No abstract

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. An urgent need in the field is a fundamental understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices in Chlamydomonas and C. elegans [2-4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or to retract cilia before entering the cell cycle [6]. Here we used our established C. elegans model to study sensoryevoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [7]. We find that males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2-carrying EVs from cilia. To our knowledge this is the first report of mechanoresponsive nature of the ciliary EV release and of ciliary EV directional transfer from one animal to another animal. Since the polycystins are evolutionarily conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other systems and biological contexts. C. elegans male mating involves stereotyped behavioral steps including response to hermaphrodite contact, location of the hermaphrodite's vulva, spicule insertion, and sperm transfer to the hermaphrodite's uterus [7]. To examine male-hermaphrodite EV-mediated interactions during mating, we paired fluorescently labeled transgenic adult males with unlabeled hermaphrodites for 24 hours (Figure 1A). Male sperm transfer was visualized with MitoTracker dye, whereas ciliary EVs were tracked via the PKD-2::GFP EV cargo protein. In all mated hermaphrodites inseminated with MitoTracker labeled sperm, we observed deposition of male-derived PKD-2::GFP EVs on the hermaphrodite vulvae (Figure 1B-C). No PKD-2::GFP EVs were found inside the hermaphrodite uterus. Location of the male-deposited EVs at the hermaphrodite's vulva is consistent with the position of a male tail during mating and suggests that EVs were released in the timeframe between successful location of the vulva and retraction of spicules post-copulation. This timeframe represents the closest contact between the male tail and the vulva area of the hermaphrodite, suggesting that the vulva may provide mechanical or chemical cues to stimulate ciliary EV release from the male. Living C. elegans males release EVs when mounted between an agarose-layered slide and a bare glass coverslip [3], with EVs usually floating close to the surface of the coverslip. To

Ciliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C . elegans , mutations in ccpp-1 , the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4 , ttll-5 , or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C . elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C . elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.

No abstract

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. The urgent need of the field is precise understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices in Chlamydomonas and C. elegans [2][3][4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or in the process of cilia retraction when cultured cells enter the cell cycle [6]. Here we used our established C. elegans model to study sensory-evoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. We find that males deposit PKD-2::GFP-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2::GFP-carrying EVs from cilia. To our knowledge this is the first report of mechanoresponsive nature of the ciliary EV release and of ciliary EV directional transfer from one animal to another animal. Since the polycystins are evolutionary conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other animals. ResultsC. elegans male mating involves stereotyped behavior steps including response to hermaphrodite contact, location of the hermaphrodite's vulva, spicule insertion, and sperm transfer to the hermaphrodite's uterus [7]. To examine male-hermaphrodite EV interactions during mating, we paired fluorescently labeled transgenic adult males with unlabeled adult hermaphrodites ( Figure 1A). Male sperm transfer was visualized with MitoTracker dye and ciliary EVs were tracked via the PKD-2::GFP EV cargo protein. After mating, we scored the hermaphrodite uterus for the presence of MitoTracker labeled male sperm. In all inseminated hermaphrodites, we observed highly localized deposition of the male-specific PKD-2-carrying EVs along the hermaphrodite vulva ( Figure 1B-C). These data demonstrate that the male directly transferred PKD-2::GFP-carrying EVs to the hermaphrodite during mating. The location of the male-deposited EVs at the hermaphrodite's vulva is consistent with the position of male

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Post-translational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates post-translational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knockdown of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in C. elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knockdown of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries. Significance Statement Combining an in vitro model of the secondary phase of spinal cord injury with shRNA knockdown, we demonstrate that the deglutamylase CCP1 protects neurons from excitotoxic 4 death. Excitotoxicity plays a role in the secondary phase of neuronal injuries, contributing to neurodegeneration. CCP1 function was previously known to be associated with cilia. We provide the first demonstration (to our knowledge) that spinal cord interneurons are ciliated. However, our data suggest that neuroprotection by CCP1 may be independent of cilia in spinal neurons. Our work supports the idea that targeting enzymes that modify tubulins, such as glutamylases and deglutamylases, might be an avenue of treatment for nervous system injuries.

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

Microtubules (MTs) are cytoskeletal elements that provide structural support, establish morphology, and act as roadways for intracellular transport in cells. Neurons extend and must maintain long axons and dendrites to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Post-translational modification of MTs is one mechanism by which neurons can regulate the cytoskeleton.The carboxypeptidase CCP1 negatively regulates post-translational glutamylation of MTs. We previously demonstrated that the CCP1 homolog in C. elegans is important for maintenance of cilia. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Furthermore, it not yet clear whether CCP1 acts on ciliary MTs in spinal cord neurons.Using an in vitro model of excitotoxic neuronal injury coupled with shRNA-mediated knockdown of CCP1, we demonstrate that CCP1 protects neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system, and knockdown of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons. Our results suggest that CCP1 acts on axonal and dendritic MTs to promote cytoskeletal rearrangements that support neuroregeneration and that enzymes responsible for glutamylation of MTs might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.