ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation

Caenorhabditis elegans dauer formation is an alternative larval developmental pathway that the worm can take when environmental conditions become detrimental. Animals can survive several months in this stress-resistant stage and can resume normal development when growth conditions improve. Although the worms integrate a variety of sensory information to commit to dauer formation, it is currently unknown whether they also monitor internal cellular damage. The Ro ribonucleoprotein complex, which was initially described as a human autoantigen, is composed of one major 60-kDa protein, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Ro60 has been shown to bind mutant 5S rRNA molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Analysis of ribosomes from a C. elegans rop-1(−) strain, which is null for the expression of Ro60, demonstrated that they contain a high percentage of mutant 5S rRNA molecules, thereby strengthening the notion of a link between the rop-1 gene product and 5S rRNA quality control. The Ro particle was recently shown to be involved in the resistance of Deinococcus radiodurans to UV irradiation, suggesting a role for the Ro complex in stress resistance. We have studied the role of rop-1 in dauer formation. We present genetic and biochemical evidence that rop-1 interacts with dauer-formation genes and is involved in the regulation of the worms' entry into the dauer stage. Furthermore, we find that the rop-1 gene product undergoes a proteolytic processing step that is regulated by the dauer formation pathway via an aspartic proteinase. These results suggest that the Ro particle may function in an RNA quality-control checkpoint for dauer formation.

Expression of the mouse cerberus-related gene, Cerr1, suggests a role in anterior neural induction and somitogenesis

The Xenopus cerberus gene encodes a secreted factor that is expressed in the anterior endomesoderm of gastrula stage embryos and can induce the formation of ectopic heads when its mRNA is injected into Xenopus embryos [Bouwmeester, T., Kim, S., Lu, B. & De Robertis, E. M. (1996) Nature (London) 382, 595–601]. Here we describe the existence of a cerberus-related gene, Cerr1, in the mouse. Cerr1 encodes a putative secreted protein that is 48% identical to cerberus over a 110-amino acid region. Analysis of a mouse interspecific backcross panel demonstrated that Cerr1 mapped to the central portion of mouse chromosome 4. In early gastrula stage mouse embryos, Cerr1 is expressed in the anterior visceral endoderm and in the anterior definitive endoderm. In somite stage embryos, Cerr1 expression is restricted to the most recently formed somites and in the anterior presomitic mesoderm. Germ layer explant recombination assays demonstrated that Cerr1-expressing somitic-presomitic mesoderm, but not older Cerr1-nonexpressing somitic mesoderm, was able to mimic the anterior neuralizing ability of anterior mesendoderm and maintain Otx2 expression in competent ectoderm. In most Lim1−/− headless embryos, Cerr1 expression in the anterior endoderm was weak or absent. These results suggest that Cerr1 may play a role in anterior neural induction and somite formation during mouse development.

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, Km ≈ 1.0 mM) also transports fructose (Km ≈ 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

We previously have demonstrated that the colonic P-ATPase α subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a β subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na+, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.

The gamma subunit of the Na,K-ATPase induces cation channel activity

The γ subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The γ subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the γ subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human γ subunit. Confocal microscopy demonstrates that the γ subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that γ colocalizes with α1 when these proteins are coexpressed. When Sf-9 cells were coinfected with α1 and γ, antibodies to the γ subunit were able to coimmunoprecipitate the α1 subunit, suggesting that γ is able to associate with α1. The γ subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the γ subunit is a functional component was supported by experiments showing γ-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.

Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein–protein interactions

Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein–protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl− currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant ΔF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.

The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42

The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks in vitro. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface.

Metabolism to a response pathway selective retinoid ligand during axial pattern formation

We report identification of 9-cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) as an in vivo retinoid metabolite in Xenopus embryos. 9-Cis-4-oxo-RA bound receptors (RARs) α, β, and γ as well as retinoid X receptors (RXRs) α, β, and γ in vitro. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-Cis-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-cis-4-oxo-RA displayed high potency for modulating axial pattern formation in Xenopus, metabolism to 9-cis-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

The deg-3 gene from the nematode Caenorhabditis elegans encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 μM and Hill coefficient (Hinh) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal–ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

Design of polyzinc finger peptides with structured linkers

Zinc finger domains are perhaps the most versatile of all known DNA binding domains. By fusing up to six zinc finger modules, which normally recognize up to 18 bp of DNA, designer transcription factors can be produced to target unique sequences within large genomes. However, not all continuous DNA sequences make good zinc finger binding sites. To avoid having to target unfavorable DNA sequences, we designed multizinc finger peptides with linkers capable of spanning long stretches of nonbound DNA. Two three-finger domains were fused by using either transcription factor IIIA for the Xenopus 5S RNA gene (TFIIIA) finger 4 or a non-sequence-specific zinc finger as a “structured” linker. Our gel-shift results demonstrate that these peptides are able to bind with picomolar affinities to target sequences containing 0–10 bp of nonbound DNA. Furthermore, these peptides display greater sequence selectivity and bind with higher affinity than similar six-finger peptides containing long, flexible linkers. These peptides are likely to be of use in understanding the behavior of polydactyl proteins in nature and in the targeting of human, animal, or plant genomes for numerous applications. We also suggest that in certain polydactyl peptides an individual finger can “flip” out of the major groove to allow its neighbors to bind shorter, nontarget DNA sequences.

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum, which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.