Multiple sensory neurons mediate starvation-dependent aversive navigation in Caenorhabditis elegans.

Animals demonstrate flexible behaviors through associative learning based on their experiences. Deciphering the neural mechanisms for sensing and integrating multiple types of sensory information is critical for understanding such behavioral controls. The soil nematode Caenorhabditis elegans avoids salt concentrations it has previously experienced under starvation conditions. Here, we identify a pair of sensory neurons, the ASG neuron pair, which in cooperation with the ASER salt-sensing neuron generate starvation-dependent salt avoidance. Animals whose sensory input is restricted to only ASER failed to show learned avoidance due to inappropriately directed navigation behaviors. However, their navigation through a salt concentration gradient was improved by allowing sensory inputs to ASG in addition to ASER. Detailed behavioral analyses of these animals revealed that input from ASG neurons is required not only for controlling the frequency of initiating a set of sharp turns (called pirouettes) based on detected ambient salt concentrations but also adjusting the migration direction during pirouettes. Optogenetic activation of ASER by ChR2 induced turning behaviors in a salt concentration-dependent manner where presence of intact ASG was important for the starvation-dependent responses. Calcium imaging of the activity of ASG neurons in freely moving worms revealed that ASG is activated upon turning behavior. Our results indicate that ASG neurons cooperate with the ASER neuron to generate destination-directed reorientation in starvation-associated salt concentration avoidance.

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

A Gustatory Neural Circuit of Caenorhabditis elegans Generates Memory-Dependent Behaviors in Na+ Chemotaxis.

Animals show various behaviors in response to environmental chemicals. These behaviors are often plastic depending on previous experiences. Caenorhabditis elegans, which has highly developed chemosensory system with a limited number of sensory neurons, is an ideal model for analyzing the role of each neuron in innate and learned behaviors. Here, we report a new type of memory-dependent behavioral plasticity in Na+ chemotaxis generated by the left member of bilateral gustatory neuron pair ASE (ASEL neuron). When worms were cultivated in the presence of Na+, they showed positive chemotaxis toward Na+, but when cultivated under Na+-free conditions, they showed no preference regarding Na+ concentration. Both channelrhodopsin-2 (ChR2) activation with blue light and up-steps of Na+ concentration activated ASEL only after cultivation with Na+, as judged by increase in intracellular Ca2+ Under cultivation conditions with Na+, photoactivation of ASEL caused activation of its downstream interneurons AIY and AIA, which stimulate forward locomotion, and inhibition of its downstream interneuron AIB, which inhibits the turning/reversal behavior, and overall drove worms toward higher Na+ concentrations. We also found that the Gq signaling pathway and the neurotransmitter glutamate are both involved in the behavioral response generated by ASEL.SIGNIFICANCE STATEMENT Animals have acquired various types of behavioral plasticity during their long evolutionary history. Caenorhabditis elegans prefers odors associated with food, but plastically changes its behavioral response according to previous experience. Here, we report a new type of behavioral response generated by a single gustatory sensory neuron, the ASE-left (ASEL) neuron. ASEL did not respond to photostimulation or upsteps of Na+ concentration when worms were cultivated in Na+-free conditions; however, when worms were cultivated with Na+, ASEL responded and inhibited AIB to avoid turning and stimulated AIY and AIA to promote forward locomotion, which collectively drove worms toward higher Na+ concentrations. Glutamate and the Gq signaling pathway are essential for driving worms toward higher Na+ concentrations.

Regulation of experience-dependent bidirectional chemotaxis by a neural circuit switch in Caenorhabditis elegans.

The nematode Caenorhabditis elegans changes its chemotaxis to NaCl depending on previous experience. At the behavioral level, this chemotactic plasticity is generated by reversing the elementary behaviors for chemotaxis, klinotaxis, and klinokinesis. Here, we report that bidirectional klinotaxis is achieved by the proper use of at least two different neural subcircuits. We simulated an NaCl concentration change by activating an NaCl-sensitive chemosensory neuron in phase with head swing and successfully induced klinotaxis-like curving. The curving direction reversed depending on preconditioning, which was consistent with klinotaxis plasticity under a real concentration gradient. Cell-specific ablation and activation of downstream interneurons revealed that ASER-evoked curving toward lower concentration was mediated by AIY interneurons, whereas curving to the opposite direction was not. These results suggest that the experience-dependent bidirectionality of klinotaxis is generated by a switch between different neural subcircuits downstream of the chemosensory neuron.

Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioral plasticity at the sensory neuron synapses of Caenorhabditis elegans.

Growing evidence suggests that sensory neuron synapses not merely pass, but actively encode sensory information and convey it to the central nervous system. The chemosensory preferences of Caenorhabditis elegans, as manifested in the direction of chemotaxis, are reversibly regulated by prior experience at the level of sensory neurons; the attractive drive is promoted by diacylglycerol (DAG) signaling, whereas the counteracting repulsive drive requires PtdIns(3,4,5)P(3) signaling. Here we report that the two opposing drives require a class IIA phosphatidylinositol transfer protein (PITP), PITP-1, which localizes to the sensory neuron synapses. In pitp-1 mutants, attraction behavior to salt is reduced, whereas conditioned repulsion from salt is eliminated: the mutants inflexibly show weak attraction behavior to salt, irrespective of prior experience. To generate flexible behavioral outputs, attraction and repulsion, PITP-1 acts in the gustatory neuron ASER and likely regulates neurotransmission from ASER, as pitp-1 mutations do not affect the ASER Ca(2+) response to sensory stimulus. Furthermore, full attraction to salt is restored in pitp-1 mutants by expression of the phosphatidylinositol transfer domain alone, and also by mutations of a DGK gene that cause accumulation of DAG, suggesting that PITP-1 serves for DAG production via phosphatidylinositol transport and, hence, regulates synaptic transmission. In addition to gustatory behavior, olfactory behaviors and osmotic avoidance are also regulated by PITP-1 in the sensory neurons that detect each sensory stimulus. Thus, PITP-1-dependent phosphatidylinositol transport is essential for sensory neuron synapses to couple sensory inputs to effective behavioral responses.

Different modes of stimuli delivery elicit changes in glutamate driven, experience-dependent interneuron response in C. elegans.

Memory-related neuronal responses are often elicited by sensory stimuli that recapitulate previous experience. Despite the importance of this sensory input processing, its underlying mechanisms remain poorly understood. Caenorhabditis elegans chemotax towards salt concentrations experienced in the presence of food. The amphid sensory neurons ASE-left and ASE-right respond to increases and decreases of ambient salt concentration in opposite manners. AIA, AIB and AIY interneurons are post-synaptic to the ASE pair and are thought to be involved in the processing of salt information transmitted from ASE. However, it remains elusive how the responses of these interneurons are regulated by stimulus patterns. Here we show that AIY interneurons display an experience-dependent response to gradual salt concentration changes but not to abrupt stepwise concentration changes. Animals with AIY intact (but AIA and AIB ablated) chemotax towards low salt concentrations similarly to wild-type animals after cultivation with low salt. ASE neurons transmit salt information about the environment through glutamatergic signaling, directing the activity of the interneurons AIY that promote movement towards favorable conditions.

Multiple p38/JNK mitogen-activated protein kinase (MAPK) signaling pathways mediate salt chemotaxis learning in C. elegans.

Animals are able to adapt their behaviors to the environment. In order to achieve this, the nervous system plays integrative roles, such as perception of external signals, sensory processing, and behavioral regulations via various signal transduction pathways. Here genetic analyses of Caenorhabditis elegans (C. elegans) found that mutants of components of JNK and p38 mitogen-activated protein kinase (MAPK) signaling pathways, also known as stress-activated protein kinase (SAPK) signaling pathways, exhibit various types of defects in the learning of salt chemotaxis. C. elegans homologs of JNK MAPKKK and MAPKK, MLK-1 and MEK-1, respectively, are required for avoidance of salt concentrations experienced during starvation. In contrast, homologs of p38 MAPKKK and MAPKK, NSY-1 and SEK-1, respectively, are required for high-salt chemotaxis after conditioning. Genetic interaction analyses suggest that a JNK family MAPK, KGB-1, functions downstream of both signaling pathways to regulate salt chemotaxis learning. Furthermore, we found that the NSY-1/SEK-1 pathway functions in sensory neurons, ASH, ADF, and ASER, to regulate the learned high-salt chemotaxis. A neuropeptide, NLP-3, expressed in ASH, ADF, and ASER neurons, and a neuropeptide receptor, NPR-15, expressed in AIA interneurons that receive synaptic input from these sensory neurons, function in the same genetic pathway as NSY-1/SEK-1 signaling. These findings suggest that this MAPK pathway may affect neuropeptide signaling between sensory neurons and interneurons, thus promoting high-salt chemotaxis after conditioning.

Single-cell transcriptional analysis of taste sensory neuron pair in Caenorhabditis elegans.

The nervous system is composed of a wide variety of neurons. A description of the transcriptional profiles of each neuron would yield enormous information about the molecular mechanisms that define morphological or functional characteristics. Here we show that RNA isolation from single neurons is feasible by using an optimized mRNA tagging method. This method extracts transcripts in the target cells by co-immunoprecipitation of the complexes of RNA and epitope-tagged poly(A) binding protein expressed specifically in the cells. With this method and genome-wide microarray, we compared the transcriptional profiles of two functionally different neurons in the main C. elegans gustatory neuron class ASE. Eight of the 13 known subtype-specific genes were successfully detected. Additionally, we identified nine novel genes including a receptor guanylyl cyclase, secreted proteins, a TRPC channel and uncharacterized genes conserved among nematodes, suggesting the two neurons are substantially different than previously thought. The expression of these novel genes was controlled by the previously known regulatory network for subtype differentiation. We also describe unique motif organization within individual gene groups classified by the expression patterns in ASE. Our study paves the way to the complete catalog of the expression profiles of individual C. elegans neurons.

Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans.

Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood. Here, we show that CASY-1, the Caenorhabditis elegans ortholog of calsyntenins/alcadeins, is essential for multiple types of learning. Through a genetic screen, we found that casy-1 mutants show defects in salt chemotaxis learning. casy-1 mutants also show defects in temperature learning, olfactory adaptation, and integration of two sensory signals. casy-1 is widely expressed in the nervous system. Expression of casy-1 in a single sensory neuron and at the postdevelopmental stage is sufficient for its function in salt chemotaxis learning. The fluorescent protein-tagged ectodomain of CASY-1 is released from neurons. Moreover, functional domain analyses revealed that both cytoplasmic and transmembrane domains of this protein are dispensable, whereas the ectodomain, which contains the LG/LNS-like domain, is critically required for learning. These results suggest that learning is modulated by the released ectodomain of CASY-1.

Caenorhabditis Elegans Exhibits Morphine Addiction-like Behavior via the Opioid-like Receptor NPR-17.

Addiction has become a profound societal problem worldwide, and few effective treatments are available. The nematode Caenorhabditis elegans (C. elegans) is an excellent invertebrate model to study neurobiological disease states. C. elegans reportedly developed a preference for cues that had previously been paired with addictive drugs, similar to place conditioning findings in rodents. Moreover, several recent studies discovered and reported the existence of an opioid-like system in C. elegans. Still unclear, however, is whether C. elegans exhibits addictive-like behaviors for opioids, such as morphine. In the present study, we found that C. elegans exhibited dose-dependent preference for morphine using the conditioned chemosensory-cue preference (CCP) test. This preference was blocked by co-treatment with the opioid receptor antagonist naloxone. C. elegans also exhibited aversion to naloxone-precipitated withdrawal from chronic morphine exposure. The expression of morphine-induced CCP and morphine withdrawal were abolished in worms that lacked the opioid-like receptor NPR-17. Dopamine-deficient mutant (cat-2 (e1112)) worms also did not exhibit morphine-induced CCP. These results indicate that the addictive function of the opioid system exists in C. elegans, which may serve as a useful model of opioid addiction.

Glutamate signaling from a single sensory neuron mediates experience-dependent bidirectional behavior in Caenorhabditis elegans.

Orientation and navigation behaviors of animals are modulated by past experiences. However, little is known about the mechanisms by which sensory inputs are translated into multi-directional orientation behaviors in an experience-dependent manner. Here, we report a neural mechanism for bidirectional salt-concentration chemotaxis of Caenorhabditis elegans. The salt-sensing neuron ASE right (ASER) is always activated by a decrease of salt concentration, while the directionality of reorientation behaviors is inverted depending on previous salt experiences. AIB, the interneuron postsynaptic to ASER, and neurons farther downstream of AIB show experience-dependent bidirectional responses, which are correlated with reorientation behaviors. These bidirectional behavioral and neural responses are mediated by glutamate released from ASER. Glutamate acts through the excitatory glutamate receptor GLR-1 and inhibitory glutamate receptor AVR-14, both acting in AIB. These findings suggest that experience-dependent reorientation behaviors are generated by altering the magnitude of excitatory and inhibitory postsynaptic signals from a sensory neuron to interneurons.

Simultaneous recording of behavioral and neural responses of free-moving nematodes C. elegans.

To reveal the neural mechanisms that control animal behavior, it is necessary to link the neural responses to behavioral changes and interpret them. We have developed a protocol to simultaneously record the behavior and neural activity of freely moving C. elegans by combining a microfluidic device and a tracking stage. Here we detail the protocol for the experiment, with an example of behavioral and neural responses of nematodes to salt concentration changes. For complete details on the use and execution of this protocol, please refer to Sato et al. (2021).

Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of C. elegans.

The ability of animals to process dynamic sensory information facilitates foraging in an ever-changing environment. However, molecular and neural mechanisms underlying such ability remain elusive. The ClC anion channels/transporters play a pivotal role in cellular ion homeostasis across all phyla. Here, we find a ClC chloride channel is involved in salt concentration chemotaxis of Caenorhabditis elegans. Genetic screening identified two altered-function mutations of clh-1 that disrupt experience-dependent salt chemotaxis. Using genetically encoded fluorescent sensors, we demonstrate that CLH-1 contributes to regulation of intracellular anion and calcium dynamics of salt-sensing neuron, ASER. The mutant CLH-1 reduced responsiveness of ASER to salt stimuli in terms of both temporal resolution and intensity, which disrupted navigation strategies for approaching preferred salt concentrations. Furthermore, other ClC genes appeared to act redundantly in salt chemotaxis. These findings provide insights into the regulatory mechanism of neuronal responsivity by ClCs that contribute to modulation of navigation behavior.

The insulin/PI 3-kinase pathway regulates salt chemotaxis learning in Caenorhabditis elegans.

The insulin-like signaling pathway is known to regulate fat metabolism, dauer formation, and longevity in Caenorhabditis elegans. Here, we report that this pathway is also involved in salt chemotaxis learning, in which animals previously exposed to a chemoattractive salt under starvation conditions start to show salt avoidance behavior. Mutants of ins-1, daf-2, age-1, pdk-1, and akt-1, which encode the homologs of insulin, insulin/IGF-I receptor, PI 3-kinase, phosphoinositide-dependent kinase, and Akt/PKB, respectively, show severe defects in salt chemotaxis learning. daf-2 and age-1 act in the ASER salt-sensing neuron, and the activity level of the DAF-2/AGE-1 pathway in this neuron determines the extent and orientation of salt chemotaxis. On the other hand, ins-1 acts in AIA interneurons, which receive direct synaptic inputs from sensory neurons and also send synaptic outputs to ASER. These results suggest that INS-1 secreted from AIA interneurons provides feedback to ASER to generate plasticity of chemotaxis.

GPC-1, a G protein gamma-subunit, regulates olfactory adaptation in Caenorhabditis elegans.

Caenorhabditis elegans genome carries two Ggamma genes, gpc-1 and gpc-2, and two Gbeta genes, gpb-1 and gpb-2. Of these, gpc-2 and gpb-1 are expressed ubiquitously and are essential for viability. Through a genetic screen, we identified gpc-1 as essential for olfactory adaptation. While wild-type animals show decreased chemotaxis to the odorant benzaldehyde after a short preexposure to the odorant, gpc-1 mutants are still attracted to the odorant after the same preexposure. Cell-specific rescue experiments show that gpc-1 acts in the AWC olfactory neurons. Coexpression of GPC-1 and GPB-1, but not GPB-2, caused enhanced adaptation, indicating that GPC-1 may act with GPB-1. On the other hand, knock down of gpc-2 by cell-targeted RNAi caused reduced chemotaxis to the odorant in unadapted animals, indicating that GPC-2 mainly act for olfactory sensation and the two Ggamma's have differential functions. Nonetheless, overexpression of gpc-2 in AWC neurons rescued the adaptation defects of gpc-1 mutants, suggesting partially overlapping functions of the two Ggamma's. We further tested genetic interaction of gpc-1 with several other genes involved in olfactory adaptation. Our analyses place goa-1 Goalpha and let-60 Ras in parallel to gpc-1. In contrast, a gain-of-function mutation in egl-30 Gqalpha was epistatic to gpc-1, suggesting the possibility that gpc-1 Ggamma may act upstream of egl-30 Gqalpha.

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression.

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Galphas proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and beta2 adrenergic receptor (beta2AR) with high sensitivity. Interestingly, we also detected constitutive activity of beta2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.

Caenorhabditis elegans DYF-11, an orthologue of mammalian Traf3ip1/MIP-T3, is required for sensory cilia formation.

Cilia and flagella play critical roles in cell motility, development and sensory perception in animals. Formation and maintenance of cilia require a conserved protein transport system called intraflagellar transport (IFT). Here, we show that Caenorhabditis elegans dyf-11 encodes an evolutionarily conserved protein required for cilium biogenesis. dyf-11 is expressed in most of the ciliated neurons and is regulated by DAF-19, a crucial transcription factor for ciliary genes in C. elegans. dyf-11 mutants exhibit stunted cilia, fluorescent dye-filling defects (Dyf) of sensory neurons, and abnormal chemotaxis (Che). Cell- and stage-specific rescue experiments indicated that DYF-11 is required for formation and maintenance of sensory cilia in cell-autonomous manner. Fluorescent protein-tagged DYF-11 localizes to cilia and moves antero- and retrogradely via IFT. Analysis of DYF-11 movement in bbs mutants further suggested that DYF-11 is likely associated with IFT complex B. Domain analysis using DYF-11 deletion constructs revealed that the coiled-coil region is required for proper localization and ciliogenesis. We further show that Traf3ip1/MIP-T3, the mammalian orthologue of DYF-11, localizes to cilia in the MDCK renal epithelial cells.

MBR-1, a novel helix-turn-helix transcription factor, is required for pruning excessive neurites in Caenorhabditis elegans.

In the developing brain, excessive neurites are actively pruned in the construction and remodeling of neural circuits. We demonstrate for the first time that the pruning of neurites occurs in the simple neural circuit of Caenorhabditis elegans and that a novel transcription factor, MBR-1, is involved in this process. We identified MBR-1 as a C. elegans ortholog of Mblk-1, a transcription factor that is expressed preferentially in the mushroom bodies of the honeybee brain. Although Mblk-1 homologs are conserved among animal species, their roles in the nervous system have never been analyzed. We used C. elegans as an ideal model animal for analysis of neuronal development. mbr-1 is expressed in various neurons in the head and tail ganglia. A comparison of the morphology of mbr-1-expressing neurons revealed that excessive neurites connecting the left and right AIM interneurons are eliminated during larval stages in wild-type but are sustained through the adult stage in the mbr-1 mutant. In addition, mbr-1 expression is regulated by UNC-86, a POU domain transcription factor, and the pruning of the excessive AIM connection is impaired in the unc-86 mutant. These findings provide an important clue for further genetic dissection of neurite pruning.

The Caenorhabditis elegans eukaryotic initiation factor 5A homologue, IFF-1, is required for germ cell proliferation, gametogenesis and localization of the P-granule component PGL-1.

Eukaryotic initiation factor 5A (eIF-5A) was originally isolated as a translation initiation factor. However, this function has since been reconsidered, with recent studies pointing to roles for eIF-5A in mRNA metabolism and trafficking [Microbiol. Mol. Biol. Rev. 66 (2002) 460; Eur. Mol. Biol. Org. J. 17 (1998) 2914]. The Caenorhabditis elegans genome contains two eIF-5A homologues, iff-1 and iff-2, whose functions in vivo were examined in this study. The iff-2 mutation causes somatic defects that include slow larval growth and disorganized somatic gonadal structures in hermaphrodites. iff-2 males show disorganized tail sensory rays and spicules. On the other hand, iff-1 mRNA is expressed in the gonad, and the lack of iff-1 activity causes sterility with an underproliferated germline resulting from impaired mitotic proliferation in both hermaphrodites and males. In spite of underproliferation, meiotic nuclei are observed, as revealed by presence of immunoreactivity to the anti-HIM-3 antibody; however, no gametogenesis occurs in the iff-1 gonads. These phenotypes are in part similar to the mutants affected in the components of P granules, which are the C. elegans counterparts of germ granules [Curr. Top Dev. Biol. 50 (2000) 155]. We found that localization of the P-granule component PGL-1 to P granules is disrupted in the iff-1 mutant. In summary, the two C. elegans homologues of eIF-5A act in different tissues: IFF-2 is required in the soma, and IFF-1 is required in the germline for germ cell proliferation, for gametogenesis after entry into meiosis, and for proper PGL-1 localization on P granules.