Abelson kinase regulates epithelial morphogenesis in Drosophila

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and α-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data

The Crk adapter protein is essential for Drosophila embryogenesis, where it regulates multiple actin-dependent morphogenic events

Small Src homology domain 2 (SH2) and 3 (SH3) adapter proteins regulate cell fate and behavior by mediating interactions between cell surface receptors and downstream signaling effectors in many signal transduction pathways. The CT10 regulator of kinase (Crk) family has tissue-specific roles in phagocytosis, cell migration, and neuronal development and mediates oncogenic signaling in pathways like that of Abelson kinase. However, redundancy among the two mammalian family members and the position of the Drosophila gene on the fourth chromosome precluded assessment of Crk's full role in embryogenesis. We circumvented these limitations with short hairpin RNA and CRISPR technology to assess Crk's function in Drosophila morphogenesis. We found that Crk is essential beginning in the first few hours of development, where it ensures accurate mitosis by regulating orchestrated dynamics of the actin cytoskeleton to keep mitotic spindles in syncytial embryos from colliding. In this role, it positively regulates cortical localization of the actin-related protein 2/3 complex (Arp2/3), its regulator suppressor of cAMP receptor (SCAR), and filamentous actin to actin caps and pseudocleavage furrows. Crk loss leads to the loss of nuclei and formation of multinucleate cells. We also found roles for Crk in embryonic wound healing and in axon patterning in the nervous system, where it localizes to the axons and midline glia. Thus, Crk regulates diverse events in embryogenesis that require orchestrated cytoskeletal dynamics

Hydrothermal interaction of solid wafers of Topopah Spring Tuff with J-13 water and distilled water at 90, 150, and 250{sup 0}C, using Dickson-type, gold-bag rocking autoclaves

The Nevada Nuclear Waste Storage Investigations Project has conducted experiments to study the hydrothermal interaction of rock and water representative of a potential high-level waste repository at Yucca Mountain, Nevada. The results of these experiments help define the near-field repository environment during and shortly after the thermal period that results from the emplacement of nuclear waste. When considered in conjunction with results contained in companion reports, these results can be used to assess our ability to accelerate tests using the surface area/volume parameter and/or temperature. These rock-water interaction experiments were conducted with solid polished wafers cut from both drillcore and outcrop samples of Topopah tuff, using both a natural ground water and distilled water as the reacting fluid. Pre- and post-test characterization of the reacting materials was extensive. Post-test identification and chemical analysis of secondary phases resulting from the hydrothermal interactions were aided by using monoliths of tuff rather than crushed material. All experiments were run in Dickson-type, gold-bag rocking autoclaves that were periodically sampled at in situ conditions. A total of nine short-term (up to 66-day) experiments were run in this series; these experiments covered the range from 90 to 250{sup 0}C and from 50 to 100 bar. The results obtained from the experiments have been used to evaluate the modeled results produced by calculations using the geochemical reaction process code EQ3/6. 31 refs., 37 figs., 7 tabs

Hydrothermal Interaction of Topopah Spring Tuff With J-13 Water as a Function of Temperature

In support of the Nevada Nuclear Waste Storage Investigations Project experiments were conducted to study the hydrothermal interaction of rock and water representative of a potential repository in tuff. These experiments provided data relevant to near-field repository conditions that can be used to: assess the ability to use accelerated tests based on the SA/V (surface area/volume) parameter and temperature; allow the measurement of chemical changes in phases present in the tuff before reaction as well as the identification and chemical analysis of secondary phases resulting from hydrothermal reactions; and demonstrate the usefulness of geochemical modeling in a repository environment using the EQ3/6 thermodynamic/kinetic geochemical modeling code. Crushed tuff and polished wafers of tuff were reacted with a natural ground water in Dickson-type gold-cell rocking autoclaves which were periodically sampled under in-situ conditions. Results were compared with predictions based on the EQ3/6 geochemical modeling code. Eight short-term experiments (2 to 3 months) at 150{sup 0}C and 250{sup 0}C have been completed using tuff from both drillcore and outcrop. Long-term experiments at 90{sup 0}C and 150{sup 0}C using drillcore polished wafers are in progress. This paper will focus on the results of the 150{sup 0}C and 250{sup 0}C experiments using drill core polished wafers. 11 references, 4 figures

The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

Actin-based protrusions are important for signaling and migration during development and homeostasis. Gain- and loss-of-function and quantitative approaches are used to define differential roles for the actin elongation factors Diaphanous and Enabled in regulating distinct protrusive behaviors in different tissues during Drosophila morphogenesis.Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous’ role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure—migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells—as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative “fingerprint”—the protrusive profile—which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

The small GTPase Rap1 and the actin-junctional linker protein Canoe/afadin are essential for the initial establishment of polarity in Drosophila, acting upstream of Bazooka/Par3 and the adherens junctions. However, feedback and cross-regulation occur, so polarity establishment is regulated by a network of proteins rather than a linear pathway.The establishment and maintenance of apical–basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being “downstream” of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway

Abelson kinase acts as a robust, multifunctional scaffold in regulating embryonic morphogenesis

Abelson family kinases (Abl) are key regulators of cell behavior and the cytoskeleton during development and in leukemia. Abl's SH3, SH2, and tyrosine kinase domains are joined via a linker to an F-actin-binding domain (FABD). Research on Abl's roles in cell culture led to several hypotheses for its mechanism of action: 1) Abl phosphorylates other proteins, modulating their activity. 2) Abl directly regulates the cytoskeleton via its cytoskeletal interaction domains, and/or 3) Abl is a scaffold for a signaling complex. The importance of these roles during normal development remains untested. We tested these mechanistic hypotheses during Drosophila morphogenesis using a series of mutants to examine Abl's many cell biological roles. Strikingly, Abl lacking the FABD fully rescued morphogenesis, cell shape change, actin regulation, and viability, while kinase dead Abl, though reduced in function, retained substantial rescuing ability in some but not all Abl functions. We also tested the function of four conserved motifs in the linker region, revealing a key role for a conserved PXXP motif known to bind Crk and Abi. We propose Abl acts as a robust multi-domain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors

Regulation of Epithelial Morphogenesis by the G Protein-Coupled Receptor Mist and Its Ligand Fog

Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells, and used RNAi-based screening to identify Mist, a Drosophila G protein-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes

Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are required for the formation and maintenance of filopodia, finger-like projections at the leading edge of migrating cells that are composed of parallel actin filaments bundled by Fascin. We imaged individual fluorescently labeled Drosophila Ena molecules on both single and Fascin-bundled actin filaments in vitro. Ena stimulates actin assembly by remaining continuously associated with the barbed end and increasing the elongation rate by approximately two- to threefold. Remarkably, the frequency and length of Ena’s processive runs are enhanced on filaments within a Fascin bundle, which drives a positive feedback cycle that allows the assembly of uniformly thick filopodia-like F-actin bundles composed of multiple filaments with aligned ends