The small GTPase RAB-18 is involved in regulating development/diapause by recruiting the intestinal cholesterol transporter NCR-1 onto the apical side in Caenorhabditis elegans.

RAB family proteins, which are small GTPases, are integral to the process of eukaryotic membrane trafficking. In the nematode, Caenorhabditis elegans, 31 RAB proteins have been identified through genome sequencing. Using an RNAi screen specifically targeting C. elegans rab genes, we identified multiple genes that are involved in the regulation of larval development, in particular, the rab-18 gene. Our molecular genetic studies resulted in several findings. First, RAB-18 predominantly functions in the intestine to regulate larval development by modulating steroid hormone signaling. Second, the C. elegans cholesterol transporter NCR-1 is a target of RAB-18 in the intestine. Third, the membrane trafficking of NCR-1 to the apical side in intestinal cells is particularly influenced by RAB-18. Finally, RAB-18 and NCR-1 possibly co-localize on membrane vesicles. Our study is the first to demonstrate the relationship between a RAB protein and a cholesterol transporter, in which the RAB protein probably drives the transporter to the apical membrane in the intestine to regulate cholesterol uptake. This study provides insight into the molecular mechanisms underlying human disease stemming from a transport defect of cholesterol and its derivative.

The G protein-coupled receptor neuropeptide receptor-15 modulates larval development via the transforming growth factor-ß DAF-7 protein in Caenorhabditis elegans.

G protein-coupled receptors (GPCRs) are a major class of membrane receptors that modulate a wide range of physiological functions. These receptors transmit extracellular signals, including secreted bioactive peptides, to intracellular signaling pathways. The nematode Caenorhabditis elegans has FMRFamide-like peptides, which are one of the most diverse neuropeptide families, some of which modulate larval development through GPCRs. In this study, we identified the GPCR neuropeptide receptor (NPR)-15, which modulates C. elegans larval development. Our molecular genetic analyses indicated the following: 1) NPR-15 mainly functions in ASI neurons, which predominantly regulate larval development, 2) NPR-15 interacts with GPA-4, a C. elegans Gα subunit, and 3) NPR-15, along with GPA-4, modulates larval development by regulating the production and secretion of the transforming growth factor-ß (TGF-ß)-like protein DAF-7. The present study is the first report to demonstrate the importance of a GPCR to the direct regulation of a TGF-ß-like protein.

The FMRFamide-like peptide FLP-1 modulates larval development by regulating the production and secretion of the insulin-like peptide DAF-28 in Caenorhabditis elegans.

The FMRFamide-like peptides (FLPs) are conserved in both free-living and parasitic nematodes. This molecular genetic study verified the relevance of the flp-1 gene, which is conserved in many nematode species, to the larval development of the free-living soil nematode Caenorhabditis elegans. Using C. elegans as a model, we found that: (1) FLP-1 suppressed larval development, resulting in diapause; (2) the secretion of FLP-1, which is produced in AVK head neurons, was suppressed by the presence of food (Escherichia coli) as an environmental factor to continue larval development; (3) the FLP-1 reduced the production and secretion of DAF-28, which is produced in ASI head neurons and is the predominant insulin-like peptide (INS) present. FLP-1 is conserved in many species of plant-parasitic root-knot nematodes that cause severe damage to crops. Therefore, our findings may provide insight into the development of new nematicides that can disturb their infection and development.

The FMRFamide-like peptide FLP-2 is involved in the modulation of larval development and adult lifespan by regulating the secretion of the insulin-like peptide INS-35 in Caenorhabditis elegans.

In the animal kingdom, neuropeptides regulate diverse physiological functions. In invertebrates, FMRFamide and its related peptides, a family of neuropeptides, play an important role as neurotransmitters. The FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families and are conserved in nematodes. Our screen for flp genes of the free-living soil nematode Caenorhabditis elegans revealed that the flp-2 gene is involved in the larval development. The gene is also conserved in plant-parasitic root-knot nematodes. Our molecular genetic analyses of the C. elegans flp-2 gene demonstrated as follows: (1) the production and secretion of FLP-2, produced in the head neurons, are controlled by environmental factors (growth density and food); (2) the FLP-2 is involved in not only larval development but also adult lifespan by regulating the secretion of one of the insulin-like peptides INS-35, produced in the intestine. These findings provide new insight into the development of new nematicides.

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin).

Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (ß-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans.

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic Caenorhabditis elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signaling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g., zebrafish).

The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cells.

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle.

Protein phosphatase 2A (PP2A) is a heterotrimer composed of single catalytic and scaffolding subunits and one of several possible regulatory subunits. We identified PPTR-2, a regulatory subunit of PP2A, as a binding partner for the giant muscle protein UNC-89 (obscurin) in Caenorhabditis elegans. PPTR-2 is required for sarcomere organization when its paralogue, PPTR-1, is deficient. PPTR-2 localizes to the sarcomere at dense bodies and M-lines, colocalizing with UNC-89 at M-lines. PP2A components in C. elegans include one catalytic subunit LET-92, one scaffolding subunit (PAA-1), and five regulatory subunits (SUR-6, PPTR-1, PPTR-2, RSA-1, and CASH-1). In adult muscle, loss of function in any of these subunits results in sarcomere disorganization. rsa-1 mutants show an interesting phenotype: one of the two myosin heavy chains, MHC A, localizes as closely spaced double lines rather than single lines. This "double line" phenotype is found in rare missense mutants of the head domain of MHC B myosin, such as unc-54(s74). Analysis of phosphoproteins in the unc-54(s74) mutant revealed two additional phosphoserines in the nonhelical tailpiece of MHC A. Antibodies localize PPTR-1, PAA-1, and SUR-6 to I-bands and RSA-1 to M-lines and I-bands. Therefore, PP2A localizes to sarcomeres and functions in the assembly or maintenance of sarcomeres.

Comparison of physiological functions of antagonistic insulin-like peptides, INS-23 and INS-18, in Caenorhabditis elegans.

In Caenorhabditis elgans, insulin-like peptides have significant roles in modulating larval diapause and adult lifespan via the insulin/IGF-1 signaling (IIS) pathway. Although 40 insulin-like peptides (ILPs) have been identified, it remains unknown how ILPs act as either agonists or antagonists for their sole receptor, DAF-2. Here we found 1) INS-23 functions as an antagonistic ILP to promote larval diapause through the IIS pathway like a DAF-2 antagonist, INS-18, 2) INS-23 and INS-18 have similar biochemical functions. In addition, our molecular modeling suggests that INS-23 and INS-18 have characteristic insertions in the B-domain, which are crucial for the recognition of the insulin receptor, when compared with DAF-2 agonists. These characteristic insertions in the B-domain of INS-23 and INS-18 would modulate their intermolecular interactions with the DAF-2 receptor, which may lead these molecules to act as antagonistic ligands. Our study provides new insight into the function and structure of ILPs.

High-resolution imaging of muscle attachment structures in Caenorhabditis elegans.

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in Caenorhabditis elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (ß-integrin) and proteins that interact in a complex with the cytoplasmic tail of ß-integrin and localize to the basal muscle cell membrane [UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin), and UNC-95], are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed "zipper-like" structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the "zipper-like" structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have "dot-like" dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

Indoles from commensal bacteria extend healthspan.

Multiple studies have identified conserved genetic pathways and small molecules associated with extension of lifespan in diverse organisms. However, extending lifespan does not result in concomitant extension in healthspan, defined as the proportion of time that an animal remains healthy and free of age-related infirmities. Rather, mutations that extend lifespan often reduce healthspan and increase frailty. The question arises as to whether factors or mechanisms exist that uncouple these processes and extend healthspan and reduce frailty independent of lifespan. We show that indoles from commensal microbiota extend healthspan of diverse organisms, including Caenorhabditis elegans, Drosophila melanogaster, and mice, but have a negligible effect on maximal lifespan. Effects of indoles on healthspan in worms and flies depend upon the aryl hydrocarbon receptor (AHR), a conserved detector of xenobiotic small molecules. In C. elegans, indole induces a gene expression profile in aged animals reminiscent of that seen in the young, but which is distinct from that associated with normal aging. Moreover, in older animals, indole induces genes associated with oogenesis and, accordingly, extends fecundity and reproductive span. Together, these data suggest that small molecules related to indole and derived from commensal microbiota act in diverse phyla via conserved molecular pathways to promote healthy aging. These data raise the possibility of developing therapeutics based on microbiota-derived indole or its derivatives to extend healthspan and reduce frailty in humans.

Twitchin kinase inhibits muscle activity.

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family's kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle.

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 µM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

Diapause is associated with a change in the polarity of secretion of insulin-like peptides.

The insulin/IGF-1 signalling (IIS) pathway plays an important role in the regulation of larval diapause, the long-lived growth arrest state called dauer arrest, in Caenorhabditis elegans. In this nematode, 40 insulin-like peptides (ILPs) have been identified as putative ligands of the IIS pathway; however, it remains unknown how ILPs modulate larval diapause. Here we show that the secretory polarity of INS-35 and INS-7, which suppress larval diapause, is changed in the intestinal epithelial cells at larval diapause. These ILPs are secreted from the intestine into the body cavity during larval stages. In contrast, they are secreted into the intestinal lumen and degraded during dauer arrest, only to be secreted into the body cavity again when the worms return to developmental growth. The process that determines the secretory polarity of INS-35 and INS-7, thus, has an important role in the modulation of larval diapause.

Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans.

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle.

In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin.

On-demand optical immobilization of Caenorhabditis elegans for high-resolution imaging and microinjection.

This paper describes a novel selective immobilization technique based on optical control of the sol-gel transition of thermoreversible Pluronic gel, which provides a simple, versatile, and biocompatible approach for high-resolution imaging and microinjection of Caenorhabditis elegans.

Suppressor mutations suggest a surface on PAT-4 (Integrin-linked Kinase) that interacts with UNC-112 (Kindlin).

Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of ß-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537-28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.

Vitamin B12 deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and reduced lifespan.

Vitamin B12 (B12) deficiency has been linked to developmental disorders, metabolic abnormalities, and neuropathy; however, the mechanisms involved remain poorly understood. Caenorhabditis elegans grown under B12-deficient conditions for five generations develop severe B12 deficiency associated with various phenotypes that include decreased egg-laying capacity (infertility), prolonged life cycle (growth retardation), and reduced lifespan. These phenotypes resemble the consequences of B12 deficiency in mammals, and can be induced in C. elegans in only 15 days. Thus, C. elegans is a suitable animal model for studying the biological processes induced by vitamin deficiency.

A Caenorhabditis elegans insulin-like peptide, INS-17: its physiological function and expression pattern.

The insulin/insulin-like growth factor-1 signaling pathway of Caenorhabditis elegans regulates larval diapause and adult lifespan through the sole insulin receptor-like protein, DAF-2. In the present study, the physiological function and expression pattern of INS-17, one of the C. elegans insulin-like peptides, were examined by disruption and overexpression of the gene, and by the use of a reporter gene. INS-17 might function as a DAF-2 antagonist in the regulation of larval diapause, but not of the adult lifespan. The reporter protein was intensively expressed during larval diapause. It showed a drastic decrease in amount after larval diapause, which matches well the physiological function of INS-17.