Parental exposure to ocean acidification impacts gamete production and physiology but not offspring performance in Nematostella vectensis.

Ocean acidification (OA) resulting from anthropogenic CO2 emissions is impairing the reproduction of marine organisms. While parental exposure to OA can protect offspring via carryover effects, this phenomenon is poorly understood in many marine invertebrate taxa. Here, we examined how parental exposure to acidified (pH 7.40) versus ambient (pH 7.72) seawater influenced reproduction and offspring performance across six gametogenic cycles (13 weeks) in the estuarine sea anemone Nematostella vectensis. Females exhibited reproductive plasticity under acidic conditions, releasing significantly fewer but larger eggs compared to ambient females after 4 weeks of exposure, and larger eggs in two of the four following spawning cycles despite recovering fecundity, indicating long-term acclimatization and greater investment in eggs. Males showed no changes in fecundity under acidic conditions but produced a greater percentage of sperm with high mitochondrial membrane potential (MMP; a proxy for elevated motility), which corresponded with higher fertilization rates relative to ambient males. Finally, parental exposure to acidic conditions did not significantly influence offspring development rates, respiration rates, or heat tolerance. Overall, this study demonstrates that parental exposure to acidic conditions impacts gamete production and physiology but not offspring performance in N. vectensis, suggesting that increased investment in individual gametes may promote fitness.

Molecular mechanisms of sperm motility are conserved in an early-branching metazoan.

Efficient and targeted sperm motility is essential for animal reproductive success. Sperm from mammals and echinoderms utilize a highly conserved signaling mechanism in which sperm motility is stimulated by pH-dependent activation of the cAMP-producing enzyme soluble adenylyl cyclase (sAC). However, the presence of this pathway in early-branching metazoans has remained unexplored. Here, we found that elevating cytoplasmic pH induced a rapid burst of cAMP signaling and triggered the onset of motility in sperm from the reef-building coral Montipora capitata in a sAC-dependent manner. Expression of sAC in the mitochondrial-rich midpiece and flagellum of coral sperm support a dual role for this molecular pH sensor in regulating mitochondrial respiration and flagellar beating and thus motility. In addition, we found that additional members of the homologous signaling pathway described in echinoderms, both upstream and downstream of sAC, are expressed in coral sperm. These include the Na+/H+ exchanger SLC9C1, protein kinase A, and the CatSper Ca2+ channel conserved even in mammalian sperm. Indeed, the onset of motility corresponded with increased protein kinase A activity. Our discovery of this pathway in an early-branching metazoan species highlights the ancient origin of the pH-sAC-cAMP signaling node in sperm physiology and suggests that it may be present in many other marine invertebrate taxa for which sperm motility mechanisms remain unexplored. These results emphasize the need to better understand the role of pH-dependent signaling in the reproductive success of marine animals, particularly as climate change stressors continue to alter the physiology of corals and other marine invertebrates.

ROR and RYK extracellular region structures suggest that receptor tyrosine kinases have distinct WNT-recognition modes.

WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.

Non-acylated Wnts Can Promote Signaling.

Wnts are a family of 19 extracellular ligands that regulate cell fate, proliferation, and migration during metazoan embryogenesis and throughout adulthood. Wnts are acylated post-translationally at a conserved serine and bind the extracellular cysteine-rich domain (CRD) of Frizzled (FZD) seven-pass transmembrane receptors. Although crystal structures suggest that acylation is essential for Wnt binding to FZDs, we show here that several Wnts can promote signaling in Xenopus laevis and Danio rerio embryos, as well as in an in vitro cell culture model, without acylation. The non-acylated Wnts are expressed at levels similar to wild-type counterparts and retain CRD binding. By contrast, we find that certain other Wnts do require acylation for biological activity in Xenopus embryos, although not necessarily for FZD binding. Our data argue that acylation dependence of Wnt activity is context specific. They further suggest that acylation may underlie aspects of ligand-receptor selectivity and/or control other aspects of Wnt function.

CKAP4 is identified as a receptor for Dickkopf in cancer cells.

The secretory protein Dickkopf-1 (DKK-1) is a known Wnt antagonist and has been shown to suppress tumorigenesis in some cancer cells; however, it is also upregulated in many types of cancer and associated with poor prognosis. Wnt-independent mechanisms by which DKK-1 promotes cancer cell proliferation are not well understood. In this issue of the JCI, Kimura and colleagues demonstrate that DKK-1 interacts with cytoskeleton-associated protein 4 (CKAP4) to promote activation of AKT. They show that both DKK-1 and CKAP4 are frequently upregulated in pancreatic and lung cancers. Importantly, targeting this interaction with an anti-CKAP4 antibody prevented tumor formation in murine xenograft models. These results identify a previously unrecognized DKK-1-mediated pathway and suggest CKAP4 as a potential therapeutic target for certain cancers.

Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-ß (TGF-ß) signaling, and mice lacking components of the TGF-ß signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-ß pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of ß4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-ß was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-ß. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-ß pathway.

Characterization of a heterodimeric Smac-based peptide that features sequences specific to both the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein.

XIAP, an important regulator of apoptosis, has emerged as a target for the development of cancer therapeutics. The homodimeric Smac protein simultaneously binds to both the BIR2 and BIR3 domains of XIAP. Peptide-based dimeric compounds that mimic the binding mode of Smac show promise as XIAP antagonists. Herein we characterize the first example of a Smac mimetic that incorporates a peptide sequence specific for BIR2. We show that the tetrapeptide motif Ala-Glu-Ala-Val has a higher affinity for BIR2 than the BIR3-specific sequence Ala-Val-Pro-Phe, and we compare the binding characteristics of a heterodimeric peptide containing both tetrapeptide motifs to those of a homodimeric peptide featuring only AVPF. Despite the enhanced affinity of AEAV (relative to AVPF) for BIR2, the heterodimeric peptide displays only a slightly higher affinity for XIAP relative to its homodimeric counterpart. Enhanced affinity of both dimers relative to the tetrapeptide AVPF is largely maintained even when the BIR2 binding groove is modified, implying that hydrophobic contacts afforded by the second peptide motif need not necessarily be made at the BIR2 binding groove to contribute substantial binding energy. Finally, we use mutagenesis to show that the difference in sequence specificity observed between the two domains is primarily owing to steric bulk introduced at the BIR2 site by lysine 206. Replacement of K206 at BIR2 with glycine, the corresponding residue in BIR3, restores the majority of the affinity for the AVPF motif exhibited by BIR3. The implications of these finding in the development of XIAP antagonists are discussed. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 122-130, 2012.