![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 16, Issue 2, 458-469, February 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705
Submitted September 29, 2004;
Revised November 10, 2004;
Accepted November 16, 2004
Monitoring Editor: Martin Chalfie
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
-toxin (PLAT) domain found in all PC-1 family members remains an enigma. Here, we report that ATP-2, the 
subunit of the ATP synthase, physically associates with the LOV-1 PLAT domain and that this interaction is evolutionarily conserved. In addition to the expected mitochondria localization, ATP-2 and other ATP synthase components colocalize with LOV-1 and PKD-2 in cilia. Disrupting the function of the ATP synthase or overexpression of atp-2 results in a male mating behavior defect. We further show that atp-2, lov-1, and pkd-2 act in the same molecular pathway. We propose that the ciliary localized ATP synthase may play a previously unsuspected role in polycystin signaling. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). This syndrome is characterized by progressive development of fluid-filled, epithelial cysts in the kidney, liver, and pancreas and accounts for 
10% of all cases of end-stage renal disease. Mutation in either the polycystic kidney disease (PKD)1 or PKD2 gene accounts for 95% of all ADPKD cases. PKD1 encodes polycystin (PC)-1, a 4302-amino acid protein with a large extracellular domain, a G protein-coupled receptor proteolytic site (GPS), 11 predicted transmembrane (TM) domains, and an intracellular C terminus. The polycystin/lipoxygenase/-toxin (PLAT) domain is located in the first cytoplasmic loop between TM1 and TM2 and has been postulated to be involved in membrane-protein or protein-protein interactions (Bateman and Sandford, 1999). This domain is conserved in all PC-1 family members and also found in a variety of membrane- or lipid-associated proteins. Polycystin-2 (PC-2, encoded by PKD2) shares homology with the transient receptor protein (TRP) channels and acts as a nonselective cation channel. Defects in PC-1 or PC-2 signaling may result in epithelial dedifferentiation and cyst formation. Several signaling pathways regulated by PC-1 and PC-2 have been identified using in vitro approaches, including G protein signaling, JAK/STAT cell cycle regulation, and mechanotransduction (Boletta and Germino, 2003), but the physiological relevance to normal and disease states remains unclear.
The nematode C. elegans is a simple but powerful animal model for studying basic molecular mechanisms underlying human ADPKD. The C. elegans LOV-1 and PKD-2 proteins are homologues of human PC-1 and PC-2 (Barr and Sternberg, 1999; Barr et al., 2001). lov-1 (location of vulva) and pkd-2 act nonredundantly in the same genetic pathway and are required for two male mating behaviors, response to hermaphrodite contact and vulva location. Accordingly, lov-1 and pkd-2 are exclusively expressed in three categories of adult male sensory neurons: the rays, the hook, and the head cephalic (CEMs), which mediate response, vulva location, and potentially other male-specific sensory behaviors, respectively (Figure 1a). LOV-1 and PKD-2 localize to the cilia of these male-specific sensory neurons. Ciliary localization of polycystins is critical for polycystin function and observed in a diversity of species ranging from C. elegans to human (Igarashi and Somlo, 2002; Watnick and Germino, 2003). LOV-1 shares a similar architecture with other PC-1 family members: a large extracellular domain (although there is no primary sequence homology between the extracellular regions of LOV-1 and PC-1), a GPS site, 11 TM domains, and the intracellular PLAT domain. Like PC-1 and PC-2, the cellular functions of LOV-1 and PKD-2 remain elusive. Based on PLAT sequence homology, a nonredundant genetic PKD pathway, and PC ciliary subcellular localization in both C. elegans and mammals, we hypothesize that the PLAT domain may perform an evolutionarily conserved role in mediating PC-1 intracellular signaling pathways.
|
In this work, we find that overexpression of the LOV-1 PLAT domain in transgenic C. elegans results in male mating behavior defects. To identify regulators or targets of LOV-1 PLAT domain, we performed a yeast two-hybrid (Y2H) screen. ATP-2 is identified as one candidate that physically interacts with the PLAT domain. We demonstrate that the barrel domain of ATP-2 is responsible for this interaction. ATP-2 and other ATP synthase components were found to localize to the cilia of male-specific sensory neurons. Over-expression of atp-2 or RNA interference (RNAi) to ATP synthase components resulted in a specific defect in the response step of male mating behavior. These results suggest that the unexpected localization of the ATP synthase to cilia plays a critical role in polycystin signaling, ciliary function, and sensory behaviors.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). him-5(e1490) was used as the wild-type (Hodgkin, 1983). Several C. elegans strains were obtained from the Caenorhabditis Genetics Center. We constructed transgenic lines by injecting plasmid DNA (100-200 ng/µl) by using standard protocols (Mello and Fire, 1995). In all experiments, we used plasmid pBX containing the wild-type pha-1(+) gene as a cotransformation marker in the pha-1(ts) strain (Granato et al., 1994).
Molecular Biology Techniques
Details of plasmid constructions are available upon request. Standard procedures were used for recombinant DNA manipulations.
Yeast Two-Hybrid
The yeast strain AH 109 (BD Biosciences Clontech, Palo Alto, CA) was used for Y2H experiments. Bait proteins were expressed in the GAL4 DNA-binding domain (DNA-BD) vector pGBKT7. A cDNA library derived from mix-staged him-5 animals was constructed in the GAL4 activation domain (DNA-AD) vector pGAD GH. The PLAT domain of C. elegans LOV-1 (amino acids 2112-2302) and human PC-1 (amino acids 3164-3349) were used as baits in Y2H hybrids. Protein-protein interactions were accessed by growth rate on SD-Leu-Trp-His-Ade high-stringency plates and -galactosidase filter assays. Both the C. elegans and human PLAT domain weakly interact with ATP-2 as judged by growth on SD-Leu-Trp-His-Ade. The interaction between the C. elegans and human PLAT domains is stronger with the animo terminus of ATP-1 (amino acids 1-209) as judged by growth on SD-Leu-Trp-His-Ade.
In Vitro Binding
In vitro-translated Y2H candidate ATP-2 protein was produced with the TNT-quick coupled transcription/translation system (Promega, Madison, WI) by using [35S]methionine (Amersham Biosciences, Piscataway, NJ). A glutathione S-transferase (GST)-fused LOV-1 PLAT domain was produced in the bacterial strain BL21(DE3) and incubated with in vitro-translated candidates proteins in 500 µl of binding buffer (20 mM Tris/Cl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.05% NP-40) at 4°C for 30 min. Then, 25 µl of glutathione-Sepharose 4B was added to the reaction and incubated at 4°C for an additional 2 h or overnight. After three washes with binding buffer, bound proteins were resuspended in 50 µl of 1x Laemmli buffer and then 10-µl samples were boiled and separated by 10% SDS-PAGE. Proteins were visualized by Coomassie staining. The dried gel was exposed for autoradiography.
Expression Analysis
Epifluorescence microscopy experiments were carried out by using a Zeiss Axioplan2 imaging system and photographed with an Orca-ER camera. Confocal experiments were carried out on a Bio-Rad MRC-1024 laser scanning confocal microscope.
Rescue of atp-2(ua2) Mutant
LB128 atp-2(ua2) unc-32(e189)/qC1 (dpy-19(e1259) glp-1(q339) hermaphrodites were crossed by him-5; myEx (ATP-2::GFP) males. F2 progeny segregating Unc and green fluorescent protein (GFP)+ were picked and maintained. Rescue of the L3 larval arrest in these lines was scored. All Unc GFP+ animals were rescued for atp-2(ua2) L3 larval arrest.
RNA Interference Assay and Mating Behaviors
For heat shock-inducible RNAi mediated by the inverted repeat (IR) genes (HS-IR-RNAi), we constructed IR genes as described previously (Tavernarakis et al., 2000). Transgenic lines carrying IR plasmids were generated. Mixstaged transgenic animals were heat shocked for 4 h at 35°C, before returning to 15°C. After 63-67 h at 15°C, L4 males were transferred to new plates for culturing another 14 h at 15°C. Mating behaviors were scored as described previously (Barr and Sternberg, 1999). For tissue-specific RNAi (TS-IR-RNAi), we constructed IRs whose transcription is under the control of the pkd-2 promoter. Transgenic lines were generated and males were scored for mating behaviors. For feeding RNAi, RNAi bacteria clones were obtained from MRC Geneservice (Cambridge, United Kingdom). Double-stranded RNA producing bacteria were grown in LB with 50 ng/ml ampicillin overnight at 37°C and seeded onto NGM agar plates, including additives (1 mM isopropyl -D-thiogalactoside, 50 ng/ml ampicillin). The next day, 6-8 L4 stage RNAi-sensitive rrf-3; him-5 hermaphrodites (Simmer et al., 2002) were transferred onto feeding plates and incubated 72 h at 15°C. L4 males were picked to new feeding plates seeded with feeding RNAi bacteria, cultured 14 h at 15°C, and the mating behaviors of adult males was scored. In all experiments, at least 24 animals were scored per experimental trial. Triplicate trials were performed for each line to obtain statistical data. All behavioral assays were done with the experimenter completely blinded to the sample.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
; White et al., 1986). Sexual dimorphism is reflected in behavior. Many of the 87 male-specific neurons mediate male mating behaviors (Figure 1a) (Liu and Sternberg, 1995). Male copulation is comprised of a stereotyped subseries of behaviors: response to hermaphrodite contact, backing along the hermaphrodite body, turning at the head or tail of the hermaphrodite, vulva location, spicule insertion, and sperm transfer. lov-1 and pkd-2 mutants are response and Lov (location of vulva) defective. Wild-type males have 95% response and vulva location efficiency, whereas lov-1 and pkd-2 mutants exhibit 25% response and vulva location efficiency (Figure 1b) (Barr and Sternberg, 1999; Barr et al., 2001).
To ascertain the function of the PLAT domain, GFP-tagged PLAT domain transgenes were introduced into wild-type animals. The mating behavior of transgenic animals is scored, with response and Lov defects being an effective readout of interference with polycystin signaling. The strong pkd-2 promoter is expressed in male-specific neurons of the head (the four CEMs) and tail (the B type neurons of rays and hook) (Figure 1a) (Barr and Sternberg, 1999; Barr et al., 2001). The pkd-2 promoter was used to drive expression of either the LOV-1 PLAT domain alone (PLAT) or a LOV-1 transmembrane segment followed by the PLAT domain (TM-PLAT) in wild-type animals. Overexpression of either PLAT or TM-PLAT interfered with response step of male mating behavior (Figure 1b). In contrast, only the TM-PLAT construct blocked both response and vulva location behaviors (Figure 1b), suggesting that PLAT-localization to the membrane is a requisite for vulva location behavior. The severity of response and Lov defects of the TM-PLAT dominant negative construct is identical to that of the lov-1 mutant (Figure 1b). The dominant negative phenotypic differences between TM-PLAT and PLAT alone may reflect differential membrane targeting ability or effector interactions. With respect to the latter possibility, TM-PLAT may bind and inhibit the function of proteins required for both vulva location and response behaviors, whereas PLAT alone may bind and inhibit the function of proteins required for response behavior.
The PLAT domain has been proposed to mediate protein-protein or protein-lipid interactions (Bateman and Sandford, 1999). An alternative possibility is that the polycystin PLAT domain functions as a ciliary localization signal. LOV-1 and PKD-2 localize to the ciliated sensory endings of male-specific neurons (Figure 1a) (Barr and Sternberg, 1999; Barr et al., 2001) and mammalian PC-1 and PC-2 localize to primary cilia of renal epithelia (Pazour et al., 2002; Yoder et al., 2002). Ciliary trafficking mechanisms are not well understood in any organism (reviewed by Rosenbaum and Witman, 2002). C. elegans is a useful model system to study protein sorting, transport, and localization (Koushika and Nonet, 2000). To test the hypothesis that the PLAT domain is responsible for ciliary targeting, we examined the subcellular localization of PLAT::GFP and TM-PLAT::GFP in wild-type males. Neither PLAT::GFP nor TM-PLAT::GFP localizes to cilia, indicating that the PLAT domain is not sufficient for ciliary targeting (our unpublished data). Combined, these behavioral and cellular analyses suggest that the PLAT domain cannot localize to the normal site of LOV-1 action (the cilium) and may act by dominantly interfering with LOV-1 and/or its effectors.
ATP-2 Interacts with the PLAT Domain of LOV-1 and Human Polycystin-1
To test the hypothesis that the PLAT domain dominantly interferes with the function of LOV-1 effectors and to identify targets of the PLAT domain, 1 x 106 cDNAs were screened for yeast two-hybrid interaction with the LOV-1 PLAT domain. Twenty plasmid-dependent interactors were isolated. The interactor ATP-2, an ATP synthase F1 subunit, was isolated twice. The mitochondrial ATP synthase (also referred to as the mitochondrial respiratory chain [MRC] complex V) is a multimeric enzyme consisting of the soluble F1 and membrane-bound F0 complexes. C. elegans ATP-2 is the subunit, or active site, of the F1 portion (Tsang et al., 2001). The MRC generates the majority of cellular ATP.
To demonstrate direct interaction between ATP-2 and LOV-1, a GST-PLAT fusion was expressed and purified from Escherichia coli. ATP-2 was in vitro transcribed and translated in the presence of radiolabeled [35S]methionine and then incubated with GST-PLAT, or GST as control, and glutathione-Sepharose 4B. Proteins were electrophoresized on SDS-PAGE, and gels were dried and exposed to film. [35S]ATP-2 binds to GST-PLAT but not GST alone, indicating that ATP-2 binds directly to the PLAT domain of LOV-1 (Figure 2a).
|
To determine whether the interaction between the PLAT domain and ATP-2 is evolutionarily conserved, we examined the association of the human PC-1 PLAT domain and C. elegans ATP-2. We find that ATP-2 also interacts with human PC-1 in a yeast two-hybrid assay (Figure 2b). We mapped the region of ATP-2 region responsible for the interaction with the PLAT domain of both human PC-1 and C. elegans LOV-1. The amino terminus of ATP-2 (amino acids 1-216), which contains a conserved barrel domain, is the minimal PLAT binding region (Figure 2B). The barrel domains of the - and -subunits are important for the assembly of the ATP synthase (Bakhtiari et al., 1999). When compared with the full-length ATP-2 protein, the barrel-containing ATP-2 fragment interacts more strongly with the PLAT domains of both PC-1 and LOV-1 (Figure 2b). That ATP-2 associates with the PLAT domains of both PC-1 and LOV-1 PLAT domain supports our hypothesis that there exists an intracellular polycystin signaling pathway conserved throughout evolution.
atp-2 Is Coexpressed with lov-1
Next, we examined the expression pattern of atp-2 by using a 1.5-kb atp-2 promoter fragment to drive GFP expression. Patp-2::GFP (P for promoter) is widely expressed throughout development in both males and hermaphrodites (our unpublished data). Consistent with the broad spatial and temporal expression, the nuclear-en-coded atp-2 gene is required for embryonic and larval development (Gonczy et al., 2000; Tsang and Lemire, 2002). To determine whether atp-2 and lov-1 are coexpressed, the male-specific neurons of the head and tail were double labeled with Patp-2::GFP and Plov-1::DsRed2 (a 3-kb lov-1 promoter driving expression of Discosoma red fluorescent protein). lov-1 and atp-2 are coexpressed in the tail ray B neurons and HOB hook neuron (Figures 3 and 5a) as well as the male-specific CEM head neurons.
|
|
ATP-2 and Other ATP Synthase Components Localize to Ray and CEM Cilia
ATP-2 is predicted to localize to the inner mitochondrial membrane with the MRC. In contrast, LOV-1 and PKD-2 proteins localize to cilia and intracellular membranes (Barr and Sternberg, 1999; Barr et al., 2001). PKD-2 and LOV-1 do not possess mitochondrial transport signals and do not localize to mitochondria (Figure 4c, c'). Furthermore, cilia do not contain mitochondria (Figure 4b, dashed box; note that in all figures, dashed boxes indicate ciliary regions of sensory neurons.). We observe the absence of mitochondria from cilia by using both fluorescence and electron microscopy. A mitochondrial targeted, cyan-shifted GFP (Ppkd-2::mitoCFP) is present in neuronal axons, cell bodies, and dendrites (Figure 4b, b') but is obviously excluded from cilia (Figure 4b). Electron micrographs of male sensory rays show that mitochondria are present only within the dendritic portion of the neuron processes but not within the cilium (David Hall, personal communication). Therefore, the interaction between the mitochondrial ATP-2 and ciliary LOV-1 protein is unexpected and surprising.
|
To determine whether ATP-2 and LOV-1 colocalize in the cell, subcellular localization of ATP-2 was examined. A 1.5-kb native atp-2 promoter was used to drive expression of the atp-2 cDNA fused to GFP (Patp-2::ATP-2::GFP). To first demonstrate that ATP-2::GFP is functional and expression/localization patterns are physiologically relevant, we introduced the Patp-2::ATP-2::GFP construct into the atp-2(ua2) deletion strain. Patp-2::ATP-2::GFP completely rescues the L3 arrest and embryo lethality phenotype of the atp-2(ua2) deletion strain (our unpublished data). ATP-2::GFP is found in a broad range of tissue types (Figure 5, a and b, left), making it difficult to ascertain ciliary localization. To look more carefully at ATP-2 subcellular localization, we examined colocalization of ATP-2 and PKD-2. LOV-1 and PKD-2 exhibit identical subcellular localization patterns (Barr and Sternberg, 1999; Barr et al., 2001). PKD-2 is expressed at much higher levels than LOV-1, making PKD-2 more useful for microscopic analysis (our unpublished data). PKD-2::DsRed2 localizes to cilia and cell bodies of ray, HOB (Figure 5a, right), and CEM neurons (Figure 5b, right). ATP-2::GFP clearly colocalizes with PKD-2::DsRed2 in CEM cilia (Figure 5b, middle). In rays and HOB, ciliary colocalization of ATP-2 and PKD-2 was ambiguous due to the expression of ATP-2::GFP in ray neurons, support cells, and hypodermis.
To specifically determine the subcellular localization of ATP-2 in male sensory neurons, the pkd-2 promoter was used to drive expression of ATP-2::GFP in the CEMs, rays, and HOB neurons (Ppkd-2::ATP-2::GFP; Figure 5, c-e). Using this cell-specific construct, ATP-2 clearly localizes to cilia in ray (Figure 5c, dashed box), CEM (Figure 5, d and e, dashed box), and HOB neurons. As expected, ATP-2::GFP also is observed in mitochondrial-like structures within dendrites, cell body, and axons (compare Ppkd-2::ATP-2::GFP in Figure 5d with Ppkd-2::mitoCFP in Figure 4b, b'), consistent with conventional mitochondrial localization.
ATP-2 is one subunit of the ATP synthase. The ATP synthase is composed of a membrane-bound proton channel (F0) and a peripheral catalytic complex (F1). We reasoned that other subunits of the ATP synthase may be present with ATP-2 in cilia. ASB-1 is a subunit of the F1 complex and ASG-2 is a membrane protein component of the F0 proton channel. We find that GFP-tagged ASB-1 and ASG-2 localize to male-specific sensory cilia as well as mitochondria (Figure 6, left and middle). The MRC is composed of four electron transporting complexes (I-IV) and the ATP synthase (complex V). NUO-1 (NADH/ubiquinone oxidoreductase) is a complex I component. In contrast to the ATP synthase, NUO-1::GFP is not found in the ciliary compartment (Figure 6, right). Thus, these distinct subcellular localizations point at an additional and nonmitochondrial related function for the ATP synthase in cilia of male-specific sensory neurons.
|
atp-2 and MRC Complex V Are Required for Male Response Behavior
We next asked whether atp-2 and other ATP synthase components are required for lov-1-mediated response and vulva location behaviors. atp-2 is an essential gene (Gonczy et al., 2000; Tsang et al., 2001). atp-2(ua2) mutants arrest at the third larval stage (L3), and both atp-2(ua2) and atp-2 RNAi result in behavioral defects, including impaired locomotion, pharyngeal pumping, defecation, and dauer formation (Gonczy et al., 2000). To circumvent earlier developmental requirements and analyze the role of atp-2 in the adult male, we lowered the activity of atp-2 with RNAi. We used two RNAi methods: feeding animals with bacteria producing double-stranded RNA (dsRNA) (Timmons et al., 2001) and in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). C. elegans neurons are largely resilient to the effects of RNAi for elusive reasons (Tavernarakis et al., 2000; Kamath et al., 2001; Timmons et al., 2001). In our hands, RNAi feeding is successful with rrf-3(pk1429) animals that are RNAi hypersensitive. IR plasmids producing dsRNA are also effectual at knocking down gene function in neurons. The latter is particularly valuable for temporal or tissue-specific gene knockdown by using appropriate promoters.
Feeding RNAi of atp-2 resulted in sterility (Ste), embryonic lethality (Emb), and larval arrest (Lva) (Table 1). Those males that survived to adulthood were response defective with intact vulva location behavior (Table 1 and Figure 7c). A response defect also was observed with atp-2 IR plasmids producing dsRNA. We used either the HS-IR-RNAi or pkd-2 promoter (TS-IR-RNAi) to drive expression of atp-2 cDNA IRs. Knockdown of atp-2 function by either HS-IR-RNAi or TS-IR-RNAi consistently resulted in response but not vulva location defects (Figure 7a, b). We also found that multicopy expression of atp-2, atp-2(xs) for excess, by using Patp-2::ATP-2::GFP (Figure 7d) or Ppkd-2::ATP-2::GFP (Figure 7e), interfered with response but not vulva location behavior. These results indicate that atp-2 is required in cell autonomous manner for response behavior and that atp-2 function may be required in the cilia of ray but not hook neurons.
|
|
An alternative possibility is that the response defect results simply from disrupting mitochondrial activity in neurons. To distinguish between mitochondrial and ciliary function, we knocked down the activity of components in the electron transport chain and the ATP synthase. Feeding RNAi of genes encoding complex I, II, III, and V produced phenotypes associated with loss of mitochondrial function (Emb, Lva, and slow growth, Gro; Table 1). However, RNAi of only components in complex V, the ATP synthase, but not other respiratory chain components affected response behavior (Figure 7c and Table 1). RNAi feeding of atp-2, asb-1, and asg-2 reduced male response behavior from 90 to 35, 67, and 54%, respectively (Table 1). Tissue-specific RNAi of asb-1, asg-2, and atp-2 produced similar response defects (Figure 7B), whereas tissue-specific RNAi of a complex III component, ZC116.2 cytochrome C, did not (our unpublished data). We observed that RNAi treatment of some predicted components in ATP synthase caused no response defects (Table 1). Perhaps the ciliary ATP synthase has a different subunit composition than the mitochondrial ATP synthase or these genes are simply not sensitive to feeding RNAi treatment. Regardless, these results indicate that the ATP synthase components ATP-2, ASB-1, and ASB-2 are specifically required in cilia of male ray neurons for response behavior.
One concern is that neurons depleted of atp-2 become generally unhealthy. We observe no neurodegeneration in male sensory neurons with HS-IR-RNAi, TS-IR-RNAi, or atp-2(xs) (our unpublished data). CEM cilia also form normally in animals overexpressing atp-2, asb-1, and asg-2 (Figures 5e and 6). We conclude that the ATP synthase is required for the function but not development of ciliated male sensory neurons.
ATP-2 Does Not Localize to AWA Cilia and Is Not Required for Olfactory Behaviors
Does the ATP-2 synthase function in all ciliated sensory neurons? Sensory cilia are found in 60 of the 302 neurons in the C. elegans hermaphrodite and in 112 of the 381 neurons in the male (Sulston et al., 1980; White et al., 1986). To test the involvement of atp-2 in other ciliated neurons, we examined ATP-2 localization and function in the amphid AWA sensory neuron pair. The AWA olfactory neurons sense attractive odorants, including diacetyl (Bargmann et al., 1993). The G protein-coupled odorant receptor ODR-10 is expressed and required in the complex, fork-shaped cilia of AWA neurons (Sengupta et al., 1996). To determine whether ATP-2 localizes to AWA cilia, we used the odr-10 promoter to drive the expression of ATP-2::GFP (Figure 8a) or ODR-10::GFP (Figure 8b). In AWA neurons, ATP-2::GFP is exclusively found in mitochondria and does not localize to cilia (Figure 8a). Two conclusions may be drawn from this result. First, the ciliary localization of ATP-2 in male-specific neurons reflects endogenous localization and is not an artifact of GFP overexpression. Second, the ciliary localization of ATP-2 is restricted to subset of sensory neurons.
|
To confirm that ATP-2 does not function in AWA olfactory neurons, we examined the effects of atp-2 tissue-specific RNAi and overexpression in the AWA olfactory neurons by using the odr-10 promoter. When the AWA neurons are killed with a laser microbeam, animals are unable to chemotax to diacetyl (Bargmann et al., 1993). Likewise, animals with abnormal AWA cilia, such as osm-5 mutants, exhibit diacetyl chemotaxis defects (Figure 8c) (Bargmann et al., 1993). Neither knockdown nor overexpression of atp-2 affected AWA-mediated diacetyl chemotactic behaviors (Figure 8c). These findings show that reducing activity of atp-2 or overexpression of atp-2 does not result in nonspecific disruption of sensory neuron function. We conclude that the ATP synthase is specifically required in ray cilia for response behavior.
atp-2 and lov-1 Act in the Same Molecular Pathway
Two simple models could explain the response-defective phenotype. In the first, the ATP synthase and polycystins act in the same molecular pathway. Alternatively, the ATP synthase and polycystins function in parallel pathways mediating response behavior. To distinguish between these possibilities, we examined the effects of atp-2 RNAi in lov-1 and pkd-2 null mutant backgrounds. We reasoned that if atp-2, lov-1, and pkd-2 act in different pathways, we would observe an additive effect and a more severe response defect than loss of either alone. We find that the response defect of lov-1(sy552) and pkd-2(sy606) mutants is not increased further by treatment with atp-2 RNAi (our unpublished data). These results show that atp-2, lov-1, and pkd-2 act in the same pathway regulating response behavior.
What is the significance of the LOV-1 and ATP-2 binding? LOV-1 may be required for ATP-2 localization to cilia or vice versa. We examined Ppkd-2::ATP-2::GFP localization in the lov-1 mutant. ATP-2::GFP ciliary localization is identical in wild-type and lov-1 mutants (our unpublished data). We conclude that lov-1 is not required for ATP-2 localization in cilia. An interesting possibility is that ATP-2 localization changes under conditions of mating stimulation by hermaphrodite partners. However, we observe no gross differences in ciliary ATP-2::GFP localization or dendritic mitochondrial distribution in virgin L4 males and mated adult males nor in wild-type versus sensory defective lov-1 mutants (our unpublished data).
Next, we compared PKD-2::GFP localization in wild-type and atp-2 RNAi-treated animals. PKD-2::GFP ciliary localization is not obviously affected by atp-2 TS-IR RNAi, although increases in dendritic PKD-2::GFP expression are observed (our unpublished data). These results indicate that ATP-2 is not essential for PKD-2 ciliary localization. We hypothesize that a common mechanism may be required for the ciliary localization of ATP-2, LOV-1, and PKD-2.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
The ATP synthase is composed of two functional domains, a catalytic F1 portion and a membrane embedded F0 portion. F1 contains multiple subunits (3,3
, 
, 
) and is bound on the membrane by its interaction with F0. F0 functions as a proton pump, and F1 uses the energy produced from proton translocation to synthesize ATP (Penefsky and Cross, 1991). The ATP synthase localizes primarily to inner mitochondria membrane (Boyer, 1997). However, the presence of ATP synthase at the cell membrane of lymphocytes, human endothelial cells, and hepatocytes has been reported (Das et al., 1994; Moser et al., 1999; Moser et al., 2001; Chang et al., 2002; Arakaki et al., 2003; Martinez et al., 2003). The polycystins also have been localized to lymphocytes, endothelial cells, and hepatocytes (Geng et al., 1996; Ibraghimov-Beskrovnaya et al., 1997; Aguiari et al., 2004). ADPKD extrarenal manifestations include liver cysts and vascular abnormalities (Igarashi and Somlo, 2002). In lymphocytes, membrane-bound ATP synthase was suggested to play a role in lymphocyte-induced tumor destruction (Das et al., 1994). Endothelial cell membrane-bound ATP synthase was proven to be active in ATP synthesis and involved in angiogenesis (Moser et al., 1999; Moser et al., 2001) and endothelial cell proliferation (Chang et al., 2002). This nonmitochondrial ATP synthase catalyzes ATP synthesis and is inhibited by angiostatin and an inhibitor of the F1 subunit (IF1), especially at low, tumor-like extracellular pH (Burwick et al., 2004). Most recently, the ATP synthase has been found in plasma membrane lipid rafts along with -tubulin (Bae et al., 2004; Li et al., 2004). Our results suggest that the ciliary localized ATP synthase may play a previously unsuspected role in polycystin signaling.
Whereas lov-1 and pkd-2 mutants are response and Lov defective, RNAi treatment of ATP synthase components or overexpression of atp-2 causes only response but not Lov mating behavior defects. LOV-1 may have tissue-specific effectors. In other words, ATP-2 may be required for polycystin-mediated signaling in ray neurons (with defects resulting in abnormal male response behaviors) but not the HOB hook neuron (as evidenced by wild-type vulva location behavior). Given the diverse clinical manifestation of ADPKD and broad distribution of the polycystins, it is likely that the polycystins will have tissue-specific regulators and targets.
We propose two models for the role of the ATP-2 within cilia. In the first model, the ATP synthase may provide the source of energy for mitochondria-less cilia. Cilia require an energy source for ATP-dependent processes such as intraflagellar transport, which is required for the assembly and maintenance of all cilia and flagella (Rosenbaum and Witman, 2002). Defects in ciliary development or signaling may result in polycystic kidney disease (Watnick and Germino, 2003). In the second model, the ATP synthase may be involved in regulating intracellular or extracellular concentrations of ATP. ATP-2 may modulate intracellular ATP and ion homeostasis by regulating ion channel and/or transporter activity, with defects resulting in abnormal secretion or absorption, culminating in cystogenesis. In both models, the ATP synthase would require a proton gradient across the ciliary membrane to synthesize ATP. In this case, the ciliary membrane is predicted to contain Na+/H+ exchangers and/or proton channels responsible for establishing the proton gradient.
The extracellular domain of LOV-1 possesses a predicted ATP/GTP binding site, or P loop, of unknown function (ASNIVGKT, amino acids 1691-1699). Extracellular ATP may bind to this site and modulate LOV-1 activity. Extracellular ATP has been shown to bind certain sites on the N-methyl-D-aspartate receptor and act as a positive allosteric modulator of channel gating (Kloda et al., 2004). In mammalian cells, extracellular ATP regulates fluid secretion and cell volume via purinergic receptors (Leipziger, 2003). In fact, ATP release and signaling is increased in PKD cell culture systems (Schwiebert and Zsembery, 2003). ATP and the PC-1 C termini have been shown to regulate chloride secretion and cyst growth in ADPKD (Wilson et al., 1999; Schwiebert et al., 2002; Hooper et al., 2003). Dysregulation of ATP synthase activity at the cell surface in ADPKD cysts may result in increases in ATP release and inappropriate chloride channel or purinergic receptor activation. Alternatively, ATP levels may regulate ATP-sensitive channels, including ATP-binding cassette transporters such as the cystic fibrosis transmembrane regulator chloride channel. Defective PC-1 calcium signaling has been implicated in ATP-stimulated chloride secretion (Wildman et al., 2003).
This report is the first to assign function the polycystin-1 PLAT domain and to demonstrate ciliary localization of the ATP synthase. The cilium is an exclusive organelle in that it is not readily accessible to proteins, in particular membrane-bound proteins (Rosenbaum and Witman, 2002). How the ATP synthase localizes to the cilium is an intriguing mechanistic question. Although we have ruled out an essential role for LOV-1 in localizing ATP-2 to cilia (and vice versa), the function of the LOV-1 and ATP-2 interaction remains to be determined. Whether the ATP synthase and PC-1 colocalize in the primary cilium of renal epithelial cells is unknown. Our studies using C. elegans as a model for ADPKD promise new avenues to understanding polycystin function and ciliary sensory signaling.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
* Corresponding author. E-mail address: mmbarr{at}pharmacy.wisc.edu.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Arakaki, N., Nagao, T., Niki, R., Toyofuku, A., Tanaka, H., Kuramoto, Y., Emoto, Y., Shibata, H., Magota, K., and Higuti, T. ((2003). ). Possible role of cell surface H(+)-ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells. Mol. Cancer Res. 1, , 931-939.
Bae, T. J., et al. ((2004). ). Lipid raft proteome reveals ATP synthase complex in the cell surface. Proteomics. 4, , 3536-3548.[CrossRef][Medline]
Bakhtiari, N., Lai-Zhang, J., Yao, B., and Mueller, D. M. ((1999). ). Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 274, , 16363-16369.
Bargmann, C. I., Hartwieg, E., and Horvitz, H. R. ((1993). ). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, , 515-527.[CrossRef][Medline]
Barr, M. M., DeModena, J., Braun, D., Nguyen, C. Q., Hall, D. H., and Sternberg, P. W. ((2001). ). The Caenorhabditis elegans autosomal dominant polycystic kidney disease gene homologs lov-1 and pkd-2 act in the same pathway. Curr. Biol. 11, , 1341-1346.[CrossRef][Medline]
Barr, M. M., and Sternberg, P. W. ((1999). ). A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401, , 386-389.[CrossRef][Medline]
Bateman, A., and Sandford, R. ((1999). ). The PLAT domain: a new piece in the PKD1 puzzle. Curr. Biol. 9, , R588-R590.[CrossRef][Medline]
Boletta, A., and Germino, G. G. ((2003). ). Role of polycystins in renal tubulogenesis. Trends Cell Biol. 13, , 484-492.[CrossRef][Medline]
Boyer, P. D. ((1997). ). The ATP synthase-a splendid molecular machine. Annu. Rev. Biochem. 66, , 717-749.[CrossRef][Medline]
Brenner, S. ((1974). ). The genetics of Caenorhabditis elegans. Genetics 77, , 71-94.
Burwick, N. R., Wahl, M. L., Fang, J., Zhong, Z., Capaldi, R. A., Kenan, D. J., and Pizzo, S. V. ((2004). ). An inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface. J. Biol. Chem. (in press).
Chang, S. Y., Park, S. G., Kim, S., and Kang, C. Y. ((2002). ). Interaction of the C-terminal domain of p43 and the alpha subunit of ATP synthase. Its functional implication in endothelial cell proliferation. J. Biol. Chem. 277, , 8388-8394.
Das, B., Mondragon, M. O., Sadeghian, M., Hatcher, V. B., and Norin, A. J. ((1994). ). A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines. J. Exp. Med. 180, , 273-281.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. ((2000). ). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, , 325-330.[CrossRef][Medline]
Geng, L., et al. ((1996). ). Identification and localization of polycystin, the PKD1 gene product. J. Clin. Investig. 98, , 2674-2682.[Medline]
Gonczy, P., et al. ((2000). ). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, , 331-336.[CrossRef][Medline]
Granato, M., Schnabel, H., and Schnabel, R. ((1994). ). pha-1, a selectable marker for gene transfer in C. elegans. Nucleic Acids Res. 22, , 1762-1763.
Hanazawa, M., Mochii, M., Ueno, N., Kohara, Y., and Iino, Y. ((2001). ). Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 98, , 8686-8691.
Hodgkin, J. ((1983). ). Male phenotypes and mating efficiency in Caenorhabditis elegans. Geneitcs 103, , 43-64.
Hooper, K. M., Unwin, R. J., and Sutters, M. ((2003). ). The isolated C-terminus of polycystin-1 promotes increased ATP-stimulated chloride secretion in a collecting duct cell line. Clin. Sci. 104, , 217-221.[Medline]
Ibraghimov-Beskrovnaya, O., et al. ((1997). ). Polycystin: in vitro synthesis, in vivo tissue expression, and subcellular localization identifies a large membrane-associated protein. Proc. Natl. Acad. Sci. USA 94, , 6397-6402.
Igarashi, P., and Somlo, S. ((2002). ). Genetics and pathogenesis of polycystic kidney disease. J. Am. Soc. Nephrol. 13, , 2384-2398.
Kamath, R. S., et al. ((2003). ). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, , 231-237.[CrossRef][Medline]
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., and Ahringer, J. ((2001). ). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, , 1-10.
Kloda, A., Clements, J. D., Lewis, R. J., and Adams, D. J. ((2004). ). Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors. Mol. Pharmacol. 65, , 1386-1396.
Koushika, S. P., and Nonet, M. L. ((2000). ). Sorting and transport in C. elegans: a model system with a sequenced genome. Curr. Opin. Cell Biol. 12, , 517-523.[CrossRef][Medline]
Leipziger, J. ((2003). ). Control of epithelial transport via luminal P2 receptors. Am. J. Physiol. 284, , F419-F432.
Li, N., Shaw, A. R., Zhang, N., Mak, A., and Li, L. ((2004). ). Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry. Proteomics 4, , 3156-3166.[CrossRef][Medline]
Liu, K. S., and Sternberg, P. W. ((1995). ). Sensory regulation of male mating behavior in Caenorhabditis elegans. Neuron 14, , 79-89.[CrossRef][Medline]
Martinez, L. O., et al. ((2003). ). Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421, , 75-79.[CrossRef][Medline]
Mello, C., and Fire, A. ((1995). ). DNA transformation. Methods Cell Biol. 48, , 451-482.[Medline]
Moser, T. L., Kenan, D. J., Ashley, T. A., Roy, J. A., Goodman, M. D., Misra, U. K., Cheek, D. J., and Pizzo, S. V. ((2001). ). Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc. Natl. Acad. Sci. USA 98, , 6656-6661.
Moser, T. L., Stack, M. S., Asplin, I., Enghild, J. J., Hojrup, P., Everitt, L., Hubchak, S., Schnaper, H. W., and Pizzo, S. V. ((1999). ). Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc. Natl. Acad. Sci. USA 96, , 2811-2816.
Pazour, G. J., San Agustin, J. T., Follit, J. A., Rosenbaum, J. L., and Witman, G. B. ((2002). ). Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr. Biol. 12, , R378-R380.[CrossRef][Medline]
Penefsky, H. S., and Cross, R. L. ((1991). ). Structure and mechanism of FoF1-type ATP synthases and ATPases. Adv. Enzymol. Relat. Areas Mol. Biol. 64, , 173-214.[Medline]
Piano, F., Schetter, A. J., Morton, D. G., Gunsalus, K. C., Reinke, V., Kim, S. K., and Kemphues, K. J. ((2002). ). Gene clustering based on RNAi phenotypes of ovary-enriched genes in C. elegans. Curr. Biol. 12, , 1959-1964.[CrossRef][Medline]
Rosenbaum, J. L., and Witman, G. B. ((2002). ). Intraflagellar transport. Nat. Rev. Mol. Cell. Biol. 3, , 813-825.[CrossRef][Medline]
Schwiebert, E. M., et al. ((2002). ). Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys. Am. J. Physiol. 282, , F763-F775.
Schwiebert, E. M., and Zsembery, A. ((2003). ). Extracellular ATP as a signaling molecule for epithelial cells. Biochim. Biophys. Acta 1615, , 7-32.[Medline]
Sengupta, P., Chou, J. H., and Bargmann, C. I. ((1996). ). odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, , 899-909.[CrossRef][Medline]
Simmer, F., Moorman, C., Van Der Linden, A. M., Kuijk, E., Van Den Berghe, P. V., Kamath, R., Fraser, A. G., Ahringer, J., and Plasterk, R. H. ((2003). ). Genome-Wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1, , E12.[Medline]
Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., and Plasterk, R. H. ((2002). ). Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr. Biol. 12, , 1317-1319.[CrossRef][Medline]
Sulston, J. E., Albertson, D. G., and Thomson, J. N. ((1980). ). The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev. Biol. 78, , 542-576.[CrossRef][Medline]
Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A., and Driscoll, M. ((2000). ). Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24, , 180-183.[CrossRef][Medline]
Timmons, L., Court, D. L., and Fire, A. ((2001). ). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, , 103-112.[CrossRef][Medline]
Tsang, W. Y., and Lemire, B. D. ((2002). ). Mitochondrial genome content is regulated during nematode development. Biochem. Biophys. Res. Commun. 291, , 8-16.[CrossRef][Medline]
Tsang, W. Y., Sayles, L. C., Grad, L. I., Pilgrim, D. B., and Lemire, B. D. ((2001). ). Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span. J. Biol. Chem. 276, , 32240-32246.
Watnick, T., and Germino, G. ((2003). ). From cilia to cyst. Nat. Genet. 34, , 355-356.[CrossRef][Medline]
White, J. G., Southgate, E., Thomson, J. N., and Brenner, S. ((1986). ). The structure of the nervous system of the nematode Caenorhabditis elegans: the mind of a worm. Phil. Trans. R. Soc. Lond. 314, , 1-340.[CrossRef]
Wildman, S. S., Hooper, K. M., Turner, C. M., Sham, J. S., Lakatta, E. G., King, B. F., Unwin, R. J., and Sutters, M. ((2003). ). The isolated cytoplasmic C-terminus of polycystin-1 prolongs ATP-stimulated chloride conductance through increased agonist stimulated calcium entry. Renal Physiol. 285, , 1168-1178.
Wilson, P. D., Hovater, J. S., Casey, C. C., Fortenberry, J. A., and Schwiebert, E. M. ((1999). ). ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys. J. Am. Soc. Nephrol. 10, , 218-229.
Yoder, B. K., Hou, X., and Guay-Woodford, L. M. ((2002). ). The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J. Am. Soc. Nephrol. 13, , 2508-2516.
This article has been cited by other articles:
|
|
 |
|
  J. Hu, S. G. Wittekind, and M. M. Barr STAM and Hrs Down-Regulate Ciliary TRP Receptors Mol. Biol. Cell, September 1, 2007; 18(9): 3277 - 3289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hu, Y.-K. Bae, K. M. Knobel, and M. M. Barr Casein Kinase II and Calcineurin Modulate TRPP Function and Ciliary Localization Mol. Biol. Cell, May 1, 2006; 17(5): 2200 - 2211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Davenport and B. K. Yoder An incredible decade for the primary cilium: a look at a once-forgotten organelle Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1159 - F1169. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
