Optimization of RNAi efficiency in PVD neuron of C. elegans.

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to its elaborate and stereotype dendrite morphology. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to knocking down genes in nervous system. In order to optimize the RNAi conditions for PVD neuron, we fed the worm strains with E. coli HT115 bacteria expressing dsRNA against mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the available sensitive backgrounds including the one expresses sid-1 under unc-119 promoter, although resulted in reduction of dendrite branching, the phenotypes were significantly modest compared to the respective loss of function mutants. In order to enhance RNAi in PVD neurons, we generated a strain that expressed sid-1 under the promoter mec-3, which exhibits strong expression in PVD. When Pmec-3::sid-1 is expressed in either nre-1(-)lin-15b(-) or lin-15b(-) backgrounds, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to the genetic background alone. Moreover, knockdown of genes playing role in dendrite regeneration in the nre-1(-)lin-15b(-), Pmec-3-sid-1[+] background resulted in significant reduction in dendrite regeneration following laser injury. The extent of dendrite regrowth due to the RNAi of aff-1 or ced-10 in our optimized strain was comparable to that of aff-1 and ced-10 mutants. Essentially, our strain expressing sid-1 in PVD neuron, provides an RNAi optimized platform for high throughput screening of genes involved in PVD development, maintenance and regeneration.

Functional Recovery Associated with Dendrite Regeneration in PVD Neuron of Caenorhabditis elegans.

PVD neuron of Caenorhabditis elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities including the control of both nociceptive touch sensation and body posture. Although both the axon and dendrites of this neuron show a regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1/MLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration through the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery in posture. Our data revealed that the axons and dendrites of PVD neurons regulate the nociception and proprioception in worms, respectively. It also revealed that dendrite and axon regeneration lead to the restoration of these differential sensory modalities.

Chromophore appended DPA-based copper(II) complexes with a diimine motif towards DNA binding and fragmentation studies.

Mixed ligand copper(II) complexes [Cu(L1)(bpy)](ClO4)21 and [Cu(L2)(bpy)](ClO4)22 (where L1 = 1-(anthracen-9-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine, L2 = 1-(pyren-1-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine and bpy = 2,2'-bipyridine) were synthesised and characterised thoroughly via different analytical and spectroscopic techniques i.e., UV-vis spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, HRMS and EPR spectroscopy. The molecular structures of the synthesised complexes were obtained using the single-crystal X-ray diffraction technique. Both complexes exhibited penta-coordinated and acquired distorted square pyramidal geometry. The redox behaviour of complexes 1 and 2 was investigated by employing cyclic voltammetry. The DNA binding study was carried out by UV-vis spectrophotometry using double-stranded salmon sperm DNA (ds-ss-DNA). The binding constant (Kb) values of 1 and 2 were 0.11 × 104 M-1 and 1.05 × 104 M-1, respectively, which indicates that 2 has better binding ability than 1. This might be due to the higher conjugative abilities with the extended surface area of the aromatic pyrene ring compared to the anthracene moiety. The fluorescence quenching experiments were also performed with EB bound DNA (EB-DNA) and Stern-Volmer constant (KSV) values were calculated as 1.23 × 105 M-1 and 1.39 × 105 M-1 for 1 and 2, respectively, suggesting that 2 showed stronger interaction with ss-DNA than 1. The molecular docking data support the DNA-binding studies, with the sites and mode of interactions against B-DNA varying with 1 and 2. Evaluation of the DNA binding properties of the complexes to linearized plasmid DNA indicated that 2 had modest DNA binding properties, which is a pre-requisite for a genotoxic agent. The effect of 1 and 2 on cell survival was analysed using HeLa cells by MTT assay and it was observed that the IC50 values of 1 and 2 were 43.7 µM and 18.6 µM, respectively. Our study paves the way for the designing of bio-inspired novel mixed metal complexes, which shows promising results for further exploration of molecular and mechanistic studies towards the development of non-platinum based economical metallodrugs.

Paraquat induced acute kidney and lung injury with a dramatic response to methylprednisolone: A case report.

Background: Paraquat poisoning is one of the leading causes of fatal poisoning in many parts of the world, especially in agricultural countries. Its high toxicity even in small amounts causes rapid damage to multiple organs, especially the kidneys, lungs, and liver, mainly through free radical-mediated injury. As no specific antidote is yet available, early diagnosis and the importance of supportive therapy are critical parts of management. Some evidence suggests a survival benefit from using immunosuppressive drugs. Case report: This case presentation concerns a 15-year-old boy from a village with a history of herbicide poisoning, later confirmed to be paraquat. Despite supportive therapy her condition continued to deteriorate with features of kidney and lung damage. The patient was then treated with methylprednisolone 500 mg daily for 5 days, along with other supportive care, and has made a remarkable recovery. Conclusions: High efficacy as an herbicide, availability and low cost make paraquat an easy-to-encounter poison for suicidal or accidental use. Its high fatality calls for urgent and effective strategies to save lives. Methylprednisolone may play a role in its treatment.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Muscleblind-1 interacts with tubulin mRNAs to regulate the microtubule cytoskeleton in C. elegans mechanosensory neurons.

Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and reduction of gentle touch sensation in mbl-1 mutant. Our data also revealed that mbl-1 is genetically epistatic to mec-7 (ß tubulin) and mec-12 (α tubulin) in regulating axon growth. Furthermore, mbl-1 is epistatic to sad-1, an ortholog of BRSK/Brain specific-serine/threonine kinase and a known regulator of synaptic machinery, for synapse formation at the correct location of the PLM neurite. Notably, the immunoprecipitation of MBL-1 resulted in the co-purification of mec-7, mec-12, and sad-1 mRNAs, suggesting a direct interaction between MBL-1 and these transcripts. Additionally, mbl-1 mutants exhibited reduced levels and stability of mec-7 and mec-12 transcripts. Our study establishes a previously unknown link between RNA-binding proteins and cytoskeletal machinery, highlighting their crucial roles in the development and maintenance of the nervous system.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site.

The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

A new method for customized fetal growth reference percentiles.

BACKGROUND: Customized fetal growth charts assume birthweight at term to be normally distributed across the population with a constant coefficient of variation at earlier gestational ages. Thus, standard deviation used for computing percentiles (e.g., 10th, 90th) is assumed to be proportional to the customized mean, although this assumption has never been formally tested. METHODS: In a secondary analysis of NICHD Fetal Growth Studies-Singletons (12 U.S. sites, 2009-2013) using longitudinal sonographic biometric data (n = 2288 pregnancies), we investigated the assumptions of normality and constant coefficient of variation by examining behavior of the mean and standard deviation, computed following the Gardosi method. We then created a more flexible model that customizes both mean and standard deviation using heteroscedastic regression and calculated customized percentiles directly using quantile regression, with an application in a separate study of 102, 012 deliveries, 37-41 weeks. RESULTS: Analysis of term optimal birthweight challenged assumptions of proportionality and that values were normally distributed: at different mean birthweight values, standard deviation did not change linearly with mean birthweight and the percentile computed with the normality assumption deviated from empirical percentiles. Composite neonatal morbidity and mortality rates in relation to birthweight < 10th were higher for heteroscedastic and quantile models (10.3% and 10.0%, respectively) than the Gardosi model (7.2%), although prediction performance was similar among all three (c-statistic 0.52-0.53). CONCLUSIONS: Our findings question normality and constant coefficient of variation assumptions of the Gardosi customization method. A heteroscedastic model captures unstable variance in customization characteristics which may improve detection of abnormal growth percentiles. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00912132.

Antioxidant conjugated metal complexes and their medicinal applications.

Antioxidants are naturally available and man-made substances have the ability to protect cells from damage due to a number of intracellular redox activities. Moreover, Antioxidants such as α-lipoic acid, curcumin and catechin are good anticancer agents. In recent years, the usage of metal complexes as therapeutic agents is gaining importance due to their useful biological properties. Most of the metal ions act as the essential components in building drug molecules that serve as medicines for cancer and neurodegenerative diseases. In particular, metals like copper, gold, ruthenium, and platinum have adequate anticancer properties at both micro- and nano-levels. Hence, conjugation of antioxidants with metals and metal-based compounds results in hybrid bioactive materials with improved anticancer properties. In this chapter, medicinal applications of antioxidant conjugated metal complexes are reviewed and discussed.

The association between first-trimester omega-3 fatty acid supplementation and fetal growth trajectories.

BACKGROUND: Prenatal omega-3 fatty acid supplementation, particularly docosahexaenoic acid and eicosapentaenoic acid, has been associated with greater birthweight in clinical trials; however, its effect on fetal growth throughout gestation is unknown. OBJECTIVE: This study aimed to examine the association between first-trimester docosahexaenoic acid and eicosapentaenoic acid supplementation and growth trajectories of estimated fetal weight and specific fetal biometrics measured longitudinally from the second trimester of pregnancy to delivery. STUDY DESIGN: In a multisite, prospective cohort of racially diverse, low-risk pregnant women, we used secondary data analysis to examine fetal growth trajectories in relation to self-reported (yes or no) first-trimester docosahexaenoic acid and eicosapentaenoic acid supplementation. Fetal ultrasonographic measurements, including abdominal circumference, biparietal diameter, femur length, head circumference, and humerus length, were measured at enrollment (8-13 weeks) and up to 5 follow-up visits. Estimated fetal weight and head circumference-to-abdominal circumference ratio (a measure of growth symmetry) were calculated. Fetal growth trajectories were modeled for each measure using a linear mixed model with cubic splines. If significant differences in fetal growth trajectories between groups were observed (global P<.05), weekly comparisons were performed to determine when in gestation these differences emerged. Analyses were adjusted for maternal sociodemographics, parity, infant sex, total energy consumption, and diet quality score. All analyses were repeated using dietary docosahexaenoic acid and eicosapentaenoic acid intake, dichotomized at the recommended cutoff for pregnant and lactating women (≥0.25 vs <0.25 g/d), among women who did not report supplement intake in the first trimester of pregnancy were repeated. RESULTS: Among 1535 women, 143 (9%) reported docosahexaenoic acid and eicosapentaenoic acid supplementation in the first trimester of pregnancy. Overall, first-trimester docosahexaenoic acid and eicosapentaenoic acid supplementation was associated with statistically significant differences (P-value <.05) in fetal growth trajectories during pregnancy. Specifically, estimated fetal weight was larger among women with docosahexaenoic acid and eicosapentaenoic acid supplementation than among those without supplementation (global P=.028) with significant weekly differences in median estimated fetal weight most apparent between 38 to 41 weeks of gestation (median estimated fetal weight difference at 40 weeks of gestation, 114 g). Differences in fetal growth trajectories for abdominal circumference (P=.003), head circumference (P=.003), and head circumference-to-abdominal circumference ratio (P=.0004) were also identified by supplementation status. In weekly comparisons, docosahexaenoic acid and eicosapentaenoic acid supplement use was associated with larger median abdominal circumference (changed from 2 to 9 mm) in midpregnancy onward (19 to 41 weeks), larger median head circumference between 30 to 33 weeks of gestation, and smaller median head circumference-to-abdominal circumference ratio in the second and third trimesters of pregnancy. There was no specific weekly difference in fetal femur length or humerus length by docosahexaenoic acid and eicosapentaenoic acid supplementation. First-trimester dietary sources of docosahexaenoic acid and eicosapentaenoic acid among women with no first-trimester docosahexaenoic acid and eicosapentaenoic acid supplementation (n=1392) were associated with differences in fetal biparietal diameter (P=.043), but not other metrics of fetal growth. At the recommended dietary docosahexaenoic acid and eicosapentaenoic acid levels compared with below-recommended levels, biparietal diameter was larger between 38 to 41 weeks of gestation. CONCLUSION: In this racially diverse pregnancy cohort, first-trimester docosahexaenoic acid and eicosapentaenoic acid supplementation was associated with significant increases in fetal growth, specifically greater estimated fetal abdominal circumference in the second and third trimesters of pregnancy.

Large-scale design and refinement of stable proteins using sequence-only models.

Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we use a high-throughput, low-fidelity assay to experimentally evaluate the stability of approximately 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We build a neural network model that predicts protein stability given only sequences of amino acids, and compare its performance to the assayed values. We also report another network model that is able to generate the amino acid sequences of novel stable proteins given requested secondary sequences. Finally, we show that the predictive model-despite weaknesses including a noisy data set-can be used to substantially increase the stability of both expert-designed and model-generated proteins.

Dendrite regeneration in C. elegans is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1.

Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects-territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.

Efficient integration of aggregate data and individual participant data in one-way mixed models.

Often both aggregate data (AD) studies and individual participant data (IPD) studies are available for specific treatments. Combining these two sources of data could improve the overall meta-analytic estimates of treatment effects. Moreover, often for some studies with AD, the associated IPD maybe available, albeit at some extra effort or cost to the analyst. We propose a method for combining treatment effects across trials when the response is from the exponential family of distribution and hence a generalized linear model structure can be used. We consider the case when treatment effects are fixed and common across studies. Using the proposed combination method, we study the relative efficiency of analyzing all IPD studies vs combining various percentages of AD and IPD studies. For many different models, design constraints under which the AD estimators are the IPD estimators, and hence fully efficient, are known. For such models, we advocate a selection procedure that chooses AD studies over IPD studies in a manner that force least departure from design constraints and hence ensures an efficient combined AD and IPD estimator.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons.

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16's inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.

A microfluidic-enabled combinatorial formulation and integrated inkjet printing platform for evaluating functionally graded material blends.

Sample library preparation is a central step in the process of evaluating materials with the general aim of efficient library formulation while minimizing resource consumption. We demonstrate here the first implementation of a microfluidic-enabled thin film sample library formulation platform with integrated inkjet printing capability for directly patterning these libraries with reduced material wastage. System development and general performance screening protocol for these patterned thin films are described. We study the combinatorial formulation capabilities of this system by focusing on some practical case studies for probing the electrical conductivity in organic, biocompatible and electroactive polymer/additive (PEDOT:PSS/DMSO and PEDOT:PSS/EG) blends. Functionally-graded thin film libraries are prepared by mixing ink components and directly dispensing the processed blends into programmed geometries using the integrated platform. Electrical and morphological characterization of these printed thin film libraries is conducted to validate the formulation efficacy of the platform. Interrogating these printed libraries, we were able to iteratively identify the location of conductivity maxima for the studied blends and corroborate the morphological basis of this enhancement with established theories.

Overview of Methods for Purification and Characterization of Metalloproteins.

Metalloproteins make up one third of all proteins and perform some of the most essential reactions on earth. The unique properties of the metal ions within these proteins, and in particular of redox-active metal ions, enables the use of a number of characterization techniques. It also necessitates unique considerations in terms of purification and characterization. In this overview, we describe the considerations and methods used for metalloprotein purification and characterization. © 2021 Wiley Periodicals LLC.

Wnt signaling establishes the microtubule polarity in neurons through regulation of Kinesin-13.

Neuronal polarization is facilitated by the formation of axons with parallel arrays of plus-end-out and dendrites with the nonuniform orientation of microtubules. In C. elegans, the posterior lateral microtubule (PLM) neuron is bipolar with its two processes growing along the anterior-posterior axis under the guidance of Wnt signaling. Here we found that loss of the Kinesin-13 family microtubule-depolymerizing enzyme KLP-7 led to the ectopic extension of axon-like processes from the PLM cell body. Live imaging of the microtubules and axonal transport revealed mixed polarity of the microtubules in the short posterior process, which is dependent on both KLP-7 and the minus-end binding protein PTRN-1. KLP-7 is positively regulated in the posterior process by planar cell polarity components of Wnt involving rho-1/rock to induce mixed polarity of microtubules, whereas it is negatively regulated in the anterior process by the unc-73/ced-10 cascade to establish a uniform microtubule polarity. Our work elucidates how evolutionarily conserved Wnt signaling establishes the microtubule polarity in neurons through Kinesin-13.