44/UNC-119/UNC-33L complex shares a stereotypic periodic pattern similar to the cortical spectrin cytoskeleton.

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UNC-44 (Ankyrin) and UNC-119 anchor the microtubule binding UNC-33L (CRMP) to the cortex

CRMPs are described as cytosolic phosphoproteins that can bind microtubules via their C-terminus (Lin et al., 2011; Niwa et al., 2017; Zheng et al., 2018). To increase subcellular resolution, we ectopically expressed UNC-33S::GFP and UNC-33L::GFP in the hypodermal seam cells and found that both efficiently bind to microtubules and that the C-terminus is indeed essential for this interaction (Figure 5A and B; Figure 5—video 1). Interestingly, we observed that upon loss of microtubule binding, UNC-33L partially relocalizes to the cortex, whereas the shorter UNC-33S does not. In neurons, immobilization of UNC-33L depends on UNC-44 and UNC-119 (Figure 3) and although expression of these proteins is mainly neuronal, low levels are also present in the hypodermal seam cells (Figure 1F; Chen et al., 2017). Indeed, we found that the cortical relocalization of UNC-33L upon loss of microtubule binding depends on UNC-44 and UNC-119 (Figure 5B; Figure 5—video 1).

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To address the dynamics of UNC-33 upon loss of microtubule binding in neurons, we expressed the three UNC-33 isoforms lacking the C-terminus (ΔC) in the PVD neuron (Figure 5—figure supplement 1A). With FRAP analysis, we found that UNC-33SΔC and UNC-33MΔC rapidly recover (Figure 5C–G), which indicates that the immobile pool of UNC-33S and UNC-33M (Figure 3N-P) is the result of microtubule binding. UNC-33LΔC however, is still highly immobile (Figure 5B, H and I). This suggests that although binding to microtubules via the C-terminus is essential for UNC-33L functioning (Figure 5—figure supplement 1B), it is not required for its immobilization. The UNC-33LΔC signal rapidly recovers in the unc-119 mutant (Figure 5J and K), which shows that UNC-119 binds to the N-terminus to immobilize UNC-33L to UNC-44. In agreement, in the unc-44 mutant UNC-33L appears to decorate the dynamic microtubules in the PVD cell body (Figure 3—video 1). Together, these results indicate that in neurons UNC-33L stably associates with microtubules via its C-terminus and to the cortex through its N-terminal interaction with UNC-119.

The UNC-44/119/33 complex immobilizes the neuronal microtubule cytoskeleton

The stability of the UNC-44/119/33 complex and the attachment of the microtubule binding UNC-33L (CRMP) to the cortex, suggests that the complex anchors microtubules in neurons. To visualize the microtubule cytoskeleton we expressed the microtubule binding protein MAPH-1 in the PVD neuron (Harterink et al., 2018) and found that microtubules are evenly distributed over the primary dendrites and the axon in wild type animals (Figure 6A). In the cell body, some microtubule mobility can be observed (Figure 6—video 1). Interestingly, in each of the unc-mutants we observed neuronal development defects and disorganization of the microtubule cytoskeleton; microtubules were no longer restricted to the primary dendrite, but penetrated the side branches as well (Figure 6A and B). Additionally, in these mutants microtubule mobility in the cell body is increased and we observed microtubule buckling at branchpoints (Figure 6—video 1). To examine the peculiar microtubule mobility, we constructed a TBA-1 (tubulin) with an N-terminal photoactivatable-GFP (PA-GFP) (Kahn et al., 2018; Patterson and Lippincott-Schwartz, 2002). Upon activating a small region in the dendrite, the cytoplasmic tubulin rapidly diffuses, while the fluorescent tubulin that is incorporated in the microtubule remains. In wild type worms, the photo-activated region hardly displaced over the course of several minutes (Figure 6C–E; Figure 6—video 2), which indicates that the neuronal microtubule cytoskeleton in a mature neuron is largely immobilized. Strikingly, this immobility was lost in the unc-44 (Ankyrin), the unc-119 and the unc-33 (CRMP) mutants (Figure 6C–E; Figure 6—video 2) and we observed rapid transport of the photo-activated region with a bias towards the cell body: In the unc-mutants: in 12/20 animals the microtubules translocated towards the cell body and in 8/20 microtubules moved in both directions. The signal from the microtubule incorporated PA-GFP-TBA-1 was generally weaker in the mutants, which might suggest the presence of fewer microtubules in the PVD dendrite. Additionally, using the microtubules plus-end marker EBP-2::GFP we observed subtle changes in microtubule plus-end dynamics and growth event frequencies in the mutants (Figure 6—figure supplement 1A–D). These results show that the UNC-44/119/33 complex functions as a cortical anchoring complex to immobilize the microtubule cytoskeleton in neurons.

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The UNC-44/119/33 complex prevents kinesin-1 dependent microtubule sliding

The displacement of the microtubules in the unc-mutants suggests that forces are exerted on the microtubule cytoskeleton, potentially by microtubule motors. Previously, unc-116 (kinesin-1) was shown to be important for dendritic microtubule organization, possibly by transporting microtubules (Yan et al., 2013). If the forces on microtubules are indeed generated by UNC-116, the unc-116 mutant should not exhibit increased microtubule dynamics, since the microtubules are still anchored to the cortex by the UNC-44/119/33 complex. Furthermore, the microtubule sliding observed in unc-mutants should be suppressed by depletion of unc-116. We used an endogenously floxed unc-116 mutant, which can knock-out unc-116 specifically in the PVD neuron lineage (Harterink et al., 2018). As anticipated, we found that loss of unc-116 did not induce microtubule mobility. More importantly, the depletion of unc-116 (kinesin-1) completely suppressed the microtubule mobility in the unc-33 (CRMP) mutant (Figure 7A–C; Figure 7—video 1), but not the microtubule polarity defect (Figure 6—figure supplement 1D) nor the morphology defects (not shown). Additionally, the partial loss-of-function mutant for the kinesin-1 adaptor, klc-2 (kinesin light chain; KLC), also suppressed the microtubule mobility of the unc-33 mutant (Figure 7A–C). This shows that in the absence of the cortical anchoring complex kinesin-1 slides microtubules through the neurites. This function appears specific to the kinesin-1 motor, as depletion of unc-104 (kinesin-3) did not suppress the microtubule mobility in the unc-33 mutant (Figure 7A–C; Figure 7—video 1).

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Direct coupling of UNC-33S (CRMP) to cortical UNC-44 (Ankyrin) rescues the unc-119 mutant

Altogether we have found that UNC-119 bridges the membrane-associated UNC-44 (Ankyrin) to the microtubule binding UNC-33L (CRMP) to immobilize the microtubule cytoskeleton (Figure 8A). While in a wild type animal the UNC-116 (kinesin-1) motor is essential for neuronal polarity (Yan et al., 2013), in the absence of either of the components of the ternary complex, UNC-116 induces robust microtubule sliding (Figure 7). We propose that the UNC-44/119/33 complex acts as a membrane-associated anchoring complex to curb kinesin-1 motor activity for proper neuronal development.

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To confirm this model, we examined whether artificial anchoring of UNC-33S (CRMP) is sufficient to balance kinesin-1 activity in the absence of the cortical anchoring complex. To do this, we fused UNC-33S, which cannot bind to UNC-119, to the GFP-nanobody (vhhGFP) and expressed it in the PVD neuron of unc-44::gfp knock-in animals (Figure 8A). Artificial coupling of UNC-33S to UNC-44 rescued the neuronal microtubule defects of the unc-33 and unc-119 mutants (Figure 8B) as well as the neuronal morphology of the unc-33 mutant (Figure 8C). Remarkably, expression of the vhhGFP::UNC-33S fusion protein in all neurons rescued the animal movement of the unc-119 mutant (Figure 8D and E and Figure 3—figure supplement 1B; Figure 8—video 1), showing that neuronal functioning is restored and that the unc-119 mutant locomotion defect is caused by a loss of UNC-33L anchoring to the cortical UNC-44 (Ankyrin) in neurons.

The C. elegans unc-119 mutant is severely paralyzed, a phenotype that can easily be rescued by reintroduction of the gene and which has extensively been exploited as a transgenesis background in C. elegans for many year (Maduro, 2015). Although it is well known that unc-119 is expressed in neurons (Maduro and Pilgrim, 1995) and to be important for neuron development (Knobel et al., 2001; Materi and Pilgrim, 2005), the molecular function underlying the severe paralysis of the mutant has remained elusive. Here we report that UNC-119 is part of a membrane anchored cytoskeleton complex that anchors the microtubule cytoskeleton to the membrane. Depletion of the cortical anchoring complex leads to dramatic microtubule sliding induced by UNC-116 (kinesin-1). We expect that this excessive microtubule sliding is the underlying cause of the microtubule polarity defects and impaired neuronal development observed in the mutants. Since UNC-116 itself is essential for dendritic minus-end out microtubule organization (Yan et al., 2013), this suggests that neuronal development relies on a fine balance between microtubule sliding and microtubule immobilization by cortical anchoring.

A proper balance of microtubule sliding and bundle immobilization was also shown to be important for neuron development in other organisms. In Drosophila, microtubules are slid over other microtubules by kinesin-1 and potentially also by cortically anchored dynein, during the earliest phases of neurite outgrowth (Del Castillo et al., 2015). To accomplish this microtubule-microtubule sliding, kinesin-1 contains an additional microtubule binding site in its tail, which when mutated leads to developmental defects in both the axon and the dendrite, without affecting kinesin-1 cargo transport (Jolly et al., 2010; Winding et al., 2016). Mutating the sequence for the presumed extra microtubule binding site in the C. elegans UNC-116 motor tail reduced the capacity of the motor to organize microtubules minus-end out (Yan et al., 2013), arguing in favor of kinesin-1 driven microtubule-microtubule sliding. This kinesin-1 dependent microtubule sliding in Drosophila supposedly is independent of the kinesin light chain adaptors (Jolly et al., 2010; Winding et al., 2016). However, we found that, similar to depletion of UNC-116 (kinesin-1), depletion of KLC-2 also was able to suppress the microtubule sliding defect in the unc-33 (CRMP) mutant. Future work will have to address the precise role of microtubule sliding by kinesin-1 in neuronal development.

UNC-119 is a highly conserved protein both in sequence and in function (Maduro et al., 2000). Interestingly, homologs for unc-119 were found to be important for neuronal development in other species as well; depletion of zebrafish UNC-119a led to various neuronal defects that are reminiscent of the C. elegans phenotypes (Manning et al., 2004) and depletion of UNC-119A in cultured rat hippocampal neurons led to reduced dendritic branching (May et al., 2014). Thus far, the mechanism of these vertebrate UNC119 homologs in neuronal development has not been investigated at the molecular level. It is well documented that the mammalian UNC119 proteins can bind to the lipid moiety of myristoylated proteins (Constantine et al., 2012). As such, UNC119 acts as a chaperone to solubilize and transport the otherwise membrane-anchored proteins to other membranes or the cilium (Konitsiotis et al., 2017; Wright et al., 2011; Zhang et al., 2011), where the interaction with the GTPases Arl2 or Arl3 allosterically releases the myristoylated cargo from UNC119 (Ismail et al., 2012; Kobayashi et al., 2003). Similar functions have been proposed for UNC-119 in C. elegans. UNC-119 was shown to interact with Arl2/3 homologues, and the unc-119 mutant has deficient transport of myristoylated proteins to the cilium as well as cilium formation defects (Warburton-Pitt et al., 2014; Zhang et al., 2011; Zhang et al., 2016). However, we observed no microtubule organization defects in mutants for the C. elegans Arl2/3 genes (results not shown). Moreover, we were able to rescue the unc-119 mutant microtubule organization and locomotion by directly attaching UNC-33 to UNC-44, demonstrating that UNC-119 functions as a structural scaffold to bring these proteins together for proper neuronal development and functioning.

In Drosophila, the kinesin-1 sliding is counteracted by the ‘mitotic’ kinesin MKLP1 (Del Castillo et al., 2015), which potentially cross-links microtubules. In mammalian neurons, the stabilization of microtubule cytoskeleton relies on various MAPs that can bind and stabilize individual microtubules and cross-link them together (Ramkumar et al., 2018). This is especially apparent in the axon, where the microtubules are regularly spaced and accumulate modifications indicative for long lived microtubules (Chen et al., 1992; Song and Brady, 2015). Less is known, however, about how such a cross-linked microtubule array is immobilized itself in the neuron. In Drosophila, the microtubule cytoskeleton is stabilized by the spectraplakin protein Shot, which binds to both microtubules and the actin cytoskeleton (Qu et al., 2017; Sanchez-Soriano et al., 2009). Although the role of Shot in setting up axon-dendrite polarity is not addressed, in the absence of Shot, axon regeneration is affected and the microtubules are less bundled, which leads to buckling of microtubules. In C. elegans we observed similar microtubule buckling in the absence of the cortical anchoring complex (Figure 6—video 1). The dynamic association of the microtubule cytoskeleton to the spectrin-actin cytoskeleton is an important factor during neuron development reviewed by Coles and Bradke (2015); Krieg et al. (2017).

Neurons typically express giant Ankyrin isoforms that may have a common evolutionary origin and that have essential functions in neuron development (Jegla et al., 2016). Interestingly, these giant Ankyrin proteins evolved several ways to connect the submembrane cytoskeleton to the microtubule network. In Drosophila, Ank2 was shown to directly bind to the microtubules (Pielage et al., 2008; Stephan et al., 2015), whereas in mammalian neurons, Ankyrin-G localizes to the axon initial segment, where it binds indirectly to microtubules via multiple EB proteins (Fréal et al., 2016; Leterrier et al., 2011). Recently it was found that the giant Ankyrin-B protein localizes to axons and that it is important for the microtubule organization in the axon (Yang et al., 2019). Since static enrichment of EB proteins is specific for the axon initial segment by binding to Ankyrin-G (Leterrier et al., 2011), it is unclear how Ankyrin-B connects to the microtubule cytoskeleton in the axon. Here, we show that in C. elegans the giant UNC-44 (Ankyrin) isoform interacts with microtubules via UNC-119 and UNC-33 (CRMP) in both axons and dendrites. Also in mammalian neurons, CRMPs are undeniably important for microtubule organization, neuronal development, and neuronal polarity (Arimura et al., 2004). This might suggest that the Ankyrin/UNC119/CRMP complex also functions as a microtubule anchor in other species. However, since UNC-119 binds to the non-conserved N-terminus of UNC-33L in C. elegans, further research is required to elucidate possible interactions and common functions of UNC-119 and UNC-33 in mammalian neurons.

In our experiments we observed that in mature neurons of wild type animals the microtubule cytoskeleton is for a large part immobile, as was seen before for other systems (Cao et al., 2020; Kahn et al., 2018; Lu et al., 2013). During neuronal development, however, growth and retraction of neurites is intimately linked to microtubule stability and dynamics (Lewis et al., 2013; Rumpf et al., 2019). Thus, it is likely that stability of the anchoring complex itself is regulated and/or that binding of the microtubules to the anchoring complex is regulated. We found that the direct coupling of UNC-33S to UNC-44 was able to bypass UNC-119 and thus to rescue the unc-119 mutant phenotype. However, whether microtubules bind continuously to UNC-33 (CRMP), or whether their binding is regulated, for example by phosphorylation (Zheng et al., 2018), is not yet known. The coordination of microtubule sliding and anchoring during neuronal development will be an important focus for further research.

Interestingly it was recently shown that CRMPs are hyper-phosphorylated in the central nervous system of Alzheimer disease patients, and the phosphorylated CRMP was observed primarily in dystrophic neurons (Mokhtar et al., 2018). In addition, this amyloid beta-dependent CRMP phosphorylation is suggested to directly impair kinesin-1 association and thus axon development in humans. As also autism associated mutations in the giant Ankyrin-B gene were found to affect the microtubule organization in the axon (Yang et al., 2019), our observation of disorganized microtubule cytoskeleton in the absence of CRMP or Ankyrin, suggest a conserved mechanism of MT stabilization across evolution. Thus, understanding how the neuronal microtubule cytoskeleton is connected to other cellular structures not only has great potential to help understand neuronal development, but may also lead to new ways to treat damaged neurons.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Arimura N
   2. Menager C
   3. Fukata Y
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Author details

1. Liu He

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration

   Competing interests

   No competing interests declared

2. Robbelien Kooistra

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing - original draft

   Contributed equally with

   Ravi Das and Ellen Oudejans

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4576-8964

3. Ravi Das

   Neurophotonics and Mechanical Systems Biology, ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Barcelona, Spain

   Contribution

   Data curation, Investigation, Visualization

   Contributed equally with

   Robbelien Kooistra and Ellen Oudejans

   Competing interests

   No competing interests declared

4. Ellen Oudejans

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Data curation, Investigation, Visualization, Methodology

   Contributed equally with

   Robbelien Kooistra and Ravi Das

   Competing interests

   No competing interests declared

5. Eric van Leen

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Johannes Ziegler

   Fast live-cell superresolution microscopy, ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Barcelona, Spain

   Contribution

   Supervision, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Sybren Portegies

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Investigation, Writing - original draft

   Competing interests

   No competing interests declared

8. Bart de Haan

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

9. Anna van Regteren Altena

   Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

10. Riccardo Stucchi

    1. Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands
    2. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands

    Contribution

    Data curation, Methodology

    Competing interests

    No competing interests declared

11. AF Maarten Altelaar

    Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands

    Contribution

    Supervision, Performed experiments

    Competing interests

    No competing interests declared

12. Stefan Wieser

    Fast live-cell superresolution microscopy, ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Barcelona, Spain

    Contribution

    Supervision, Methodology

    Competing interests

    No competing interests declared

13. Michael Krieg

    Neurophotonics and Mechanical Systems Biology, ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Barcelona, Spain

    Contribution

    Supervision, Writing - review and editing

    Competing interests

    No competing interests declared

14. Casper C Hoogenraad

    1. Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands
    2. Department of Neuroscience, Genentech, Inc, South San Francisco, United States

    Contribution

    Supervision, Writing - original draft

    For correspondence

    c.hoogenraad@uu.nl

    Competing interests

    No competing interests declared

15. Martin Harterink

    Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    m.harterink@uu.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8256-6651

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