concertina: Biological Overview | References
Gene name - concertina
Synonyms - Gα12/13
Cytological map position - 2L
Function - signaling
Keywords - G-protein, necessary to trigger acto-myosin contractility during gastrulation, required for RhoGEF2 and consequent MyosinII apical recruitment, Ric-8 interacts with inactive Cta and directs its localization within the cell
Symbol - cta
FlyBase ID: FBgn0000384
Genetic map position - chr2L:22,981,816-22,992,951
Classification - G-alpha: Alpha subunit of G proteins (guanine nucleotide binding)
Cellular location - cytoplasmic
|Recent literature||Xie, S., Mason, F. M. and Martin, A. C. (2016). Loss of Galpha12/13 exacerbates apical area-dependence of actomyosin contractility. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27489340
During development, coordinated cell shape changes alter tissue shape. In the Drosophila ventral furrow and other epithelia, apical constriction of hundreds of epithelial cells folds the tissue. Genes in the Galpha12/13 pathway coordinate collective apical constriction, but the mechanism of coordination is poorly understood. Coupling live-cell imaging with a computational approach to identify contractile events, this study discovered that differences in constriction behavior are biased by initial cell shape. Disrupting Galpha12/13 exacerbates this relationship. Larger apical area is associated with delayed initiation of contractile pulses, lower apical E-cadherin and F-actin levels, and aberrantly mobile Rho-Kinase structures. These results suggest that loss of Galpha12/13 disrupts apical actin cortex organization and pulse initiation in a size-dependent manner. It is proposed that Galpha12/13 robustly organizes the apical cortex despite variation in apical area to ensure the timely initiation of contractile pulses in a tissue with heterogeneity in starting cell shape.
Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. This study reports the function of a common pathway comprising the heterotrimeric G proteins Gα12/13 (Concertina), Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog (Poor gastrulation, Pog & CG31660) was identified as being required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. It is proposed that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators (Kerridge, 2016).
During tissue morphogenesis, cells rearrange their contacts to invaginate, intercalate, delaminate or divide. During Drosophila gastrulation, invagination of the presumptive mesoderm in the ventral region of the embryo and of the posterior midgut requires apical cell constriction, a geometric deformation that occurs in different organisms. Elongation of the ventral-lateral ectoderm requires cell intercalation, a general topological deformation associated with junction remodelling. In the ectoderm, the so-called 'vertical junctions', oriented along the dorsal-ventral axis, shrink, followed by extension of new 'horizontal' junctions along the anterior-posterior axis. Despite differences in the cell deformations associated with intercalation and apical constriction, recent studies revealed that both processes require myosin II (MyoII) contractility. Cell shape changes rely on the pulsatile activity of MyoII in the apical-medial cortex, whereby MyoII undergoes cycles of assembly and disassembly allowing stepwise deformation1. Moreover, each step of deformation is stabilized and thereby retained, contributing to the irreversibility of tissue morphogenesis. In the mesoderm, each phase of apical area constriction mediated by MyoII pulses is followed by a phase of shape stabilization involving persistence of medial MyoII. In the ectoderm, medial-apical MyoII pulses flow anisotropically towards vertical junctions resulting in steps of shrinkage that are stabilized by a planar-polarized pool of junctional MyoII. This ratchet-like behaviour of MyoII is regulated by the Rho1-Rok pathway and requires quantitative control over MyoII activation. Low Rho1/Rok activity fails to form actomyosin networks, intermediate activation establishes MyoII pulsatility and high activation confers stability. The signalling mechanisms that cause stepwise activation of MyoII by Rho1 remain unknown. It is also unclear whether different pathways for Rho1 activation operate in the mesoderm and in the ectoderm as indeed Rho1 can be activated by numerous signalling mechanisms or whether a common pathway might exist (Kerridge, 2016).
Tissue-specific factors can result in polarized shape changes by signalling through cell surface receptors. For instance, in Drosophila ectoderm, pair rule genes encoding transcription factors control planar-polarized enrichment of MyoII through the combinatorial expression of the surface proteins Toll2, Toll6 and Toll8 in stripes. Likewise, in the mesoderm, Twist and Snail induce expression of Fog, a secreted ligand, and a G-protein-coupled receptor (GPCR) Mist (methuselah-like 1), which is reported to transduce Fog. The downstream G protein Gα12/13 (known as Concertina (Cta) in Drosophila) is required for RhoGEF2 and thereby MyoII apical recruitment. As RhoGEF2 is a known GEF for Rho1, the requirement of Gα12/13 for RhoGEF2 apical recruitment suggests that GPCRs and G-protein signalling mediate MyoII activation through the Rho1 pathway. These considerations prompted asking whether G-protein signalling directly controls the different regimes of MyoII dynamics (pulsatility and/or stability) in the mesoderm and planar polarized activation of Rho1 and MyoII in the ectoderm (Kerridge, 2016).
This study reports the function of the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and regulating MyoII dynamics both in the mesoderm and in the ectoderm. Receptor activation, through the GEF activity of the GPCR, converts Gα from an inactive GDP-bound state, in a complex with Gβγ, to an active GTP-bound state. This results in dissociation of Gβγ, enabling binding of both Gα-GTP and Gβγ to their respective effectors for signalling. This study found that Gα12/13 and the Gβ13F/Gγ1 complex constitute distinct signalling modules, which regulate MyoII dynamics medial-apically and/or junctionally in a tissue-dependent manner. A ubiquitously expressed GPCR called Smog, was found to be required for cell shape changes associated with both mesoderm invagination and ectoderm elongation. During these morphogenetic events, Smog functions with other GPCRs, Mist in the mesoderm and an as yet unknown GPCR in the ectoderm, to activate the Rho1-Rok pathway. This results in stepwise activation of Rho1 and MyoII, ensuring irreversible cell shape changes (Kerridge, 2016).
First, this study reports that Gα12/13 and Gβ13F/Gγ1 function as distinct signalling modules that control Rho1 and MyoII in different domains. Gα12/13 activates medial-apical MyoII through its effector RhoGEF2 both in the ectoderm and the mesoderm. In mammals, p115-RhoGEF interacts directly with Gα12 suggesting that this may be a conserved signalling module. In contrast, Gβ13F/Gγ1 activates MyoII both at cell junctions and in the medial-apical domain. This modularity may provide distinct regulatory mechanisms for the activation of MyoII in different subcellular compartments owing to the existence of different molecular effectors of Gα-GTP and Gβγ. Second, stepwise activation of Rho1 by multiple GPCRs and their ligands determines the emergence of a pulsatile regime medial-apically, or stable activation. In the mesoderm, Smog and Mist GPCRs, together with high expression of their ligand Fog, ensure stabilization and rapid (<5 min) accumulation of MyoII ensuring apical constriction. In the ectoderm, low Fog expression and thus lower activation of Gα12/13 and RhoGEF2 is responsible for intermediate medial-apical activation of MyoII and pulsatility. Indeed, Fog, constitutively active Gα12/13QL and RhoGEF2 overexpression all lead to stable accumulation of MyoII instead of pulsation, similar to constitutively active RhoV14 (Kerridge, 2016).
Interestingly, the same receptor Smog controls MyoII activation in different subcellular domains during intercalation and apical constriction begging the question of how activation of Gα12/13 and Gβγ is differentially achieved in the ectoderm and the mesoderm. The polarization of Smog activation is to some extent imparted by the ligand. Fog/Smog regulates medial-apical accumulation of MyoII in the two tissues: Fog induces medial Rho1 and Rok activation in the mesoderm and ectoderm and, when ectopically expressed in the ectoderm, it can increase Rho1 and Rok in the medial cortex. This argues that another mechanism results in junction-specific activation of Smog, Gβ13F/Gγ1, Rho1 and Rok in the ectoderm (Kerridge, 2016).
It is possible that an unknown ectoderm-specific ligand activates Smog specifically at junctions. Junctional localization of the Rho1 pathway by Smog may also be imparted by subcellular processing of Smog signalling, such as localization/activation of downstream effectors of Gα12/13 and Gβγ. The recently identified Toll receptors required for MyoII planar-polarized activation may bias Smog signalling although the molecular mechanisms remain unclear. This could be through localization of RhoGEFs. In the mesoderm, the transmembrane protein T48 localizes RhoGEF2 apically through binding to its PDZ domain, and is required for apical MyoII activation in parallel with Smog, Gα12/13 and Gβγ. Similarly, other GEFs may be required for junctional Rho1 activation by Smog (Kerridge, 2016).
What might be the advantage of having multiple GPCRs? Gastrulation sets the foundation for all other future processes in development and hence requires robustness. GPCRs with similar functions yet subtle differences such as ligand specificity may offer advantages compared with single ligand-receptor pairs. For instance, high cortical tension associated with mesoderm invagination may require multiple GPCRs activating parallel pathways to attain efficiency of the process. Moreover, multiple GPCRs may concede tissue-specific regulation of the common G-protein subcellular pathways. Finally, multiple GPCRs can allow stepwise activation of MyoII. Although activation by one GPCR is sufficient to induce pulsatility, more GPCRs are required to shift the actomyosin networks to more stable regimes (Kerridge, 2016).
The discovery that Smog and heterotrimeric G protein activate Rho1 and MyoII in two different morphogenetic processes provides a potentially general molecular framework for tissue mechanics. It is proposed that different developmental inputs tune a common GPCR/G-protein signalling pathway to direct specific patterns and levels of Rho1 activation. Quantitative control specifies the regime of MyoII activation through Rho1, namely pulsatility or stability of MyoII. Modular control defines the subcellular domains where MyoII accumulates (medial-apical or junctions) depending on molecular effectors. How developmental signals tune GPCR signalling will be important to decipher (Kerridge, 2016).
The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets have been reported. To identify Gα12 determinants that mediate specific protein interactions, a structural and evolutionary comparison was used between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway (Montgomery, 2014).
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. 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. 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).
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. 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. 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 G-protein (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. 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). Nematoda and vertebrate Ric-8 has GEF activity and positively regulates HGP signalingin vivo and in vitro. 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 (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. 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).
In many species, olfactory transduction is triggered by odorant molecules that interact with olfactory receptors coupled to heterotrimeric G-proteins. The role of G-protein-linked transduction in the olfaction of Drosophila is currently under study. This study supplies a thorough description of the expression in the olfactory receptor organs (antennae and maxillary palps) of all known Drosophila melanogaster genes that encode for G-proteins. Using RT-polymerase chain reaction, this study analyzed 6 Gα (Gs, Gi, Gq, Go, Gf, and concertina), 3 Gβ (Gβ5, Gβ13F, and Gβ76C), and 2 Gγ genes (Gγ1 and Gγ30A). All Gα protein-encoding genes showed expression in both olfactory organs, but Gf mRNA was not detected in palps. Moreover, all the Gβ and Gγ genes are expressed in antennae and palps, except for Gβ76C). To gain insight into the hypothesis of different G-protein subunits mediating differential signaling in olfactory receptor neurons (ORNs), immunohistochemical studies were performed to observe the expression of several Gα and Gβ proteins. Gs, Gi, Gq, and Gβ13F subunits were found to display generalized expression in the antennal tissue, including ORNs support cells and glial cells. Finally, complete coexpression was found between Gi and Gq, which are mediators of the cyclic adenosine monophosphate and IP3 transduction cascades, respectively (Boto, 2010).
Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help in the understanding of animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. This study reports the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, these data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction (Fox, 2007).
Regulation of apical constriction during Drosophila VF formation is a paradigm for how signal transduction directs morphogenesis. This study identified Abl as a novel regulator of this process. The results suggest that Abl acts in parallel to the known signaling pathway that promotes apical myosin activation by helping to organize a continuous apical actin network. Furthermore, the results help to explain the greater severity of the RhoGEF2-mutant phenotype relative to other VF mutants by suggesting that RhoGEF2 plays crucial roles in both myosin and actin regulation (Fox, 2007).
Previous work established myosin as a key output of RhoGEF2 signaling during mesoderm internalization. However, ambiguities remained regarding the circuitry of this pathway, since the RhoGEF2 phenotype is much more severe than that of cta or fog mutants, suggesting that a simple linear pathway is unlikely. The data suggest that RhoGEF2 plays dual roles in actin and myosin regulation, and thus its inactivation has more severe effects (Fox, 2007).
From these data, a mechanistic model was developed for the regulation of apical constriction during VF formation. The regulation of actin localization by Abl and RhoGEF2 promotes organization of the apical actin network in constricting cells. It is suggested that Abl regulates actin by actively downregulating cortical Ena in mesoderm, thus leading to polarized actin accumulation, similar to the role that it was shown to play in follicle cells. RhoGEF2 plays a distinct, Cta-independent role in the effective assembly of organized apical actin. While RhoGEF2 and Abl are modulating actin assembly, the mesodermal transcription machinery activates Fog-Cta signaling, apically stabilizing RhoGEF2. This allows the efficient activation of apical myosin. Coupling of these two cues -- an organized apical actin ring at AJs and stable apical myosin activation -- cooperate to ensure highly coordinated actomyosin constriction throughout the sheet of mesodermal cells in a short timeframe (Fox, 2007).
This model helps explain the mutant phenotypes observed in this and previous studies. In abl mutants, Fog-Cta allow RhoGEF2 stabilization and myosin contraction, but the lack of organized mesodermal actin in these mutants, which results from inappropriate Ena regulation, prevents the uniform assembly of actin-based contractile rings. cta mutants lack a stabilizing signal for RhoGEF2, preventing uniform apical myosin activation and uniform constriction. However, some cells can constrict without Fog-Cta, accumulating apical myosin levels comparable to those in wild type. In RhoGEF2 mutants, the combined failure to stabilize/activate myosin and a lack of organized apical actin severely compromises apical constriction. The similarity between RhoGEF2 and cta;abl mutants supports this model, as both processes should be compromised (Fox, 2007).
The model suggests that organized apical actin is an essential prerequisite for cell constriction. Although both Abl and RhoGEF2 regulate actin localization, the data argue that each acts independently. First, actin defects arise during cellularization, when Abl and RhoGEF2 have non-overlapping localizations. Second, whereas Abl clearly acts through Ena, loss of RhoGEF2 disrupts actin without altering Ena localization. Finally, Abl is not a Rho effector in S2 cells (Fox, 2007).
Several unanswered questions remain. With respect to abl, a major question is why do some cells apically constrict while others fail? This phenotype resembles the cellularization defects of abl mutants, in which only some cells fail to reorganize actin into furrows. However, all cells exhibit excess apical Ena and thus form abnormally long, apical microvilli. Perhaps, in some cells, furrow actin assembly drops below a crucial threshold and furrows fail. In the VF, the absence of Abl may have similar effects. VF defects could result from both competition for cellular actin and recruitment of other regulators (e.g. the formin Diaphanous) to ectopic locations, preventing their action in VF formation. This may reduce actin assembly into contractile rings. When constriction initiates, stochastic variations in ring strength may lead some rings to fail, leading to unconstricted cells. Future work is needed to identify the full set of actin regulators involved, and to assess how they work. Interestingly, recent work implicates Abl in epithelial-mesenchymal transitions. Whereas Abl disrupts VF formation, Twist is normally localized in abl mutants, suggesting that this major regulator of such transitions is not an Abl target in flies (Fox, 2007).
The data also reveal the importance of mesodermal Ena downregulation. This may result from increased mesodermal Abl activity, suggested by elevated levels of mesodermal Abl relative to non-mesoderm; however, this remains to be tested. It is also necessary to identify the mechanism by which Abl regulates Ena. In some places, such as the syncytial blastoderm, Abl localizes to sites where Ena is normally absent and, in the absence of Abl, ectopic Ena is found at these sites. This suggests that Abl actively antagonizes Ena localization. At other times and regions, however, such as the leading-edge during dorsal closure, Abl co-localizes with Ena, and thus may hold it in an inactive state. In VFs, Abl localizes to the apical-lateral cortex, and Ena localizes to this site in its absence. Further studies of Abl action will be needed to clarify the mechanisms by which it downregulates Ena (Fox, 2007).
Interestingly, manipulating mammalian Ena/VASP can affect cell contractility Thus, Ena-downregulation may permit proper VF cell contractility. Testing this hypothesis will be important (Fox, 2007).
The results also raise questions regarding RhoGEF2. The model suggests that RhoGEF2 acts via two mechanisms, only one of which is Cta-dependent. Perhaps another upstream cue acts on RhoGEF2 to promote actin organization. Because RhoGEF2 mutants have actin-organization defects in all cells, this regulator may act in all cells prior to gastrulation. However, the data do not rule out a second mesoderm-specific RhoGEF2 regulator acting in parallel to Cta. Although Rho-Kinase is a potential Rho effector with respect to myosin, another effector may regulate actin organization. Attractive candidates are the Formins, which reorganize actin in many processes (Fox, 2007).
The data strengthen the idea that different cytoskeletal regulators direct distinct morphogenetic processes. Both Abl and Fog regulate mesodermal apical constriction but are dispensable for germband cell-cell intercalation. Thus, although both processes require dynamic myosin reorganization, distinct regulators act in each (Fox, 2007).
The picture becomes more complex when considering other roles of Fog, Cta and RhoGEF2. All are required for internalization of the posterior midgut and salivary glands, but these cells internalize in abl mutants. Thus, different types of apical constriction may be regulated differently. It will be interesting to explore the roles of Fog, Cta and RhoGEF2 during dorsal closure, which requires Abl (Fox, 2007).
This work supports mechanistic connections between VF formation and neural tube closure. Both involve actin-based apical constriction to internalize a sheet of cells into a tube. Mice lacking Abl and Arg kinases have neural tube defects, and actin organization in neuroepithelial cells appears altered; interestingly, these cells have ectopic actin that is less polarized than normal, similar to what was observed in abl-mutant VFs. Furthermore, double-mutant analysis suggests that mammalian Ena plays a role in neural tube closure in conjunction with Profilin. Thus, Abl-Ena signaling may represent a conserved mechanism of actin regulation during apical constriction. New mechanistic insights can now be pursued in mammals (Fox, 2007).
Rho also regulates neural tube closure. Mice lacking p190RhoGAP have neural tube defects. Interestingly, p190RhoGAP is an Arg substrate in the brain, suggesting possible direct links between Abl and Rho in apical constriction. The role of Drosophila p190RhoGAP in the VF has yet to be examined, but RhoGAP68F is implicated in VF formation. Future work in both flies and mice will provide further mechanistic insights into conserved mechanisms of apical cell constriction (Fox, 2007).
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 shows 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).
Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs), have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Gα subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation. Using Drosophila S2 cells as a model system, this study shows that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, it was found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. It is proposed that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Gα activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development (Rogers, 2004).
The cellular functions of the microtubule plus-end binding protein EB1 has been characterized in Drosophila S2 cells; this protein plays an important role in regulating microtubule dynamics and in the assembly and dynamics of the mitotic spindle (Elliott, 2005). In order to learn more about EB1's functions, attempts were made to identify EB1 binding partners with affinity purification. Recombinant Drosophila GST-EB1 bound to glutathione Sepharose beads was used as an affinity chromatography matrix to bind interacting partners from S2 cell extracts. Bound proteins were eluted from the beads and separated by SDS-PAGE, and excised bands were subjected to tryptic digestion and mass spectrometry fingerprinting (Rogers, 2004).
Twenty 'EB1-specific' proteins were identified over the course of five independent pull-down experiments. However, of these, only six candidates were identified in all five trials: CLIP190, the Drosophila ortholog of vertebrate CLIP-170, which localizes to the plus ends of microtubules, Orbit/MAST, a microtubule plus-end-associated protein that interacts with CLIP-170, nonmuscle myosin II heavy chain, the minus-end-directed kinesin, Ncd, Shortstop, a member of the spectraplakin family of actin/microtubule cross-linking proteins, and DRhoGEF2. This paper focuses on DRhoGEF2 for further study (Rogers, 2004).
The association of DRhoGEF2 with EB1 in vitro raised the possibility that this protein may localize to the tips of microtubules. To test this idea, polyclonal antibodies were generated against the C-terminal 720 amino acid residues of DRhoGEF2. These antibodies recognized a ~280 kDa polypeptide on immunoblots of S2 cell extracts; this polypeptide was eliminated after DRhoGEF2 RNAi treatment, indicating that the antibodies were reacting with the correct polypeptide (Rogers, 2004).
By immunofluorescence, anti-DRhoGEF2 antibodies recognized punctate structures distributed throughout the cell. Superimposed upon this punctate pattern, however, were short (~1 μm) linear tracks that colocalized with the tips of microtubules. Moreover, immunofluorescent staining of DRhoGEF2 in S2 cells expressing low amounts of EB1-EGFP indicated that these two proteins colocalize exactly at microtubule tips. In the perinuclear region of many cells, DRhoGEF2 antibodies also stained larger spots that costained with γ-tubulin, a centrosome marker. Depletion of DRhoGEF2 with RNAi eliminated antibody staining of all of these structures in S2 cells. Thus, these immunofluorescence experiments reveal that DRhoGEF2 exists in three pools within S2 cells: punctate throughout the cell, at microtubule tips, and on centrosomes (Rogers, 2004).
DRhoGEF2 tagged with green fluorescent protein (GFP) was tested to examine its dynamic behavior through time-lapse imaging with a spinning-disk microscope. As predicted from immunofluorescence data, 'comet-like' structures of DRhoGEF2-GFP moved from the cell center toward the periphery in a manner that was very similar to that observed for EB1-GFP. In many cells, an intense spot of DRhoGEF2-GFP was observed near the perinuclear region. This spot likely corresponded to the centrosome staining because the tips of nucleated microtubules emanated from this point in a radial pattern. Microtubule dynamics are essential for this movement because it could be eliminated with either 10 μM colchicine or 10 μM taxol. Thus, it is concluded that DRhoGEF2 associates with the tips of growing microtubules and exhibits plus-end tracking that is qualitatively similar to that described for EB1 (Rogers, 2004).
Because DRhoGEF2 was isolated based upon its association, direct or indirect, with EB1, EB1 was deleted from cells with RNAi and whether the association of DRhoGEF2 with microtubule tips was examined. In cultures treated with control dsRNA, scoring of fixed cells stained for DRhoGEF2 and microtubules revealed that 94% of the cells (n = 300) had DRhoGEF2 associated with the microtubule tip. In contrast, in S2 cells treated for 7 days with EB1 dsRNA, only 5% of the cells retained DRhoGEF2 at the plus ends. These results demonstrate that targeting of DRhoGEF2 to growing microtubule plus ends is an EB1-dependent process (Rogers, 2004).
To further understand DRhoGEF2 functions, how overexpression and depletion of the protein affects the morphology of S2 cells was examined. When S2 cells are plated on concanavalin A, they adopt a 'fried-egg' appearance with a dome-like central domain defined by the nucleus and perinuclear organelle-rich region and an extended, symmetrical lamella. In contrast, overexpressing DRhoGEF2 caused many cells to adopt a smaller, contracted footprint on the substrate and to become significantly taller than control cells. These overexpressing cells formed a skirt of abnormally large membrane ruffles that tapered to the base of a raised, organelle-rich compartment, and the overall morphology resembled a 'bonnet' shape. This result suggests that DRhoGEF2 can induce contractility, in agreement with the genetic phenotype of DRhoGEF2 mutations (Rogers, 2004).
Several genetic studies implicate DRhoGEF2 as a positive regulator of Rho1. To test whether Rho activation is involved in generating the unusual phenotype associated with DRhoGEF2 overexpression, cells were transfected with constitutively active Rho1V14 and transfected cells were identified with an antibody raised against Drosophila Rho1. As predicted, most of the Rho1V14-expressing cells duplicated the morphology produced by DRhoGEF2 overexpression. In order to next test if inhibition of Rho1 prevented DRhoGEF2-induced shape change, DRhoGEF2-EGFP was transfected into cells that had been treated with Rho RNAi. Depletion of Rho1 by RNAi produced large multinucleate cells that did not contract in response to DRhoGEF2 overexpression. In contrast, RNAi inhibition of the other six Rho family members did not block DRhoGEF2-induced contraction (Rogers, 2004).
Active Rho is known to stimulate nonmuscle myosin II, and a genetic interaction has been demonsrated between DRhoGEF2 and myosin II during Drosophila morphogenesis. One well-characterized mechanism by which Rho1 activates myosin II is Rho kinase (DROK in Drosophila) stimulation, which activates the motor by phosphorylating the myosin light chain and by inactivating myosin light chain phosphatase. In order to determine if DROK is indeed downstream of DRhoGEF2, DROK was depleted with RNAi or kinase activity was inhibited with Y-27632, a pharmacological inhibitor, and then cell morphology was examined after DRhoGEF2 overexpression. Both treatments significantly reduced the numbers of cells exhibiting the contracted morphology. From these data, it is concluded that DRhoGEF2 changes S2 cell morphology through Rho1 and its downstream effector, DROK (Rogers, 2004).
To confirm that myosin II is a downstream effector in the DRhoGEF2 pathway in this system, the behavior of GFP-tagged myosin II in control S2 cells on concanavalin A was compared with that of S2 cells overexpressing DRhoGEF2. To perform this analysis, a stable cell line was generated expressing the myosin II regulatory light chain (RLC), known by Drosophila nomenclature as Spaghetti squash, under the control of the gene's endogenous promoter. Ectopic expression of RLC-GFP did not produce observable defects in actin organization or behavior; its distribution exactly coincided with the myosin II distribution determined by immunofluorescence staining of the same cells. RLC-GFP typically incorporated into punctae in the cell periphery and into higher-order structures in the central region of the cells. Time-lapse spinning-disk confocal microscopy revealed that punctae of RLC-GFP formed in the distal cell periphery and then translocated centripetally at a constant rate of 4.0 ± 0.3 μm/min toward the cell center. Such behavior of RLC-GFP is qualitatively very similar to the behavior of fluorescently labeled myosin II in cultured mammalian cells (Rogers, 2004).
Upon overexpression of DRhoGEF2, punctae of RLC-GFP were rarely observed. Instead, the majority of RLC-GFP signal was present in circular 'purse string' structures surrounding the organelle-dense region at the center of the cell. Time-lapse observation revealed that peripheral formation of RLC-GFP punctae and retrograde flow were infrequent in DRhoGEF2-overexpressing cells and that these RLC-GFP-containing purse strings were stable over a span of hours. The location and concentration of the myosin II suggests that actomyosin contraction is responsible for producing the bonnet-shaped appearance of these cells. From these observations, it is propose that DRhoGEF2 regulates myosin II dynamics and contractility in S2 cells (Rogers, 2004).
Genetic analyses of epithelial-sheet invagination in the early Drosophila embryo suggest that DRhoGEF2 may act downstream of the heterotrimeric Gα protein Concertina (Cta). To examine directly whether Concertina can activate DRhoGEF2, cells were transfected either with Myc-tagged wild-type Cta or Myc-tagged Cta bearing a constitutively activating point mutation (R277H) that inactivates GTPase activity, and the morphology of the transfected cells was examined. Cells expressing Myc-Cta were morphologically indistinguishable from untransfected cells, and only 3% of cells displayed a mildly contracted phenotype. In contrast, the majority of cells expressing Myc-CtaR277H exhibited the contracted morphology and myosin II purse string reminiscent of DRhoGEF2 overexpression. Similar results were obtained with three other constitutively activated Concertina constructs. However, the shape change was prevented in 88% of these cells (if they were pretreated for 7 days with dsRNA so that DRhoGEF2 was depleted. These results suggest that Concertina can act upstream of DRhoGEF2 to regulate S2 cell morphology (Rogers, 2004).
Next it was determined whether activation of DRhoGEF2 through Concertina affected its association with the microtubule cytoskeleton. Cells expressing Myc-Cta or Myc-CtaR277H were fixed and double stained for the Myc epitope tag and for DRhoGEF2. Overexpression of wild-type Concertina did not affect DRhoGEF2 association with microtubule plus ends or with the centrosome. However, constitutively activated Concertina resulted in DRhoGEF2 dissociation from microtubule tips; only 10% of the cells showed any colocalization of DRhoGEF2 with microtubule plus ends. Instead, DRhoGEF2 exhibited a diffuse staining pattern throughout the cell; this pattern likely represents association with the plasma membrane. Targeting of EB1 to the plus ends was not perturbed by CtaR277H, suggesting that Concertina signaling regulates the interactions between DRhoGEF2 and factors at microtubule tips (Rogers, 2004).
In an attempt to identify novel cellular factors that interact with EB1, this study unexpectedly discovered that DRhoGEF2, a key regulator of morphogenesis in Drosophila, associates with the tips of growing microtubules. This interesting type of intracellular motility required EB1 in a manner analogous to the EB1-dependent microtubule plus-end tracking of the vertebrate adenomatous polyposis coli (APC) tumor suppressor protein. This finding represents the first example of a regulator of the actin cytoskeleton that tracks along microtubule plus ends. Moreover, the dissociation of DRhoGEF2 from microtubule tips upon activation of Concertina also represents the first example of a regulated association of a protein with the microtubule plus end (Rogers, 2004).
The dissection of the DRhoGEF2 pathway at a cellular level is also consistent with genetic studies of Drosophila morphogenesis. These studies implicate Concertina in myosin II contractility through the Rho/Rho kinase pathway. The Rho1/Rho kinase/myosin II system is a widely employed module for bundling and contraction of actin filaments; it is involved in the formation of adhesion structures and stress fibers, retraction of the trailing edge in migrating cells, muscular contraction, morphogenetic cell shape changes, and construction of the cleavage furrow at the end of mitosis. Context- and location-specific activation of the Rho1/Rho kinase/myosin II module is likely to reside in the activation of specific RhoGEFs, over 20 of which reside within the Drosophila genome. This hypothesis is consistent with observations that inhibition of Rho1 or its downstream effectors causes a dramatic cytokinesis failure in S2 cells and embryos, but inhibition of DRhoGEF2 does not. Instead, DRhoGEF2 has been implicated in morphogenetic cell shape changes only in epithelial cells. Thus, it is believed that the signaling pathway that was engineered in S2 cells recapitulates events involved in the cellular shape changes preceding gastrulation in Drosophila blastula epithelia cells (Rogers, 2004).
However, in Drosophila development, this signaling pathway must be activated in a polarized manner by an unidentified receptor and its ligand so that myosin contraction occurs locally at the apical surface. In such a setting of asymmetric signaling, it is proposed that the intracellular transport of DRhoGEF2 on microtubule plus ends may play an important role in localized activation of the pathway. It is speculated that inactive DRhoGEF2 interacts with the tips of microtubules, whereupon these growing microtubules deliver 'packets' of DRhoGEF2 in the vicinity of the actin cortex. If DRhoGEF2 does not receive an activating input, it diffuses back into the cytoplasm to begin the transport cycle anew. However, if DRhoGEF2 is delivered to a subcortical region containing a high concentration of receptor-activated Concertina, DRhoGEF2 can locally activate the Rho1/Rho kinase/myosin II module. Moreover, because DRhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains, microtubule-delivered DRhoGEF2 may be retained at the cortex if activated by Concertina. Although a microtubule-assisted activation of the Rho pathway during cellular shape changes during morphogenesis (such as in epithelial cells) is proposed, similar models that account for small GTPase activation during cellular motility have been suggested as well (Rogers, 2004).
In principle, interactions between DRhoGEF2 and its cortical activators could occur through diffusion within the cytoplasm. The evolution of this elaborate microtubule polymerization-based transport mechanism undoubtedly reflects some important property of the signaling pathway that is not yet understood. Perhaps the amount of DRhoGEF2 carried on the tip of a microtubule represents some quanta -- a critical concentration of the protein required either to respond to upstream inputs or to locally activate Rho1 in a cortical subdomain. This idea is supported by the observation that, at very low expression levels and without Concertina signaling, DRhoGEF2-GFP efficiently tracks microtubule ends without activating cellular contraction. Alternatively, it is possible that interaction with EB1 or some other protein at the microtubule plus end primes DRhoGEF2 for activation at the cortex. A third possibility is that microtubule dynamic instability is not uniform within a polarized cell but is locally modulated in order to deliver DRhoGEF2 to the cortex in a nonrandom manner. Testing between these hypotheses will require identification of the signaling components (i.e., the ligand-receptor pair) that act upstream of Concertina, reconstitution of the complete pathway in S2 cells, and the selective disruption of the association of DRhoGEF2 with microtubule tips in Drosophila embryos (Rogers, 2004).
A single Rho GTPase family member is capable of initiating several different processes, including cell cycle regulation, cytokinesis, cell migration, and transcriptional regulation. It is not clear, however, how the Rho protein selects which of these processes to initiate. Guanine nucleotide exchange factors (GEFs), proteins that activate Rho GTPases, could be important in making this selection. This study shows that in vivo, DRhoGEF2, a GEF that is ubiquitously expressed and specific for Rho1, is reiteratively required for epithelial folding and invagination, but not for other processes regulated by Rho. The limitation of DRhoGEF2 function supports the hypothesis that the GEF selects the outcome of Rho activation. DRhoGEF2 exerts its effects in gastrulation through the regulation of Myosin II to orchestrate coordinated apical cell constriction. Apical myosin localization is also regulated by Concertina (Cta), a Gα12/13 family member that is thought to activate DRhoGEF2 and is itself activated by a putative ligand, Folded gastrulation (Fog). Fog and Cta also play a role in the morphogenetic events requiring DRhoGEF2, suggesting the existence of a conserved signaling pathway in which Fog, Cta, and DRhoGEF2 locally activate Myosin for epithelial invagination and folding (Nikolaidou, 2004).
During Drosophila gastrulation, mesodermal precursors are brought into the interior of the embryo by formation of the ventral furrow. The first steps of ventral furrow formation involve a flattening of the apical surface of the presumptive mesodermal cells and a constriction of their apical diameters. In embryos mutant for folded gastrulation (fog), these cell shape changes occur but the timing and synchrony of the constrictions are abnormal. A similar phenotype is seen in a maternal effect mutant, concertina (cta). fog encodes a putative secreted protein whereas cta encodes an α-subunit of a heterotrimeric G protein. It has been proposed that localized expression of the fog signaling protein induces apical constriction by interacting with a receptor whose downstream cellular effects are mediated by the cta Gαprotein (Morize, 1998).
In order to test this model, fog was ectopically expressed at the blastoderm stage using an inducible promoter. In addition, the constitutive activation of Cta protein was examined by blocking GTP hydrolysis using both in vitro synthesized mutant alleles and cholera toxin treatment. Activation of the Fog/Cta pathway by any of these procedures results in ectopic cell shape changes in the gastrula. Uniform fog expression rescues the gastrulation defects of fog null embryos but not cta mutant embryos, arguing that cta functions downstream of fog expression. The normal location of the ventral furrow in embryos with uniformly expressed fog suggests the existence of a fog-independent pathway determining mesoderm-specific cell behaviors and invagination. Epistasis experiments indicate that this pathway requires snail but not twist expression (Morize, 1998).
The folded gastrulation (fog) gene is required during Drosophila gastrulation for two morphogenetic movements, formation of the ventral furrow and invagination of the posterior midgut primordium. fog coordinates cell shape changes during these invaginations by inducing apical constriction of cells in spatially and temporally defined manners. fog is expressed in the invagination primordia in a pattern that precisely precedes the pattern of constrictions. Overexpression of fog in the dorsoanterior region of the embryo induces ectopic constrictions, indicating localization of fog transcripts may define domains of cell shape changes. fog encodes a novel protein with a putative signal sequence but no potential transmembrane domains. It is suggested that fog functions as a secreted signal that activates the G protein α subunit encoded by concertina in neighboring cells. These analyses indicate that cell-cell communication ensures the rapid, orderly progression of constriction initiations from the middle of invagination primordia out toward the margins (Costa, 1994).
The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, this study identified two phases in this process: first, a slow stochastic phase in which some individual cells begin to constrict and, second, a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations (Sweeton, 1991).
The Drosophila concertina(cta) gene encodes a G α-like protein. The CTA protein is only 35-44% identical to any of the previously characterized Drosophila G α proteins. CTA is more similar to mouse proteins G α 12 and G α13. CTA is necessary to coordinate cell shape changes during gastrulation. Mutant embryos fail to undergo proper gastrulation. The central region of the ventral zone fails to undergo the rapid transition to a groove. Some cells constrict their apices and move their nuclei basally, but this process seems poorly coordinated. Patches of cells that have failed to undergo apical constriction are left amid cells that have changed shape appropriately. An abnormal furrow is ultimately formed in many mutant embryos, but it lacks the depth and length of the furrow in wild type. At the posterior end, mutants fail to constrict enough cells to form the shallow cup in which the pole cells sit. Formation of the cephalic fold remains normal. In spite of the gastrulation defect, mutants have normal polarity. Cuticles have normal denticle belts and other cuticular features. The cuticle has holes at the anterior and posterior ends, presumably because of improper morphogenetic movements. While removal of maternal cta produces these developmental defects, zygotic elimination has no effect on viability. In early embryos, highly abundant amounts of messenger RNA are found throughout the cytoplasm. Upon blastoderm formation the level of messenger RNA decreases and it remains low throughout gastrulation until the extended germband stage. Then a higher level of CTA mRNA accumulates in the mesoderm, presumably from zygotic expression. In the ovary, mRNA first appears in the germarium. Throughout oogenesis, the accumulation of messenger is restricted to the germline and is observed in both the nurse cells and the oocyte. It is thought that cta acts through the cytoskeleton, perhaps through modification of actin polymerization (Parks, 1991)
Search PubMed for articles about Drosophila Concertina
Boto, T., Gomez-Diaz, C. and Alcorta, E. (2010). Expression analysis of the 3 G-protein subunits, Gα, Gβ, and Gγ, in the olfactory receptor organs of adult Drosophila melanogaster. Chem Senses 35: 183-193. PubMed ID: 20047983
Costa, M., Wilson, E. T. and Wieschaus, E. (1994). A putative cell signal encoded by the folded gastrulation gene coordinates cell shape changes during Drosophila gastrulation. Cell 76: 1075-1089. PubMed ID: 8137424
Elliott, S. L., Cullen, C. F., Wrobel, N., Kernan, M. J. and Ohkura, H. (2005). EB1 is essential during Drosophila development and plays a crucial role in the integrity of chordotonal mechanosensory organs. Mol Biol Cell 16: 891-901. PubMed ID: 15591130
Fox, D. T. and Peifer, M. (2007). Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila. Development 134(3): 567-78. Medline abstract: 17202187
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: 1099-1105. PubMed ID: 16228011
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
Kerridge, S., Munjal, A., Philippe, J. M., Jha, A., de Las Bayonas, A. G., Saurin, A. J. and Lecuit, T. (2016). Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis. Nat Cell Biol 18(3): 261-70. PubMed ID: 26780298
Montgomery, E. R., Temple, B. R., Peters, K. A., Tolbert, C. E., Booker, B. K., Martin, J. W., Hamilton, T. P., Tagliatela, A. C., Smolski, W. C., Rogers, S. L., Jones, A. M. and Meigs, T. E. (2014). Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation. Mol Pharmacol 85: 586-597. PubMed ID: 24435554
Morize, P., Christiansen, A. E., Costa, M., Parks, S. and Wieschaus, E. (1998). Hyperactivation of the folded gastrulation pathway induces specific cell shape changes. Development 125: 589-597. PubMed ID: 9435280
Nikolaidou, K. K. and Barrett, K. (2004). A Rho GTPase signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation. Curr Biol 14: 1822-1826. PubMed ID: 15498489
Parks, S., and Wieschaus. E. (1991). The Drosophila gastrulation gene concertina encodes a Gα-like protein. Cell 64: 447-458. PubMed Citation: 1899050
Peters, K. A. and Rogers, S. L. (2013). Drosophila Ric-8 interacts with the Galpha12/13 subunit, Concertina, during activation of the Folded gastrulation pathway. Mol Biol Cell 24: 3460-3471. PubMed ID: 24006487
Rogers, S. L., Wiedemann, U., Hacker, U., Turck, C. and Vale, R. D. (2004). Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol. 14(20): 1827-33. Medline abstract: 15498490
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date revised: 2 April 2016
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