Involvement of guanine nucleotide exchange factor xLARG in epiboly of cells of the animal pole of Xenopus laevis embryos

The development of multicellular organisms is a complicated coordinated process of the movement of groups of embryonic cells, which is controlled by many regulatory systems. At present little is known about the regulation of the earliest manifestations of the movement in the embryogenesis: epiboly and radial intercalation. The coordinators of these processes may be small GTPases of the Rho family and their activators, the factors of exchange of guanylic nucleotides. It has been shown in this work that the overexpression of the factor of exchange of guanylic nucleotides xLARG in Xenopus laevis embryos leads to an increase in the amount of the active form of xLARG. In addition, an increase in the expression of xLARG disturbs the process of radial intercalation. The data obtained suggest that xLARG is involved in maintaining the xLARG activation level necessary for the occurrence of epiboly.     

Flotillins control zebrafish epiboly through their role in cadherin‐mediated cell–cell adhesion

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E‐cadherin‐mediated cell–cell junctions. These results provide the first in vivo evidence that Flotillins regulate E‐cadherin‐mediated cell–cell junctions to allow epiboly progression.     

Dynamic regulation of LARG in blastopore closure and archenteron formation during Xenopus laevis gastrulation

Rho GTPases have important roles in regulating cell migration and are activated by Rho-specific guanine nucleotide exchange factors (RhoGEFs). However, the role of leukemia-associated RhoGEF (LARG), responding to G12/13 family, has not been studied in vertebrate development. Here, the in vivo biochemical function of LARG was examined during early embryonic development in African frog Xenopus laevis. Gain-of-function study was performed by injecting the RNA of full-length xLARG to 2 cell-stage embryos. The ectopic expression of this protein resulted in the defect of blastopore closure during early embryogenesis. Expression of the dominant-negative form caused the defect in cell movement and following archenteron formation during late gastrulation, which is represented by the blister formation in the ventral side of the embryos. The phenotype was rescued by co-expressing the mutant with Rho or wild type xLARG, confirming the specificity of the dominant-negative activity of xLARG mutant. In this study, I showed for the first time that the spatiotemporal expression of xLARG is very dynamic and specifically regulated in early Xenopus embryonic development and xLARG may mediate Gα₁₃ signal to activate Rho to exert its function in gastrulation movement and archenteron formation. My results implicate that the dynamic regulation of maternal and zygotic xLARG expression and its biochemical activity is necessary for proper gastrulation.     

Geometry of movement of the outer surface of the embryo during <Emphasis Type="Italic">Xenopus</Emphasis> gastrulation

The surface of Xenopus laevis embryos was marked with carbon particles, after which the location of mark groups was recorded by time-lapse video imaging and subsequent image analysis until their disappearance in the depth of gastric invagination. Measuring the distances between individually identifiable marks whose size is smaller than the size of a single cell makes it possible to quantitatively analyze the geometry of collective cell movement without any external coordinate system. During the dorsal blastopore lip (DBL) formation, the invagination of surface cells fundamentally differs from the preceding and subsequent lateromedial (LM) intercalation, being associated with a decrease in the meridional distance and an increase in the latitudinal distance between the marked surface sites. The sites that began to move towards the DBL later overtake the areas that started movement earlier, which leads to a “plug” in the movement of cells. Pushing the “plug” into the inner layers by changing the DBL shape becomes the rate-limiting stage of gastrulation; then, the directed cell movement is replaced by epiboly based on LM intercalation when the marks remaining on the outer surface of the marginal zone diverge along its meridians without directed migration towards the blastopore. As a result, directional movement of cells and LM intercalation become successive phases of collective cell movement, and the entire morphogenesis of DBL is the direct consequence of epiboly deceleration occurring upon gastric invagination.     

Mutations in half baked/E-cadherin block cell behaviors that are necessary for teleost epiboly

Epiboly, the spreading of the blastoderm over the large yolk cell, is the first morphogenetic movement of the teleost embryo. Examining this movement as a paradigm of vertebrate morphogenesis, we have focused on the epiboly arrest mutant half baked (hab), which segregates as a recessive lethal, including alleles expressing zygotic-maternal dominant (ZMD) effects. Here we show that hab is a mutation in the zebrafish homolog of the adhesion protein E-cadherin. Whereas exclusively recessive alleles of hab produce truncated proteins, dominant alleles all contain transversions in highly conserved amino acids of the extracellular domains, suggesting these alleles produce dominant-negative effects. Antisense oligonucleotides that create specific splicing defects in the hab mRNA phenocopy the recessive phenotypes and, surprisingly, some of the ZMD phenotypes as well. In situ analyses show that during late epiboly hab is expressed in a radial gradient in the non axial epiblast, from high concentrations in the exterior layer of the epiblast to low concentrations in the interior layer of the epiblast. During epiboly, using an asymmetric variant of radial intercalation, epiblast cells from the interior layer sequentially move into the exterior layer and become restricted to that layer; there they participate in subtle cell shape changes that further expand the blastoderm. In hab mutants, when cells intercalate into the exterior layer, they tend to neither change cell shape nor become restricted, and many of these cells 'de-intercalate' and move back into the interior layer. Cell transplantation showed all these defects to be cell-autonomous. Hence, as for the expansion of the mammalian trophoblast at a similar developmental stage, hab/E-cadherin is necessary for the cell rearrangements that spread the teleost blastoderm over the yolk.     

Effect of diabetes on guanine nucleotide exchange factor activity in skeletal muscle and heart

The present study examined the effects of diabetes and insulin treatment of diabetic rats on the activity of the protein synthesis initiation factor, the guanine nucleotide exchange factor. In extracts from gastrocnemius and psoas muscles from two-day diabetic rats, guanine nucleotide exchange factor activity was reduced to 80% and 67% of control values, respectively. Insulin treatment (2 h) restored guanine nucleotide exchange factor activity to control values in both muscles. In contrast, guanine nucleotide exchange factor activity was unchanged in extracts from either soleus muscle or heart from diabetic rats compared to controls. Also, insulin treatment did not increase guanine nucleotide exchange factor activity in extracts from soleus and heart. The results suggest that the diabetes-induced impairment in peptide-chain initiation in fast-twitch skeletal muscle (i.e. gastrocnemius and psoas) is related to an inhibition of guanine nucleotide exchange factor activity and that slow-twitch muscle is spared from the effect on initiation due to the preservation of guanine nucleotide exchange factor activity.     

The large N-terminal domain of Cdc25 protein of the yeast Saccharomyces cerevisiae is required for glucose-induced Ras2 activation

The Saccharomyces cerevisiae CDC25 gene encodes a guanine nucleotide exchange factor for Ras proteins whose catalytic domain is highly homologous to Ras-guanine nucleotide exchange factors from higher eukaryotes. In this study, glucose-induced Ras activation and cAMP response were investigated in mutants lacking the N-terminal domain of Cdc25 or where the entire CDC25 coding sequence was substituted by an expression cassette for a mammalian guanine nucleotide exchange factor catalytic domain. Our results suggest that an unregulated, low Ras guanine nucleotide exchange factor activity allows a normal glucose-induced cAMP signal that appears to be mediated mainly by the Gpr1/Gpa2 system, but it was not enough to sustain the glucose-induced increase of Ras2-GTP normally observed in a wild-type strain.     

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.     

Multicellular computer simulation of morphogenesis: blastocoel roof thinning and matrix assembly in Xenopus laevis

In the blastocoel roof (BCR) of the Xenopus laevis embryo, epibolic movements are driven by the radial intercalation of deep cell layers and the coordinate spreading of the overlying superficial cell layer. Thinning of the lateral margins of the BCR by radial intercalation requires fibronectin (FN), which is produced and assembled into fibrils by the inner deep cell layer of the BCR. A cellular automata (CA) computer model was developed to analyze the spatial and temporal movements of BCR cells during epiboly. Simulation parameters were defined based on published data and independent results detailing initial tissue geometry, cell numbers, cell intercalation rates, and migration rates. Hypotheses regarding differential cell adhesion and FN assembly were also considered in setting system parameters. A 2-dimensional model simulation was developed that predicts BCR thinning time of 4.8 h, which closely approximates the time required for the completion of gastrulation in vivo. Additionally, the model predicts a temporal increase in FN matrix assembly that parallels fibrillogenesis in the embryo. The model is capable of independent predictions of cell rearrangements during epiboly, and here was used to predict successfully the lateral dispersion of a patch of cells implanted in the BCR, and increased assembly of FN matrix following inhibition of radial intercalation by N-cadherin over-expression.     

The N-terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor

The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal guanine nucleotide exchange factor (GEF) domain. In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.     

Rap1 signaling in endothelial barrier control

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell–cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.     

GEF-mediated GDP/GTP exchange by monomeric GTPases: A regulatory role for Mg2+?

GTPases share highly conserved guanine nucleotide-binding domains and fulfill diverse functions through a common molecular switch. An inactive GDP-bound protein is turned on by a guanine nucleotide exchange factor (GEF) that catalyzes exchange of GTP for GDP, but unfortunately little is known about the mechanism of GEF action. A common mechanism for GDP/GTP exchange can be envisioned wherein GEFs activate monomeric GTPases through transient disruption of Mg²⁺ coordination in the nucleotide-binding pocket while stabilizing a nucleotide-free (and cation-free) conformation. After guanine nucleotide exchange, Mg²⁺ coordination is restored to complete the conformational switch to the active GTP-bound state. Evidence in the literature highlighting an important regulatory role for Mg²⁺ in the mechanism of GEF-mediated GDP/GTP exchange by monomeric GTPases is summarized. BioEssays 20 :516–521, 1998. © 1998 John Wiley & Sons, Inc.     

Lck regulates Vav activation of members of the Rho family of GTPases.

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.     

Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin

Fibronectin (FN) is reported to be important for early morphogenetic movements in a variety of vertebrate embryos, but the cellular basis for this requirement is unclear. We have used confocal and digital time-lapse microscopy to analyze cell behaviors in Xenopus gastrulae injected with monoclonal antibodies directed against the central cell-binding domain of fibronectin. Among the defects observed is a disruption of fibronectin matrix assembly, resulting in a failure of radial intercalation movements, which are required for blastocoel roof thinning and epiboly. We identified two phases of FN-dependent cellular rearrangements in the blastocoel roof. The first involves maintenance of early roof thinning in the animal cap, and the second is required for the initiation of radial intercalation movements in the marginal zone. A novel explant system was used to establish that radial intercalation in the blastocoel roof requires integrin-dependent contact of deep cells with fibronectin. Deep cell adhesion to fibronectin is sufficient to initiate intercalation behavior in cell layers some distance from the substrate. Expression of a dominant-negative beta1 integrin construct in embryos results in localized depletion of the fibronectin matrix and thickening of the blastocoel roof. Lack of fibronectin fibrils in vivo is correlated with blastocoel roof thickening and a loss of deep cell polarity. The integrin-dependent binding of deep cells to fibronectin is sufficient to drive membrane localization of Dishevelled-GFP, suggesting that a convergence of integrin and Wnt signaling pathways acts to regulate radial intercalation in Xenopus embryos.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

The solution structure and dynamics of the DH‐PH module of PDZRhoGEF in isolation and in complex with nucleotide‐free RhoA

The DH‐PH domain tandems of Dbl‐homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho‐family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH‐PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small‐angle X‐ray scattering (SAXS), and hydrogen‐deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH‐PH tandem of RhoA‐specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH‐PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide‐free RhoA exhibits elevated dynamics when in complex with DH‐PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.     

Kinetic analysis of M2 muscarinic receptor activation of Gi in Sf9 insect cell membranes.

A steady-state kinetic mechanism describing the interaction of M(2) muscarinic acetylcholine receptors and the guanine nucleotide-binding protein G(i)alpha(2)beta(1)gamma(3) are presented. Data are consistent with two parallel pathways of agonist-promoted GTPase activity arising from receptor coupled to a single or multiple guanine nucleotide-binding proteins. An aspartate 103 to asparagine receptor mutation resulted in a receptor lacking the ability to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate) or guanosine triphosphate hydrolysis by the G protein. An aspartate 69 to asparagine receptor mutant was able to catalyze agonist-specific guanine nucleotide exchange and GTPase activity. A threonine 187 to alanine receptor mutation resulted in a receptor that catalyzed guanine nucleotide exchange comparable with wild-type receptors but had reduced ability to stimulate GTP hydrolysis. A tyrosine 403 to phenylalanine receptor mutation resulted in an increase in agonist-promoted GTPAse activity compared with wild type. The observation that the threonine 187 and tyrosine 403 mutants promote guanine nucleotide exchange similarly to wild type but alter GTPase activity compared with wild type suggests that the effects of the mutations arise downstream from guanine nucleotide exchange and may result from changes in receptor-G protein dissociation.     

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

Localization of the Translational Guanine Nucleotide Exchange Factor eIF2B: A Common Theme for GEFs?

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stressdependent controls target this eIF2B-recycling step allowing a re-programming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.