LeBoeuf, Brigitte et al. (2010) Worm Breeder's Gazette "A library of expression clones and cellular manipulation tools"

Garcia, Rene, LeBoeuf, Brigitte

[Worm Breeder's Gazette, 2010]

LeBoeuf, Brigitte et al. (2021) International Worm Meeting "UPRmt required for anal depressor symmetry and male muscle remodeling"

Garcia, Luis Rene, LeBoeuf, Brigitte

[International Worm Meeting, 2021]

The mitochondrial unfolded protein response (UPRmt) is normally studied in the context of stress. When mitochondrial function is perturbed, the transcription factor ATFS-1 is trafficked to the nucleus, instead of the mitochondria, to promote gene expression involved in metabolic remodeling and repair of mitochondrial damage. However, the UPRmt is also necessary for normal development under normal conditions. One cell that uses UPRmt is the anal depressor (adp), which is required for defecation; when the adp contracts, waste is expelled from the anus. The H-shaped muscle has left and right sarcomeres that span from the dorsal hypodermis to the rectal epithelium. We used atfs-1 tissue-specific CRISPR to drive Cas9 expression in the enteric muscles, including the adp, and discovered that the muscle no longer makes the symmetrical H shape, but instead has hyper-expanded asymmetric sarcomeres. This suggests that the adp uses UPRmt to establish symmetry during early development. Furthermore, in L4 males, the adp remodels to change its form and function from a defecation muscle, with a dorsal/ventral sarcomere, to an anterior/posterior-oriented sex muscle. The remodeling process also requires atfs-1, since reduction in its function disrupts the disassembly of its dorsal/ventral sarcomere. Additionally, ATFS-1 is expressed in developing male adps, but is below detection in the hermaphrodite muscle, suggesting that ATFS-1 has sexually dimorphic functions during post-embryonic developmental. We asked if artificially inducing UPRmt via ethidium bromide (EtBr), while over-expressing ATFS-1 in the nucleus, could lead to adp defects. While EtBr treatment delays development in both sexes and impairs male tail development, it does not affect adp remodeling, indicating that while too little ATFS-1 prevents muscle remodeling, excess nuclear ATFS-1 is tolerated. Future work will focus on what impact ATFS-1 has on metabolic function in the adp and what role the previously identified WNT/Ca2+ pathway has on regulating UPRmt-controlled metabolism.

LeBoeuf, Brigitte L. et al. (2011) International Worm Meeting "K+ channel interplay modifies male mating response."

Garcia, L. Rene, LeBoeuf, Brigitte L.

[International Worm Meeting, 2011]

A large population of K+ channels provides a way for organisms to maintain and modify cell excitability, allowing for appropriate behavioral responses to various stimuli. C. elegans male mating is a complex behavior that requires precise regulation to be successfully completed. Males must locate a hermaphrodite, properly position their tales on the hermaphrodite cuticle, locate the vulva, insert their copulatory spicules, and transfer sperm. We focus on the spicule insertion step of mating, and have identified three K+ channels expressed in the sex circuit that regulate the timing of insertion: ether-a-go-go (EAG)/EGL-2, EAG-related gene (ERG)/UNC-103, and big current (BK)/SLO-1. 79% of a population of males lacking all three channels displays spontaneous spicule protraction (Prc) in the absence of mating cues. Our research indicates the function of these three K+ channels is modified by the presence or absence of the others. The loss of SLO-1 alone results in 70% instance of Prc, while a slo-1(lf) egl-2(0) double mutant is 30% Prc. This decrease in the mutant phenotype is due to an up-regulation of unc-103 in the absence of egl-2, as a transgenically expressed dominant-negative form of unc-103 returns the Prc phenotype to 72%. Additionally, the rg432 allele of slo-1 affects the Prc penetrance in an unc-103(0);egl-2(0) background. 58% of unc-103(0);egl-2(0) males are Prc; that percentage is reduced to 35% in unc-103(0);slo-1(rg432) egl-2(0) males. slo-1(rg432) is a point mutation in an intron located in a region that contains variable splicing and could result in up-regulation of specific isoforms that preferentially reduce male sex muscle excitability. In support of isoform-specific regulation, we determined that particular isoforms of slo-1 are more capable of rescuing the Prc defect than others. Our work suggests that while the male sex muscles attempt to compensate for the loss of one K+ channel by up-regulating others, various factors exist that determine the success of such mechanisms. Future work will explore how K+ channel expression changes over time due to loss of channel function. This will allow us to dissect the relationship between the K+ channels that control appropriate sensory-motor responses.

LeBoeuf, Brigitte L. et al. (2013) International Worm Meeting "Sensory neurons override recurrent motor programs to induce ejaculation during mating."

Garcia, L. Rene, LeBoeuf, Brigitte L.

[International Worm Meeting, 2013]

Siring progeny requires sustained spicule insertion into the hermaphrodite vulva coupled to sperm transfer. Coordinated signaling maintains the male's copulatory spicules over the vulva and couples genital position with repetitive spicule thrusts necessary to breach the vulva. Breaking out of this loop requires penetration, but this alone is insufficient to promote sperm transfer, as ectopic protraction does not lead to ejaculation. Males readily reverse ectopic spicule protraction and return to their interest in mating. However, after intromission and ejaculation, males are not immediately interested in mating again. To elucidate the circuit that controls ejaculation and how it is integrated with the circuits promoting vulva location and spicule insertion, we used optogenetics, cell ablation, and calcium imaging in free-moving males during mating. To achieve consistent ectopic ejaculation, we had to photo-stimulate using channelrhodopisin2 in a large circuit including muscles, tail and head neurons. Playing a vital role are neurons previously identified to promote vulva location and spicule insertion behaviors, but also synapse the somatic gonad. G-CaMP3 calcium imaging revealed that although gonadal activity and sperm initiation is coordinated with spicule insertion, it is not enough to allow sperm release. Efficient exit of the sperm out of the cloaca requires SPV and SPD sensory neurons that send their processes through the spicules and are exposed at the spicule tips. Following insertion and sperm initiation, calcium transients occur throughout the intestines and sex and body wall muscles. The transients could be a result of the seizure-like state that occurs in the posterior third of the male following insertion, allowing for sperm movement. After retraction, the male takes several minutes to recover, a period that is shortened when the male's ability to transfer sperm is reduced. Integrating one additional component to the circuitry that maintains spicule location at the vulva and facilitates insertion allows male mating behavior to be completed.

LeBoeuf, Brigitte et al. (2015) International Worm Meeting "Investigation of the EGL-2/EAG K+ channel PAS domain's role in responding to environmental changes."

Garcia, L. Rene, LeBoeuf, Brigitte, Gonclaves, Jimmy

[International Worm Meeting, 2015]

The highly conserved ether-a-go-go (EAG) K+ channel (also known as Kv10.1 and KCHN1) contains an n-terminal Per-Arnt-Sim (PAS) domain that interacts with a cyclic nucleotide-binding homology (CNBh) domain. These channels regulate egg-laying, chemotaxis and male mating in C. elegans and are involved in cognition, seizures, development and cancer in humans; however the functional role for the PAS and CNBh domains remain unidentified. In other contexts, the PAS domain has different functions, one of which is a nutrient sensor. Thus, we are interested in the role of the PAS domain in the egl-2-encoded EAG K+ channel. We are utilizing a variety of molecular and behavioral approaches to address this question. Using CRISPR, we tagged the genomic EAG K+ channel gene egl-2 by inserting YFP to a region between the CNBh domain and the c-terminus. The cells that express the CRISPR-engineered K+ channel in egl-2:YFP worms match transgenic animals that express promoter:YFP constructs. Our previous research identified egl-2 up-regulation as an important aspect of both aging and response to transient food deprivation. To confirm this construct is useful in measuring variable metabolic states, we measured fluorescent levels in both aging and transiently food-deprived males. We found that fluorescence increases with age or after a transient period of food deprivation, similar to our previous reports using transgenic promoter:marker constructs. Interestingly, while EGL-2:YFP is expressed uniformly in sex muscles, the protein is trafficked to the ciliated tips of sensory neurons in both the head and tail. This suggests EAG K+ channels regulate membrane excitability in cilia, perhaps responding to environmental signals through their PAS domains. To address this possibility, we are constructing a version of EAG with the channel removed and the circular permutated GFP inserted in its place, in an attempt to create a construct that will change fluorescence when the animal experiences different nutrient conditions. .

LeBoeuf Brigitte et al. (2010) Biology of the C. elegans Male, Madison, WI "Transient Starvation Produces Lasting Effects on Behavior via EAG K+ channel Expression and Interaction with CaMKII"

Garcia L Rene, LeBoeuf Brigitte

[Biology of the C. elegans Male, Madison, WI, 2010]

An organism experiences varying states of food availability throughout its lifespan. Periods of extreme nutrient deprivation are known to have lasting consequences for an organism, but the mechanisms via which a period of transient starvation affect behavior are unknown. We study the lasting effects of transient starvation using C. elegans male mating behavior. Males must maintain their copulatory spicules inside their tail until they penetrate the hermaphrodite vulva to successfully transfer sperm. Deleting the ether-a-go-go related gene (ERG) K+ channel unc-103 interferes with the tight regulation required to maintain appropriate spicule position until the vulva is penetrated, as ~30% of males lacking this K+ channel protract their spicules in the absence of mating cues. This protraction constitutive (Prc) phenotype induced by loss of unc-103 is suppressed when newly adult males are starved for as little as 3 hrs. In addition, feeding starved unc-103(0) males does not reverse the effects of transient starvation, and the Prc phenotype remains suppressed indefinitely. Therefore, starvation is activating a mechanism responsible for down-regulating sex muscle excitability, and this mechanism remains active even upon return to abundant nutrient conditions. We show that expression of the ether-a-go-go (EAG) K+ channel EGL-2 is necessary for the lasting effects of starvation. We grew unc-103(0) and unc-103(0);egl-2(0) males on plates with and without E. coli in the presence of the translation inhibitor cycloheximide. 34% of unc-103(0) and 74% of unc-103(0);egl-2(0) males starved for 18 hrs and then fed for 7 hrs display the Prc phenotype, indicating that translation of egl-2 is required for the lasting effects of transient nutrient deprivation on behavior. In addition, EGL-2's function is regulated by the calcium/calmodulin-dependent protein kinase II (CaMKII) unc-43. We show that EGL-2 and UNC-43 interact in vitro using yeast two-hybrid and protein binding assays and that mutating potential CaMKII binding sites on EGL-2 changes channel activity. In particular, mutating the EGL-2 S567 to F increases channel activity under abundant nutrient conditions while reducing the channel's ability to function when food is limited. We propose that CaMKII is binding to EGL-2 to attenuate channel activity during food abundance, but this binding is a necessary step in producing the EGL-2 modifications that lead to lasting channel activity during and after a period of transient starvation.

LeBoeuf, Brigitte et al. (2019) International Worm Meeting "The WNT/Ca2+ and WNT/PCP signaling pathways function in the male tail to regulate sex-specific anal depressor remodeling during development."

LeBoeuf, Brigitte, Garcia, L. Rene, Chen, Xin

[International Worm Meeting, 2019]

Determining the mechanisms responsible for programmed muscle remodeling provides insights for how remodeling occurs in pathologies such as heart disease and cancer. The male anal depressor (anal dep) muscle undergoes structural and functional reorganization during the L4 larval stage. Two dorsal/ventral sarcomeres that control defecation in hermaphrodites and L1-L3 males are disassembled and reorganized into four anterior/posterior sarcomeres that adjust spicule position to allow sperm transfer during mating. To determine what genes control sarcomere remodeling, we screened developmental mutants and identified a role for WNT ligands egl-20 and lin-44, as well as frizzled receptor lin-17, in regulating anal dep disassembly. lin-17 is required in both the anal dep as well as the underlying rectal epithelium. lin-44 interacts with lin-17 in the rectal epithelium, where downstream signaling involves the planar cell polarity (PCP) pathway. In contrast, egl-20 interacts with lin-17 in the anal dep. Secreted, non-cell autonomous egl-20, as well as both secreted and membrane-tethered cell autonomous egl-20, regulate anal dep disassembly. We determined that the Ca2+ signaling pathway is downstream of egl-20/lin-17 in the anal dep. We performed a mutant screen for disassembly defects and discovered egl-8/PLC-? is required for normal sarcomere disassembly, and also identified WNT/Ca2+ components including goa-1, itr-1, unc-68, and calcineurin further promote anal dep remodeling. Additionally, we showed that calcium levels increase in wild type male anal deps during L4, and that this increase is not present in males lacking the WNT signaling controlled by egl-20. We determined that egl-8;egl-20 double mutants are similar to either single mutant, indicating they are in the same pathway, and that egl-8 functions in the anal dep to regulated disassembly. To determine that the Ca2+ increase we see in the muscle is downstream of WNT, we artificially increased the Ca2+ levels in egl-20 males using the C. elegans version of DREADD, a modified muscarinic receptor that responds to Clorozipine N-Oxide. Increasing Ca2+ over the developmental stages of the male results in males with normal anal depressors, indicating that it is the increase in Ca2+ levels promoted by egl-20 in the anal dep that are important for sarcomere rearrangement.

LeBoeuf, Brigitte L. et al. (2009) International Worm Meeting "Identifying circuits that respond to changing conditions: CaMKII interacts with EAG K+ channels to reduce C. elegans male sex muscle excitability when food is scarce."

Garcia, L. Rene, LeBoeuf, Brigitte L.

[International Worm Meeting, 2009]

Organisms respond to conditions of food scarcity by up-regulating behaviors that help locate new food sources and down-regulate non-essential behaviors. Behaviors are down-regulated through circuits that reduce the organisms'' response to stimuli, and include the down-regulation of neurons and muscles that produce a behavior. Reducing the activity of excitable cells lessens seizure susceptibility, but the molecular circuits that accomplish this are not well understood. When the ether-a-go-go related gene K+ channel/unc-103 is mutated in C. elegans, male sex muscles seize, resulting in premature protraction of the copulatory spicules. Our genetic analysis demonstrates that CaMKII/unc-43 and unc-103''s paralog, egl-2, function in the sex muscles to reduce excitability when males are food deprived. We show using Yeast Two-Hybrid and in vitro binding assays that UNC-43 and EGL-2 directly interact. This interaction depends on a serine at amino acid 567, located in a potential CaMKII binding site on the EGL-2 c-terminus. Mutating S567 to F results in an active EGL-2 channel that can suppress unc-103(0)-induced muscle seizures, a function that the canonical gain-of-function allele (n693) of egl-2 does not possess. Since an egl-2(0) allele does not disrupt mating behavior, we hypothesize that under well-fed conditions EGL-2 is inactive, while when food is scarce, phosphorylation by UNC-43 activates the channel, reducing excitability. Two types of Ca2+ channels produce the muscle contractions that control spicule protraction, and under conditions where food is scarce, the effects of Ca2+ influx from both these channels needs to be attenuated. The channels are the cell-membrane located L-type voltage-gated Ca2+ channel (L-VGCC) and the sarcoplasmic-reticulum-membrane located ryanodine receptor (RyR). L-VGCCs induce a tonic sex muscle contraction that causes spicule protraction. RyRs are responsible for rhythmic contractions that enable the male to penetrate the tightly closed hermaphrodite vulva. We used acetylcholine agonists arecoline and levamisole that induce calcium influx via L-VGCCs and RyRs, respectively, to determine what UNC-43 and EGL-2 regulate. egl-2(gf) males display reduced response to arecoline but not levamisole. unc-43(n468gf) males display a reduced response to both agonists. We hypothesize that UNC-43 attenuates the effects of L-VGCCs through activation of EGL-2 and RyRs in a separate pathway.

Brigitte L LeBoeuf et al. (2004) West Coast Worm Meeting "Identifying and Characterizing a Mutation Involved in Male Mating Behavior"

Brigitte L LeBoeuf, L Rene Garcia

[West Coast Worm Meeting, 2004]

C. elegans males have a set of stereotypical mating behaviors that can be used to dissect the genetic and molecular pathways involved in behavior. Males mate by locating and positioning their spicules over the hermaphrodite's vulva, inserting their spicules, and then transferring sperm. To study how spicule insertion behavior is regulated, we isolated 5 mutations: sy557, sy558, sy559, sy574, and rg2 that cause the male's spicules to protrude from their bodies in the absence of mating cues. sy557 is located in unc-103 , whereas the other 4 affected genes have not been identified. UNC-103 is an erg-like K+ channel that is postulated to control male mating behavior. UNC-103 is expressed in many neurons in the head, ventral cord, and male tail, but sy557 seems to only affect spicule protraction. sy557 causes 66% of males to display the Pr otraction c onstitutive (Prc) phenotype. To understand how unc-103 interacts with other genes that control the timing of spicule protraction, we made double mutant combinations between unc-103(sy557) and the other mutations. unc-103(sy557); sy558 and unc-103(sy557); sy559 males are 100% Prc, suggesting that these combinations of mutations lead to a deficit in behavioral control that is too difficult to overcome. In contrast, the unc-103(sy557); sy574 double mutant males are 78% Prc, similar to the 66% of sy557 and 54% of sy574 . Since unc-103 might function in the same pathway as the gene affected by sy574 , we are identifying the gene that is affected by sy574 to understand how unc-103 may regulate behavior. sy574 maps to a 570 kb region on LG IV between unc-24 and dpy-20 . We have narrowed the region to between SNPs K08F4 and R102. Candidate genes that are located in this region include unc-43, gpa-7, tax-6, and cal-4 . Mutations in unc-43, gpa-7, and tax-6 complement sy574 , indicating that the mutation is not in these genes.

Brigitte LeBoeuf et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "unc-103 (ERG-like K+ channel) and unc-43 (CaMKII) inhibit male spicule muscle contractions prior to mating"

Brigitte LeBoeuf, Rene Garcia

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

We are using male mating to determine how a complex behavior is regulated at the molecular level. Males mate by locating the hermaphrodite"s vulva and inserting their copulatory spicules so they can transfer sperm. Successful insertion requires the sex muscles to contract at the appropriate time, a process unc-103 regulates. Deleting this ERG-like K+ channel causes ~40% of males to spontaneously protract their spicules. unc-103 is expressed broadly in muscles and neurons, however we found that expressing unc-103 DNA in the spicule muscles can rescue the premature protraction defect. Although unc-103 regulates the spicule muscles prior to mating, ~60% do not display spontaneous protraction, suggesting that a parallel pathway regulates this behavior. We identified unc-43 CaMKII as a component of this pathway. ~50% of unc-43(sy574) males display spontaneous protraction; when combined with unc-103(0), ~100% of double mutant males display the defect. Though unc-43 is a widely-expressed protein that regulates many behaviors, the sy574 allele only perturbs spicule protraction, allowing us to determine how this signaling molecule regulates one behavior. Spicule protraction is mediated by calcium influx into the sex muscles; to control this behavior, we found that unc-43 is suppressing calcium influx from extracellular, but not intracellular stores. A loss-of-function mutation in the egl-19 L-type Ca2+ channel suppresses unc-43(sy574)-induced protraction, but a deletion in the unc-68 ryanodine receptor does not. unc-43 is possibly suppressing calcium influx by mediating the function of the EAG-like K+ channel egl-2 and the effects of unc-64 syntaxin. A gain-of-function mutation in egl-2 is able to suppress the unc-43(sy574) phenotype, as is a reduction-of-function mutation in unc-64. Now that we know what is working with unc-43, we wanted to determine where it is regulating spicule protraction. We ablated the SPC motor neuron that controls protraction and the accessory anal depressor muscle that contacts the sex muscles. unc-43 is probably functioning in both neurons and muscles, as ablating both the SPC and the anal depressor rescues unc-43(sy574)-induced spicule protraction. We propose that unc-43 is controlling spicule protraction timing in both muscles and neurons via a pathway parallel to unc-103.