Progesterone induces meiosis through two obligate co-receptors with PLA2 activity.

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, its detailed signal transduction remains elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/ß hydrolase domain-containing protein 2) as an essential mPRß co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires both P4 and mPRß. This PLA2 activity bifurcates P4 signaling by inducing mPRß clathrin-dependent endocytosis and producing lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing the ABHD2 PLA2 activity that requires both mPRß and ABHD2 as obligate co-receptors.

Correction: Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

[This corrects the article DOI: 10.1371/journal.pbio.3000901.].

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis.

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260-275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

miRNA-dependent regulation of STIM1 expression in breast cancer.

Store-operated Ca2+ entry (SOCE) has been shown to be important for breast cancer metastasis in xenograft mouse models. The ER Ca2+ sensor STIM1 and Orai plasma membrane Ca2+ channels molecularly mediate SOCE. Here we investigate the role of the microRNA machinery in regulating STIM1 expression. We show that STIM1 expression is regulated post-transcriptionally by the miRNA machinery and identify miR-223 and miR-150 as regulators of STIM1 expression in the luminal non-aggressive MCF7 breast cancer cell line. In contrast, STIM1 expression in the more aggressive basal triple-negative MDA-MB-231 cell line is not significantly modulated by a single miRNA species but is rather upregulated due to inhibition of the miRNA machinery through downregulation of Ago2. Consistently, overexpression of Ago2 results in decreased STIM1 protein levels in MDA-MB-231 cells. Clinically, STIM1 and Ago2 expression levels do not correlate with breast cancer progression, however in the basal subtype high STIM1 expression is associated with poorer survival. Our findings show that STIM1 expression is differentially regulated by the miRNA machinery in different cell types and argue for a role for this regulation in breast cancer.

Spatially restricted subcellular Ca2+ signaling downstream of store-operated calcium entry encoded by a cortical tunneling mechanism.

Agonist-dependent Ca2+ mobilization results in Ca2+ store depletion and Store-Operated Calcium Entry (SOCE), which is spatially restricted to microdomains defined by cortical ER - plasma membrane contact sites (MCS). However, some Ca2+-dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca2+ tunneling, mediates the uptake of Ca2+ flowing through SOCE into the ER followed by release at distal sites through IP3 receptors. Here we show that Ca2+ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca2+ release when stores are full, SOCE shows little effect in raising mitochondrial Ca2+, and Ca2+-tunneling is completely inefficient. In contrast, gKCa displays a similar sensitivity to Ca2+ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca2+ release. These results show that in contrast to the previously described long-range Ca2+ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca2+ rise within the cortical region of the cell to activate a specific subset of effectors.

The VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling.

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRß (PAQR8) partner that is required for mPRß plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.

Store-Operated Ca2+ Entry in Oocytes Modulate the Dynamics of IP3 -Dependent Ca2+ Release From Oscillatory to Tonic.

Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3 Rs at distant sites, a process we refer to as "Ca2+ teleporting." Therefore, sustained SOCE activation in the presence of an IP3 -dependent "leak" pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095-1103, 2017. © 2016 Wiley Periodicals, Inc.

The Ca2+-activated Cl- channel Ano1 controls microvilli length and membrane surface area in the oocyte.

Ca(2+)-activated Cl(-) channels (CaCCs) play important physiological functions in epithelia and other tissues. In frog oocytes the CaCC Ano1 regulates resting membrane potential and the block to polyspermy. Here, we show that Ano1 expression increases the oocyte surface, revealing a novel function for Ano1 in regulating cell morphology. Confocal imaging shows that Ano1 increases microvilli length, which requires ERM-protein-dependent linkage to the cytoskeleton. A dominant-negative form of the ERM protein moesin precludes the Ano1-dependent increase in membrane area. Furthermore, both full-length and the truncated dominant-negative forms of moesin co-localize with Ano1 to the microvilli, and the two proteins co-immunoprecipitate. The Ano1-moesin interaction limits Ano1 lateral membrane mobility and contributes to microvilli scaffolding, therefore stabilizing larger membrane structures. Collectively, these results reveal a newly identified role for Ano1 in shaping the plasma membrane during oogenesis, with broad implications for the regulation of microvilli in epithelia.

Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway.

A STIM1-dependent 'trafficking trap' mechanism regulates Orai1 plasma membrane residence and Ca²âº influx levels.

The key proteins mediating store-operated Ca(2+) entry (SOCE) are the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the plasma membrane Ca(2+)-selective channel Orai1. Here, we quantitatively dissect Orai1 trafficking dynamics and show that Orai1 recycles rapidly at the plasma membrane (Kex≃0.1 min(-1)), with ∼40% of the total Orai1 pool localizing to the plasma membrane at steady state. A subset of intracellular Orai1 localizes to a sub-plasmalemal compartment. Store depletion is coupled to Orai1 plasma membrane enrichment in a STIM1-dependent fashion. This is due to trapping of Orai1 into cortical ER STIM1 clusters, leading to its removal from the recycling pool and enrichment at the plasma membrane. Interestingly, upon high STIM1 expression, Orai1 is trapped into STIM1 clusters intracellularly, thus preventing its plasma membrane enrichment following store depletion. Consistent with this, STIM1 knockdown prevents trapping of excess Orai1 into limiting STIM1 clusters in the cortical ER. SOCE-dependent Ca(2+) influx shows a similar biphasic dependence on the Orai1:STIM1 ratio. Therefore, a STIM1-dependent Orai1 'trafficking trap' mechanism controls Orai1 plasma membrane enrichment and SOCE levels, thus modulating the SOCE 'bandwidth' for downstream signaling.

Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest.

Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSßγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest.

The Xenopus TRPV6 homolog encodes a Mg(2+) -permeant channel that is inhibited by interaction with TRPC1.

The TRP gene family encodes primarily cation non-selective, Ca2+ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca2+ selective and are expressed in epithelia where they are important in Ca2+ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca2+ , and displays a high permeability to Mg2+ . The oocyte does not exhibit functional TRPV6-like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions.

How to make a good egg!: The need for remodeling of oocyte Ca(2+) signaling to mediate the egg-to-embryo transition.

The egg-to-embryo transition marks the initiation of multicellular organismal development and is mediated by a specialized Ca(2+) transient at fertilization. This explosive Ca(2+) signal has captured the interest and imagination of scientists for many decades, given its cataclysmic nature and necessity for the egg-to-embryo transition. Learning how the egg acquires the competency to generate this Ca(2+) transient at fertilization is essential to our understanding of the mechanisms controlling egg and the transition to embryogenesis. In this review we discuss our current knowledge of how Ca(2+) signaling pathways remodel during oocyte maturation in preparation for fertilization with a special emphasis on the frog oocyte as additional reviews in this issue will touch on this in other species.