InteractiveFly: GeneBrief

ric8a: Biological Overview | References

Gene name - ric8a

Synonyms - ric-8

Cytological map position -

Function - signaling

Keywords - G-protein α-subunit binding, cell polarity, asymmetric cell division, mitotic spindle localization, gastrulation

Symbol - ric8a

FlyBase ID: FBgn0028292

Genetic map position - chrX:9150224-9152708

Classification - Guanine nucleotide exchange factor synembryn

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. This study shows that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. Mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. It is proposed that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding (Kanesaki, 2013).

The coordinated movement of cells is one of the foundations of tissue morphogenesis. The forces driving the cellular movements are generated by surface dynamics, such as rearrangements of cell adhesions and changes of the contractility of cortical acto-myosin networks. However, the surface mechanics resisting deformation forces and maintaining cortical integrity are not well understood (Kanesaki, 2013).

The shape of the cell surface can change dynamically. One notable surface feature is the bleb, a spherical protrusion of the plasma membrane observed in diverse cellular processes such as locomotion, division, and apoptosis. Formation of blebs is driven by hydrostatic pressure in the cytoplasm. According to the current model, blebbing starts with local compression of the cytoskeletal network and proceeds according to a subsequent increase of the pressure (Charras, 2005). The compression of the cytoskeleton is mediated by the contractile force of non-muscle myosin II (MyoII). Though it has been shown that various cells, such as germ line and cancer cells, utilise blebs for their motility, the role of blebs and the mechanism of blebbing in tissue morphogenesis are still largely unclear (Kanesaki, 2013).

Invagination of a cellular layer is one of the common events in tissue morphogenesis. In gastrulation in Drosophila, ventral cells of the blastoderm embryo invaginate and then differentiate to mesoderm. The process of mesoderm invagination can be grossly divided into two sequential steps: apical constriction and furrow internalisation. During apical constriction, ventral cells collectively contract their apices and consequently form a shallow furrow on the embryo. During furrow internalisation, the ventral furrow becomes deeper and the layer of cells becomes engulfed in the embryonic body. The molecular and cellular mechanisms underlying apical constriction have been studied extensively. The change of cellular shape is mediated by integrated functioning of the cortical acto-myosin network and cellular adherens junction complex. The force driving the constriction is generated by pulsed contractility of MyoII (Martin, 2009). The tensile force from individual cells is transmitted to epithelial tissue through the adherens junction, and the tissue generates feedback force that leads to anisotropic constriction of ventral cells (Kanesaki, 2013).

Heterotrimeric Gprotein (HGP) has an important role in apical constriction in Drosophila gastrulation. Signaling triggered by the extracellular ligand folded gastrulation (fog) promotes surface accumulation of MyoII in ventral cells, and the Fog signaling is mediated through an HGP α subunit encoded by concertina (cta). HGP belongs to the GTPase family, and its activity is regulated by multiple factors, including guanine nucleotide exchange factor (GEF). A previous study showed that ric-8 mutation results in a twisted germ-band due to abnormal mesoderm invagination (Hampoelz, 2005). ric-8 was first identified as a gene responsible for synaptic transmission in Caenorhabditis elegans, and was shown to interact genetically with EGL-30 (C. elegans Gαq) (Miller, 2000). Nematoda and vertebrate Ric-8 has GEF activity and positively regulates HGP signalingin vivo and in vitro (Afshar, 2004; Gabay, 2011; Tall, 2003). In Drosophila, Ric-8 is essential for targeting of HGPs toward the plasma membrane and participates in HGP-dependent processes such as asymmetric division of neuroblasts (David, 2005; Hampoelz, 2005; Wang, 2005; Kanesaki, 2013 and references therein).

In this study, the precise role of ric-8 in mesoderm invagination was investigated. It was found that cortical stability of ventral cells is impaired in a ric-8 mutant. By a combination of genetic and pharmacological analyses, blebbing of ventral cells was found to be induced by either disruption of cortical actin or mutation of ric-8. It is suggested that HGP signaling constitutively organises cortical actin, thereby reinforcing the resistance of cells against deformation (Kanesaki, 2013).

Ventral cells intrinsically exhibit a few small blebs during mesoderm invagination. This indicates that surface contraction during apical constriction induces blebbing even in normal invagination. This study found that Ric-8 and HGP signaling are required for suppression of abnormally large blebs, and for the stabilisation of the cortex in invaginating cells. The physical mechanism underlying blebbing has been studied extensively in cultured cells. The contractile force of the acto-myosin network causes an increase of hydrostatic pressure in the cytoplasm, which leads to detachment of the plasma membrane from the cortical actin layer. The dynamics of blebs observed in ric-8 ventral cells were similar to those reported in cultured cells in terms of time and size, suggesting that the mechanisms underlying blebbing in these two systems are conserved (Kanesaki, 2013).

The average size of blebs changes as development proceeds: blebs become larger during furrow internalisation. Immuno-fluorescence imaging revealed that MyoII is abnormally accumulated beneath enlarged blebs in the ric-8 mutant. This correlation suggests that MyoII acts to induce an increase of hydrostatic pressure. Although MyoII is an indispensable factor for apical constriction, its activity can also cause malformation of the cells. How MyoII accumulates abnormally in the ric-8 mutant remains unclear. It cannot be ruled out that other processes of mesoderm invagination, such as mechanical stress from surrounding cells, also contributes to the enlargement of blebs. During apical constriction, epithelial tissue generates tension along the anterior-posterior axis, and ventral cells undergo constriction in an anisotropic manner. Similar force may also be generated at the tissue level during furrow internalisation, causing the cells there to be squeezed, and consequently increasing the intracellular pressure. Blebbing in the ric-8 mutant may be a consequence of abnormal cytoskeletal networks and physical stress acting cell to cell. In normal situations, cells would resist such physical stress and maintain the surface integrity, thereby supporting correct morphogenetic movements (Kanesaki, 2013).

This study demonstrates that HGP signaling has two functions in mesoderm invagination: induction of the apical constriction via MyoII accumulation and maintenance of the cellular surface via organisation of cortical actin. Although Fog is required for apical constriction, F-actin is organised in a Fog-independent manner, suggesting that these two functions are regulated in different ways. cta mutants and G-iα65A mutants showed similar phenotypes regarding cortical actin, suggesting that these Gα paralogs have overlapping functions. Because the Drosophila genome encodes 6 Gα subunits and 5 of them are expressed in early embryos, the contribution of G α paralogs other than Cta and G-iα65A to the suppression of blebbing cannot be rule out. The finding that ric-8, Gβ13F, and Gγ1 mutants showed blebbing, a hallmark of severely disturbed cortical actin, supports the idea that multiple HGP pathways control cortical actin redundantly. However, currently it is not known whether those signaling pathways act on the same downstream effectors. Considering that most blastoderm cells showed a dispersed signal of GFP-Moesin in the mutants, HGPs appear to be rather constitutive regulators of cortical actin organisation. Nevertheless, the abnormality of the cortex does not affect the morphology of the 'standstill' cells that do not carry out the inward movement. Thus, HGPs are required to reinforce the cortex so that the cells can endure the stress generated during tissue folding (Kanesaki, 2013).

It was previously reported that ric-8 is required for Drosophila gastrulation (Hampoelz, 2005). This study extensively investigated mesoderm invagination and found that apical constriction is indeed compromised in the ric-8 mutant. Based on the observation of Fog-dependent MyoII accumulation, it is concluded that ric-8 is required for Fog-Cta signaling. It is unlikely that this phenotype is a secondary consequence of the disorganised F-actin in the ric-8 mutant, because actin was organised normally in the fog mutant embryo and ectopic Fog expression induced cell flattening even in late B-treated embryos. These findings instead suggested that Fog-Cta signaling and actin organisation are separate pathways and Ric-8 is involved in both pathways (Kanesaki, 2013).

Given that HGPs constitutively regulate F-actin, the signaling seems to be active in most blastoderm cells. Some unknown extracellular ligand and its receptor thus appear to be expressed to activate HGPs. It is also possible that cytoplasmic HGP regulators such as Pins, Loco, or other RGS proteins are involved in the activation. In the formation of the blood-brain barrier in Drosophila, Pins and Loco positively regulate HGP signaling. Embryos mutant for Pins also show abnormal cellular movements during mesoderm invagination. It is also intriguing to hypothesise that Ric-8 participates in the activation of HGPs through its GEF activity, which has been characterised both in vivo and in vitro. This hypothesis suggests the possibility that HGPs are endogenously activated. Future analysis of the responsible cytoplasmic regulators may clarify the mechanism of HGP regulation, and may give new insights regarding the intricate network of HGP signaling in animal development (Kanesaki, 2013).

How might HGP be functionally linked to actin polymerisation? Since G α12/13 participates in the activation of Formin family proteins in mammalian fibroblasts and a human Formin inhibits the formation of blebs in a prostate cancer cell line, a candidate factor regulating actin filaments downstream of HGP could be Diaphanous (Dia), a Drosophila Formin. Although it has been shown that organisation of actin via Dia is required for ventral furrow invagination, it is unclear whether Dia is also required for cortical stability during morphogenesis. Considering that Dia is an actin nucleator, it is speculated that Dia might act in the assembly of the actin meshwork and thereby reinforce the cortex. Indeed, it was observed that the dia mutant embryos showed cellular deformation during gastrulation, suggesting the functional relevance of the actin nucleator in the suppression of blebs. Further analysis will be required to clarify the functions of Dia (Kanesaki, 2013).

Previous studies demonstrated that ventral cells form a particular type of F-actin meshwork that makes a basic frame for apical constriction. RhoA- and Abelson-mediated signaling is required for organisation of the apical F-actin meshwork, while the Fog-Cta pathway is not. Thus, it is surprising that the mutants for HGPs, including Cta, showed a defect of cortical actin. HGP signaling may organise only a moiety of F-actin which is distinct from the one specifically accumulated at apices. HGP signaling regulates the organisation of cortical actin and mediates the establishment of the blood-brain barrier in Drosophila , suggesting that this function of HGPs is rather common in fly embryogenesis (Kanesaki, 2013).

Drosophila Ric-8 interacts with the Galpha12/13 subunit, Concertina, during activation of the Folded gastrulation pathway

Heterotrimeric G-proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G-protein-coupled receptors (GPCRs). However, Gα subunits may also be activated in a non-canonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. This study used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger acto-myosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. This study describes a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. It was shown that Ric-8 regulates this pathway through a physical interaction with Cta, and that Ric-8 preferentially interacts with inactive Cta and directs its localization within the cell. This system was also used to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility (Peters, 2013).

A novel assay was established for testing potential Fog pathway components, and it was found that in Drosophila tissue culture Ric-8 is required for pathway activation and not only binds the Gα12/13, Cta, but preferentially binds inactive Cta, CtaGA. A role was defined for Ric-8 as an escort/scaffold for CtaGA by using artificially induced localization of Ric-8 to the mitochondria. Upon Ric-8 translocation it was found that CtaGA co-localizes with ectopically localized Ric-8, while the cellular localization of wild-type and constitutively active Cta were unaffected. Additionally, when Ric-8 was mis-targeted to the mitochondria, cells were impaired in their ability to constrict in response to Fog application. Further, evolutionarily conserved residues were identified within Ric-8 potentially important for 1) establishing a Ric-8/Cta binding interface 2) nucleotide specific recognition of Cta, and 3) successful G-protein signaling downstream of Fog (Peters, 2013).

The novel cell-based assay was ideal for examining Fog-induced activation of the Rho pathway, due to the ease in which it was possible to deplete cells of specific proteins using RNAi, the rapidity of screening multiple genes simultaneously, and the ability to visualize pathway activation using simple microscope-based examination. This assay opens numerous possibilities for the identification of other pathway components, including the unidentified GPCR involved in transduction of the Fog signal, as well as investigation of general cellular functions such as mechanochemical force production and regulation of the acto-myosin cytoskeleton. Additionally, although not highlighted in this study, it was possible to view Fog-induced contractility in real-time. This allows for further investigation of pathway components that specifically affect the kinetics of Fog responsiveness, and/or the longevity and persistence of pathway activation. In Drosophila, and other systems, Ric-8 modulates the behavior of Gα subunits during asymmetric cell divisions (reviewed in (Hinrichs et al., 2012)). Due to its role in establishing asymmetry in dividing cells and subsequently controlling cell proliferation rates, Ric-8 has become of interest to the field of cancer biology (Luo et al., 2008; Muggerud et al., 2009). This model cell culture system provides a streamlined approach for further investigation into parsing out the complicated signaling networks involved in establishing these disease states (Peters, 2013).

Previous work has implicated Ric-8 as a chaperone during Gα biosynthesis to stabilize nascent protein production, and in turn as an essential factor in Gα membrane targeting. This function of Ric-8 has been shown to affect the stability of all classes of mammalian Gα subunits. Given the necessity of Ric-8 in mammalian systems for Gα stabilization and membrane localization it is likely that Ric-8 acts similarly in Drosophila, as evidenced by the mis-targeting of Gαi and Cta, in the absence of Ric-8, to the cortex of the epithelium of Drosophila embryos and the mis-localization of Cta in Drosophila tissue culture cells. However, unlike Gαi, the levels of Cta are not dramatically affected in the absence of Ric-8; additionally, some rescue was seen in cells depleted of endogenous Ric-8, overexpressing constitutively active Cta, indicating that at least a small amount of Cta is localized correctly and functional. Therefore, while plasma membrane levels of Cta are affected by Ric-8 overall levels of protein are not. One possibility, given constitutively active Cta was still able to rescue, is that Ric-8 could be important for Gα cycling at the site of receptor activation, which is thought to be important for spatial regulation of Gα signaling (Peters, 2013).

Though signaling nodes involving GPCRs, Gα subunits, and Ric-8 have been extensively studied there is little known about the structure of Ric-8 and how it interacts with Gα. A predicted model of Ric-8 was used as a conceptual basis to visualize mutants, and key conserved residues important for Cta binding, nucleotide specificity, and execution of productive G-protein pathway activation were identified. Based on these data the structure/function assay of Ric-8 identified four cluster mutations, mutants 1, 9, 10 and 13, that inhibited CtaGA binding, of which three: 1, 9, and 13, also failed to rescue Fog-induced constriction to wild-type levels. Of these four mutants, only mutant 1 (in the N-terminus of Ric-8) was found to have an inhibitory effect on binding to wild-type, constitutively active, and constitutively inactive Cta, while mutants 9, 10 and 13 (in the C-terminus of Ric-8) were only deficient in binding inactive Cta. The Itoh lab found that a truncated version consisting of the N-terminal half (residues 1-301) of Ric-8 was sufficient to bind Gαq. In accordance with these data, it is suggested that residues in mutant 1 are important for non-nucleotide specific Cta interaction, while residues in mutants 9, 10 and 13 confer nucleotide specific recognition of Cta. This study presents the first evidence of specific residues within Ric-8 facilitating interaction with a Gα (Peters, 2013).

Several mutants had effects in only the binding or contractile assay. Mutant 10 inhibited binding, while mutants 6-8 prevented Fog-induced constriction. Mutant 10 was able to modestly rescue cellular constriction but exhibited decreased binding to Cta, implying this mutant is still functional but perhaps folded in a manner unproductive for robust binding to Cta; this may be due to its proximity to mutant 13. Mutants 6-8 are capable of binding Cta, but not rescuing Ric-8 function downstream of pathway activation. While the function of mutant clusters 6-8 is unclear, it is tempting to hypothesize that this region is a potential site for Ric-8 GEF activity (Peters, 2013).

In the early dividing C.elegans embryo, Drosophila melanogaster neuroblasts and epithelium and several mammalian tissue culture cell lines Ric-8 localizes Gα subunits to the plasma membrane. The current data suggest there is an additional level regulating Gα localization that is dependent on the nucleotide-bound state of Gα. This study has identified a cluster of residues that may facilitate this interaction with Cta. Clustered Ric-8 mutants, deficient in binding CtaGA in immunoprecipitation assays, when tagged with a sequence directing them to the mitochondria had varying effects in their ability to ectopically localize CtaGA. Mito-Ric-8 mutant 1 did not recruit CtaGA to its ectopic location at the mitochondria, while Mito-Ric-8 mutants 9, 10, and 13 triggered mitochondrial mis-localization of CtaGA. Interestingly, mutants 9, 10 and 13 exhibited decreased binding to constitutively inactive Cta, CtaGA, but not wild-type nor constitutively active Cta, CtaQL. This implies that these residues may confer temporally regulated nucleotide specific recognition sites for Cta (Peters, 2013).

Based on characterization of Ric-8, and data from the literature, the following model is proposed. Ric-8 acts to initially chaperone the folding of Cta, allowing Cta, Gβ13F, and Gγ1 to form a complex that is then transported to the plasma membrane. Upon Fog/GPCR interaction, GTP-bound Cta is released from the Gβγ heterodimer, and interacts with RhoGEF2 (via its RGS domain), causing hydrolysis of GTP to GDP. Specific, evolutionarily conserved residues regulate the binding of GDP-bound Cta to Ric-8, or alternatively Ric-8 stabilizes a nucleotide-free version of Cta. This allows Cta to bypass destruction and be re-inserted into the Fog pathway to activate downstream targets (Peters, 2013).

Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of G&alpha:i depends on Partner of Inscuteable (Pins). This study establishes that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Gαi, regulates cortical localization of the subunits G&alpha: and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells. This demonstrates, first, that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and, second, that a pI-specific activity tilts the spindle in the absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI-specific activity for two reasons. First, Fz signalling is still active in the pins and Gαi mutants as the spindle was correctly oriented along the antero-posterior axis in these mutants. Second, Fz accumulates at the posterior apical cortex of pI cells and this accumulation of Fz is maintained in Gαi pI cells. It is therefore envisaged that, although orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. fz,pins double mutants were analyzed to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium. Therefore, in the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells, indicating that Fz may also tilt the spindle in wild-type cells along their apico-basal axis. To test this, spindle orientation was analyzed in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells. Together, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric-8a/Pins/Gαi-dependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arises from this balance. Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex. The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (David, 2005).

In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR-Gαi complex, which is the homologue of the Pins-Gαi complex. These data demonstrate that, in the dividing pI cell, Ric-8a is required for asymmetric localization of Pins, Baz and Numb and for mitotic-spindle positioning. It is proposed that these activities of Ric-8a depend on an unexpected function of Ric-8a: localizing Gαi and G<β13F at the plasma membrane. This study of ric-8a also revealed that, in the pI cell, ric-8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype that was observed in ric-8a pI cells could be accounted for by some persistence of the Ric-8a protein in somatic clones. Alternatively, an intriguing possibility is that ric-8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling. ric-8a loss of function would thereby affect both the Fz- and Gαi-dependent activities exerted on the spindle, resulting in a milder apico-basal tilt (David, 2005).

Importantly, developmental processes ranging from gastrulation, neural-tube closure, neurogenesis and retina formation to asymmetric segregation of cell-fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral, etc) and parallel to the plane of the epithelium. This study shows that, in dividing pI cells, these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the antero-posterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz- and Gαi-dependent activities are likely to act through forces pulling on astral microtubules. Fz and heterotrimeric G signalling are implicated in mitotic-spindle positioning during both symmetric and asymmetric cell division. The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to understanding of the mechanisms that control mitotic-spindle positioning (David, 2005).

Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins; Ric-8 complexes with Pins through their mutual interactions with Gα

Asymmetric division of Drosophila neuroblasts (NBs) and the C. elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Gα, RIC-8, which has recently been characterized in C. elegans and in mammals. The Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pI cells. Ric-8 is necessary for membrane targeting of Gαi, Pins and Gβ13F, presumably by regulating multiple Gα subunit(s). Ric-8 forms an in vivo complex with Gαi and interacts preferentially with GDP-Gαi, which is consistent with Ric-8 acting as a GEF for Gαi. Ric-8 complexes with Pins through their mutual interactions with Gα. Comparisons of the phenotypes of Gαi, Ric-8, Gβ13F single and Ric-8;Gβ13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Gαi activity. In addition, Gβγ acts to restrict Gαi (and GoLoco proteins) to the apical cortex, where Gαi (and Pins) can mediate asymmetric spindle geometry (Wang, 2005).

In neuroblasts (NBs), two apically localized protein cassettes (Bazooka, Par3-DmPar6-DaPKC0 and Gα-Partner of Inscuteable [Pins, a GDP dissociation inhibitor (GDI) of Gα]), which are linked by Inscuteable (Insc) -- mediate all aspects of NB asymmetric division. These two conserved protein cassettes are spatially separated in pI cells of the sensory organ precursor (SOP) lineage: Pins-Gα localizes to the anterior, whereas Baz-Par-6-DaPKC localizes to the posterior cortex. In both Drosophila and C. elegans asymmetry models, the activation of heterotrimeric G-protein signalling apparently occurs via a receptor-independent mechanism that is mediated by proteins containing GoLoco/GPR (G-protein regulatory) motifs with GDI activity (for example, Drosophila Pins and nematode GPR1/2), which can compete with Gβγ for GDP-Gα. With respect to the spindle geometry of Drosophila NBs, Gβ13FGγ1 seems to have a more crucial role than Gα and Pins in this process. By contrast, Gα subunits, GOA-1 and GPA-16, and the GoLoco proteins GPR1/2 are essential in C. elegans, for the generation of a net posterior force that is necessary for asymmetric spindle positioning. Gβγ, in contrast, does not play a positive role in this process. More recently, RIC-8, a novel non-receptor guanine nucleotide-exchange factor (GEF) for Gα, has been shown to be required for asymmetric spindle positioning in the C. elegans zygote (Afshar, 2004; Couwenbergs, 2004; Tall, 2003; Hess, 2004). This study characterizes the role of the Drosophila Ric-8 homologue in neural progenitor asymmetric division (Wang, 2005).

Database searches of rat Ric-8A identified a putative Drosophila homologue, Ric-8 (CG15797, at cytological position 8D10 of the X chromosome), which shares ~31% amino-acid identity with rat Ric-8A. Ric-8 RNA is ubiquitously expressed with an abundant maternal component. In glutathione S-transferase (GST) pull-down assays, GST-Ric-8 interacts directly with Gα in vitro. In co-immunoprecipitation experiments using embryonic extracts, Ric-8, similarly to Pins and Gβ13F, interacts strongly with Gα when GDP has been added in excess, but interacts poorly with Gα in the presence of excess GTP-gammaS. This indicates that Ric-8 preferentially interacts with GDP-Gα. These interactions are consistent with Ric-8 acting as a GEF for Gα, similarly to its mammalian and nematode homologues (Wang, 2005).

To ascertain that the in vitro binding of Ric-8 with Gα reflects an in vivo association, co-immunoprecipitation experiments were performed using embryonic extracts. Ric-8 was detected in immunocomplexes when precipitation was performed with anti-Gα but not with the pre-immune control, indicating that Ric-8 complexes with Gα in vivo. To further substantiate this interaction using a different approach, protein extracts from wild-type embryos were incubated with agarose beads coupled to bacterially expressed MBP-Gα or MBP protein. Ric-8 was detected in the bound complex with MBP-Gα but not MBP (Wang, 2005).

In Drosophila NBs, Gα is present in at least two mutually exclusive complexes: a heterotrimeric complex with Gβ13F, or with a GoLoco-containing protein, Pins, which acts as a GDI for, and can directly interact with Gαi. Conventional G-protein-coupled receptors (GPCRs) promote nucleotide exchange on the Gαi-Gβγ heterotrimeric complex, whereas the mammalian non-receptor GEF RIC-8A cannot act on the heterotrimer. To explore the molecular context in which Ric-8 might act on Gα, whether Ric-8 can complex with Pins or Gβ13F was examined in Drosophila using co-immunoprecipitation experiments with embryonic extracts. When precipitations were performed using anti-Ric-8, Pins was specifically detected in the immunocomplex; in precipitations using anti-Pins, Ric-8 was also specifically detected. No direct interaction was observed with Ric-8 and Pins in the in vitro binding assays, indicating that Ric-8 complexes with Pins through their mutual interactions with Gα. To confirm these findings using a different approach, wild-type embryonic extracts were incubated with agarose beads coupled to bacterially expressed MBP-Ric-8 fusion protein. Pins but not Gβ13F was found in the bound complex with MBP-Ric-8. Thus, Ric-8 preferentially binds to the GDP-Gα-Pins complex, a similar finding to that seen in C. elegans embryos. This is in contrast to conventional GPCRs, which act on the heterotrimeric complex (Wang, 2005).

To determine the effects of ric-8 loss of function, several mutant alleles were isolated by imprecise excision of a P-element, EY05996. ric-8P587 removes the entire coding region (-953 bp to +1853 bp; ric-8 transcriptional start is +1), whereas ric-8P340 contains a larger deletion with unsequenced breakpoints. Both maternal and zygotic components were removed in the ric-8P340 and ric-8P587 germline clones (GLCs). These mutant embryos showed indistinguishable phenotypes, indicating that both are null alleles. Experiments were performed using embryos that were derived from ric-8P587 GLCs (Wang, 2005).

Gα shows punctated, cytosolic distribution in dividing and non-dividing NBs of ric-8 GLCs, in contrast to the apical cortical crescents seen in wild-type NBs. Pins also seemed to be cytosolic, which is consistent with findings that is required for the recruitment of Pins to the cortex. The issue of whether Gβ13F is also required for membrane targeting of Gα was examined using a newly generated anti-Gα antibody, as it was unclear whether the reported inability to detect Gα in Gβ13F mutant NBs by immunofluorescence was due to low sensitivity of the previously available antibody. The specificity of this new antibody was demonstrated by the absence of immunoreactivity in Gαi mutant embryos or nota in both immunofluorescence and Western experiment. It was found that Gα was uniformly localized on the cortex of Gβ13F GLC NBs, with clearly reduced intensity compared with the wild type. Pins was also uniformly cortical in Gβ13F GLC NBs, which indicates that the residual Gα on the cortex is sufficient to recruit Pins. The localization of Gα and Pins in blastoderm embryos that were derived from ric-8 and Gβ13F GLCs lends further support to these findings. Strikingly, Gα and Pins localized as punctated, cytosolic 'spots' in ric-8 GLC embryos, whereas in both wild-type and Gβ13F GLC embryos, Gα was membrane associated. Therefore, ric-8, but not Gβ13F, is crucial for the membrane targeting of Gα in NBs and other cell types (Wang, 2005).

In ric-8 GLC NBs, Insc was cytosolic. Baz and aPKC localized non-uniformly/asymmetrically on the cortex, but with reduced intensity and often as broader crescents, indicating that residual polarity cues remained. Mira crescents were often mislocalized in metaphase ric-8 NBs; mitotic domain 9 cells failed to re-orient their spindle by 90°, indicating that ric-8 is required for spindle re-orientation in cells of mitotic domain 9. These defects are similar to those seen in Gαi mutant NBs. Ric-8 is also required for the asymmetric division of pI cells. In ric-8 mutant metaphase pI cells, Gα and Pins did not form the anterior cortical crescents. Similarly, in Gαi metaphase pI cells, the anterior crescent of Pins did not form. In both ric-8 and Gα mutants, the Pon crescent was undetectable or significantly reduced. Nevertheless, Pon localized at the anterior cortex in anaphase pI cells of both mutants (Wang, 2005).

Antibodies specific for Ric-8 were generated against the amino-terminal (aa 1-150) or carboxy-terminal (aa 425-573) region of Ric-8. Ric-8 was localized to the cytoplasm of NBs throughout the cell cycle, even though Gα was seen as an apical crescent in mitotic NBs. However, interestingly, Ric-8 was also observed as 'spot'-like structures at the apical cortex of metaphase NBs, partially colocalizing with the Gα, indicating that their interaction might occur on the cytosolic face of the plasma membrane or in the cytoplasm. Similarly, in pI cells, Ric-8 was also cytosolic throughout the cell cycle (Wang, 2005).

ric-8 GLCs also exhibit abnormal gastrulation, in addition to defects in asymmetric divisions. Since gastrulation defects were also seen in Gβ13F and Gγ1 GLC embryos but not in Gαi embryos, the relationship was examined between ric-8 and Gβ13F. During cellular blastoderm formation, Gβ13F is delocalized from the cortex and is largely cytosolic in ric-8 GLC embryos, indicating that ric-8 is required for cortical localization of Gβ13F during these early stages. Consistently, Gβ13F is also largely cytosolic in NBs throughout the various stages of the cell cycle in stage-10 embryos derived from ric-8 GLCs. Given that Gαi loss of function alone does not disturb Gβ13F localization and Gβ13F does not complex with Ric-8, it was hypothesized that Ric-8 mediates the cortical localization of Gβ13F through its regulation of another Gα subunit. To further explore this possibility, it was asked whether Ric-8 can complex with Pins in embryos devoid of maternal and zygotic Gαi. If there was another Gα subunit involved, it might allow Ric-8 to complex with Pins by interacting with both, even in the absence of Gα. Indeed, Ric-8 complexes with Pins in the absence of Gα. Given that Ric-8 does not display a direct interaction with Pins, these data indicate that an, as yet unidentified, Gα subunit that is also regulated by Ric-8 may act (possibly in conjunction with Gα) to mediate Gβ13F cortical localization (Wang, 2005).

Gβ13F protein levels in ric-8 GLCs are significantly reduced compared with wild-type embryos; Gα and Pins levels remain unaffected. By contrast, Gα protein levels in Gβ13F GLCs are reduced, whereas Ric-8 levels do not change in or Gβ13F GLCs. Gβγ might normally be in excess; therefore, despite the reduction in Gβγ levels in ric-8 mutants, sufficient cytosolic levels may remain to stabilize normal levels of Gα. These data indicate that Ric-8 is required only for membrane targeting of Gα but not its stability; Gβ13F is required for the stability of Gα but not for its membrane targeting. In addition, Ric-8 is involved in both membrane association and the stability of Gβ13F, possibly by acting through another Gα subunit (Wang, 2005).

The requirement of Ric-8 for cortical localization and stability of Gβ13F prompted an examination of whether NB spindle geometry and difference in daughter-cell size are severely disrupted in ric-8 mutants, as shown for Gβ13F GLCs. In telophase NBs of wild-type stage-10 embryos, the ratio of ganglion mother cell (GMC) and NB (GMC/NB) diameter never exceeded 0.8 (average ratio = 0.42. By contrast, a hallmark of Gβ13F or Gγ1 loss is the high frequency of divisions that generate daughters of approximately equal size. These cells are telophase NBs in which the GMC diameter/NB diameter ratio was 0.8 or more (for Gβ13F NBs, 64% of divisions were similar sized with an average GMC/NB ratio of 0.82. The residual size asymmetry which remained was shown to be due to the reduced levels of asymmetrically localized Par proteins. However, a surprising observation was that, although cortical Gβ13F localization was disrupted in ric-8 mutant NBs, only 16% of telophase NBs divided into two similar-sized daughter cells, similar to those observed in Gαi mutant NBs. Thus, ric-8 GLC NBs did not display a phenotype similar to that of Gβ13F loss-of-function mutants. Further removal of Baz (by RNA interference) in ric-8 GLCs resulted in similar-sized division in 94% of NBs, indicating that partially localized Baz (Par proteins) can provide some asymmetry cues in ric-8 mutant NBs. Therefore, Ric-8 probably acts in the same pathway as Gα to redundantly regulate the difference in daughter-cell size in the Baz pathway. It was shown previously that in Gβ13F mutants, the number of abdominal Even-skipped positive lateral (EL) neurons in stage-15 embryos was severely decreased, presumably because a high frequency of similar-sized divisions rapidly reduces the cell volume of daughter NBs, resulting in early cessation of divisions. It was found that wild-type embryos produced an average of 9.0 EL neurons per abdominal hemisegment at stage 15; both ric-8 GLCs and Gαi mutants showed a similar reduction of EL neurons. By contrast, Gβ13F GLC embryos showed a more marked reduction in the numbers of EL neurons. These data indicate that, with respect to both numbers of EL neurons and NB daughter-cell size asymmetry, ric-8 and mutants exhibit similar phenotypes that are less severe than those seen in Gβ13F mutants (Wang, 2005).

Two alternative explanations are envisioned for why ric-8 and Gβ13F mutants have different effects on the asymmetric size of the daughter cells. (1) The generation of functional Gβγ may occur even in the absence of ric-8 function, despite the majority of the molecules being cytosolic. (2) Alternatively, the severe phenotypes seen in Gβ13F or Gγ1 mutant NBs may be an indirect consequence caused by the uniform cortical distribution of Gα (and Pins); the failure of ric-8 GLC NBs to exhibit a marked decrease in asymmetric daughter size would be because Gα and Pins are both cytosolic in ric-8 mutants and presumably inactive. To distinguish between these possibilities, ric-8, Gβ13F double mutant GLC embryos were made in which both ric-8 and Gβ13F would be completely removed. Interestingly, the double mutant GLC NBs exhibited phenotypes similar to those of ric-8 GLC NBs rather than Gβ13F GLC NBs. In double GLC NBs, Gα and Pins are cytosolic, whereas Baz localized non-uniformly/asymmetrically on the cortex. Only 24% of NBs divided into two similar-sized daughter cells. These observations indicate that the cytoplasmic Gβγ in ric-8 GLC NBs is non-functional and further suggests that the marked decrease in the difference in daughter-cell size of Gβ13F GLC NBs is an indirect consequence of the uniform cortical localization of Gα (and Pins) (Wang, 2005).

These data indicate that ric-8 mutants mediate asymmetric division of NBs and SOPs by regulating heterotrimeric G-protein localization. ric-8 acts at the top of a hierarchy for the sequential membrane/cortical localization of the apical proteins Gαi-Pins-Insc. The role of Ric-8 in membrane targeting of Gα is novel. Interestingly, Ric-8 also promotes cortical localization of Gβ13F in Drosophila. These data raise the possibility that this may be mediated indirectly by additional substrate(s) of Ric-8, which are presumably additional Gα subunit(s). Rat Ric-8A interacts with multiple brain membrane Gα subunits, including Gα13, Gαo, Gαq and Gα1,2. It is therefore speculated that Ric-8 may control the localization and stability of Gβ13F by regulating multiple Gα subunits. Precedence for a role of Gα in Gβγ membrane localization has been reported in mammalian cells (Wang, 2005).

This analyses of ric-8, Gαi, and ric-8;Gβ mutants support the view that, in NBs, cortically localized Gα mediates asymmetric spindle geometry and asymmetric daughter-cell size, which is positively regulated by Ric-8, and that an important role of Gβγ is to restrict Gα from the basal cortex. In the absence of Gβγ, the GoLoco/Gα complex expands from its normal apical localization, becomes uniformly cortical and can largely override the residual polarity cues that are provided by the asymmetrically localized, but drastically reduced levels of, Par proteins to greatly reduce spindle asymmetry and the difference in daughter size. The residual asymmetry that is present in the absence of Gβ13F is lost following further removal of Par function. The negative regulation of Gαi by Gβ13F in Drosophila NBs is similar to that in the C. elegans zygote, in which excess Gα activity was observed following loss of function of Gβ or Gγ. The findings that ric-8 mutants are genetically epistatic to mutants, both with respect to Gαi-Pins localization and to spindle geometry, are different from those reported in C. elegans embryos, in which inactivation of Gβγ alleviates the requirement for RIC-8 in asymmetric division. This indicates that different mechanisms of heterotrimeric G-protein regulation are present in the asymmetric division of nematode embryos and Drosophila NBs. These findings are consistent with a model in which Ric-8 has a crucial role in Gα activity by localizing the GoLoco/Gα complex onto the cortex and/or generating GTP-Gα as a GEF to mediate spindle geometry. Ric-8 also regulates the cortical localization and activity of Gβ, possibly through its regulation of multiple Gα subunits; Gβ acts to restrict Gα localization only to the apical cortex. Gα subunits that are asymmetrically localized at the apical cortex, in conjunction with Par proteins, mediate asymmetric spindle geometry and differences in daughter-cell size (Wang, 2005).

Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G proteins

Heterotrimeric G proteins act during signal transduction in response to extracellular ligands. They are also required for spindle orientation and cell polarity during asymmetric cell division. This study show that, in Drosophila, both functions require the Gα interaction partner Ric-8. Drosophila Ric-8 is a cytoplasmic protein that binds both the GDP- and GTP-bound form of the G-protein α-subunit Gαi. In ric-8 mutants, neither Gαi nor its associated β-subunit Gβ13F are localized at the plasma membrane, which leads to their degradation in the cytosol. During asymmetric cell division, this leads to various defects: apico–basal polarity is not maintained, mitotic spindles are misoriented and the size of the two daughter cells becomes nearly equal. ric-8 mutants also have defects in gastrulation that resemble mutants in the Gα protein concertina or the extracellular ligand folded gastrulation. These results indicate a model in which both receptor-dependent and receptor-independent G-protein functions are executed at the plasma membrane and require the Ric-8 protein (Hampoelz, 2005).

How could Ric-8 act in localizing heterotrimeric G-proteins to the plasma membrane? Myristoylation of Gα subunits at the N-terminus was shown to be essential for their cortical targeting. As mutations in Drosophila N-myristoyltransferase lead to more widespread defects during embryogenesis, it is unlikely that Ric-8 participates in lipid modification. In vertebrates, G-proteins are only translocated to the plasma membrane following association of the heterotrimer. When the amount of Gαi is insufficient, βγ-subunits fail to leave the endoplasmic reticulum. In ric-8m/z mutants, Gβ13F is no longer found in the Gαi immunoprecipitates, indicating that the mislocalization of G-protein subunits might actually be due to a failure to assemble the heterotrimer. As Ric-8 binds to Gαi but not to the fully assembled heterotrimer, it is speculated that it might be a chaperone or assembly factor that is needed for Gαi to bind Gβγ-subunits (Hampoelz, 2005).

In C. elegans, membrane localization of GOA-1 (Gαo) does not depend on RIC-8. Although localization of the Gβ13F homologue GPB-1 or the Gα-subunit GPA-16 in ric-8 mutant worms remains to be tested, this result could be explained by differences in the way that heterotrimeric G-proteins are assembled in the two organisms. The guanine nucleotide-exchange factor (GEF) activity that has been detected for Ric-8 in vitro can not easily be reconciled with a role in membrane localization of G-protein subunits. No GEF domain has been found in Ric-8 and a deletion analysis can therefore not be used to address the in vivo relevance of this activity. In any case, GEF activity can not explain ric-8 function in receptor-dependent G-protein signalling, where the receptor itself catalyses GDP/GTP exchange in the ligand-bound state. In addition, several of the current data indicate that the C. elegans model cannot be applied to Drosophila: first, this study showed that the GoLoco domains of Pins can dissociate the heterotrimer even in the absence of Ric-8 in vitro. This is not unexpected, since previous experiments using a yeast assay have already demonstrated the receptor (and Ric-8)-independent ability of a Pins homologue to dissociate heterotrimeric G-protein complexes. Second, the C. elegans model does not explain ric-8 function in gastrulation, which is independent of Pins. Finally, it was shown that Ric-8 does not help, but actually inhibits, the formation of Pins–Gαi complexes. Analysing G-protein localization in ric-8 mutant vertebrate cells will be of particular interest to test which of the two proposed functions is present in higher organisms (Hampoelz, 2005).

Resistance to inhibitors of cholinesterase 8A catalyzes release of Gαi-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Gαi-GDP complexes

Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in C. elegans and Drosophila have placed RIC-8 (Ric8a in Drosophila) in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the G alphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of G alphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and G alphai regulate a similar process. Mammalian Ric-8A dissociates G alphai-GDP/LGN/NuMA complexes catalytically, releasing activated G alphai-GTP in vitro. Ric-8A-stimulated activation of G alphai causes concomitant liberation of NuMA from LGN. It is concluded that Ric-8A efficiently utilizes GoLoco/G alphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Gαi-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division (Tall, 2005).

Models are envisioned in which one cellular function of Ric-8A is to dissociate Gαi-GDP/GoLoco complexes by stimulation of nucleotide exchange. G protein control of asymmetric cell division involves cycling of Gαi between its GDP- and GTP-bound forms, as evidenced by the fact that (in C. elegans) both RIC-8 and RGS7 influence the pathway in opposed fashion. It remains speculative whether Gαi-GDP/GoLoco or the production of Gαi-GTP from a GoLoco scaffold activates signaling. It stands to reason that Gαi-GTP must dissociate from GoLoco at some point during signaling. If multiple rounds of cycling between Gαi-GDP/GoLoco and liberated Gαi-GTP are required to complete cell division, then Ric-8A-stimulated dissociation of a Gαi/GoLoco complex could be responsible for either terminating or activating the signal. In either context, RGS-facilitated hydrolysis of GTP by Gα ensues. The resultant Gαi-GDP could rebind to GoLoco (and not βγ) to complete one round of the cycle. Rapid cycling of this process may be necessary to regulate the pulling forces on microtubules appropriately during a round of chromosome segregation (supporting information on the PNAS web site, for these proposed models). Regulation of other Gα or Gα/GoLoco-mediated signaling pathways by Ric-8A is also worth considering, given the number of distinct Gα binding partners of mammalian Ric-8A and Ric-8B and the many processes that appear to be regulated by RIC-8 in C. elegans (Tall, 2005).

RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. Mutating a regulator of G protein signaling, RGS-7, results in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Gαo-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, it was found that RIC-8 stimulates GTP binding by Gαo and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Gαo, demonstrating that Gαo passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector (Hess, 2004).

The heterotrimeric G proteins that control C. elegans spindle movements operate via an activation/inactivation cycle different from the signal transduction G protein cycle. Two redundant Gαo-related Gα proteins, GOA-1 and GPA-16, along with the Gβ subunit GPB-1 and the Gγ subunit GPC-2, are required for proper spindle movements in C. elegans embryos. Activation of these G proteins is thought to be receptor independent, since (1) it occurs in the one-cell C. elegans zygote, which is encased by an impermeable egg shell, so that no source of an extracellular ligand is obvious, and (2) a set of nontransmembrane proteins have been identified that appear to activate the G proteins in lieu of transmembrane receptor(s). Removal of any of these activators results in spindle movement defects similar to those in embryos lacking the Gα proteins. The activators include the 97% identical GPR-1 and GPR-2 proteins, which contain a GPR/GoLoco motif that binds GOA-1 in its GDP bound form. The involvement of Gαo and GPR/GoLoco proteins in mitotic spindle control appears to be evolutionarily conserved, since the GPR/GoLoco motif protein PINS acts with a Gαi/o protein to control asymmetric neuroblast divisions in Drosophila, the mammalian GPR/GoLoco protein LGN regulates mitotic spindle organization, and the mammalian Gαo protein is found associated with the mitotic spindle in cultured cells. In C. elegans, GPR-1/2 proteins form a complex with the coiled-coil protein LIN-5, which localizes GPR-1/2 to the cell cortex and mitotic spindle. An additional nonreceptor activator that controls C. elegans centrosome movements is RIC-8, whose mammalian ortholog Ric-8A was recently shown to act in vitro as a guanine nucleotide exchange factor for G proteins including Gαo (Hess, 2004).

Fundamental issues regarding the mechanism of asymmetric spindle positioning remain unresolved: (1) all models propose that asymmetric microtubule forces are generated by greater G protein activity at the posterior than at the anterior pole of the zygote, but it remains unclear how such asymmetric G protein activity is generated; (2) alternative models have been proposed in which either a Gα·GDP/GPR complex or Gα·GTP is the active G protein species that promotes microtubule forces, but it remains to be established which of these species are actually generated and active; (3) the mechanism by which an active G protein controls microtubule forces is unknown (Hess, 2004).

This study shows that an RGS protein, RGS-7, controls asymmetric movements of the mitotic spindle. RGS-7 affects force on the anterior but not the posterior spindle pole, suggesting that it is a source of asymmetric G protein function. In vitro, RIC-8 promotes GTP binding by Gαo, while RGS-7 acts as a Gαo GTPase activator, demonstrating that Gαo is present in its GTP bound form as part of its receptor-independent activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 apparently promotes G protein function. RGS-7 could serve dual roles as both a Gαo inactivator and a Gαo effector so that its net function is to promote microtubule force (Hess, 2004).


Search PubMed for articles about Drosophila Ric-8

Afshar, K., Willard, F. S., Colombo, K., Johnston, C. A., McCudden, C. R., Siderovski, D. P. and Gonczy, P. (2004). RIC-8 is required for GPR-1/2-dependent Gα function during asymmetric division of C. elegans embryos. Cell 119: 219-230. PubMed ID: 15479639

Charras, G. T., Yarrow, J. C., Horton, M. A., Mahadevan, L. and Mitchison, T. J. (2005). Non-equilibration of hydrostatic pressure in blebbing cells. Nature 435: 365-369. PubMed ID: 15902261

David, N. B., Martin, C. A., Segalen, M., Rosenfeld, F., Schweisguth, F. and Bellaiche, Y. (2005). Drosophila Ric-8 regulates G&alpha:i cortical localization to promote G&alpha:i-dependent planar orientation of the mitotic spindle during asymmetric cell division. Nat Cell Biol 7: 1083-1090. PubMed ID: 16228010

Gabay, M., Pinter, M. E., Wright, F. A., Chan, P., Murphy, A. J., Valenzuela, D. M., Yancopoulos, G. D. and Tall, G. G. (2011). Ric-8 proteins are molecular chaperones that direct nascent G protein alpha subunit membrane association. Sci Signal 4: ra79. PubMed ID: 22114146

Hampoelz, B., Hoeller, O., Bowman, S. K., Dunican, D. and Knoblich, J. A. (2005). Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G proteins. Nat. Cell Biol. 7(11): 1099-1105. 16228011

Hess, H. A., et al. (2004). RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans. Cell 119: 209-218. 15479638

Kanesaki, T., Hirose, S., Grosshans, J. and Fuse, N. (2013). Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation. Mech Dev 130: 132-142. PubMed ID: 23085574

Martin, A. C., Kaschube, M. and Wieschaus, E. F. (2009). Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457: 495-499. PubMed ID: 19029882

Miller, K. G., Emerson, M. D., McManus, J. R. and Rand, J. B. (2000). RIC-8 (Synembryn): a novel conserved protein that is required for G(q)alpha signaling in the C. elegans nervous system. Neuron 27: 289-299. PubMed ID: 10985349

Peters, K. A., Rogers, S. L. (2013) Drosophila Ric-8 interacts with the Galpha12/13 subunit, Concertina, during activation of the Folded gastrulation pathway. Mol Biol Cell. PubMed ID: 24006487

Tall, G. G., Krumins, A. M. and Gilman, A. G. (2003). Mammalian Ric-8A (synembryn) is a heterotrimeric G protein guanine nucleotide exchange factor. J. Biol. Chem. 278: 8356-8362. PubMed ID: 12509430

Tall, G. G. and Gilman, A. G. (2005). Resistance to inhibitors of cholinesterase 8A catalyzes release of Gαi-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Gαi-GDP complexes. Proc Natl Acad Sci U S A 102: 16584-16589. PubMed ID: 16275912

Wang, H., Ng, K. H., Qian, H., Siderovski, D. P., Chia, W. and Yu, F. (2005). Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins. Nat. Cell Biol. 7(11): 1091-8. 16228012

date revised: 14 October 2013

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