KLP-7/Kinesin-13 orchestrates axon-dendrite checkpoints for polarized trafficking in neurons

Abstract

The polarized nature of neurons depends on their microtubule dynamics and orientation determined by both microtubule-stabilizing and destabilizing factors. The role of destabilizing factors in developing and maintaining neuronal polarity is unclear. We investigated the function of KLP-7, a microtubule depolymerizing motor of the Kinesin-13 family, in axon-dendrite compartmentalization using PVD neurons in Caenorhabditis elegans. Loss of KLP-7 caused a mislocalization of axonal proteins, including RAB-3, SAD-1, and their motor UNC-104, to dendrites. This is rescued by cell-autonomous expression of the KLP-7 or colchicine treatment, indicating the involvement of KLP-7-dependent microtubule depolymerization. The high mobility of KLP-7 is correlated to increased microtubule dynamics in the dendrites, which restricts the enrichment of UNC-44, an integral component of Axon Initial Segment (AIS) in these processes. Due to the loss of KLP-7, ectopic enrichment of UNC-44 in the dendrite potentially redirects axonal traffic into dendrites that include plus-end out microtubules, axonal motors, and cargoes. These observations indicate that KLP-7-mediated depolymerization defines the microtubule dynamics conducive to the specific enrichment of AIS components in dendrites. This further compartmentalizes dendritic and axonal microtubules, motors, and cargoes, thereby influencing neuronal polarity.

• Neuronal polarization ensures distinct compartmentalization of molecules within the neuron and is required for its proper function. It is unclear how microtubule destabilization factors participate in the process.

• Using PVD neurons in C. elegans, the authors establish that KLP-7 (Kinesin-13) specifically regulates axon-dendrite compartmentalization. KLP-7-dependent microtubule depolymerization limits the formation of the axon initial segment (AIS), preventing the entry of axonal molecules into the dendrites.

• The study advances the understanding of how cytoskeletal regulators define compartmentalization in neurons required for the maintenance of the molecular repertoire of neurites and their identity.

INTRODUCTION

The polarized structure and composition of the neurons enable directional information transfer in a neural circuit. Microtubules are one of the crucial determiners of neuronal polarization (Barnes and Polleux, 2009; Marín et al., 2010; Kuijpers and Hoogenraad, 2011; Yogev and Shen, 2017). Dynamics and organization of microtubules define neuronal migration, selective intracellular trafficking, organelle positioning, signaling hubs, and force generators in the developing neurons (Tolić-Nørrelykke, 2008; Hirokawa et al., 2010; De Forges et al., 2012; Subramanian and Kapoor, 2012). Microtubules are made of α and β tubulin heterodimers equilibrating between phases of polymerization and depolymerization that define their polarity, dynamics, and organization during neuronal morphogenesis (Sakakibara et al., 2013; Horio et al., 2014; Kapitein and Hoogenraad, 2015; Kelliher et al., 2019; Gudimchuk and McIntosh, 2021).

In the context of neuronal polarity, regulators of microtubule stabilization have been well investigated (Baas et al., 1991, 2016; Witte et al., 2008; Polleux and Snider, 2010) for example, MAP2C and TRIM46 stabilize the microtubule array and are important for the axon initiation (Dehmelt et al., 2006; Van Beuningen et al., 2015). Similarly, microtubule assembly regulator Collapsin Response Mediator Protein 2 (CRMP2) regulates the compartmentalization of axons and dendrites (Arimura et al., 2004; Yoshimura et al., 2005; Maniar et al., 2012). Microtubule destabilizers like Stathmin/Op18 are locally inactivated in the nascent axon during development (Watabe-Uchida et al., 2006). Similarly, a microtubule severing protein, Katanin is finely regulated for the proper axon development (Karabay et al., 2004; Yu et al., 2005; Qiang et al., 2006). Conditional loss of microtubule depolymerizing KIF2/Kinesin-13 in the rodent hippocampus and KLP-7 in Caenorhabditis elegans caused the formation of ectopic branches and loss of axon-dendrite compartmentalization (Homma et al., 2003, 2018; Ghosh-Roy et al., 2012; Puri et al., 2021). The roles and mechanisms of microtubule depolymerizers in the process of neuronal polarization are yet to be investigated in detail.

PVD neurons are highly polarized neurons with a well-defined axon and highly stereotyped dendritic arbor. These are mechanosensory neurons for proprioception, harsh touch, and cold temperature sensation (Way and Chalfie, 1989; Chatzigeorgiou et al., 2010; Albeg et al., 2011; Brar et al., 2024). Previous studies have elucidated the role of various regulators, including Ankyrin (UNC-44), CRMP2 (UNC-33), UNC-119, Patronin (PTRN-1), Ninein (NOCA-2), and TIAM-1 in the process of neuronal polarity (Maniar et al., 2012; He et al., 2020, 2022; Lin et al., 2022). Loss of function of these genes caused aberrant microtubule organization and mislocalization of the axonal cargoes into the dendrites (Maniar et al., 2012; He et al., 2020, 2022; Lin et al., 2022). It is unclear how microtubule depolymerizing factors such as KLP-7 or EFA-6 influence axon-dendrite compartmentalization.

In this study, we have found a specific role of KLP-7/Kinesin-13 in the proper compartmentalization of axons and dendrites in the PVD neurons. Using a reporter of microtubule dynamics, EBP-2::GFP, we have found that KLP-7 maintains an increased level of microtubule dynamics and minus-end-out microtubule organization in the major dendrite of the PVD neuron. In the absence of KLP-7, the axonal cargoes, including RAB-3, SAD-1, and the motor UNC-104 populate the dendrites. This correlates with decreased microtubule dynamics, increased population of plus-end-out microtubules, and ectopic accumulation of the AIS component UNC-44 in dendrites. Our study suggests that KLP-7 optimizes microtubule dynamics in PVD neurites critical to preventing ectopic enrichment of AIS components in the dendrites.

RESULTS

KLP-7/Kinesin-13 regulates the axon-dendrite compartmentalization in the PVD neurons

KLP-7 is a motor of the Kinesin-13 family required for the dynamic instability of the microtubules through depolymerization (Desai et al., 1999; Srayko et al., 2005; Trofimova et al., 2018). Human orthologue of KLP-7, KIF2 has been associated with clinical manifestations of cortical development, epilepsy, microcephaly, and Autism Spectrum Disorder (Poirier et al., 2013; Yuen et al., 2015; Tian et al., 2016; Cavallin et al., 2017; Costain et al., 2019). Conditional knockout of KIF2 in rodents resulted in defects in neuritogenesis, neuronal maturation, function, and survival (Ogawa and Hirokawa, 2015; Homma et al., 2018; Ruiz-Reig et al., 2022). Similar anatomical defects were also observed in the touch neurons of C. elegans, where the loss of the klp-7 gene resulted in the formation of ectopic neurites with axonal markers (Puri et al., 2021). The mechanism by which Kinesin-13 regulates the axonal and dendritic identities is not clear.

We investigated aspects of neuronal polarity in the loss of function mutant of klp-7 (Kinesin-13). We checked the distribution of the axonal marker, RAB-3 as a reporter for neuronal polarity in the PVD neuron (Figure 1A). PVD neurons of C. elegans have a well-defined axon running ventrally and fasciculated with the ventral nerve cord and a highly stereotyped dendritic arbor with orthogonal branches (Albeg et al., 2011; Sundararajan et al., 2019). The mCherry::RAB-3 reporter appears punctate mostly in the ventral nerve cord region in the control (wildtype) background (Figure 1A). Unlike in wildtype, mCherry::RAB-3 was also present in the dendrites of the klp-7 null mutant (Figure 1B-C). Estimation of the dendritic branches containing mCherry::RAB-3 showed an accumulation in 11.5% of the dendritic branches of klp-7(0) as compared with 3.5% in the wildtype (Figure 1D). Though the loss of klp-7 caused localization of mCherry::RAB-3 throughout the PVD neuron, including the minor dendrite, it was more pronounced in the distal region of the major dendrite where klp-7 null mutant had ∼29% dendrites with mCherry::RAB-3 as compared with 11.8% of the wildtype (Supplemental Figure S1A). This accumulation was prominent in the tertiary branches of klp-7(0) with 33.2% occupancy (Supplemental Figure S1B). Interestingly, quaternary branches of the klp-7 null mutant also showed mislocalization of mCherry::RAB-3 (Supplemental Figure S1B). Moreover, the precedence of mCherry::RAB-3 in the dendrites as observed by the relative density and intensity of these particles, was also increased in the null mutant of klp-7 (Figure 1, E and F). Though klp-7 mutant dendrites showed remarkably increased occupancy by RAB-3 particles as observed by the intensity and density of the particles, the axons did not show any difference to the wildtype (Supplemental Figure S1, C and D). As we did not observe complete misrouting of RAB-3 to the dendrites, we speculate that higher accumulation in the dendrites may be because of an increased anterograde transport in the mutant.

FIGURE 1:

KLP-7 is required for the proper axon-dendrite compartmentalization in the PVD neurons. (A) Representative images and schematics of the PVD neuron expressing soluble GFP (green) and mCherry::RAB-3 (magenta), with the region around the cell body (dashed box) magnified below. Schematic depicts crosshair reference points of body symmetry (A- anterior, P- posterior, D- dorsal, V- ventral). (B and C) Representative images of the whole PVD neuron expressing soluble GFP (green) and mCherry::RAB-3 (magenta) in the wildtype (wt), and loss of function mutants of klp-7(0), efa-6(0), and double mutant of klp-7(0) and efa-6(0) with the regions around the cell body (dashed box) magnified and schematized in (C). The axonal and dendritic distributions of mCherry::RAB-3 in (C) are marked by the blue arrows and yellow arrowheads, respectively. Schematic in (C) shows the distribution of mCherry::RAB-3 (red dots) in the dendritic arbor of various mutants. (D) Quantification of the dendritic branches showing accumulation of mCherry::RAB-3 in the wildtype (wt), and loss of function mutants of klp-7(0), efa-6(0), and double mutant of klp-7(0) and efa-6(0) normalized to the total number of dendritic branches represented as a percentage. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.05*, 0.001***, ns (not significant). Number of animals assessed (n) is mentioned along the X-axis. (E and F) Relative particle density (E) and mean intensity (F) of the particles of mCherry::RAB-3 quantified for the dendrites (major and minor) with respect to the axon of PVD neurons of wildtype (wt), klp-7(0), efa-6(0), and klp-7(0);efa-6(0) double mutant. Comparison of means within a category was done using ANOVA and Bonferroni Test and p > 0.05*, 0.001***. Number of worms assessed (n) is mentioned along the X- axis.

As KLP-7 is a microtubule catastrophe factor, we asked whether any other microtubule-catastrophe factor would also regulate the axon-dendrite compartmentalization. We investigated another mutant that has a loss of function of efa-6. Apart from the conventional role in membrane trafficking and actin regulation (Franco et al., 1999), in C. elegans, EFA-6 has a microtubule depolymerizing domain that causes the catastrophe of the microtubules near the cell cortex (O’Rourke et al., 2010). Loss of EFA-6 causes the cells to have long microtubules and has been phenotypically correlated to spindle defects, embryonic lethality, and dlk-1-independent axon regeneration (O’Rourke et al., 2010; Chen et al., 2015; Qu et al., 2019). Unlike the loss of function mutant of klp-7, a null mutant of efa-6 did not mislocalize RAB-3 to the dendrites (Figure 1, B and C). Measurement of the number of dendrites with RAB-3 and distribution of RAB-3 in the primary dendrites of the PVD neuron of efa-6(0) were equivalent to those of wildtype (Figure 1, D–F; Supplemental Figure S1, A and B). The axon-dendrite distinction seems to be preserved in this mutant like the wildtype. Furthermore, we did not observe a synergism between klp-7 and efa-6 in the double mutant as compared with the klp-7 mutant alone (Figure 1, B–F; Supplemental Figure S1, A–D). Only in the posterior dendrite (minor), the effect of klp-7(0) seemed suppressed by efa-6(0) in the density of RAB-3 particles, indicating a differential requirement of KLP-7 in PVD neurites (Figure 1D; Supplemental Figure S1, A and C). Although microtubule depolymerization by KLP-7 and EFA-6 in the dendrites could be mechanistically different, the role of KLP-7 in neuronal polarity is highly specific.

Cell-autonomous role of KLP-7 in maintaining selective traffic in axons and dendrites

We observed another presynaptic marker SAD-1 kinase, which is required for neuronal polarity and presynaptic development (Crump et al., 2001). SAD-1::GFP was expressed as part of the same transgene as mCherry::RAB-3 (Maniar et al., 2012). Similar to RAB-3, SAD-1 also appears punctate and enriched in the Ventral Nerve Cord region of the PVD neurons (Maniar et al., 2012). Loss of klp-7 also resulted in the mislocalization of SAD-1 to the PVD dendrites (Figure 2A). This mislocalization was observed in nearly 8% of the dendrites (Figure 2B). To further assess the role of KLP-7 in SAD-1 localization, we expressed the KLP-7 cDNA (15 ng) under the PVD-specific promoter as an extrachromosomal array transgene. PVD-specific expression of the KLP-7 in the wild-type background did not show any SAD-1 localization to the dendrites (Figure 2, A and B). However, in the mutant background, expression of this transgene rescued the SAD-1 mislocalization phenotype (Figure 2, A and B).

FIGURE 2:

KLP-7 is necessary and sufficient to maintain polarized trafficking in the axons and dendrites. (A) Representative images and schematics of PVD neuron expressing SAD-1::GFP (green) and mScarlet (magenta) in the wildtype (wt), and loss of function mutant of klp-7(0), PVD specific overexpression of 15 ng klp-7b isoform (klp-7[+]), and PVD specific rescue of klp-7(0) labeled as klp-7(0);klp-7[+]. The axonal and dendritic distributions of SAD-1::GFP are marked by the blue arrows and yellow arrowheads, respectively. Schematic shows the distribution of SAD-1::GFP (red dots) in the dendritic arbor of various genotypes. (B) Quantification of the dendritic branches showing accumulation of SAD-1::GFP in the wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue normalized to the total number of dendritic branches represented as a percentage. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.01**, 0.001***, ns (not significant). Number of animals assessed (n) is mentioned along the X-axis. (C and D) Relative particle density (C) and mean intensity (D) of the particles of SAD-1::GFP quantified for the dendrites (major and minor) with respect to the axon of PVD neurons of the wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue. Comparison of means within a category was done using ANOVA and Bonferroni Test and p > 0.05*, 0.01**, 0.001***. Number of worms assessed (n) is mentioned along the X- axis. (E and F) Representative images (E) and quantification (F) of the distribution of endogenously UNC-104::GFP (green) in the dendrites and axons of PVD neurons expressing mScarlet (magenta). Enrichment of UNC-104::GFP in the dendrites in (E) are marked by yellow arrowheads. Comparison of absolute intensities of UNC-104::GFP in the dendrites and axons per unit of their lengths was done between wildtype (wt) and mutant klp-7(0) using ANOVA and Bonferroni Test. p < 0.05*, ns (not significant). Number of worms assessed (n) is mentioned along the X- axis.

We also scored the density and intensity of SAD-1 particles in the dendrites and axons of PVD neurons. Overexpression of the klp-7 transgene in the klp7(0) background partially restored the relative localization of SAD-1 in the dendrites with respect to the axons (Figure 2, C and D). The density and intensity of the dendritic SAD-1 particles were restored to the wildtype levels in the rescue of klp-7(0) (Supplemental Figure S2, A and B). Interestingly, due to the expression of klp-7 transgene in either wildtype or klp-7 mutant background, the density and mean intensity of the SAD-1 particles were drastically reduced in the axonal process (Supplemental Figure S2, A and B), possibly due to a disruption in its anterograde transport. This was strengthened by the live observation of mCherry::RAB-3 particles in PVD neurons of wild type and klp-7(0) worms. In the wild type, RAB-3 particles were present in the primary dendrites of a small proportion of animals. These particles were mobile, however, long-duration runs were not observed (Supplemental Movie S1). Interestingly, we observed a robust anterograde movement in the klp-7(0), in the primary, secondary, and ectopic branches of PVD neurons (Supplemental Movie S2).

Movie S1 (linked to Figure 2).

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Live observation of mCherry::RAB-3 in the PVD neurons of wildtype animals.

Movie S2 (linked to Figure 2).

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Live observation of mCherry::RAB-3 in the PVD neurons of klp-7(0) animals.

To understand how KLP-7 could be regulating the process, we investigated the motor UNC-104, which is required for the anterograde trafficking of presynaptic components, including RAB-3 and SAD-1 (Hall and Hedgecock, 1991; Yonekawa et al., 1998; Maniar et al., 2012). We observed UNC-104::GFP expressed under its endogenous promoter as a GFP knock-in transgene (Cong et al., 2021). Due to its ubiquitous neuronal expression, UNC-104::GFP was observable in multiple neuronal processes, including motor neuron commissures and sublateral nerve cords (Zhou et al., 2001; Kumar et al., 2010; Li et al., 2016; Cong et al., 2021). Based on neurite traces generated from the constitutive PVD reporter, mScarlet, we analyzed the intensities of UNC-104::GFP in the dendrites of PVD neurons. In the loss of function mutant of klp-7, we observed a marked increase in the UNC-104::GFP intensity in the dendritic regions of the PVD neurons (Figure 2, E and F) while the axonal intensity remains unchanged. This correlates with the ubiquitous localization of SAD-1 or RAB-3 in the klp-7(0) mutant. This suggested that KLP-7 regulates the entry of axonal traffic into the dendrites through a predominantly axonal motor, thereby defining the checkpoint to segregate the axonal and dendritic proteins.

Increased microtubule stability due to loss of klp-7 affects the axon-dendrite compartmentalization

Microtubule organization in the neurons is important for the differential trafficking of the cargoes in the axonal versus dendritic compartments (Kapitein et al., 2010; Kapitein and Hoogenraad, 2011; Maeder et al., 2014). Previous studies have established the causal effect of microtubule stabilization on axon specification by pharmacological perturbation of the microtubules through Paclitaxel to induce multiple neurites with axonal identity (Xiao et al., 2006; Witte et al., 2008). On the other hand, microtubule destabilizers like Nocodazole and Colchicine could reduce the formation of dendrite-like processes (Witte et al., 2008; Puri et al., 2021).

As we observed a cell-autonomous role of klp-7 to restrict the entry of axonal cargoes such as SAD-1 or RAB-3 in the PVD dendrites, we speculated that loss of klp-7 might have increased the stability of microtubules in dendrites, thereby routing the axonal motors and cargoes into the dendrites. To understand whether KLP-7 mediated microtubule instability defines the dendritic identity, we used pharmacological upregulation of microtubule instability by Colchicine. Previously, Colchicine exposure has been used to suppress the effects of microtubule-stabilizing mutations in C. elegans (Kirszenblat et al., 2013; Puri et al., 2021). We used a concentration of 1 mM Colchicine, which has been shown to affect the microtubule dynamics and function of the gentle touch receptor neurons (Chalfie and Thomson, 1982; Bounoutas et al., 2011; Lockhead et al., 2016; Puri et al., 2021). In the klp-7 loss of function mutant, the prevalence of RAB-3 in the dendrites decreased significantly with exposure to Colchicine (Figure 3, A–C). We observed a marked reduction in the RAB-3 occupancy in the proportion of dendrites as well as the localization of RAB-3 particles in klp-7(0) dendrites upon Colchicine treatment (Figure 3, B and C). This is likely due to a balance between Colchicine mediated microtubule depolymerization with respect to stabilized microtubules in klp-7(0) mutant. To further strengthen this, we exposed the wildtype worms to the same dose of Colchicine. As the dendrites of wildtype PVD neurons had negligible localization of RAB-3, Colchicine treatment did not alter this distribution (Supplemental Figure S3, A–C). However, the axonal distribution of RAB-3 particles in wildtype neurons was remarkably reduced upon Colchicine exposure (Supplemental Figure S3C). This suggested that KLP-7 maintains an optimal level of microtubule dynamics in the dendrites to prevent the entry of axonal cargoes like RAB-3 into dendrites.

FIGURE 3:

KLP-7-dependent microtubule organization defines the axon-dendrite compartmentalization. (A) Representative images of the klp-7(0) mutant following the treatment with Mock (Ethanol) and Colchicine (1 mM). The axonal and dendritic distributions of mCherry::RAB-3 are marked by the blue arrows and yellow arrowheads, respectively. Schematic shows the distribution of mCherry::RAB-3 (red dots) in the dendritic arbor of klp-7(0) upon Colchicine treatment. (B and C.) Percentage of the dendritic branches with respect to the total number of dendritic branches showing accumulation of mCherry::RAB-3 (B) and density of mCherry::RAB-3 particles in the primary dendrite (C) of the klp-7(0) mutant in mock and Colchicine treated conditions. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.05*, 0.01**. Number of animals assessed (n) is mentioned along the X-axis.

Differential regulation of microtubule dynamics by Kinesin-13 is correlated to its mobility in neuronal compartments

Kinesin-13 is a key determinant of dynamic instability (Desai et al., 1999; Srayko et al., 2005; Ogawa et al., 2017; Trofimova et al., 2018). To understand the microtubule dynamics and organization regulated by KLP-7 in PVD neurons, we observed the dynamics of the plus tips of the microtubules using the EBP-2::GFP marker. EBP-2::GFP binds to the plus ends of microtubules and shows comet-like movement in a time-lapse acquisition (Stepanova et al., 2003; Ghosh-Roy et al., 2012). To assess the EBP-2::GFP dynamics, we generated the kymographs from segments (orange dotted lines) on the major and minor dendrites and axons of PVD neurons originating at the cell body (Figure 4A). In the kymographs, based on the angle, distance, and duration of the traces, polymerization rates and polarity were assessed (Figure 4A).

FIGURE 4:

Microtubule dynamics and polarity in the PVD dendrites perturbed by loss of klp-7 function. (A) Representative images of endogenously expressed EBP-2::GFP and PVD neuron expressing mScarlet. Different neurites used for assessing EBP-2::GFP comet dynamics are depicted with orange segmented lines. The origin of the segmented line is marked by a circle, which refers to the proximal region in the kymographs depicted below with plus and minus end out microtubules marked in cyan and magenta, respectively. Traces in the kymographs are measured for the length and time of polymerization, which yield the polymerization rate and angle that provides the orientation of the comets. (B) Kymographs with their respective schematics showing the trajectories of EBP-2::GFP comet in the PVD major dendrite, minor dendrite, and the axonal compartments of the wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue. Schematics show the orientation of the comets in the plus-end-out (cyan) and minus-end-out (magenta) directions. (C) Rates of polymerization were quantified as the covered distance divided by the duration of the EBP-2::GFP comets in the PVD major dendrite, minor dendrite, and the axon of the wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.001***. Number of animals assessed (n) is mentioned along the X-axis. (D) Representative image showing the major, minor, and axonal compartments to assess the distribution of GFP::KLP-7 (5 ng extrachromosomal array) in the PVD neuron. (E and F) Representative images (E) of FRAP assay of GFP::KLP-7 in various compartments quantified as recovery profiles (F). Number of animals assessed (n) is mentioned in the legend. Comparison of maximum recovery was done using ANOVA and Bonferroni Test. p < 0.01**. Data points represent Mean±S.D. (G–I) Relative orientation of the microtubules is represented as a fraction polarity of EBP-2::GFP comets in the plus-end-out (cyan) and minus-end-out directions (magenta) in the PVD major dendrite (G), minor dendrite (H), and the axon (I) of the wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.001***, ns (not significant). Number of animals assessed (n) is mentioned along the X-axis.

In the wild-type neurons, the rates of polymerization were higher in both the major and minor dendrites as compared with the axons (Figure 4, B and C), indicating higher microtubule dynamics in the dendrites. In the klp-7(0) mutant, not only the rates of polymerization were decreased globally, but also the distinction between the axon and dendrites became obsolete (Figure 4C). KLP-7 can affect the rates of polymerization by affecting the depolymerization on the microtubule plus ends and regulating the availability of α and β tubulin dimers for new polymerization events. Indirectly, other microtubule dynamics regulators like CLASP or loss of Mini Spindles (Msps) can accumulate on the plus ends (Li et al., 2011; Moriwaki and Goshima, 2016) in the absence of Klp10-dependent depolymerization as observed for Drosophila microtubules can also participate in the process. Overexpression of the klp-7 transgene also reduced the rates of polymerization in the dendrites, whereas in the klp-7(0) background, it rescued the rates of polymerization (Figure 4C). The major dendrite of both the klp-7 overexpression (klp-7[+]) and the rescue (klp-7(0);klp-7[+]) showed higher dynamic microtubules than in the axons (Figure 4C). These results support that KLP-7-mediated depolymerization maintains the dynamic microtubules in the major dendrite of the PVD neurons.

We investigated the presence and mobility of GFP::KLP-7 in the major dendrite, minor dendrite, and the axon of the PVD neuron (Figure 4, D–F). Localization of GFP::KLP-7 was equivalent in all these compartments based on the normalized intensities (Figure 4D; Supplemental Figure S4A). Mobility of Kinesin-13/KLP-7 has been correlated to its activity in vitro and in the gentle touch neurons (Helenius et al., 2006; Tan et al., 2006; Puri et al., 2021). Unlike other cargo-carrying motors, the mobility of Kinesin-13/ Mitotic Centromere-Associated Kinesin (MCAK)/ KLP-7 is not a direct readout of its depolymerization activity but its unidimensional diffusive movement on the microtubule lattice (Helenius et al., 2006; Tan et al., 2006). However, Kinesin-13 mediated depolymerization is significantly facilitated by its movement on the microtubule lattice, indirectly correlating to its ability to scout and bind the microtubule ends for depolymerization (Helenius et al., 2006). Therefore, we assessed the mobility of GFP::KLP-7 in the primary dendrites and axon (Figure 4E) by Fluorescence Recovery After Photobleaching (FRAP) assay as an indirect readout of its activity in these compartments correlated with the differential microtubule dynamics. The FRAP assay revealed that the mobility of GFP::KLP-7 is significantly lesser in the axon with respect to the other compartments (Figure 4, E and F). Quantification of the maximum recovery from the recovery profiles showed a considerably small mobile fraction in the axon as compared with the major dendrite, and minor dendrite (Figure 4F). Moreover, a comparison of the halftimes of the recovery profiles also showed a delayed recovery in the AIS as compared with the major dendrite (Supplemental Figure S4, B and C). This is in line with the previous observations in wild-type PLM neurons where activity or mobility of KLP-7 was comparatively lower in the anterior process akin to the axon (Puri et al., 2021). The differential nature of KLP-7 in different compartments of the neuron allows these compartments to acquire a distinct microtubule organization (Puri et al., 2021).

Kinesin-13 regulates the relative orientation and distribution of dynamic microtubules in the major dendrite

Apart from microtubule dynamics, microtubule orientation is also a determiner of neuronal polarity or polarized trafficking in the neuron. The major dendrite showed minus end-out microtubules with the comets moving towards the cell body, whereas the minor dendrite and the axon showed plus end-out orientation of the microtubules (Figure 4B). This is similar to previous studies measuring the EBP-2::GFP comets in PVD neurons (Maniar et al., 2012; Harterink et al., 2018).

In the klp-7(0) mutant, the major dendrite showed a significant number of comets that are moving in the opposite direction as also visible in the kymographs (Figure 4B). We estimated the percentage of EBP-2::GFP comets in either direction for each kymograph. Based on the percentage of the EBP-2::GFP comets, the major dendrite of the wildtype had a minus end-out orientation of the microtubules, whereas the mutant has a mixed orientation (Figure 4G). On the other hand, klp-7 loss of function did not perturb the plus end-out orientation of the microtubules in the minor dendrite or the axon (Figure 4, G–I). We also quantified the density of comets in either direction, which is the number of comets per unit length of the major dendrite and per unit time of acquisition. There was a significant increase in the plus-end out microtubules in the major dendrite (Supplemental Figure S4D) and not in the minor dendrite (Supplemental Figure S4E) or axon (Supplemental Figure S4F). As the minor dendrite and the axons have plus-end out microtubules, any perturbation in the plus-end out comets is unnoticeable. The comet orientation and density were comparable to wildtype upon expression of klp-7 cDNA in the wildtype or klp-7(0) backgrounds (Figure 4, G–I; Supplemental Figure S4, D–F).

Interestingly, we observed an appearance of the EBP-2::GFP comets in the higher-order branches in the klp-7 loss of function mutant (Supplemental Figure S4G). In the wild type, EBP-2::GFP comets are present in the primary dendrites and the axon, whereas, in the klp-7(0) mutant, these comets also went into secondary branches and sometimes to tertiary branches (Supplemental Figure S4G). This could be due to the ingression of stabilized microtubules in the higher-order branches. Consequentially, it may increase the anterograde traffic of the RAB-3 or SAD-1 vesicles to the higher-order dendrites. Previous results have elucidated that increased stabilization of microtubules in the klp-7(0) results in the loss of the minus-end-out microtubule population as visualized by the minus-end reporter PTRN-1 (Puri et al., 2021). We checked the presence of the minus end protein PTRN-1::tagRFP in the PVD dendrites to understand whether the minus ends are perturbed due to the loss of the klp-7 gene. The density of the PTRN-1::tagRFP was comparable between the wildtype and klp-7(0) neurons (Supplemental Figure S4, H and I). However, we observed cohesive patches of PTRN-1::tagRFP instead of punctae in the minor dendrite and axonal segments of wild-type neurons. Similar regions were distinctly observable in the major dendrite of klp-7(0) neurons (Supplemental Figure S4H). Based on the comet densities and PTRN-1::tagRFP punctae, we concluded that KLP-7 is maintaining the microtubule polarity possibly by forming a dendritic checkpoint that regulates the entry of the plus end-out microtubules into the major dendrite.

Axon Initial Segment (AIS) protein UNC-44 accumulates in the dendrites of klp-7(0)

Previous studies have explored the dynamic microtubules in the PVD dendrites and deciphered Ankyrin (UNC-44), CRMP2 (UNC-33), UNC-119, Kinesin-1 (UNC-116), Patronin (PTRN-1), and Ninein (NOCA-2) as the major regulators of microtubule dynamics (Maniar et al., 2012; Yan et al., 2013; Taylor et al., 2015; He et al., 2020, 2022). Among these, the Ankyrin family protein, UNC-44 is a major organizer of the AIS that also helps the formation of the parallel bundle of plus-end-out microtubules in the axon through axolemmal immobilization of UNC-33 (CRMP2; He et al., 2020). Apart from the diffusion barrier function of the AIS, it acts as a sorting filter for the intracellular cargoes between the somatodendritic and axonal compartments (Burack et al., 2000; Al-Bassam et al., 2012; Farías et al., 2015; Leterrier, 2018; Eichel et al., 2022). The reliability of these functions is significantly influenced by various factors, including the structure and dimensions of the cargo, the condition of the microtubules, the interactions between cargo molecules and the cortical cytoskeleton, and the specificity of cargo-motor interactions, among others (Nakata and Hirokawa, 2003; Lewis et al., 2009; Kuijpers et al., 2016; Balasanyan et al., 2017). In C. elegans, the loss of AIS components like UNC-44, UNC-119, and UNC-33 resulted in the loss of axon-dendrite compartmentalization (Maniar et al., 2012; He et al., 2020). It is unclear whether KLP-7 somehow regulates the components of the AIS defining the axon-dendrite compartmentalization in PVD.

To explore the role of KLP-7 in the maintenance of the AIS, we imaged a GFP reporter of UNC-44 in the PVD neurons (He et al., 2020). Based on the molecular constitution and function of the mammalian AIS, a homologous structure has been defined in the laterodistal region of the PVD axon (Figure 5A). UNC-44::GFP expressed under its own promoter has ubiquitous expression and is present in all the neurites of PVD neurons (Figure 5B). Mean intensity quantification of UNC-44::GFP in the primary dendrites and AIS showed a remarkable enrichment in the AIS region (Figure 5, B and C). The intensity was highest in the AIS region (cyan arrowheads, Figure 5, B and C). In the klp-7(0) mutant, the bona fide AIS (cyan arrowhead) did not show a similar degree of enrichment (Figure 5C). However, in the klp-7(0) mutant, the levels of UNC-44::GFP in both the primary dendrites and axon were comparable to those in wild type neurons (Figure 4C). This indicated that differential enrichment of UNC-44 in the AIS as compared with the other neurites requires klp-7. Overexpression of the klp-7 isoform caused a drastic decrease in the UNC-44 enrichment at the AIS with comparable intensities in the dendrites (Figure 5C). In the rescue worms, UNC-44::GFP intensity in the AIS was slightly improved, however, it was not significantly different than the dendritic intensity. This could be due to the formation of bona fide AIS coinciding with or preceding the expression of the klp-7 transgene under the ser-2 (prom3) promoter (Tsalik et al., 2003), thus partially rescuing the phenotype.

FIGURE 5:

Specific enrichment of AIS protein, UNC-44 is dependent on klp-7 function. (A) Schematic showing the dendrites (major and minor), ectopic branches, and AIS compartments to assess the distribution of UNC-44::GFP in a typical PVD neuron magnified in the middle panel. (B) Representative images of the PVD neuron in wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue expressing mScarlet and single copy insertion of UNC-44::GFP under its endogenous promoter. Yellow and cyan arrowheads depict the ectopic branches and AIS of the PVD neuron. (C) Mean intensities of UNC-44::GFP in the dendrites (major and minor) and AIS of PVD neurons of wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.01**, 0.001***, ns (not significant). Number of animals assessed (n) is mentioned along the X-axis. (D) Ectopic branches emanating from the cell bodies of wildtype (wt), mutant klp-7(0), klp-7[+] overexpression, and klp-7(0);klp-7[+] rescue neurons were assessed for their enrichment and quantified as the mean intensity of UNC-44::GFP. Comparison of means was done using ANOVA and Bonferroni Test. p < 0.01**, 0.001***, ns (not significant). Number of animals assessed (n) is mentioned along the X-axis.

Interestingly, loss of klp-7 also results in the formation of ectopic branches that emanate from the cell body (Figure 5B, orange arrowheads). The occurrence of such ectopic branches was quite lower in the wildtype (Supplemental Figure S4A). As compared with the ectopic branches in the wildtype neurons, the klp-7(0) neurons showed an enrichment comparable to that of the AIS (Figure 5B). Around 70% of the ectopic branches of the klp-7(0) showed AIS-like enrichment as compared with 25% of the wildtype ectopic branches (Supplemental Figure S4A). UNC-44::GFP intensities in the ectopic branches of the PVD neurons of klp-7(0) were comparatively higher, than that of wildtype (Figure 5D). In the overexpression and rescue transgenic, the ectopic branches were similar in UNC-44::GFP intensity as those in wildtype (Figure 5D). The formation of AIS coincides with the formation of a polarized array of microtubules by increased stabilization and fasciculation, which are codependent to ensure polarized sorting of the cargoes between axonal and dendritic compartments (Fréal et al., 2019). Increased microtubule stabilization in klp-7 null mutant may enrich UNC-44 in a non-specific manner to PVD dendrites, which causes aberrant sorting of RAB-3, its motor UNC-104, and plus end out microtubules. Recent evidence of ectopic accumulation of TRIM46 in the Kif2a knockout hippocampal neurons (Ruiz-Reig et al., 2022) also supports this hypothesis.

The relationship between the proportion of major dendrites with mislocalization of presynaptic cargoes RAB-3/SAD-1, along with the increased ratio of plus-end out microtubules indicated a dependence on KLP-7 depletion for these traits (Figure 6A). Together with this, the formation of ectopic AIS-like dendritic processes in KLP-7 loss of function (Figure 6, B and C) showed how dendrites can attain axon-like identity due to the depletion of KLP-7 function.

FIGURE 6:

KLP-7 mediated axonal checkpoint determining axonal sorting of cargoes and microtubules. (A) Proportion of major dendrites with characteristic features of dendrite and axon differentiated based on microtubule polarity, RAB-3, and SAD-1 densities in the wildtype (wt) and klp-7(0) mutant represented as a stacked bar graph. The criteria are defined by the mean value of each feature in the wildtype major dendrite as mentioned in the legend. The filled and open areas in each stacked bar represent the proportions with dendrite and axon-like characteristics, respectively. Comparison between wt and klp-7(0) was done using Fisher’s exact test. p < 0.01**, 0.001***. Number of animals assessed (n) is mentioned along the X-axis. (B) Microtubule depolymerization activity of Kinesin-13 (KLP-7) maintains differential microtubule dynamics in the dendrites and axons. The presence of dynamic microtubules due to this activity prevents the enrichment of Ankyrin (UNC-44) in dendrites of wild-type neurons. Specific enrichment of UNC-44 in the AIS ensures trafficking of plus end out microtubules, axonal motor (UNC-104), and presynaptic cargoes into the axon only. (C) Graphical representation of wildtype and klp-7(0) PVD neurons. The absence of microtubule depolymerization in the klp-7(0), decreases the microtubule dynamics in dendrites as well as axons leading to ectopic enrichment of UNC-44. Enrichment of UNC-44 in the AIS of wildtype or dendrites of klp-7(0) directs the axonal traffic of plus end microtubules and axonal motor, UNC-104, and presynaptic cargoes (RAB-3 and SAD-1) in these compartments. Accumulation of AIS component UNC-44, plus end-out microtubules, axonal motor UNC-104, and presynaptic cargoes RAB-3/SAD-1 in processes other than axon attributes axon-like identity to such processes.

DISCUSSION

Our study demonstrates that KLP-7/Kinesin-13 plays a crucial role in regulating the compartmentalization of axons and dendrites in the PVD neurons. Previous studies indicated the importance of Kinesin-13 in establishing dendritic identity (Homma et al., 2018). In this study, we shared a mechanistic angle on how the activity of KLP-7 is related to the differential level of microtubule dynamics in dendrite versus axon. Increased microtubule instability driven by KLP-7 is key to preventing the accumulation of AIS components in the dendrites, delimiting the entry of axonal proteins into dendrites (Figure 6). This provides new insight into how microtubule instability and AIS element Ankyrin/UNC-44 are antagonistically related to control neuronal polarity (Figure 6).

KLP-7/Kinesin-13 dependent microtubule organization regulates the axon dendrite compartmentalization

Neuronal polarization commences with axon specification (Dotti et al., 1988; Higgs and Das, 2022) determined by microtubule stabilization and bundling (Witte et al., 2008; Van Beuningen et al., 2015). The stability of the microtubules imparts strength to the growing axon, provides a platform for other cytoskeletal interactions, promotes selective trafficking of molecules, and generates force for changes in morphology during growth (Hawkins et al., 2010; Kapitein and Hoogenraad, 2011). It is unclear how microtubule catastrophe factors, which also contribute to the dynamic instability of microtubules participate in neuronal polarization.

Loss of Kinesin-13/KIF2/KLP-7 causes an aberrant arborization of neurons with defects in axon-dendrite compartmentalization and premature degeneration (Homma et al., 2018; Puri et al., 2021; Ruiz-Reig et al., 2022). Conditional KIF2 knockout animals also showed behavioral defects like epilepsy (Homma et al., 2018; Partoens et al., 2021). Mechanistic insight into these phenotypes due to the loss of Kinesin 13 was lacking. We found that microtubule stabilization in the loss of function of KLP-7 (Kinesin-13) could lead to the formation of multiple axon-like neurites during development, similar to that observed upon Taxol treatment (Witte et al., 2008). We showed other microtubule depolymerizers like EFA-6 did not affect the axon-dendrite compartmentalization. We could significantly suppress the defect of the klp-7 null mutant by Colchicine treatment and fully rescue by cell-autonomous expression of klp-7 transgene, indicating additional roles of KLP-7 besides microtubule stabilization in preserving the axon-dendrite compartmentalization.

KLP-7 is a microtubule depolymerizing enzyme, and under the Wnt signaling, its differential activity in the PLM neurites regulates its microtubule organization (Desai et al., 1999; Puri et al., 2021). but it was unclear how KLP-7 organizes the microtubules in the PVD neurons. We showed that loss of klp-7 resulted in decreased polymerization and a mixed orientation of microtubules in the major dendrite, which typically has minus end-out microtubule polarity. In other neurites with plus end out polarity, that is, minor and axon, the polymerization dynamics were affected, but the polarity of the microtubules was unchanged by klp-7 deletion. In the klp-7 null mutant, apart from an increase in the plus-end out EBP-2::GFP comets, we observed a concomitant decrease in the minus-end out microtubules, which rules out the possibility of specific depolymerization of plus-end out microtubules by KLP-7 in the major dendrite. Furthermore, overexpression of klp-7 did not perturb the polarity of microtubules in the major dendrite. It could be an effect of global disruption of intraneuronal trafficking of cargoes, including axonal cargoes and microtubules.

KLP-7 negatively regulates the formation and integrity of the AIS-like sorting filters in the PVD neurites

Previous studies have explored neuronal polarity in the PVD neurons and have found the role of UNC-44, UNC-33, UNC-119, TIAM-1, PTRN-1 and NOCA-2 (Maniar et al., 2012; He et al., 2020, 2022; Lin et al., 2022). In the loss of function mutants of these genes, besides the mislocalization of axonal cargoes in the dendrites, microtubule polarity in the neurites was also perturbed (Maniar et al., 2012; He et al., 2020, 2022; Lin et al., 2022). Additional mechanisms like mislocalization of UNC-104 to dendrites, defects in Kinesin-1 mediated microtubule sliding, loss of cytoskeletal interactions, and possible defects in microtubule nucleation were also associated with these mutants (Maniar et al., 2012; He et al., 2020, 2022; Lin et al., 2022). It was unclear how microtubule depolymerizing enzymes such as KLP-7 coordinate with these molecules to regulate the microtubule organization and neuronal polarity.

An aberrant transport of axonal proteins in the major dendrite in the klp-7(0) could be due to the ectopic formation of AIS-like sorting filter in these dendrites. The AIS serves two critical functions in neurons: 1.) Acting as a diffusion filter to regulate the movement of cytoplasmic and membrane proteins (Winckler et al., 1999; Nakada et al., 2003; Albrecht et al., 2016; Eichel et al., 2022), 2.) Sorting intracellular cargo between the axonal and somatodendritic compartments both before and within the AIS (Burack et al., 2000; Al-Bassam et al., 2012; Farías et al., 2015; Leterrier, 2018; Eichel et al., 2022). The effectiveness of these functions relies heavily on factors such as the cargo structure and size, the condition of microtubules, interactions between cargo molecules and the cortical cytoskeleton, and the specificity of cargo-motor interactions (Nakata and Hirokawa, 2003; Lewis et al., 2009; Kuijpers et al., 2016; Balasanyan et al., 2017). Previous studies in the C. elegans system have demonstrated that UNC-44 is essential for the function of the AIS, which restricts axonal cargoes such as RAB-3 and somatodendritic cargoes such as DMA-1 to their respective compartments (Maniar et al., 2012; He et al., 2020; Eichel et al., 2022). Loss of UNC-44 also perturbs the microtubule orientation in the dendrites towards an increase in the plus end out microtubules, which may support the movement of anterograde cargoes (Maniar et al., 2012; He et al., 2020). These results indicate that the axonal microtubules, motors, and cargoes prefer the neurites where UNC-44 is likely to be in a membrane-anchored complex with UNC-119 and UNC-33L (large isoform; He et al., 2020).

Although the AIS protein Ankyrin/UNC-44 was ubiquitously localized to all neurites of PVD, normalized intensity of this protein in the wildtype PVD neurons showed a marked enrichment in the bona fide AIS defined as per the molecular constitution akin to mammalian AIS (Maniar et al., 2012; He et al., 2020). Loss of klp-7 affected the selective enrichment of UNC-44::GFP in the AIS with additional enrichment of UNC-44::GFP in dendritic branches. This was conspicuous in the ectopic branches emanating from the cell bodies of PVD neurons. Though this anatomical feature was rare in wildtype animals, these branches showed an excess of UNC-44::GFP in the deletion mutant of klp-7. This result corroborated previous observations of mosaic arbors in Drosophila class IV ddaC neurons upon Patronin RNAi with ectopic accumulation of AIS components and hippocampal neurons of Kif2A conditional knockout rodents with ectopic TRIM46 enrichment (Ruiz-Reig et al., 2022; Thyagarajan et al., 2022).

Formation of AIS in the axon correlates well with the decreased dynamics of microtubule polymerization and GFP::KLP-7. In developing anterior PVD dendrite, the UNC-44/UNC-33 complex is excluded from the distal MTOC where dynamic microtubules are prevalent (Liang et al., 2024). In our study, in the absence of catastrophe factor KLP-7, microtubules are relatively stable and the complex of UNC-44/UNC-119/UNC-33/nondepolymerizing microtubules is probably stabilized and forms a sorting structure akin to AIS. Our results of klp-7 overexpression, which perturbed the bonafide localization of UNC-44::GFP at the AIS strengthened this hypothesis. AIS-type accumulations of UNC-44 in the dendrites may serve as conduits for axonal traffic. Due to loss of klp-7, axon-specific motor UNC-104 was significantly increased in the dendrites indicating most of the axonal traffic to be also present in the dendrites. KLP-7 activity in the dendrites regulates the microtubule polymerization and dynamics, which further prevent the formation and integrity of the AIS-like sorting filters thereby restricting the traffic of axonal microtubules, motors, and cargoes. The conditions that allow axonal traffic into any neurite appear to be the presence of AIS components, and stable and plus-end out microtubules which are fulfilled in the klp-7(0) dendrites.

Overall, our study sheds light on the intricate mechanisms underlying the regulation of axon-dendrite compartmentalization through KLP-7-mediated microtubule regulation and AIS formation and integrity (Figure 6, B and C).

MATERIALS AND METHODS

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C. elegans culturing, constructs, and transgenesis

The worms were cultured on the Nematode Growth Medium (NGM) seeded with OP50 bacteria at 20°C. Mutants and transgenics were crossed, and homozygosity of mutant and transgene alleles was confirmed by genotyping and phenotyping. The mutant and transgenes are mentioned in Supplemental Table S1. The allele kyIs445 [pdes-2::mCherry::RAB-3 (0.5 ng/µl); pdes-2::SAD-1::GFP (2 ng/µl); podr-1::DsRed (30 ng/µl)] is a gift from Cory Bargmann lab, ebp-2(wow47 [ebp-2::gfp::3xflag]) is a gift from Feldman lab and. To generate shrEx483, young adult worms were microinjected with 5 ng/µl of pser2prom3::gfp::klp-7b, 10 ng/µl of pser2prom3::mScarlet, and 50 ng/µl of pttx-3::rfp. The construct of pser2prom3::gfp::klp-7b was generated using the InFusion cloning. Similarly, shrEx490 was generated by microinjecting 15 ng/µl of pser2prom3:: klp-7b, 10 ng/µl of pser2prom3::mScarlet, and 2 ng/µl of pmyo-2::mCherry.

Sample preparation

Neuronal polarity was assayed in L4 staged worms for both control and mutant conditions. The worms were harvested after a definite period and, are mounted on 7.5% agarose pads using a cocktail of 0.1% Tricaine and 10 mM Tetramisole.

For imaging of microtubule-associated End Binding Protein (EBP) comets, the worms were mounted on 10% agarose pads in a mounting medium of 0.1µm polystyrene beads. A maximum of five to seven worms were mounted simultaneously for imaging. For FRAP assays, the mounting was done in a mixture of 0.1% Tricaine and 10 mM Tetramisole as described before.

Worms were grown on Colchicine (1 mM) containing NGM plates because hatching for the pharmacological perturbation of the microtubules.

Microscopy

Worms were imaged on the Nikon A1R confocal using 60X/1.4NA oil objective at a voxel scale of 0.414 µm x 0.414 µm x 1.0 µm. A large area scanning was enabled to image the whole worm. To image multiple reporters sequential channel imaging was used. For some ubiquitously expressed reporters like UNC-104::GFP, and UNC-44::GFP, the imaging was carried out at a voxel scale of 0.09 µm x 0.09 µm x 0.125 µm using 60X/1.4NA oil objective.

Live imaging of EBP comets was carried out on the Zeiss Observer.Z1 inverted microscope equipped with Yokogawa CSU-X1 spinning disk unit and Photometrics Evolve camera. Imaging was carried out on a 100X/1.4NA oil objective at a temporal resolution of one frame per second for 2 min.

mCherry::RAB-3 particles were observed using Zeiss Observer.Z1 inverted microscope equipped with Yokogawa CSU-X1 spinning disk unit and Photometrics Evolve camera. Imaging was done on a 100X/1.4NA oil objective at a temporal resolution of 1.4 frames per second for 2 min.

FRAP assays were carried out on the Nikon A1R confocal using 60X/1.4NA oil objective at a voxel scale of 0.09 µm × 0.09 µm × 0.2 µm. A circular region of 5-µm diameter was chosen for the photobleaching, and the entire acquisition was carried out at a frame rate of one frame per second. Before photobleaching, five frames were collected, photobleaching was carried out for 5 s, and post photobleaching acquisition was carried out for a duration of 2 min.

Analysis

Neuronal polarity has been assayed by the percentage of dendrites with mCherry::RAB-3, density of mCherry::RAB-3 punctae, and population of worms with dendritic SAD-1::GFP in the primary dendrites. The percentage of dendrites and population distribution has been assessed manually by locating punctae over a particular threshold intensity. This threshold has been set by the intensity of the mCherry::RAB-3 punctae present in the axon (ventral nerve cord) in the wildtype neurons. The density and mean intensities of mCherry::RAB-3 or SAD-1::GFP punctae have been assessed by the line profile of intensities in the primary dendrites and peak finding tool using the BAR plugin in ImageJ (Fiji). The density of the punctae was calculated by the number of punctae divided by the length of the neurite while the peak intensities averaged for all punctae in the assessed neurite yield the mean intensity. Relative density and mean intensity were obtained by normalizing dendritic values with axonal values. PTRN-1::tagRFP punctae were also analyzed for their density in the primary dendrites using the line profile and peak finding tool using the BAR plugin in ImageJ. For quantification of neuro-prevalent reporters like UNC-104::GFP and UNC-44::GFP in the PVD neurites, we used the constitutive reporter mScarlet for tracing neurites using the SNT plugin in ImageJ (Fiji). From the linescan profile of each neurite for the reporters (UNC-104::GFP or UNC-44::GFP), intensities and lengths of the neurite were obtained. The total of the intensities was divided by the length of the neurite to obtain the intensity per unit length of the neurite.

Timelapse acquisitions of EBP-2::GFP comets were analyzed in ImageJ by extracting kymographs from different neurites of PVD neurons. The traces in the kymographs were assessed using the line tool to obtain the angle, length, and duration. Slopes of the traces obtained from the angles were used for the orientation of the comets. The length of the traces was divided by their duration to obtain the rates of polymerization.

The images and videos were quantified using ImageJ. All the images were background-corrected and normalized to the maximum. Intensities obtained from the FRAP region were normalized to prebleach and postbleach intensities and plotted against time. The FRAP curves were fitted to the following single exponential equation using the Levenberg-Marquardt iteration algorithm in Origin:

The maximum recovery amplitude (I_max) and the half-time (t_1/2 = ln 0.5/–τ) parameters were obtained from the fit.

Intensities in a given neurite were quantified from a line ROI spanning across multiple optical sections and normalized with a constitutive marker.

Data representation and statistics

Quants obtained from ImageJ were fed into Excel and Origin to get statistically relevant data. The data has been plotted as violin and box-whisker plots with individual values. The mean value is represented by black squares within the plots. Significance values of p < 0.05*, 0.01**, and 0.001*** are marked within the plots between the compared datasets. Relevant nonsignificant comparisons have been marked by “ns.” The number of organisms tested is mentioned in the respective plots. Each experimental dataset has been obtained from a minimum of N = 3 experiments carried out across multiple generations of the worm strain or condition. Parametric data were compared using ANOVA and Bonferroni test. Nonparametric datasets were compared using Fisher’s exact test and are mentioned in the figure legends.

Abbreviations used:

AIS

axon initial segment

ANOVA

analysis of variance

BAR

broadly applicable routines plugin

CGC

Caenorhabditis elegans center

CLASP

cytoplasmic linker-associated proteins

CRISPR

clustered regularly interspaced short palindromic repeats

CRMP2

collapsin response mediator protein 2

DBT

department of biotechnology

DMA-1

dendrite morphology abnormal-1

EBP-2

end binding protein-2

EFA-6

exchange factor for Arf-6

FRAP

fluorescence recovery after photobleaching

GFP

green fluorescent protein

IA

India Alliance

KIF2

kinesin family member 2

KLP-7

kinesin-like protein-7

Klp10

kinesin like protein 10

MAP2C

microtubule associated protein 2C

MCAK

mitotic centromere-associated kinesin

Msps

mini spindles

NBRC

National Brain Research Centre

NGM

nematode growth medium

NIH

National Institute of Health

NOCA-2

Non-centrosomal microtubule array-2

Op18

oncoprotein 18

PLM

posterior lateral microtubule neuron

PTRN-1

patronin homolog-1

PVD

posterior ventral process D

RAB-3

Ras-associated binding homolog-3

ROI

Region of Interest

SAD-1

synapses of amphid defective-1

SNT

simple neurite tracer

TIAM-1

tumor invasion and metastasis factor homolog-1

TRIM46

tripartite motif containing 46

UNC-104

uncoordinated-104

UNC-119

uncoordinated-119

UNC-33

uncoordinated-33

UNC-44

Uncoordinated-44