Characterizing human KIF1Bß motor activity by single-molecule motility assays and Caenorhabtidis elegans genetics.

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1Bß is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1Bß lead to a wide range of neuropathies. While previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1Bß remain elusive. In this study, we determined the motile parameters of KIF1Bß through single-molecule motility assays. We find that the C-terminal region of KIF1Bß has an inhibitory role in the motor activity. Alphafold2 prediction suggests that the C-terminal region blocks the motor domain. Additionally, we established simple methods for testing the axonal transport activity of human KIF1Bß using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1Bß activities both in vitro and in vivo, that is caused by a Charcot-Marie-Tooth-disease-associated Q98L mutation.

The FHA domain is essential for the autoinhibition of KIF1A/UNC-104.

KIF1A/UNC-104, a member of the kinesin superfamily motor proteins, plays a pivotal role in the axonal transport of synaptic vesicles and their precursors. Drosophila melanogaster UNC-104 (DmUNC-104) is a relatively recently discovered Drosophila kinesin. Although some point mutations that disrupt synapse formation have been identified, the biochemical properties of DmUNC-104 protein have not been investigated. Here, we prepared recombinant full-length DmUNC-104 protein and determined its biochemical features. We analyzed the effect of a previously identified missense mutation in the forkhead-associated (FHA) domain, called bristly (bris). The bris mutation strongly promoted the dimerization of DmUNC-104 protein, whereas wild-type DmUNC-104 was a mixture of monomers and dimers. We further tested the G618R mutation near the FHA domain which was previously shown to disrupt the autoinhibition of C. elegans UNC-104. The biochemical properties of the G618R mutant recapitulated those of the bris mutant. Finally, we found that disease-associated mutations also promote the dimerization of DmUNC-104. Collectively, our results suggest that the FHA domain is essential for the autoinhibition of KIF1A/UNC-104, and that abnormal dimerization of KIF1A is linked to human diseases.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Transient accumulation and bidirectional movement of KIF13B in primary cilia.

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein-2 motors. Nematodes additionally employ a cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs.

BACKGROUND: Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. RESULTS: We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. CONCLUSIONS: Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

Characterization of TAG-63 and its role on axonal transport in C. elegans.

Model organisms are increasingly used to study and understand how neurofilament (NF)-based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG-63 as a NF-like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG-63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti-NEFH antibody detects a band at the predicted molecular weight of TAG-63 in Western blots of whole worm lysates and this band cannot be detected in tag-63 knockout worms. A transcriptional tag-63 reporter expresses in a broad range of neurons, and various anti-NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC-104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine-labeled in vitro reconstituted TAG-63 filaments disintegrate upon drug exposure. Speeds of UNC-104 motors are diminished in tag-63 mutant worms with visibly increased accumulations of motors along axons. UNC-104/TAG-63 and SNB-1/TAG-63 not only colocalize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals. In summary, we identified and characterized TAG-63 in C. elegans, and demonstrate that lack of this protein limits axonal transport efficiencies. Additionally, this study would aid in developing NF-related disease models in the future.

Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition.

In kinesin-3, the coiled-coil 1 (CC1) can sequester the preceding neck coil (NC) for autoinhibition, but the underlying mechanism is poorly understood. Here, we determined the structures of the uninhibited motor domain (MD)-NC dimer and inhibited MD-NC-CC1 monomer of kinesin-3 KIF13B. In the MD-NC-CC1 monomer, CC1 is broken into two short helices that unexpectedly interact with both the NC and the MD. Compared with the MD-NC dimer, the CC1-mediated integration of NC and MD not only blocks the NC dimer formation, but also prevents the neck linker (NL) undocking and the ADP release from the MD. Mutations of the essential residues in the interdomain interaction interface in the MD-NC-CC1 monomer restored the MD activity. Thus, CC1 fastens the neck domain and MD and inhibits both NC and NL. This CC1-mediated lockdown of the entire neck domain may represent a paradigm for kinesin autoinhibition that could be applicable to other kinesin-3 motors.

A Model for Chemomechanical Coupling of Kinesin-3 Motor.

Introduction: Kinesin-3 motor, which is in the monomeric and inactive form in solution, after cargo-induced dimerization can step on microtubules towards the plus end with a high velocity and a supperprocessivity, which is responsible for transporting the cargo in axons and dendrites. The kinesin-3 motor has a large initial landing rate to microtubules and spends the majority of its stepping cycle in a one-head-bound state. Under the load the kinesin-3 motor can dissociate more readily than the kinesin-1 motor. Methods: To understand the physical origin of the peculiar features for the kinesin-3 motor, a model is presented here for its chemomechanical coupling. Based on the model the dynamics of the motor under no load, under the ramping load and under the constant load is studied analytically. Results: The theoretical results explain well the available experimental data under no load and under the ramping load. For comparison, the corresponding available experimental data for the kinesin-1 motor under the ramping load are also explained. The predicted results of the velocity, dissociation rate and run length versus the constant load for the kinesin-3 motor are provided. Conclusions: The study has strong implications for the chemomechanical coupling mechanism of the kinesin-3 dimer. The origin of the kinesin-3 dimer in the predominant one-head-bound state is due to the fact that the rate of ATP transition to ADP in the trailing head is much larger than that of ADP release from the MT-bound head. The study shows that the kinesin-3 ADP-head has an evidently longer interaction distance with microtubule than the kinesin-1 ADP-head, explaining why in the initial ADP state the kinesin-3 motor has the much larger landing rate than the kinesin-1 motor and why under the load the kinesin-3 motor can dissociate more readily than the kinesin-1 motor. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00795-1.

Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A in vivo.

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in Caenorhabditis elegans Posterior Lateral Mechanosensory neurons. The synaptic vesicle anterograde kinesin motor UNC-104/KIF1A regulates this vesicle behavior at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behavior at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight toward the asynaptic end. This suggests that the neuron optimizes UNC-104 levels on the cargo surface to maximize the fraction of vesicles entering the branch and minimize the fraction going to the asynaptic end.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans5,6 and in EVs in the mouse embryonic node and isolated from human urine.7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Cilia-derived extracellular vesicles (EVs) contain signaling proteins and act in intercellular communication. Polycystin-2 (PKD-2), a transient receptor potential channel, is a conserved ciliary EVs cargo. Caenorhabditis elegans serves as a model for studying ciliary EV biogenesis and function. C. elegans males release EVs in a mechanically-induced manner and deposit PKD-2-labeled EVs onto the hermaphrodite vulva during mating, suggesting an active release process. Here, we study the dynamics of ciliary EV release using time-lapse imaging and find that cilia can sustain the release of PKD-2-labeled EVs for a two-hour duration. Intriguingly, this extended release doesn't require neuronal synaptic transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The ciliary kinesin-3 motor KLP-6 is necessary for both initial and extended ciliary EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dihydroceramide desaturase DEGS1/2 ortholog TTM-5 is highly expressed in the EV-releasing sensory neurons, localizes to cilia, and is required for sustained but not initial ciliary EV release, implicating ceramide in ciliary ectocytosis. The study offers a comprehensive portrait of real-time ciliary EV release, and mechanisms supporting cilia as proficient EV release platforms.

KIF13A-A Key Regulator of Recycling Endosome Dynamics.

Molecular motors of the kinesin superfamily (KIF) are a class of ATP-dependent motor proteins that transport cargo, including vesicles, along the tracks of the microtubule network. Around 45 KIF proteins have been described and are grouped into 14 subfamilies based on the sequence homology and domain organization. These motors facilitate a plethora of cellular functions such as vesicle transport, cell division and reorganization of the microtubule cytoskeleton. Current studies suggest that KIF13A, a kinesin-3 family member, associates with recycling endosomes and regulates their membrane dynamics (length and number). KIF13A has been implicated in several processes in many cell types, including cargo transport, recycling endosomal tubule biogenesis, cell polarity, migration and cytokinesis. Here we describe the recent advances in understanding the regulatory aspects of KIF13A motor in controlling the endosomal dynamics in addition to its structure, mechanism of its association to the membranes, regulators of motor activity, cell type-specific cargo/membrane transport, methods to measure its activity and its association with disease. Thus, this review article will provide our current understanding of the cell biological roles of KIF13A in regulating endosomal membrane remodeling.

Force generation of KIF1C is impaired by pathogenic mutations.

Intracellular transport is essential for neuronal function and survival. The most effective plus-end-directed neuronal transporter is the kinesin-3 KIF1C, which transports large secretory vesicles and endosomes.1-4 Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients.5-8 In contrast to other kinesin-3s, KIF1C is a stable dimer and a highly processive motor in its native state.9,10 Here, we establish a baseline for the single-molecule mechanics of Kif1C. We show that full-length KIF1C molecules can processively step against the load of an optical trap and reach average stall forces of 3.7 pN. Compared with kinesin-1, KIF1C has a higher propensity to slip backward under load, which results in a lower maximal single-molecule force. However, KIF1C remains attached to the microtubule while slipping backward and re-engages quickly, consistent with its super processivity. Two pathogenic mutations, P176L and R169W, that cause hereditary spastic paraplegia in humans7,8 maintain fast, processive single-molecule motility in vitro but with decreased run length and slightly increased unloaded velocity compared with the wild-type motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles. Our results show how its mechanics supports KIF1C's role as an intracellular transporter and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

Neurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Sensory cilia act as a specialized venue for regulated extracellular vesicle biogenesis and signaling.

Ciliary extracellular vesicle (EV) shedding is evolutionarily conserved. In Chlamydomonas and C. elegans, ciliary EVs act as signaling devices.1-3 In cultured mammalian cells, ciliary EVs regulate ciliary disposal but also receptor abundance and signaling, ciliary length, and ciliary membrane dynamics.4-7 Mammalian cilia produce EVs from the tip and along the ciliary membrane.8,9 This study aimed to determine the functional significance of shedding at distinct locations and to explore ciliary EV biogenesis mechanisms. Using Airyscan super-resolution imaging in living C. elegans animals, we find that neuronal sensory cilia shed TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites: the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. A big unanswered question in the EV field is how cells sort EV cargo. Here, we show that two EV cargoes- CIL-7 and PKD-2-localized and trafficked differently along cilia and were sorted to different environmentally released EVs. In response to mating partners, C. elegans males modulate EV cargo composition by increasing the ratio of PKD-2 to CIL-7 EVs. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional Diversity.

In addition to their force-generating motor domains, kinesin motor proteins feature various accessory domains enabling them to fulfill a variety of functions in the cell. Human kinesin-3, Kif14, localizes to the midbody of the mitotic spindle and is involved in the progression of cytokinesis. The specific motor properties enabling Kif14's cellular functions, however, remain unknown. Here, we show in vitro that the intrinsically disordered N-terminal domain of Kif14 enables unique functional diversity of the kinesin. Using single molecule TIRF microscopy, we found that Kif14 exists either as a diffusible monomer or as processive dimer and that the disordered domain (1) enables diffusibility of the monomeric Kif14, (2) renders the dimeric Kif14 super-processive and enables the kinesin to pass through highly crowded areas, (3) enables robust, autonomous Kif14 tracking of growing microtubule tips, independent of microtubule end-binding (EB) proteins, and (4) is sufficient to enable crosslinking of parallel microtubules and necessary to enable Kif14-driven sliding of antiparallel ones. We explain these features of Kif14 by the observed diffusible interaction of the disordered domain with the microtubule lattice and the observed increased affinity of the disordered domain for GTP-bound tubulin. We suggest that the disordered domain tethers the motor domain to the microtubule providing a diffusible foothold and a regulatory hub, tuning the kinesin's interaction with microtubules. Our findings thus exemplify pliable protein tethering as a fundamental mechanism of molecular motor regulation.

GSK3ß Impairs KIF1A Transport in a Cellular Model of Alzheimer's Disease but Does Not Regulate Motor Motility at S402.

Impairment of axonal transport is an early pathologic event that precedes neurotoxicity in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), a causative agent of AD, activate intracellular signaling cascades that trigger phosphorylation of many target proteins, including tau, resulting in microtubule destabilization and transport impairment. Here, we investigated how KIF1A, a kinesin-3 family motor protein required for the transport of neurotrophic factors, is impaired in mouse hippocampal neurons treated with AßOs. By live cell imaging, we observed that AßOs inhibit transport of KIF1A-GFP similarly in wild-type and tau knock-out neurons, indicating that tau is not required for this effect. Pharmacological inhibition of glycogen synthase kinase 3ß (GSK3ß), a kinase overactivated in AD, prevented the transport defects. By mass spectrometry on KIF1A immunoprecipitated from transgenic AD mouse brain, we detected phosphorylation at S402, which conforms to a highly conserved GSK3ß consensus site. We confirmed that this site is phosphorylated by GSK3ß in vitro Finally, we tested whether a phosphomimic of S402 could modulate KIF1A motility in control and AßO-treated mouse neurons and in a Golgi dispersion assay devoid of endogenous KIF1A. In both systems, transport driven by mutant motors was similar to that of WT motors. In conclusion, GSK3ß impairs KIF1A transport but does not regulate motor motility at S402. Further studies are required to determine the specific phosphorylation sites on KIF1A that regulate its cargo binding and/or motility in physiological and disease states.

Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins.

Primary cilia are antenna-like sensory organelles that regulate a substantial number of cellular signalling pathways in vertebrates, both during embryonic development as well as in adulthood, and mutations in genes coding for ciliary proteins are causative of an expanding group of pleiotropic diseases known as ciliopathies. Cilia consist of a microtubule-based axoneme core, which is subtended by a basal body and covered by a bilayer lipid membrane of unique protein and lipid composition. Cilia are dynamic organelles, and the ability of cells to regulate ciliary protein and lipid content in response to specific cellular and environmental cues is crucial for balancing ciliary signalling output. Here we discuss mechanisms involved in regulation of ciliary membrane protein trafficking and signalling, with main focus on kinesin-2 and kinesin-3 family members.

Cell-Specific α-Tubulin Isotype Regulates Ciliary Microtubule Ultrastructure, Intraflagellar Transport, and Extracellular Vesicle Biology.

Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range of pathologies called ciliopathies. Ciliary specialization in form and function is observed throughout the animal kingdom, yet mechanisms generating ciliary diversity are poorly understood. The "tubulin code"-a combination of tubulin isotypes and tubulin post-translational modifications-can generate microtubule diversity. Using C. elegans, we show that α-tubulin isotype TBA-6 sculpts 18 A- and B-tubule singlets from nine ciliary A-B doublet microtubules in cephalic male (CEM) neurons. In CEM cilia, tba-6 regulates velocities and cargoes of intraflagellar transport (IFT) kinesin-2 motors kinesin-II and OSM-3/KIF17 without affecting kinesin-3 KLP-6 motility. In addition to their unique ultrastructure and accessory kinesin-3 motor, CEM cilia are specialized to produce extracellular vesicles. tba-6 also influences several aspects of extracellular vesicle biology, including cargo sorting, release, and bioactivity. We conclude that this cell-specific α-tubulin isotype dictates the hallmarks of CEM cilia specialization. These findings provide insight into mechanisms generating ciliary diversity and lay a foundation for further understanding the tubulin code.