Rho-LIM kinase signaling regulates ecdysone-induced gene expression and morphogenesis during Drosophila metamorphosis

The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).

The malformed legs in DlimkD522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity. Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast city (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).

Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by Slam

Essential for proper function of small GTPases of the Rho family, which control many aspects of cytoskeletal and membrane dynamics, is their temporal and spatial control by activating GDP exchange factors (GEFs) and deactivating GTPase-activating-proteins (GAPs). The regulatory mechanisms controlling these factors are not well understood, especially during development, when the organization and behaviour of cells change in a stage dependent manner. During Drosophila cellularization Rho signalling and RhoGEF2 are involved in furrow canal formation and the organization of actin and myosin. This study analyzed, how RhoGEF2 is localized at the sites of membrane invagination.The PDZ domain is necessary for localization and function of RhoGEF2, and Slam was identified as a factor that is necessary for RhoGEF2 localization. It was also demonstrated that Slam can recruit RhoGEF2 to ectopic sites. Furthermore the PDZ domain of RhoGEF2 can form a complex with Slam in vivo, and Slam transcripts and protein colocalize at the furrow canal and in basal particles. Based on these findings, it is proposed that accumulation of slam mRNA and protein at the presumptive invagination site provides a spatial and temporal trigger for RhoGEF2-Rho1 signalling (Wenzl, 2010).

RhoGEF2 is an essential regulator of Rho1 activity during many different stages of Drosophila development including cellularization. However, little has been known about the events and factors that control RhoGEF2 localization and subsequent Rho1 activation at the furrow canal. This study assigned a new function to the PDZ domain of RhoGEF2 in being sufficient and required for furrow canal localization. The pattern and the dynamics of furrow canal localization of different PDZRG2 containing constructs are very similar to that of endogenous RhoGEF2 thereby reflecting the behaviour of the full-length protein during cellularization. The domain could be used to effectively target other proteins like RFP, Myc or GST to the furrow canal. Thus despite being a multidomain protein, furrow canal localization depends ultimately only on residues that assure the structural integrity of the ligand recognition site of the PDZ domain. It was reported previously that the RhoGEF2 PDZ domain is involved in the subcellular localization of RhoGEF2 during apical constriction of mesodermal cells in gastrulation. It has been suggested that a direct interaction between the PDZ domain and the PDZ binding motive at the C-terminus of the apically localized transmembrane protein T48 is involved in the recruitment of RhoGEF2 to the apical site of the cells. However, it is clear that this interaction is not essential for apical RhoGEF2 localization, since this localization is lost only in T48/cta double mutants (Wenzl, 2010).

By using immunoprecipitations from staged embryonic extracts it was possible to show that a transgenic 4xPDZRG2-myc6 construct can physically interact with Slam in vivo. Of course this does not directly proof that Slam also interacts with full-length endogenous RhoGEF2. Nevertheless different arguments are presented that support the assumption that a physical interaction between Slam and RhoGEF2 underlies the observed functional relationship between these two factors in cellularizing embryos. The PDZ domain is the critical element that mediates the localization of RhoGEF2 at the furrow canal where it colocalizes with Slam. It was shown that this PDZ domain can form a complex with Slam in vivo. Further in vivo experiments confirmed that furrow canal localization of RhoGEF2 depends on slam in a dosage dependent manner which supports the biochemical findings. Moreover Slam can recruit RhoGEF2 to ectopic sites in embryos as well as in S2 cells and aspects of the RhoGEF2 mutant phenotype can be observed in slam deficient embryos. Overall it is reasonable to conclude that there may be a direct or indirect interaction between Slam and RhoGEF2 during formation of the cellular blastoderm. This interaction would be mediated by the PDZ domain of RhoGEF2. The data also demonstrate that slam acts upstream of RhoGEF2 (Wenzl, 2010).

The molecular function of slam has remained unknown, although the essential role of this gene in cellularization is well established (Merrill, 1988). It has been proposed that Slam is involved in membrane traffic, since in slam mutants the polarized insertion of membrane is disturbed. This study describes an additional cell biological function of slam in being a developmental switch that temporally and spatially controls Rho activity in blastoderm embryos by regulating the subcellular localization of the Rho1 activator RhoGEF2. Thus by proposing the existence of a protein complex containing RhoGEF2 and Slam, physiological and molecular function of Slam can be linked (Wenzl, 2010).

PDZ domains often interact with the C-termini of transmembrane proteins. There are different classes of PDZ binding motifs that can be classified according to their amino acid composition. Although not being a transmembrane but a membrane associated protein, Slam possesses a potential class II PDZ binding motif at its C-terminus. However, this motif seems to be dispensable for the recruitment of RhoGEF2 by Slam to ectopic sites. This is consistent with the fact that a slam allele with a mutated C-terminus rescues the cellularization phenotype of slam deficient embryos. In addition this allele is able to recruit RhoGEF2 to the furrow canal membrane. Furthermore RhoGEF2 to be still present although with reduced levels at the furrow canal in germline clones of a C-terminally truncated slam allele slamwaldo1 (Wenzl, 2010).

Besides the interaction between Slam and the PDZ domain of RhoGEF2, an interaction between Slam and Patj was observed in co-IPs from staged embryonic extracts. This is consistent with the fact that both proteins almost perfectly colocalize during cellularization at the furrow canal as well as in basal particles. Furthermore a functional relation between Slam and Patj is seen, since Patj levels at the furrow canal are reduced in embryos that are zygotically deficient for slam. Patj is a conserved protein that contains 4 PDZ domains and was previously reported to be able to interact with Crumbs in vitro and in vivo during epithelial polarity establishment later in development. However, the importance of this interaction remains unclear, since embryos that are maternally and zygotically mutant for Patj have been reported to develop until adulthood without obvious phenotypes. This would argue against an essential role of Patj during cellularization. As shown by another report, the mutants used in the study still expressed a truncated Patj protein that contained the first PDZ domain thus it is likely that residual Patj function was still present. Zygotic Patj null mutants, in which the coding sequence of Patj was removed completely, died during second instar larval stage, indicating that Patj is an essential gene. Therefore it would be worth to generate maternal Patj null mutants to investigate the role of this protein during cellularization in more detail. Nevertheless the interaction between Patj and Slam seems to depend mainly on the C-terminus of Slam, since in slamwaldo1 mutants Patj levels at the furrow canal are strongly reduced. Thus it is possible that the putative PDZ binding motif at the C-terminus of Slam is important for a direct interaction with one of the PDZ domains of Patj. The Slam Patj interaction also shows that besides controlling RhoGEF2 localization Slam has other independent functions, which could account for the strikingly stronger cellularization phenotype of slam mutants compared to the weaker phenotype of RhoGEF2 deficient embryos (Wenzl, 2010).

RhoGEF2 also functions in different epithelial invagination processes like salivary gland formation or in the establishment of the epithelium in the wing imaginal disc of Drosophila L3 larvae. It appears likely that the subcellular localization of the protein is controlled by genes encoding different receptors that are expressed during different developmental stages in a tissue specific manner like slam or T48 which would allow a very precise temporal and spatial regulation of Rho activity by employing the same ubiquitously expressed activating factor. RhoGEF2 also has a function in the maternally controlled formation of the metaphase furrows during the cleavage divisions 10-13 of the syncytial blastoderm stage and it was shown that localization of the protein to these furrows depends on maternal components of the recycling endosome. The start of zygotic slam expression at the onset of cellularization thus could assure that sufficient levels of RhoGEF2 and thus Rho activity become associated with the membrane tip during invagination. At the same time the metaphase furrows that have recently been shown to be rather active endocytic membrane domains are transformed into a domain forming the furrow canal, which were reported to be much more inactive and stable (Wenzl, 2010).

This study also shows that slam transcripts exhibit a new and unique mRNA localization pattern. A significant portion of slam mRNA is associated with the furrow canal membrane domain. Surprisingly the initial processes that ensure a local restriction of Rho activity would be the proper localization of the slam RNA/protein particles. The asymmetrical localization of transcripts within a cell often linked with localized translation is an important mechanism for the spatial regulation of gene activity. Apical localization of transcripts during cellularization has been described for a number of genes including wg, run and ftz. This study showed that the transcripts are transported to localize to the apical cytoplasm of the cells of the cellular blastoderm. However, little is known about the functional importance of this transcript localization. The localization of slam transcripts might also include a basal to apical transport step, since large basal particles were seen containing slam mRNA and protein in cellularizing embryos. It has been reported previously that apical Rho activity during posterior spiracle formation is mediated in part by RhoGEF64C. The transcript of this gene does localize to the apical membrane of the epithelial cells which undergo apical constriction and subsequent invagination. The mechanisms that ensure the association of transcripts with a specific membrane domain remain to be solved and slam would offer a good system to study this question. Future studies will show whether and how the localization of slam mRNA is involved in defining the sites for membrane invagination and what other functions are served by slam besides initiating Rho signalling (Wenzl, 2010).

Taken together, a model is proposed for the developmental control of Rho1 signalling at the furrow canal, in that the slam RNA-protein particles are targeted to the prospective site of membrane invagination at the onset of cellularization. Slam would have several functions, mainly initiating the formation of the furrow canal as a distinct membrane domain by regulating membrane traffic and at the same time it would recruit and restrict RhoGEF2 and maybe other factors to this domain. After reaching a critical concentration the GEF activity would be activated by a yet unknown mechanism. Rho1 would be converted into its GTP-bound form and downstream targets like Dia or Rho-kinase would be activated. Consistent with this model is the observation that the dose-dependent activity of Slam, both higher or lower than normal levels, directly corresponds to the amount of RhoGEF2 protein and the speed of cellularization as for example shown by the local injection of slam RNA (Wenzl, 2010).

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. This study shows that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis results in cell intercalation defects. A pathway is delineated that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, evidence is provided that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin (Levayer, 2011).

Epithelial tissues have a robust architecture that is essential for their barrier function. This barrier function depends on their ability to build adhesive contacts at adherens junctions through the recruitment and stabilization of E-cadherin (E-cad), β-catenin (β-cat) and α-catenin (α-cat) by actin filaments (F-actin). During development, epithelia are also extensively reshaped by remodelling of cell contacts. This plasticity is essential for morphogenesis during embryogenesis and organogenesis. Work in the past decade showed that this requires force generation by actomyosin networks and their anchoring at cell junctions by E-cad/β-cat/α-cat complexes. Thus, E-cad plays a pivotal role in junction robustness and plasticity by mediating both adhesion (cohesion) and tension transmission (remodelling). Understanding what controls the distribution and dynamics of E-cad/β-cat/α-cat complexes is therefore key to understanding cell packing and the mechanics of tissue morphogenesis. Disruption of this balance marks key steps in the progression of solid tumours. The loss of epithelial organization during the epithelial to mesenchymal transition is an extreme example in which E-cad endocytosis causes the loss of adhesion and tension transmission at the cell cortex (Levayer, 2011).

The early development of the Drosophila embryo is a powerful system to study epithelial morphogenesis. Spatial regulation of force generation by actomyosin networks and force transmission to adhesion by E-cad both contribute to apical cell constriction in the invaginating mesoderm1, and cell intercalation in the elongating ectoderm called the germ band. Germ-band extension (GBE) is driven by cell intercalation in the ventrolateral region, whereby cells exchange neighbours through planar polarized junction remodelling, namely shrinkage of 'vertical' junctions (that is, junctions oriented along the dorsoventral axis). Intercalation is powered by non-muscle Myosin-II (Myo-II): anisotropic actomyosin contractile flows from the medial apical region to 'vertical' junctions drive junction shrinkage. The shortening of vertical junctions is stabilized by Myo-II at the cortex. Actomyosin contractility is transmitted at the cortex by E-cad complexes through β-cat. Interestingly, E-cad/β-cat/α-cat complexes also exhibit a planar polarized distribution complementary to that of Myo-II: E-cad is less abundant in shrinking 'vertical' junctions. This E-cad polarity is also required to orient actomyosin flows to 'vertical' junctions. It is unknown what controls the planar polarized distribution of E-cad. This may depend on Rho kinase (ROCK), which is required for the polarized distribution of Par3 (Levayer, 2011).

This study shows that the planar polarized distribution of E-cad is also controlled by an upregulation of clathrin- and dynamin-mediated endocytosis at adherens junctions, in particular in 'vertical' junctions of intercalating cells. Blocking endocytosis causes the loss of E-cad planar polarization and a block of intercalation. This led to an investigation of the mechanisms that control planar polarized upregulation of clathrin-mediated endocytosis (CME) of E-cad at adherens junctions. Activation of WASP (Wiscott-Aldrich Syndrome Protein) and the Arp2/3 (Actin-Related Protein 2/3) complex by Cdc42 controls the branched actin polymerization that is required for vesicular scission. This study identified an additional pathway controlling the initiation of E-cad endocytosis through the recruitment of the AP2 (Adaptor Protein 2) complex and clathrin. This recruitment is driven by lateral clustering of E-cad that relies on unbranched actin polymerization induced by Dia, and the presence of Myo-II. Dia and Myo-II are both activated by the guanine exchange factor RhoGEF2 (Levayer, 2011).

This study has delineated two distinct roles for actin in E-cad endocytosis. Dia and Myo-II control the initiation of E-cad endocytosis by enrichment of clathrin and AP2 in an E-cad-dependent manner. This is tightly spatially regulated in the ventrolateral region and in 'vertical' junctions during cell intercalation by cortical RhoGEF2 localization, an activator of Dia and Myo-II in Drosophila embryos. This is distinct from the role of branched actin polymerization by Arp2/3, which promotes vesicular scission similarly to dynamin. At later stages of development, this depends on WASP and is controlled by Cdc42, aPKC (atypical protein kinase C) and Cip4 (Cdc42-interacting protein 4. In early embryos, as WASP is inhibited by the JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription), Scar instead plays a critical role in vesicular scission. Inhibition of Arp2/3 in scar mutants and its constitutive activation (artificially induced by myrWASP) did not affect clathrin and AP2 concentration at adherens junctions, unlike Dia. The different tiers of regulation of E-cad endocytosis by Arp2/3 and Dia may reflect different roles for actin in constitutive versus regulated E-cad endocytosis. Certain situations require a rapid change in the rate of endocytosis, and may do so by tuning the rate of initiation by clathrin and AP2. It will be interesting to see whether rapid collapse of adherens junctions during epithelial to mesenchymal transition relies on a similar process (Levayer, 2011).

Crosslinking E-cad with an IgG is sufficient to promote dorsal endocytosis of E-cad by upregulating the concentration of clathrin, similarly to Dia activation, even following inhibition of Dia, Myo-II or RhoGEF2. Considering the highly correlated localizations of E-cad complexes with AP2 and RhoGEF2, it is proposed that Dia and Myo-II control the initiation of E-cad endocytosis by inducing lateral clustering of E-cad, similar to Fc receptor clustering during phagocytosis or nanoclusters of GPI (glycosylphosphatidylinositol)-anchored proteins. This may have been co-opted by the pathogen Listeria, whose entry into epithelial cells requires E-cad endocytosis. This mechanism may also require specific 'priming' of E-cad, by ubiquitylation as in mammals, although these tyrosines are not conserved in flies. Importantly, the mechanism of AP2 recruitment by E-cad remains unknown in all systems (Levayer, 2011).

Inhibition of E-cad endocytosis increased E-cad levels and disrupted its planar polarized distribution. Myo-II also accumulated in the medial apical region of cells. The GBE defects in shi-ts mutants or following clathrin inhibition are the result of the altered distribution of actomyosin tensile forces. E-cad/β-cat/α-cat complexes affect the lateral flow of medial actomyosin pulses and Myo-II polarized junctional accumulation, presumably through the regulation of tension transmission within the medial network and/or at the junctions. The medial accumulation of Myo-II when E-cad endocytosis is inhibited may thus reflect an inhibition of actomyosin flow towards the cortex. These results emphasize the interplay between actomyosin contractile dynamics and E-cad adhesive complexes during epithelial morphogenesis (Levayer, 2011).

Cordoba, S. and Estella, C. (2014). The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development. PLoS Genet 10: e1004621. PubMed ID: 25329825

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

Protein Interactions

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo

Morphogenesis of the Drosophila embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. The guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. It is proposed that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. These results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors (Padash Barmchi, 2005; full text of article).

Guanine nucleotide exchange factors regulate the activity of the small GTPase Rho1, which is thought to act as a molecular switch in a broad spectrum of morphogenetic processes that require a complex reorganization of the actin cytoskeleton. However, the manner in which different aspects of Rho1 function are regulated by RhoGEFs is not well understood. This study found that DRhoGEF2 protein is broadly distributed in epithelia during oogenesis and embryonic development and concentrated at the apical surface of cells, suggesting that it may regulate Rho1 throughout morphogenesis. The defects of DRhoGEF2 mutants are less severe than those of Rho1 mutants, suggesting that DRhoGEF2 regulates specific aspects of Rho1 function (Padash Barmchi, 2005).

DRhoGEF2 has been shown to regulate cell shape changes during gastrulation, and DRhoGEF2 is implicated in epithelial folding during imaginal disc development, a process that depends on cell shape changes that are similar to those driving invagination of the germ layers. This paper shows that DRhoGEF2 regulates cytoskeletal reorganization and function during pole cell formation and blastoderm cellularization. All of these processes require the contraction of actomyosin rings. It is proposed that DRhoGEF2 regulates Rho1 activity during cell shape changes requiring actomyosin contractility. The results support the hypothesis that individual RhoGEFs may regulate specific aspects of Rho1 function during development (Padash Barmchi, 2005).

Interestingly, DRhoGEF2 has been found to be nonessential during cytokinesis, which also involves the function of contractile actin rings. The function of Rho1 during cytokinesis is regulated by the RhoGEF pebble that initiates actin ring assembly. In pebble mutants, cytokinesis is blocked at mitotic cycle 14 and subsequent mitoses occur without cytokinesis, creating polyploid, multinucleated cells. Although large multinucleated cells are also observed in DRhoGEF2 mutants at the extended germ band stage it is not clear whether these cells are caused by a block in cytokinesis or are caused by earlier defects during cellularization. In contrast to pebble, DRhoGEF2 may not be required for the assembly of actin rings, but may play a nonessential role in the separation of daughter cells. This is reminiscent of observations during cellularization. Although the function of actin rings appears compromised throughout cellularization, the data suggest that some contractile activity remains that leads to the basal closure of blastoderm cells and is responsible for the cellularized appearance of DRhoGEF2 mutants at the onset of gastrulation (Padash Barmchi, 2005).

At the retracted germ band stage, DRhoGEF2 is enriched at the apical cortex of cells in the leading edge of the lateral epidermis, which is consistent with the view that it may regulate Rho1 during dorsal closure. Rho1 function is essential for dorsal closure, and the cuticles of zygotic Rho1 mutants show dorsal holes. In DRhoGEF2 mutants, the lateral epithelial sheets closed the embryo dorsally. This does not exclude the possibility that constriction of actin cables may contribute to dorsal closure and that DRhoGEF2 may play a role in this process. Overall, the data suggest that DRhoGEF2 function may not be essential for the generation of contractile force, but rather regulate the temporal and spatial coordination of actomyosin contractility (Padash Barmchi, 2005).

During syncytial nuclear divisions and cellularization, DRhoGEF2 is localized specifically at the invaginating furrows. In DRhoGEF2 mutants, actin is irregularly distributed and metaphase furrow formation is less uniform than in the wild type. The defects in furrow formation lead to mitotic defects and the subsequent elimination of abnormal nuclei from the cortex so that, at the onset of cellularization, ~20% of the nuclei have been lost. These phenotypes are reminiscent of the defects seen in mutants of the nonreceptor tyrosine kinase Abelson (Abl). The abnormalities in actin distribution observed in abl mutants are likely caused by the mislocalization of Dia, which leads to ectopic actin polymerization at the apical end of cells. Changes in Dia distribution were not observed in DRhoGEF2 mutants, suggesting that DRhoGEF2 may regulate actin distribution by a different mechanism . Perturbations in actin distribution are observed throughout early development in DRhoGEF2 mutants. During cellularization, significant amounts of actin fail to redistribute to the base of the furrow canal. These observations show that one of the roles of DRhoGEF2 is to regulate furrow assembly. The defects in actin distribution also affect the pole cells, which fail to reorganize their cortical actin cytoskeleton and remain embedded in the somatic nuclear layer rather than sitting on top of it. Consequently, they are obliterated during invagination of the cellularization front (Padash Barmchi, 2005).

It is speculated that DRhoGEF2 may have a function in the assembly of actin cables by regulating the association of actin with other proteins such as myosin II. The mislocalization of actin observed in DRhoGEF2 mutants may be caused by failure of actin to associate with myosin. Interestingly, although myosin II is present at the metaphase furrows, it plays no essential role in their formation, and this suggests that the function of DRhoGEF2 in furrow assembly may be independent of actomyosin contractility (Padash Barmchi, 2005).

Phenotypic analysis suggests that DRhoGEF2 regulates actomyosin contractility during cellularization. Previously, the actin-binding protein Bnk has been implicated in the regulation of contractile forces. In bnk mutants, actin hexagons detach from each other and constrict prematurely. Based on this phenotype, it is suggested that, during the slow phase, cortical actin hexagons are linked to each other through Bottleneck (Bnk), and that actomyosin constriction causes the network to contract as a whole, thereby pulling the membrane front inwards. Once the cellularization front has reached the base of the nuclei and Bnk is degraded, actin hexagons detach from each other and contract as individual rings, thereby closing the blastoderm cells basally. It is proposed that DRhoGEF2-mediated activation of Rho1 may regulate the force that keeps actin hexagons under tension. Bnk counteracts contraction during the slow phase by linking individual actin rings to each other. Degradation of Bnk during the fast phase releases individual actin rings, and the DRhoGEF2-mediated contractile force now contributes to basal closure. Therefore, DRhoGEF2 and bnk act in concert to coordinate actin ring contraction during cellularization. In DRhoGEF2-bnk double mutants, the actin network disintegrates progressively, suggesting that DRhoGEF2 and bnk may play an additional role in the assembly or stabilization of actomyosin filaments (Padash Barmchi, 2005).

It has been proposed that actin network contraction contributes to the inward movement of the furrow canal. Although the data suggest that network tension is severely reduced in DRhoGEF2 mutants, the rate of membrane invagination is unaffected. This is consistent with reports on the role of myosin II during cellularization, suggesting that network tension may not contribute to membrane invagination. In the wild type, actin rings squeeze the nuclei slightly and push them basal-wards as the actin network moves over them. This may contribute to the parallel alignment of astral microtubules surrounding the nuclei and to nuclear elongation. In DRhoGEF2 mutants, nuclei are wider than in the wild type and irregularly aligned. It is proposed that network tension may create an ordered hexagonal array of actin rings that contributes to a parallel alignment of nuclei during cellularization. The force moving the actin network inward may be created by plus end-directed tracking of actin on astral microtubules and by membrane insertion as previously suggested. These observations suggest that actomyosin contractility plays a role in the spatial coordination of cytoskeletal function during cellularization (Padash Barmchi, 2005).

Two effector pathways have been implicated in the transduction of Rho1 activation to the actin cytoskeleton. During cytokinesis, which is mechanistically related to cellularization, a linear pathway including profilin and Dia have been proposed to link Rho1 to the contractile actomyosin ring. The maternally supplied Dia plays a role in a spectrum of cytoskeletal functions during early embryogenesis that also require DRhoGEF2 function, such as metaphase furrow formation, pole cell formation, and cellularization. Dia is localized at the cellularization front and is necessary for the recruitment of cytoskeletal components such as the actin-binding protein anillin and the septin homologue Pnut. The phenotypes of dia mutants suggest that dia is necessary for the assembly of contractile actin rings at sites of membrane invagination (Padash Barmchi, 2005).

The similarities between dia and DRhoGEF2 mutants might suggest dia as a downstream effector of DRhoGEF2. However, the defects of dia mutants are morphologically different from those of DRhoGEF2 mutants. In dia mutants, metaphase furrows do not form and contractile rings at the base of polar cytoplasmic buds fail to assemble. During cellularization, actin fails to condense into individual rings, and the network disintegrates during the second phase of cellularization. In DRhoGEF2 mutants actin rings form and remain largely intact but fail to constrict. In addition, the temporal and spatial localization of Dia and Pnut to the cellularization front was unaffected in DRhoGEF2 mutants and dia was not required for the localization of DRhoGEF2. These findings do not exclude that DRhoGEF2 activity may in part be mediated by dia, however, they suggest that some dia-dependent aspects of Rho1 function are still active in DRhoGEF2 mutants and that another pathway may be involved in transduction of the DRhoGEF2 signal (Padash Barmchi, 2005).

A well-characterized pathway regulating actomyosin contractility in mammalian cells and in C. elegans links Rho1 to actin via Rho kinase, the regulatory subunit of myosin light chain phosphatase (MBS) and myosin II. Rho kinase-mediated phosphorylation inhibits the activity of MBS and induces a conformational change in myosin II allowing it to form filaments that promote sliding of antiparallel actin filaments. The data are consistent with a model in which DRhoGEF2 regulates the association of actin with myosin II, thereby stabilizing actomyosin cables. It is proposed that failure to activate the Rho kinase pathway may compromise the recruitment of actin into contractile cables. This may destabilize actin cables and lead to the mislocalization of actin and to the defects in actomyosin contractility observed in DRhoGEF2 mutants. The Drosophila homologue of Rho kinase, Drok, and myosin II have recently been identified as downstream effectors of DRhoGEF2 during the regulation of actomyosin contractility in Schneider (S2) cells. In addition, myosin II is required for basal closure of blastoderm cells and the myosin II heavy chain encoded by zipper (zip) interacts genetically with DRhoGEF2. These data support the model that DRhoGEF2 may regulate actomyosin contractility through the Rho kinase pathway. Mutants in Drok and Drosophila myosin light chain phosphatase have been identified, however, their role during early embryogenesis has not been reported. Interestingly, inhibition of Drok activity by injection of the specific Rho kinase inhibitor Y-27632 into embryos before cellularization disrupts the localization of myosin II. Similar observations have been made in Drok mutant cell clones in imaginal discs. By contrast, DRhoGEF2 mutants reveal no significant changes in the localization of myosin II during cellularization. It is possible that the differences in myosin II localization between DRhoGEF2 and Drok mutants are due to different mechanisms of action at the molecular level. In mammalian cells myosin II phosphorylation is required for the generation of contractile force but not for its localization. Further investigations will be necessary to resolve how the DRhoGEF2 signal is transduced to the cytoskeleton (Padash Barmchi, 2005).

Little is known about the events that regulate the specific subcellular localization and activation of DRhoGEF2. It has recently been shown that DRhoGEF2 particles are transported from the cytoplasm to the cell periphery by tracking microtubule plus ends in Drosophila S2 cells. DRhoGEF2 particles have been observed during syncytial development that may be involved in a similar process in the embryo. It is speculated that DRhoGEF2 may be delivered to specific membrane subdomains at the cellularization front by microtubules. The G-protein α-subunit encoding gene concertina (cta) has been shown to regulate the dissociation of DRhoGEF2 from microtubules. cta has been implicated in the activation of DRhoGEF2 during gastrulation, but is not required during cellularization. It has been suggested that the force moving the actin network inward may be generated by plus end-directed crawling of actin on astral microtubules. It is speculated that DRhoGEF2 may regulate actin ring constriction during cellularization while associated with the tip of astral microtubules by recruiting Rho1 to the site of actin rings (Padash Barmchi, 2005).

DRhoGEF2 is concentrated in actin-rich regions throughout development and the human orthologue of DRhoGEF2, PDZ-RhoGEF, has been shown to bind to actin directly. Although the domain structure of DRhoGEF2 and PDZ-RhoGEF is very similar, the actin-binding region of PDZ-RhoGEF is not conserved in DRhoGEF2. Nevertheless, the localization of DRhoGEF2 is consistent with the view that it may associate with actin, however, further experiments are needed to corroborate this theory (Padash Barmchi, 2005).

Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2

A hallmark of epithelial invagination is the constriction of cells on their apical sides. During Drosophila gastrulation, apical constrictions under the control of the transcription factor Twist lead to the invagination of the mesoderm. Twist-controlled G protein signaling is involved in mediating the invagination but is not sufficient to account for the full activity of Twist. A Twist target was identified, the transmembrane protein T48, which acts in conjunction with G protein signaling to orchestrate shape changes. Together with G protein signaling, T48 recruits adherens junctions and the cytoskeletal regulator RhoGEF2 to the sites of apical constriction, ensuring rapid and intense changes in cell shape (Kolsch, 2007).

Apical constriction of cells can contribute to the invagination of epithelia, such as during gastrulation or organogenesis, and the closure of wounds. In the Drosophila embryo, apical constrictions occur along the ventral side of the blastoderm epithelium, leading to the formation of the ventral furrow and the invagination of the mesoderm. Proteins necessary for the mechanics of these cell shape changes include the Rho guanosine 5'-triphosphate-exchange factor RhoGEF2 and a heterotrimeric G protein. Whereas RhoGEF2 is essential for furrow formation, disruption of the heterotrimeric G protein, such as by loss of its α subunit Concertina (Cta), leads to a delay but no lasting defects in mesoderm morphogenesis. These maternally supplied proteins must be activated under the control of the zygotic genome in the embryo (Kolsch, 2007).

Twist is the zygotic transcriptional activator that is essential for the cell shape changes that produce the ventral furrow. One of its targets is the transcriptional repressor Snail, which is also essential for mesodermal morphogenesis (Kolsch, 2007).

However, the cell biological events responsible for the cell shape changes must ultimately be regulated by targets that are not transcription factors. Of the known Twist targets, only one, folded gastrulation (fog), is involved in mediating shape changes. Mutants in fog, which codes for a secreted peptide, show the same defects as embryos lacking Cta. Fog is therefore thought to act in the same pathway as Cta, which is referred to as Fog/Cta signaling (Kolsch, 2007).

Fog/Cta signaling is thought to cause changes in the actin cytoskeleton in conjunction with RhoGEF2. Recruitment of myosin from basal to apical in constricting ventral cells is partly dependent on Fog/Cta and absolutely dependent on RhoGEF2. Furthermore, the mammalian homologs of RhoGEF2 and Cta interact. Finally, binding of Drosophila RhoGEF2 to microtubules by means of EB1 is disrupted by activated Cta. Given that myosin recruitment and apical constriction are reduced but not abolished in the absence of Fog/Cta, there must be other factors regulated by Twist that explain its effects on apical constriction (Kolsch, 2007).

In a screen for genes that mediate the zygotic control of gastrulation, the region uncovered by the chromosomal deficiency Df(3R)TlP was found to be necessary for the proper formation of the ventral furrow. Phenotypic analysis and molecular mapping of a set of overlapping deficiencies identified the gene T48 as being responsible for the defects seen in Df(3R)TlP. T48 is expressed in the mesoderm. It codes for a predicted protein with a signal peptide and a potential transmembrane domain. When an internally hemagglutinin-tagged T48 protein (T48HA) was expressed in embryos, it localized at the peripheries of blastoderm cells, consistent with a close association with or insertion into the plasma membrane. Optical cross-sections showed that T48HA is targeted to the apical membrane (Kolsch, 2007).

No other structural motifs are recognizable in the protein. However, the C-terminal amino acid sequence -Ile-Thr-Thr-Glu-Leu (-ITTEL) conforms to the class I consensus for peptides that interact with PDZ domains. T48 has no obvious human ortholog but shows some similarity to the intracellular part of Fras1, which also has a PDZ-binding motif. To find candidates for PDZ domains that might interact with T48, the putative PDZ-binding sequence was analyzed with an algorithm designed to determine the PDZ domains that show the optimal fit for any given peptide. Of the predicted interactors, RhoGEF2 was particularly interesting in view of its role in ventral furrow formation. Furthermore, the mammalian ortholog of RhoGEF2 has been shown to bind to Plexin-B1 by means of a PDZ-binding motif (-Val-Thr-Asp-Leu) very similar to that of T48 (Kolsch, 2007).

Whether the C terminus of T48 is indeed able to interact with RhoGEF2 was tested. A 35S-labeled C-terminal peptide of T48 preferentially coprecipitated with the PDZ domain of RhoGEF2 rather than those of other PDZ domain-containing proteins, in contrast to Crumbs, which was used as a control and which preferentially coprecipitated with PDZ domains from its physiological interaction partner Stardust, as well as Bazooka. In Schneider S2 cells, a green fluorescent protein (GFP)-tagged RhoGEF2 PDZ domain or full-length RhoGEF2 was localized in the cytoplasm or formed intracellular aggregates when expressed alone, but localized to the plasma membrane when coexpressed with T48. In both assays, the interaction required the presence of the -ITTEL motif and was not seen with other PDZ domains. Thus, T48 interacts with RhoGEF2 by means of its PDZ-binding motif and is able to enrich RhoGEF2 to the plasma membrane (Kolsch, 2007).

To understand the function of T48 during gastrulation, the subcellular localization of RhoGEF2 and its dependence on T48 were studied in the developing embryo. Before gastrulation, the apical surfaces of the blastoderm epithelium are dome shaped and the developing adherens junctions are located subapically. RhoGEF2 is associated with the basally located furrow canals, whereas Armadillo is found just below this site and at a subapical position of the lateral cell membranes (Kolsch, 2007).

After cellularization was completed, these distributions changed specifically in ventral cells. Even before morphological changes occurred, RhoGEF2 and Armadillo disappeared from the basal ends. Subsequently, Armadillo disappeared from its subapical site and accumulated apically. A weak association of RhoGEF2 with the apical plasma membrane was seen at this stage (Kolsch, 2007).

As cells begin to flatten apically, high levels of both RhoGEF2 and Armadillo accumulate apically. Although they concentrated in the same region of the cell, Armadillo was restricted to the cell junctions, whereas RhoGEF2 was often more enriched between these sites. Notably, movement of the adherens junctions occurred not only in constricting cells but also in the more lateral mesodermal cells that flattened and became stretched on their apical sides (Kolsch, 2007).

To examine whether these processes depend on T48, stage-selected T48 mutant embryos were stained. Loss of RhoGEF2 and Armadillo from the basal side was unaffected in these embryos, as was the apical concentration of Armadillo. The cells flatten apically and lengthen, but the absence of constrictions results in a thick placode rather than an indentation. Localization of RhoGEF2 to the apical membrane is slightly delayed and possibly reduced. T48 therefore contributes to but is not essential for the recruitment of RhoGEF2 to the apical membrane. This is consistent with the observation that furrow formation is not completely abolished, but only delayed or weakened. Therefore other mechanisms were examined that might participate in RhoGEF2 localization (Kolsch, 2007).

As in the case of T48, mutations in the Fog/Cta pathway delay but do not abolish apical constriction and furrow formation. It was therefore considered whether Fog/Cta signaling might cooperate with T48 to recruit RhoGEF2. In embryos lacking Cta, the recruitment of RhoGEF2 was weakened. Combining mutations in cta and T48 resulted in much more notable effects. These cta,T48 embryos failed to make a furrow; the lack of apical constrictions was mirrored by a failure to accumulate RhoGEF2 apically. Thus, T48 and Fog/Cta signaling act in parallel to concentrate RhoGEF2 apically (Kolsch, 2007).

Severe defects were also observed in the behavior of the adherens junctions in the double-mutant embryos. Armadillo staining disappeared from its tight subapical localization but did not reaccumulate apically. Thus, movement of the junctions is not simply mediated by a tensile force from the constricting actin cytoskeleton: an independent step of at least partial disassembly must occur. It is speculated that this might be controlled by Snail, which regulates the disassembly of cell junctions in vertebrates. It was found that the disassembly of Armadillo from the subapical position was indeed blocked in snail (but not in twist) mutant embryos. Thus, Snail acts in parallel to Twist to direct the disassembly of subapical junctions, a process to which currently unknown Twist targets may also contribute (Kolsch, 2007).

Having observed that T48 and Fog/Cta activation are required for the apical localization of RhoGEF2 and Armadillo, whether T48, like Fog/Cta signaling, was able to trigger their relocalization in other cells was also tested. Ubiquitous expression of T48 in the embryo led to a concentration of RhoGEF2 at the apical membranes of lateral cells. Armadillo localization in ectodermal cells was no longer restricted to a distinct subapical domain but extended to the apical end of the lateral membranes in many cells. When T48 was coexpressed with activated Cta, this effect was slightly enhanced, and some embryos showed morphological defects (Kolsch, 2007).

With T48, a missing factor has been found in the control cascade from transcriptional regulation by Twist to the cell biological mediators of furrow morphogenesis. Two Twist targets, Fog and T48, appear to act in separate pathways that converge on RhoGEF2, which integrates the signal to activate myosin and modify the actin cytoskeleton. This model shows the maternally supplied RhoGEF2 is largely attached to microtubules by means of EB1. The onset of Twist expression has two effects. Fog is synthesized, which triggers the activation of Cta. This in turn releases RhoGEF2 from the microtubules that, by analogy to its vertebrate homologs, may bind to Cta through its RGS domain, allowing some myosin activation and constriction. In parallel, T48 is synthesized and targeted to the apical membrane, where it acts to concentrate RhoGEF2 through its PDZ-binding motif. In the absence of Fog-mediated displacement of RhoGEF2 from EB1, T48 can probably still recruit sufficient freely diffusible RhoGEF2 to allow slow constriction. Only when both mechanisms fail are the downstream events of constriction and junction reassembly abolished completely (Kolsch, 2007).

The utilization of Gα12/13 proteins and a microtubule-bound RhoGEF have also been reported in vertebrate gastrulation. The absence of an obvious homolog of T48 in vertebrates might suggest that this element of the control mechanism is unique to Drosophila gastrulation. However, the PDZ-binding motif in Plexin-B1 is similar to that of T48 and acts during neuronal growth cone remodeling by recruiting PDZ-RhoGEF. Therefore, this mechanism of controlling cell shape may operate in a variety of systems (Kolsch, 2007).

Apical secretion in epithelial tubes of the Drosophila embryo is directed by the formin-family protein Diaphanous

Apical localization of filamentous actin (F-actin) is a common feature of epithelial tubes in multicellular organisms. However, its origins and function are not known. This study demonstrates that the Diaphanous (Dia)/Formin actin-nucleating factor is required for generation of apical F-actin in diverse types of epithelial tubes in the Drosophila embryo. Dia itself is apically localized both at the RNA and protein levels, and apical localization of its activators, including Rho1 and two guanine exchange factor proteins (Rho-GEFs), contributes to its activity. In the absence of apical actin polymerization, apical-basal polarity and microtubule organization of tubular epithelial cells remain intact; however, secretion through the apical surface to the lumen of tubular organs is blocked. Apical secretion also requires the Myosin V (MyoV) motor, implying that secretory vesicles are targeted to the apical membrane by MyoV-based transport, along polarized actin filaments nucleated by Dia. This mechanism allows efficient utilization of the entire apical membrane for secretion (Massarwa, 2009).

Apical localization of F-actin is a general feature of tubular epithelial structures. It has been observed in mammalian MDCK cells forming tubes in three-dimensional cell culture, in the cytoplasm underlying the apical membrane facing the lumen in mammalian secretory organs, such as the lacrimal gland, and in the different epithelial tubes of the Drosophila embryo. The lower level of gene duplication in Drosophila, and the ability to follow the consequences of targeted gene inactivation in the tubular structures, allowed identification of the mechanism responsible for nucleating the actin terminal web at the apical side of epithelial tube cells. This study has demonstrate that Dia, which is known to promote the formation of linear actin filaments, is responsible for producing this actin network in Drosophila embryonic tubular structures. Despite differences in the diameter and function of the different tubular organs, the polarized apical actin cables formed by Dia appear to have a common role in trafficking secretory vesicles to the apical tube surface (Massarwa, 2009).

While the role of Dia in promoting apical secretion spans the entire duration of tracheal morphogenesis, two other Formin-homology proteins act at very specific junctions of Drosophila tracheal morphogenesis. Formin 3 participates in the generation of a continuous dorsal trunk tube by promoting vesicular trafficking in the fusion cells of each metamer, perpendicular to the tube lumen. Another Formin domain protein, DAAM, promotes the organization of F-actin in rings around the circumference of the tracheal tube, at the final stages of tracheal morphogenesis (Massarwa, 2009).

It is likely that each of the three Formin domain proteins is regulated by distinct activators that are concentrated at different sites. The localized activation of Formin 3 may eventually lead to polarized vesicle movement, similar to Dia, but toward a different membrane. The activation of DAAM may be necessary for the localized synthesis of F-actin, which will modify the contours of the apical membrane, and thus define the shape of chitin layered on top. The function of Dia stands out, since it is required throughout tracheal development, and is also involved in morphogenesis of other tubular organs (Massarwa, 2009).

The mechanism of localized activation of Dia operates after apical-basal polarity of the cells has been established. Thus, no defects were observe in overall polarity in dia mutant embryos. It seems that the steps upstream to Dia activation utilize the existing polarity at multiple tiers in order to trigger Dia at a highly restricted position. The two Rho-GEF proteins, Gef2 and Gef64C, exhibit a tight apical localization in the cells forming the tubes. The single Rho1 protein, which is downstream to the Rho-GEFs, is again tightly localized to the apical surface in tubular structures. Binding of Rho1 to Dia leads to an opening of the autoinhibited form of Dia and to the formation of a Dia dimer representing the active form (Goode, 2007). Since GTP-bound Rho1 is the immediate activator of Dia, it is particularly important that Rho1 be embedded in the apical membrane, to ensure spatially restricted nucleation of actin polymerization. In C. elegans, a GEF and a Rho protein were shown to be essential for the development of the lumen of the excretory cell. It will be interesting to determine if a Dia-family protein is subsequently activated to promote secretion (Massarwa, 2009).

Dia is also apically localized, both at the mRNA and protein levels. Elimination of the dia 3'UTR demonstrated a persistence of apical protein localization, even when mRNA localization was lost, suggesting that there are two parallel and independent mechanisms for apical localization. The multiple tiers of apical localization assure that activated Dia will be highly restricted to the apical surface (Massarwa, 2009).

It is interesting to note that, while Gef2, Gef64C, Rho1, and Dia proteins are broadly expressed, partially due to maternal contribution of mRNA, they exhibit apical localization only in the tubular structures. This raises the possibility that genes that are specifically expressed in the tubular organs contribute to the apical localization. Alternatively, apical localization may rely directly on the specific phospholipid composition of the apical tube membranes. It will be interesting to determine if a common mechanism is responsible for the apical localization of the different proteins in the pathway, and if this mechanism relies on components that are restricted to the tubular organs (Massarwa, 2009).

The cellular machinery which dictates the apical localization of Rho-GEFs/Rho1/Dia appears to be in place early on. For example, expression of Dia-GFP in the trachea demonstrated apical localization of the protein already at the stage when the tracheal pits are formed. Yet, generation of the polarized actin cables by Dia, and their utilization for secretion, takes place at a later stage, and follows a stereotypic temporal order in the different tracheal branches. What triggers activation of Dia, following the apical localization of the different components? This study has demonstrated that both Gef2 and Gef64C are required to trigger Rho1, which activates Dia. While the activity of the two Rho-GEFs is similar, both have to accumulate to a critical level in order to activate the system. Thus, no secretion takes place when either of them is missing, or when each of them is present at half dose. The delay in activation of Dia and in secretion, may be explained by the time required to accumulate sufficient levels of Rho-GEF proteins. When the system was 'short circuited' by expression of activated Rho1, which was properly localized to the apical surface, Dia-dependent apical secretion was observed already at early stages of tracheal pit formation (Massarwa, 2009).

The results identify Drosophila MyoV (Didum) as a primary motor for apical trafficking of secretory vesicles along the polarized, Dia-nucleated actin cables in tubular organs. When the activity of MyoV was compromised in the tubular epithelia, apical secretion of cargos requiring Dia-generated actin cables was abolished. In contrast, since MyoV operates downstream to Dia, the actin cables themselves remained intact. An analogous role for MyoV has been recently demonstrated during trafficking of Rhodopsins to photoreceptor rhabdomers (Li, 2007). The functional link between the Dia pathway and MyoV was demonstrated by the ability of myoV RNAi to suppress constitutively activated Rho1 or Dia phenotypes. These results further support the direct link between Dia and apical secretion (Massarwa, 2009).

The polarized actin network formed via the nucleating activity of Dia can account for the final phase of secretory vesicle transport to the apical plasma membrane. Class V myosins, such as MyoV, are known to be involved in transfer of vesicles from microtubules to cortical actin networks, suggesting that polarized microtubule arrays may promote the long-range trafficking of the secretory vesicles from their sites of formation to the cell cortex. Consistent with this scenario, a polarized arrangement was demonstrated of microtubulesin tube epithelial cells, the minus ends of which are in close proximity to the apical membrane, which remains intact in the absence of Dia. The universality of this system is highlighted by similarities to polarized secretion in budding yeast, where Myo2p-mediated transport of secretory vesicles into the bud utilizes Dia-generated actin bundles as tracks, in order to deposit the compounds for polarized cell growth (Massarwa, 2009).

When early steps in the secretory pathway are compromised by reducing the activity of the COPII or COPI complexes, accumulation of cargo is observed within the cells and reduced amounts are detected in the lumen. Since these manipulations block an early and global process of secretion, all cargo vesicles are affected (Tsarouhas, 2007). However, after exit from the Golgi, it appears that distinct classes of vesicles are generated, each containing a different set of cargos, and trafficked by a distinct mechanism. One class of vesicles contains chitin-modifying enzymes (such as Verm or Serp), and is targeted to the septate junctions. When the structure of the septate junctions was compromised, these proteins failed to be secreted. Another class of vesicles may contain transmembrane proteins that are deposited in the apical membrane, such as Crb (Massarwa, 2009).

This study now uncovers a third class of cargo vesicles. Several distinct cargos that are secreted to the apical lumen rely on Dia for their secretion. These cargos include the 2A12 antigen, Pio, and the artificial rat ANF-GFP construct. In the absence of Dia, these proteins failed to be secreted to the lumen, but also did not accumulate within the epithelial tube cells. It is believed that when secretion is disrupted, the vesicles are efficiently targeted for lysosomal degradation, since a block of lysosomal targeting facilitated intracellular accumulation of vesicles that failed to be secreted. Inability to secrete Pio resulted in tracheal defects that were similar to pio mutant embryos. Additional defects of dia pathway mutant embryos, such as highly convoluted tracheal branches, may stem from the absence of additional, yet unknown, proteins in the lumen. The mechanisms underlying the incorporation of distinct cargos into different secretory vesicles, as well as the recognition of each vesicle type by different motors and trafficking scaffolds, remain unknown (Massarwa, 2009).

In conclusion, this work has uncovered a universal mechanism, which operates in very different types of tubular epithelial structures in Drosophila. The conserved feature of an apical F-actin network in tubular epithelia of diverse multicellular organisms, and the high degree of conservation of the different components generating and utilizing these actin structures, strongly suggests that this polarized secretion mechanism is broadly used across phyla. The ability to generate polarized actin cables that initiate at the apical membrane provides an efficient route for trafficking vesicles by MyoV, leading to their fusion with the apical membrane and secretion. It is likely that different pathological situations manifested in aberrant formation of epithelial structures, or their utilization for secretion once the tubular organ is formed, represent defects in different components of this pathway. For example, it was shown that mutations in MyoVa in humans disrupt actin-based melanosome transport in epidermal melanocytes (Massarwa, 2009).

RhoGef2: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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