MINIREVIEW Molecular and genetic characterization of osmosensing and signal transduction in the nematode Caenorhabditis elegans Keith P. Choe and Kevin Strange Departments of Anesthesiology, Pharmacology and Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA Introduction Regulation of intracellular and extracellular solute and water balance is a fundamental requirement for meta- zoans. The volume of animal cells is regulated by accu- mulation and loss of inorganic ions, primarily Na + , K + and Cl – , and specialized organic solutes termed organic osmolytes [1,2]. The effector mechanisms that mediate volume regulatory changes in the intracellular levels of these solutes are generally well-defined [1]. However, little is known about how animal cells detect volume changes and transduce those signals into regu- latory responses. Extracellular solute and water balance in animals is maintained by behavioral responses and by the func- tion of the kidney and kidney-like organs, and extra- renal organs, such as the insect hindgut, fish and crustacean gills and the mammalian intestine. As with cell volume perturbations, the molecular mechanisms by which animals detect extracellular osmotic and ionic disturbances are not fully defined. In addition, little is known about how the activities of various osmoregulatory solute and water transport pathways are coordinately regulated during osmotic challenges. The nematode Caenorhabditis elegans provides numerous experimental advantages for defining the Keywords mechanosensing osmoregulation; osmotic stress; organic osmolytes; Ste20 kinases; TRP channels; WNK kinases Correspondence K. Strange, Vanderbilt University Medical Center, T-4202 Medical Center North, Nashville, TN 37232-2520, USA Fax: +1 615 343 3916 E-mail: kevin.strange@vanderbilt.edu (Received 2 July 2007, accepted 30 August 2007) doi:10.1111/j.1742-4658.2007.06098.x Osmotic homeostasis is a fundamental requirement for life. In general, the effector mechanisms that mediate cellular and extracellular osmoregulation in animals are reasonably well defined. However, at the molecular level, little is known about how animals detect osmotic and ionic perturbations and transduce them into regulatory responses. The nematode Caenorhabd- itis elegans provides numerous powerful experimental advantages for defin- ing the genes and integrated gene networks that underlie basic biological processes. These advantages include a fully sequenced and well-annotated genome, forward and reverse genetic and molecular tractability, and a rela- tively simple anatomy. C. elegans normally inhabits soil environments where it is exposed to repeated osmotic stress. In the laboratory, nema- todes readily acclimate to and recover from extremes of hypertonicity. We review recent progress in defining the molecular mechanisms that underlie osmosensing and associated signal transduction in C. elegans. Some of these mechanisms are now known to be highly conserved. Therefore, studies of osmosensing in nematodes have provided, and will undoubtedly continue to provide, new insights into similar processes in more complex organisms including mammals. Abbreviations dsRNA, double stranded RNA; GCK-3, germinal center kinase-3; GFP, green fluorescent protein; OSR1, oxidative-responsive 1; PASK, proline-alanine-rich Ste20-related kinase (also known as SPAK); RNAi, RNA interference; Ste20, sterile-20; TRP, transient receptor potential; TRPV, TRP-vanilloid; WNK, with-no-lysine (K). 5782 FEBS Journal 274 (2007) 5782–5789 ª 2007 The Authors Journal compilation ª 2007 FEBS molecular bases of physiological processes including osmoregulation [3,4]. Worms have a short life cycle (2– 3 days at 25 °C), produce large numbers of offspring by sexual reproduction and can be cultured easily and inex- pensively in the laboratory. Sexual reproduction occurs by self-fertilization in hermaphrodites or by mating with males. Self-fertilization allows homozygous animals to breed true and greatly facilitates the isolation and main- tenance of mutant strains whereas mating with males allows mutations to be moved between strains. The reproductive and laboratory culture characteristics of C. elegans make it an exceptionally powerful model sys- tem for forward genetic analysis. Mutagenesis and genetic screening allows the unbiased identification of genes underlying a biological process of interest, allows genes to be ordered into pathways, and can provide important and novel mechanistic insights into the molecular structure and function of proteins. In addition to forward genetic tractability, C. ele- gans also has a fully sequenced and well-annotated genome. The genomic sequence and virtually all other biological data on this organism are assembled in read- ily accessible public databases (e.g. WormBase; http:// www.wormbase.org). Numerous reagents including mutant worm strains and cosmid and YAC clones spanning the genome are freely available through pub- lic resources. Creation of transgenic worms is relatively easy, inexpensive and rapid, requiring little more than injection of transgenes into the animal’s gonad or bombardment with DNA-coated microparticles. C. ele- gans gene expression can be specifically and potently targeted for knockdown using RNA interference (RNAi), either at the single worm level by injection of double stranded RNA (dsRNA), or at the population level by feeding worms dsRNA-producing bacteria. Libraries of dsRNA feeding bacteria are now available that allow for over 90% of the genome to be screened for a particular phenotype. Finally, C. elegans is a highly differentiated animal but is comprised of < 1000 somatic cells. This relatively simple anatomy greatly facilitates the study of biological processes and has made it possible to trace the lineage of every adult cell beginning with the first cell division. C. elegans inhabits surface soil and decaying organic matter that undergoes periodic desiccation. Under lab- oratory conditions, worms can readily survive and acclimate to extreme osmotic stress [5,6]. In addition, these animals have well developed sensory mechanisms that allow them to detect and avoid hypertonic envi- ronments [7]. Our goal in this minireview is to summa- rize what is currently known about the molecular mechanisms of osmotic stress resistance, osmosensing and signal transduction in C. elegans. Osmoregulatory organs in C. elegans C. elegans has simple ‘kidney’ that is comprised of three cells, the excretory cell, the duct cell and the pore cell [8]. Destruction of any of these cells by laser abla- tion causes the animal to swell with fluid and die [9]. The excretory cell is a large, H-shaped cell that sends out processes both anteriorly and posteriorly from the cell body. A fluid-filled excretory canal is sur- rounded by the cell cytoplasm. The basal pole of the cell faces either the pseudocoel or hypodermis whereas the apical membrane faces the excretory canal lumen. An excretory duct connects the excretory canal to the outside surface of the worm. The duct is formed from cuticle that is continuous with the animal’s exoskele- ton. A duct cell surrounds the upper two-thirds of the duct and a pore cell surrounds the lower third. The excretory cell is a single-cell ‘epithelium’ that secretes solutes and water into the excretory canal. Duct cells may also play an important role in solute and water transport. The apical surface area of duct cells is greatly amplified by extensive invaginations and the cytoplasm is filled with mitochondria, suggesting that it may be involved in solute transport, possibly selective solute reabsorption [8]. The worm ‘skin’ or hypodermis is an epithelium that underlies a thick cuticle composed of collagens. Gap junctions connect the excretory cell to the hypodermis, suggesting an interaction between the two cell types important for whole animal osmoregulation. In addi- tion, several studies have suggested a role for the hypodermis in osmoregulation. For example, a recent study suggests that fibroblast growth factor signaling in the hypodermis regulates whole animal fluid balance [10]. The intestine of adult C. elegans is comprised of 20 epithelial cells that function in digestion and nutrient absorption. In addition, the intestine is in direct con- tact with the external environment and thus could readily mediate osmoregulatory exchanges of solutes and water. Recent studies support the notion that the intestine plays an important role in whole animal osmoregulation [11,12]. Behavioral avoidance of osmotic stress Nematodes sense the external environment in part through a pair of openings in the cuticle on their head termed amphids. Eight neurons associate directly with each amphid pore and contact the external environ- ment via dendrites that terminate in sensory cilia. Another four sensory neurons have dendrites that associate with a support cell termed the amphid sheath K. P. Choe and K. Strange Osmosensing and signaling in C. elegans FEBS Journal 274 (2007) 5782–5789 ª 2007 The Authors Journal compilation ª 2007 FEBS 5783 cell. Axons extend from the cell bodies of the amphid neurons to the central nervous system where they make synaptic contacts with other neurons. Laser abla- tion studies have demonstrated that the amphid neu- rons function in thermosensation, chemosensation and mechanosensation [13]. C. elegans is attracted to low concentrations of salts, sugars and other chemicals. However, at high concen- trations, worms initiate an avoidance response to these substances. Culotti and Russell [7] concluded that the repellent effect of these solutes is due to hypertonicity rather than to the solutes themselves. Using forward genetic analysis, Culotti and Russell [7] identified osm mutants that were osmotic avoidance defective. osm-9 mutants are defective in their ability to detect not only hypertonic media, but also mechanical pertur- bation (nose touch), volatile repellents and chemical attractants [14,15]. The OSM-9 protein is a 937-amino acid member of the transient receptor potential (TRP) family of cation channels. TRP channels are divided into TRPC, TRPV, TRPM, TRPML, TRPP, TRPN and TRPA subfamilies. All TRP channels are com- prised of six predicted transmembrane domains and intracellular N- and C-termini. Functional TRP chan- nels are formed from homomeric or heteromeric asso- ciation of four TRP subunits. TRP channels function in diverse physiological processes, including sensory transduction, epithelial transport of Ca 2+ and Mg 2+ , Ca 2+ signaling and modulation of membrane potential [16,17]. OSM-9 was the first TRP-vanilloid (TRPV) channel to be identified at the molecular level. Green fluores- cent protein (GFP) reporter studies demonstrated that OSM-9 localizes in the superficial sensory cilia of amphid neurons where it could directly detect envi- ronmental hypertonicity, mechanical force, and chemi- cal attractants and repellents [14]. Unfortunately, OSM-9 has not yet been successfully expressed in a heterologous system where it can be functionally characterized. However, consistent with the role of osm-9 in osmosensation and mechanosensation, mam- malian TRPV4 has been shown to be activated by hypotonic stress when expressed heterologously [18]. TRPV4 is expressed in circumventricular organs of the mammalian central nervous system where it appears to play a role in detecting plasma osmolality and regulating the secretion of the systemic osmoregu- latory hormone vasopressin [19]. Interestingly, even though OSM-9 and TRPV4 only share 26% amino acid identity, TRPV4 rescues the defects in osmotic avoidance and nose touch behaviors when it is expressed in the amphid neurons of osm-9 mutant worms [20]. Mutations in TRPV4 that affect its functioning as an ion channel eliminate or reduce its ability to rescue, indicating that cation flux through the channel is likely the proximal signal that mediates osmosensing and mechanosensing [20]. The mechanism by which OSM-9 and TRPV4 detect hypertonic media and nose touch are unclear. However, it is possible that the channels are regulated via mechanical forces transmitted directly through the lipid bilayer and ⁄ or cytoskeletal attachments. ocr-2, odr-3 and osm-10 are also expressed in amp- hid sensory neurons and are essential for the osmotic avoidance behavior. OCR-2 is another TRPV family member and it colocalizes with OSM-9 in amphid neuron sensory cilia [21]. Interestingly, both proteins must be present for either of them to localize prop- erly, suggesting that OSM-9 and OCR-2 form a het- eromeric channel and ⁄ or function in a multiprotein signaling complex [21]. odr-3 encodes a Ga protein and null mutations in this gene prevent detection of nose touch, volatile repellents and hypertonicity [22]. These phenotypes are very similar to those of osm-9 mutants, suggesting that ODR-3 may function with the channel to regulate the response to external stim- uli. osm-10 encodes a novel cytosolic protein that is essential for detection of hypertonicity, but not nose touch or volatile repellents [23], suggesting that it may be involved in discriminating osmotic from other stimuli that activate OSM-9 and ⁄ or OCR-2. Although more work is needed to understand if and how OSM-9, OCR-2, ODR-3 and OSM-10 function together to mediate osmotic avoidance, studies on these proteins illustrate the power of forward genetic analysis in C. elegans as a means to identify rapidly and in an unbiased manner genes involved in osmo- sensing. Protein damage triggers organic osmolyte accumulation Measurements of internal osmolality have not been made on C. elegans because of the animal’s small size. However, other nematodes that have been stud- ied are hyper-osmoconformers that maintain an inter- nal osmolality slightly higher than that of the environment [24]. This is not surprising considering that nematodes lack a rigid skeleton. Instead, internal turgor or hydrostatic pressure inflates a flexible cuticle and gives the animal rigidity that is necessary for locomotion. The soil and decaying organic environments of C. elegans are osmotically unstable and the animal undoubtedly experiences periods of desiccation and rehydration in its native habitat. In the laboratory, Osmosensing and signaling in C. elegans K. P. Choe and K. Strange 5784 FEBS Journal 274 (2007) 5782–5789 ª 2007 The Authors Journal compilation ª 2007 FEBS worms readily acclimate to extreme hypertonic stress [5,6]. When exposed to hypertonic media, C. elegans rapidly loses water and up to 50% of its body volume. If the shrinkage is severe, worms become paralyzed due to loss of turgor pressure [5,25] (Fig. 1A). Worms exposed to nonlethal hypertonic stress recover their volume within a few hours by yet to be characterized mechanisms. During long-term exposure to hypertonic- ity, C. elegans synthesizes and accumulates large quan- tities of the compatible organic osmolyte glycerol (Fig. 1B) [5,11]. Signaling pathways that regulate organic osmolyte accumulation in animal cells are poorly defined. To begin identifying the signaling mechanisms that regu- late glycerol synthesis, we performed a genome-wide RNA interference screen for genes that regulate osmosensitive gene expression [11]. Expression of the gene gpdh-1, encoding glycerol-3-phosphate dehydro- genase-1, an enzyme that catalyzes a rate-limiting step of glycerol synthesis, increases dramatically in C. elegans following exposure to hypertonic stress [5] (Fig. 1B). To assess gpdh-1 expression in vivo,we generated a strain of worms that expresses GFP dri- ven by the gene’s promoter. GFP expression in this strain is virtually undetectable unless the worms are exposed to hypertonic media. Using this reporter strain and a library of RNAi feeding bacteria [26], we screened for gene knockdowns that activate gpdh-1. This screen identified 122 genes whose knockdown induced gpdh-1 expression in the absence of hypertonic stress. These genes are termed regula- tors of glycerol-3-phosphate-dehydrogenase (rgpd) expression [11]. rgpd gene functions fell into six defined categories; extracellular matrix, signaling, metabolism, protein trafficking, transcriptional regulation and protein homeostasis, as well as a group of genes with unas- signed functions. Interestingly, genes predicted to func- tion in cellular protein homeostasis are the largest class of rgpd genes (54 out of 122 total rgpd genes). These include genes that encode proteins required for RNA processing, protein synthesis, protein folding and protein degradation. Knockdown of these genes is expected to increase intracellular levels of damaged and denatured proteins. Damaged proteins in turn act as a signal to activate glycerol accumulation (Fig. 2). Glycerol functions to stabilize protein structure and its accumulation would allow cells to lower intracellular ionic strength, which can disrupt protein synthesis and folding [11]. Interestingly, protein damage caused by other stres- sors, such as heat shock and oxidative stress, do not activate gpdh-1 expression, demonstrating that osmotic stress causes a form of protein damage that selectively induces glycerol accumulation [11]. Osmotic stress has been shown previously to disrupt new protein synthesis [27,28], which likely results in the accumulation of incomplete and aberrantly folded polypeptides in the cytoplasm. The majority of the protein homeostasis genes identified in our RNAi screen function in RNA processing, protein synthesis and cotranslational pro- tein folding [11]. These observations are consistent with a model in which gpdh-1 expression is specifically activated by osmotically induced disruption of new protein synthesis and cotranslational folding rather than by denaturation of existing proteins (Fig. 2). Such a mechanism would allow cells to discriminate between osmotically induced and other forms of stress-induced protein damage [11]. 00.524 6 Hours in hypertonic media 365 mM NaCl51 mM NaCl A B 0 6 12 18 24 30 36 42 48 0 2 4 6 8 10 0 100 200 300 400 500 600 700 800 Hours in hypertonic media Worm glycerol (nmoles/mg protein) Relative gpdh-1 expression Fig. 1. Response of C. elegans to acute and chronic hypertonic stress. (A) Images of a single worm on agar containing 51 m M NaCl and after acute transfer to agar containing 365 m M NaCl. Note that the worm initially shrinks and becomes paralyzed. Complete vol- ume recovery occurs within 2–3 h and full mobility is regained. Scale bar ¼ 200 lm. (B) Changes in gpdh-1 expression and whole animal glycerol levels in worms exposed to hypertonic stress. gpdh-1 expression was assessed using a GFP reporter driven by the gene’s promoter. Data are replotted from Lamitina et al. [5,11]. K. P. Choe and K. Strange Osmosensing and signaling in C. elegans FEBS Journal 274 (2007) 5782–5789 ª 2007 The Authors Journal compilation ª 2007 FEBS 5785 Role of cuticle collagens in regulating organic osmolyte accumulation Four of the genes identified in our RNAi screen encode the collagens DPY-7, DPY-8, DPY-9 and DPY-10, which play important roles in formation of the C. elegans cuticle. Loss-of-function mutations in these genes induce gpdh-1 expression [11] and glycerol accumulation [11,29]. Mutations in dpy genes also cause a short and fat, dumpy phenotype, which is thought to reflect changes in cuticle structure [30]. DPY collagens are secreted proteins and given their role in cuticle formation, they almost certainly function extracellularly to regulate glycerol accumula- tion. Interestingly, our RNAi screen of gpdh-1 expression also identified ten genes that are predicted to encode secreted proteins [11]. Mutant alleles of three of these genes, osr-1, osm-7 and osm-11, have been characterized and shown to cause constitutive accumulation of glycerol [25,29]. osr-1 and osm-7 are expressed in the hypodermis [25,29]. Epistasis analy- sis suggests that OSR-1 functions with calmodulin- dependent protein-kinase II and a mitogen-activated protein kinase cascade to regulate glycerol accumula- tion [25,29]. Collagens and other secreted proteins are essential components of a C. elegans mechanosensorory com- plex that detects tactile stimuli [31]. Similarly, DPY collagens and OSR-1, OSM-7 and OSM-11 could func- tion in the cuticle to detect and transduce hypertonic stress-induced mechanical signals that regulate glycerol accumulation. Further characterization of secreted proteins that regulate gpdh-1 expression will help define their role in osmosensing. With-no-lysine (K) (WNK) and Ste20 kinases regulate hypertonic stress responses C. elegans germinal center kinase-3 (GCK-3) is a mem- ber of the GCK-VI subfamily of sterile-20 (Ste20) ser- ine-threonine protein kinases that includes vertebrate oxidative-responsive 1 (OSR1) and proline-alanine-rich Ste20-related kinase (PASK; also known as SPAK) [32]. GCK-VI kinases are expressed in transporting epithelia [33]. We recently demonstrated that GCK-3 binds to and regulates a cell volume sensitive ClC Cl – channel in C. elegans [34]. Mammalian OSR1 and PASK bind to and phosphorylate members of the SLC12 cation-Cl – cotransporter family in response to cell volume changes [35]. Taken together, these data suggest that GCK-VI kinases may play a role in cell and systemic osmoregulation [36]. We recently examined the role of GCK-3 in whole animal osmotic homeostasis [12]. GFP reporter analy- sis demonstrated that gck-3 is expressed in multiple locations including osmoregulatory tissues. Systemic RNAi of gck-3 almost completely prevents acute vol- ume recovery and chronic survival in 400 mm NaCl, demonstrating that the kinase is essential for systemic osmoregulation. Using tissue-specific RNAi, we also demonstrated that GCK-3 functions in the hypodermis and intestine to mediate acute volume recovery and survival during hypertonic stress. These two tissues are Fig. 2. Model for regulation of gpdh-1 expression by disruption of protein homeo- stasis. Hypertonic stress induced water loss causes elevated cytoplasmic ionic strength, which in turn disrupts new protein synthesis and cotranslational protein folding. Damaged proteins function as a signal that activates gpdh-1 expression and glycerol synthesis. Glycerol replaces inorganic ions in the cyto- plasm and functions as a chemical chaper- one that aids in the refolding of misfolded proteins. Loss of function of protein homeo- stasis genes also causes accumulation of damaged proteins and activation of gpdh-1 expression. Green arrows and red lines indi- cate activation and inhibition, respectively. Osmosensing and signaling in C. elegans K. P. Choe and K. Strange 5786 FEBS Journal 274 (2007) 5782–5789 ª 2007 The Authors Journal compilation ª 2007 FEBS in contact with the environment and likely mediate osmoregulatory exchanges of ions and water. We pro- pose that GCK-3 regulates ion and water uptake mechanisms in these two tissues to mediate acute sys- temic volume recovery following water loss and shrink- age (Fig. 3). After volume recovery, accumulation of the compatible osmolyte glycerol (see above) replaces inorganic ions absorbed during acute volume recovery [11]. Interestingly, survival of gck-3(RNAi) worms is much lower in animals exposed to high NaCl versus high sorbitol [12]. Acute volume recovery was similar in the presence of the two solutes. This suggests that in addition to regulating solute and water uptake mechanisms required for acute volume recovery, GCK-3 may also regulate transport processes responsi- ble for excretion of a chronic NaCl load. Using a yeast two-hybrid screen, we identified WNK-1 as a protein that physically interacts with GCK-3 [12]. WNK protein kinases are serine ⁄ threo- nine protein kinases that lack a conserved lysine resi- due in the catalytic domain [37]. Humans have four WNK kinases and rare mutations in WNK1 and WNK4 cause an autosomal dominant form of hyper- tension [38]. WNK1 and WNK4 control blood pressure by regulating the activity of ion transport pathways that mediate salt transport in distal renal tubules [39]. Several recent studies have demonstrated that WNK1 and WNK4 bind to, phosphorylate, and activate PASK and OSR1 [39]. In C. elegans, systemic RNAi of WNK-1 decreases acute volume recovery and survival in a manner qualitatively similar to GCK-3 RNAi. The effects of RNAi for WNK-1 and GCK-3 in the same worms are not additive, suggesting that the kinases function in a common pathway to regulate systemic osmoregulation. We hypothesize that WNK-1 functions upstream from GCK-3 in a manner similar to that proposed for its mammalian homologues (Fig. 3) [39]. Almost nothing is known about what functions upstream from WNK kinases to sense osmo- tic stress in animals. Our study provides the founda- tion for genetic analysis of the WNK-1 ⁄ GCK-3 pathway that regulates hypertonic stress responses. Conclusions and future perspectives C. elegans has proven to be an exceptionally powerful model system for defining the genes and gene networks that underlie basic biological processes such as devel- opment, neural function and sensory physiology. The worm normally inhabits osmotically unstable soil environments and is thus well-suited for studies of osmosensing and associated signal transduction mecha- nisms. Little is known about the molecular bases of these processes in animals. The forward and reverse genetic and molecular tractability of C. elegans has already provided unique insights into TRP channel physiology, regulation of organic osmolyte accumula- tion and WNK and Ste20 kinase signaling. C. elegans will undoubtedly continue to provide new understand- ing of how animals detect and protect themselves from osmotic stress. Given the fundamental and conserved nature of osmotic stress resistance, studies on C. ele- gans will clearly provide new and important insights that are applicable to all animals. Acknowledgements This work was supported by NIH grants DK61168 and DK51610. K.C. was supported by NIH NRSA GM77904. References 1 Strange K (2004) Cellular volume homeostasis. Adv Physiol Educ 28, 155–159 2 Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high Fig. 3. Model of GCK-3 and WNK-1 function in the hypodermis and intestine of C. elegans. Hypertonic shrinkage activates osmosen- sors that in turn activate WNK-1. WNK-1 binds to and likely acti- vates GCK-3 by phosphorylation. We predict that GCK-3 then regulates solute and water transport pathways that mediate acute volume recovery. K. P. Choe and K. 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P. Choe and K. Strange 5784