O'Hagan, Robert et al. (2009) International Worm Meeting "Genetic and Physiological Investigation of a TRPP Ion Channel Complex Needed for Male Mating."

Barr, Maureen, O'Hagan, Robert

[International Worm Meeting, 2009]

TRP ion channels are important in peripheral sensory neurons that detect environmental cues, central neurons of the mammalian brain where they associate with molecules needed for synaptic plasticity, and in non-neuronal tissues such as the kidney, where they function in sensation of the internal environment. A TRP polycystin (TRPP) ion channel composed of PKD1 and PKD2 localizes to cilia and is essential for normal human kidney function. This TRPP complex has been proposed to directly transduce the mechanical forces created by renal fluid flow, and mutations in the genes encoding these subunits cause polycystic kidney disease. However, we lack a thorough understanding of the molecular and physiological functions of TRP channels in these diverse tissues. In an effort to understand the molecular and physiological functions of TRP ion channels, we are studying the TRPP ion channel complex subunits LOV-1 and PKD-2, the C. elegans homologs of PKD1 and PKD2. This complex, which is needed for proper male sex attraction and mating behaviors, localizes to cilia in the HOB hook neuron, the RnB ray neurons in the tail, and in the CEM neurons in the head. The sensory function and localization of this complex in cilia may be conserved in C. elegans male neurons and human renal epithelia. Using in situ electrophysiology, we are examining the functions of male specific sensory neurons and the LOV-1/PKD-2 TRPP complex. Because these ciliated neurons are thought to perform sensory roles, we will observe responses to mechanical and/or chemical stimuli. Comparison of the responses of sensory neurons in wild-type and lov-1 or pkd-2 mutants should allow us to clarify the function of this TRPP complex and to determine if it is directly or indirectly activated by sensory stimuli. The TRPP complex is localized to cilia and ER and may be performing separable roles in these locations. To determine the effect of mislocalization of this complex, we will test animals lacking the function of regulators of the abundance or localization of the LOV-1/PKD-2 complex, such as the kinesin KLP-6, the inositol 5-phosphatase CIL-1, and the calcineurin TAX-6. We are currently mapping and cloning another molecule needed for TRPP complex localization, called cil-6, which is mutated in my12 and my22 animals. These cil-6 mutants have defects in PKD-2 localization and male mating behavior.

O'Hagan, Robert et al. (2019) International Worm Meeting "The Tubulin Code: Writers, erasers, and readers specialize cilia."

Zhang, Winnie, Bellotti, Sebastian, Smith, Harold, De Stasio, Elizabeth, Nguyen, Ken, Gu, Amanda, O'Hagan, Robert, Morash, Maggie, Hall, David, Power, Katlin, Golden, Andrew, Barr, Maureen

[International Worm Meeting, 2019]

In eukaryotic cells, microtubules (MTs) act as highways, and molecular motors, such as kinesins, are cargo trucks. The "Tubulin Code" posits that tubulin isotypes and tubulin post-translational modifications such as polyglutamylation (addition of glutamate side-chains) act like signposts that regulate the activity of motors and the structure of MTs (Verhey and Gaertig, 2007). Cilia from all eukaryotes share conserved 9+0 or 9+2 MT ultrastructures and well-characterized molecular motors, and are therefore a powerful model with which to discover the mechanisms by which the Tubulin Code acts. Cephalic male (CEM) cilia possess a unique architecture: 9 MT doublets splay into 18 singlets, which is essential for motor-based transport and the release of ciliary extracellular vesicles. We showed that in CEM cilia, the a-tubulin TBA-6 and the glutamylase TTLL-11 write the Tubulin Code, and the deglutamylase CCPP-1 acts as an eraser, while the kinesin-3 KLP-6 and the kinesin-2 OSM-3 are readers of the Tubulin Code [Morsci and Barr, 2011; O'Hagan et al, 2011; Silva et al, 2017; O'Hagan et al, 2017]. In amphid channel cilia, the Tubulin Code performs a different function. In ccpp-1 amphid channel cilia, hyperglutamylation produces ciliary degeneration as detected by dye-filling and transmission electron microscopy. In humans and mice, mutations in Ccp1, the homolog of C. elegans ccpp-1, result in neurodegeneration. To discover molecules involved ciliary MT stability, we took candidate and forward genetic screening approaches to find modifiers of the ccpp-1 Dyf phenotype. We find that the ccpp-1 ciliary degeneration phenotype was suppressed by mutation in any of three glutamylases ttll-4, ttll-5, and ttll-11 or the never in mitosis kinase nekl-4 (see poster by K. Power). NEKs have been implicated in ciliary length regulation and human ciliopathies. This work should uncover novel pathways that establish, maintain, and modify MT architecture and motor function.

O'Hagan, Robert et al. (2011) International Worm Meeting "CCPP-1 Is A Putative Tubulin Deglutamylase That Regulates The Function And Stability Of Neuronal Sensory Cilia."

Swoboda, Peter, Hall, David H., Barr, Maureen M., Piasecki, Brian P., Silva, Malan, Nguyen, Ken C.Q., Phirke, Prasad, O'Hagan, Robert

[International Worm Meeting, 2011]

When assembled into microtubules (MTs), tubulins have protruding C-terminal tails that accumulate post-translational modifications (PTMs) such as detyrosination and polyglutamylation. PTMs have long been known to mark stable MTs. Although their cellular function and significance are not yet clear, PTMs have been proposed to act as signposts that guide various kinesin and dynein motors to transport their cargoes to specific destinations. For example, trafficking of KIF5C, a mouse kinesin-1 motor, is affected by detyrosination, and KIF1A, a mouse kinesin-3, is affected by polyglutamylation. In cilia, axonemal MTs are especially prone to PTMs, which we propose regulate MT stability as well as the function of motors that drive intraflagellar transport. We have found that perturbation of a PTM profoundly affects cilia in sensory neurons in C. elegans. We identified a mutation in C. elegans that affects ccpp-1, which encodes a cytosolic carboxypeptidase that deglutamylates microtubules in a subset of neuronal sensory cilia. Loss of CCPP-1 function causes defective localization and abundance of the polycystin PKD-2 and a kinesin-3, KLP-6, in male-specific neurons. Loss of CCPP-1 also causes increased velocity of motile kinesin-2 OSM-3::GFP puncta in the cilia of male-specific neurons. Loss of CCPP-1 had cell-specific effects on the level of polyglutamylation detected by a monoclonal antibody. Mutations in ccpp-1 cause defective dye-filling of sensory neurons that becomes progressively more severe with age, suggesting that sensory neurons construct cilia that degenerate over time. Ultrastructural analysis of mutants showed that some cilia had defective MT structures and some cilia were fragmented or absent. Others have shown that loss of CCP1, a homolog of CCPP-1, in mice causes degeneration of brain neurons such as Purkinje neurons in the cerebellum, olfactory mitral cells, and retinal photoreceptors, and also causes sperm immotility. Although all affected cells are ciliated, the impact of loss of CCP1 on cilia has not been explored in mutant mice. We propose that neurodegeneration caused by loss of CCP1 in mammals may represent a novel ciliopathy in which cilia are formed normally but degenerate, depriving the cell of cilia-based signal transduction that is necessary for maintenance of neurons.

O'Hagan, Robert et al. (2021) International Worm Meeting "Understanding Interactions Between Microtubule-Associated Proteins And Post-Translational Modifications Of Microtubules In Sensory Neurons"

Toyos, Yandis, Avrutis, Alexandra, Ross, Nicole, O'Hagan, Robert, Cuyuche, Kaylee

[International Worm Meeting, 2021]

Microtubules (MTs) act as tracks upon which molecular motors, such as kinesins and dyneins, travel to transport essential materials throughout cells. However, the mechanisms that regulate MT structure and motor functions to ensure that diverse cargos are appropriately delivered to particular regions or organelles within cells is not completely understood. In neurons, regulation of MT-based transport may be especially important because they extend elongated axons and dendrites. Although all MTs are made by assembly of alpha and beta tubulins, they are not uniform. The Tubulin Code hypothesis suggests that MTs can be specialized by incorporating different alpha and beta tubulins, as well as addition of reversible post-translational modifications (PTMs) such as glutamylation (addition of glutamate side chains to alpha or beta tubulin C-terminal tails). We previously found that glutamylases that add glutamylation to MTs (TTLL-4, TTLL-5, and TTLL-11) and a deglutamylase that reduces glutamylation (CCPP-1) influence MT structure and the activity of particular motors in sensory neuronal cilia in a cell-specific manner. Particular tubulins are also essential for regulating the structure of MTs in neuronal sensory cilia. Therefore, the MT tracks themselves encode information that regulates their own structure and stability, as well as the function of kinesin motors. Non-motor microtubule-associated proteins (MAPs) act as another mechanism to regulate the MT cytoskeleton, but the function of many MAPs is not yet known. MT binding of some MAPs, such as mammalian Tau, has been found to be sensitive to MT glutamylation in vitro, suggesting that these two layers of MT regulation may interact. To understand the genetic interactions of MAPs and the Tubulin Code, we observed the localization of fluorescently-tagged MAPs, such as the C. elegans Tau homolog PTL-1 and the DCX family member/RP1L1 homolog F27C1.13, combined with mutations that disrupt particular tubulin genes or regulators of MT glutamylation. Both Tubulin Code PTMs and MAPs are involved in neurodegenerative diseases in humans. Mutation of a human ccpp-1 homolog is associated with infantile-onset neurodegeneration, Tau is implicated in Alzheimer's Disease and other Tauopathies, and mutation of RP1L1 is associated with occult macular dystrophy, resulting in progressive blindness. Both PTL-1 and F27C1.13 were expressed in ciliated sensory neurons, suggesting that C. elegans might be used to model the human diseases associated with these genes. Our results support the hypothesis that PTMs and MAPs are essential cytoskeletal regulators that act in concert to regulate and specialize neuronal MTs.

O'Hagan, Robert et al. (2015) International Worm Meeting "Identification of the pathways by which post-translational microtubule glutamylation regulates ciliary maintenance in C. elegans."

Bellotti, Sebastian, Barr, Maureen, O'Hagan, Robert, Morash, Maggie, Zhange, Winnie

[International Worm Meeting, 2015]

Microtubules form dynamic networks in cells and display diverse post-translational modifications (PTMs), including polyglutamylation. The Tubulin Code posits that these PTMs regulate microtubule stability and the properties of molecular motors that use microtubules as tracks for intracellular transport (Verhey and Gaertig, 2007). The enzymes that regulate polyglutamylation are now known: Tubulin tyrosine ligase-like (TTLL) proteins add glutamate side chains, while cytoplasmic carboxypeptidases (CCPs) deglutamylate microtubules (Janke 2011). We sought to find molecules that regulate the PTM enzymes themselves and the downstream molecules that mediate their effects on microtubule stability and traffic.Nematode sensory cilia, which contain highly modified microtubules, provide an ideal model for studying how microtubule PTMs function in vivo. Mutations in ccpp-1, encoding a CCP deglutamylase, result in progressively degenerating amphid cilia (O'Hagan et al 2011). To identify other molecules that function in polyglutamylation-mediated microtubule stability, we performed a standard EMS F2 Suppressor Screen in C. elegans for novel mutations that suppress the ciliary deterioration of ccpp-1 mutants. From our screen of over 100,000 haploid genomes, 15 isolates were confirmed to suppress the ciliary degeneration of ccpp-1. We are performing complementation tests to determine which suppressors are mutations in ttll genes and whole genome sequencing to identify causal mutations.A better understanding of the CCPP-1 pathway and how polyglutamylation controls ciliary and microtubule stability will have important implications for human health and will provide potential drug targets for ciliopathies, which cause blindness, respiratory issues, male infertility, and polycystic kidney disease in humans. We hope that identification of our mutations may shed light on ciliopathies for which genetic causes are currently unknown.

O'Hagan, Robert et al. (2015) International Worm Meeting "Post-translational microtubule glutamylation levels control ciliary motor transport, microtubule structure, and cytoskeletal stability."

Hall, David, Morash, Margaret, Barr, Maureen, Nguyen, Ken, Bellotti, Sebastian, Silva, Malan, O'Hagan, Robert

[International Worm Meeting, 2015]

Post-translational modifications (PTMs) added to microtubules (MTs) may act as a "Tubulin Code" that guides the activities of kinesins, dyneins, and other MT-binding proteins. Ciliary MTs are highly decorated with PTMs, but the functions of PTMs in cilia are largely unknown. We previously showed that loss of CCPP-1, a predicted MT deglutamylase, caused defective localization of the ciliary receptor PKD-2 and the kinesin-3 motor KLP-6, and abnormally fast movement of the kinesin 2 OSM-3, in male-specific B-type neuronal cilia. In amphid channel cilia, ccpp-1 mutants displayed a progressive Dyf phenotype and deterioration of MT structure. Loss of a CCPP-1 homolog in mice also affects ciliated cells, causing degeneration of cerebellar Purkinje neurons, olfactory mitral cells, and retinal photoreceptors, and also causes sperm immotility. Therefore, MT glutamylation may play a conserved role in cilia and flagella. Here we show that select Tubulin Tyrosine Ligase-Like (TTLL) MT glutamylases oppose the activity of CCPP-1. Mutations in ttll-4, ttll-5, or ttll-11 suppressed the ccpp-1 progressive Dyf phenotype, but did not suppress PKD-2 ciliary localization defects. The ttll-11 mutation suppressed ccpp-1 effects on kinesin-3 KLP-6 localization and kinesin-2 OSM-3 velocity. Ciliary MT structure typically contains doublets composed of A- and B- tubules. Ultrastructural analysis revealed loss of ciliary B-tubules in ccpp-1, while ttll-11 mutants displayed abnormally long ciliary doublet regions. MT glutamylation reduced B-tubule stability in both neuronal types, despite differences in structural details. We hypothesize that glutamylation targets MTs for degradation by MT-severing enzymes. To elucidate the pathways by which glutamylation controls ciliary MT stability, we performed a screen for suppressors of the ccpp-1 Dyf phenotype. Our screen should identify molecules upstream and downstream of MT glutamylation. Our data suggest that, in cilia, MT glutamylation is part of a Tubulin Code that regulates ciliary transport of molecular motors and sensory receptors and controls axonemal structure.

Robert O'Hagan et al. (2007) International Worm Meeting "Regulation of Dopamine Signaling in the Modulation of Locomotion."

Bob Horvitz, Robert O'Hagan

[International Worm Meeting, 2007]

Although genes encoding dopamine receptors and proteins involved in dopamine biosynthesis, uptake, and vesicle loading are known, mechanisms that regulate dopaminergic signaling are not well understood. Genetic studies of a dopamine-modulated behavior might elucidate mechanisms that regulate dopaminergic signaling. When well-fed hermaphrodites encounter a bacterial lawn, they slow their locomotion by approximately 30%. This behavior, called the basal slowing response, requires dopaminergic signaling: animals mutant in cat-2, which encodes the tyrosine hydroxylase needed for dopamine biosynthesis, are defective in basal slowing behavior. However, animals that have been food-deprived for 30 minutes slow their locomotion by about 70% upon entry into a bacterial lawn, a behavior called the enhanced slowing response. Dopamine plays only a minor role in the enhanced slowing response, suggesting that food-deprivation might suppress dopaminergic signaling and promote the use of other neurotransmitters to mediate locomotory slowing. In other words, dopamine signaling is prone to regulation by the recent history of feeding. We hope to understand how sensation of bacteria evokes dopamine release and how dopaminergic signaling is regulated by the experience of food deprivation. Screens for altered basal slowing responses might identify novel genes involved in processes such as: sensory transduction; dopamine vesicle loading, priming, and fusion; and regulation of dopamine receptors or their downstream effectors. Screens for animals that lack basal slowing behavior are difficult because of the small magnitude of the response. Instead, we are screening for mutants that exhibit an increased basal slowing response. We mutagenized cat-2 mutants carrying a cat-2-rescuing extrachromosomal array and looked for animals that slowed excessively. Because this array is inherited by a fraction of progeny, we can determine if the increased basal slowing of isolates depends on cat-2 and thus on dopamine. We are characterizing several isolates with cat-2-dependent increased basal slowing responses. In the future, we plan to use in vivo electrophysiological techniques to record from neurons in the dopaminergic circuit to observe mechanisms that regulate quantal content, vesicular release, and the abundance and activity of receptors. We also plan to compare the physiology of wild-type animals and mutant isolates from our screens.

Robert O'Hagan et al. (2005) International Worm Meeting "The roles of MEC proteins in the production of mechanoreceptor currents"

Martin Chalfie, Robert O'Hagan

[International Worm Meeting, 2005]

Screens for touch insensitive (Mec) mutants have identified several genes needed for mechanosensation. The proteins encoded by these genes have been assigned putative roles based on their sequence and genetic interactions. Confirmation of these roles, however, has proven challenging. Recently, the roles of components of the touch transduction channel in touch receptor neurons have been confirmed using in vivo whole-cell electrophysiology [OHagan et al. Nat Neurosci 8, 43-50 (2005)]. In these experiments, specific missense mutations of the DEG/ENaC proteins MEC-4 or MEC-10 [encoded by mec-4(u2) and mec-10(u20), respectively] caused a dramatic change in the selectivity of mechanoreceptor currents (MRCs) recorded in PLM touch cells in response to mechanical stimulation, confirming that MEC-4 and MEC-10 form the pore of a touch transduction complex. The absence of MEC-4, or MEC-2 and MEC-6, which are also members of the transduction channel complex, abolished MRCs, confirming that they are required for transduction. However, the role originally assigned to the large diameter microtubules unique to the touch cellsintracellular tether points thought to be crucial for mechanical gating of ion channelsappears to be invalid, as MRCs (with reduced amplitude) were still observed in touch cells in mec-7 -tubulin null mutants lacking large diameter microtubules.We have recently tested several other genes needed for mechanosensory behavior using in vivo electrophysiology. The extracellular matrix proteins, MEC-1, MEC-5, and MEC-9, which localize the transduction channels along the touch cell neurite [Emtage et al. Neuron 44, 795-807 (2004)], are required for generation of MRCs. In addition, mutation of mec-14, encoding a putative touch channel regulator with similarity to oxidoreductase-like proteins (including Shaker channel -subunits) also causes a drastic reduction in the amplitude of MRCs. In contrast, a putative null mutation of mec-18 causes no significant change in MRCs in L4 and young adult animals. This finding suggests that MEC-18 is needed after transduction.Hence, by using in vivo electrophysiology, we can assess the roles of genes involved in touch sensation by distinguishing between their requirement in mechanotransduction versus subsequent processes such as propagation of the signal down the neurite, transmission to other neurons in the touch circuit, or habituation.

Robert O'Hagan et al. (2003) International Worm Meeting "In vivo recordings of sensory transduction currents in C. elegans touch cells"

Robert O'Hagan, Martin Chalfie, Miriam B Goodman

[International Worm Meeting, 2003]

Genetic screens have identified 12 genes predicted to encode components of a mechanotransduction complex needed to sense touch along the body. We have recently shown that co-expression of the products of four of these genes (mec-4, mec-10, mec-2, mec-6) in Xenopus oocytes produces an amiloride-sensitive sodium current. However, evidence that these proteins are needed for an amiloride-sensitive channel that responds to mechanical perturbation in vivo has been lacking. We have now recorded from PLM cells in vivo and find that touch evokes an inwardly-rectifying current that is likely to be carried by sodium and appears to be blocked by amiloride. Forces as small as 100 nN are sufficient to elicit an inward current in PLM. This current is unlikely to be a mechanical artifact, since larger forces fail to evoke any obvious change in membrane current in other neurons in the tail. Transduction current adapts rapidly during sustained stimulation. In a manner reminiscent of receptor potentials in Pacinian corpuscles in mammals, both positive and negative changes in force produce an inward current. Preliminary studies also suggest that this current is abolished by mutation of mec-4. We are currently examining the response in cells that lack this and other proteins in the degenerin channel complex, as well as other proteins implicated in the function of these cells. These experiments should allow us to identify touch cell genes needed for the initial transduction event as well as those that act subsequently.

Robert O'Hagan et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Regulation of Dopamine Signaling in the Modulation of Locomotion"

Bob Horvitz, Robert O'Hagan

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Although genes that encode dopamine receptors and other proteins involved in dopamine biosynthesis, uptake, and vesicle loading are known, we lack a clear understanding of how dopaminergic signaling is regulated. Genetic studies of the basal slowing response, a dopamine-mediated behavior of C. elegans, might elucidate mechanisms that regulate dopaminergic signaling. When well-fed hermaphrodites encounter a bacterial lawn, they slow their locomotion by approximately 30%. This behavior is called the basal slowing response and requires dopaminergic signaling. However, if animals are food-deprived for 30 minutes, they slow their locomotion by about 70% upon entry into a bacterial lawn. Dopamine plays a minor role in execution of this behavior, called the enhanced slowing response, suggesting that food-deprivation might suppress dopaminergic signaling and promote the use of other neurotransmitters to mediate locomotory slowing. We hope to understand how sensory stimulation causes dopamine release and how dopaminergic signaling is regulated. Screens for altered basal slowing responses might identify novel genes involved in processes such as sensory transduction; dopamine vesicle loading, priming, and fusion; and regulation of dopamine receptors or their downstream effectors. However, screens for animals with defects in basal slowing behavior are difficult because of the small magnitude of the response. For this reason, we first screened for mutants that exhibit an increased basal slowing response. We mutagenized cat-2 deletion mutants carrying cat-2 in an extrachromosomal array. This cat-2 mutant lacks the tyrosine hydroxylase needed for biosynthesis of dopamine and is defective in basal slowing behavior; however, the transgene rescues basal slowing behavior. Since this array is inherited by a fraction of the progeny, we can confirm that the increased basal slowing of isolates depends on cat-2 and endogenous dopamine. We are currently characterizing an isolate with a cat-2-dependent increased basal slowing response. In the future, we will perform screens for animals with defects in basal slowing by mutagenizing strains with increased basal slowing responses. We will characterize mutants identified in these screens using in vivo electrophysiological recordings of dopamine-secreting neurons and dopamine-responsive cells.