Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.     

Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.     

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.     

Serrano (Sano) Functions with the Planar Cell Polarity Genes to Control Tracheal Tube Length

Epithelial tubes are the functional units of many organs, and proper tube geometry is crucial for organ function. Here, we characterize serrano (sano), a novel cytoplasmic protein that is apically enriched in several tube-forming epithelia in Drosophila, including the tracheal system. Loss of sano results in elongated tracheae, whereas Sano overexpression causes shortened tracheae with reduced apical boundaries. Sano overexpression during larval and pupal stages causes planar cell polarity (PCP) defects in several adult tissues. In Sano-overexpressing pupal wing cells, core PCP proteins are mislocalized and prehairs are misoriented; sano loss or overexpression in the eye disrupts ommatidial polarity and rotation. Importantly, Sano binds the PCP regulator Dishevelled (Dsh), and loss or ectopic expression of many known PCP proteins in the trachea gives rise to similar defects observed with loss or gain of sano, revealing a previously unrecognized role for PCP pathway components in tube size control.     

A Highlights from MBoC Selection: Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p

Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase–formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p–Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via “polarisome” components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.     

Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1-5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.     

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.     

Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins.     

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.     

A Phosphatidylinositol-Transfer Protein and Phosphatidylinositol-4-phosphate 5-Kinase Control Cdc42 to Regulate the Actin Cytoskeleton and Secretory Pathway in Yeast

The actin cytoskeleton rapidly depolarizes in yeast secretory (sec) mutants at restrictive temperatures. Thus, an unknown signal conferred upon secretion is necessary for actin polarity and exocytosis. Here, we show that a phosphatidylinositol (PI) transfer protein, Sfh5, and a phosphatidylinositol-4-phosphate 5-kinase, Mss4, facilitate Cdc42 activation to concomitantly regulate both actin and protein trafficking. Defects in Mss4 function led to actin depolarization, an inhibition of secretion, reduced levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] in membranes, mislocalization of a pleckstrin homology domain fused to green fluorescent protein, and the mislocalization of Cdc42. Similar defects were observed in sec, myo2-66, and cdc42-6 mutants at elevated temperatures and were rescued by the overexpression of MSS4. Likewise, the overexpression of SFH5 or CDC42 could ameliorate these defects in many sec mutants, most notably in sec3Δ cells, indicating that Cdc42-mediated effects upon actin and secretion do not necessitate Sec3 function. Moreover, mutation of the residues involved in PI binding in Sfh5 led to the mislocalization and loss of function of both Sfh5 and Cdc42. Based upon these findings, we propose that the exocytic signal involves PI delivery to the PI kinases (i.e., Mss4) by Sfh5, generation of PI(4,5)P₂, and PI(4,5)P₂-dependent regulation of Cdc42 and the actin cytoskeleton.     

Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6

Epidermal Growth Factor-receptor (Egfr) signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Here we provide evidence for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90 degrees rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90 degrees. Here we show that rlt is a rotation-specific allele of the inhibitory Egfr ligand Argos and that modulation of Egfr activity shows defects in ommatidial rotation. Our data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. We provide further evidence for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway.     

An InCytes from MBC Selection: Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1⁺ as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745–2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1ΔN) or the SAM domain (pob1ΔSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1ΔN, and pob1ΔSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.     

Wdpcp,a PCP Protein Required for Ciliogenesis,Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.     

Kermit Interacts with Gαo,Vang, and Motor Proteins in Drosophila Planar Cell Polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue – the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.     

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.     

Progress and challenges in understanding planar cell polarity signaling

During development, epithelial cells in some tissues acquire a polarity orthogonal to their apical–basal axis. This polarity, referred to as planar cell polarity (PCP), or tissue polarity, is essential for the normal physiological function of many epithelia. Early studies of PCP focused on insect epithelia (Lawrence, 1966 [1]), and the earliest genetic analyses were carried out in Drosophila (Held et al., 1986; Gubb and Garcia-Bellido, 1982 [2,3]). Indeed, most of our mechanistic understanding of PCP derives from the ongoing use of Drosophila as a model system. However, a range of medically important developmental defects and physiological processes are under the control of PCP mechanisms that appear to be at least partially conserved, driving considerable interest in studying PCP both in Drosophila and in vertebrate model systems. Here, I present a model of the PCP signaling mechanism based on studies in Drosophila. I highlight two areas in which our understanding is deficient, and which lead to current confusion in the literature. Future studies that shed light on these areas will substantially enhance our understanding of the fascinating yet challenging problem of understanding the mechanisms that generate PCP.     

Syndecan-4 assists Mycobacterium tuberculosis entry into lung epithelial cells by regulating the Cdc42, N-WASP,and Arp2/3 signaling pathways

Syndecan-4 (SDC4) is a transmembrane heparin sulfate proteoglycan that regulates inflammatory responses, cell motility, cell adhesion and intracellular signaling. In this study, we found that overexpression of SDC4 promoted the infection efficiency of Mycobacterium tuberculosis (Mtb), whereas knockdown of SDC4 reduced the infection efficiency, suggesting that SDC4 assisted Mtb infection of epithelial cells. We also observed that Mtb infection affected the F-actin/G-actin ratio, which was also correlated with SDC4 expression levels. Analysis of the Cdc42, N-WASP, and Arp2/3 signaling pathways during Mtb infection revealed that knockdown of Cdc42 and N-WASP or the addition of ZCL278, Wiskostatin or CK636 (blockers of Cdc42, N-WASP, and Arp2/3, respectively) significantly exacerbated Mtb infection in lung epithelial cells. Taken together, our data indicate that SDC4 assists Mtb infection of epithelial cells by regulating the Cdc42, N-WASP, and Arp2/3 signaling pathways, which regulate the polymerization of the actin cytoskeleton.