BIOLOGICAL OVERVIEW

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 (Rogers, 2002). 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-Cta^R277H 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-Cta^R277H 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 Cta^R277H, 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).

GENE STRUCTURE

cDNA clone length - 7380 (transcript variant E)

Bases in 5' UTR - 422

Exons - 8

Bases in 3' UTR - 340

PROTEIN STRUCTURE

Amino Acids - 2559

Structural Domains

DRhoGEF2 was identified in a genetic screen for Rho signaling pathway components in Drosophila. DRhoGEF2 was found to encode a Rho-specific guanine nucleotide exchange factor (Barrett, 1997). The same gene, DRhoGEF2 was similarly identified in an independent screen carried out by Hacker (1998), a screen designed to characterize the maternal effects of zygotic lethal mutations. The gene was independently cloned by Werner (1997). DRhoGEF2 encodes a protein that contains a PDZ domain near the amino terminus, and a cystine-rich butterfly motif in the central region that is present in isoforms of protein kinase C and the mouse Dbl family oncoprotein Lfc. The C-terminal region of DRhoGEF2 contains an extensive region of homology with two separate protein motifs characteristic of the Dbl family of oncoproteins. The first motif, termed the Dbl homology domain promotes the exchange of guanine nucleotides within Rho family GTPases. The second domain, juxtaposed to the Dbl homology domain and C-terminal to it, is a Pleckstrin homology domain (Hacker, 1998).

The Guanine nucleotide exchange factor RhoGEF2 possesses potential lipid (pleckstrin homology) and protein-protein (PDZ, RGS, and DH [Dbl homology]) interaction domains (Rogers, 2004)

date revised: 2 January 2023

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