Protein Interactions

A Drosophila gene (properly termed Rho-type guanine exchange factor - FlyBase ID: FBgn0015803) has been cloned that has substantial sequence homology to a distinct class of proto-oncogenes: these include DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately downstream of the Rho/RacGEF. Rho/RacGEFs are known to catalyze the dissociation of GDP from the Rho/Rac subfamily of Ras-like GTPases, thus activating the target Rho/Rac. Members of the Rho/Rac subfamily regulate organization of the actin cytoskeleton, which controls the morphology, adhesion and motility of cells. Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest, message is most abundant in furrows and folds of the embryo where cell shapes are changing and the cytoskeleton is likely to be undergoing reorganization (Werner, 1997).

Mammalian Rho-kinase/ROK alpha, one of the targets of Rho, has been shown to bind to Rho in GTP-bound form and to phosphorylate the myosin light chain (MLC) and the myosin-binding subunit (MBS) of myosin phosphatase, resulting in the activation of myosin. Thus, Rho-kinase/ROK alpha has been suggested to play essential roles in the formation of stress fibers and focal adhesions. A two-hybrid analysis demonstrates that Drosophila Rho-associated kinase interacts with the GTP-bound form of the Drosophila Rho1 at the conserved Rho-binding site. Rok can phosphorylate MLC and MBS, preferable substrates for bovine Rho-kinase, in vitro. These results suggest that Rok is an effector of Rho1 (Mizuno, 1999).

Exchange factors for Rho proteins are known to activate their downstream targets through direct binding to Rho GTPases. Hence, the molecular basis for the observed genetic interaction between pebble and Rho1 may be a physical interaction between Pebble and Rho1 proteins. To test this hypothesis, a yeast two-hybrid assay was carried out. Fusions of Pbl (full-length and amino-terminally truncated) as well as Drosophila Rho proteins, Rho1, Rac1, and Cdc42, were constructed with both the GAL4 DNA-binding domain (DBD) and GAL4 activation domain (AD). Plasmids were transformed in various combinations, and the resulting colonies were tested for the ability to activate HIS3 and lacZ reporters. Only colonies that carry plasmids expressing both Pbl (full-length or DeltaPbl325-853, amino-terminally truncated Pbl) and Rho1 are able to grow on a medium that lacks His, Leu, and Trp, suggesting that the HIS3 gene is induced. Therefore, it is suggested that Pbl and Rho1 proteins interact in vivo and that this interaction is specific for Rho1, but not for Rac1 or Cdc42. Furthermore, the DH domain, but not the amino terminus of Pbl (containing BRCT domains and NLS), is essential for this interaction, because it is abolished by a small deletion within the DH domain, but not by the amino-terminal truncation of Pbl. These results indicate that Pbl and Rho1 form a protein complex in vivo, and that the basis for the genetic interaction between pbl and Rho1 may be a direct interaction between the two proteins (Prokopenko, 1999).

To test the physical interaction between Drosophila Rok and various Rho-GTPases, a pull-down assay using GST-GTPase fusion proteins and in vitro translated Rok was used. Rok binds to the constitutively active form of Drosophila RhoA, but not to constitutively active Rac1 or Cdc42. Mutating a key amino acid within the effector-binding domain (T37A) abolishes the interaction with Rok. These results suggest that Rok is an effector specific for Drosophila RhoA (Winter, 2001).

The Rho GTPases regulate many different cellular and developmental processes, and activation of Rho GTPase signaling is mediated through interaction with the Dbl homology (DH) protein domain. The expression pattern is described of DrhoGEF3 (cytological position 61B1-B3), which encodes a new member of the DH domain protein family from Drosophila and is a homolog of the human protein hPEM-2. During gastrulation and germ band extension, DrhoGEF3 exhibits a segmented expression pattern. DrhoGEF3 is subsequently expressed in the visceral mesoderm, at the sites of muscle attachment and in specific groups of sub-epidermal cells. The possible function of such a dynamically expressed signaling molecule is discussed (Hicks, 2001).

Initially, during the blastoderm stage, DrhoGEF3 expression occurs in a pattern of circumferential stripes. Upon gastrulation a close association between these stripes and several early morphogenetic events become apparent. At the anterior trunk region DrhoGEF3 is initially expressed in the cells flanking the cephalic furrow, which later move into the furrow during its invagination. DrhoGEF3 expression is also detectable in cells anterior to the invaginating posterior midgut that undergo extensive shape changes. As the germ band extends, DrhoGEF3 becomes expressed in a segmented pattern that is similar to genes known to be required for segmentation, such as engrailed. Indeed, the stripes of DrhoGEF3 expression, which are 3-4 cell diameters wide, partially overlap the posterior edge of the stripe of engrailed expression in each segment. Upon the completion of germ band extension, DrhoGEF3 is specifically expressed in scattered groups of sub-epidermal cells within each segment. At this stage DrhoGEF3 is also detectable in the visceral mesoderm, and expression within this tissue persists as the germ band retracts. Finally, following the completion of germ band retraction and dorsal closure, DrhoGEF3 expression is detected at the sites of muscle attachment (Hicks, 2001).

Mutations in dpix (rho-type guanine exchange factor) were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dpix encodes dPix, a Rho-type guanine nucleotide exchange factor (RtGEF) homologous to mammalian Pix. dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations led to decreased synaptic levels of the PDZ protein Dlg, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Thus, a Rho-type GEF and Rho-type effector kinase regulate postsynaptic structure (Parnas, 2001).

Spire links rho family signaling to the actin cytoskeleton

spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. Spire also contains two domains with similarity to the actin monomer-binding WH2 domain, and Spire binds to actin in the interaction trap system and in vitro. In addition, Spire interacts with the rho family GTPases RhoA, Rac1 and Cdc42 in the interaction trap system. This evidence supports the model that Spire links rho family signaling to the actin cytoskeleton (Wellington, 1999).

Previous work has shown that in spir mutants, the microtubules bundle at the cortex prematurely, during stage 8, and this bundling of the microtubules is accompanied by rapid, microtubule-dependent swirling of the cytoplasm. Both the bundling of microtubules and cytoplasmic streaming are normally seen later in stage 10 wild-type oocyte. A bi-directional signaling process occurs between the oocyte and the posterior follicle cells to establish the posterior pole of the egg. Phenotypes indicative of a defect in this signaling process include transformation of the posterior follicle cells into an anterior follicle cell fate; misorganization of the microtubules at stage 6; localization of Oskar mRNA to the center of the oocyte; localization of Bicoid mRNA to the posterior pole, and premature cytoplasmic streaming. The posterior follicle cell fates are established correctly in spir. In contrast to a previous report, no central spot of Oskar mRNA staining is observed in spir mutants. Finally, Bicoid mRNA localization appears relatively normal in spir. These results suggest that signaling between the posterior follicle cells and the oocyte is not abnormal in spir mutants. In spir mutant oocytes, microtubules sometimes show abnormal distributions during stage 6, but it is believed that this probably reflects an earlier manifestation of the known spir microtubule defect (Wellington, 1999 and references therein).

WH2 domains, like those found in Spir, have been found in the Wiskott-Aldrich syndrome protein (WASP), verprolin, Scar-1 (see Drosophila SCAR), and a number of other proteins of unknown function. The WH2 domains of N-WASP and of Scar1 have been shown to bind directly to G-actin in vitro. In addition, Spir binds to unpolymerized actin in vitro. Although Spir is capable of binding to actin monomers through its WH2 domain, no defects in the actin cytoskeleton have been observed in spir mutants, suggesting a number of possibilities. The defects may be in actin structures that are difficult to observe, such as the cell cortex. In fact, spir phenotypes can be mimicked by treatment with cytochalasin D, a drug that affects the polymerization state of actin; defects in the actin cytoskeleton in cytochalasin D-treated oocytes have not been observed. Alternatively, spir may act downstream of the actin cytoskeleton and, thus, not change it. Finally, in vitro experiments have shown that, in the absence of the neighboring cofilin homology and acidic domains, the two WH2 domains of N-WASP have either no effect on or slowly depolymerize filamentous actin. Since Spir is lacking the cofilin homology and acidic domains, Spir may have only minor or no effect on filamentous actin (Wellington, 1999 and references therein).

It is becoming more apparent that a relationship exists between the actin cytoskeleton and premature microtubule-dependent cytoplasmic streaming. The premature cytoplasmic streaming phenotype of spir can be mimicked by the addition of cytochalasin D, a drug which depolymerizes actin filaments. Additional evidence that the actin cytoskeleton is involved in repressing microtubule bundling and streaming comes from analysis of mutant phenotypes of genes linked to the actin cytoskeleton. In addition to spir, mutants in chickadee, which encodes profilin and capu, which is thought to bind to profilin, also exhibit these microtubule behaviors. While phenotypes for cdc42 and rac1 have been described during oogenesis, their effects on patterning during oogenesis are unknown. The finding that Spir interacts with rho family GTPases suggests that at least one of the rho family GTPases is functioning in patterning. Further genetic and biochemical studies will be required to determine the nature of Spir's interaction with rho family GTPases in vivo. The analysis of spir suggests that rho family GTPAses and actin function with Spir in patterning the Drosophila oocyte. Further studies on spir should elucidate the role of rho family GTPases and the actin cytoskeleton in patterning during oogenesis (Wellington, 1999).

Pkn protein kinase is a Rho/Rac effector target required for dorsal closure

Among the putative Rho/Rac effector targets in mammals are the protein kinase N/protein kinase C-related kinase (PKN/PRK) family of serine/threonine kinases. PKN (also referred to as PRK1) and the closely related protein PRK2 together account for the vast majority of Rho-binding autokinase activity detected in most mammalian tissues. The carboxy-terminal catalytic domains of these kinases are highly homologous to the PKC family kinases, but they possess unique amino-terminal regulatory sequences, including three leucine zipper-like repeats shown to be important for the interaction with the Rho GTPase. These proteins also interact detectably with the Rac GTPase, suggesting that they may be shared effector targets of the Rho and Rac GTPases. Despite the identification of closely related PKN homologs in several organisms, the precise biological function of these putative Rho targets remains unknown. The ability of the Rho GTPases to regulate cell morphology and motility suggests that these proteins and their associated signaling pathway components are likely to perform functions essential to the normal morphogenesis of developing multicellular organisms. Dorsal closure (DC) does not require new cell divisions, and appears to depend solely on dramatic cell shape changes within a subset of epidermal cells. These shape changes are initially restricted to two symmetric rows of epidermal cells, known as the leading edge (LE) cells, and are followed by the stretching of the more lateral epidermal cells, ultimately resulting in the meeting of the two rows of LE cells at the dorsal midline. Three classes of genes have been implicated in DC; namely, the Rho family of GTPases; the c-Jun amino (N)-terminal kinase (JNK) cascade components, including the decapentaplegic (dpp) signaling pathway genes, and several membrane-associated proteins. The recently described Drosophila loss-of-function Rho1 mutant is defective for DC, and homozygous mutant embryos exhibit an obvious hole in the dorsal-anterior portion of the larval epidermis. Although Rac1 loss-of-function mutants have yet to be reported, overexpression of Rac1N17, a dominant-negative form of Rac1, in developing Drosophila embryos, results in a DC defect. Similar results have also been reported for Cdc42N17 (Lu, 1999 and references).

Four Drosophila homologs of the mammalian JNK pathway genes [hemipterous (hep; JNKK), basket (JNK), Djun, and kayak (Dfos)] are all required for DC. Mutations in any of those genes result in a dorsal-open phenotype very similar to that seen in either the Rho1 mutants or embryos overexpressing dominant-negative Rac1 or Cdc42. Furthermore, disruption of the JNK pathway in hep mutants abolishes the expression of two independent downstream target genes of Djun, dpp and puckered (a MAP kinase phosphatase), which are also required for DC. The hep mutation also blocks an increase in dpp expression in the LE cells induced by expression of activated Rac1V12 in those cells. These results have led to a model for DC in which Rac1 (and possibly Cdc42) signals through the JNK pathway to activate the expression in LE cells of Dpp, a secreted ligand of the TGF-beta receptor. In turn, Dpp relays an instructive signal to initiate stretching of the more lateral epidermal cells. The signaling role of Rho1 or downstream effector targets of Rho1 in this process is unknown. A Drosophila homolog of the mammalian PKN family kinases, Drosophila Pkn is shown to bind specifically to both Rho1 and Rac1 GTPases in a GTP-dependent manner, and its kinase activity is promoted by both interactions, suggesting that Rho1 and Rac1 GTPases can utilize Pkn as a downstream effector target. Both Rho1 and Rac1 bind Pkn through the amino-terminal region of the protein. Significantly, it appears that Rho1 and Rac1 interact with Pkn through distinct binding sites. A loss-of-function mutation in the Drosophila Pkn gene leads specifically to a DC defect during embryogenesis. However, this Pkn-mediated DC pathway is independent of the Rac-JNK-Dpp pathway; rather, the Pkn-mediated DC pathway appears to act coordinately with the Rac-JNK-Dpp pathway to regulate epidermal cell shape changes during morphogenesis of the Drosophila embryo (Lu, 1999 and references).

The predicted Drosophila Pkn protein sequence is closely related to both human PKN and PRK2 (60% overall identity to both) and has all of the conserved features found in other PKN family members, including the amino-terminal negative regulatory pseudosubstrate motif, the three leucine zipper repeats (HR1a, HR1b, and HR1c) that mediate GTPase binding, a central conserved region of unknown function found in the PKC-eta kinases, and the carboxyl-terminal PKC-like kinase domain (Lu, 1999).

The expression pattern of the Drosophila Pkn gene is highly dynamic during embryogenesis. An in situ hybridization analysis of wild-type embryos reveals that Pkn mRNA is abundant at the blastodermal stage, suggesting that it is maternally loaded. At stage 13, when DC is normally initiated, the most prominent expression is seen in the dorsal LE cells and in two pairs of discontinuous stripes on the epidermis of each segment. However, the expression becomes more restricted in later stages and can only be detected in the anterior and posterior spiracles, the pharynx, and the mouth tip at stage 16 (Lu, 1999).

Although homozygous Pkn mutant embryos do not exhibit obvious developmental defects prior to stage 13, ~10% of them die as embryos with an obvious hole in the anterior region of the dorsal epidermis. This dorsal-open phenotype closely resembles the DC defects observed previously with loss of function of the Rho1 gene and several components of the Rac-mediated JNK cascade. To examine the requirement for maternally loaded Pkn mRNA, germ-line clone (GLC) mutants of Pkn were generated: ~50% of the mutant embryos derived from these clones are found to display the same DC defect. Phenotypic analysis of these embryos with various histological markers reveals that they are correctly patterned and do not exhibit any detectable defects of the central or peripheral nervous system or the somatic musculature, suggesting that Pkn is not required prior to DC (Lu, 1999).

According to the current working model of DC, the Rac1 and Cdc42 GTPases activate the downstream JNK cascade kinases to induce the expression of several genes in the LE cells, including dpp. Thus, DPP mRNA expression in the LE cells is a convenient assay of activation of the JNK cascade in vivo. Because Pkn functions biochemically as a Rac1 effector target, and mutations in Pkn cause a DC defect similar to that seen with JNK pathway mutants, it was of interest to determine whether Pkn is required for the previously established Rac-JNK-Dpp pathway. Therefore, the expression of DPP mRNA was examined in Pkn mutant embryos. Mutations in the JNK cascade gene hep result in embryos that fail to express detectable levels of DPP mRNA in the LE cells. In contrast to the loss of dpp expression seen in hep mutant embryos, expression of dpp in the LE cells of Pkn mutants is not detectably affected, indicating that Pkn is not required for the previously reported Rac-JNK-Dpp pathway. This conclusion was also verified by examination of the expression of beta-galactosidase from a puckered-lacZ enhancer trap, which has been shown previously to be eliminated in a hep mutant background. Pkn mutant embryos that exhibit an obvious dorsal-open phenotype retain normal levels of puckered-lacZ expression, confirming that the Rac-JNK pathway leading to transcriptional activation is not detectably affected by the absence of Pkn (Lu, 1999).

The shapes of epidermal cells of Pkn mutant embryos were examined. All epidermal cells adopt an unstretched polygonal shape following an initial apparently normal LE cell stretching, similar to that seen in the JNK pathway mutants, such as hep. Thus, it appears that while both Rac-JNK- and Pkn-mediated signals are required for the stretching of epidermal cells required for DC, they are associated with distinct pathways. Because the expression of Pkn mRNA is enriched specifically in the LE cells prior to DC, the possibility that Pkn expression is regulated by the JNK pathway was examined. However, Pkn expression is unaffected in hep mutant embryos (Lu, 1999).

Accumulating evidence suggests that the Rac1 GTPase, as well as some of the JNK pathway components, performs a function in the LE cells that may be distinct from the regulation of gene expression, but is necessary for the stretching of the LE cells. Because Pkn is a biochemical effector of the Rac1 GTPase that mediates DC, but is not required for dpp or puckered gene expression, the possibility was tested that the Pkn mutant interacts genetically with the JNK pathway component, basket. Removal of one copy of the basket gene from a Pkn mutant GLC background significantly increases the frequency of dorsal-open embryos, suggesting that both Pkn and JNK activities converge at some point to affect a related aspect of the DC process (Lu, 1999).

The Rho1 GTPase has also been implicated in DC. Null alleles of Rho1 exhibit a DC defect that closely resembles that seen in Pkn mutants although the relevant Rho1-mediated pathway in this process has not been established. Because the cell shape changes in the LE cells appear to initiate the DC process, and Pkn expression is enriched in the LE cells just prior to DC, the possibility was explored that a Rho1-Pkn signal mediates shape changes in those cells. To start, the requirement for Rho1 activity was examined specifically in the LE cells. A dominant-negative form of Rho1 (Rho1N19) is expressed in the LE cells of wild-type embryos. More than 60% of these embryos display a dorsal-open phenotype very similar to that seen in the Rho1, Pkn, and JNK pathway mutants. Similar to Rho1 and Pkn mutants, but not to a hep mutant, embryos expressing Rho1N19 in the LE cells exhibit normal levels of DPP mRNA expression in the LE cells. Moreover, all of the epidermal cells in these embryos ultimately adopt an unstretched polygonal shape following a normal initial LE cell stretching at early stage 13. This result demonstrates clearly that expression of the dominant-negative Rho1 in the LE cells does not cause a dorsal-open phenotype by nonspecifically blocking the Rac-JNK-Dpp pathway and suggests that a Rho1-mediated second instructive signal is generated in the LE cells, which together with Dpp, is required for the stretching of the more lateral-ventral cells (Lu, 1999).

These results are consistent with a role for a Rho1-Pkn signal that contributes to the stretching of the LE cells. To establish a specific requirement for Pkn in the LE cells, a transgenic line was established in which a putative dominant-negative form of Pkn (kinase deficient) is expressed under the control of a UAS element. By crossing this line to a line harboring the LE-GAL4 driver, it was determined that a small percentage of embryos exhibit a DC defect that is indistinguishable from that seen in Pkn or Rho1 loss-of-function mutants. Genetic interaction between Rho1 and Pkn was examined. Removal of one copy of the Rho1 gene (a null allele) from the Pkn mutant GLC background results in an increase in the frequency of dorsal-open embryos from 55% to 68%. The fact that a reduction of Rho1 activity enhances the frequency of DC defects observed in a Pkn-null background suggests that Rho1 most likely interacts with at least one additional downstream target that is also required for DC. Consistent with this hypothesis, it was observed that heat shock-induced Pkn expression is not sufficient to rescue the Rho1 mutant DC defect (Lu, 1999).

Currently, little is known regarding the biological role of the mammalian PKN family proteins. Expression of a dominant-negative (kinase-deficient) form of human PRK2 in microinjected fibroblasts results in the disruption of actin stress fibers, suggesting a normal role for PRK2 in regulating Rho-mediated actin reorganization. Both human PKN and PRK2 undergo partial caspase-mediated proteolysis during apoptosis, suggesting a potential role for these kinases in the morphological changes in cells during programmed cell death. Significantly, both Rho and Rac GTPases have been implicated previously in apoptosis. A closely related homolog of PRK2 in starfish is highly expressed and specifically phosphorylated in oocytes during meiotic maturation, suggesting a possible role in that process. Human PKN, which is highly expressed in brain, has been found to be enriched in the neurofibrillary tangles associated with Alzheimer's disease and can phosphorylate the Tau protein as well as the Neurofilament L protein, suggesting a potential role for PKN in neuronal degeneration. Despite these observations, the precise biological function of these prominent Rho/Rac effector targets remains unclear. The results described here provide evidence for a biological function for this putative Rho/Rac-effector target in embryonic development and indicate a specific role for Drosophila Pkn in regulating cell shape changes required for tissue morphogenesis. Previous studies have revealed a role for the Drosophila Rho1 GTPase in several developmental processes, including gastrulation, establishment of tissue polarity, and DC. However, Pkn is required only for DC, indicating that Pkn is not required for all Rho1-mediated activities related to morphogenesis. Thus, it seems likely that additional Rho-associated downstream effector targets mediate the function of Rho in these other developmental processes. Possibly, the ability of Pkn to serve as a target of both activated Rho and Rac GTPases accounts for a specialized role for this protein in DC, which appears to require both Rho- and Rac-mediated signaling pathways. Significantly, gastrulation, a morphogenetic process that is also largely dependent on cell shape changes, requires Rho1 but not Rac1 GTPase activity and is unaffected by the absence of Pkn (Lu, 1999 and references).

The fact that homozygous zygotic Pkn mutants that undergo normal DC ultimately die during larval development suggests that Pkn may mediate additional functions of Rho/Rac signaling that are utilized in post-embryonic development. However, no obvious developmental defects are found in somatic homozygous Pkn mutant clones in the adult eye, and the loss-of-function Pkn mutant does not suppress developmental defects associated with overexpression of Rho1 or Rac1 in the developing fly eye. Together with the observation that these GTPases appear to play a role in establishing normal ommatidial polarity, these results suggest that it is unlikely that Pkn is mediating the tissue polarity functions of Rho and Rac in eye development. However, a role for Pkn in additional morphogenetic processes that take place during larval development cannot be excluded (Lu, 1999).

Although the precise role of Pkn in DC is not clear, for the following reasons, it appears likely that Pkn is transducing a Rho-dependent signal in the LE cells: (1) Pkn expression is enriched in the LE cells of stage 13 embryos; (2) the activated Rho1 GTPase binds to and activates the Pkn kinase in vitro; (3) loss-of-function Rho1 and Pkn mutants exhibit a very similar dorsal-open phenotype that is associated with a defect in stretching of the LE cells; (4) in both Rho1 and Pkn mutant embryos, expression of DPP mRNA in the LE cells is apparently normal; and (5) expression of the Rho1N19 and the PknKD mutants specifically in the LE cells (where Pkn expression is highly enriched just prior to DC) results in dorsal-open phenotypes that are indistinguishable from the Pkn mutant phenotype. The fact that expression of Rho1N19 specifically in the LE cells leads to a cell nonautonomous stretching defect in the more lateral epidermal cells (despite normal dpp expression in the LE cells), suggests that Rho1 mediates a second, Dpp-independent, instructive signal. Possibly, this signal is also mediated by a Rho1-Pkn interaction. However, the finding that removal of one copy of Rho1 from a Pkn mutant GLC background (a genetic null) increases the frequency of dorsal-open embryos suggests that Rho1 is probably performing an additional, Pkn-independent, function in DC. In support of this hypothesis, it was found that the heat shock-Pkn transgenic construct is unable to rescue the DC defect in Rho1 mutant embryos. What thus far remains unknown is the Rho effector target that mediates the ability of Rho to regulate gene expression via activation of the serum response factor, which is a known downstream target of Rho activation in mammalian cells. Hence, it is possible that this second instructive signal is induced by a Rho-mediated transcriptional pathway, which is clearly distinct from the Rac-mediated transcriptional pathway. It is worth noting that these results do not exclude the possibility that the cell shape changes associated with the lateral epidermal cells require an additional direct (cell-autonomous) role for the Rho1-Pkn pathway in those cells as well. It is clear, however, that the role of Rho1 in the LE cells during DC is distinct from that of Rac1 (Lu, 1999).

In light of the fact that Drosophila Pkn interacts equally well with the activated Rac GTPase, it is possible that a Rac-Pkn interaction also contributes to DC. However, the lack of a Rac1 loss-of-function mutant in Drosophila makes it difficult to examine the specific role of that interaction. Because the JNK pathway mutants are also associated with a defect in stretching of the LE cells, it has been suggested that components of the JNK pathway may mediate a Rac-dependent cell stretching signal that is unrelated to transcriptional regulation. It is difficult to imagine how Pkn could transduce a signal from Rac to this JNK-mediated cell shape change pathway and yet not be required for the Rac-JNK transcriptional pathway. However, it is possible that Pkn can transmit a Rac signal independent of the JNK-Dpp pathway. Indeed, recent evidence suggests that the Drosophila gene Myoblast city, which is required for DC, encodes a Rac-specific activator that does not appear to regulate dpp expression (Nolan, 1998). This observation suggests that Rac may perform multiple functions in dorsal closure (Lu, 1999).

Significantly, there does seem to be some cross-talk between the Pkn-mediated signaling pathway and the JNK pathway. Removal of one copy of basket from a Pkn mutant GLC background significantly increases the frequency of dorsal-open embryos. This result suggests that some component of JNK cascade signaling is sensitive to the activity of Pkn. Taken together with the fact that Rho1 generates a JNK-Dpp independent signal in the LE cells that is required for DC, it is clear from these studies that distinct but coordinated signaling pathways mediated by the Rho and Rac GTPases within the LE cells are essential for normal DC, and that Pkn is a strong candidate for an effector that mediates signals downstream of both GTPases (Lu, 1999).

PlexinB interacts with RhoA during axon pathfinding

Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for RacGTP binding. The interaction of PlexB with RacGTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters RacGTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to RacGTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).

Several lines of evidence suggest that RhoA is involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).

PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).

The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rhorev220 and RhoAl(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).

The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating RacGTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).

Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of RacGTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).

The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).

RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion

The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established. Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system. The identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, is presented that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42 (Bashaw, 2001).

To identify signaling molecules involved in controlling axon guidance decisions, chimeric receptor overexpression phenotypes were used to perform a sensitized genetic screen. Overexpression of the Robo-DCC chimeric receptor (Robo's extracellular domain fused to DCC's cytoplasmic domain) leads to dose-dependent CNS axon guidance defects in which axons abnormally cross the CNS midline, and also results in reduced viability. The EP collection (a collection of P-element inserts that allow GAL4-dependent misexpression of flanking genes) was screened for genes that, when overexpressed pan-neurally in combination with Robo-DCC, would enhance the viability defects of the chimera. Such genes could play a role in DCC-mediated attractive axon guidance, or alternatively could function in parallel attractive-signaling pathways. This study characterizes of one of the genes identified in this screen (Bashaw, 2001).

Expression of EP3035 dramatically enhances the axon guidance defects of the Robo-DCC chimera, leading to a significant increase in ectopic midline crossing. Molecular characterization of the genomic region adjacent to EP3035 has revealed a large transcription unit that encodes a novel member of the Dbl family of guanine nucleotide exchange factors (GEFs) specific for the Rho family of small GTPases, GEF64C. In addition to the canonical Dbl and pleckstrin homology (PH) domains, GEF64C also contains several proline-rich motifs, including a sequence similar to the Enabled EVH1 domain binding site (LPLPP). RNA in situ analysis on EP3035/ElavGal4 embryos confirms that EP3035 drives overexpression of the GEF64C transcript. In addition, the genetic enhancement of Robo-DCC by EP3035 can be phenocopied by expressing a UAS GEF64C transgene, confirming that the enhancement is due to GEF64C expression. Protein expression analysis in wild-type embryos, using an mAb to GEF64C, reveals broad, low level expression of this GEF, with some enrichment in the CNS. The specificity of the GEF64C mAb is demonstrated by comparing embryos expressing full-length UASGEF64C under control of elavGAL4, with those expressing a COOH-terminal truncation, UASGEF64CDeltaC, which removes the mAb epitope. Robust CNS expression can be seen in animals with the wild-type transgene, while only the low-levels characteristic of wild-type expression can be seen in animals with the truncated transgene (Bashaw, 2001).

Since GEF64C was identified in a gain of function screen, it was of interest to assess the consequence of loss of GEF64C function on midline axon guidance. Point mutations were generated in the GEF and three independent alleles were sequenced. Two of these alleles, GEF64C1 and GEF64C29, result in premature stop codons, while a third generates a missense mutation at the COOH terminus of the protein. Embryos carrying mutations in GEF64C were examined with an antibody that labels all CNS axons (mAb BP102). No major defects were discovered in the GEF64C mutants: the longitudinal connectives and commissural axon bundles were comparable to those seen in wild-type animals. RNA interference using a fragment of GEF64C double-stranded RNA also failed to reveal strong axon guidance defects, arguing against maternal contribution as an explanation for the absence of a mutant phenotype (Bashaw, 2001).

Genetic redundancy could explain the modest consequences of removing GEF64C; indeed, examination of the Drosophila genome reveals that there are ~22 GEFs specific for Rho family GTPases, a number of which appear to be expressed in the embryonic CNS. This raises the possibility that multiple GEFs function during midline guidance and that disrupting just one has limited effect. For example, mutations in Drosophila trio, another Rho GEF with well-established roles in regulating axon outgrowth, cause only minor disruptions in the CNS axon scaffold, whereas they have more profound effects in combination with other mutations that affect midline axon guidance. Alternatively, it is possible that GEF64C mutations do cause defects in subsets of CNS neurons, but that these defects are not readily apparent when all axons are visualized simultaneously (Bashaw, 2001).

To further investigate a potential requirement for GEF64C in midline axon guidance, the effects of removing GEF64C function were examined in animals that carried mutations in the frazzled (fra) gene, which encodes the Drosophila homolog of the DCC-attractive Netrin receptor. Mutations in fra cause a range of defects in CNS axon guidance consistent with its role in attracting commissural axons to the midline. fra; GEF64C double mutant embryos exhibit a marked enhancement of the guidance defects typically observed in fra mutants; there is a substantial reduction in commissure thickness and a greater number of segments where commissures fail to form. Thus, in the fra mutant background where normal axon attraction to the midline is partially defective, loss of GEF64C exacerbates these defects, suggesting an endogenous role for GEF64C in attractive guidance at the midline. It should be noted that this double mutant analysis does not provide evidence of GEF64C's involvement in DCC signaling, nor does it preclude such a role. Dose-sensitive genetic and biochemical interactions between fra and GEF64C, which could suggest a direct involvement in DCC signaling, have not thus far been observed (Bashaw, 2001).

In contrast to the modest effects of loss of GEF64C function, pan-neural overexpression of GEF64C (using EP3035 or UASGEF64C) results in a dramatic, dose-dependent, gain of function phenotype, in which many axons abnormally project across the midline. The commissures are thicker and there is a commensurate reduction in the longitudinal axon tracts. This phenotype suggests that GEF64C expression promotes axon attraction to the midline. The point mutations in GEF64C were introduced on the EP3035 chromosome, allowing for GAL4 overexpression of the mutant alleles. None of the mutant alleles, nor the UASGEF64CDeltaC transgene (a deletion of the Dbl and PH domains), were capable of generating the gain of function phenotype, indicating that the abnormal midline crossing is due to GEF64C expression, and that this effect requires the intact Dbl and PH domains. Examination of gain of function embryos with antibodies to Wrapper, a marker for midline glia, indicates that the guidance defects caused by GEF64C overexpression are not a secondary consequence of nonautonomous perturbations of midline glial cell survival or migration (Bashaw, 2001).

The GEF64C overexpression phenotype is qualitatively similar to the phenotype of mutations in the Robo receptor, raising the possibility that GEF64C promotes attraction to the midline by interfering with Robo repulsion. Several observations argue against this idea. (1) There are significant differences between the GEF64C gain of function and robo loss of function phenotypes: robo mutations have more profound effects on the growth cones that pioneer the ipsilaterally projecting FasII-positive posterior corner cell (pCC) pathway than does GEF64C overexpression. (2) Overexpression of GEF64C does not appear to affect Robo protein expression or localization. (3) In terms of genetic predictions based on the function of commissureless (comm), Comm downregulates Robo receptors on commissural axons. In comm mutants no axons cross the midline; in robo;comm double mutants, the phenotype is like robo. Thus, if GEF64C overexpression were blocking robo function, the GEF64C gain of function should be at least partially epistatic to mutations in comm -- this is not the case. For these reasons, it is believed that GEF64C overexpression exerts its effects through stimulation of an attractive signaling pathway, rather than through inhibition of Robo repulsion (Bashaw, 2001).

The GEF64C gain of function phenotype suggests that by increasing the expression of an attractive signaling molecule, it is possible to overcome the normal repulsive signals that are present at the midline. To determine whether GEF64C expression would also allow axons to cross the midline in genetic backgrounds where axons are biased toward being repelled, GEF64C was coexpressed with a hyperactive mutant form of the Robo receptor: RoboY-F. Pan-neural expression of UASroboY-F results in a commissureless phenotype, in which no axons cross the midline. If in this roboY-F background GEF64C expression is simultaneously driven, many commissural axons are now able to cross the midline, and some segments appear to be nearly wild-type. Thus, even in this artificially repulsive background, GEF64C can allow significant axon growth to and across the midline, raising the exciting possibility that finding ways to stimulate the activity of functionally homologous mammalian GEFs could promote regrowth of injured axons in the adult CNS (Bashaw, 2001).

How does expression of GEF64C promote axon attraction? One likely scenario is that it exerts its effects by specifically activating one or more of the Rho-family GTPases. There are six RhoGTPases in the fly genome: Rac1, Rac2, Mtl, RhoA, RhoL, and Cdc42. Reasoning that genetically limiting the downstream target should suppress the GEF64C gain of function phenotype, use was made of the GEF64C gain of function phenotype and dominant negative GTPase transgenes for Rac1, RhoA, and Cdc42 to determine which, if any, of these GTPases are the downstream target(s) of GEF64C. Based largely on the differential effects of Rac and Rho on neurite extension (Rac promotes extension and Rho promotes retraction), it has been proposed that during axon guidance Rac could play a role in attractive responses, whereas Rho could stimulate repulsion. Therefore it was predicted that the GEF64C gain of function phenotype would depend on Rac activity, but not on Rho. Surprisingly, the opposite appears to be true; the RhoA dominant negative strongly suppresses the GEF64C gain of function, whereas the Rac1 and Cdc42 dominant negatives have little or no effect. This observation argues against the simplest form of the model that Rac mediates attraction, and Rho mediates repulsion (Bashaw, 2001).

To test if the specificity of GEF64C for RhoA seen in these genetic experiments is also observed in independent assays for GEF64C function, in vitro binding and guanine nucleotide exchange assays were performed. Glutathione S-transferase (GST) pull down experiments indicate that GEF64C can bind equally well to Rac1, RhoA, and Cdc42, whereas GEF64C acts as an in vitro exchange factor for Rac and Rho (exhibiting a modest preference in catalyzing the exchange of GDP for GTP on Rho, relative to Rac) but does not have exchange activity for Cdc42. Such promiscuity in the in vitro association of GEFs with small GTPases has been observed for many RhoGEFs, including Vav and Trio. To further examine the function of GEF64C, its effects on the actin cytoskeleton in cultured fibroblasts were determined. Microinjection of a GEF64C expression vector into quiescent, serum-starved Swiss 3T3 cells resulted in a dramatic stimulation of actin stress fiber formation relative to control cells, a phenotype indicative of Rho activation. Coinjection of GEF64C and C3 transferase, a protein inhibitor specific for Rho, completely blocks GEF64C's ability to induce stress fibers, arguing further that GEF64C functions by activating Rho (Bashaw, 2001).

The reciprocal loss and gain of function genetic data presented here support a role for GEF64C in promoting axon attraction to the CNS midline. Overexpression of GEF64C can overcome the normal repulsive signals present at the midline, and can even drive attraction to the midline in a background where Robo repulsion is abnormally strong. Surprisingly, genetic and cell culture evidence suggest that these attractive effects are meditated through the activation of RhoA, but not Rac. These findings present a paradox. Previous evidence from a number of different experimental systems is consistent with the general idea that Rac and Cdc42 are positive regulators of neurite outgrowth and that Rho is a negative regulator. These observations on axon outgrowth have been extended to axon guidance, suggesting that Rac and Cdc42 would mediate attractive guidance responses and Rho would mediate repulsion, and have led to the investigation of the role of the Rho GTPases in the regulation of axon guidance. For example, mammalian Ephexin, a GEF for RhoA, has been implicated in the repulsive responses mediated by Eph receptors, and the repulsive effects Drosophila Plexin B, a member of the Semaphorin receptor family, also appear to be mediated by RhoA (Bashaw, 2001).

These findings suggest that the opposite is also possible; namely, that RhoA may also play a role in attraction, and argue against a single generalizable function for the Rho GTPases in axon guidance. The simplest interpretation of these data is that GEF64C promotes midline attraction through the stimulation of an attractive signaling pathway. However, it should be noted that until GEF64C function is linked to a known receptor/ligand system, it remains a formal possibility that GEF64C expression could exert its effects through enhancing the activity of an unknown repellant present in the lateral CNS. Because such a model would predict strong defects in the longitudinal extension of FasII-positive axons, something that was not observed, the idea that GEF64C activates an attractive pathway is favored. Nevertheless, discovering how the Rho GTPases can elicit different and even opposite axon guidance responses in different contexts is a major challenge for the future, and promises to enrich the understanding of molecular mechanisms of axon guidance in the developing nervous system (Bashaw, 2001).

A Rho GTPase signaling pathway, in conjunction with concertina and folded gastrulation, is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation

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. 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 Galpha12/13 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).

If the guanine nucleotide exchange factor (GEF) is important in selecting the outcome of activating Rho, then its function should be limited to a subset of those associated with the GTPase. To address this possibility, the in vivo function of DRhoGEF2 was investigated. Two hypomorphic alleles, DRhoGEF2PX6 and DRhoGEF2PX10, in combination with null alleles of DRhoGEF2, give adults that have crumpled and/or blistered wings. Earlier in development, the DRhoGEF24.1/DRhoGEF2PX6 wing discs appear buckled rather than conforming to the stereotypical folding pattern observed in the wild-type. This malformation is not a result of either improper patterning or loss of apico-basal polarity. It must therefore be caused by disruption of another mechanism -- for example, the propagation of a localized signal that brings about folding in specific places. To test this hypothesis, clones of DRhoGEF21.1 cells spanning a fold were generated (Nikolaidou, 2004).

In large mutant clones that are less influenced by physical constraints, the folds fail to follow the line of the fold in wild-type tissue. Bifurcation of folds does not occur in wild-type discs, supporting the idea that the mutant tissue is unable to respond to a localized signal to fold. Although the clonal and DRhoGEF24.1/DRhoGEF2PX6 mutant tissues do appear folded, the irregularity of the folds indicates that this is probably a consequence of passive folding, as is seen in the gastrulation mutants and murine neurulation mutants that fail to invaginate tissue appropriately (Nikolaidou, 2004).

The possibility was investigated that other events involving epithelial invagination or folding might also require DRhoGEF2 activity. One such event is the invagination of a placode to form a salivary gland tube on both sides of the embryo. Combinations of dominant-negative alleles with a putative null allele of DRhoGEF2 showed that in 93% of embryos some or all of the salivary-gland cells fail to invaginate and instead remain on the outside. Because maternally provided DRhoGEF2 is vital for epithelial invagination in gastrulation, this and the above two phenotypes represent three examples of the requirement for DRhoGEF2 in epithelial-layer morphogenesis (Nikolaidou, 2004).

If DRhoGEF2 is participating in the selection of the cell's response to activated Rho, then its function should be limited. Rho is known to play a role in cytokinesis, cell cycle regulation and planar polarity. The large size of clones of DRhoGEF2, equivalent numbers of cells in twin wild-type and mutant clones, and normal polarity of mutant tissue indicate that unlike Rho, DRhoGEF2 is not required for any of these processes, nor is it required for apico-basal polarity. No significant defects were seen in the gross morphology of the nonepithelial tissues of muscles and neurons in late-stage DRhoGEF24.1/DRhoGEF26.5 and DRhoGEF24.1/DRhoGEF25.1 embryos. In addition, the normal cell cycle control shows that the convolution of DRhoGEF2 mutant wing discs is not a result of excessive proliferation (Nikolaidou, 2004).

Although the possibility exists that DRhoGEF2 has a function not addressed, it seems likely that its role is confined to the control of epithelial morphogenesis. This limit of DRhoGEF2 function suggests that it is important in selecting a role for Rho only in epithelial morphogenesis, whereas other GEFs would activate Rho in other processes; for example Pebble activates Rho primarily in cytokinesis, and Trio acts on Rac in neuronal outgrowth (Nikolaidou, 2004).

To study in more detail the mechanism by which DRhoGEF2 affects epithelial morphogenesis, the possible targets of DRhoGEF2 activation have been considered. One of these is myosin II. During gastrulation, Zipper (Zip), the heavy chain of myosin II, appears to accumulate on the apical side of the mesodermal precursors in the ventral furrow (VF). To address the possibility that apical myosin localization is required for other invagination events, salivary-gland formation was analyzed in embryos expressing the myosin light chain, Spaghetti squash (Sqh), as a fusion with green fluorescent protein (Sqh-GFP). Although Sqh-GFP is present at the cortex of all the cells, it is concentrated at the apical surface of salivary-gland precursors that are about to invaginate or are in the process of invaginating. Sqh-GFP does not accumulate apically until invagination, as demonstrated by the lack of apical localization in cells that are present more anteriorly in the placode but that will invaginate later (Nikolaidou, 2004).

It is not clear if this apical myosin accumulation is present in time to contribute to apical constriction. To resolve this question, the localization of Sqh-GFP was observed in the invaginating VF during gastrulation. In wild-type cells, Sqh-GFP is maintained at the tip of the growing membrane that forms between the nuclei during cellularization, the process immediately prior to gastrulation. At the end of cellularization, Sqh-GFP begins to decrease on the basal side and accumulate on the apical side of the ventral cells, i.e., only those that will constrict apically. This redistribution of myosin precedes apical cellular constriction, suggesting that it contributes to the process. Basally located Sqh-GFP is subsequently lost, and the apical levels increase (Nikolaidou, 2004).

In DRhoGEF2 germline clone-derived (GLC) embryos (i.e., those lacking maternal DRhoGEF2), Zip, the myosin heavy chain, is lost from the basal side of cells in the developing VF, but it accumulates at much lower levels on the apical side than it does in the wild-type. These results imply that a signal through DRhoGEF2 is needed in order for the ventral cells to induce apical Zip localization. In contrast, relocalization of β-heavy spectrin occurred normally in DRhoGEF2 GLC embryos, indicating that cell polarity is maintained in these cells and that at least some forms of protein relocalization, especially that of a protein that is found in close proximity to Zip (Nikolaidou, 2004).

The possibility was considered that myosin localization is also regulated by other components of the DRhoGEF2 signaling pathway. By analogy to the mammalian and C. elegans orthologs and as a result of genetic studies , DRhoGEF2 is thought to participate in a signal transduction pathway, which is called here the DRhoGEF2 signaling pathway, initiated by Folded gastrulation (Fog) and propagated by Concertina (Cta). Mutations in both these genes result in gastrulation defects. In embryos derived from cta mutant mothers, a low level of Zip accumulates on the apical side only of apically constricting cells in the invaginating VF. This is also true in DRhoGEF2 GLC embryos. In contrast, there is no apical myosin apparent in the cells that do not constrict their apical surface. These data clearly link the presence of apical myosin with apical constriction and indicate that in gastrulation this is controlled by the DRhoGEF2 signaling pathway. The link between DRhoGEF2 and Myosin is also supported by the documented genetic interactions between DRhoGEF2 and zip in leg and wing development (Nikolaidou, 2004).

It is not clear how DRhoGEF2 influences the apical accumulation of myosin. It could act via the Rho effector Rho kinase. When activated by Rho in mammalian cells, Rho kinase is responsible for revealing the actin binding site on the regulatory light chain of myosin II. Thus, in DRhoGEF2 mutants, a possible failure in the activation of Rho1 and Rho kinase would result in the inability of myosin to bind actin (Nikolaidou, 2004).

If DRhoGEF2 is required reiteratively for epithelial morphogenesis, it is hypothesized that Fog and Cta might also be used reiteratively. mRNA for fog is expressed in invaginating tissue during gastrulation and salivary-gland formation, suggesting that Fog also participates in invagination of the salivary gland. At present there are conflicting reports regarding the role of fog in salivary gland formation. This study finds that some or all cells fail to invaginate in 90% of the embryos. Because invagination in gastrulation is cell autonomous, it is considered more likely that this phenotype results from a lack of fog in these cells rather than because of earlier developmental defects (Nikolaidou, 2004).

The possibility was addressed that the pathway is also important in wing development. Initial descriptions indicate that Fog and Cta play no role in this process. However, demonstrating a previously undisclosed role for Fog and Cta, combinations of mutations in fog or cta and DRhoGEF2 result in synergistic effects on wing development. Together, these results point to the reiterative use of the DRhoGEF2 signaling pathway in development to bring about epithelial folding or invagination (Nikolaidou, 2004).

Preliminary data indicate that the folds in the wing disc are brought about by apical cell constriction. It is therefore proposed, because both gastrulation and salivary gland invagination also involve apical cell constriction, that this is a major aspect of DRhoGEF2 function. The location of the folds in wing discs is highly stereotypical, which would suggest that specific signals are activated in these locations to initiate folding. One candidate for this signal is Fog, which is perhaps acting in conjunction with a second signal to bring about epithelial folding in the wings. In gastrulation, fog and cta are essential, but their phenotypes are not as strong as that observed after the removal of maternal DRhoGEF2, again indicating the requirement for additional signals that activate DRhoGEF2. The nature of this additional signal, or signals, remains elusive (Nikolaidou, 2004).

Citron Kinase is an essential effector of the Pbl-activated Rho signalling pathway in Drosophila

Pebble (Pbl)-activated RhoA signalling is essential for cytokinesis in Drosophila melanogaster. The Drosophila citron gene, [a. k. a. sticky (sti)], encodes an essential effector kinase of Pbl-RhoA signalling in vivo. Drosophila citron is expressed in proliferating tissues but is downregulated in differentiating tissues. Citron can bind RhoA and localisation of Citron to the contractile ring is dependent on the cytokinesis-specific Pbl-RhoA signalling. Phenotypic analysis of mutants showed that citron is required for cytokinesis in every tissue examined, with mutant cells exhibiting multinucleate and hyperploid phenotypes. Strong genetic interactions were observed between citron and pbl alleles and constructs. Vertebrate studies implicate at least two Rho effector kinases, Citron and Rok, in cytokinesis. By contrast, no evidence was found of a role for the Drosophila ortholog of Rok in cell division. It is concluded that Citron plays an essential, non-redundant role in the Rho signalling pathway during Drosophila cytokinesis (Shandala, 2004).

RNA interference-mediated silencing of sticky/citron in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such Sticky-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes, suggesting that these GTPases antagonistically regulate Sticky functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. Antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis (D'Avino, 2004).

Citron has been proposed to act downstream of Rho in the regulation of cytokinesis. However, little in vivo evidence has been found to support this proposition. To test whether Citron participates in Rho signalling, genetic interactions were examined between citron and a known regulator of the Rho pathway, the Rho-GEF-encoding gene, pebble. The first assay chosen was the ability to modify the moderate citron embryonic PNS phenotype. pbl mutants were chosen rather than Rho mutants because Pbl appears to be a specific Rho activator for cytokinesis, whereas loss of Rho also affects many other processes. Removing one copy of pbl in cit mutants results in a significant reduction in the overall number of cells in the PNS, while most of the remaining cells (52%) appear to be multinucleate. Therefore, a mild reduction in Pbl-mediated Rho activation during cytokinesis results in a significant enhancement of the cit mutant embryonic PNS defects (Shandala, 2004).

A complementary approach monitored whether under- or over-expression of citron could modify a loss-of-Pbl phenotype. Since strong pbl phenotypes arise too early and are too drastic to be of use, an RNAi construct was generated to inhibit Pbl synthesis later in development. Expression of this pblRNAi construct in the posterior half of the wing resulted in a decrease in the size of the corresponding region. Analysis of the affected area revealed that more than 67% of cells produce multiple hairs in contrast to the invariably single-haired cells in wild-type, a phenotype observed when cytokinesis is blocked, for example by inhibition of RacGAP50C. As expected, co-staining of pupal wings with phalloidin and the DNA stain Hoechst 33258 revealed that the pblRNAi-expressing cells were abnormally large and typically multinucleate, resembling the embryonic phenotype of pbl mutants. The intermediate nature of the en-GAL4>UAS-pblRNAi wing size and multiple hair phenotypes allowed detection of enhancement and suppression by prospective interactors. To test the specificity of this assay system, the pblRNAi phenotype was examined in a RhoA/+ background. Significant diminution of the pbl-depleted region of the wing shows that the pblRNAi phenotype is enhanced by removal of one copy of wild-type RhoA, as seen in other genetic assays for pbl function. The multiple hair phenotype was quantified in a defined wing region posterior to vein L5. A significant increase in the proportion of multihaired cells from 67% to 84% upon loss of one copy of RhoA shows that this assay could detect reductions in the dose of cytokinesis effector genes. Removal of one copy of wild-type citron also reduces the size of the posterior half of the wing in en-GAL4>UAS-pblRNAi flies and enhances the multiple hair phenotype. Identical effects were observed in Df(3)iro-2 heterozygous mutants . The genetic interactions between loss of function citron and pbl phenotypes support the role of Citron as a Rho effector in cytokinesis. Ectopic expression of citron in various Drosophila tissues generates no dramatic phenotype in wild-type or pblRNAi backgrounds, suggesting that the activity of Rho is rate limiting for Citron function (Shandala, 2004).

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation.

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitant with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. Both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. These results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane (Grosshans, 2005).

This morphological analysis of the mutant phenotypes reveals a new function of RhoGEF2 and dia in the formation of the furrow canal. This function is consistent with the co-localisation of both proteins with F-actin at the furrow canal and the reduced amounts of F-actin in RhoGEF2 and dia mutants. Biochemical analysis demonstrates actin polymerisation by Dia and thus supports the model that RhoGEF2 and Dia organise actin filaments to control the formation of the furrow canals. Furthermore, evidence is provided that the previously characterised genes nullo and sry- alpha act in a genetic pathway in parallel to RhoGEF2 and dia, suggesting that they control two distinct aspects of furrow canal formation. This conclusion is based on the assumption that amorphic situtations were used in these experiment. The possibility cannot be excluded that RhoGEF2 and dia stabilise the furrow canal rather than control its initial formation. A function in the formation is supported by the observation that the proportion of nuclei in multinuclear cells does not increase in the course of cellularisation (Grosshans, 2005).

The following arguments support the hypothesis that RhoGEF2 and dia act in the same genetic pathway that controls spatially restricted assembly of actin filaments. In both dia and RhoGEF2 mutants the morphology of the furrow canal is disrupted. The furrow canals are much larger than normal and filled with cytoplasmic blebs. Both proteins are localised at the furrow canal and both precede the appearance of the cellularisation front. The localisation of both proteins does not depend on F-actin. However, they are directly or indirectly involved in the assembly of F-actin since the amount of F-actin is reduced at the furrow canal of the mutant embryos. The strongest argument for a functional connection is that Dia localisation at the furrow canal depends on RhoGEF2 during the early phase of cellularisation. Rho1 may mediate this functional link by direct interactions with RhoGEF2 and Dia. However, the findings do not show that RhoGEF2 exclusively functions via dia. Other targets of Rho1-GTP, like citron kinase, protein kinase N or Rho kinase may be activated in parallel to Dia. Although a reduction of MyoII at the furrow canal was observed during the first half of cellularisation in embryos from RhoGEF2 germline clones, correspondingly lower MyoII levels are also observed in embryos from dia germline clones, which indicates that the reduction of MyoII may be a consequence of reduced F-actin levels. Consistent with the reduction of F-actin at the furrow canal, levels of MyoII were also reduced in the mutant embryos. In contrast to the reduction at the furrow canal, cortical F-actin appeared to be increased in some embryos from dia germline clones. This increase was variable and not observed in all of the experiments, however (Grosshans, 2005).

The difference in the RhoGEF2 and dia mutant phenotypes clearly shows that dia has additional functions and may be controlled by other not yet identified factors besides RhoGEF2. Whereas RhoGEF2 mutants pass through the cleavage cycles without obvious defects, dia is involved in formation of pole cells and pseudo cleavage furrows. As a possible consequence of these additional functions, dia mutants in contrast to RhoGEF2 mutants often have a more disrupted F-actin array, larger furrow canals and a more disturbed cellularisation than RhoGEF2 mutants. Furthermore in the early phase of cellularisation Dia localisation depends on RhoGEF2, whereas later, after the furrow has formed, Dia becomes enriched to a certain degree at the cellularisation front independently of RhoGEF2. One gene that may act in parallel to RhoGEF2 to control Dia localisation is Abl. Embryos from Abl germline clones have reduced amounts of Dia at the furrow canal and show a disrupted F-actin array similar to that observed in dia and RhoGEF2 mutants. However, the molecular link between Abl and Dia is elusive and no abnormalities in the morphology of the furrow canal in Abl mutants have been described. Thus Dia may be controlled and activated by multiple pathways including RhoGEF2 among others (Grosshans, 2005).

It is not known how the position of the invaginating plasma membrane is determined. RhoGEF2 and Dia are not likely to be part of a pattern formation process, but their localisation reflects an early readout of this pattern, since the nuclei and centrosomes are properly arranged in RhoGEF2 and dia mutants. RhoGEF2 and Dia proteins are early markers for these sites and precede furrow canal formation because specific staining was detected for both Dia and RhoGEF2 when the nuclei were still spherical and when the cellularisation front was not yet visible. Other factors beside RhoGEF2 and Dia are also involved in furrow canal formation, because many furrow canals still form in RhoGEF2 and dia mutants, which indicates that there is genetic redundancy (Grosshans, 2005).

At present it can only be speculated about which factors and mechanisms are responsible for RhoGEF2 localisation. Candidates may be among the group of genes involved in furrow canal formation. However, for all of these mutations no ultrastructural analysis has been reported that would allow the morphological defect to be defined and their function for furrow canal formation to be compared with the function of RhoGEF2 and dia. Among this group are Rab11 and nuf, which encode a GTPase of the recycling endosome and its putative effector. Considering the assumed biochemical activities, it is conceivable that vesicle targeting is important for transporting factors to the site of membrane invagination. This raises the possibility that RhoGEF2 is transported by such vesicles to the sites of membrane infolding. Analysis of RhoGEF2 protein distribution in nuf and Rab11 mutants and the phenotype of double mutants may address this hypothesis. Alternatively, RhoGEF2 may be transported to the site of the future furrow canal along microtubules that form open baskets around the nuclei, or other recruiting factors may precede at the site of membrane invagination (Grosshans, 2005).

Furthermore, slow as molasses (slam) is required for timed formation of the furrow and invagination of the membrane in the first half of cellularisation. Like Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation precedes furrow canal formation. Slam may act by recruiting MyoII to the furrow canal, but the biochemical activities of Slam have not been defined. Although the membrane does not invaginate initially in slam mutants, a complete F-actin array is visible. Thus despite the overlapping localisation of Slam, RhoGEF2 and Dia, their functions are clearly distinguishable (Grosshans, 2005).

How do RhoGEF2 and Dia act in furrow canal formation? If the biochemical activity of Dia is considered to nucleate actin filaments and the enlarged and labile furrow canals in the dia mutants, it is conceivable that Dia organises and assembles a coat of F-actin at the site of membrane invagination and furrow canal formation. The coat of F-actin may be important for the compactness and stability of the furrow canal to prevent infoldings of the cytoplasm. Such a function may be related to the function of F-actin in endocytic events. The subset of actin filaments controlled by RhoGEF2 would not significantly contribute to pulling in the plasma membrane, since membrane invagination proceeds with normal speed in RhoGEF2 mutants. Alternatively, RhoGEF2 and Dia may perform their function independently of actin polymerisation. Although the amount of F-actin is reduced in the mutants, the possibility that the polymerisation activity of Dia is not required for all or part of its function cannot be excluded. Dia may also influence the organisation of microtubules, as interactions of mDia1 with microtubules and EB1, a microtubule-associated protein, have been described (Grosshans, 2005).

The differences in protein localisation and mutant phenotypes of RhoGEF2 and nullo suggest that they have distinct activities. In contrast to the frequently missing furrow canals in single mutants, their complete absence in embryos lacking both gene functions clearly implies, however, that their functions are redundant from a genetic point of view. These results show that RhoGEF2 and dia are required for the formation of a compact and stable furrow canal. If one of the two pathways is disturbed, the furrow canal can still form, albeit with a lower and variable efficiency that depends on the conditions. For example the nullo phenotype is strongly temperature sensitive. However, if both pathways are affected, furrow canals do not form at all. Future studies will resolve how the actin filaments are involved in bending the plasma membrane that leads to the furrow canal and will further demonstrate how RhoGEF2 protein is expressed in the hexagonal array to serve as a template for local actin polymerisation (Grosshans, 2005).

crossveinless-c, a RhoGAP for Rho1 and Rac1, is required for actin reorganisation during morphogenesis

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In a search for genes that regulate Drosophila morphogenesis, several lethal alleles have been isolated of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and it is proposed that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions (Denholm, 2005).

Removal of any of the Rac candidates, Rac1, Rac2 or Mtl, and reduction or removal of Cdc42 failed to modify the cv-cM62 phenotype in the Malpighian tubules. By contrast, 50% of cv-cM62 mutant embryos additionally homozygous for Rho172R and 20% of cv-cM62 mutant embryos heterozygous for Rho172R had a phenotype significantly less severe than that of the cv-cM62 homozygote alone. The Malpighian tubules of doubly mutant embryos undergo convergent extension movements to some extent, such that they resemble weaker alleles of cv-c. These data strongly suggest that Rho1 is a substrate for Cv-c in the Malpighian tubules (Denholm, 2005).

Genetic interations were examined in the embryonic epidermis. Dorsal closure defects occur in 82% of embryos doubly mutant for Rac1 and Rac2. However, if Rac1,Rac2 embryos are additionally mutant for cv-cM62, 37% of these embryos are rescued, indicating that Rac GTPases are substrates for Cv-c during dorsal closure (Denholm, 2005).

The posterior spiracle phenotype in cv-c embryos was not suppressed by any of the RhoGTPase mutants, possibly because maternal contribution of the substrate GTPase is sufficient to provide full activity in this tissue. However, it was unexpectedly found that embryos mutant for both Rac1 and Rac2 do not decrease, but enhance the cv-cM62 posterior spiracle phenotype. One possible explanation for this interaction comes from observations in cell culture, where it has been shown that Rac activity downregulates Rho activation. If Rac normally functions to inhibit Rho in the posterior spiracle, then loss of Rac and Cv-c together, would lead to hyperactivity in Rho1 and this would lead to the enhancement of the cv-c phenotype. In support of this, cv-c-like posterior spiracle phenotypes were seen with low penetrance in Rac1 Rac2 embryos (Denholm, 2005).

Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. This study demonstrates that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD nucleates actin, but the activity of the other isoforms has not been addressed. This study finds that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. It is proposed that Rho1, Cappuccino and Spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments (Rosales-Nieves, 2006).

The results indicate that Rho1 regulates the timing of ooplasmic streaming by regulating the microtubule/microfilament crosslinking that occurs at the oocyte cortex. In this model, crosslinking antagonizes the formation of the dynamic subcortical microtubule arrays that are required for ooplasmic streaming. It is proposed that activated Rho1 transduces a signal during stages 8-10b that promotes the crosslinking activity of Capu and SpireC by preventing binding of SpireD to both Capu and SpireC. Rho1 then becomes inactivated at stage 10b, presumably by a signalling event, allowing SpireD to bind to Capu and SpireC, thereby inhibiting microtubule/microfilament crosslinking. When signalling through this pathway or the level of Capu and/or Spire protein is reduced through mutation, ooplasmic streaming occurs constitutively from stage 8 up to and through stage 13, resulting in the severe patterning defects that are observed in these mutants. That SpireD also inhibits the crosslinking activity of SpireC indicates that a parallel regulatory mechanism exists for SpireC-mediated crosslinking. Although a role for Rho1 in regulating actin nucleation by Capu and Spire cannot be ruled out, the mechanism established in this study by which Spire and Rho1 regulate the crosslinking activity of Capu does not seem pertinent to actin nucleation. Viewed in light of the fact that the P597T mutation in the FH2 domain, which is encoded by the capu2F allele, does not affect actin-nucleation activity but is less efficient at crosslinking microtubules and microfilaments, the crosslinking activity describe in this study seems to be an important aspect of how ooplasmic streaming is regulated in vivo (Rosales-Nieves, 2006).

The data have several broader implications. The finding that Capu and Spire regulate each other’s activity indicates an explanation for the conserved co-expression of these two de novo actin-nucleation factors, both of which create linear actin filaments and are required to mediate the same developmental events. Moreover, this work establishes Rho1 as a direct regulator of a broader group of actin-nucleating proteins, and is the first evidence for how the activities of Spire and Capu are regulated to coordinate the ooplasmic streaming events in vivo. The direct interaction between Rho1 and Capu indicates an additional level of complexity to this mechanism. It is, therefore, possible that Rho1 may simultaneously regulate the nucleation and crosslinking activities of Capu through an, as yet unclear, mechanism. Further investigation of this will require the expression of full-length Capu constructs that contain the relevant binding site (Rosales-Nieves, 2006).

To date, much work has been devoted to understanding the role of formins, and more recently Spire, in controlling actin dynamics and nucleation. However, diaphanous-related formin proteins also have profound effects on microtubule dynamics and stability, with recent evidence indicating that these effects are, at least in some cases, independent of the actin-nucleation function. The data presented in this study indicate that direct regulation of microtubule architecture may be a property that is common to a larger subset of formins, as well as to at least one of the Spire protein isoforms. The distinct mechanism by which Spire and Capu regulate microtubule/microfilament crosstalk is consistent with the highly specialized function of these proteins in regulating germline development in Drosophila. Indeed, the mammalian homologue of Capu, formin-2, is also required only in the female germline, where it regulates proper chromosome segregation, which is another process that involves intimate coordination of microtubule and microfilament dynamics. Recently, a mutation at the formin-2 locus has been implicated in unexplained female infertility in humans. Therefore, Capu and Spire seem to be elements of a highly conserved cassette that is required for the earliest stages of metazoan development. Precisely how the activity of these proteins is coordinated with developmental signalling circuits to allow for the proper regulation of ooplasmic streaming or chromosome segregation will certainly provide interesting areas for future work (Rosales-Nieves, 2006).

The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. This study analyzes a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, it is shown that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. The results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system (Matusek, 2006).

Drosophila DAAM is required to organize an array of parallel running actin cables beneath the apical surface of the tracheal cells that define the taenidial fold pattern of the cuticle. The actin ring pattern corresponds exactly to that of the taenidial fold pattern, and it is proposed that the actin rings organized by DAAM define the position of taenidial fold formation. The genetic interaction and epistasis data are consistent with a model that DAAM activity is regulated by RhoA. In addition, DAAM works together with the non-receptor tyrosine kinases Src42A and Tec29 to regulate the actin cytoskeleton of the Drosophila tracheal system (Matusek, 2006).

The basic structure of the insect tracheal system is a highly conserved tubular network in every species. The most important function of this network is to allow oxygen flow to target cells. Thus, tracheal tubes need to be both rigid enough, to ensure continuous air transport, and flexible enough along the axis of the tubes, to prevent the break down of the tube system when body parts or segments move relative to each other. These requirements are mainly ensured by the tracheal cuticle, which covers the luminal surface of the tubes and displays cuticle ridges (making the overall structure similar to the corrugated hose of a vacuum cleaner). Analysis of DAAM mutants provides the first direct evidence that this hypothesis is correct. The data demonstrate that in the absence of DAAM the taenidial fold pattern is severely disrupted, often leading to the collapse of the tubes and to discontinuities in the tubular network. In addition, the analysis revealed that the remarkably ordered cuticle pattern, displayed in the wild-type trachea tubes, depends on DAAM-mediated apical actin organization. Apical actin is organized into parallel-running actin cables, much the same way teanidial folds run in the cuticle. Significantly, the formation of these actin bundles precedes the onset of cuticle secretion, and the number and phasing of the actin rings correspond exactly to that of the taenidial folds in the cuticle. Thus these studies revealed a novel aspect of apical actin organization in the tracheal cells that has not been appreciated before (Matusek, 2006).

The DAAM gene encodes a novel member of the formin family of proteins, involved in actin nucleation and polymerization. Consistent with this, DAAM is colocalized with apical actin in the tracheal cells, and the activated form of DAAM is able to induce actin assembly when expressed in tracheal cells and in other cell types (unpublished). In DAAM mutant tracheal cells, apical actin is still detected, albeit at a somewhat lower level than in wild type, but the bundles formed in the mutant are much shorter and thinner than in wild type, and often appear to be crosslinked to each other. Most strikingly, global actin organization is almost completely lost, although some local order can still be detected. Remarkably, the cuticle pattern in mutant tracheal cells still follows the underlying aberrant actin pattern. Overall, in DAAM mutants, both the tracheal cuticle and the apical actin pattern resemble a mosaic of locally ordered patches that failed to be coordinated and aligned with each other and the axis of the tracheal tubes. It is thus proposed that the apical actin bundles play a key role in patterning the tracheal cuticle by defining the place of taenidial fold formation. Regarding the function of DAAM, the results suggest that the major role of this formin in the tracheal cells is to organize the actin filaments into parallel running actin rings or spirals instead of simply executing the well characterized formin function related to actin assembly. However, whether this is a direct effect on actin organization, and thus represents a novel formin function, needs to be further elucidated. An alternative model could be that DAAM is primarily required for actin polymerization but tightly coupled to an actin 'organizing' protein. In such scenario, the polymerization activity should be a redundant requirement, whereas the link to the organizing protein would be a DAAM-specific function, thereby explaining the presence of unorganized actin bundles in DAAM mutant tracheal cells (Matusek, 2006).

In the case of the main tracheal airways, which are multicellular along their periphery, it is striking that in wild type the run of the actin bundles is perfectly coordinated across cell boundaries. In addition, the run is always perpendicular to that of the tube axis. How does DAAM ensure the coordination of these two aspects of actin organization? Because the DAAM protein and the apical actin cables are both found at the level of the adherens junctions, it is possible that DAAM regulates the coordination of the actin cables at the cell boundaries through a direct interaction with junctional protein complexes. However, other explanations are also possible, and further experiments will be required to elucidate the molecular mechanism of this regulatory function. The fact that actin cables normally run perpendicular to the tube axis seems to suggest that tracheal cells are able to sense a global orientation cue and align their actin bundles accordingly. The nature and source of this cue is unknown, as is the mechanism by which DAAM is involved in the read-out of this signal. Nevertheless, it is interesting that in DAAM and btl-Gal4/UAS-C-DAAM mutant trachea, the main pattern of the cuticle phenotype is often changing from one segment to the other, suggesting that the effect of the 'global' orientation cue is limited to metameric units (Matusek, 2006).

Sequence comparisons of FH2 proteins suggest a close phylogenetic relationship between the DRF, FRL and DAAM subfamilies. Members of these three subfamilies have a high level of conservation in the FH2 domain, and importantly, also in the region of the GBD and DAD domains, suggesting that the FRL and DAAM family formins are also regulated by autoinhibition and RhoGTPases, like the DRFs. Further evidence is presented in support of this view. First, DAAM and RhoA display a strong genetic interaction. Second, C-DAAM (an N-terminally truncated form of DAAM) behaves like an activated form much the same way DRF family formins behave. Third, epistasis experiments with C-DAAM and RhoA suggest that RhoA acts upstream of DAAM. Thus, the data support the model in which DAAM, at least in the Drosophila tracheal system, is regulated by autoinhibition that can be relieved by RhoGTPases (Matusek, 2006).

This conclusion, however, contradicts the observation that human DAAM1 is required for Wnt/Fz/Dvl dependent RhoA activation in cultured cells and that xDaam1 appears to mediate Wnt-11 dependent RhoA activation in Xenopus embryos. These results suggested that DAAM functions upstream of RhoA in non-canonical Wnt/Fz-PCP signaling. An explanation for these distinct conclusions might be related to the fact that DAAM, in contrast to xDaam1, does not appear to be required for Fz/Dsh-PCP signaling. Hence, it is possible that the Drosophila ortholog is regulated in the same way as the DRF formins, while the vertebrate family members can be regulated in a different way, once bound by Dsh/Dvl and recruited into PCP signaling complexes (Matusek, 2006 and references therein).

Genetic interactions with the hypomorphic DAAMEx1 allele identified two non-receptor tyrosine kinases, Src42A and Tec29, as strong interacting partners. Although both of these kinases play multiple roles during embryogenesis, single mutants for both affect the tracheal cuticle pattern in a similar way to DAAM. These results suggest that DAAM and the Src family kinases work together to regulate the actin cytoskeleton and cuticle pattern in tracheal cells. Although it is not known whether DAAM physically binds Src42A and/or Tec29, it has been established that the FH1 region of DRFs and other formins can bind SH3 domains, including those of the Src family kinases. In agreement with these data that DAAM acts upstream of Src42A and Tec29 in tracheal cells, cytoskeleton remodeling and SRF activation mediated by mouse Dia1 and mouse Dia2 requires Src activity. Moreover, a recent report suggests that RhoD and human DIA2C regulate endosome dynamics through Src activation, proposing that Src activity is stimulated via human DIA2C dependent recruitment to early endosomes. Similarly, the Limb deformity protein (a formin) interacts with Src on the perinuclear membranes of primary mouse fibroblasts. Based on these examples, it is speculated that in Drosophila tracheal cells the RhoA/DAAM/Src module may not only be required to organize apical actin bundles, but additionally it might represent a link to secretory vesicles and to the regulation of exocytosis. Future studies will be required to test this hypothesis, and to unravel the mechanisms whereby DAAM family formins and Src family kinases contribute to cytoskeletal remodeling in the Drosophila tracheal system and in other tissues (Matusek, 2006).

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

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).

Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. This paper shows that, contrary to previous models, the Drosophila CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, the Sti Citron-Nik1 homology domain was found to interact with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion causes an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. It is proposed that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics (Bassai, 2011).

Current models propose that in human cells, RhoA recruits and then activates CIT-K, which in turn phosphorylates MRLC at the CF. Results from this study indicate that, at least in Drosophila, this model is incorrect. Indeed, Sti recruitment to the CF does not require interaction with RhoA, and instead, Sti appears necessary to maintain proper RhoA localization at the CF specifically in late cytokinesis. In addition, Sti depletion causes an increase and not a decrease of mono- and diphosphorylated MRLC at the cleavage site in late cytokinesis. Because MRLC phosphorylation is controlled by RhoA signaling, it is proposed that aberrant RhoA localization after Sti depletion might cause an increase of phosphorylated MRLC at the CF. MRLC phosphorylation has been described to control actin dynamics, and thus, this model could well explain the CR disorganization observed after Sti knockdown. Why does Sti depletion cause an increase of MRLC phosphorylation? The simplest hypotheses are that Sti kinase activity could, directly or indirectly, promote the activity of an MRLC phosphatase or inhibit RhoA and/or an MRLC kinase. Identification of Sti targets will be necessary to fully comprehend how this kinase controls RhoA localization during cytokinesis and to distinguish between the aforementioned hypotheses (Bassai, 2011).

Rho1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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